PLATELET-INSPIRED NANOMEDICINE FOR THE HEMOSTATIC
MANAGEMENT OF BLEEDING COMPLICATIONS IN
THROMBOCYTOPENIA AND TRAUMA
by
DASHAWN A. HICKMAN
Submitted in partial fulfillment of the requirement for the degree of
Doctor of Philosophy
Department of Pathology
CASE WESTERN RESERVE UNIVERSITY
January, 2019
CASE WESTERN RESERVE UNIVERSITY
SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
DaShawn A. Hickman
candidate for the degree of Doctor of Philosophy*.
Committee Chair Nicholas Ziats, PhD
Committee Member Anirban Sen Gupta, PhD
Committee Member James Anderson, MD, PhD
Committee Member Agata Exner, PhD
Committee Member Howard Meyerson, MD
Committee Member Clive Hamlin, PhD
Date of Defense August 16, 2018
*We also certify that written approval has been obtained for any proprietary material contained therein.
Dedication
I would like to dedicate my dissertation to my family who have always supported me and encouraged me to be great, especially my parents and siblings; my friends for setting the bar high and keeping my spirits higher, the MSTP entering class of 201l and my labmates.
i Table of Contents Dedication ...... i
List of Tables ...... ix
List of Figures ...... x
Acknowledgements ...... xvi
List of Abbreviations ...... xvii
Abstract ...... xxvii
Chapter 1 : Bleeding Complications in Thrombocytopenia and Trauma ...... 1
1.1 Introduction ...... 1
1.2 Trauma ...... 2
1.3 Thrombocytopenia...... 4
1.4 Complex Mechanism of Hemostasis ...... 7
1.5 Management of Traumatic Bleeding ...... 10
1.6 Acknowledgments ...... 12
1.7 References ...... 12
Chapter 2 : Externally Administered (Topical and Intracavitary) Biomaterials and
Advanced Technologies for Management of Traumatic Bleeding ...... 28
2.1 Introduction ...... 28
2.2 Tourniquets ...... 28
2.3 Naturally derived biomaterials for hemostatic applications ...... 30
ii 2.3.1 Absorptive and passively interactive materials in hemostatic technologies ...... 31
2.3.2 Bioactive materials in hemostatic technologies ...... 35
2.4 Synthetically derived hemostatic materials ...... 52
2.5 Compression bandage technologies ...... 57
2.6 Combination systems and advanced technologies ...... 61
2.7 Hemostatic materials and technologies for intracavitary applications...... 62
2.8 Discussion ...... 63
2.9 Acknowledgments ...... 66
2.10 References ...... 66
Chapter 3 : Intravenously Administered Biomaterials and Advanced Technologies for
Management of Traumatic Non-compressible Hemorrhage ...... 110
3.1 Introduction ...... 110
3.2 Naturally derived intravascular pro-coagulant materials ...... 111
3.3 Synthetically derived pro-coagulant systems ...... 114
3.4 Materials and technologies to enhance clot strength and stability ...... 116
3.5 Materials and technologies for vascular embolization ...... 122
3.6 Natural platelet-derived systems ...... 124
3.7 Synthetic biomaterials-based platelet mimics and substitutes ...... 134
3.8 Discussion ...... 139
3.9 Acknowledgments ...... 143
iii 3.10 References ...... 143
Chapter 4 : Designing an Ideal Platelet-inspired System for the Intravenous
Management of Traumatic Bleeding...... 166
4.1 Introduction ...... 166
4.2 Current State of Traumatic Bleeding Management ...... 167
4.3 Designing an Ideal System ...... 169
4.4 Design of SynthoPlateTM Nanoparticle System ...... 171
4.5 SynthoPlateTM In Vitro Evaluation ...... 173
4.6 In Vivo Evaluation of SynthoPlateTM In Thrombocytopenia Model ...... 174
4.7 Discussion ...... 178
4.8 Acknowledgments ...... 180
4.9 References ...... 181
Chapter 5 : Intravenous administration of synthetic platelets (SynthoPlate) in a mouse liver injury model of uncontrolled hemorrhage improves hemostasis ...... 189
5.1 Introduction ...... 189
5.2 Materials and Methods ...... 190
5.2.1 Animals ...... 190
5.2.2 SynthoPlate Manufacture ...... 190
5.2.3 Liver Laceration Model of Uncontrolled Hemorrhage and SynthoPlate Evaluation ...... 191
5.2.4 In Vivo Biodistribution of SynthoPlate Particles ...... 193
5.2.5 Statistics...... 194
iv 5.3 Results ...... 194
5.3.1 SynthoPlate Transfusion as a Pretreatment Results in Decreased Blood Loss and Improved
Hemodynamics During Acute, Severe Hemorrhagic Shock ...... 194
5.3.2 Systemic Biodistribution SynthoPlate Particles Following Liver Laceration ...... 196
5.3.3 SynthoPlate Particles Reduce Blood Loss and Improve Hemodynamics in Mice in
Hemorrhagic Shock ...... 198
5.4 Discussion ...... 198
5.5 Authorship...... 203
5.6 Acknowledgement ...... 203
5.7 Disclosure ...... 203
5.8 References ...... 204
Chapter 6 : Intravenous synthetic platelet (SynthoPlateTM) nanoconstructs reduce bleeding and improve ‘golden hour’ survival in a porcine model of traumatic arterial hemorrhage ...... 207
6.1 Introduction ...... 207
6.2 Results ...... 212
6.2.1 SynthoPlateTM stability in storage ...... 212
6.2.2 SynthoPlateTM sterilization and its effects ...... 213
6.2.3 SynthoPlateTM cytotoxicity analysis ...... 217
6.2.4 In vivo Safety and Biodistribution Studies in Pigs ...... 217
6.2.5 Hemostatic Efficacy Evaluation in Femoral Artery Bleeding Model ...... 221
6.3 Discussion ...... 226
6.4 Materials and Methods ...... 230
v 6.4.1 Materials ...... 230
6.4.2 Manufacture of SynthoPlateTM ...... 232
6.4.3 Long term Storage-in-Suspension Studies ...... 233
6.4.4 Studies on SynthoPlateTM sterilization and its biofunctional effects ...... 233
6.4.5 In vitro cytotoxicity assay with SynthoPlateTM ...... 235
6.4.6 In vivo Safety and Biodistribution Studies in Pigs ...... 236
6.4.7 Femoral Artery Bleeding Model in Pigs ...... 239
6.4.8 Hemostatic Efficacy Evaluation in Femoral Artery Bleeding Model ...... 240
6.4.9 Statistical Analysis ...... 241
6.5 Acknowledgements...... 241
6.6 Author Contributions ...... 242
6.7 Competing Financial Interests ...... 242
6.8 References ...... 243
Chapter 7 : Trauma-targeted Delivery of Tranexamic Acid for Augmenting Hemostasis
...... 255
7.1 Abstract ...... 255
7.2 Introduction ...... 257
7.3 Materials and Methods ...... 261
7.3.1 Materials ...... 261
7.3.2 TXA-loaded targeted nanovesicle (TTNV) development and characterization ...... 262
7.3.3 In vitro analysis of the effect of free TXA and TTNVs on tPA-induced fibrinolysis ...... 264
7.3.4 In vivo safety of FMP-decorated empty nanovesicles (TNVs) and TXA-loaded TTNVs ...... 266
7.3.5 Development of rat liver hemorrhage traumatic injury model ...... 267
vi 7.3.6 Evaluation of TTNVs in rat liver hemorrhage model ...... 268
7.3.7 Statistical analysis ...... 269
7.4 Results ...... 270
7.4.1 Characterization of TTNVs and TXA release kinetics ...... 270
7.4.2 ROTEM analysis of the effect of TXA-loaded liposomes in human blood ...... 271
7.4.3 In vivo safety evaluation of various doses of FMP-decorated liposomes ...... 274
7.4.4 Effect of clot-targeted delivery of TXA-loaded nanovesicles in rat liver injury model ...... 276
7.5 Discussion ...... 280
7.6 Conclusion ...... 284
7.7 Acknowledgement ...... 285
7.8 References ...... 286
Chapter 8 : Design and Administration of Nanoparticles to Prevent Pseudoallergic
Responses in Intravenous Hemostatic Management of Bleeding ...... 296
8.1 Introduction ...... 296
8.2 Materials and Methods ...... 298
8.2.1 Materials ...... 298
8.2.2 Manufacture of SynthoPlateTM 1.0 ...... 299
8.2.3 Manufacture of SynthoPlateTM 1.5 ...... 300
8.2.4 Manufacture of SynthoPlateTM 2.0 ...... 300
8.2.5 In vivo Safety Studies in Pigs...... 300
8.2.6 Femoral Artery Bleeding Model in Pigs ...... 303
8.2.7 Hemostatic Efficacy Evaluation in Femoral Artery Bleeding Model ...... 304
8.2.8 Statistical Analysis ...... 304
vii 8.3 Results ...... 305
8.3.1 Synthesis of SynthoPlate PEG variants ...... 305
8.3.2 Evaluation of SynthoPlate PEG variants ...... 305
8.3.3 Evaluation of SynthoPlate Administration Protocol Safety ...... 309
8.3.4 Evaluation of SynthoPlate Administration Protocol Efficacy ...... 310
8.4 Discussion ...... 312
8.5 References ...... 314
Chapter 9 : Conclusion and Future Directions ...... 319
9.1 Conclusion ...... 319
9.2 Future Directions ...... 322
9.2.1 Nanovesicle Design ...... 322
9.2.2 Nanoparticle Evaluation ...... 323
9.2.3 Nanoparticle Technology Translation ...... 324
9.3 References ...... 324
Bibliography ...... 327
viii List of Tables
Table 1-1. Classification of hemorrhagic shock. Reproduced with permission from
Cannon, JW10, Copyright Massachusetts Medical Society...... 4
Table 1-2. Principles of damage control resuscitation. Reproduced with permission from Cannon, JW10, Copyright Massachusetts Medical Society...... 5
Table 2-1 Topical and Externally Administered Hemostatic Biomaterials from
Natural Sources ...... 50
Table 2-2 Topical and Externally Administered Hemostatic Biomaterials from
Synthetic Sources ...... 59
Table 8-1. Characteristics of SynthoPlate PEG variants ...... 299
ix List of Figures
Figure 1-1. Pathobiology of Hemorrhagic Shock...... 2
Figure 1-2 Schematic of the complex mechanism of blood vessel hemostasis ...... 8
Figure 2-1 Representative chemical structures of cotton (cellulose) biopolymers
and its derivatives ...... 34
Figure 2-2 Schematic of concomitant roles of thrombin and fibrin(ogen) in
propagating the formation of hemostatic clots via activation of platelets and
formation of fibrin mesh...... 36
Figure 2-3 Multiscale schematic representation of fibrillar collagen structure ...... 40
Figure 2-4 Chemical structures of some polysaccharide polymers ...... 48
Figure 3-1. Selected results from hemostasis-relevant studies carried out with
PolyP-coated silica particle systems (A and B) and thrombin-loaded CaCO3-based
particles mixed in TXA-NH3+ (C, D and E) ...... 117
Figure 3-2 Schematic of fibrinogen conversion to fibrin and assembly of cross-
linked fibrin biopolymeric mesh catalyzed by thrombin (FIIa) and FXIIIa, and
hemostatic materials and technologies inspired by these mechanisms...... 120
Figure 3-3 Selected results from hemostatic studies carried out with platelet-like
microgel particles (PLPs, A-D) and with polySTAT injectable polymer systems (E-
G) ...... 122
Figure 3-4 Schematic of platelet’s injury site-selective adhesion and aggregation
mechanism, and various hemostatic technologies inspired by these mechanisms...... 125
Figure 3-5 Selected results from hemostatic studies with infusible platelet
membrane (IPM) technology ...... 128
x Figure 3-6 Selected results from studies involving technologies that leverage platelet’s bleeding site-selective adhesion mechanisms ...... 131
Figure 3-7 Selected results from studies carried out with various fibrinogen-coated particle designs...... 133
Figure 3-8 Selected results from studies carried out with various particle platforms surface-decorated with fibrinogen-relevant RGD or H12 peptides to mimic platelet aggregation mechanism ...... 138
Figure 4-1. Platelet aggregometry studies where undecorated latex beads, or H12 peptide-decorated latex beads, or latex beads co-decorated with H12 peptides and rGPIbα fragments were added to ADP-activated platelets ...... 170
Figure 4-2. Manufacture and characterization of SynthoPlate constructs...... 171
Figure 4-3. Ex vivo aggregometry studies for SynthoPlate with mouse platelets...... 172
Figure 4-4. Effect of SynthoPlate on secondary hemostasis ...... 174
Figure 4-5. Development of thrombocytopenia model in mouse and evaluation of
SynthoPlate capability in correcting tail transection bleeding time in thrombocytopenic mouse...... 175
Figure 4-6. Characterization of SynthoPlate‐mediated hemostatic clot in transected tail of thrombocytopenic mouse...... 176
Figure 4-7. Evaluation of systemic pro‐coagulant risk and biodistribution of
SynthoPlate in mice...... 177
Figure 5-1 Experimental timeline, liver laceration model, and SynthoPlate mechanism ...... 192
xi Figure 5-2 Analysis of SynthoPlate pretreatment on blood loss and hemodynamics
during hemorrhagic shock...... 195
Figure 5-3 In vivo biodistribution of SynthoPlate particles ...... 197
Figure 5-4 Analysis of SynthoPlate transfusion on blood loss and hemodynamics
as a rescue strategy in acute, hemorrhagic shock ...... 199
Figure 6-1 Schematic representation of SynthoPlateTM design and mechanism ...... 209
Figure 6-2 Lipid-to-peptide bioconjugation schemes (along with corresponding
chemical structures) for synthesizing DSPE-PEG-VBP, DSPE-PEG-CBP and
DSPE-PEG-CMP molecules, that were used to manufacture the SynthoPlateTM
nanoconstruct...... 210
Figure 6-3 [A] SynthoPlateTM diameter analysis over a 6-month period and [B]
Analysis after sterilization with filtration or E-beam. [C] Representative fluorescent images and quantitative analysis of surface-averaged fluorescence intensity of adhered SynthoPlateTM. [D] Aggregometry analysis ...... 212
Figure 6-4 Representative Dynamic Light Scattering (DLS) data of SynthoPlateTM
size distribution characterization over a 6-month period ...... 213
Figure 6-5 Bacteriostatic and fungistatic analysis of SynthoPlateTM suspension
exposed to E-beam sterilization and challenged with 6 different organisms ...... 214
Figure 6-6 Representative Lumi-Aggregometry raw data from studies on
SynthoPlateTM (fresh unsterilized versus sterilized) interaction with platelets
(resting or agonist-activated) in PRP ...... 215
Figure 6-7 MTT-based metabolic activity analysis data of human 3T3 fibroblasts
in culture incubated with SynthoPlateTM ...... 216
xii Figure 6-8 [A] Average vitals and [B] average blood lab values over the course of the experiment of uninjured pigs upon administration of unmodified particles or
SynthoPlateTM [C] Analysis of C3:C3a plasma concentration ratios in blood [D]
Biodistribution analysis from harvested organs...... 218
Figure 6-9 Representative hematoxylin and eosin (H&E) stained histology images
(32x magnification) of organ samples from pigs treated with saline, unmodified particles or SynthoPlateTM particles ...... 220
Figure 6-10 Schematic representation of pig femoral artery hemorrhage model setup ...... 221
Figure 6-11 Hemostatic efficacy analysis in injured pigs ...... 222
Figure 6-12 Schematic representation and representative images of hemostasized injury site in the femoral artery of pigs treated with SynthoPlateTM or saline or control particles: [A] Representative hematoxylin and eosin (H&E), [B] bright field, and [C-E] fluorescent images of the site of injury (transected artery) with the injured vessel components and hemostatic clot in view for injured pig treated with
SynthoPlateTM ...... 224
Figure 6-13[A] Complement (C3:C3a) analysis data on drawn blood from pigs and
[B] biodistribution data from pigs subjected to femoral artery injury and administered intravenously with unmodified particles or SynthoPlate nanoconstructs...... 225
Figure 6-14 [A] Representative ex vivo Lumi-aggregometry analysis data and [B]
ROTEM analysis data (CT, MCF and A10 parameters) of blood samples drawn
xiii from pigs after being subjected to femoral artery injury and administered
intravenously with saline or unmodified particles or SynthoPlate nanoconstructs...... 227
Figure 7-1. Cellular and molecular components of hemostasis regulation ...... 258
Figure 7-2. Ascorbic Acid-based reaction for assaying TXA, and representative
calibration curve using this assay, based on which TXA release kinetics from
nanovesicles was estimated...... 264
Figure 7-3. Representative instrument set-up, characteristic profile, actual
instrument and relevant measured parameters in Rotational Thromboelastometry
(ROTEM) analysis of whole blood ...... 265
Figure 7-4. Representative confocal images showing red fluorescent FMP- decorated nanovesicles can bind and localize with activated platelets within
crosslinked fibrin-rich clots formed from PRP in presence of thrombin ...... 269
Figure 7-5. [A] Schematic of the manufacturing process of TTNVs [B] Envisioned targeted delivery of TXA at the trauma site from TTNVs [C] Representative DLS characterization results [D] Representative encapsulation efficiency of TXA in
TTNVs [E] Representative release profile of TXA from TTNVs ...... 271
Figure 7-6. ROTEM analysis of clot characteristics of whole blood spiked with tPA and treated with saline (no TXA), free TXA or TXA loaded within nanovesicles ...... 272
Figure 7-7. In vivo safety evaluation with TNVs as well as TTNVs ...... 274
Figure 7-8. [A] Schematic of rat liver injury hemorrhage model set-up [B] Post- injury vitals of animals [C] Blood loss analysis post-injury and treatment
xiv administration [D&E] 1-hr and 72-hr survival analysis post-injury and treatment administration ...... 275
Figure 7-9. Representative images from histopathologic analysis (H & E staining) of excised tissue sections from liver injured rats administered with various treatments ...... 277
Figure 7-10. Representative confocal microscopy images of immunostained tissue sections from injured liver site ...... 280
Figure 8-1 Schematic representation of pig femoral artery hemorrhage model setup
...... 303
Figure 8-2 Vital analysis in safety pigs administered SynthoPlate PEG variants ...... 307
Figure 8-3 Images of pig skin responses after SynthoPlate PEG variant administration ...... 308
Figure 8-4 Analysis of C3a plasma concentration in blood drawn from pigs ...... 309
Figure 8-5 Vital analysis in safety pigs administered SynthoPlate 2.0 at 50ml
(bolus) or 500ml (infusion) ...... 310
Figure 8-6 Vital analysis in injury pigs administered SynthoPlate 2.0 at 50ml
(bolus) or 500ml (infusion) ...... 311
Figure 8-7 Hemostatic efficacy analysis in injured pigs ...... 312
xv Acknowledgements
I would like to thank Dr. Anirban Sen Gupta for allowing me to join his lab, for supporting me in carrying out this great research during my PhD and for being a better mentor than I could have asked for. I would like to acknowledge my committee members past and present
(Drs. Nicholas Ziats, James Anderson, Agata Exner, Howard Meyerson, Clive Hamlin,
Nicole Steinmetz and Jeffrey Capadona) for their advice and guidance throughout this entire process. I am grateful to the Medical Scientist Training Program, and American
Heart Association for supporting me during my PhD.
xvi List of Abbreviations
Abbreviation Full Name
A10 amplitude 10 minutes after CT
AAAS American Association for the Advancement of Science
ADP adenosine di-phosphate
Ala alanine (A)
Alb albumin
ANOVA analysis of variance
aPC activated protein C
APTT activated partial thromboplastin time
Arg arginine (R)
Asp aspartic acid (D)
ATLS Advanced Trauma Life Support
BCOP blood colloidal osmotic pressure
BEH ethylene bridged hybrid
BL black
bpm beats per minute
bpm breaths per minute
BSA bovine serum albumin
C-A-T® Combat Application Tourniquet
Ca++ Calcium
CARPA complement activation-related pseudoallergy
xvii CBP collagen binding peptide
CD cluster of differentiation
cDNA complementary deoxyribonucleic acid
CFT clot formation time
CO2 carbon dioxide
CP control particles
CRASH-2 Clinical Randomization of an Antifibrinolytic in Significant Hemorrhage 2 trial CT clotting time
Cys cystine (C)
DAPI 4′,6-diamidino-2-phenylindole
DFSD Dry Fibrin Sealant Dressing
DHPE dihexadecanoyl-sn-glycero-3-phosphoethanolamine
DIC disseminated intravascular coagulation
dL deciliter
DLS dynamic light scattering
DMEM Dulbecco’s Modified Eagle Medium
DMSO dimethyl sulfoxide
DSPC 1,2-Distearoyl-sn-glycero-3-phosphocholine
DSPE 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine
EACA e-aminocaproic acid
e-beam electron beam
EC endothelial cell
xviii EDTA ethylenediamine tetraacetic acid
EKG electrocardiogram
ELAD elastic adhesive
ELISA enzyme-linked immunosorbent assay
EMT Emergency Medical Tourniquet
EU endotoxin units
FBS fetal bovine serum
FDA Federal Drug Administration
FDPs fibrin degradation products
FFP fresh frozen plasma
Fg fibrinogen
FII coagulation factor II/ prothrombin
FIIa activated coagulation factor IIa/ thrombin
FIII coagulation factor III/ tissue factor/thrombokinase/ thromboplastin
FITC fluorescein isothiocyanate
FIX coagulation factor IX
FIXa activated coagulation factor IX
FMP fibrinogen mimetic peptide
FV coagulation factor V
FVa activated coagulation factor V
FVII coagulation factor VII
FVIIa activated coagulation factor VII
xix FVIII coagulation factor VIII
FX coagulation factor X
FXa activated coagulation factor X
FXI coagulation factor XI
FXIa activated coagulation factor XI
FXIII coagulation factor XIII
FXIIIa activated coagulation factor XIII
Gln glutamine (Q)
Glu glutamic acid (E)
Gly glycine (G)
GMP good manufacturing practice
GP glycoprotein
GRF gelatin-resorcin-formalin
H&E hematoxylin and eosin
Hct hematocrit
Hg hemoglobin
His histidine (H)
HLA human leukocyte antigen
HPMA N-(2-Hydroxypropyl) methacrylamide
HR heart rate
HUS hemolytic urine syndrome
I.V. Intravenous
xx IACUC Institutional Animal Care and Use Committee
ICU Intensive Care Unit
IgE immunoglobulin E
Ile isoleucine (I)
INR international normalized ratio
IPM infusible platelet membrane
IRB Institutional Review Board
ITP Immune Thrombocytopenia
kg kilograms
L Liter
LAL limulous ameobocyte lysate
Leu leucine (L)
LI30 lysis index after 30 minutes
Lys lysine (K)
mal maleimide
MALDI-TOF matrix assisted laser desorption ionization-time of flight
MAP mean arterial pressure
MATTERs Military Application of Tranexamic Acid in Trauma Emergency Resuscitation study MCF maximum clot firmness
MCHC mean corpuscular hemoglobin concentration
MCV fL mean corpuscular volume in femtoliters
mg miligram
xxi min minutes
ml mililiters
ML maximum lysis
mmHg milimeters of mercury
mPEG methoxy(polyethylene glycol)
mRNA messenger ribonucleic acid
MW molecular weight
MWCO molecular weight cutoff
NHLBI National Heart Lung and Blood Institute
NHSMA N-hydroxysuccinimide methacrylate
NIGMS National Institute of General Medical Sciences
NIH National Institutes of Health
nm nanometer
NS normal saline
oC degrees Celsius
ORC oxidized regenerated cellulose
PAI-1 plasminogen activator inhibitors-1
PAI-2 plasminogen activator inhibitors-2
PAMPer Prehospital Air Medical Plasma clinical trial
PaO2 partial pressure of oxygen
PATCH Pre-hospital Anti-fibrinolytic for Traumatic Coagulopathy and Haemorrhage
xxii PBS phosphate buffered saline
PC platelet concentrates
PEG poly (ethylene glycol)
PEO poly (ethylene oxide)
PF4 platelet factor 4
PGA poly (glycolic acid)
Phe phenylalanine (F)
Plg plasminogen
PLGA poly (lactic co-glycolic acid)
PLL poly (L-lysine)
PLPs platelet-like particles
Plt platelet
pNIPAm- poly (N-isopropyacrylamide-co-acrylic acid) AAc poly-HEMA poly (2-hydroxyethyl methacrylate)
PolyP polyphosphate
PoyP-SNP PolyP loaded in silica nanoparticles
PPO poly (propylene oxide)
PPP platelet poor plasma
Pro proline (P)
PROPPR Pragmatic, Randomized, Optimal, Platelet and Plasma Ratios clinical trial
PRP platelet rich plasma
PS penicillin-streptomycin
xxiii PS phosphatidylserine
PT prothrombin time
QC-ACS Quikclot-Advanced Clotting Sponge
QCG Quikclot Combat Gauze
R&D research and development
RBC red blood cells
RDH Rapid Deployment Gauze
RDW-CV red blood cell distribution width- coefficient of variation
RES reticulo-endothelial system
RGDS arginine-glycine-aspartic acid-serine
rGP recombinant glycoprotein
RhB rhodamine B
RMT Ratcheting Medical Tourniquet
ROTEM rotational thromboelastometry
rpm revolutions per minute
SaO2 oxygen saturation
SC Synthocyte particles
SD standard deviation
SDS sodium dodecyl sulfate
Ser serine (S)
serpin serine protease inhibitor
SK streptokinase
xxiv SMC smooth muscle cell
SNP silica nanoparticles
SOF-TT Special Operations Forces Tactical Tourniquet
sPLA2 secreted phospholipase A2
SpO2 peripheral capillary oxygen saturation
STAAMP Study of Tranexamic Acid during Air Medical Prehospital Transport
TACTIC Trans-agency Collaboration for Trauma Induced Coagulopathy consortium
TAFI thrombin-activated fibrinolysis inhibitor
TBS tris-buffered saline
TCCC Tactical Combat Casualty Care
TCNVs TXA-loaded control (untargeted) nanovesicles
TCP thrombocytopenia
TEMPO 2,2,6,6-tetramethylpiperidine-1-oxyl radical
TF tissue factor
THPTA tris(3-hydroxypropyltriazolylmethyl)amine
Thr threonine (T)
TNV targeted nanovesicles
tPA tissue plasminogen activator
Trp tryptophan (W)
TTNV TXA-loaded targeted nanovesicles
TTP thrombotic thrombocytopenic purpura
TXA tranexamic acid
xxv Tyr tyrosine (Y)
U.S. United States
g microgram
UHCMC𝜇𝜇 University Hospitals Cleveland Medical Center
uPA urokinase
UPLC ultra high performance liquid chromatography
USA United States of America
USAISR US Army Institute of Surgical Research
UV ultraviolet
Val valine (V)
VBP von Willebrand Factor binding peptide
vWF von Willebrand Factor vWF-A1
WBC white blood cells
WHO World Health Organization
WT wild type
WWII World War II
xxvi Platelet-inspired Nanomedicine for the Hemostatic Management of Bleeding
Complications in Thrombocytopenia and Trauma
Abstract
by
DASHAWN A. HICKMAN
Traumatic non-compressible hemorrhage and coagulopathy remain leading causes of civilian and military mortalities, especially in pre-hospital and limited resource scenarios.
Patients with thrombocytopenia are at an even higher risk due to their low number of platelets, a major blood component involved in clotting. Current clinical strategy to treat this involves massive transfusion of whole blood or blood components (e,g. RBC: platelet: plasma at 1:1:1 ratio), but such blood products present issues of limited availability and portability, high risk or contamination and short shelf-life, that substantially limit their widespread use in resource-limited scenarios. Therefore, there remains a significant need for intravenous hemostatic products that allow hemorrhage control and coagulopathy mitigation, while avoiding the above issues associated with blood-derived products. To address this need, my research has focused on developing an I.V.-administrable platelet- inspired nanomedicine system for the hemostatic management of bleeding complications in thrombocytopenia and trauma. To this end, the primary focus of this dissertation is on evaluating the mechanism, safety and efficacy of platelet-inspired nanomedicine systems
xxvii in the hemostatic management of clinically relevant bleeding complications. My overall
hypothesis is that the design of nanoscale delivery systems with platelet-inspired
heteromultivalent surface-modifications, can enable injury site-specific biointeractions and
drug delivery to enhance platelet-mediated hemostatic processes, leading to decreased
blood loss and increased survival. I have tested this hypothesis in two platelet-inspired
nanomedicine designs: (i) in a synthetic nanoparticle based functional mimic of platelets
(termed SynthoPlateTM) that can leverage and amplify the body’s physiological clotting
mechanisms specifically at the bleeding site and (ii) in a platelet-targeted nanovehicle for
injury-site selective delivery of hemostasis augmenting drugs. The SynthoPlateTM
technology was designed and evaluated in (i) tail-bleeding model in thrombocytopenic mice, (ii) femoral artery injury model in pigs, and (iii) liver resection hemorrhage model in mice, while the injury site-targeted hemostatic drug delivery strategy was evaluated in
liver resection hemorrhage model in rats. Beyond this, I have also investigated ways to
refine nanomedicine design to improve systemic safety, by altering surface charge and
hydrophilicity, as well as modulating the administration protocol. In summary, my research
has established the potential for platelet-inspired nanomedicines for the hemostatic
management of bleeding complications. I believe that these nanomedicine technologies
have significant potential for clinical translation in the future phases of this research.
xxviii Chapter 1: Bleeding Complications in Thrombocytopenia and Trauma
Some content based on: Hickman DA, Pawlowski CL, Sekhon UDS, Marks J, Gupta AS. Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding. Adv Mater. 2017 Nov 22. doi: 10.1002/adma.201700859.
1.1 Introduction
Blood, a fluid connective tissue composed of red blood cells (RBCs), white blood cells
(WBCs), platelets, and non-cellular liquid (plasma) containing salts, nutrients and proteins,
is present at a volume of approximately 5 liters in the average human body (70 kg body
weight). Blood is responsible for transport of gases and nutrients to tissues as well as
providing immune surveillance and hemostatic responses as needed. Hence, loss of blood
can result in a variety of pathologic scenarios that can lead to tissue morbidities and
mortalities. For example, in traumatic injuries in both battlefield and civilian conditions,
significant blood loss from truncal, junctional and internal non-compressible injuries can
result in significant pre-hospital (and potentially preventable) mortalities stemming from
hypothermia, coagulopathy, infection, acidosis and multiple organ failure1–6. Also, certain
congenital or disease-associated conditions (e.g. coagulation factor deficiencies and
platelet dysfunctions) as well as drug-induced effects (e.g. bone marrow suppression due to chemotherapy and radiotherapy in cancer patients) can put certain patient populations at high bleeding risks7–9. Therefore, management of various bleeding complications remains
a highly significant clinical area, and substantial interdisciplinary research efforts are
focused on the development of materials and technologies for efficient hemostatic
management of bleeding.
1
Figure 1-1. Pathobiology of Hemorrhagic Shock. Reproduced with permission from Cannon, JW10, Copyright Massachusetts Medical Society.
1.2 Trauma
Trauma is an injury that can lead to severe disability or death and these injuries can vary
in severity and location. As a leading cause of mortality worldwide with a U.S. morbidity
of over 45 million each year, trauma represents 30% of ICU admissions11,12. Trauma is
expected to rise to the third leading cause of disability worldwide by 203013. Mechanisms
2 of trauma include motor vehicle collisions, motorcycle accidents, jumps/falls greater than
20 feet, stabbings, gunshot wounds, plane crashes, blast injuries and being crushed. An
analysis of over 500 prehospital deaths from trauma revealed that ~30% of the injuries
were potentially survivable given current in-hospital treatment options14. Of those
potentially survivable deaths, more than half of the patients died from hemorrhage which
lead to shock14,15. Hemorrhagic shock is a severe loss of blood that leads to poor tissue perfusion and oxygen delivery. On a cellular level, the lack of oxygen forces cells to switch to anaerobic metabolism which leads to the accumulation of lactic acid, inorganic phosphates and free radicals16. If reperfusion does occur, this could lead to ischemia- reperfusion injury by flushing this build-up of anaerobic metabolic by-products to healthy tissue17. If normal blood flow is not re-established, the cells will eventually undergo
necrosis and apoptosis. On a tissue level, this hypoperfusion can lead to end-organ damage and multi-organ failure. Figure 1-1 depicts how massive loss of blood causes simultaneous activation of the hemostasis and fibrinolytic systems, and compensatory mechanisms that lead to the phenotype of coagulopathy, hypothermia, and acidosis which result in even more dysregulation and eventual death.10
Four classes of shock are described by the Advanced Trauma Life Support (ATLS) manual
produced by the American College of Surgeons (Table 1-1). Class 1 hemorrhage is defined
as having blood volume loss up to 15% with a normal blood pressure, pulse pressure and
respiratory rate. Class II hemorrhage is a 15-30% blood loss with tachycardia (100-
120bpm), tachypnea (20-30bpm), normal blood pressure and narrowed pulse pressure.
Class III involves 30-40% blood loss, tachycardia (120-140bpm), tachypnea (30-40bpm),
3
Table 1-1. Classification of hemorrhagic shock. Reproduced with permission from Cannon, JW10, Copyright Massachusetts Medical Society.
confusion. Class IV hemorrhage patients have lost more than 40% of their blood volume
leading to a significant drop in blood pressure and mental status with a compensatory
increase in heart rate (greater than 140bpm), a narrowed pulse pressure, and tachypnea
(greater than 35bpm). They will also present with cold and pale skin and a delayed capillary
refill18. Management of trauma patients is done on a case by case basis, but major priorities
for all patients include volume resuscitation and hemorrhage control (Table 1-2). Current methods emphasize the use of red blood cells, plasma and platelets10.
1.3 Thrombocytopenia
Risk of hemorrhage in trauma is increased in patients with thrombocytopenia.
Thrombocytopenia is a diagnosis given with a platelet count below the lower limit of
normal (<150,000/µL [150 x 109/L] for adults). There are three levels of thrombocytopenia; mild (platelet count 100,000 to 150,000/µL), moderate (50,000 to 99,000/µL), and severe
(<50,000/µL)19. While a normal platelet count range is 150,000 to 450,000/µL with means
of 266,000 and 237,000/µL for men and
4 Table 1-2. Principles of damage control resuscitation. Reproduced with permission from Cannon, JW10, Copyright Massachusetts Medical Society.
women respectively20, most individuals have little variation in their platelet count
(differences in platelet count greater than 98,000/µL occurred in less than 0.1% of
people)20. This wide population range and narrow individual range is important to note
because a small proportion of the population (2.5%) will have a baseline platelet count
lower than normal range, but will have no clotting issues. At the same time, an individual
can have a clinically significant decrease in platelet count and still be in the normal
population normal range.
Platelets are derived from megakaryocytes in the bone. Major causes of thrombocytopenia
include decreased platelet production in the bone marrow; peripheral platelet destruction
5 or consumption; dilution; and sequestration. Bone disorders that can be caused by nutrient deficiencies, myelodysplastic syndromes, infections and sepsis that lead to dysregulation
of megakaryocytes can lead to decreases in platelet production21. Platelet destruction can
be caused by antibodies in immune thrombocytopenia (ITP). ITP is an autoimmune disease
where the body creates antibodies targeting its own platelets leading to their destruction by
the body’s immune cells. This mechanism can also be drug induced by drugs such as
heparin and quinine22. Drugs and treatments such as chemotherapy or radiation can
suppress the bone marrow and lead to low platelet counts. Platelets can be consumed by
thrombus in disorders such as disseminated intravascular coagulation (DIC), thrombotic
thrombocytopenic purpura (TTP), and hemolytic urine syndrome (HUS) where
dysregulated coagulation leads to depletion of circulating platelets. Massive transfusions
can also lead to the dilutions of platelets and a low platelet count. About 1/3 of the blood
platelets reside in the spleen and in cases of splenomegaly, the number of platelets in the
spleen can increase, decreasing the total amount of circulating platelets, without decreased
the total platelet count leading to thrombocytopenia23.
There are a number of principles used to guide the management of patients with thrombocytopenia depending on the etiology of their low platelet count, but one consistent principle is in the emergency management of bleeding. Critical bleeding in a patient with severe thrombocytopenia requires immediate platelet transfusion24.
6 1.4 Complex Mechanism of Hemostasis
The word ‘hemostasis’ was coined by ancient Greeks from the terms haíma (meaning blood) and stasis (meaning stoppage) to describe the phenomenon of blood stagnation when alum came in contact with a wound25. Although scattered evidences of utilizing blood products and application of coagulation/hemostasis concepts are found in ancient Greek and Roman history, as well as in reports of transfusion and organ transplants around late
18th and early 19th centuries, the modern concept of hemostatic mechanisms is credited to the seminal report written by Paul Morawitz in 190526,27. In his report, Morawitz emphasized the role of platelets, ‘thrombokinase’ (now known as tissue factor), calcium, prothrombin and fibrinogen in promoting blood coagulation. Later in the 20th century, additional coagulation factors were identified and characterized, and the concepts of coagulation being guided by a cascade of enzymes and co-factors (i.e. the intrinsic and extrinsic coagulation cascades) leading to the final output of fibrin formation (common pathway of cascades) were put forward28,29. According to the current (and still evolving) understanding of the process, the body’s natural mechanisms of hemostasis are rendered by a complex, spatio-temporally regulated sequence of responses involving a combination of cellular (e.g. platelets and tissue factor-bearing cells) and plasma (e.g. coagulation factors) components, as depicted in the schematic of Figure 1-2.
In the absence of injury, healthy endothelial cells lining the luminal wall of blood vessels avoid blood clotting by secretion of heparin-like molecules, thrombomodulin, nitric oxide and prostacyclin, as well as, sterically hindering adsorption of clot-relevant proteins on the vessel wall due to presence of endothelial glycocalyx. Tissue injury and bleeding result in
7
Figure 1-2 Schematic of the complex mechanism of blood vessel hemostasis. Vessel injury can lead to endothelial activation and denudation resulting in secretion and deposition of von Willebrand Factor (vWF) and exposure of collagen at the injury site, as well as, exposure of tissue factor (TF) bearing cells at the site; vWF and collagen exposure allows platelet adhesion and activation, while TF exposure allows extrinsic pathway of coagulation to propagate and produce moderate amounts of thrombin (FIIa) that activates other coagulation factors in the intrinsic pathway; activated platelets aggregate via fibrinogen (Fg) mediated interaction with platelet surface integrin GPIIb-IIIa to form a platelet plug (primary hemostasis) that staunches bleeding; the surface of aggregated active platelets exposes negatively charged phospholipids that allow co-localization and further activation of coagulation factors to form the prothrombinase (FVa + FXa + FII) complex in presence of calcium (Ca++), leading to amplified generation of thrombin (FIIa) that breaks down fibrinogen (Fg) to fibrin; fibrin self-assembles and undergoes further crosslinking by action of FXIIIa to form a dense biopolymeric mesh that forms the hemostatic clot and arrests flow of blood components (secondary hemostasis).
endothelial damage, leading to vasoconstriction to reduce blood flow out of the injury site and secretion/exposure of pro-coagulant proteins and factors. Platelets rapidly respond to the bleeding site by undergoing adhesion (primarily to von Willebrand Factor and sub- endothelial collagen), activation and inter-platelet aggregation at the site to form the platelet plug30–33, a process commonly known as ‘primary hemostasis’. In tandem, exposed
8 sub-endothelial collagen, von Willebrand factor (vWF) secreted from injured endothelium and activated platelets, and tissue factor (Coagulation Factor III, formerly known as
thrombokinase or thromboplastin) on sub-endothelial matrix and localized leukocytes lead
to initiation, amplification and propagation of the coagulation cascade, culminating in
thrombin-catalyzed formation of fibrin from fibrinogen, a process commonly known as
‘secondary hemostasis’34–36. Critical upstream steps of this process (e.g. conversion of
prothrombin to thrombin) are greatly amplified by anionic lipids (phosphatidylserines) on
the membrane of active platelets as well as by anionic polymers (polyphosphates or PolyP)
secreted by active platelets37–44. Pro-hemostatic active platelets also secrete molecules
adenosine di-phosphate (ADP) and platelet factor 4 (PF4) that can modulate hemostatic
mechanisms. The fibrin, formed as the final product of the coagulation cascade, associates
into a cross-linked biopolymeric mesh facilitated by activated coagulation factor XIII
(FXIIIa) and platelet-secreted polyphosphate (PolyP) to secure the platelet plug and other
blood components at the bleeding site and form the final clot45,46. The clot-incorporated
active platelets also facilitate clot retraction and healing mechanisms47–50. Post-healing of
the injury site, the mature fibrin clot is lysed through the action of plasmin, which is
generated from the zymogen plasminogen on the surface of the fibrin, as well as, on
neighboring cell surfaces by the action of tissue plasminogen activator (tPA) or urokinase
(uPA)51. The tPA is produced from endothelial cells while uPA is produced from
monocytes, macrophages and urinary epithelium. Plasmin-induced proteolysis of fibrin
results in formation of soluble fibrin degradation products (FDPs), which can have some immunomodulatory and chemotactic functions relevant to healing phases. In healthy individuals the clot formation and fibrinolytic systems are highly regulated to ensure
9 hemostatic balance, and any dysregulation can lead impaired or weak clot formation (poor
hemostasis and re-bleeding) or overly strong occlusive clot growth (thrombosis). For
example, the plasminogen activators (tPA and uPA) and plasmin action are regulated by
local high concentration of serine protease inhibitor (serpin) molecules like plasminogen
activator inhibitors-1 (PAI-1), plasminogen activator inhibitor-2 (PAI-2) and α-2
antiplasmin52, as well as non-serpin molecules like α-2 macroglubulin and thrombin- activated fibrinolysis inhibitor (TAFI)53,54. Fibrin-bound tPA shows greatly enhanced catalytic efficiency of plasminogen activation compared to solution phase tPA55. In
parallel, plasmin is protected from inhibition by α-2 antiplasmin upon binding to fibrin56.
Thus, a combination of feedback mechanisms of fibrin formation and fibrin destruction
maintains the precise spatiotemporal regulation of hemostasis.
1.5 Management of Traumatic Bleeding
Based on this complex concert of mechanisms in clotting, biomaterials-based approaches
to render and augment hemostasis have focused on mimicking and leveraging the various
mechanistic aspects, including constriction (pressure), platelet (primary hemostasis)-
relevant components and coagulation (secondary hemostasis)-relevant components57. The optimum requirements for a hemostatic material (and technology) is that it (i) should be applicable and adaptable to a variety of actively bleeding wounds, (ii) should act rapidly to reduce blood loss and maintain this hemostatic condition for long durations if needed, (iii) should be easily manufacturable, sterilizable and portable, (iv) should be stable under a variety of atmospheric and ambient conditions, (v) should be easily usable by non- specialized personnel if needed and (vi) should be reasonably biocompatible so as to not
10 induce any short-term or long-term adverse effect in the body57. To this end, for externally
visible and accessible (often compressible) injuries, a variety of biomaterials-based
technologies in the form of powders, bandages, sprays, foams, gels, tourniquets and
tamponades have been developed58–64. In contrast, for management of internal (often non-
compressible) bleeding, the clinical gold standard is the transfusion of whole blood or
blood components (RBC, plasma and platelets)65–70, and use of fibrinogen concentrate or
recombinant coagulation factors in selected groups of patients (e.g. recombinant Factor
VIIa used in hemophilia patients)71–75. Patients with thrombocytopenia in the intensive care
unit can range from 8%-67%. Up to 30% of these patients may require platelet transfusions,
primarily for purposes of hemorrhage prophylaxis70. The importance and early
administration of these clinical gold standards has been validated by recent clinical trials.
The Prehospital Air Medical Plasma (PAMPer) clinical trial evaluated a total of 501 patients and concluded that prehospital administration of thawed plasma was safe and
resulted in lower 30-day mortality and a lower median prothrombin time ratio than
standard-care resuscitation76. The PROPPR (Pragmatic, Randomized Optimal Platelet and
Plasma Ratios) trial demonstrated the importance of platelets in particular for the
management of traumatic bleeding. This study determined that early platelet administration
is associated with improved hemostasis and reduced mortality in traumatic bleeding
patients compared to patients who received blood products without platelets77. Despite these data, donor-derived blood and its components often have limited availability, require meticulous type matching, pose issues of high pathologic contamination or immunogenic risks, and have limited portability and short shelf-life. These issues are particularly challenging for transfusion of platelets, which present challenges of alloimmunization and
11 refractoriness, have a high bacterial contamination risk and have a shelf-life of only 3-7
days78–85. Therefore, significant research efforts are currently being focused on minimizing
contamination and improving storage life of platelets86–89 as well as on developing in vitro bioreactor technologies for production of ‘donor-independent platelets’ from cultures of precursor cells90–92. In parallel, robust interdisciplinary research efforts are being directed
on developing biomaterials-based technologies that can be administered intravenously and
can mimic, amplify and leverage various mechanistic components of hemostasis to rapidly
staunch bleeding. The next two chapters will comprehensively and critically review the various externally used and internally applicable hemostatic technologies in terms of materials, clotting mechanisms and current state-of-art, and discuss the challenges and opportunities associated with these technologies to help interdisciplinary advancement of the field.
1.6 Acknowledgments
This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the
National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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27 Chapter 2: Externally Administered (Topical and Intracavitary) Biomaterials and
Advanced Technologies for Management of Traumatic Bleeding
Content based on: Hickman DA, Pawlowski CL, Sekhon UDS, Marks J, Gupta AS. Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding. Adv Mater. 2017 Nov 22. doi: 10.1002/adma.201700859.
2.1 Introduction
Externally administered hemostatic treatment of an injury or lesion is applicable to simple
cuts and bruises, exposed and puncture wounds, surgical lacerations, and heterogenous
blunt trauma. These scenarios result in various degrees of bleeding and tissue damage, and
hence the development of appropriate hemostatic materials and technologies is driven by
the need to quickly staunch bleeding, absorb shed blood, cover and protect the injury,
prevent contamination and provide a suitable environment for healing. These materials and
strategies include topical use of tourniquets, dressings, bandages, foams, powders and gels,
as well as, administering materials in an intracavitary fashion. A wide variety of materials
and technologies have been developed, pre-clinically evaluated and clinically translated in this area as discussed in the following sections.
2.2 Tourniquets
As described previously, a blood vessel’s first natural response to injury is spasm and construction, leading to reduction in blood flow (stypsis) and facilitating the subsequent cellular and biomolecular clotting mechanisms. Therefore, a number of hemostatic materials and technologies have focused on augmenting this process by collapsing blood vessels with application of pressure to allow platelet activity, coagulation cascade and
28 formation of a fibrin clot. Beyond applying manual pressure with fingers and palm, a widely used technology in this category is that of tourniquets. A tourniquet is essentially a circumferentially constrictive bandage that can restrict blood supply to extremities. In ancient Roman history, Galen of Pergamon (129 - 200 AD) was known to use this method for stopping blood flow and was criticized for its use by those who feared that tourniquets would increase blood loss from a wound. In the early 16th century, the famous Prussian military physician Hans von Gersdorff had described tourniquet use in amputation surgery1. Towards the end of the 16th century, Wilhelm Fabry of German surgical science
fame, reportedly also used tourniquets in amputation surgery, utilizing mechanical
tightening2. Historically in such uses, tourniquets have been reported to present certain
negative consequences, especially in the context of denying blood supply to extremities
hereby causing ischemia and infarction as well as causing mechanical nerve damage.
Despite this criticism, over time tourniquets have become an adapted and accepted
hemostatic technology in pre-hospital management of bleeding in the military (i.e. in combat wounds), as emphasized by documents of the Tactical Combat Casualty Care
(TCCC) of the US military and reviewed recently by Lakstein et al and Kragh et al3,4.
Before being adapted as a tactical mainstay, most tourniquets were made of an improvised
stick with a cloth or a silicone elastic strip. However, due to material inferiority and risks
of slippage (defective fastening), in the mid 2000s significant R&D efforts were dedicated
to tourniquet design and standard-of-usage. The first result was a technology named
Combat Application Tourniquet (C-A-T®, North American Rescue, USA), which is essentially a composite improvisation of the ‘cloth with turning stick for tightening’ approach (aka Spanish windlass design) that can be used with minimal effort (e.g. with one
29 hand)5. Continued studies on tourniquet design, ease of usage in complex time-limiting
environment, patient comfort, hemostatic efficacy and reduction of tissue morbidity and
mortality have led to other tourniquet technologies such as Emergency Medical Tourniquet
(EMT, Delfi Medical Innovations, Canada), Special Operations Forces Tactical Tourniquet
(SOF-TT), Ratcheting Medical Tourniquet (RMT, M2 Inc, USA) etc. The EMT tourniquet
is formed of circumferentially usable bladder that can go around the limb, a clamp that
limits the inflated portion while holding the bladder close to the limb, and an inflator bulb
equipped with a connector tube and twist cap. Hence, the design is similar to a blood
pressure cuff except for the clamp component. The SOF-TT tourniquet is similar in design
principle to the C-A-T system (i.e. the Spanish windlass design) with an aluminum-based
stick. The RMT system requires use of a ratcheting lever instead of a windlass stick for
tightening the tourniquet. The usefulness of tourniquets to reduce blood loss (and hence
morbidity/mortality) by simple yet efficient application of styptic pressure is responsible
for its current consideration as a standard-of-care component for emergency responders in
civilian trauma6,7.
2.3 Naturally derived biomaterials for hemostatic applications
The environment has been a dependable source for a wide variety of materials like cotton, collagen, gelatin, silk, elastin, fibrin etc., that have found extensive applications in biomedical areas of device coatings, tissue adhesives and sutures, tissue repair and regeneration, cell encapsulation, drug and gene delivery etc.8. A variety of naturally
derived materials have also found significant applications in the area of hemostasis and
bleeding management. Some of these materials have only absorption and passive
30 interaction properties, while others have active biointeractions to promote hemostatic
mechanisms. Absorptive and passively interactive materials impart partial hemostasis merely by wound site coverage, absorption of blood and exudates, and subsequent protection, but do not contain any specific component that promote or augment hemostasis or biologically protect from bacterial infection. Bioactive materials and dressings are systems that adhere to the bleeding tissue to either render hemostasis-stimulating properties by themselves or by virtue of components embedded in them that facilitate hemostatic mechanisms and prevent infection. The following sections will provide a descriptive review of absorptive, passively interactive and bioactive materials derived from natural sources that have various hemostatic applications, and at the end Table 2-1 will provide an
‘at-a-glance’ summary of naturally derived hemostatic materials along with representative technology names, characteristic evaluations and current application status.
2.3.1 Absorptive and passively interactive materials in hemostatic technologies
While the function of a tourniquet is to augment the vasoconstriction and stypsis at the bleeding site, the function of a bandage or dressing is to directly cover and protect the site from further contamination and tissue damage, allow exchange of gas and fluids (blood and exudate absorption and removal), potentially prevent infection and allow healing in the long term. To this end, sterile wound dressings and absorbent pads have been made of various materials including cotton gauze or cotton pad, oxidized cellulose material, nylon/rayon/polyester variants, tulle gauze, semi-permeable porous polymer membranes and foams, hydrocolloidal and hydrofiber materials, and amorphous hydrogels. Cotton is a cellulosic polymer that differs from wood cellulose by the fact that it has a much higher
31 degree of polymerization and crystallinity than wood cellulose. Cellulose is a
homopolysaccharide of glucopyranose, polymerized through β-glucosidic bonds9,10.
Cotton contains about 90% of this cellulosic polymer along with a small amount of
hydrophobic waxes and pectin, while wood contains about 40-50% cellulosic polymer. The
US military standard field dressing consists of two layers of gauze wrapped over densely
packed cotton, such that it can absorb a large volume of blood while the cotton strands are
thought to trigger platelet activation and aggregation due to high hydrophilicity, negative
surface charge and surface energy11. The cotton gauze or pad, when directly placed over a
bleeding injury, may provide high blood absorption, partial hemostatic reaction (thrombin
generation via contact pathway) and additional tamponade effect with adjunctive
compression components, but they can adhere to the wound tissue which may be an issue
during their subsequent removal. Nonetheless, in recent years interesting research is being
focused on modulating the hemostatic characteristics of cotton dressings and gauze by
modifying them with other hemostatic or strengthening materials like kaolin mineral,
chitosan, viscose, rayon etc.11,12. Oxidized cellulose (OC) derived from cotton and
oxidized regenerated cellulose (ORC) derived usually from wood pulp, refer to
manipulation of the cellulose structure where primary and secondary alcohol moieties are
oxidatively converted to aldehyde, ketone or carboxyl groups, which significantly changes
the physico-chemical and mechanical properties of OC and ORC compared to native
cellulose13–17. The use of OC and ORC as hemostatic dressing materials in surgery was reported around WWII18–20, and since then its widespread application as a surgical wound
dressing material has established its biocompatibility, bactericidal and hemostatic
properties10,21–26. The acidic pH of these oxidized cellulose materials and the negative
32 charge are thought to impart platelet activation, aggregation and intrinsic pathway of
coagulation mechanisms. These materials are also reported to be biodegradable via
enzymatic (glycosidase-based) and macrophagic processes27,28. Recent advancement of
these materials involves modification of the material matrix with other hemostatic agents
like fibrin29. Like cotton gauze and pads, the OC and ORC materials can present the issue of adherence to the bleeding tissue, that may pose some logistical difficulties during their subsequent removal.
The adherence issue can be resolved by making materials non-adherent yet absorbent in dressings, e.g., paraffin-soaked tulle gauze and various polymer-based, hydrogel-based, hydrofiber-based and hydrocolloid-based dressings. Tulle dressings (Tulle Gras in French, meaning ‘oily tulle’) are essentially open weave cloth gauze soaked in paraffin (petroleum jelly), balsam and olive oil to impart hydrophobicity and non-adherence to bleeding wound site30. These materials are usually used for low exudate wounds and can be further modified
by impregnating with anti-septic agents like chlorhexidine gluconate to impart infection-
protection. The cotton pad, gauze and tulle materials are traditionally considered as
‘passive wound dressing’ materials since their primary function is to fully cover the wound
for physical and mechanical protection, while allowing exudate absorption and fluid exchange. For high exudate situation, cellulose-based hydrogels have also been developed.
For example, amorphous hydrogel materials consisting of mostly water with about 2-3%
33
Figure 2-1 Representative chemical structures of cotton (cellulose) biopolymers and its derivatives that have undergone extensive research in the development of hemostatic technologies like gauze and wound dressings.
of a gel-forming polymer such as sodium carboxymethylcellulose, modified starch or
sodium alginate, along with about 15-20% propylene glycol (a humectant and
preservative), have been developed for wound dressing and hemostatic applications31,32.
These dressings, along with synthetic polymer-based hydrocolloid and hydrofiber systems
(discussed later in this review), are highly suitable for burn wounds and necrotic or sloughy wound beds, since the water content can cool down the injury site to aid in comfort and
34 healing. The gels are often used with a secondary dressing material like perforated or gas-
permeable plastic film that prevents the water content of the hydrogel from evaporating
outward but rather donated towards the wound. Similar hydrated water-donating property
can be achieved with corboxymethylcellulose-based hydrogelic particles dispersed within
polyurethane film or foam (for hydrocolloids) or carboxymethylcellulose-based fibers
manufactured into non-woven pads or ribbons33. Both of these systems are useful for
hemostatic action, protection and healing maintenance of heavily exudating deep wounds.
Figure 2-1 shows representative chemical structures of common cellulose-based materials
that are used in hemostatic wound-dressing applications. It is important to note here that
although cotton and oxidized cellulose system are traditionally included in the ‘passive
dressing’ group, they can be argued to possess a degree of bioactivity due to their ability
to potentially stimulate primary (platelet activation and/or aggregation) and secondary
(contact activation of intrinsic coagulation pathway) hemostatic mechanism components.
However, a systematic study of the hemostatic mechanisms triggered by these materials is
yet to be reported, and most studies report performance output in terms of extent of
hemostasis, blood loss and tissue morbidity evaluation.
2.3.2 Bioactive materials in hemostatic technologies
Thrombin, Fibrinogen and Fibrin: Thrombin, fibrinogen and fibrin (along with active platelets), are the critical components for forming the hemostatic clot, as shown in Figure
2-2. Consequently, fibrin which is the protein formed as the end product of the coagulation cascade via reaction of thrombin on fibrinogen, has become an important bioactive material to be used in hemostatic applications. Reports from the early 20th century suggest fibrin to
35 be superior to cotton in terms of hemostatic capacity34–36, and since then fibrin dressings
and sealants have become one of the most-studied hemostatic material, especially in the
surgery field37–41. In early reports by Grey and Harvey, fibrin was used as pre-polymerized
material processed into tamponade and plaque-like devices for treatment of bleeding in
parenchymal organs34,35. These products demonstrated hemostatic capacity but had
reduced capacities of tissue-integration since the fibrin was already pre-polymerized.
Figure 2-2 Schematic of concomitant roles of thrombin and fibrin(ogen) in propagating the formation of hemostatic clots via activation of platelets and formation of fibrin mesh.
Around WWII, Cronkite et al and Tedrick et al reported the usage of fibrinogen with
thrombin to produce fibrin in situ in relevance to skin transplant procedures42,43, which has
led to the modern-day usage of this precursor mixture for fibrin-based hemostatic products.
Fibrin can be used in dry condition where animal- or human-sourced thrombin and
fibrinogen are freeze-dried, processed into powder, foam, fleece, etc. and impregnated into a secondary bandage or carrier dispersant system to be applied directly onto the bleeding site41,44–49. The secondary dressing can be a passive polymeric strip like silicone with an
absorbent vicryl mesh backing or another bioactive but mechanically more robust material
like collagen sheet50–52. The materials design rationale for all these systems is that upon
contact with an actively bleeding site, the fibrinogen and thrombin in the dressing will
36 interact to form fibrin in situ, leading to a hemostatic effect. In all evaluations so far, these
fibrin(-generating) dressings have shown superior hemostatic performance compared to
passive dressings, possibly because of their pro-coagulant bioactivity49,53–58. Fibrin
adhesives can also be used topically in a liquid form, where the freeze-dried fibrinogen and thrombin components are reconstituted in sterile saline immediately before administration, often through a specially manufactured dual-barrel injection device37,38,59,60. These liquid sealants can have high degree of tissue adherence due to physico-chemical (electrostatic,
hydrogen and covalent bonding) interactions as well as mechanical integration into the
tissue and can be applied to heterogenous injury sites due to their form-filling nature. The dry fibrin dressings and the liquid fibrin adhesive sealants have been reported to contain varying degrees of fibrinogen and thrombin, depending upon products from various companies and laboratories61,62. These compositionally different products have been tested for assessing variations in hemostatic performance, tensile strength, tissue adhesiveness etc. In some cases, no significant difference was found in the output performances, while in other cases changing the fibrinogen concentration showed some effect in mechanical
strength and tissue adherence63–66. The fibrin-forming precursor systems (dry or liquid) can
also contain other pro-coagulant materials like Factor XIII (a fibrin cross-linking
transglutaminase enzyme) as well as anti-fibrinolytic agents like aprotinin and tranexamic
acid (TXA, discussed in detail later in the next chapter) to modulate clot formation speed
and clot strength/stability67–69. Although such modifications may provide marginal benefits, the significance of such benefits have not been systematically evaluated and statistically established.
37 The lingering issue for a long time with such fibrinogen and thrombin-based products has
been the risk of immunogenicity and viral contamination, since the components are sourced
from animal (bovine, porcine) or human pooled blood. Bovine thrombin preparations have
been implicated in immunogenic reactions and increased risk of adverse clinical outcomes
following its use in surgical procedures70. Fibrin based products that were mass-produced around 1944-45 to meet the needs during WWII were withdrawn in 1946 because of reports of hepatitis transmission71. As a progression of that, in the late 1970s many plasma-sourced fibrinogen-based products, originally clinically approved for hemostatic and surgical procedures, were recalled by the FDA. In the last decade, due to the emergence and establishment of rigorous blood screening, serological testing and pathogen (bacteria, virus) reduction/inactivation technologies, plasma-sourced products including fibrin systems have undergone a revival36,41,71. Through rigorous research conducted at several
laboratories, including studies led by the US Army Institute of Surgical Research
(USAISR), products like TachoComb and TachoSil (Nycomed, Austria) and Dry Fibrin
Sealant Dressing (DFSD, developed by American Red Cross with USAISR) have become
an important component in the current state-of-art toolbox for hemostatic management of traumatic and surgical bleeding36,41,49. Fibrin’s natural spatio-temporally regulated
characteristics of biodegradation and wound healing are also responsible for its continued
popularity in the milieu of hemostatic materials72. In recent years, there have also been
reports on sourcing the fibrinogen and thrombin from salmon73–77 as well as developing recombinant versions of such coagulation proteins78–81. For example, RecothromTM
(ZymoGenetic Inc, USA) is a fully recombinant human thrombin that has been clinically
approved as a topical hemostatic agent to treat oozing blood and capillary bleeding and can
38 be used in conjunction with other wound dressings. A transgenic approach has also been
utilized to develop proteins such as fibrinogen in the milk of other mammals for potential pharmacotherapeutic use82. Recombinant and transgenic versions of thrombin and
fibrinogen are under pre-clinical and clinical evaluation, and can potentially resolve the
availability and immunogenicity issues that are otherwise associated with human or animal
plasma-resourced products. As for liquid fibrin sealants and adhesive products, although
they remain a relevant material in surgery, their widespread pre-hospital use (e.g. in the
battlefield) has been somewhat restricted due to time-consuming rehydration and mixing process of the lyophilized powders for in situ delivery. Furthermore, the resultant fibrin is capable of hemostasis in low volume and pressure bleeding scenarios but not heavy traumatic bleeding. In recent years, autologous fibrin generation technologies like
CryoSeal (Asahi Kasei Medical Co., Tokyo, Japan) and Vivostat (Vivostat A/S, Denmark)
have been reported that utilize small volumes of patient’s own plasma to generate fibrin
sealant in the operating room83. These sealants have shown significant clinical promise as
hemostatic materials during spine and sternum surgeries84–87. Fibrin has also been
developed into ‘foam’ technologies for spray-based hemostatic use as sealants for heavy parenchymal bleeding88–90. Fibrin sealants have also been combined with other hemostatic
materials like oxidized cellulose-based Surgicel® (Ethicon, USA) to cumulatively enhance
hemostatic capability in surgical applications91. Due to their potential for externally
injectable and space-filling properties, the liquid versions of fibrin sealant products may
find use in intracavitary hemostatic applications.
39 Collagen and Gelatin: Another important bioactive material in hemostatic applications is collagen and its denatured variant, gelatin (multiscale schematic structure shown in Figure
2-3). Collagen is the most abundant structural protein found in the extracellular matrices
of many connective tissues in mammals, making up about 25-35% of the whole-body
protein content92,93. Fibrous collagen is present in the sub-endothelial matrix (Type III
collagen mainly, with Type IV in the basement membrane) and upon injury (including
endothelial disruption and denudation), collagen is exposed to circulating blood
components. Von Willebrand Factor secreted from injured endothelium and activated
platelets can bind and self-associate on exposed collagen. Platelets can bind to self-
associated vWF via platelet surface GPIbα receptor protein, and can also bind to collagen
directly via platelet surface GPIa/IIa and GPVI receptor proteins94–96. These adhesion
mechanisms of platelets at the injury site lead to further activation of localized platelets,
ultimately leading to active platelet aggregation at the site (primary hemostasis) as well as
Figure 2-3 Multiscale schematic representation of fibrillar collagen structure where triple-helical microfibrils formed of Gly-X-Y amino acid repeat units assemble in staggered orientation to form collagen fibrils, which in turn further assemble to form high molecular weight collagen fibers and bundles; denaturation of collagen disrupts this assembled structures to form gelatin, which can be partly reassembled into helical components to form lower molecular weight gels.
40 augmentation of coagulation cascade events on the active platelet membrane (secondary
hemostasis)97,98. The direct effect of collagen on coagulation factor localization and activation has also been reported, e.g. for FXII and FIX99. These physiological mechanisms have inspired the utilization of bio-derived collagen as a topical hemostatic material. In
1960s and 1970s, Hait et al had reported on the capability of isolated bovine collagen to adhere effectively to bleeding surfaces and promote hemostasis100–102. Such properties have
eventually led to development of a wide variety of collagen-based products in sheet,
powder, foam and fiber forms to be utilized as a topical hemostatic material, especially in
surgical applications103–108. Representative commercially available technologies that utilize
such collagen forms are AviteneTM (Davol Inc, USA), HelistatTM (Integra LifeSciences,
USA), InstatTM (Ethicon, J & J, USA) etc. Collagen-based materials and technologies have also been studied for liquid form sealants. For example, a liquid composite spray consisting of microfibrillar bovine collagen, bovine thrombin and autologous plasma named
CoStasis® (Cohesion Technologies, USA) was reported to render efficient hemostasis
when externally administered in pre-clinical animal bleeding models (e.g. liver, spleen and
kidney bleeding) and was subsequently clinically approved for surgical applications109–112.
As with animal-sourced fibrin, animal-sourced collagen can pose immunogenic risks, and
therefore research into reducing immunogenicity and infectivity has led to the popularity
of a low-immunogenic collagen-derived material called gelatin113–117. Gelatin can be
formed by thermal denaturation or irreversible hydrolysis of collagen and is extensively
used in the food industry118. Gelatin was also been found to retain hemostatic properties
like collagen119,120. Gelatin has been mixed into fibrin dressings to enhance the mechanical stability of the material72. Gelatin-based solid materials in spongy and powder form (e.g.
41 GelFoam®, Pfizer, USA) have been evaluated for hemostatic dressings in surgical procedures but have shown limited efficacy in controlling severe bleeding121–123. Gelatin
has also been evaluated as a material component in liquid hemostatic sealants, e.g. in
products like Floseal® (Baxter, USA) where it was combined with thrombin to form a composite hemostatic sealant matrix124. In these composite gelatin-based sealants, the
gelatin component is made up of collagen-derived gelatin cross-linked by glutaraldehyde
and ground into macroscopic particles, which is mixed with bovine thrombin in a special
syringe for intra-operative administration125–128. Another gelatin-based liquid sealant
material has been reported as gelatin-resorcin-formalin glue (GRF glue), where gelatin, resorcinol, formaldehyde and glutaraldehyde are mixed in an aqueous medium for application to a bleeding site such that the aldehydes promote reaction and integration with tissue, gelatin promotes hemostatic mechanisms and resorcinol provides bacteriostatic action129–131. This material has been reported in extensive use in acute aortic dissection procedures but has also raised issues of formaldehyde-associated toxicity. Similar to liquid fibrin-based sealants, collagen-based and gelatin-based liquid sealants have also been evaluated for heavy hemorrhage treatment and these may also find use in intracavitary hemostasis applications. In recent years, there is also growing interest in recombinant collagen and gelatin materials that may potentially find application in future hemostatic technologies that pose reduced immunogenic risks106,132,133.
Polysaccharide derivatives: Aside from materials and technologies based on fibrin
(fibrinogen + thrombin), collagen and gelatin, a third prominent category of bioactive
materials in hemostatic applications is that of natural polysaccharides in native or modified
42 forms134. One prominent material in this category is alginate, which is a linear
polysaccharide derived from brown algae and is comprised of blocks of (1,4)-linked β-D- mannuronate (M) and α-L-guluronate (G) residues where the blocks may be consecutive
G (i.e. GGGGGG), consecutive M (i.e. MMMMMM) or alternating G and M residues (i.e.
GMGMGM)135,136. The negatively charged uronic acid chains of alginate can gel in
presence of a cation such as calcium (Ca++), and this sequestration of Ca++ is thought to be
responsible for the hemostatic property of these gels since Ca++ is a co-factor for platelet
activation as well as several coagulation cascade reactions137,138. In pre-clinical animal
model studies as well as in clinical investigation, alginate-based dressings have shown
superior hemostatic performance compared to traditional gauze in low-to-moderate bleeding scenarios139–141. The hydrogel state of alginate dressings is suitable to keep the
wound-bed moist for healing and provide comfort during dressing changes. Besides the
traditional hydrogel form, the alginate material can also be made into micro/nano particles
as well as micro/nano fiber using suitable processing techniques, and these solid forms
have been recently evaluated in hemostatic applications. Alginate microspheres loaded
with the anti-fibrinolytic agent tranexamic acid or the pro-coagulant agent thrombin, have
shown promising hemostatic capabilities in vitro and in vivo in pre-clinical models142,143.
Currently a large number of alginate-based wound dressings are clinically approved for
surgical and hemostatic applications, e.g. Algosteril® (Johnson & Johnson, USA) and
KALTOSTAT® (Conva Tec, UK). Alginate material has also been combined with other
hemostatic materials (collagen, gelatin, oxidized cellulose, other polysachharides etc.) for
the development of composite hemostat technologies that may provide enhanced treatment
capabilities compared to mono-component systems. For example, highly absorbent
43 collagen-alginate and gelatin-alginate dressings have shown superior wound treatment
capabilities144–146. Alginate fibers have also been combined or co-spun with other polysachharides (e.g. chitosan) and natural polymers (e.g. gelatin), to enhance hemostatic and anti-bacterial effects136,147.
Along with alginate, chitosan itself has attained prominence in the area of hemostatic dressings and technologies. Chitin is a hard nitrogenous polysaccharide composed of β
(1,4)-linked 2-acetamido-2-deoxy-β-D-glucose (N-acetylglucosamine), and it is the second most ubiquitous natural polysaccharide on earth (after cellulose), usually found in the exoskeleton as well as in the internal structure of invertebrates (crustaceans, shellfish etc.)148. Enzymatic or alkaline de-acetylation of chitin results in the linear polysaccharide
chitosan, which can essentially remain as a co-polymer of N-acetylglucosamine and glucosamine depending on the degree of de-acetylation. The ability of chitosan to facilitate coagulation was reported first in the early 1980s by Malette et al149, and this has led to
extensive investigation of chitosan in various forms for hemostatic materials and technologies. The hemostatic ability of positively charged chitosan is thought to stem from
its electrostatic interaction with negatively charged cell membranes of RBCs, resulting in
RBC agglutination as a ‘physical’ mechanism of hemostatic plug formation150,151. Chitosan has also been reported to enhance adhesion, activation and aggregation of platelets (i.e. enhancement of primary hemostatic mechanisms) as well as to be able to adsorb fibrinogen from plasma, as well as, to trigger complement activation152–156. The platelet stimulating
effect of chitosan has been attributed to Ca++ mobilization. Chitosan is an acidic polyelectrolyte where about 50% de-acetylation of chitin results in a chitosan system with
44 pKa of ~7.5 (i.e. soluble in water). Therefore, modulating the de-acetylation degree
provides a control to modulate chitosan physico-mechanical as well as chemical properties.
Chitosan can be made into films, fibers, hydrogels, lyophilized particulates and solutions, and all of these forms have demonstrated hemostatic capabilities in various studies. For example, chitosan-based layered materials with interconnected open porous structures and high specific surface area were used to develop a highly effective hemostatic dressing named HemCon® (HemCon Medical Technologies, Oregon, USA) that was extensively evaluated by USAISR investigators for management of traumatic hemorrhage, resulting in the technology being added to the metric of hemostatic strategies for US military71,157,158.
This material however is rigid and inflexible, which may make it difficult to be applied
over complex injuries. In further progress of this technology, the material has shown great
clinical success in both military and civilian populations and a flexible version
(Chitoflex®, HemCon Medical Technologies) has been developed for further
evaluation159–161. Particulate (granulated, powder) forms of chitosan have also been utilized
in developing hemostatic technologies. A prominent example is a technology named
Celox® (MedTrade Products Ltd, UK), which utilizes chitosan granules and flakes to
provide high contact surface area for interact with blood162. Upon direct administration at
the wound site, blood interaction with this material is known to result in swelling of the
granules for hydrogelic absorptive effect, and the chitosan contact promotes multiple
mechanistic components of hemostasis. This material has been extensively evaluated for
hemostatic management of heavy bleeding wounds (liver blunt trauma, arterial puncture
bleeding, groin laceration etc.), showing highly effective hemostasis and reduced
occurrence of re-bleeding163–166. Along with HemCon®, Celox® is now a prominent
45 component of hemostatic management in pre-hospital and hospital scenarios. Chitosan has also been utilized to make hydrogel systems in situ by reacting thiol-modified chitosan with maleimide-modified ε-polylysine, and these materials have shown promising hemostatic capabilities added with tissue-adhesive properties of polylysine167. There are
also several recent reports on developing foams with chitosan or mixture of chitosan with
other materials (e.g. gelatin etc.), for potential application in hemostatic technologies168–
170. Chitosan gauze has also been recently reported to be coated with N-(2-
Hydroxypropyl)methacrylamide (HPMA) based synthetic fibrin-strengthening polymer
(e.g. PolySTAT) for enhanced hemostatic action151. The PolySTAT material itself will be
discussed in the context of fibrin-strengthening materials, later in Chapter 3. A chitosan
analog, poly(-N-acetyl glucosamine), produced by a fermentation process and isolated
from controlled, aseptic, microalgal cultures grown on a defined culture medium, has also
gained reputation as a hemostatic material in gel form, fiber slurry and membrane form171–
173. Thromboelastographic evaluation of this material, along with other analogous materials
like chitin, chitosan etc. demonstrated reduced clot induction time when mixed with blood.
The membrane form prepared by lyophilization and sheet-pressing of this material was
added to a backing of standard hemostatic gauze, resulting in a hemostatic technology named Rapid Deployment Gauze (RDH, Marine Polymer Technologies, USA) that was
evaluated extensively for its hemostatic capability in severe traumatic bleeding174,175. The
hemostatic efficacy shown by this technology in pre-clinical and clinical studies has resulted in its clinical approval and its adaptation as an important component in the bleeding injury management strategies in pre-hospital scenarios. Porous polysaccharide microparticles developed from processed potato starch (dextrin) have also been studied for
46 potential hemostatic applications, especially as a technology named TraumaDEX®
(Medafor Inc., USA) that can be directly applied to bleeding wounds. When evaluated in
pre-clinical bleeding models, this material and technology has demonstrated hemostatic efficacy comparable to standard gauze dressing. In similar studies, the hemostatic efficacy of TraumaDEX® was also found to have no statistical difference compared to
Celox®176,177. Altogether, bio-derived polysaccharides, like alginate, chitosan and dextrin,
form an important materials category for hemostatic applications, and a large variety of
technologies comprising of films, sheets, membranes, powders, microparticulates and
liquid forms have been developed from these materials, many of which have become
clinically approved systems for bleeding management in pre-hospital as well as hospital scenarios in military and civilian settings. Figure 2-4 shows representative chemical structures of relevant polysaccharide materials used in hemostatic dressings described above.
Minerals and zeolites: As mentioned previously, the ability of alum to facilitate hemostasis is by augmenting vasoconstriction and stypsis, which essentially is a mechanical augmentation of hemostasis. Certain microporous aluminosilicate minerals
(also called zeolites) have shown the ability for bioactive augmentation of hemostatic mechanisms. The most famous material of this category is Quikclot® (Z-Medica Inc.,
USA), a granular zeolite technology, that has been shown to render efficient hemostasis in severe hemorrhage from arteries, liver injuries and groin injuries in multiple animal models178–181. The high hemostatic efficacy of this material was also demonstrated in
clinical studies and this has resulted in its approval for use in combat casualties with severe
47
Figure 2-4 Chemical structures of some polysaccharide polymers, namely alginate, chitosan and dextrin that have been extensively used in development of hemostatic bandages and dressings.
bleeding182. The material is thought to promote rapid hemostasis via a combination of
super-absorbent property (which removes aqueous volume from blood and concentrates
clotting factors), platelet activation capacity and coagulation factor activation capability
(contact activation of intrinsic hemostatic pathway)183–185. However, the interaction of this
zeolite material with blood (aqueous medium) is substantially exothermic, resulting in
48 sudden rise in local temperature, which was found to cause tissue damage and debilitation186,187. In this aspect some research is being directed towards modulating the composition and structure of these zeolite minerals to retain its hemostatic property while reducing the exothermic side-effects188–191. In recent years, Quikclot® has been used to modify the matrix of standard gauze and sponge used in US military, resulting in a technology named Quikclot Combat Gauze (QCG) or Quikclot-Advanced Clotting Sponge
(QC-ACS). The exothermic side effect in this technology has been reduced by replacing the active ingredient zeolite with kaolin192–194. This new mineral-impregnated dressing material has shown similar hemostatic effect as the mineral itself and is currently an important component of tactical hemostatic strategies for the US military195. Another mineral based technology is WoundStat® (TraumaCare Inc., USA) that uses a smectite mineral and super-absorbent polymers196,197. This technology has shown highly efficient hemostasis in topical treatment of arterial bleeding and had gained much popularity as a field hemostat in US military198,199. However, further safety studies on the material revealed several systemic side effects, including vascular endothelial injury, transmural damage, systemic thrombotic and embolic risks, due to microscopic residues of WoundStat remaining in the wound and blood vessels161,199. This has resulted in the military avoiding the usage of this material and replacing it with the QCG technology mentioned previously.
49
Table 2-1 Topical and Externally Administered Hemostatic Biomaterials from Natural Sources
50 51 2.4 Synthetically derived hemostatic materials
While a large volume of research has been conducted on naturally derived materials for
developing bleeding management technologies, a small number of synthetically derived
systems, especially certain polymers and peptides, have also been developed and evaluated
for hemostatic applications. One such polymer category is that of basic poly(amino acids)
like polylysine. During the 1950s DeVries et al reported that basic poly(amino acids) like
polylysine, polyarginine etc. augment generation of thrombin and fibrin and also retard
fibrinolysis166,200,201. It was postulated that the cationic nature of polylysine facilitates
complexation and activation of certain coagulation factors202. This kind of report has
possibly guided the use of polylysine as a cationic polyeletrolyte to be mixed with chitosan
for hemostatic applications, but stand-alone polylysine based technologies have not
undergone much development possibly because of biocompatibility and cytotoxicity issues
of cationic polymers203. Poly(alkylene oxides), e.g. poly (ethylene oxide) (PEO) and
poly(propylene oxide) (PPO), have been investigated as hemostatic materials since they
are already well-established biocompatible synthetic polymers in biomaterials
applications. For example, Wang et al and Wellisz et al have reported on the hemostatic
capability of a PEO-PPO-PEO block copolymer based waxy material (now marketed as
Ostene®, Baxter USA) to render hemostasis in orthopedic surgeries204,205. This material
acts much like natural bone wax (a mixture of beeswax with paraffin or petroleum jelly) in
the context of facilitating hemostasis by a tamponade mechanical effect rather than a
biochemical augmentation of coagulation mechanisms. A similar block copolymer made of poly(ethylene glycol)-b-poly(dihydroxyacetone) has also been reported recently by
Spector et al. to have promising mechanical hemostatic properties206. A mixture of tetra-
52 succinimidyl-derivatized and tetra-thiol-derivatized poly(ethylene glycol) has been recently evaluated as a hydrogel material for hemostatic liquid sealant207. In these studies,
it was shown that this material can crosslink with tissues and is capable of rendering a
mechanical hemostat effect by virtue of sealing a puncture hole in a rabbit artery bleed
model. Several successive evaluations in pre-clinical models and in clinical trails have led to approval of this material as a surgical sealant named CoSeal® (Baxter, USA)208,209. A
similar material has been developed by co-polymerizing poly(ethylene glycol) with poly(α- hydroxy acid) diacrylate, and after evaluation in vitro and in vivo, this material is currently marketed as AdvaSeal® (Ethicon Inc., USA). Thus, poly(ethyelene oxide) based synthetic external hemostatic sealants have undergone considerable clinical translation, especially in surgical bleeding applications.
Another class of synthetic polymers that has gained clinical significance in tissue sealant applications is poly(cyanoacrylates). This class of polymers was reported to have excellent tissue-adhesive properties via polar interactions with the tissue and was therefore used as a sutureless tissue sealant as early as the 1940s. Synthetic cyanoacrylates (e.g. 2-octyl
cyanoacrylate) under the product name Dermabond® (Ethicon Inc., New Jersey, USA),
was approved in the 1990s by the FDA for skin closure. Since then, this class of tissue
sealant has been widely used for surgical wound closure and hemostat applications116,210–
213. Long chain poly(cyanoacrylates) sealants have shown reduced tissue toxicity and have
progressed into clinically approved technologies like Histoacryl® (TissueSeal LLC, USA)
and GLUture® (World Precision Instruments) for topical applications in surgeries213,214.
Cyanoacrylate based tissue sealants have also been combined with tourniquet-based
53 procedures to evaluate their ability to staunch bleeding from larger hemorrhagic injuries215.
It should be noted that cyanoacrylates technically do not possess inherent ‘hemostatic’
property in the classical sense, but rather their mode of action is through physical sealing,
mechanical barrier and wound closure. Several other well-known polymeric biomaterials used in biomedical devices like sutures, contact lenses, coatings, drug delivery systems etc. have been investigated for hemostatic applications. For example, poly(glycolic acid) or
PGA is an established biomaterial that is used in a variety of clinically approved bioerodible devices like sutures and implants, and this polymer has been used to develop a hemostatic felt dressing. This felt (Soft PGA Felt, Aventis Behring, Germany) was evaluated in surgical hemostatic treatment of liver resection surgeries in patients, showing promising results as a hemostatic technology option combined with fibrin glue216. Poly(2- hydroxyethyl methacrylate) or poly-HEMA is an established biomaterial which had originally become known for its use in contact lens devices. Porous particles made from this polymer has been reported to have the capability of reducing blood loss in endovascular occlusion surgeries217. Poly(acrylic acid) is another synthetic polymer
biomaterial which was used to develop a hemostatic technology named Feracryl® by
Russian scientists in the 1980s218. This material was developed by reaction of acrylic acid
with Mohr’s salt in an aqueous medium and had a small percentage of iron(III) coordinated
with the polymer. In evaluation in vitro and in vivo, this material was found to be non-toxic
with promising hemostatic ability and this has led to development of several acrylic acid
based hemostatic technologies in Russia but not much has been reported elsewhere
globally. Carr et al have reported on a microporous poly(acrylamide) gel in the
development of a super-absorbent hemostatic technology named BioHemostat®
54 (Hemodyne Inc., USA), which showed great promise in the treatment of heavy bleeding
injuries219. The hemostatic ability of this material was attributed to its capacity of absorbing
high amount of fluids from the wound and expanding to exert a mechanical tamponade
effect on the wound. Casey et al. have recently reported on the development of a series of
cationic acrylamide hydrogels that demonstrated the capability of activating coagulation
factors that may aid in clot formation and hemostasis220. Another tissue sealant technology based on albumin-glutaraldehyde (e.g. BioGlue®, CryoLife Inc., Georgia, USA), is available in the United States for surgical adhesive applications in open surgical repair of large vessels (such as aorta, femoral and carotid arteries)221. In recent years, mussel-
inspired biomimetic materials design approaches have led to several interesting novel
classes of tissue-adhesive synthetic polymers, including citric-acid based, catechol based and hyperbranched poly(aminoester) based systems, which may find potential use as hemostats and sealants222–224. These studies and reports establish the promise of tissue- interactive synthetic polymeric systems for the engineering of hemostatic technologies, that can staunch bleeding through mechanical as well as biochemical mechanisms.
Recently, several research groups have reported on a synthetic peptide termed RADA16-I
(essentially a 4-mer repeat of Arginine-Alaninine-Aspartate-Alanine) that can self- assemble into supramolecular structures and can gel in presence of blood to arrest blood cells and promote a coagulatory effect225–228. The molecular mechanism of such hemostatic
action of this synthetic peptide material is thought to be more due to gelation and tissue
adhesion, and the presence of any additional biochemical involvement in the coagulation
cascade is currently unclear. Besides being used directly as absorbent dressing, sealant or
tamponade systems, synthetic polymers are also used as scaffold or backing components
55 in many hemostat technologies. Examples of these were previously described in
technologies where polyurethane films and foams were used as scaffold material for
dressings. In other composite applications, polymers like polypropylene and polyurethane
have been used as scaffold materials for impregnating chitosan material229,230. Dressings
made of polymer films, foams and amorphous hydrogels are considered to be ‘interactive
wound dressings’ as they are mostly transparent to allow monitoring of wound status, while
maintaining non-adherent and absorbent properties. The polymer film wound dressings are
usually made of semi-permeable polyurethane membrane with an acrylic adhesive backing,
while he polymer foam wound dressings are formed of soft, open cell, hydrophobic polyurethane foam sheet, approximately 6–8mm thick, with a high capacity of exudate absorption231. The film systems are usually used for low exudate wounds while the foam systems can be used for heavy bleeding and high exudate wounds. Polymer-based semi-
permeable dressings and foams also have the advantage of preventing bacteria and fluid
transfer at the injury site while allowing regulated transfer of air and moisture, such that
the wound bed can be kept optimally moist to aid comfort and healing. However, if the
wound site produces high amount of exudate, its build-up may negatively affect the
adjoining tissue (e.g. maceration). Altogether, a variety of synthetic polymeric biomaterials
have become stand-alone or integrative components of many hemostatic materials and
technologies, as they provide advantages of customizing chemistry for stimulating pro-
coagulant mechanisms while reducing immunogenicity risks otherwise associated with
some of the bio-derived materials.
56 2.5 Compression bandage technologies
The aspect of applying pressure to cause ‘stypsis’ and the aspect of applying bandages and
dressings (made from natural or synthetic biomaterials) to allow absorptive and interactive
mechanisms at the bleeding site, have been integrated to result in compression bandage
technologies. The simplest versions of such technologies consist of cotton gauze pads
placed with manual pressure over the bleeding wound, but this material as well as
procedure is unreliable due to variations in severity of injuries and external manual pressure
needed for efficient hemostatic compression232,233. A marginal improvement of this
technology is found in the Army Field Bandage where a thick pad of cotton is contained
within layers of gauze and tying straps are attached to this material to help fastening and
tightening of pressure234. A further improvement is found in the emergency field bandage
(aka ‘the Israeli bandage’, First Care Products) originally designed and developed by Israeli
military serviceman Ben Bar-Natan, which consists of an elastic bandage sewn over a
sterile non-adherent absorbent pad material. The bandage is equipped with a pressure bar
through which the wrapping material may be inserted, reversed in direction and tightened
over the bleeding site to apply tourniquet-like compression235. Unlike the direct placement
of gauze or gauze-wrapped cotton pad on the bleeding injury, which can make removal of
the bandage material cumbersome and risky, the utilization of non-adherent material along
with the compression bar made this bandage design a welcome improvement in
management of traumatic wounds in the military. Another military-tested compression
bandage is the CinchTight bandage system (H&H Medical Corporation, USA), which is
available in a variety of sizes and consists of long elastic wrapping sewn over a sterile non-
adherent absorbent pad and equipped with a metal hook (instead of the pressure bar found
57 in the Israeli bandage) through which the elastic wrap can be manipulated to wrap-around
and tighten for compressive force on the wound site. Another relatively recent technology
in the mix of compression bandages is the Elastic Adhesive (ELAD) bandage developed
by Dr. Sody Naimer in Israel236. In this design, the non-adherent sterile absorbent pad is
laminated over by a long self-adherent polyethylene strip that allows for flexible wrapping
around various tissue/organ morphologies while the transparency of the strip allows for
direct observation of the dressing-covered wound site to ensure that bleeding has been
reduced. In all the above designs, the main purpose is to provide a combination of
compression plus sterile coverage of the wound that can reduce bleeding to some extent,
and this reduction may be sufficient to reduce the tissue morbidity or mortality risk until
the patient can be attended to in a medical facility. The elastic wrap-around materials in
many cases are proprietary in composition, but it is apparent that most of these materials
are possibly latex or nylon or CobanTM (3M, USA) type non-woven elastic composites or
polyethylene-based stretchable polymers. Table 2-2 provides an ‘at-a-glance’ summary of synthetically derived hemostatic biomaterials along with representative technology names, characteristic evaluations and current application status.
58 Table 2-2 Topical and Externally Administered Hemostatic Biomaterials from Synthetic Sources
59 60
2.6 Combination systems and advanced technologies
The biggest challenge in external management of bleeding is the treatment of severely
bleeding wounds where the injury is heterogenous and the hemorrhage is often non-
compressible. This is significant in both civilian and military trauma cases where severe
exsanguination results in high mortality237. Therefore, while a staggering volume of
research endeavors, technology development and clinical translation have been dedicated
to development of hemostatic systems over the past century, the current focus is on efficient utilization of these systems as well as development of new systems to specifically treat non-compressible (often intracavitary) hemorrhage. This has not only prompted research in new materials and technologies, but also establishment of suitable pre-clinical animal models where the efficacies of these technologies can be evaluated and compared in a standardized way in terms of clotting mechanisms, time-to-clot, clot strength and stability, and survival (damage control resuscitation)232,238,239. The recent comparison studies using
such established animal models have provided interesting insight in the pros and cons of
various hemostatic materials. For example, in a comparison of WoundStat® (mineral
powder) verus Celox® (chitosan powder) using a heavy-bleeding arteriotomy model in
pigs, it was found that WoundStat® conferred better hemostasis, but Celox® conferred
better tissue-compatibility161. This can have potential impact on using these materials to
treat heavy-bleeding intracavitary wounds where the material is supposed to interact with
61 heterogenous tissue beds and would either biodegrade (e.g. for chitosan) or need to be
removed (e.g. for mineral) over time. In another study comparing QCG versus Celox®
versus chitosan-impregnated gauze versus HemCon® in porcine bleeding model, the QCG gauze was shown to have a superior performance than the rest, and it was also evident that such technologies used by a trained medical personnel (e.g. military trained in tactical combat casualty care or TCCC course) results in improved hemostasis compared to non- medical personnel240. Combining two different hemostatic materials/mechanisms may
provide a cumulative advantage over using each material by itself. This is evident in the
superior performance of DFSD (fibrinogen and thrombin impregnated dressing on polymer
mesh backing) and QCG (Quikclot zeolite impregnated gauze) compared to fibrin only or
Quikclot® zeolite only49,183. Another combination material example is a technology named
TraumaStat® (OreMedix, USA), where chitosan was combined with silica and polyethylene to form a flexible gauze that integrated the tissue adhesiveness of chitosan with the coagulation factor activating capacity (contact activation) of silica, to render superior hemostatic capability compared to just chitosan-based systems241,242.
2.7 Hemostatic materials and technologies for intracavitary applications
Hemostatic materials in gel, particulate, pelleted and other flexible forms may be more
useful in treating certain highly lethal hemorrhagic injuries like deep ballistic puncture
wounds and intracavitary blunt injuries where the standard-of-care is to rapidly administer
‘packing and absorbent’ material into the injury volume to staunch bleeding. The benefit of such ‘space-filling’ hemostatic materials was demonstrated by Kheirabadi et al using fibrin sealant foam and FloSeal® in a liver hemorrhage model (bleeding within abdominal
62 cavity) model243. To this end, in recent years several interesting biomaterials and advanced
technologies for intracavitary (or complex wound) hemostatic applications have been
reported. One such technology is made of plant-derived cellulose sponge pellets coated
with chitosan that are compressed as a starting form and upon absorption of blood (aqueous
medium), expand axially within a very short period of time (~ 20 seconds)244. As a result,
this material is capable of combining pro-coagulant (because of chitosan), absorptive
(because of cellulose) and tamponade (because of expanding) effects, to render efficient
hemostasis. After rigorous evaluation, this technology has recently undergone FDA-
approval under the name XSTAT® (RevMedX, Orgeon, USA)245. Self-expanding foams
made of in situ forming polyurethane mixtures have also been reported in the context of
evaluating hemostatic capability in heavy bleeding wounds246,247. In another recent
approach, an algae-based gel product developed by Landolina et al from alginate and other
polysachharides was shown to rapidly staunch bleeding in open wounds in soft tissue and
is being currently marketed in the veterinary as well as prospective human trauma hemostat
uses under the names Vetigel® and Traumagel® (Cresilon Inc. formerly Suneris Inc.,
USA)248. Translation of this material in hemostatic treatment of bleeding injuries in civilian
and military population would require further testing in established animal models, along
with determination of the biocompatibility and spatio-temporal fate of the material at the injury site.
2.8 Discussion
Bleeding due to injuries, trauma, surgical procedures and congenital or drug-induced coagulation disorders, can lead to a variety of health risks. Therefore, bleeding risks and
63 incidents need to be rapidly treated249,250. In many bleeding injuries, the body’s inherent
mechanisms become insufficient to rapidly staunch bleeding, and this necessitates the
administration of certain materials and technologies to facilitate, augment, compensate or
mimic the natural mechanisms of hemostasis. In the previous sections, I have
comprehensively reviewed the various materials and technologies currently approved, as
well as, under research for hemostatic applications. The bulk of hemostatic materials
research has been directed towards technologies for management externally accessible
bleeding scenarios, ranging from fibrin and thrombin glues, to zeolite powders, to bioactive
dressings, foams, gels, tourniquets and self-expanding tamponades. In recent years, there
has been a concerted effort to evaluate this large volume of technologies in standardized
bleeding models, especially for heavy-bleeding injuries. These efforts have been mostly led by the US military’s medical research divisions, since improving military survival through rapid hemostasis on the battlefield has become of paramount importance due to continued wars and conflicts over the last decade. In this context, Kheirabadi has recently described the ideal characteristics of externally administered hemostatic dressings251.
Arnaud et al reported on the comparative evaluation of ten clinically approved hemostatic
dressings in a groin transection model in pigs, by monitoring mean arterial blood pressure,
rate and time of survival, blood loss and post-treatment re-bleeding194,195. In these studies,
it was found that Quikclot® ACS (zeolite impregnated flexible absorbent sponge), Celox®
(chitosan granule), WoundStat® (smectic mineral on flexible polymer) and X-sponge®
(kaolin impregnated flexible gauze) all have comparable superior hemostatic capabilities
relative to standard gauze or rigid hemostatic technologies like HemCon®. Rall et al carried
out similar comparative evaluation of several such dressings in a pig arterial bleeding
64 model, and found that Quikclot®, Celox® and HemCon® do not have statistically significant differences in their hemostatic capabilities, and they all meet the current established standards put forward by the Committee on Tactical Combat Casualty Care for point-of-
care bleeding management252. Rall et al essentially concluded that the lack of clear
superiority of any of these hemostatic technologies is an indication that current clinically
approved hemostatic dressing technology has potentially reached a plateau for efficacy. A
potential improvement of this scenario may be achieved by the latest volume-filling technologies like XSTAT®, Traumagel® etc., especially to manage complex junctional,
intracavitary or open wound bleeding. Many of the current clinically approved or newer pre-clinically evaluated technologies contain bioactive materials for augmenting coagulation (minerals, collagen, chitosan, alginate, fibrinogen/fibrin etc.). Some of these may be biodegradable and resorbable, while some may require subsequent removal before surgical procedures. In this aspect, it is extremely important to study the biodegradation/resorption pathways, risk of residual materials post-removal and short and long term thrombo-inflammatory responses of resorbed or residual material. Almost all evaluations of the hemostatic materials and technologies so far have been in short term hemostatic capability and resultant survival, with limited focus on biodegradation
/resorption /removal pathways and long-term in vivo effects. Multi-disciplinary efforts involving biomedical and materials engineers, emergency medical and surgical personnel and physiology/pathology experts should be dedicated to such evaluations in the long term.
65 2.9 Acknowledgments
This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the
National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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109 Chapter 3: Intravenously Administered Biomaterials and Advanced Technologies for
Management of Traumatic Non-compressible Hemorrhage
Content based on: Hickman DA, Pawlowski CL, Sekhon UDS, Marks J, Gupta AS. Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding. Adv Mater. 2017 Nov 22. doi: 10.1002/adma.201700859.
3.1 Introduction
As emphasized previously, bleeding can cause a variety of debilitating health issues, especially in traumatic and surgical settings. The majority of hemorrhage-related deaths in
the military and civilian populations occur from non-compressible and heavy intracavitary hemorrhage that cannot be completely managed by tourniquets, external dressings and bandages1,2. Certain dressings and bandages have undergone advancement in materials
components as well as technology designs to improve the treatment options for such heavy
bleeding, especially in intracavitary applications, as reviewed in the previous chapter.
However, intravenous fluid resuscitation and subsequent blood component transfusion
currently remains the primary option for improving survival of civilian and battlefield
casualties with heavy truncal and junctional hemorrhage and traumatic brain injuries, as
described previously3–5. Unfortunately, such procedures are often logistically challenging
in austere pre-hospital (e.g. first responder) conditions, as well as, in medical facilities
without access to blood components, due to the limitations of blood availability and
portability, lack of appropriately trained personnel, high contamination risk, short shelf-
life etc. Especially for platelets, the major hemostatic cellular component of blood, these
issues present severe functional and logistical barriers, and therefore the development of
intravenously transfusable semi-synthetic or synthetic platelet analogues that can provide
110 platelet’s hemostatic functions without its biologic issues and availability/accessibility limitations, has become an exciting area of biomaterials research. In parallel, synthetic mimicry of certain other non-cellular coagulatory components of blood have also gained momentum in recent years. All of these materials and technologies are to be intravenously administered and are currently in the pre-clinical evaluation stage, with anticipated translation not only for pre-hospital management of civilian and military trauma but also for transfusion applications in surgery and hematology/oncology areas in hospital settings.
3.2 Naturally derived intravascular pro-coagulant materials
As described in Chapter 1, overall hemostasis is a combined output of platelet plug formation (primary hemostasis) and coagulation cascade leading to crosslinked fibrin biopolymer formation (secondary hemostasis) at the bleeding site. Therefore, while one arm of materials research has focused on technologies that leverage and mimic platelet functions, the other arm has focused on various molecular aspects of modulating the initiation, propagation or stabilization of the coagulation cascade components and final fibrin clot. In modulating the molecular aspects of the coagulation cascade, the most obvious approach is to intravenously administer fibrinogen as well as coagulation factors.
In a bleeding patient, fibrinogen levels can be replenished by directly administering fibrinogen concentrate, or cryoprecipitate (a mixed product containing fibrinogen, factor
VIII, von Willebrand factor, and FXIII). These fibrinogen products have been extensively studied in pre-clinical animal models as well as in selected clinical trials and significant benefit of these products in hemostatic management of traumatic and surgical bleeding have been demonstrated6–9. Further clinical studies are needed to establish the short-term
111 and long-term systemic safety of such products. In the field of coagulation factors, FVIII
and FIX concentrates isolated from pooled donor plasma became commercially available
in the 1970s for the treatment of hemophilia, but were found to transmit the risk of viral
hepatitis10,11. In transfusion medicine, the current clinical mainstay of factor replenishment
is the use of fresh frozen plasma (FFP), which is rich in multiple coagulation factors
(prothrombin or FII, FV, FVII, FIX and FX)12–14. However, FFP also suffers from issues
of availability, pathogenic contamination risks and immunologic side-effect risks10,15,16.
These issues have led to the research and development of a variety of coagulation factors
using recombinant technologies during the past two decades10,17–21. For example, following
the cloning of the human FVIII mRNA in 1984, a variety of recombinant FVIII proteins
have been developed, such as Kogenate® (Bayer), Recombinate® (Baxter), Refacto®
(Wyeth Pharmaceuticals), and ADVATE® (Baxter)22,23. All these recombinant FVIII preparations have demonstrated significant hemostatic efficacy in treating bleeding risks in patients with hemophilia A. Similarly, the cloning of FIX from a human liver cDNA library has led to development of recombinant FIX (e.g. BeneFix®, Wyeth
Pharmaceuticals) for bleeding risk treatment in haemophilia B patients24–26. It is important
to note here that the use of Factor VIII and Factor IX have been reported to induce
development of inhibitory alloantibodies in at least 30% of treated patients after multiple
prophylactic dosages, and this has led to the development of one of the most notable
recombinant coagulation factor products, the recombinant Factor VIIa (FVIIa)17,18. As was
depicted in Figure 1-2, this coagulation factor can interact with Tissue Factor to generate
moderate amounts of thrombin (Factor IIa) in the extrinsic pathway of coagulation cascade.
Factor VIIa (and thrombin) can also reportedly activate FIX to FIXa, which subsequently
112 takes part in activation and complexation with other coagulation factors (FXa and FVIIIa)
on the surface of activated platelet membrane in presence of Ca++ to form the
prothrombinase (FXa + FVa + FII) complex, further amplifying the generation of thrombin
(Figure 1-2) via the common pathway of coagulation27,28. Thrombin further activates
localized platelets and also breaks down fibrinogen to form fibrin, the final coagulation
cascade product. Therefore, the central theme in utilization of recombinant or plasma- derived FVIIa to improve hemostasis is to augment the generation of thrombin while bypassing the tenase amplification step, and this has proven highly beneficial in hemostatic treatment of patients with congenital coagulation factor defects, hematologic malignancies, thrombocytopenia and traumatic bleeding. FVII was originally cloned in the late 1980s from human liver and Hep G2 cells and has subsequently led to the development of the recombinant FVIIa product NovoSeven® (Novo Nordisk)29–31. This product was originally
approved clinically to treat hemophilia patients where lack of FVIII (Hemophilia A) or FIX
(Hemophilia B) affects the intrinsic pathway of thrombin generation, and therefore
intravenous administration of FVIIa was postulated to compensate for this by generating
thrombin via the tissue factor (extrinsic) pathway32,33. NovoSeven® has also been recently
approved for use in patients with Glanzmann Thrombasthenia where patient’s platelets
have qualitative or quantitative deficiencies that affect their ability to bind to fibrinogen
via integrin GPIIb/IIIa. For patients with thrombocytopenia (platelet deficiencies), it has
been demonstrated that combining FVIIa therapy with platelet transfusion may provide
additive benefit, since the common pathway of coagulation cascade that leads to amplified
thrombin generation and fibrin formation is facilitated by the presence of activated platelet
membrane18. It is important to note here that the hemostatic promise shown by intravenous
113 FVIIa has also led to its incorporation in externally applicable dressings like gelatin sponge
or microporous polysaccharide spheres, to improve the overall hemostatic capacity of these
technologies34,35. The clinical benefit of recombinant FVIIa in trauma, intracranial
bleeding, and other heavy bleeding surgeries are also being explored36–39. For example,
two randomized, placebo-controlled, double-blind trials (one in blunt trauma and one in
penetrating trauma) have been carried out to evaluate the efficacy and safety of
recombinant FVIIa as adjunctive therapy for hemostatic management of trauma patients,
and the results have indicated a reduction in need of blood component transfusion in the
patients, along with systemic safety of the FVIIa in the dose ranges studied38.
3.3 Synthetically derived pro-coagulant systems
In recent years, research approaches have also focused on mimicking and leveraging the
body’s coagulation mechanisms using intravascularly applicable synthetic biomaterials.
For example, in the work of Morrissey and colleagues, it was demonstrated that inorganic
polyphosphate polymers (PolyP) secreted from activated platelets is capable of facilitating
activation of FXI to FXIa and FV to FVa, which subsequently results in the prothrombinase
complex leading to thrombin generation and back-activation of other coagulation factors40–
42. This property of platelet-derived PolyP was recently leveraged by Kudela et al to create
silica nanoparticles coated with this PolyP polymer (PolyP-SNP), which demonstrated promising hemostatic characteristics in vitro in the context of augmenting thrombin generation and reducing clotting time in thromboelastographic evaluation with pooled plasma43. To advance this technology towards safe clinical translation as an intravenous
hemostat, an important design requirement would be to control the exposure of the PolyP-
114 coated surface to coagulation factors selectively at the bleeding site such that the thrombin
and fibrin augmentation can be localized at that site while avoiding systemic off-target pro- coagulant effects. An additional challenge may be the maintenance of PolyP activity over reasonable periods of time, since these polyphosphate polymers are known to be rapidly deactivated by phosphatases in circulation. Masking the PolyP coating on particles from phosphatase action in circulation and then programming in a mechanism to have it exposed
(unmasked) selectively at the bleeding site can lead to a unique target-responsive hemostatic particle design. The PolyP material itself has shown additional interesting properties regarding enhancement of fibrin clot strength and stability, in that it can inhibit fibrinolysis by affecting thrombin-activated fibrinolysis inhibitor (TAFI), as well as, increase fibrin fiber thickness40. This property may be beneficial to intravenous hemostasis
applications if the clot formation can be directed selectively at the bleeding site, especially
in non-compressible hemorrhage (e.g. in trauma). In another interesting recent report,
Baylis et al have reported on ‘self-propelled’ particles made from calcium carbonate
+ (CaCO3) mixed with protonated tranexamic acid (TXA-NH3 ), where the propulsion
+ occurs due to carbon dioxide generation via the reaction between CaCO3 and TXA-NH3
in the milieu44. These particles were reported to be capable of being propelled through
blood against convective forces, and external application of these particles in several pre- clinical bleeding models (mice and pigs) demonstrated promising tissue-penetration to deliver loaded thrombin for hemostatic clot promotion. The particles were reported to have safe biodistribution when intravenously injected in mice. However, it was unclear from the report whether the particles, reportedly about 10μ in diameter, were designed for actual safe intravascular applications systemically, since solid particles of such large diameter are
115 known to pose occlusive risks in the microvasculature. Figure 3-1 shows selected results
from hemostasis-relevant studies carried out with PolyP-coated silica particle systems
+ (Figure 3-1A and B) and the ‘self-propelled’ CaCO3 particles mixed with TXA-NH3 and
thrombin payloads (Figure 3-1C, D and E). Figure 3-1A demonstrates that PolyP loaded in silica nanoparticles (PolyP-SNP) accelerates thrombin generation compared to SNP alone while Figure 3-1B demonstrates that PolyP delivered via SNP particles (PO3 in SNP)
reduces clotting time of blood (i.e. speeds up coagulation) compared to PolyP in solution.
Figure 3-1C shows representative fluorescence and scanning electron micrograph images
of CaCO3 particles along with a schematic of administration of such ‘self-propelled’
+ particles loaded with thrombin in TXA-NH3 medium to a liver puncture site. Figure 3-1D
shows fluorescence-based confirmation (green fluorescence) of particles in the liver injury
model in mice, while Figure 3-1E shows significant reduction of blood loss in the mouse
liver injury model when such ‘self-propelled’ particles were used to deliver thrombin deep
into the injury site.
3.4 Materials and technologies to enhance clot strength and stability
As described previously in Chapter 1, in physiological scenarios hemostasis is a highly
spatiotemporally regulated balance between clot formation and clot lysis45. However, in
pathological scenarios, dysregulated fibrinolysis can cause substantial hemostatic
abnormality (e.g. trauma-induced coagulopathy) leading to sub-optimal stoppage of
bleeding (weak fibrin clot and hyper-fibrinolysis) as well as disseminated intravascular
coagulopathy (DIC) risks46. In such cases, drugs, biomaterials and technologies that can
reduce fibrinolysis or increase fibrin strength (and stability) can provide significant
116 therapeutic benefit. One such drug is the antifibrinolytic agent tranexamic acid (TXA), which is a synthetic derivative of the amino acid lysine that inhibits fibrinolysis by blocking the lysine binding sites on plasminogen47,48. TXA has recently undergone extensive clinical studies as an intravenous hemostat, e.g. in the Clinical Randomization of an
Antifibrinolytic in Significant Hemorrhage 2 (CRASH-2) trial49,50. These studies have
Figure 3-1. Selected results from hemostasis-relevant studies carried out with PolyP-coated silica particle + systems (A and B) and thrombin-loaded CaCO3-based particles mixed in TXA-NH3 that can self-propel themselves into wound depth (C, D and E). A demonstrates that PolyP loaded in silica nanoparticles (PolyP- SNP) accelerates thrombin generation compared to SNP alone while B demonstrates that PolyP delivered via SNP particles (PO3 in SNP) reduces clotting time of blood (i.e. speeds up coagulation) compared to PolyP in solution; C shows representative fluorescence and scanning electron micrograph images of CaCO3 particles along with a schematic of experimental set-up to administer thrombin-loaded ‘self-propelled’ particles in + TXA-NH3 medium to a liver puncture site; D shows presence of green fluorescent particles deep within the liver injury site in mice; E demonstrates that liver injury site-localized delivery of thrombin-loaded ‘self- propelled’ CaCO3 particles in TXA-NH3+ results in significant reduction of blood loss, compared to non- propelled thrombin delivery or control treatment. Figure components adapted and reproduced with permission43,44. Copyright 2015, John Wiley & Sons Inc. and 2015, AAAS Science Advances CC-BY NC.
117 established that TXA administration can reduce exsanguination and thus the number of
transfusions, and also reduce mortality risks in trauma and surgery patients. However,
CRASH-2 trial studies were performed in civilian hospitals and did not have specificity
towards measures of coagulopathy, injury severity or mechanism of injury, which may not
be fully applicable to combat trauma. Therefore, TXA has been further studied for
hemostatic potential in severe combat trauma, e.g. in the Military Application of
Tranexamic Acid in Trauma Emergency Resuscitation (MATTERs) study51. These studies
have further concluded that the use of TXA with blood component–based resuscitation in
treating combat injury results in improved coagulation outcomes and survival in patients
requiring massive transfusion. Based on such promising findings, further studies with TXA
for mitigating hemorrhagic complications are currently ongoing, e.g. the Pre-hospital Anti- fibrinolytics for Traumatic Coagulopathy and Haemorrhage (PATCH) trial, the Study of
Tranexamic Acid during Air Medical Prehospital Transport (STAAMP) trail and the
efforts of the Trans-agency Collaboration for Trauma Induced Coagulopathy (TACTIC)
consortium52, that are expected to enhance the optimal therapeutic approaches involving
TXA.
It has also been well established that when platelets interact with fibrin polymers within blood clots, the biomechanical contraction of clot-associated active platelet cytoskeleton facilitates the retraction and stiffening of clots53,54. This property of platelets was used as
an inspiration to produce synthetic platelet-like particles (PLPs) that were formed of
deformable ultra-low crosslinked poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-
AAc) gel microparticles surface-modified with fibrin protofibril-specific antibody
118 motifs55. These PLPs were tested in vitro by flowing them in thrombin-activated platelet- poor plasma (PPP) within endothelialized microfluidic devices, and the particles demonstrated formation of fibrin. It should be noted that in this study the PPP was already thrombin-activated, and classically it is the thrombin (and not platelets) that break down the plasma fibrinogen to fibrin. Platelets essentially augment this process by providing the active platelet surface for the formation of prothrombinase complex and PLPs were not reported to have such active ‘fibrin-generating’ surface but rather a ‘fibrin-binding’ surface. However, it was not clear from the studies how the thrombin-activated PPP itself
could not form sufficient fibrin, but the presence of the PLP particles in that thrombin-
activated PPP somehow resulted in fibrin formation at levels similar to that of platelet-rich
plasma. Nonetheless, the PLPs demonstrated fibrin-retracting property when incorporated
in clots, although this action occurred over much longer time-scales compared to retraction
time-scales shown by natural platelets. For in vivo studies, the PLPs were injected in rats
and 5 min after injection the rat femoral vein was injured to bleed. The PLPs were found
to get incorporated in the clots at the vein injury site and the bleeding time for PLP-injected
animals were found to be only slightly lower (~120 sec) than non-injected animals (~150 sec) and comparable to FVIIa-injected animals. Further evaluation of this technology will be necessary regarding evaluation of long-term biodistribution (higher than 5 min circulation), systemic side-effects and performance in animal models with platelet dysfunctions (e.g. thrombocytopenia, coagulopathy etc.).
Other reported literature on polymeric material for intravenous hemostat application was inspired by the physiological action of the transglutaminase coagulation factor FXIIIa,
119 which strengthens and stabilizes the fibrin clot by creating intra- and inter-fiber cross-links
Figure 3-2 Schematic of fibrinogen conversion to fibrin and assembly of cross-linked fibrin biopolymeric mesh catalyzed by thrombin (FIIa) and FXIIIa, and hemostatic materials and technologies inspired by these mechanisms.
through amide bond formation between the lysine and glutamic acid residues of self-
assembled fibrin protofibrils (native mechanism schematic shown in Figure 3-2)56,57. This
polymer, termed polySTAT, was formed by incorporating fibrin-binding peptide domains
on a linear water-soluble (hydroxyethyl)methacrylate (HEMA) and N-hydroxysuccinimide
methacrylate (NHSMA) co-polymer backbone [p(HEMA-co-NHSMA)]58. The fibrin-
binding peptide itself was adapted from the work previously reported by Kolodziej et al
regarding phage display-based development of fibrin-specific peptides59. The polySTAT
polymer was able to incorporate homogenously within fibrin clots when added to
fibrinogen in presence of thrombin. Rheometric and thromboelastographic
characterization of mechanical properties of polySTAT-enriched fibrin clot compared
against fibrin clots enriched with recombinant FXIIIa or with polySCRAM (control
polymer containing scrambled non-specific peptide) showed that the polySTAT polymer
enhanced clot formation, strengthening and stabilization capacities at levels significantly
higher than the control polymer and comparable to the physiologically relevant
120 recombinant FXIIIa activity. In a rat femoral artery bleeding model of trauma-induced coagulopathy, intravenous administration of the polySTAT polymer significantly enhanced clotting strength and stability, reduced re-bleeding and blood loss and rendered efficient stabilization of blood pressure with reduced need of volume resuscitation.
Biodistribution of the polymer indicated that the material is mostly cleared from plasma and sequestered in the kidney over time, which may be indicative of its circulation safety regarding off-target effects. Dose response effects of this polymer as well as its long-term effects from accumulation in the kidney need to be further investigated. Figure 3-3 shows representative results from hemostatic studies carried out with PLP particles (Figure 3-3A-
D) and with polySTAT polymer systems (Figure 3-3E-G). Figure 3-3A shows magnified images of fibrin-binding soft microgel platelet-like particles (PLPs), natural platelets
(PRP), non-binding control microgel particles (S11–ULCs), and fibrin-binding rigid polystyrene particles (H6-PS) within fibrin matrices 1 hr post fibrin-polymerization while
Figure 3-3B shows the calculated spread area of PLPs, natural platelets, S11-ULCs and
H6-PS particles within these fibrin matrices, demonstrating that soft PLPs have platelet- like spreading behavior (compared to control non-binding and control rigid particles).
Figure 3-3C and D show bleeding time and blood loss analysis data in rat femoral vein injury model where administration of PLPs resulted in reduced bleeding time as well as reduced blood loss similar to administration of FVIIa (compared to saline vehicle or control particles). Figure 3-3E shows the homogenous incorporation of the fibrin-strengthening polySTAT polymer (green fluorescence) within native fibrin matrix (red fluorescence).
Figure 3-3F shows that polySTAT incorporation and strengthening within fibrin results in reduced clot lysis over time (i.e. increased clot strength and stability) compared to control
121 saline and control polymer (polySCRAM). Figure 3-3G demonstrates that intravenous administration of polySTAT results in significant reduction of blood loss rate in a rat femoral artery hemorrhage model.
Figure 3-3 Selected results from hemostatic studies carried out with platelet-like microgel particles (PLPs, A-D) and with polySTAT injectable polymer systems (E-G). A shows magnified images of fibrin-binding soft ultra-low crosslinked microgel particles (PLPs), natural platelets (PRP), non-binding control microgel particles (S11–ULCs), and fibrin-binding rigid polystyrene particles (H6-PS) within fibrin matrices 1 hr post fibrin polymerization and B shows the calculated spread area of PLPs, PRP platelets, S11-ULCs and H6-PS particles within the fibrin matrices, demonstrating that soft PLPs have spreading behavior comparable to natural platelets (PRP) and greater than control particles; C and D show bleeding time and blood loss analysis data in rat femoral vein injury model where administration of PLPs resulted in reduced bleeding time and blood loss comparable to administration of FVIIa and significantly lower than treatment with vehicle or control particles; E shows the homogenous incorporation of the fibrin-binding polySTAT polymer (green fluorescence) within native fibrin matrix (red fluorescence); F shows that polySTAT incorporation within fibrin results in crosslinking based strengthening of fibrin and thus reduction of clot lysis over time (i.e. increased clot stability) compared to treatment with control saline and control polymer (polySCRAM); G demonstrates that intravenous administration of polySTAT results in significant reduction of blood loss rate in a rat femoral artery hemorrhage model. Figure components adapted and reproduced with permission.55,58 Copyright 2014, Macmillan Publishers Ltd and 2015, AAAS.
3.5 Materials and technologies for vascular embolization
Another interesting area of using synthetic materials in intravascular hemostatic applications, is the area of blood vessel embolization that is often used to staunch internal bleeding in aneurysms, gastro-intestinal bleeds etc.60–62. Catheter-mediated deployment of
122 permanent metallic coils or synthetic liquid polymers (e.g. Onyx, an ethylene-vinyl alcohol copolymer dispersed in dimethyl sulfoxide solvent) has already been clinically approved for such site-localized embolic applications60,63,64. Metallic coils and Onyx polymers have
been reported to present issues of material migration, secondary tissue damage, off-target
embolization, incomplete occlusion, biocompatibility issues etc.61,65. To resolve some of
these issues, Avery et al recently reported on development of a shear-thinning polymer system formed from a nanocomposite of gelatin and silica nanoparticles, that can be injected into vascular compartment for embolic effect66,67. The unique rheological
properties of this material, rendered by charge based ‘physical hydrogel’ interactions
between the silica and gelatin materials, allows for shear-driven (e.g. through a pressurized catheter) introduction into blood vessel due to shear-thinning of the material and upon release of pressure the nanocomposite can quickly recover its solid like behavior to render embolization. This unique polymeric system has shown promising embolic efficacy in small and large animal models, to staunch bleeding. Gelatin is a collagen-derived degradation product and therefore can directly activate platelets68. Silica, because of its
negative charge, is known to activate coagulation factors69. Therefore, while these materials attributes may be beneficial in augmentation of embolic and hemostatic effects selectively at the injury site, presence of these materials in systemic circulation as a result of polymer nanocomposite degradation, may pose systemic pro-thrombotic and pro- coagulant risks. Therefore, future advancement of this material and embolic technology would require in depth evaluation of its long-term systemic safety (including degradation products), clearance mechanisms, off-target effects on systemic hemostatic components
(e.g. platelets and coagulation factors) and effects on immune and complement system.
123 3.6 Natural platelet-derived systems
The platelet membrane presents multiple macromolecular entities that participate in
various aspects of primary (platelet adhesion and aggregation) and secondary (fibrin mesh
formation) hemostasis. Figure 3-4 shows a schematic of platelet’s hemostatically relevant surface-interactive adhesion and aggregation mechanisms and the technologies inspired therefrom. The GPIb-IX-V complex as well as GPIa/IIa and GPVI are responsible for
platelet adhesion at the bleeding site by binding to vWF and collagen, respectively70–73.
The platelet surface integrin GPIIb-IIIa, in its activated conformation, is responsible for
binding blood protein fibrinogen by which platelets can bridge with each other to form a
platelet plug74,75. Active platelet surface also expresses P-selectin (by secretion from
platelet α granules), which can bind to glycoprotein ligands on other platelets and
leukocytes at sites of platelet aggregation and inflammation76,77. The surface of active
platelets also present a high extent of anionic phospholipids (e.g. phosphatidylserines
translocated from inner leaflet to outer leaflet of the membrane) that, in presence of calcium
ion (Ca++), facilitates co-localization and activation of several coagulation factors to form
the extrinsic and intrinsic tenase as well as the prothrombinase complex, ultimately leading
to the conversion of Factor II (prothrombin) to Factor IIa (thrombin) which in turn breaks
down fibrinogen to fibrin. Activated platelets also secrete several granule contents, e.g.
PolyP and vWF that can modulate and amplify various aspects of coagulation. Because of
such multifactor role of platelets in clot promotion, transfusion of platelet concentrates
(PC) has become an important clinical component in the prophylactic management of
bleeding risks, as well as, emergency treatment of traumatic hemorrhage78,79. However, the
PC products at room temperature pose high risks of bacterial contamination, and also are
124
Figure 3-4 Schematic of platelet’s injury site-selective adhesion mechanisms (platelet GPIbα of the GPIb- IX-V complex binding to vWF, and GPIa-IIa as well as GPVI binding to collagen) and aggregation mechanism (fibrinogen-mediated bridging of active platelet surface integrin GPIIb-IIIa), and various hemostatic technologies inspired by these mechanisms.
known to undergo activation and degranulation during transport and storage, resulting in
very short shelf-life (3-5 days)80,81. Therefore, a significant volume of research is currently
being directed towards reducing contamination risks by pathogen reduction technologies82–
84, as well as, enhancing the platelet storage stability and shelf-life via cold-storage (4oC),
freezing and lyophilization85–92. In parallel, several research efforts have been directed
towards development of hemostatic technologies that use a variety of platelet-derived
biological components.
Since platelet’s hemostatic properties are significantly mediated by various membrane
components, platelet membranes isolated from outdated platelet concentrates have been
utilized to develop phospholipid vesicles, about 0.5 μ in diameter, through a serial process
of differential centrifugation, freeze-thawing, pasteurization and sonication, and these vesicles have been evaluated as intravenously administered hemostat under the product
125 name ‘infusible platelet membrane’ (IPM CyplexTM, Cypress Bioscience, San Diego,
California)93. Evaluation of IPM has been carried out in vitro, in vivo in pre-clinical models
and in multiple phases of clinical studies, but it is yet to be approved by the FDA for clinical
use, due to certain issues. For example, since the membrane is isolated from allogeneic
platelet concentrates, the pathogenic contamination risks associated with platelet
concentrates may also be present in IPM products, which in turn require rigorous
purification and treatment to ensure safety. It has been reported that such treatments can
lead to loss of functional components in the membrane that are otherwise needed for
hemostatic mechanisms. Several antigens (HLA I and II) and integrin GPIIb-IIIa have been
reported to be lost in IPM due to purification and pathogen reduction treatments94. While
loss of HLA can be viewed as an advantage in the context of the ability to use IPM without
the need for blood type matching, the loss of GPIIb-IIIa renders inability to form the fibrinogen-mediated platelet plug and thereby affects primary hemostatic capability of
IPM. Interestingly, presence of pro-coagulant phospholipid function was reported to be retained on IPM surface, implying that secondary hemostatic mechanisms may still be rendered by these vesicles95. Also, partial functionality of the GPIb-IX-V complex and P- selectin was reported to be retained in flow cytometric analysis of IPM, suggesting that primary hemostatic function may be partly possible by this product94. In pre-clinical animal
models, IPM has shown promising hemostatic efficacy (usually measured by reduction in
blood volume loss or improvement in time for blood to clot from a controlled injury),
without systemic toxicity and thromboembolic risks96–98. These promising studies have led
to clinical evaluation of IPM in healthy as well as thrombocytopenic patients. In early phase
clinical studies, transfusion of IPM showed hemostatic capability comparable to
126 transfusion of allogeneic platelet concentrates and did not show major adverse effects of
immune response and refractoriness. Clinical efficacy of IPM products, especially in
patients who show refractory immune response to repeated platelet transfusions as well as
in patients who are thrombocytopenic with multiple other indications, is yet to be
established. Efficacy in non-compressible hemorrhage (e.g. in trauma) is also yet to be
established. A variant of the IPM strategy named Thrombosomes® (CellPhire, Maryland
USA) was recently reported, where freeze-dried platelets stabilized in trehalose were prepared from a pool of Group O in-date leukoreduced apheresis platelet units99,100. This
product was tested for its physical and surface characteristics, systemic distribution,
toxicity, sequestration and hemostatic efficacy in vitro as well as in vivo in pre-clinical animal models of bleeding (in rabbits, beagles and rhesus macaque), and the results reported were quite promising in terms of in vitro platelet aggregatory and pro-coagulant capacity and in vivo systemic circulation and safety. In vivo hemostatic efficacy has been reported for this product in terms of reducing bleeding in pre-clinical animal models.
Interestingly, isolated platelet membrane-based technologies have also been recently reported by several research groups in the context of coating drug delivery vehicles (e.g. polymeric and silica nano/microparticles) to demonstrate targeted action in treating cancer and bacterial infection, thereby emphasizing the potential utilization of platelet’s role in multiple pathologies for unique treatment strategies101–104. Figure 3-5 shows selected
results from hemostatic studies with IPM, where addition of IPM in thrombocytopenic
blood perfused ex vivo in rabbit aorta segments dose-dependently increased fibrin
deposition at low and high shear rates (shown in Figure 3-5A) and in vivo
127
Figure 3-5 Selected results from hemostatic studies with infusible platelet membrane (IPM) technology, where (A) addition of IPM in thrombocytopenic blood perfused ex vivo in rabbit aorta segments dose- dependently increased fibrin deposition at low and high shear rates and (B) in vivo administration of IPM in thrombocytopenic rabbits resulted in significant reduction of bleeding time. Figure components adapted and reproduced with permission.97,98 Copyright 2000, John Wiley & Sons Inc. and 2012, IJPSR.
administration of IPM in thrombocytopenic rabbits resulted in reduction of bleeding time
(shown in Figure 3-5B).
Instead of using intact isolated platelet membrane to form freeze-dried vesicles or coated
particle systems, some approaches have focused on incorporating specific platelet surface
protein and receptor components in lipidic or polymeric particle systems. The earliest
report of this approach was in a product called ‘plateletsome’, where deoxycholate-induced extraction of platelet membrane fraction with multiple protein components including GPIb,
GPIIb/IIIa and GPIV was incorporated into the membrane of liposomal vesicles formed of sphingomyelin, phosphatidylcholine and monosialylganglioside using reverse-phase
evaporation sonication technique105. This product was tested for hemostatic capability and
demonstrated substantial reduction of tail-bleeding in thrombocytopenic rats while
showing systemic safety. However, no further reports have been available about this
product after the mid 1990s. In an analogous approach, Takeoka and colleagues from Keio
128 University and Waseda University in Japan developed recombinant GPIbα (rGPIbα) and
GPIa-IIa (rGPIa-IIa) proteins and conjugated them on the surface of liposomes, latex beads
or albumin particles to render synthetic particulate systems with platelet-inspired vWF- binding and collagen-binding capabilities106–109. In vitro, these designs have shown
efficient binding to vWF-coated surfaces as well as collagen surfaces in presence of soluble
vWF, with more binding evident at higher shear rates, thereby closely mimicking the
mechanisms of natural platelet adhesion at high shear. Takeoka and colleagues have also
investigated leveraging the co-operative activity of platelet surface motifs that facilitate
hemostasis in a physiological scenario. Platelets render primary hemostasis by a co-
operative interplay between pro-adhesive (vWF-binding and collagen-binding) and pro-
aggregatory (fibrinogen-mediated GPIIb-IIIa binding) functionalities110,111. Takeoka et al
demonstrated that platelet’s pro-adhesive mechanisms of vWF-binding and collagen- binding could be combined on a synthetic particle surface by conjugating rGPIbα and rGPIa-IIa motifs together on the outer leaflet of liposomes108,109. The rGPIbα motif was
utilized to enable reversible binding to vWF under shear flow, while the rGPIa-IIa motif
was utilized to enable adhesion stabilization by concomitant binding to collagen.
Compared to liposomes bearing rGPIbα only or rGPIa-IIa only, those bearing a
combination of these motifs showed significantly higher adhesion and retention on
collagen-coated surfaces in presence of soluble vWF under high shear flow conditions. In another recent interesting approach, Doshi et al created spherical polystyrene as well as crosslinked discoid albumin particles, surface-decorated either with recombinant vWF-A1
domain fragment or recombinant platelet GPIbα domain fragment to mimic platelet-to-
vWF interaction under flow112. These particle systems were evaluated in vitro where the
129 discoid particles showed better adhesion than spherical particles on target surfaces, and the
particles could get incorporated within platelet thrombi on collagen-coated surfaces in
microfluidic chambers. Figure 3-6 shows selected study results of the above-described
technologies that leverage platelet’s adhesion mechanisms. Aggregometry studies shown
in Figure 3-6A demonstrates that addition of rGPIbα-decorated albumin microsphere particles significantly enhances that percent (%) aggregation of platelets in presence of vWF and ristocetin, compared to without ristocetin, thereby confirming that the aggregation enhancement is due to the binding of rGPIbα to ristocetin-induced unfolded vWF. Figure 3-6B shows that polystyrene microparticles surface-decorated with vWF’s
A1 domain or GPIbα fragment can undergo shear-responsive enhanced adhesion on GPIbα
–coated or vWF-coated microfluidic surfaces respectively. Figure 3-6C shows a schematic of rGPIbα-coated red fluorescent liposomes flowed over vWF-coated surfaces, with the results demonstrating enhanced adhesion and accumulation of these liposomes over time at higher shear, mimicking the shear-responsive interaction of platelet GPIbα to vWF in platelet’s adhesion mechanism.
The designs described above represent primarily the adhesive component of platelet function in hemostasis, but do not have the ability to aggregate among themselves or site- selectively amplify the aggregation of active platelets. Platelet aggregation is a function of fibrinogen-mediated bridging of integrin GPIIb-IIIa on active platelets and therefore certain designs have focused on leveraging this mechanism by coating particles with fibrinogen. In the 1980s, Coller et al reported conjugation of fibrinogen on acrylonitrile beads, where the resultant beads enhanced agglomeration of ADP-activated platelets in
130
Figure 3-6 Selected results from studies involving technologies that leverage platelet’s bleeding site- selective adhesion mechanisms. A shows platelet aggregometry studies to demonstrate that addition of rGPIbα-decorated albumin microsphere particles significantly enhances the aggregation level of platelets in presence of vWF and ristocetin, compared to without ristocetin, thereby confirming that the aggregation enhancement is due to the binding of rGPIbα to ristocetin-induced unfolded vWF; B shows that polystyrene microparticles surface-decorated with vWF’s A1 domain or platelet’s GPIbα fragment can undergo shear- responsive enhanced adhesion on GPIbα–coated or vWF-coated surfaces respectively, under flow, mimicking platelet-relevant adhesion mechanisms; C shows an experimental schematic of flowing rGPIbα- decorated red fluorescent liposomes over vWF-coated surfaces, with the corresponding fluorescence microscopy results demonstrating enhanced adhesion and accumulation of these liposomes over time at higher shear, mimicking the shear-responsive interaction of platelet GPIbα to vWF. Figure components adapted and reproduced with permission109,112. Copyright 2012, John Wiley & Sons Inc. and 2000, American Chemical Society.
vitro113. Fibrinogen-coated systems were also reported in the early 1990s by Agam and
Livne, where erthyrocytes (RBCs) were coated with fibrinogen, and these modified RBCs showed induction of platelet aggregation in vitro and reduction of prolonged bleeding time in thrombocytopenic rats in vivo114. In the mid 1990s, Yen et al reported a biomaterials technology named ‘ThrombospheresTM’ consisting of albumin microspheres coated with fibrinogen, and these particles rendered aggregation of active platelets in vitro as well as
131 reduction of bleeding time in an ear-punch model in rabbits in vivo115,116. A similar design
termed ‘SynthocytesTM’ was reported by Levi et al in the late 1990s, where fibrinogen-
coated albumin microcapsules were able to reduce the bleeding time as well as blood
volume loss in an ear-punch bleeding model and abdominal surgical incision model in
thrombocytopenic rabbits117. In vitro platelet-aggregating capacity of the fibrinogen-coated
albumin particles under shear flow conditions was also reported in early 2000 by the
research group from Waseda University and Keio University in Japan118. Another similar
design of fibrinogen-coated albumin particles was reported under the product name
FibrinoplateTM by a company called Advanced Therapeutics & Co, but further information
about this product was not available after 2011. Figure 3-7 shows selected results from studies carried out with various fibrinogen-coated particle designs. Figure 3-7A shows the effect of administering fibrinogen-coated albumin microcapsules (SynthocyteTM) in
reducing ear punch bleeding time in thrombocytopenic rabbits, where induction of
thrombocytopenia (TCP) significantly increased bleeding time from normal and
administration of SynthocyteTM particles (SC) could significantly reduce bleeding time in
these animals, compared to administration of control particles (CP) or saline. Figure 3-7B
shows scanning electron micrograph of the SynthocyteTM particles incorporated with
platelets and fibrin in hemostatic clots. Figure 3-7C shows the ability of fibrinogen-coated
albumin particles to bind to platelet-immobilized surfaces in absence versus presence of
Ca++ ions, compared to binding of unmodified albumin particles, while Figure 3-7D shows representative fluorescence microscopy images of these binding studies. These results
establish that fibrinogen-coated particles can interact and bind with active (e.g. Ca++
activated) platelets under flow environment, possibly via interaction with platelet surface
132
Figure 3-7 Selected results from studies carried out with various fibrinogen-coated particle designs. A shows the effect of administering fibrinogen-coated albumin microcapsules (SynthocyteTM) in reducing ear punch bleeding time in thrombocytopenic rabbits, where induction of thrombocytopenia (TCP) significantly increased bleeding time from normal levels and administration of SynthocyteTM particles (SC) could significantly reduce bleeding time in these animals, compared to administration of control particles (CP) or saline; B shows scanning electron micrograph of the SynthocyteTM particles incorporated with platelets and fibrin in hemostatic clots; C shows the ability of fibrinogen-coated albumin particles to bind to platelet- immobilized surfaces in absence versus presence of Ca++ ions, compared to binding of unmodified albumin particles and D shows representative fluorescence microscopy images of these binding studies, confirming high binding of fibrinogen-coated particles and establishing that these particles can interact with active platelets under flow environment, possibly via interaction with platelet surface integrin GPIIb-IIIa, thereby mimicking and amplifying the active platelet aggregation component of hemostasis. Figure components adapted and reproduced with permission117,118. Copyright 1999, Macmillan Publishers Ltd and 2001, American Chemical Society.
integrin GPIIb-IIIa. Thus, these technologies seem to efficiently leverage and amplify the active platelet aggregation component of hemostasis by means of a ‘super-fibrinogen’ design. Off-target systemic thromboembolic risks can be a possible issue with such designs, and therefore rigorous in vivo pre-clinical studies are needed to establish safety and clearance.
133 3.7 Synthetic biomaterials-based platelet mimics and substitutes
Creating platelet-inspired designs by utilizing isolated platelet membranes, fibrinogen
coatings and platelet surface-relevant recombinant protein fragments can present issues of batch-to-batch variation in bioactivity, protein stability and solubility, potential immunogenicity and high cost. Therefore, a significant research interest has been focused on utilization of synthetic ligand systems (e.g. peptides) decorated on semi-synthetic or synthetic particle platforms that can render hemostatic mechanisms of platelets while providing advantages of controllable and scalable manufacture, consistent bioactivity, long shelf-life and reduced biological/immunological side effects. To this end, the majority of research approaches has been focused in the area of mimicking fibrinogen-binding to active platelet surface integrin GPIIb-IIIa by decorating particle surfaces with fibrinogen-relevant synthetic peptide ligands, in essence once again rendering a ‘super-fibrinogen’ type design.
Fibrinogen is a hepatic protein, approximately 340,000 in molecular weight and present in the blood at concentration ranges of 200-400 mg/dL. Fibrinogen has an approximately 45 nm long macromolecular structure consisting of two outer D-domains, each connected by a coiled-coil segment to its central E domain. The molecule contains two sets of three polypeptide chains, termed Aα, Bβ and γ, which are joined together in the N-terminal E domain by five symmetrical disulfide bridges75,119. Upon activation of platelets in a
hemostatic (or thrombotic) environment, the surface integrin GPIIb/IIIa assumes a ligand-
binding conformation that can bind to fibrinogen via the Aα chain tetrapeptide Arg-Gly-
Asp-Ser (RGDS) and the γ chain 12-residue peptide His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-
Gly-Asp-Val (HLGGAKQAGDV)120,121. Since these peptide sequences are present on both
termini of fibrinogen, the fibrinogen molecule can act as a bridging system between active
134 platelets (schematic shown previously in Figure 3-4), resulting in aggregation of these
platelets to form the primary hemostatic plug. Based on these mechanisms, one of the
earliest semi-synthetic designs was that of ‘Thromboerythrocytes’ reported by Coller et al,
where RGD sequence Ac-CGGRGDF-NH2 was covalently conjugated to the surface amino
group of RBCs using a heterobifunctional crosslinking reagent (N-maleimido-6-
aminocaproyl ester of 1-hydroxy-2-nitrobenzene4-sulfonic acid)122. These RGD-decorated
RBCs showed binding to active platelet integrin GPIIb-IIIa, were capable of co-
aggregating with activated platelets and could bind to active platelets on collagen-coated
surfaces. Decoration of synthetic particle platforms (latex beads, poly-lactic acid based
polymeric particles etc.) with RGD sequences (e.g. CGGRGDF on latex beads and GRGDS
on poly-lactic acid-based particles) have been reported by Takeoka and colleagues123 as
well as Lavik and colleagues124,125, in the context of mimicking and amplifying the
fibrinogen-mediated interactions with integrin GPIIb-IIIa on activated platelets.
Interestingly, in the studies conducted by Takeoka et al with CGGRGDF-decorated particle systems, it was found that these particles could promote activation (and subsequent aggregation) of inactive platelets123. Similar effects of linear RGDS-containing peptides
have also been reported by Du et al and Beer et al120,126. Furthermore, the RGD tri-peptide sequence is known to have ubiquitous cell- and matrix-recognition capabilities127,128. In
fact, this sequence has been reported in a variety of research including targeting to tumor
cells, endothelial cells, bone cells etc. beyond its interaction with platelets. Therefore,
utilizing CGGRGDF or GRGDS type of linear ubiquitous RGD peptides to mimic
hemostasis-relevant fibrinogen-mimetic platelet aggregation mechanisms on synthetic
platelet designs may pose major systemic thromboembolic risks (due to activation of
135 circulating resting platelets) as well as render cross-reactivity with other cellular entities in
vivo that may affect clinical translation. Because of the issues associated with these linear
ubiquitous RGD peptides, Takeoka and colleagues have also researched utilizing
fibrinogen γ-chain relevant peptides, specifically the sequence HLGGAKQAGDV
otherwise known as the H12 peptide, to decorate particle platforms. This peptide was used
to modify the surface of latex beads, albumin particles and liposomes, and the resultant
systems were evaluated for their platelet-inspired pro-aggregatory hemostatic activities129–
133. In vitro, the H12-decorated latex beads showed minimal aggregation effect on inactive
platelets, suggesting that this peptide may confer improved systemic safety compared to
RGDS peptides. Albumin microparticles surface-decorated with H12 peptides were able to
enhance platelet aggregation on collagen-immobilized surfaces in vitro and improve ear
punch bleeding time in thrombocytopenic rabbits and tail bleeding time in
thrombocytopenic rats, both by approximately 50%. Liposomes surface-decorated with
H12 peptides showed similar hemostatic capability, and encapsulating ADP in such
liposomes enabled augmentation of platelet activation and aggregation as well as correction
of ear bleeding time in thrombocytopenic rabbits and tail bleeding time in
thrombocytopenic rats. These studies however did not provide any information regarding
the overall biodstribution of the particles. Figure 3-8 shows selected results from in vitro
and in vivo studies that have been carried out using particle designs surface-decorated with
fibrinogen-relevant RGD or H12 peptides to mimic and amplify fibrinogen-mediated
platelet aggregation. Figure 3-8A shows platelet aggregometry studies with RGD peptide- decorated RBCs (Thromboerythrocytes), where unmodified control erythrocytes were unable to enhance aggregation of ADP-activated platelets and Thromboerythrocytes were
136 unable to enhance aggregation of inactive platelets or integrin GPIIb-IIIa-blocked platelets, but Thromboerythrocytes were able to significantly enhance the aggregation of ADP- activated platelets. Figure 3-8B shows representative fluorescent images as well as quantitative surface-coverage data of H12 peptide-decorated, or RGD peptide-decorated or undecorated fluorescently-labeled latex beads suspended in reconstituted blood binding to platelet-immobilized surfaces, indicating the enhanced binding capability of H12- decorated and RGD-decorated beads to platelets. Figure 3-8C shows the capability of H12 peptide-decorated PEGylated albumin particles (H12-PEG-Alb) to significantly reduce ear punch bleeding time upon intravenous administration in thrombocytopenic rabbits, compared to administration of control undecorated PEG-albumin (PEG-Alb) particles.
Figure 3-8D shows the capability of H12 peptide-decorated PEGylated liposomal vesicles
(H12-PEG-vesicles) to significantly reduce tail bleeding time in thrombocytopenic rats, compared to administration of control undecorated PEG-vesicles. Figure 3-8E shows quantitative bleeding time reduction data and representative fluorescence image of hemostasized vessel, where administration of RGD-decorated polymeric nanoparticles
(GRGDS-PLGA-PLL particles) significantly reduced bleeding time in a rat femoral artery injury model, compared to administration of undecorated particles or saline.
Regarding use of peptides to mimic platelet’s adhesion mechanisms, an early demonstration is found in reports from Gyongyossy-Issa and colleagues involving the development of platelet GPIbα-relevant 15-mer peptides that have binding capability to vWF134. These peptides were tethered onto liposomal surfaces stabilized via cross-linking,
and the resultant liposomal constructs, 150-200 nm in diameter, were tested as ‘synthetic
137
Figure 3-8 Selected results from studies carried out with various particle platforms surface-decorated with fibrinogen-relevant RGD or H12 peptides to mimic platelet aggregation mechanism. A shows platelet aggregometry studies with RGD-decorated RBCs (Thromboerythrocytes), where unmodified control erythrocytes were unable to enhance aggregation of ADP-activated platelets and Thromboerythrocytes were unable to enhance aggregation of inactive platelets or integrin GPIIb-IIIa-blocked platelets, but they could significantly enhance the aggregation of ADP-activated platelets, thereby mimicking fibrinogen and GPIIb- IIIa mediated platelet aggregation mechanism; B shows representative fluorescent images as well as quantitative surface-coverage data of H12 peptide-decorated, or RGD peptide-decorated or undecorated fluorescently-labeled latex beads suspended in reconstituted blood and flowed over platelet-immobilized surfaces, indicating the enhanced GPIIb-IIIa-binding capability of H12-decorated and RGD-decorated beads to platelets; C shows the capability of H12-decorated PEGylated albumin particles (H12-PEG-Alb) to significantly reduce ear punch bleeding time upon intravenous administration in thrombocytopenic rabbits, compared to administration of control undecorated PEG-albumin (PEG-Alb) particles; D shows the capability of H12-decorated PEGylated liposomal vesicles (H12-PEG-vesicles) to significantly reduce tail bleeding time in thrombocytopenic rats, compared to administration of control undecorated PEG-vesicles; E shows quantitative bleeding time reduction data and representative fluorescence image of hemostasized vessel, where administration of RGD-decorated polymeric nanoparticles (GRGDS-PLGA-PLL particles)
138 significantly reduced bleeding time in a rat femoral artery injury model, compared to undecorated particles or saline. Figure components adapted and reproduced with permission122–124,129,130. Copyright 1992, American Society for Clinical Investigation; 2003, Elsevier; 2008, John Wiley & Sons Inc.; 2005, American Chemical Society and 2009, AAAS.
platelet substitutes’135. Although the biochemical characterizations of these constructs have been reported, the actual hemostatic efficacy evaluation of these constructs in vivo have not been reported in any pre-clinical animal model.
3.8 Discussion
Regarding mimicry of platelet functions, one straightforward approach that several research groups have employed involves detergent-based extraction of natural platelet’s membrane and then coating synthetic particles with this membrane material. Since many of platelets’ hemostatic mechanisms are associated with its surface biology, this approach, theoretically, can capture and leverage this surface biology on synthetic platforms for hemostatic (and drug delivery) applications. Additionally, due to a significant history of research on the evaluation of infusible platelet membrane (IPM) vesicles as intravenous hemostats, the membrane-coated particle approaches can potentially streamline their design optimization. One concern in this approach is the loss of bioactivity of certain platelet surface motifs that is rendered by the various membrane extraction and purification steps, which may in turn lead to capturing only a part of hemostatic mechanisms or presenting batch-to-batch inconsistency in hemostatic function. Another concern is that the limited availability of platelet concentrates may in turn affect the availability of extractable platelet membranes and this can become a logistical barrier to translation. Such issues can be potentially resolved by utilizing recombinant protein fragments or small peptide ligands
139 that can mimic the functions of platelets’ hemostasis-relevant surface components without having to mimic the complete surface membrane and complex biology. Examples of this approach are seen in designs that utilize recombinant GPIbα, GPIa-IIa and vWF-A1 protein fragments, as well as, in strategies that employ vWF-binding, collagen-binding and integrin
GPIIb-IIIa-binding peptides to decorate synthetic particle platforms.
A variety of particle fabrication technologies to modulate physico-mechanical and geometric parameters have been established in recent years, and this is expected to benefit the incorporation of such parameters in refining the materials and technologies for synthetic platelet research. Upon adhesion and activation at the injury site due to physical
(shear) and biochemical (receptor-based signaling cascades) triggers, platelets transform from the ‘resting’ biconvex discoid morphology to an ‘activated’ stellate pseudopodal morphology with significant reduction in modulus and remain irreversibly adhered at the injury site for hemostasis. This phenomenon can inspire the development of materials with environment-responsive physico-chemical and mechanical properties for development of platelet-inspired hemostats. Regarding utilization of semi-synthetic and synthetic particle based systems for designing platelet surrogates, it is critical to study and mitigate systemic risks associated with the particle material itself, as well as, with the surface chemistries and ligands utilized for particle surface decoration. In previous sections, the potential risks associated with using ubiquitous cross-reactive ligands (e.g. RGDS) have been discussed.
In the same note, every ligand motif should be studied both in free form as well as particle- tethered form for systemic effects on platelets (e.g. by aggregometry) and plasma (e.g. by thrombin generation assays). In addition, the base materials used for particle construction
140 should be chosen carefully to ensure minimal toxicity and high biocompatibility. It is also important to determine the spatiotemporal fate of these particles and materials once administered in vivo, in the context of biodegradation, clearance mechanisms and elimination pathways. Maintaining systemic safety while rendering targeted hemostatic action at the bleeding site within an appropriate time window and undergoing effective clearance, will be the key to success for any of these technologies.
Regarding utilization of coagulation cascade components for intravascular hemostasis, the direct use of recombinant coagulation factors is clinically well established in selective groups of patients. The bulk of these approaches are essentially directed at amplifying the generation of thrombin through tissue-factor pathway or common pathway of coagulation, such that the thrombin can convert fibrinogen to fibrin to facilitate clotting. It is important to note that the generation of thrombin is significantly enhanced in presence of active platelet membrane via formation of tenase complex (FVIIIa + FIXa) and prothrombinase complex (FXa + FVa) on the phosphatidyl serine-rich membrane surface. Therefore, use of coagulation factors in patients with platelet deficiency or dysfunction may have only limited benefit towards hemostasis. In comparison, use of materials and technologies that can promote activation of certain coagulation cascade components independent of platelet presence may show some benefit towards subsequently amplifying hemostasis. One such interesting material is the PolyP coating on particles that attempts to mimic the action of polyphosphates secreted from active platelets by activating FXI (to FXIa) and FIX (to
FIXa) en route to activation of FX and subsequently amplifying thrombin generation through the tenase and prothrombinase complexes on active platelet surface. While this
141 mechanism and technology have generated substantial interest in recent hemostasis research, it would be important to limit the action of PolyP-coated particles at the bleeding site, to avoid systemic hypergeneration of thrombin and fibrin. One potential approach to shielding of the PolyP coating can be the use of a mask that may be cleaved site-selectively by bleeding site relevant enzyme action, thereby exposing the PolyP only locally to amplify coagulation cascade activation and propagation. Precise regulation of these mechanisms will be important to avoid systemic effects. The same rationale holds true for technologies that may attempt to coat synthetic or semi-synthetic particle platforms with tissue factor or negatively charged phospholipids. Other pro-coagulant technologies that strengthen the fibrin clot (e.g. fibrin-crosslinking microgel particles or FXIIIa action mimicking polymers) may have to depend upon sufficient prior generation of fibrin at the bleeding site to be able to significantly benefit in vivo from their mechanism of fibrin-strengthening.
This in turn would require prior sufficient generation of thrombin and resultant conversion of fibrinogen in a site-selective manner at the bleeding site, which in turn requires the involvement of tissue factor-dependent thrombin generation and platelet-dependent thrombin amplification pathways at the site. Therefore, future research should be directed at modularly combining the hemostasis mimicking and enhancing mechanisms of the different materials and technologies to find an optimum system that (i) promotes and enhances active platelet or platelet-inspired aggregation at the bleeding site, (ii) augments thrombin generation and fibrin formation selectively at that site and (iii) strengthens the formed fibrin biopolymeric network to reduce clot lysis and amplify hemostasis. While individual components of such systems are currently under pre-clinical research, these and potential integrated systems need to be rigorously studied for biodistribution, systemic
142 safety and site-selective hemostatic action in multiple well-characterized bleeding models in vivo. Through robust interdisciplinary research between materials scientists, chemical and biomedical engineers, molecular biologists, biochemists and clinicians, future advancement of hemostatic materials and technologies can be envisioned to revolutionize bleeding treatment and transfusion medicine approaches.
3.9 Acknowledgments
This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the
National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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165 Chapter 4: Designing an Ideal Platelet-inspired System for the Intravenous
Management of Traumatic Bleeding
Content based on: Hickman DA, Pawlowski CL, Sekhon UDS, Marks J, Gupta AS. Biomaterials and Advanced Technologies for Hemostatic Management of Bleeding. Adv Mater. 2017 Nov 22. doi: 10.1002/adma.201700859.
Shukla M, Sekhon UD, Betapudi V, Li W, Hickman DA, Pawlowski CL, Dyer MR, Neal MD, McCrae KR, Sen Gupta A. In vitro characterization of SynthoPlate™ (synthetic platelet) technology and its in vivo evaluation in severely thrombocytopenic mice. J Thromb Haemost. 2017 Feb;15(2):375-387. doi: 10.1111/jth.13579. PubMed PMID: 27925685; PubMed Central PMCID: PMC5305617.
4.1 Introduction
The past two chapters have been a comprehensive review of the various topical, intra-
cavitary and intravenous hemostatic technologies in terms of materials, mechanisms and
state-of-art, and discussed challenges and opportunities to help advancement of the field.
For externally accessible injuries, a variety of natural and synthetic biomaterials have
undergone robust research, leading to hemostatic technologies including glues, bandages,
tamponades, tourniquets, dressings and pro-coagulant powders. In contrast, treatment of
internal non-compressible hemorrhage still heavily depends on transfusion of whole blood
or blood’s hemostatic components (platelets, fibrinogen and coagulation factors).
Transfusion of platelets poses significant challenges of limited availability, high cost,
contamination risks, short shelf-life, low portability, performance variability and
immunological side-effects, while use of fibrinogen or coagulation factors provides only
partial mechanisms for hemostasis. With such considerations, significant interdisciplinary
research endeavors have been focused on developing materials and technologies that can
166 be manufactured conveniently, sterilized to minimize contamination and enhance shelf-
life, and administered intravenously to mimic, leverage and amplify physiological
hemostatic mechanisms. We have developed an I.V.-administrable ‘synthetic platelet’
nanoconstruct (SynthoPlateTM), that can mimic and amplify the body’s natural hemostatic
mechanisms specifically at the bleeding site while maintaining systemic safety. We have also begun to explore ways to exploit SynthoPlateTM’s lipid core for injury-site specific
delivery of hemostasis augmenting drugs such as Tranexamic Acid (TXA, anti-fibrinolytic
agent), recombinant coagulation factors, polyphosphates etc.
4.2 Current State of Traumatic Bleeding Management
While externally administered and intra-cavitary hemostatic materials and technologies
have significantly improved the treatment options for accessible injuries, emergency or
prophylactic management of internal non-compressible hemorrhage is still heavily
dependent on transfusion of whole blood or blood components (RBC, plasma and
platelets). These transfusion methods and products are essentially meant to compensate for
blood volume loss and to facilitate or augment physiological inherent mechanisms of
primary and secondary hemostasis. Along with blood transfusion, there are a few
pharmaceutical compounds like desmopressin, aprotinin and tranexamic acid (TXA) that
have been either approved or under clinical trial for intravenous administration to augment
hemostasis1. These pharmaceutical compounds act essentially as facilitators or stabilizers of hemostatic pathway components, but are not directly pro-hemostatic agents. For
example, desmopressin acts by mimicking the function of natural vasopressin to
systemically increase the plasma concentration of Factor VIII and vWF, and therefore can
167 be used to treat mild hemophilia and vWF disease2. However, meta-analysis reports of
controlled clinical trials with desmopressin have indicated that such systemic amplification
of coagulation facilitators may pose a risk of off-target thrombosis3,4. Aprotinin is a broad-
spectrum inhibitor of serine proteases, and therefore its use in hemostasis is debatable since
it can block pro-hemostatic factors (e.g. thrombin) as well as anti-coagulatory factors (e.g
activated protein C) and fibrinolytic factors (e.g. plasmin)5. Nonetheless, the use of this
agent has shown benefit in management of bleeding complications in several surgical
procedures6,7. As described previously, TXA does not promote clotting but rather maintains clot strength and stability by preventing its plasmin-induced breakdown8. This property has
shown benefits in preventing bleeding related morbidities and mortalities9. Along with the
use of these pharmaceutical agents, blood component (especially platelet) transfusion
remains the primary intravascular strategy in rendering hemostasis, and the use of blood
products present significant challenges due to limited availability, high cost of purification
and storage, contamination risks, and immunological side-effects. While efficient blood-
banking protocols and modern pathogen reduction technologies work relentlessly to
resolve some of these challenges in locations with suitable medical facilities, the challenges
persist in remote locations and austere battlefield conditions. This has triggered robust
research endeavors in development of semi-synthetic or synthetic materials-based systems
that can provide the hemostatic functions of blood components while circumventing the
issues associated with natural blood products. In the context of hemostasis, these research
endeavors are currently directed towards materials that capture the biology or functions of
platelets and the coagulation cascade components.
168 4.3 Designing an Ideal System
Prompt resuscitation and point-of-injury treatment of ongoing hemorrhage is of upmost importance to improve survival10,11. In recent years, resuscitation of bleeding trauma patients has shifted away from crystalloid fluids to balanced blood product transfusion12–
14. Important in the current resuscitation strategies is the transfusion of pooled platelets,
which have been shown to be a critical component of modern hemostatic resuscitation
strategies15,16. As mentioned in Chapter 1, the PAMPer trial prehospital adminstration of thawed plasma resulted in lower 30-day mortality and a lower median prothrombin time
than standard-care resuscitation while the PROPPR trial demonstrated that early platelet
administration is associated with improved hemostasis and reduced mortality in traumatic
bleeding patients compared to patients who received blood products without platelets17,18.
However, platelet transfusions are fraught with several challenges including increased risk
of bacterial contamination in storage, platelet activation/degranulation during storage, and
a limited shelf life of only 5 days to 7 days19–21. As well, platelet transfusions account for
25% to 30% of all transfusion reactions while only accounting for around 10% of blood
product transfusions19,22. Finally, given the challenging requirements for storage of pooled platelets, there is very limited availability of platelets outside of large central blood banks, which presents a major problem for hemorrhage resuscitation in remote civilian locations
and austere battlefield conditions19,22. This has prompted significant research in platelet
substitutes that may resolve the above issues while providing platelet-inspired hemostatic action.
169 For an ideal design of a platelet-inspired hemostatic particle, the injury site-selective adhesive and aggregatory mechanisms of platelets should be functionally mimicked and integrated on a biocompatible, intravenously injectable particle platform. To this end,
Takeoka and colleagues have demonstrated the possibility of combining the platelet- inspired pro-adhesive function with pro-aggregatory function by either co-conjugation of rGPIbα and H12 peptide on the same latex bead, or mixing rGPIbα-decorated latex beads with H12 peptide-decorated latex beads23. When the two motifs were conjugated onto the surface of the same bead, the large size of the rGPIbα motif sterically masked the action of the smaller H12 peptide, and therefore the effect seen was mostly of platelet-inspired adhesion but no aggregation (Figure 4-1). In contrast, when each motif was conjugated to separate beads and the beads were mixed together, the resultant mixture significantly enhanced the aggregation and surface-coverage of platelets on collagen-coated surfaces in presence of soluble vWF under shear flow conditions, when compared to H12 peptide- decorated beads only. These studies suggested that if platelet-relevant pro-adhesive and pro-aggregatory functionalities could be combined on a single particle platform without the issue of mutual steric interference, the resultant design would have superior hemostatic capacity compared to designs that bear only pro-adhesive or only pro-aggregatory function.
Figure 4-1. Platelet aggregometry studies where undecorated latex beads, or H12 peptide-decorated latex
170 beads, or latex beads co-decorated with H12 peptides and rGPIbα fragments were added to ADP-activated platelets and the results showed that the co-decorated beads had no additional aggregation enhancement effects, possibly due to the masking of small H12 peptides by the large rGPIbα fragments.
4.4 Design of SynthoPlateTM Nanoparticle System
Rationalizing from this idea, we have utilized the technique of heteromultivalent surface-
modification to decorate synthetic particle surfaces simultaneously with vWF-binding peptides (VBP), collagen-binding peptides (CBP) and active platelet GPIIb-IIIa-binding fibrinogen-mimetic peptides (FMP), to create a unique synthetic platelet design, the
SynthoPlateTM 24,25. The VBP, Thr-Arg-Tyr-Leu-Arg-Ile-His-Pro-Glu-Ser-Trp-Val-His-
Glu-Ile (TRYLRIHPQSWVHQI) was derived from the C2 domain (residues 2303-2332) of the coagulation factor FVIII, based on reports of FVIII binding domains on vWF26. The
CBP sequence was a single chain 7-mer repeat of the Glycine(G)- -Proline(P)-
Figure 4-2. Manufacture and characterization of SynthoPlate constructs.
171
Hydroxyproline(O) tri-peptide (i.e. -[GPO]7-) that has helicogenic affinity to fibrillar
collagen but minimal ability to activate platelets via platelet collagen receptors GPIa/IIa
and GPVI (hence no systemic thrombotic risk)27,28. The FMP sequence was the cyclic RGD
peptide cyclo-CNPRGDY[-OEt]RC, which was reported to have high affinity and
selectivity for active platelet GPIIb-IIIa but minimal affinity for resting GPIIb-IIIa (hence
reduced systemic thromboembolic risk) as well as minimal selectivity for other common
cell integrins (hence reduced cross-reactivity in vivo)29. Each peptide was conjugated to a
poly(ethylene glycol)-terminated lipid (i.e. PEG-lipid) molecule via amide, thioether or
‘click’ chemistry to form lipid-PEG-peptide conjugates25. Resultant conjugates could be
self-assembled and extruded into ~150nm unilamellar liposomal vesicles such that the
Figure 4-3. Ex vivo aggregometry studies for SynthoPlate with mouse platelets.
172 vesicle’s outer leaflet had heteromultivalent decorations of VBP, CBP and FMP motifs
(Figure 4-2). Due to the small size of the peptides and adequate PEG spacer length, they
did not sterically interfere with respective bioactivities. This integrative synthetic platelet
design (bearing VBP + CBP + FMP peptides together) was named SynthoPlateTM. The
SynthoPlateTM nanoparticles were able to demonstrate platelet-inspired hemostasis-
relevant bioactivities in vitro.
4.5 SynthoPlateTM In Vitro Evaluation
We observe, utilizing aggregometry studies with mouse platelets, that SynthoPlate does
not aggregate or activate resting platelets and that SynthoPlate can rescue aggregation
when added to diluted platelets (Figure 4-3). Looking more closely at the classical
secondary hemostasis pathway, thrombin generation assays were performed. These studies
demonstrated that SynthoPlate does not have the ability to rapidly and spontaneously
activate coagulation co-factors in plasma to generate thrombin. In the presence of activated platelets, it does slightly enhance the generation of thrombin likely by recruiting and clustering activated platelets (Figure 4-4A). In parallel plate flow chamber experiments
with plasma in the presence of platelet agonist (ADP), flowing over a ‘vWF + collagen’
surface, larger clots are generated in the presence of SynthoPlate (Figure 4-4B and C). D‐
dimer analysis of the generated/deposited clots on the ‘vWF + collagen’ surface confirms
that the clots resulting from enhanced clot formation in the SynthoPlate‐added group are
indeed rich in fibrin, and that the process does not just involve fibrinogen binding to
recruited active platelets (Figure 4-4D).
173
Figure 4-4. Effect of SynthoPlate on secondary hemostasis
4.6 In Vivo Evaluation of SynthoPlateTM In Thrombocytopenia Model
We showed that SynthoPlate significantly reduced tail transection bleeding time in normal mice at superior levels compared to particles decorated with pro-adhesive (VBP + CBP) peptides only or pro-aggregatory (FMP) peptides only30–32. We then
174
Figure 4-5. Development of thrombocytopenia model in mouse and evaluation of SynthoPlate capability in correcting tail transection bleeding time in thrombocytopenic mouse.
developed a thrombocytopenia mouse model to evaluate SynthoPlate capability in
correcting tail transection bleeding time in thrombocytopenic mouse. Mice underwent
dose‐dependent depletion of platelets resulting in a corresponding increase in bleeding time
after tail transection (10 μg of antibody cocktail dose resulted in ~90% platelet depletion and a corresponding four‐fold to five‐fold increase in bleeding time as compared with normal mice) (Figure 4-5A). Severely thrombocytopenic (TCP) mice injected with
175 phosphate‐buffered saline (PBS) or control (unmodified) particles at various doses show
no improvement (reduction) in bleeding time, but TCP mice injected with SynthoPlate
vesicles show a dose‐dependent significant improvement in bleeding time, with the highest
dose (1000 nL−1, equivalent to the normal murine platelet count) reducing the bleeding
Figure 4-6. Characterization of SynthoPlate‐mediated hemostatic clot in transected tail of thrombocytopenic mouse.
time to ~150s (close to the bleeding time of ~100s for normal mice) (Figure 4-5B).
Immunofluorescence analysis showed that the SynthoPlateTM particles could co-localize with injured endothelium and locally available active platelets to enhance primary hemostasis (platelet recruitment and aggregation) and could in effect also enhance
176 secondary hemostasis (fibrin generation) (Figure 4-6). In addition, it was demonstrated
that SynthoPlateTM was systemically safe (no systemic pro-thrombotic or pro-coagulant
risk) and could be eliminated mostly via liver over time (Figure 4-7)25. The size, shape and
stiffness aspects of platelets influence their vascular margination towards the wall under a hemodynamic flow environment33,34. Based upon such observations, the heteromultivalent
surface- modification strategy used for SynthoPlateTM
Figure 4-7. Evaluation of systemic pro‐coagulant risk and biodistribution of SynthoPlate in mice.
177
particles, could also be adapted to other particle platforms (e.g. discoid albumin particles)
to integrate platelet-relevant hemostatic surface chemistry with platelet’s fluid dynamic
margination-influencing biophysical (e.g. geometric) properties35.
4.7 Discussion
It is important to note here that platelet’s hemostasis-relevant surface mechanisms are driven by multiple interactions occurring in tandem, and therefore the design of a synthetic platelet mimic can significantly benefit from incorporating such heterotypic interactions via materials engineering. This aspect was first emphasized in studies where multiple recombinant protein fragments (e.g. rGPIbα that binds vWF and rGPIa/IIa that binds collagen) were combined on latex beads or albumin particles. The recombinant technology usually involves high cost, and the large size of these recombinant protein fragments often poses mutual steric interference to their respective bioactivity when conjugated together on micro- or nano-scale particles. Protein fragments may also pose risks of immunogenicity over time. These issues can be potentially resolved by identifying and utilizing small peptides instead of protein fragments that can perform the same platelet-inspired functions.
It was demonstrated in our recent reports that platelet’s hemostatically relevant adhesive and aggregatory functions could be efficiently captured via heteromultivalent modification of lipidic and polymeric particles with multiple types of small peptides. Such approaches may allow easy manufacture and scale up by leveraging modern technologies of automated peptide synthesis, purification and high throughput particle manufacture methods. These strategies can also incorporate biophysical components of particle design (e.g. shape, size,
178 flexibility etc.) that mimic the fluid dynamic behavior of platelets regarding their
interaction with the RBC volume and efficient margination to vessel wall. Exploiting such
biophysical parameters for particle design can open new doors not only for synthetic
platelet technologies, but also for efficient platforms in vascular drug delivery36.
While such heteromultivalent integrative designs demonstrate combinatorial functional advantages in mimicking platelet mechanisms, in advancement and translation of such technologies, there may be potential challenges regarding large-scale manufacturing of such multi-component designs especially in the context of Good Manufacturing Practice
(GMP) scale process development37. Also, these liposome-based and albumin-based
particle designs as well as polymeric nanoparticle-based designs described previously, should be extensively studied for dose limitations in terms of toxicity (i.e. maximum tolerance), systemic risks, and possible immune response risks. Biodistribution is an important technique in formulating drugs as it assists in determining the pharmacokinetic profile and toxicity. There are limitations to utilizing any type of biodistribution protocol.
In Figure 4-7, data is presented that was obtained by intravenously injecting RhB labeled particles into the mice and allowing the particles to circulate for 2hrs. After this time, the mice were euthanized, the organs were collected and homogenized, and the RhB labeled
particles were extracted and quantified via UPLC. The concentration of the nanoparticles
(mg of nanoparticles/mg of tissue) in each organ,based off a calibration curve, and the
injected dose was utilized to determine the percent biodistribution in each organ.
Unfortunately, this protocol only allows us to determine the biodistribution at one time
point of a given animal and each sample must undergo considerable preparation to be
179 analyzed. In this same study, separate mice were injected with radiolabeled SynthoPlate
3 111 ( H tagged cholesteryl ester in the vesicle membrane or encapsulated InCl3 in the vesicle core). These mice were whole body imaged by single photon emission computed
111 tomography for assessment of the InCl3-labeled particle distribution or killed for
harvesting of organs and blood for scintillation counting of 3H-labeled particles. This
protocol allowed for real-time kinetic analysis of particle distribution in a single animal.
Unfortunately, the use of radioactive tracers in these protocols require extra institutional
approval, experimental constraints, safety precautions and waste processing.
In addition to biodistribution studies, in order to utilize the nanoparticle designs discussed
as intravenous hemostat technologies, it would be critical to carry out dose escalation
studies in appropriate in vivo models, along with systemic monitoring of hemodynamics
and vitals. For example, it has been demonstrated in the field of nanoparticle-based drug
delivery research that certain liposome-based formulations can result in complement- activation related pseudoallergy (CARPA) reactions38,39 depending on dose, particle size
and surface-charge and administration speed.
In the following chapters, I will describe the warranted studies that I carried out to validate
and characterize the efficacy and risk of our ‘synthetic platelet surrogate’ designs and the
methods established to enhance the efficacy and mitigate the risks of our designs.
4.8 Acknowledgments
This work was supported by the National Heart Lung and Blood Institute (NHLBI) of the
180 National Institutes of Health under award numbers R01 HL121212 (PI: Sen Gupta). The content of this publication is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
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0002/ejnm-2012-0002.xml
188 Chapter 5: Intravenous administration of synthetic platelets (SynthoPlate) in a mouse
liver injury model of uncontrolled hemorrhage improves hemostasis
Content based on: Dyer MR, Hickman DA, Luc N, Haldeman S, Loughran P, Pawlowski CL, Sen Gupta A, Neal MD. Intravenous administration of synthetic platelets (SynthoPlateTM) in a mouse liver injury model of uncontrolled hemorrhage improves hemostasis. J. Trauma Acute Care Surg. 2018; (March). doi: 10.1097/TA.0000000000001893. PMID: 29538234
5.1 Introduction
Traumatic hemorrhage continues to be a leading cause of preventable death in civilian and battlefield scenarios. Given the challenges previously described associated with platelet transfusions, there is significant clinical interest in extending the shelf-life and hemostatic viability of platelets via cold storage and lyophilization1,2, as well as in developing
transfusable synthetic platelet substitutes that allow long-term storage and platelet-inspired hemostatic action3–5. To this end, we have developed a synthetic platelet nanotechnology,
namely SynthoPlate, that incorporates the proadhesive and proaggregatory properties of native platelets by virtue of decorating a combination of platelet function-mimicking peptides (von Willebrand Factor-binding peptides [VBP], collagen binding peptides or collagen binding peptide [CBP] and active platelet GPIIb-IIIa–binding fibrinogen-mimetic peptides [FMP]) on a biocompatible lipid vesicle platform6. SynthoPlate is shelf-stable, portable, and easily administered, addressing many of the challenges and limitations of human platelet usage in remote and prehospital settings. Detailed chemistry of these peptide decorations and functions, in vitro characterization of the resultant SynthoPlate technology and its promising hemostatic effect in correcting tail bleeding time in normal
as well as thrombocytopenic mice, have been reported previously7,8. Building on these
189 findings, we hypothesized that SynthoPlate treatment would result in decreased blood loss
in acute, hemorrhagic shock. To test this, we used a validated, reproducible, and high- throughput model of murine liver injury that results in uncontrolled intraperitoneal
hemorrhage and severe shock9.
5.2 Materials and Methods
5.2.1 Animals
C57BL/6 WT mice (8–12 weeks, male) were purchased from the Jackson Laboratories
(Bar Harbor, ME). Mice were housed in accordance with University of Pittsburgh
(Pittsburgh, PA) and National Institutes of Health (Bethesda, MD) animal care guidelines
in specific pathogen-free conditions with 12-hour light-dark cycles and free access to
standard feed and water. All animal experiments were approved conducted in accordance
with the guidelines set forth by the Animal Research and Care Committee at the University
of Pittsburgh.
5.2.2 SynthoPlate Manufacture
SynthoPlate is made of multiple lipid, lipid-peptide, and cholesterol components, as has
been described previously7,8. 1,2-Distearoyl-sn-glycero-3-phosphocholine, 1,2-Distearoyl-
sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000] (DSPE-
mPEG1000), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-
[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Mal), and 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG2000-
Azide) were purchased from Avanti Polar Lipids (Alabaster, AL). Rhodamine B-
dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE-RhB) was purchased from
190 Invitrogen (Carlsbad, CA). The peptides VBP (sequence CTRYLRIHPQSWVHQI), CBP
(sequence C[GPO]7) and FMP (sequence cyclo-{Pra}CNPRGD{Tyr(OEt)}RC) were custom-synthesized by GeScript (Piscataway, NJ). vWF-binding peptide and CBP were
10 conjugated to DSPE-PEG2000-Mal using thiol-maleimide coupling and FMP was
conjugated to DSPE-PEG2000-azide via copper-catalyzed alkyne-azide cycloaddition
“click” chemistry (CuCAAC)11. The conjugates were purified by dialysis and characterized
by MALDI-TOF mass spectrometry. Cholesterol was purchased from Sigma-Aldrich
(Saint Louis, MO). The lipid vesicle fabrication technique of “film rehydration and
extrusion” was used to manufacture SynthoPlate as described previously2,4.
Heteromultivalently decorated SynthoPlate vesicles were produced and dynamic light
scattering and electron microscopy characterization indicated fresh-made vesicles were approximately 200 nm in diameter as we have previously reported7.
5.2.3 Liver Laceration Model of Uncontrolled Hemorrhage and SynthoPlate Evaluation
We developed and validated a murine model of uncontrolled hemorrhage using a liver
laceration9. A schematic and timeline of the experimental design is presented in Figure
5-1. Briefly, mice were anesthetized with sodium pentobarbital (70 mg/kg) via
intraperitoneal injection, and real-time hemodynamic monitoring is achieved through femoral arterial cannulation. A midline laparotomy is performed, preweighed absorption
191 triangles are inserted away from the liver, and 75% of the left middle lobe of the liver is
Figure 5-1 Experimental timeline, liver laceration model, and SynthoPlate mechanism. (A) Experimental design and timeline for pretreatment studies. (B) Experimental Design and timeline for rescue studies. (C) The liver laceration procedure is performed through a midline laparotomy and 75% of the left middle lobe is lacerated. This results in uncontrolled intraperitoneal hemorrhage and associated hemorrhagic shock. SynthoPlate particles, a novel synthetic platelet nanotechnology, mimic the adhesion, and aggregatory properties of native platelets by decorating lipid vesicles with platelet functioning-mimicking peptides including include a vWF-binding peptide (VBP), a CBP and an active platelet GPIIb-IIIa–binding FMP. These peptides are those able to bind to sites of hemorrhage and other activated platelets to assist and amplify hemostasis.
lacerated. Mice were treated via tail-vein injection with SynthoPlate particles (30 mg/kg), control particles (unmodified particles, 30 mg/kg), or normal saline (0.9%) either 30 minutes before (pretreatment) to or 1 minute after (rescue) performing the liver laceration.
Animal surgeons were blinded to treatment groups. For rescue strategy experiments, the tail vein was cannulated with a mouse tail vein catheter (SAI Infusion Technologies), and the treatment was administered through the catheter. The dose for SynthoPlate infusion
192 was determined from prior work in tail transection bleeding models, with final infusion
volumes of 250 μL to 300 μL7. When administered in the rescue setting, the SynthoPlate infusion was followed by a flush of normal saline, equal volume, to ensure particles were infused into the circulation. To determine the effect of SynthoPlate on acute blood loss mitigation, mice were sacrificed 20 minutes following liver laceration. Blood loss was calculated by assessing the difference in the weight of absorption triangles pre- and post-
liver laceration. The weight of the resected liver was determined immediately following
sacrifice. Hemodynamic data were collected using DMSI-400 (Micro-Med) software.
5.2.4 In Vivo Biodistribution of SynthoPlate Particles
In consideration of a theoretical risk of systemic off-target sequestration of SynthoPlate resulting in microvascular complications, we sought to evaluate the biodistribution and determine clearance of SynthoPlate following hemorrhagic shock in mice subjected to liver laceration as described above. Tissue homogenates were analyzed using ultra high- performance liquid chromatography (UPLC). For this, organs (brain, heart, lung, liver, injured liver, kidney, and spleen) were harvested at the time of sacrifice, snap-frozen in liquid nitrogen, dried in a lyophilizer, and dry weight was recorded. The organs were homogenized at 4,000 rpm for two cycles of 25 seconds using a BeadBug Microtube
Homogenizer (Benchmark Scientific, Edison, NJ) with 3.0-mm high-impact zirconium beads. Samples were shaken overnight at 750 rpm at 37 °C in a 1:1 solution of methanol/chloroform to extract the RhB-conjugated lipids. These samples were centrifuged at 12,000g for 20 minutes, and the supernatant, containing RhB-labeled lipids from SynthoPlate, was collected. The RhB-labeled lipids were resolved using Waters
193 Acquity UPLC system (column: Waters BEH C8 1.7 μm) and analyzed with a fluorescence
detector (Waters Corporation, Milford, MA) using excitation wavelength 560 nm and emission wavelength of 580 nm. The uptake of SynthoPlate in the organs was determined using an RhB fluorescence-based calibration curve for SynthoPlate and reported as the
percent of injected dose as per calibration curve.
5.2.5 Statistics
The primary objective for this study was to determine the efficacy of SynthoPlate transfusion to reduce blood loss in acute, hemorrhagic shock compared with control
particles and normal saline (0.9%). Secondary endpoints were hemodynamic analysis and
biodistribution. Based on pilot data, to detect a decrease in blood loss of 20% resulting in
a treatment effect size of 1.5, at 80% power, and a significance level of 0.05, we determined
that eight mice per group would be needed for the primary endpoint analysis. All data are
presented as mean ± SD for n ≥ 3 unless stated otherwise in the figure legends. Statistical
significance was determined with the two-tailed Student's t test or one-way analysis of variance with Bonferroni post hoc test using Graph Pad Prism software (GraphPad). A p value of less than 0.05 was considered significant.
5.3 Results
5.3.1 SynthoPlate Transfusion as a Pretreatment Results in Decreased Blood Loss and
Improved Hemodynamics During Acute, Severe Hemorrhagic Shock
We evaluated the capability of SynthoPlate to improve hemostasis, as determined by blood
loss, in a severe model of trauma and hemorrhagic shock, using a validated murine model
194 of uncontrolled hemorrhage induced by liver laceration (Figure 5-1).19 As demonstrated in
Figure 5-2A, mice pretreated 30 minutes before liver laceration with SynthoPlate showed a significant decrease in acute blood loss following liver laceration, compared with mice treated with control particles or normal saline (0.86 ± 0.16 g control particle [CP] vs. 0.84
± 0.13 g normal saline [NS] vs. 0.68 ± 0.09 g SynthoPlate). To ensure the differences in blood loss between treatment groups was due to SynthoPlate
Figure 5-2 Analysis of SynthoPlate pretreatment on blood loss and hemodynamics during hemorrhagic shock. Mice were pretreated 30 minutes before liver laceration with either CP, SynthoPlate, or 0.9% NS. Pretreatment with SynthoPlate results in a significant reduction in blood loss acutely (20 minutes) after liver laceration (n = 12 CP, n = 14 SP and NS) (A). As a measure of control for degree of hemorrhage between the different treatment groups the weight of the resected liver was weight to ensure the same degree of liver injury, as demonstrated in (B) no significant difference between the liver injury between any treatment group was identified. All mice underwent arterial cannulation and real-time hemodynamic monitoring, shown in (C) mice that received SynthoPlate took significantly longer to develop hypotension (defined as <40 mm Hg)
195 compared with mice treated with control particles or saline (n = 8 per group). Representative tracings of the MAP between mice treated with SynthoPlate particles compared to control particles demonstrating the improved hemodynamics and delay to develop hypotension (D). No difference in time to hypotension was noted between SynthoPlate or control particle treated mice (n = 11 CP, n = 12 SP, n = 11 NS) (F). *p < 0.005 **p < 0.01 ***p < 0.05. MAP, mean arterial pressure.
transfusion and not secondary to differences in levels of severity of liver injury, the weight of the segment of liver resected during the laceration was determined. There were no significant differences between treatment groups with regard to the weight of the resected liver (Figure 5-2B, 0.32 ± 0.05 g CP vs. 0.33 ± 0.03 g NS vs. 0.30 ± 0.04 g SynthoPlate).
All animals had femoral arterial lines placed for real-time hemodynamic monitoring, and as a secondary endpoint, we compared the time elapsed following liver laceration to development of hypotension, defined as a mean arterial pressure less than 40 mm Hg. As shown in Figure 5-2C-D, the time to develop hypotension is significantly prolonged in mice pretreated with SynthoPlate, and hypotension was prevented in some mice pretreated with SynthoPlate compared with mice treated with control particles or normal saline (168.3
± 106.6 seconds CP vs. 137 ± 58 seconds NS vs. 546.7 ± 329.8 seconds SynthoPlate).
5.3.2 Systemic Biodistribution SynthoPlate Particles Following Liver Laceration
We sought to evaluate the biodistribution-organ distribution, particle clearance, and off- target sequestration of SynthoPlate particles in the setting of our hemorrhagic shock model.
Ultra high-performance liquid chromatography was performed to determine the percent of injected SynthoPlate particle extracted from an organ harvested following liver laceration.
We found that in the acute hemorrhagic shock setting, SynthoPlate particles are distributed most frequently in the lung, liver, and in the injured liver site (Figure 5-3), although the overall sequestration of these particles in uninjured sites was low. As inferred from Figure
196 5-3, immediately following hemorrhage, only approximately 25% of the injected
SynthoPlate particles are cleared into the various organs, suggesting that most of the transfused SynthoPlate particles remain in circulation and available for continued assistance in hemorrhage control over time, if needed.
Figure 5-3 In vivo biodistribution of SynthoPlate particles. The systemic biodistribution of SynthoPlate particles following liver laceration and hemorrhagic shock was assessed using two different methods. SynthoPlate particle concentration in each organ was determined with UPLC and then expressed as a percent of the overall dose that was injected before liver laceration. As shown, the liver is most common organ the SynthoPlate particles are found, which is consistent with prior data the demonstrated in uninjured mice the liver and spleen were the major organs for SynthoPlate clearance (n = 6).
197 5.3.3 SynthoPlate Particles Reduce Blood Loss and Improve Hemodynamics in Mice in
Hemorrhagic Shock
After determining efficacy in reducing blood loss and improving hemodynamics in a pretreatment setting, we next sought to evaluate efficacy in a rescue model where
SynthoPlate transfusion was performed after liver laceration to more accurately model traumatic hemorrhage (Figure 5-1). Mice transfused with SynthoPlate following liver laceration experienced significantly less blood loss compared to either CP- or NS-treated mice (Figure 5-4A, 0.89 ± 0.17 g CP vs. 0.92 ± 0.19 g NS vs. 0.69 ± 0.18 g SynthoPlate).
The weights of the resected livers were determined to control for model severity.
Consistent with the findings in the pretreatment experiments, there were no significant differences in the weight of the resected liver between the treatment groups (Figure 5-4B,
0.33 ± 0.05 g CP vs. 0.31 ± 0.05 g NS vs. 0.30 ± 0.06 g SynthoPlate). In contrast to the pretreatment experiments, SynthoPlate transfusion following liver laceration did not result in any hemodynamic differences between the SynthoPlate group compared to control particles or normal saline (Figure 5-4C, 543 ± 208 seconds CP vs. 486 ± 127 seconds NS vs. 510 ± 202 SynthoPlate).
5.4 Discussion
We present here the potential applicability of an intravenous synthetic platelet nanotechnology (SynthoPlate) as a primary resuscitative agent in mitigating traumatic hemorrhage in a murine model of uncontrolled hemorrhage. Our data demonstrate the ability of SynthoPlate particles to assist in primary hemorrhage control in a setting of severe bleeding, in a pretreatment setting as well as a rescue strategy. These data are
198
Figure 5-4 Analysis of SynthoPlate transfusion on blood loss and hemodynamics as a rescue strategy in acute, hemorrhagic shock. Mice underwent liver laceration and were transfused either CP, SynthoPlate, or 0.9% NS following the liver laceration. Administration of SynthoPlate following liver laceration led a significant reduction in blood loss compared to control particles or saline (n = 11 CP, n = 12 SP, n = 11 NS) (A). As a control for the degree of hemorrhage, the weight of the resected liver was determined and as demonstrated in (B) there were no significant differences between the treatment groups. Finally, hemodynamic monitoring did not reveal any significant differences in the hemodynamic profiles between the treatment groups, as assessed by time to develop hypotension (C). *p < 0.05.
particularly striking when it is considered that the SynthoPlate particles are administered as a single-dose injection without other sources of fluid resuscitation during the hemorrhage period.
SynthoPlate particles can improve hemostasis by direct mimicry and amplification of platelet's primary hemostatic mechanisms of injury site-specific adhesion and aggregation
199 resulting in “in effect” enhancement of available platelet procoagulant surface to also
amplify secondary hemostasis (Figure 5-1). While SynthoPlate has been evaluated
previously, both via in vitro characterization and in vivo via tail vein bleeding assays, we
sought to evaluate the hemostatic capabilities in a more severe model of bleeding with
resultant shock physiology. We initially chose to evaluate the effects of SynthoPlate
transfusion in a pretreatment setting in the severe hemorrhage model in mice because our previous studies had shown hemostatic benefit of SynthoPlate pretreatment in murine models of subacute bleeding (tail transection), and we hypothesized that this benefit is conserved when bleeding became more acute (hemorrhagic trauma)7,8. After seeing
promising results of hemostatic and hemodynamic benefit in pretreatment evaluation, we
moved to evaluation in a model more reflective of rescuing from traumatic hemorrhage,
that is, transfusion after the liver injury was performed. Again, in this setting, we noted
improved blood loss in mice transfused with SynthoPlate.
SynthoPlate transfusion in the pretreatment setting resulted in improved hemodynamics,
as measure by the time elapsed from liver laceration to a drop to a mean arterial pressure
below 40 mm Hg. In contrast to pretreatment transfusion, the hemodynamic effects of
SynthoPlate transfusion following liver laceration did not result in a different
hemodynamic profile compared to either control particles or saline. In fact, the
hemodynamic profiles between the two experimental strategies are quite different as all
treatment groups in rescue experiments took longer to develop hypotension. We rationalize
this difference between experiments may be due to delivery of the particles. To ensure the
particles, either SynthoPlate or control, are delivered and completely removed from the
200 catheter, a saline flush is performed immediately after injecting the particle dose. This is
in effect a secondary fluid bolus delivered to the mouse which may alter the time to
hypotension as compared to the pretreatment strategy.
We also emphasize here that several “synthetic platelet” designs have been developed and
reported in the past few decades, but these designs have been very rarely evaluated in an
in vivo model of traumatic uncontrolled hemorrhage. The majority of past studies with
synthetic platelet designs have involved particles that only bear the aggregatory function of platelets (e.g., liposomal, albumin or polymeric particles coated with fibrinogen or fibrinogen-relevant Arginylglycylaspartic acid peptides), and these have been evaluated
mostly as pretreatment in platelet dysfunction (e.g., thrombocytopenia) models using tail-
bleeding and ear-punch injuries in mice, rats, or rabbits12,13. Only one specific design made from polymeric nanoparticles decorated with a generic RGD peptide has been studied in a more severe injury (femoral artery bleed) as a pretreatment (administered 5 minutes before injury) and a rescue treatment in blast trauma (administered 5 minutes after blast injury) in mice, showing promising results14,15. Therefore, our current studies with SynthoPlate
administered intravenously as pretreatment (30 minutes before injury), as well as a rescue
treatment in a mouse model of uncontrolled liver hemorrhage, are essentially one of the
very few to show the feasibility and potential of such a technology in mitigating severe
hemorrhage in both prophylactic (e.g., in elective surgery) and emergency (e.g., trauma) framework.
201 Previous work has demonstrated that intravenous injection of SynthoPlate particles in
uninjured mice does not lead to systemic pro-thrombotic risks, as evidenced by a lack of
generation of pro-thrombin fragments or D-dimer fragments in circulation8. Also, 2 hours following injection in uninjured mice, clearance of SynthoPlate particles from circulation was found to be approximately 15%, with the liver and spleen being primarily responsible for clearance8. Analysis of systemic biodistribution of SynthoPlate particles during
hemorrhage showed the liver as the major clearing site for circulating SynthoPlate particles
consistent with the prior studies in uninjured mice. Finally, we quantified the clearance of
SynthoPlate from circulation in the setting of acute hemorrhage and found that only
approximately 25% of injected particle dose was cleared at the time of sacrifice. This
suggests that most the SynthoPlate transfused remains in circulation and can provide
hemostatic assistance in areas with ongoing bleeding.
While our findings certainly present evidence for the beneficial effects of SynthoPlate
transfusion on reducing blood loss in severe hemorrhagic shock, our work is not without
limitations. First, our study was powered to detect differences in blood loss and therefore
other important endpoints in hemorrhagic shock such as survival could not be adequately
addressed in this current study. Second, our comparison groups were control particles and
normal saline. More evidence is suggesting early administration of blood products,
including packed red blood cells, fresh frozen plasma, and platelets have benefits over
crystalloid administration. Future studies that compare SynthoPlate transfusion against
platelet transfusion and in conjunction with platelet transfusion are needed and a planned
area of work.
202 Despite these limitations, we present evidence for the effectiveness in two different strategies, pretreatment and rescue strategies. Pretreatment effectiveness presents the potential for use of SynthoPlate in the setting of thrombocytopenia and platelet dysfunction
(e.g., uremia) to reduce blood loss during planned procedures. Rescue therapy effectives highlights an exciting potential for the use of SynthoPlate as part of a resuscitation protocol, in conjunction with platelets or as a substitute, in acute bleeding.
In conclusion, these data show great promise for the use of SynthoPlate as a viable intravenous resuscitative product for resuscitative mitigation of hemorrhagic shock by decreasing blood loss and stabilizing blood pressure.
5.5 Authorship
M.R.D., D.H., N.L., S.H., C.P., A.S.G., and M.D.N. designed the research study. D.H.,
N.L., C.P., and A.S.G. fabricated the SynthoPlate particles. M.R.D., D.H., and S.H. conducted the experiments. M.R.D., D.H., S.H., and P.L. acquired data. M.R.D., D.H.,
P.L., A.S.G., and M.D.N. analyzed the data. M.R.D., A.S.G., and M.D.N. wrote the article.
5.6 Acknowledgement
We would like to thank Ms. Lauryn Kohut for her expertise and assistance with development of the liver laceration model.
5.7 Disclosure
M.D.N. has the following financial relationships to disclose: Consultant, External
Scientific Advisor for Anticoagulation Science for Janssen Pharmaceuticals (Johnson &
203 Johnson), Trauma Advisory Board, CSL Behring, and US Patent 9,072,760 TLR4 inhibitors for the treatment of human infectious and inflammatory disorders (issued to
Neal, Wipf, Hackam, Sodhi). ‘SynthoPlate’ technology is associated with the US Patent
9107845 and the SynthoPlate trade mark is registered currently with USPTO (ASG, CP).
This work is supported by U.S. National Institutes of Health grants 1 R35 GM119526-01
and UM1HL120877-01, support from the Vascular Medicine Institute at the University of
Pittsburgh, the Hemophilia Center of Western Pennsylvania, and the Institute for
Transfusion Medicine, as well as the American Association for the Surgery for Trauma
Research award (all to M.D.N.), U.S. National Institutes of Health grants 5R01 HL121212
(to A.S.G.) and U.S. National Institutes of Health grant 1S10OD019973-01 (Dr. Simon C.
Watkins).
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206 Chapter 6: Intravenous synthetic platelet (SynthoPlateTM) nanoconstructs reduce
bleeding and improve ‘golden hour’ survival in a porcine model of traumatic
arterial hemorrhage
Content based on: Hickman DA, Pawlowski CL, Shevitz A, Luc NF, Kim A, Girish A, Marks J, Ganjoo S, Huang S, Niedoba E, Sekhon UDS, Sun M, Dyer M, Neal MD, Kashyap V, Sen Gupta A. Intravenous synthetic platelet (SynthoPlate) nanoconstructs reduce bleeding and improve “golden hour” survival in a porcine model of traumatic arterial hemorrhage. Sci Rep. 2018;(February):1–14. doi: 10.1038/s41598-018-21384-z. PMCID: PMC5814434
6.1 Introduction
In remote civilian locations and austere battlefield conditions, uncontrolled traumatic
hemorrhage remains one of the leading causes of mortality1–7. In such scenarios,
transfusion of whole blood or blood components (RBCs, plasma and platelets) can
significantly improve survival8–10. Extensive trauma resuscitation studies have indicated substantial benefits of early platelet transfusion to treat hemorrhagic shock and enhance survival possibilities8–13, however, the limited availability and portability, need for blood
type matching, special storage requirements, high risks of bacterial contamination at room
temperature and very short shelf-life (3-5 days at room temperature) of platelets, present severe logistical challenges for their applicability in pre-hospital scenarios14–24. These
issues have led to robust research efforts in improving the storage stability, portability and
availability of platelets, e.g. via lyophilization and cold-storage, as well as, developing
pathogen reduction technologies to reduce contamination25–32. However, these approaches
have only marginally improved the pre-hospital applicability of platelets, and the research
continues to find alternative platelet-based options, e.g. Isolated Platelet Membrane (IPM
207 CyplexTM) and Thrombosome® 33–35. These technologies involve isolation, purification,
sterilization, temperature-stabilization and lyophilization of outdated platelets or platelet-
derived membrane to utilize their residual hemostatic bioactivity. However, such
processing can lead to significant ‘loss-in-function’ of hemostatically relevant glycoprotein on the platelet membrane surface36,37, which can potentially lead to batch-to-batch
functional inconsistencies. Due to such challenges with natural platelet-based products, a
parallel area of research has focused on the development of synthetic platelet surrogates38–
40. The design requirements for such platelet surrogates are: (i) large scale in vitro
manufacturability, (ii) sterilizability without compromising biofunctional properties, (iii)
long-term storage as suspension or lyophilized powder, (iv) easy portability, (v)
intravenously administrable ‘on demand’ in pre-hospital scenarios, (vi) post-administration circulation safety without systemic risks, (vii) ability to mimic and amplify endogenous hemostatic mechanisms selectively at the bleeding site, and (viii) biodegradation and safe elimination from the body. Also, such synthetic platelet surrogates can potentially avoid the need for blood type matching and can be used more universally, since they would not bear blood antigens.
As comprehensively reviewed in chapters 2 and 3 and in several review articles39–41,
several ‘synthetic platelet’ designs have been reported in the past that primarily mimic
platelet’s aggregatory capability only via surface- decoration of polymeric, lipidic or albumin particles with fibrinogen (Fg) or Fg-relevant peptides. While these designs have shown some promise as intravenous hemostats, the majority of them have only been reportedly evaluated in small animal bleeding models and only in a pre-treatment
208 framework, i.e. particle administration before bleeding injury. No synthetic platelet design
Figure 6-1 Schematic representation of SynthoPlateTM design and mechanism, showing nanoconstructs heteromutlivalently decorated with VBP, CBP and FMP motifs to render platelet-inspired interactions with vWF, collagen and active platelet integrin GPIIb-IIIa respectively, and thus amplify platelet-mediated primary hemostatic mechanisms at the injury site. Illustration by Erika Woodrum.
has yet been reported regarding evaluation in a large animal model, that is also in an
emergency/rescue framework (i.e. particle administration after bleeding injury) in
traumatic bleeding. We have developed a synthetic platelet surrogate, SynthoPlateTM, by leveraging a clinically relevant biocompatible liposomal platform that is heteromultivalently surface-decorated with three types of peptides: von Willebrand Factor
(vWF)-binding peptide (VBP) and collagen-binding peptide (CBP) to mimic platelet’s bleeding site-selective adhesion mechanisms and active platelet integrin GPIIb-
209
Figure 6-2 Lipid-to-peptide bioconjugation schemes (along with corresponding chemical structures) for synthesizing DSPE-PEG-VBP, DSPE-PEG-CBP and DSPE-PEG-CMP molecules, that were used to manufacture the SynthoPlateTM nanoconstruct.
IIIa-binding fibrinogen-mimetic peptide (FMP) to mimic activated platelet aggregation
mechanism (Figure 6-1)42–44. This integrative biomimetic approach to combine platelets’
adhesion and aggregation functions on a single particle platform is highly unique and can
also be adapted to other particle platforms beyond the SynthoPlateTM’s liposomal
platform45. The chemical components and manufacturing details for SynthoPlateTM are provided in the Methods section and the relevant chemical synthesis schemes are shown in
Figure 6-2. We have previously reported the detailed biofunctional and mechanistic characterization of SynthoPlateTM nanoconstructs46 and have also demonstrated that the
heteromultivalent integration of platelet-inspired adhesion and aggregation mechanisms
210 on the same particle platform results in higher hemostatic capability compared to designs
bearing ‘adhesion only’ or ‘aggregation only’ functions43,45. We further demonstrated that
prophylactic administration of SynthoPlateTM in normal as well as thrombocytopenic mice
could augment hemostatic capability in a non-trauma bleeding model (bleeding time reduction in murine tail transection)43,45. Building on these studies, here we sought to
investigate the translational promise and hemostatic capability of SynthoPlateTM in
emergency management of traumatic exsanguinating hemorrhage in a pilot scale study in
large animal (pig) model. For this, we first characterized the effect of long-term storage (in
saline) on SynthoPlateTM stability, as well as, the effect of sterilization on SynthoPlateTM
stability and biofunction in vitro. Next, for in vivo studies, we adapted a femoral artery
uncontrolled hemorrhage model in pigs, that has been utilized for evaluation of hemostatic
dressings47,48. In traumatic exsanguinating hemorrhage, significant number of patients succumb within the first 1-2 hours, and hemostatic intervention within this window (known
as the ‘golden hour’) can significantly improve survival10,49,50. Our model results in the loss
of ~1 liter of blood within 15-20 minutes, resulting in 75% mortality within first 45 min.
Therefore, we utilized this model to test the SynthoPlateTM technology I.V.-administered
immediately following injury, and the animals were observed for up to 2 hours to monitor
blood loss, blood pressure, heart rate and survival. The systemic safety and biodistribution
of SynthoPlateTM was also characterized. Administrations of saline or of unmodified
211 (control) particles were used as comparison groups.
Figure 6-3 [A] SynthoPlateTM diameter (size distribution) analysis over a 6-month period demonstrated that the particles retained their size within 20% of their starting diameter when stored in a 0.9% NaCl solution at 25oC, indicating long term stability; [B] After sterilization with filtration or E-beam, SynthoPlateTM showed minimal alteration in size (diameter) compared to fresh-made (unsterilized) samples, indicating that the sterilization did not affect particle stability; [C] Representative fluorescent images and quantitative analysis of surface-averaged fluorescence intensity of adhered SynthoPlateTM showing that sterilized SynthoPlateTM (red) maintained its ability to adhere to ‘collagen + vWF’-coated surface at levels similar to fresh-made (unsterilized) SynthoPlateTM, indicating that sterilization did not affect the biofunctional properties of VBP and CBP; [D] Aggregometry analysis showed that neither fresh-made (unsterilized) or sterilized SynthoPlateTM induced spontaneous aggregation of platelets without agonist (w/o ADP), but both unsterilized and sterilized SynthoPlateTM markedly increased platelet aggregation in the presence of ADP, indicating that sterilization did not affect the biofunctional property of FMP on and also SynthoPlateTM did not have any thrombotic risk towards resting platelets.
6.2 Results
6.2.1 SynthoPlateTM stability in storage
Liposomal nanoconstruct instability is reflected by large alterations in size (diameter), stemming from particle disassembly, aggregation and clustering. Dynamic Light Scattering
(DLS) analysis was used to monitor SynthoPlateTM stability in saline suspension over a 6- month period. Fresh-made liposome batches show a size (diameter) variability of +/- 15% in laboratory scale manufacture. As shown in Figure 6-3A, our analysis showed that when
212 stored as a suspension in 0.9% NaCl at room
temperature, SynthoPlateTM particles showed
additional size variability over time of about +/- 5%,
thus maintaining their size within an overall
variability of +/- 20% of their starting diameter for
up to 6 months. Representative DLS data at Month 1,
Month 3 and Month 6 are shown in Figure 6-4. Also,
by visual inspection, there were no signs of particle
aggregation or settlement out of solution. This
suggests that SynthoPlateTM is amenable to long- Figure 6-4 Representative Dynamic Light term storage under these conditions and indicates Scattering (DLS) data of SynthoPlateTM their potential for pre-hospital availability as a small size distribution characterization over a 6- volume suspension without compromising stability. month period; data is shown for 1 month,
3 month and 6 month time points,
demonstrating that there is minimal 6.2.2 SynthoPlateTM sterilization and its effects alteration in particle diameter and SynthoPlateTM sterilization studies by filtration (0.2 therefore indicating particle stability in µm filter) as well as E-beam exposure (25 and 40 storage (saline suspension at 25oC). kGy) were carried out with 6 different challenge organisms, namely Staphylococcus aureus, Kocuria rhizophila, Clostridium sporogenes,
Bacillus subtilis spizizenii, Candida albicans and Aspergillus brasiliensis, as described in
the Methods section. For all organisms tested, SynthoPlateTM itself did not have any bacteriostatic or fungistatic activity, and this data is shown in Figure 6-5. SynthoPlateTM
solutions sterilized by either filtration or by E-beam exposure showed no visible growth of
213 microorganisms, suggesting effective sterilization by all methods tested. The endotoxin
Figure 6-5 Bacteriostatic and fungistatic analysis of SynthoPlateTM suspension exposed to E-beam sterilization and challenged with 6 different organisms, namely Staphylococcus aureus, Kocuria rhizophila, Clostridium sporogenes, Bacillus subtilis spizizenii, Candida albicans and Aspergillus brasiliensis, that were allowed to grow in appropriate conditions for up to 5 days. T= test, C= control, N = No growth, P = positive growth and N/A = positive growth established.
bioburden of the concentrated SynthoPlateTM formulation (1 x 105 moles lipid per ml) was
measured to be 3.5-5 EU/ml as per a standard chromogenic limulous ameobocyte lysate
(LAL) assay (see Methods for details). When diluted in sterile saline to the in vivo dosing level of 5 x 104 moles lipid per ml, this equates to a dose of ~0.83 EU/kg, which is
substantially less than the endotoxin safety threshold of ≤ 5 EU/kg for in vivo usage51.
Therefore, our SynthoPlateTM preparation can be considered safe for in vivo usage.
Furthermore, after sterilization by either filtration or E-beam, SynthoPlateTM particles showed statistically insignificant variability in size compared to fresh-made (unsterilized)
SynthoPlateTM (Figure 6-3B), suggesting that the sterilization methods did not affect
particle integrity, size and stability.
To test whether sterilization affects the platelet-inspired adhesive function of CBP and
VBP moieties on SynthoPlateTM, Rhodamine B-labeled (red fluorescent) SynthoPlateTM
214 particles in saline suspension, unsterilized or sterilized, were incubated on ‘vWF +
Figure 6-6 Representative Lumi-Aggregometry raw data from studies on SynthoPlateTM (fresh unsterilized versus sterilized) interaction with platelets (resting or agonist-activated) in platelet-rich-plasma (PRP); results indicate that neither unsterilized nor sterilized SynthoPlateTM has any activating and aggregatory effect on resting platelets (top group of traces w/o ADP), while both unsterilized and sterilized SynthoPlateTM are capable of enhancing the aggregation of activated platelets (bottom group of traces with ADP) above that seen for PRP only. These results also demonstrate that sterilization does not affect the pro-aggregatory function of SynthoPlateTM on activated platelets.
collagen’ coated glass coverslips in a 12-well plate with stirring at 120 rpm, following
which the coverslips were washed and imaged (experimental details provided in Methods
section). Representative images of Rhodamine B (red) fluorescent adhered SynthoPlateTM,
as well as, statistical data of fluorescence intensity analysis from multiple images per
condition (5 images per condition), are shown in Figure 6-3C. As evident from the results,
sterilized SynthoPlateTM particles retained their capability to adhere to ‘collagen + vWF’-
coated surfaces at levels similar to freshly prepared SynthoPlateTM particles, indicating that
sterilization by filtration or E-beam does not affect the platelet-inspired adhesive activity
of VBP and CBP moieties on SynthoPlateTM. To test whether sterilization affects the ability
215 of FMP moieties on SynthoPlateTM to interact with active platelet integrin GPIIb-IIIa for
amplifying platelet aggregation, lumi-aggregometry assays were performed using platelet
rich plasma (PRP) isolated from citrated human whole blood. These assays were performed
with 100% PRP or PRP diluted 50% (v/v) with platelet poor plasma (PPP) supplemented
with fresh made (unsterilized) or sterilized (filtration and E-beam) SynthoPlateTM particles,
with or without addition of platelet agonist ADP (experimental details in Methods section).
In these studies, neither unsterilized nor sterilized SynthoPlateTM particles were found to
induce any spontaneous platelet aggregation in the absence of ADP, but both were able to
markedly enhance platelet aggregation in the presence of ADP, as shown in Figure 6-3D.
Representative raw data from aggregometry studies are shown in Figure 6-6. Therefore,
these results indicate that sterilization by filtration or E-beam does not affect the platelet- inspired aggregatory activity of FMP moieties on SynthoPlateTM. Hence, altogether these
results establish that SynthoPlateTM
can be effectively sterilized without
affecting its platelet-inspired
bioactivity, which is important in
the context of its future translation
through large-scale manufacturing
and sterilization without
compromising its hemostatic
Figure 6-7 MTT-based metabolic activity analysis data of function. human 3T3 fibroblasts in culture incubated with 0.5 nM SynthoPlateTM (dose relevant to in vivo studies) or 1 nM SynthoPlateTM (double of in vivo dose) showed no statistical difference compared to cell metabolic activity in culture media only, indicating that SynthoPlateTM is not cytotoxic at the dose used.
216 6.2.3 SynthoPlateTM cytotoxicity analysis
SynthoPlateTM was incubated with 3T3 human fibroblasts (see Methods for culture
conditions and viability analysis) at doses relevant to the pig dose used in the current
studies (0.5 nM) and double the dose (1 nM). Cell viability was analyzed by standard MTT
based metabolic assay and compared to cell cultures without SynthoPlateTM incubation (i.e. culture media only). As shown in Figure 6-1. the cell metabolic activity was not affected by SynthoPlateTM incubation (i.e. no statistical difference), indicating that there is no
cytotoxicity associated with SynthoPlateTM at the dose (as well as double the dose) used in vivo in the current studies.
6.2.4 In vivo Safety and Biodistribution Studies in Pigs
The systemic safety of SynthoPlateTM in pigs within the ‘golden hour’ (60 min) time-frame, was tested by administering the SynthoPlateTM dose in un-injured pigs intravenously through the jugular vein. Dose was calculated by
5 10 ÷ 50 = 1 10 / , 12 11 where∗ a typical𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 unit𝑝𝑝𝑝𝑝𝑝𝑝 of platelets𝑑𝑑𝑑𝑑𝑑𝑑𝑑𝑑 for 𝑚𝑚human𝑚𝑚 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 transfusion𝑠𝑠𝑠𝑠 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 𝑣𝑣 𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣𝑣contains ≥ 3∗ x 1011𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝𝑝 platelets. Since𝑚𝑚𝑚𝑚
actual platelets are ~10 times larger (in diameter) than SynthoPlate, we decided to scale the transfusion of SynthoPlate (or control particles) by 1 order of magnitude, to 5 x 1012
particles per dose. Administrations of saline or unmodified (control) particle dose were
used as comparison. Two animals per treatment group (i.e. 6 total) were used for these
studies. The effects of the treatments on vitals, blood chemistry, complement (C3 to C3a)
activation, platelet function (aggregation) and blood clot viscoelastic properties (using
217 ROTEM) were examined for the 60-minute post-administration period (experimental
Figure 6-8 [A] Average vitals over the course of the experiment of uninjured pigs and [B] average blood lab values over the course of the experiment of uninjured pigs upon administration of unmodified particles or SynthoPlateTM, showed that there are no statistically significant differences in values compared to saline administration, indicating that SynthoPlateTM administration has no detrimental physiological effect; [C] Analysis of C3:C3a plasma concentration ratios in blood drawn from pigs administered with unmodified particles or SynthoPlateTM showed no statistically significant alterations of this ratio compared to saline administration groups, indicating that in the dose and administration protocol used, SynthoPlateTM did not have complement activation (and CARPA) risk; [D] Biodistribution analysis from harvested organs indicated a similar distribution profile for unmodified particles and SynthoPlateTM, with a majority of particles cleared through the liver.
details in Methods section). Sixty minutes post treatment administration, pigs were euthanized and major organs (lungs, heart, liver, kidney, spleen) were harvested for histological (using H&E stain) and biodistribution (using UPLC on tissue homogenate) analysis. As shown in Figure 6-8A, following administration of unmodified particles or
SynthoPlateTM in uninjured pigs, none of the animals showed any substantial alteration in their vitals compared to saline. Ex vivo analysis of arterial blood collected from the pigs post treatment administration showed no significant changes in the blood analysis
218 parameters between saline and the two particle treatment groups (Figure 6-8B), indicating
that the SynthoPlateTM dose did not affect physiological parameters of blood in vivo. Also,
as shown in Figure 6-8C, no significant alterations in the C3:C3a plasma concentration
ratios were found in animals administered with unmodified particles or SynthoPlateTM. It
is important to note here that complement ratio alterations of 4-fold or greater are reported
to be a risk indicator for drastic activation of complement system and deleterious reactions
such as complement activation-related pseudoallergy (CARPA)52–54. SynthoPlateTM (as well as control particles) did not show any such effect compared to saline. Therefore, altogether these results suggest that within our experimental dose and animal observation window, administration of SynthoPlateTM did not cause any alarming systemic risks. The
biodistribution analysis of SynthoPlateTM (as well as unmodified particles) in uninjured
pigs is shown in Figure 6-8D, demonstrating that both types of particles have similar organ
distribution profile during the 1-hour circulation period. Within this window, most particles
of either type were found to be cleared to the liver, possibly by the reticulo-endothelial
system (RES), as this mechanism is known for clearance of PEG-ylated liposomes55 (base
particle for the design of SynthoPlateTM nanoconstructs). Interestingly ~30 % of the
injected dose of particles was found to still remain in the systemic circulation (blood),
suggesting that this amount can be potentially still available after the 1-hour period to keep localizing at the injury site for hemostatic action. Hematoxylin and eosin staining-based
histopathological analysis of harvested organs from SynthoPlateTM-injected un-injured animals showed no signs of off-target thrombi in the tissue samples (Figure 6-9).
219 Combining these findings with our previous report that SynthoPlateTM does not activate
(and aggregate) resting circulating platelets and also does not spontaneously trigger
Figure 6-9 Representative hematoxylin and eosin (H&E) stained histology images (32x magnification) of organ samples from pigs treated with saline, unmodified particles or SynthoPlateTM particles, show that SynthoPlateTM (as well as unmodified particles) does not cause any thrombotic risks in organs; scale bar of 200 μm shown in bottom row kidney image is applicable to all histology images.
220 thrombin (and fibrin) generation in plasma43,45, we rationalize that our SynthoPlateTM dose was safe in pigs, with minimal systemic pro-thrombotic, pro-coagulant and complement activation risks.
Figure 6-10 Schematic representation of pig femoral artery hemorrhage model setup. A CO2 sensor was placed at the end of the endotracheal tube and mechanical ventilation was provided, EKG electrodes were placed on the pig’s limbs, a pulse-oximeter probe was placed on the pig’s mouth, an esophageal temperature probe was placed to measure core temperature, an angiocatheter was placed in the carotid artery to acquire invasive blood pressure and also withdraw blood samples for ex vivo analysis, an angiocatheter was placed in the internal jugular vein to deliver saline (or nanoparticle treatments) via an infusion pump.
6.2.5 Hemostatic Efficacy Evaluation in Femoral Artery Bleeding Model
The pig femoral artery hemorrhage model is described in detail in the Methods section and an experimental schematic is shown in Figure 6-10. Briefly, an acute hemorrhagic injury
(near transection) was caused in the femoral artery with a 3.5 mm arterial punch and 1-
221 minute following injury, saline, unmodified (control) particles or SynthoPlateTM particles
Figure 6-11 Hemostatic efficacy analysis in injured pigs shows that [A] pigs administered with SynthoPlateTM had a reduced blood loss rate (blue) compared to those treated with saline (red) and unmodified particles (green), especially within first 30 min post treatment administration, where blood loss rate in SynthoPlateTM treated pig was significantly lower than those treated with unmodified (control) particles and saline (** p<0.01); [B] SynthoPlateTM administration in pigs also resulted in significantly lower total blood loss compared to saline administration (* p<0.05); [C] SynthoPlateTM administration in pigs resulted in maintenance and stabilization of a higher average mean arterial blood pressure (MAP) over time (data points shown for every 10 min); [D] SynthoPlateTM administration in pigs resulted in a significant enhancement of survival, with 100% surviving the first 60 min (golden hour) and 75% surviving the additional 60 min (p < 0.05), compared to administration of saline (25% survival by 60 min and 0% survival by ~90 min) or unmodified particles (50% survival by 90 min, 25% survival at 120 min).
were administered as a bolus dose through the jugular vein. Four animals per treatment group (i.e. 12 total) were used for these studies. Vitals, blood loss, hemodynamic
222 parameters and survival were monitored in real time for 60 minutes (golden hour) and an
additional 60 minutes (i.e. total observation for 120 min). During these observations, blood was also drawn through a carotid angiocatheter to run platelet aggregometry and ROTEM
analysis ex vivo. At the end of experiments, pigs were euthanized (unless they had already
succumbed earlier from their injury), and organs and clot were harvested for histological
and fluorescence-based distribution analysis (experimental details in Methods section). As shown in Figure 6-11A, bolus administration of SynthoPlateTM immediately following
femoral artery injury, resulted in substantial reduction of the blood loss rate over time. It is
important to note here that the condition of the saline-treated pigs rapidly declined after
injury as indicated by their mean arterial pressure (Figure 6-11C) and 75% of them died
before 60 minutes (Figure 6-11D); therefore, blood loss rate for this group appears to approach zero after this time point (Figure 6-11A). Meanwhile, pigs injected with unmodified (control) particle dose continued to undergo low rates of bleeding due to a delay in destabilization of mean arterial pressure (MAP) (Figure 6-11B), but ultimately one of them succumbed while actively bleeding around 60 minutes and two other died around 100 minutes (Figure 6-11D), though bleeding stopped in both of them at around
60 minutes possibly due to hemorrhage-induced hypotension (Figure 6-11A and C).
SynthoPlateTM –treated pigs exhibited significantly lower blood loss rates than comparison
groups during the first 30 min and achieved full hemostasis (at 15 min, 18 min, 30 min and
80 min for the 4 pigs respectively) such that the blood loss rate for this group also became
zero by the 80-minute time point (Figure 6-11A). Furthermore, these animals exhibited
stabilized MAP (Figure 6-11C) throughout the 120-minute period. Total blood loss for
SynthoPlateTM-treated pigs (normalized to body weight and time survived) was also found
223 to be significantly lower to comparison treatment (Figure 6-11B). As shown in Figure
6-11D, SynthoPlateTM-treated animals showed a significantly improved survival, with
Figure 6-12 Schematic representation and representative images of hemostasized injury site in the femoral artery of pigs treated with SynthoPlateTM or saline or control particles: [A] Representative hematoxylin and eosin (H&E) stained histology image (32x magnification) and [B] representative bright field image (10x magnification) of the site of injury (transected artery) with the injured vessel components (EC: endothelial cell, SMC: smooth muscle cell) and hemostatic clot in view for injured pig treated with SynthoPlateTM; [C1] representative fluorescence image (10x magnification) of FITC-anti-CD42b (green fluorescence) stained platelets, [C2] Rhodamine B-labeled (red fluorescence) SynthoPlateTM particles and [C3] overlay of C1 and C2, in the same field of view as the brightfield image B demonstrating that red fluorescent SynthoPlateTM is co-localized with green fluorescent platelets at the site of injury within the hemostatic clot; [D1, D2 and D3] are similar representative images for injured pig treated with saline, and [E1, E2 and E3] are similar representative images for injured pig treated with unmodified (control) particles showing that some platelets indeed localize at the injury site to promote hemostasis but presence of control particle is minimal at the site and therefore control particles do not have the ability to augment hemostasis; scale bar of 50 μm shown in C1 is applicable to all fluorescent images.
100% surviving the golden hour (first 60 minutes) and 75% maintaining survival at the end of 120 minutes. In comparison, 75% of the saline-treated animals died within the first 60 min and 75% of the unmodified (control) particle-treated animals progressively died during the 60-120 min period. Immunohistochemistry and fluorescence imaging analysis of harvested clots from the hemostasized femoral artery of SynthoPlateTM-treated animals revealed significant incorporation of SynthoPlateTM in the platelet-rich clot at the site of
224 injury (yellow overlay of green
platelets and red SynthoPlateTM), but
no such red fluorescent particle
presence was found at the arterial
injury site for unmodified (control)
particle-treated pigs (representative
fluorescence images for the three
treatments shown in Figure 6-12).
Also, similar to the safety experiments
in uninjured pigs, during the golden
hour (60 min) there were no drastic
changes in the C3:C3a plasma
concentration for SynthoPlateTM-
treated or unmodified particle-treated
animals compared to saline-treated
animals (Figure 6-13). Furthermore, Figure 6-13[A] Complement (C3:C3a) analysis data on the biodistribution of the unmodified drawn blood from pigs and [B] biodistribution data from
TM pigs subjected to femoral artery injury and administered particles and SynthoPlate (analyzed
intravenously with unmodified particles or SynthoPlate at the point of pig death or euthanasia) nanoconstructs. were similar (Figure 6-13).
Aggregometry studies showed that pigs treated with SynthoPlateTM had a stable platelet aggregation profile while pigs treated with unmodified (control) particle demonstrated a trend for decreasing platelet aggregation
225 and pigs treated with saline showed a trend for platelet hyper-aggregation (Figure 6-14A).
ROTEM analysis of blood revealed no presence of coagulopathy in SynthoPlateTM or saline
treated animals, as indicated by clotting time (CT), maximum clot firmness (MCF), and
amplitude 10 minutes after CT (A10) values from EXTEM and FIBTEM assays that all
(Figure 6-14B)56. Unmodified particle treated fall within ‘normal’ reference range, based
on criteria established in previous studies animals did begin to show signs of coagulopathy
at the 120-minute time point as indicated by MCF and A10 EXTEM and FIBTEM values
that are below the reference range (Figure 6-14B). Altogether, the data suggest that
SynthoPlateTM has the ability to reduce blood loss rate as well as total blood loss (Figure
6-11A and B), stabilize MAP (Figure 6-11C) and increase overall survival (Figure 6-11D)
after traumatic hemorrhage injury, due to the ability of SynthoPlateTM to enhance active platelet recruitment and aggregation to form hemostatic clot at the injury site and prevent coagulopathy.
6.3 Discussion
Traumatic exsanguinating hemorrhage continues to be a major cause of military and civilian fatalities in pre-hospital settings, where blood components have limited availability15,16. In such scenarios, volume resuscitation with saline or plasma expanders
shows only limited benefit57. Currently, studies are being directed at improving the
survivability in such conditions by anti-fibrinolytic agents like tranexamic acid (TXA) as
well as by improving blood component storage, portability and availability through cold-
storage and lyophilization27,58–63. Time is critical in mitigation of exsanguinating
226
Figure 6-14 [A] Representative ex vivo Lumi-aggregometry analysis data and [B] ROTEM analysis data (CT, MCF and A10 parameters) of blood samples drawn from pigs after being subjected to femoral artery injury and administered intravenously with saline or unmodified particles or SynthoPlate nanoconstructs.
hemorrhage since significant mortalities occur within 1-2 hours of injury, and hemorrhage control within this time can significantly improve survival49,50. Therefore, intravenously
227 transfusable synthetic RBC and platelet surrogates that can be administered early at point- of-injury can have tremendous benefit in emergency mitigation of hemorrhagic shock39–41.
In the area of synthetic platelet surrogates, past approaches have focused mostly on
mimicking only the aggregatory mechanism of platelets by decorating micro- and
nanoparticles with fibrinogen or fibrinogen-relevant peptides, essentially creating a ‘super-
fibrinogen’ construct39,41. However, platelet’s native hemostatic action is a co-operative
concert of injury site-selective adhesion and aggregation mechanisms64. Hence in our
design, we hypothesized that integrating these two mechanisms on a single particle
platform will allow a superior mimicry of platelet’s hemostatic mechanisms selectively at
the bleeding site while minimizing off-target effects. We previously tested this hypothesis
via heteromultivalent decoration of liposomal as well as albumin-based nanoparticles with
VBP, CBP and FMP motifs and successfully validated the superior hemostatic efficacy of this integrative design compared to ‘adhesion only’ and ‘aggregation only’ designs43,45.
SynthoPlateTM is a refined liposome-based version stemming from this integrative design44, and we have recently demonstrated its hemostatic efficacy in a non-trauma
bleeding model in severely thrombocytopenic mice46. Building on this, here we
investigated the translational promise of the SynthoPlateTM technology in vitro
(sterilization effects and storage stability) and in vivo (large animal hemorrhage model).
Our studies demonstrate that sterilization of SynthoPlateTM by either filtration or E-beam
does not affect the product’s stability and platelet-inspired biofunctionalities. Our studies
also indicate that long-term storage in suspension does not affect SynthoPlateTM stability.
228 Altogether, these data suggest that sterile suspensions of SynthoPlateTM can potentially
allow long-term storage and availability for hemorrhage mitigation in pre-hospital settings.
Our in vitro and in vivo studies clearly demonstrate that the SynthoPlateTM dose did not
have any cytotoxicity and systemic risks in the experimental conditions tested. Future
studies will be directed at assessing dose escalation-based pharmacology and toxicology
analysis, as well as, evaluating the potential of multiple doses. Following arterial injury
and bolus administration of SynthoPlateTM (or control) particles, our results show that
compared to saline (0% survival), SynthoPlateTM treatment renders 100% survival during
the ‘golden hour’. Interestingly, unmodified (control) particles showed ~50% survival
during this first hour, however, survival for this treatment group fell to 25% during the
second hour of the experimental window. This can be attributed to the fact that the
unmodified particles seemed to transiently improve MAP, possibly due the colloidal nature
of injected liposome suspension transiently increasing blood colloidal osmotic pressure
(BCOP). However, since unmodified particles were incapable of biofunctionally
enhancing hemostasis, these animals continued to bleed at a higher rate and succumbed. In
contrast, a combination of hemodynamic stabilization and hemostatic enhancement
significantly improved the survival of the SynthoPlateTM-treated pigs within and beyond
the ‘golden hour’. It is to be noted that, because of MAP stabilization and longer survival
time, the blood loss rate data Figure 6-11A) apparently suggests that the SynthoPlateTM- treated animals continued to bleed (albeit at a very low rate) up to and beyond the ‘golden hour’. However, this is a ‘survival bias’, since majority of injured animals treated with saline or unmodified particles essentially could not recover from being rapidly
229 hypotensive, which resulted in ‘apparent reduction’ of blood loss rate and ultimately death.
Interestingly, several ‘externally applicable’ hemostatic dressings were recently evaluated
in a pig femoral artery hemorrhage model, that showed overall survival to be 50-60% in
the 1-hour period47. Considering that in our studies, administering only a single bolus
intravenous dose of SynthoPlateTM, with no additional dressings, resulted in 100% survival
of the pigs for 60 minutes (and 75% for 120 min), we rationalize that the superior
hemostatic capability of SynthoPlateTM can be potentially combined with externally
applied hemostatic dressings, to further improve the hemostatic outcomes in pre-hospital
scenarios. We also note that in the current studies the treatments were administered within
1 minute after injury and we have not yet evaluated the ‘administration time window’ for
SynthoPlateTM following injury to determine at what point of delayed administration its
hemostatic benefit may fail to increase survival. Our future studies will further evaluate
these possibilities in porcine trauma models. Our current findings therefore strongly
demonstrate the potential of SynthoPlateTM as a viable synthetic platelet surrogate for
point-of-injury management of traumatic hemorrhagic.
6.4 Materials and Methods
6.4.1 Materials
The lipid components, namely, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]
(DSPE-mPEG1000), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-
[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Mal) and 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG2000-
230 Azide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Rhodamine B- dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE-RhB) was purchased from
Invitrogen (Carlsbad, CA, USA). The peptides CTRYLRIHPQSWVHQI (VBP), C[GPO]7
(CBP) and cyclo-{Pra}CNPRGD{Tyr(OEt)}RC (FMP) were custom-synthesized by
Genscript (Piscataway, NJ, USA). VBP and CBP were conjugated to DSPE-PEG2000-Mal
65 via thiol-maleimide coupling and FMP was conjugated to DSPE-PEG2000-azide via
copper-catalyzed alkyne-azide cycloaddition (CuCAAC)66 (schematics shown in
Supplementary Figure S.1), conjugates were purified by dialysis and characterized by
MALDI-TOF mass spectrometry. Sterile normal saline solution (0.9% NaCl) was
purchased from Baxter (Deerfield, IL, USA). Cholesterol, rat tail type I collagen, copper(II)
sulfate (CuSO4), Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), sodium
ascorbate, decaethylene glycol monododecyl (C12E10) and the Mammalian Cell Lysis Kit
were from Sigma-Aldrich (Saint Louis, MO, USA). Ethylenediamine Tetraacetic Acid
(EDTA), cellulose dialysis tubing (MWCO 2k and 3.5k), phosphate buffered saline (PBS),
chloroform, methanol, LAL Chromogenic Endotoxin Quantification kit, Penicillin-
Streptomycin (PS), DMEM without phenol red and Vybrant® MTT Cell Proliferation
Assay Kit were purchased from Fisher Scientific (Pittsburgh, PA, USA). Citrated human
whole blood was drawn from healthy, aspirin-refraining donors via venipuncture according
to IRB approved protocols. Adenosine di-phosphate (ADP) and soluble calf skin type I
collagen were from Bio/Data Corporation (Horsham, PA, USA), and human vWF (FVIII-
free) was from Hematologic Technologies (Essex Junction, VT, USA). For rotational
thromboelastometry (ROTEM) analysis, all reagents were purchased from ROTEM
(Munich, Germany). For in vivo studies on pigs, Yorkshire Farm Pigs were purchased from
231 Shoup Investments Ltd. Telazol, isoflurane and pentobarbital were obtained from Patterson
Veterinary (Greely, CO, USA). The Beadbug Mircotube Homogenizer was purchased from
Benchmark Scientific (Edison, NJ, USA). The Guinea Pig Complement C3 ELISA kit and
Human Complement C3a des Arg ELISA kit were from Abcam (Cambridge, MA, USA).
Mouse-derived embryonic fibroblast cell line 3T3 was provided graciously by the
laboratory of Eben Alsberg, Case Western Reserve University. High glucose Dulbecco’s
Modified Eagle Medium (DMEM) and Fetal Bovine Serum (FBS) were from American
Type Culture Collection (Manassas, VA, USA).
6.4.2 Manufacture of SynthoPlateTM
SynthoPlateTM was manufactured using ‘film rehydration and extrusion’ technique as
42,44 described previously . Briefly, DSPC, cholesterol, DSPE-PEG2000-VBP, DSPE-
PEG2000-CBP, DSPE-PEG2000-FMP and DSPE-mPEG1000 were homogeneously mixed at
0.475, 0.45, 0.0125, 0.0125, 0.025 and 0.025 mole fractions, respectively, in 1:1 chloroform:methanol. Solvent was removed via rotary evaporation, and the thin lipid film was rehydrated with normal saline solution (0.9% NaCl) at a concentration of 1 x 105 moles lipid per mL. This lipid suspension was subjected to 10 freeze/thaw cycles and subsequent extrusion through 200nm pore diameter polycarbonate membrane using a pneumatic extruder (Northern Lipids, Burnaby, Canada) to create heteromultivalently decorated
SynthoPlateTM vesicles. Dynamic light scattering (DLS) and electron microscopy
characterization indicated fresh-made vesicles were ~200nm in diameter.
232 6.4.3 Long term Storage-in-Suspension Studies
In order to evaluate the stability of SynthoPlateTM under storage, particles were
manufactured as described above, packaged into polypropylene tubes, purged with
nitrogen gas and stored for up to 6 months at room temperature (~25°C). Particle size
distribution was measured weekly via Dynamic Light Scattering (DLS). This data was
considered as reflective of particle stability over time, since large variations in size would
be indicative of particle instability.
6.4.4 Studies on SynthoPlateTM sterilization and its biofunctional effects
SynthoPlateTM samples were evaluated at Lexamed (Toledo, OH, USA) for sterilization
via filtration and E-beam exposure. First, it was necessary to validate the sterilization
methods by demonstrating that SynthoPlateTM itself does not inhibit the growth of common contaminating organisms and hence does not interfere with the standard sterilization assays. For this, SynthoPlateTM samples were inoculated with 6 different challenge
organisms, namely Staphylococcus aureus, Kocuria rhizophila, Clostridium sporogenes,
Bacillus subtilis spizizenii, Candida albicans and Aspergillus brasiliensis, and allowed to
grow in appropriate conditions for up to 5 days. Any differences in growth profiles between
samples with or without SynthoPlateTM over the 5-day period were recorded. Next,
SynthoPlateTM samples were sterilized by filtration in 0.2 µm filter and by E-beam
exposure at both 25 and 40 kGy doses. Sterility was assessed by observation of the samples
for any visible growth of microorganisms over the course of 7 days. Furthermore,
endotoxin burden in the formulation was evaluated using a standard chromogenic limulous
ameobocyte lysate (LAL) assay. Briefly, nanoparticle samples were incubated with 0.5%
233 (v/v) of 1.5% C12E10 detergent for 20 minutes at 65°C followed by 1 hour at room
temperature to solubilize the lipid components of SynthoPlateTM and release any masked
endotoxin67. Endotoxin burden in the detergent-treated SynthoPlateTM samples was
quantified using the LAL assay.
To assess whether sterilization affects SynthoPlateTM particle stability, post-sterilization
particle size distribution was measured by DLS and compared to fresh-made (unsterilized) particles. Furthermore, in order to measure whether sterilization affects the platelet- inspired biofunctional abilities of SynthoPlateTM, specific adhesion and aggregation assays
were performed as adapted from our previous reports43,46. To test whether sterilization
affects the platelet-inspired adhesive function of CBP and VBP moieties on SynthoPlateTM
(to collagen and vWF, respectively), acid washed glass coverslips were coated with a
solution of 1:1 vWF:collagen, and 200µl RhB-labeled SynthoPlateTM suspensions (0.4
mg/ml), un-sterilized or sterilized, were incubated on these coverslips in a 12-well plate
for 30 minutes at 37oC on a shaker set at 100 rpm. Following this, SynthoPlateTM
suspension was removed, and loosely bound particles were further washed away with PBS.
The coverslips were then mounted onto microscope slides and imaged (using Zeiss
AxioObserver inverted fluorescence microscope) for adhered SynthoPlateTM (Rhodamine
B fluorescence, λex=560 nm, λem=580 nm). To test whether sterilization affects the ability
of FMP moieties to bind to active platelet integrin GPIIb-IIIa to amplify platelet aggregation, platelet lumi-aggregometry assays were performed using a PAP-8E (Bio/Data
Corp, Horsham, PA, USA) instrument. For this, human whole blood was collected in
citrated tubes from healthy consenting donors in accordance with protocols and guidelines
234 approved by the Institutional Review Board (IRB) of University Hospitals Cleveland
Medical Center (UHCMC, IRB Number 12-16-06). Informed consent was obtained from
all subjects as per this IRB-approved guideline. The whole blood was centrifuged at 150
x g for 15 minutes to obtain platelet rich plasma (PRP). Some of the PRP was further
centrifuged at 2500 x g for 25 minutes to obtain platelet poor plasma (PPP). Aggregometry
studies were carried out at 37°C with stirring at 1200 rpm. ADP-mediated aggregation was
assessed with 225µl of 100% PRP or PRP diluted 50% (v/v) with PPP supplemented with
fresh-made (un-sterilized) or sterilized (filtration and E-beam) SynthoPlateTM particles
with a particle concentration of 5 x 1010/mL, with or without adding agonist (2 x 10-5 M
ADP). These adhesion and aggregation results of SynthoPlateTM post-sterilization were
compared to that for fresh-made (unsterilized) SynthoPlateTM.
6.4.5 In vitro cytotoxicity assay with SynthoPlateTM
3T3 cells were grown in DMEM supplemented with 10% FBS and 1% PS. Cultures were
maintained in a humidified atmosphere of 5% CO2 at 37°C. 3T3 cells were seeded into 96
well plates at 5000 cells/well. Within 24 hours, media was removed and seeded wells were
treated with media containing SynthoPlateTM particles versus media without the particles.
SynthoPlateTM particles were applied at 0.5 nM and 1 nM concentrations, which
correspond to the dose administered to pigs and double the dose administered to the pigs respectively. All treatments were suspended in phenol red free DMEM and an n=8 was maintained for all test groups. The treated cells were incubated at normal culture conditions
for 24 hours. After 24 hours, a standard MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was used to assess metabolic viability of the cells. In
235 this assay, metabolic activity (i.e. cell viability) reduces the MTT dye to insoluble formazan
which has a purple color, and the intensity of this can be assessed by colorimetric (i.e. UV-
Visible spectrometry) technique to reflect cell viability. For this, post incubation with or
without SynthoPlateTM, media was removed and fresh phenol red free media and MTT
stock solution were applied to all wells. The cells were incubated at normal culture
conditions for 4 hours. After 4 hours, a solution of SDS-HCl was mixed into all wells. and
incubated for a final period of 4 hours. Finally, the formazan intensity (i.e. cell metabolic viability) was quantified with UV-visible spectrometry, measuring absorbance at 570 nm.
6.4.6 In vivo Safety and Biodistribution Studies in Pigs
All in vivo porcine studies were carried out in accordance with relevant guidelines and
regulations approved by Case Western Reserve University Institutional Animal Care and
Use Committee (IACUC, Protocol Number 2015-0135). Yorkshire Farm Pigs 25-36kg
were acclimated to the laboratory space for 48 hours. Pigs were fasted for 12 hours before
experiments but had free access to water. Pigs were sedated with Telazol (6-8mg/kg)
injected intramuscularly, intubated with an endotracheal tube and anesthetized with
isoflurane (1-5% to effect). A CO2 sensor was placed at the end of the endotracheal tube.
Mechanical ventilation was provided to keep end-tidal CO2 and respiration rate initially at
normal values. EKG electrodes were placed on the pig’s limbs. A pulse-oximeter probe
was placed on the pig’s mouth (cheek or tongue). An esophageal temperature probe was
placed to measure core temperature, which was maintained between 36oC-38oC with a water-filled warming blanket. An angiocatheter was placed in the carotid artery to acquire invasive blood pressure and withdraw blood samples for ex vivo testing. Another
236 angiocatheter was placed in the internal jugular vein to deliver saline or particle treatments.
The 50ml dose of SynthoPlateTM (or control particles) to be administered in the pigs was
1X1011 particles/ml (per calculation shown in Supplementary Section S.4) followed by a
450ml saline infusion. Invasive arterial blood pressure, CO2, SpO2, temperature, and heart
rate were recorded every 30 seconds for the first 10 minutes, every minute for the next 20
minutes, and every 5 minutes thereafter for a total of 60 minutes. Arterial blood was drawn
via the carotid artery angiocatheter at baseline, 30 minutes, and 60 minutes. At the end of
experiments, pigs were euthanized with an I.V. overdose of pentobarbital (0.22ml/kg).
In order to evaluate the systemic safety of SynthoPlateTM in pigs within the ‘golden hour’
time-frame, without any confounding effects from injury, treatments were administered (at
the bolus dose stated previously) in a pilot group of non-injured animals (total n=6; saline:
n=2, unmodified particles: n=2, SynthoPlateTM: n=2), and the animals were observed for 1 hour. The surgeons were blinded to the administered treatments, and blinding was ensured by a researcher who assigned a randomly generated code to each treatment. The effects of the administered treatments on vitals, blood chemistry, platelet function (aggregation), and blood clot viscoelastic properties were examined. It has been reported that nanoparticles
(including liposomes) can sometimes lead to a hypersensitivity reaction and complement activation (known as complement activation-related pseudoallergy or CARPA)52.
Therefore, the risk for such reactions was assessed ex vivo. For this, specific complement
activation marker (C3 to C3a) was measured in the blood drawn from the pig at baseline
as well as 30 and 60 minutes post administration of saline, unmodified particles or
SynthoPlateTM. In complement activation, C3 is cleaved into C3a and C3b leading to a
237 decrease in plasma C3 levels and an increase in plasma C3a levels, and thus the C3:C3a
ratio can indicate such risks. For these studies, platelet poor plasma was isolated from pig
blood samples by centrifuging for 25 minutes at 2500 x g and measurements of C3 and
C3a were carried out using Guinea Pig Complement C3 ELISA kit and Human
Complement C3a des Arg ELISA kit respectively (Abcam, Cambridge, MA, USA). Pig
whole blood samples were also analyzed for pH, calculated base excess, carbon dioxide
(CO2), calculated bicarbonate, partial pressure of oxygen (PaO2), oxygen saturation (SaO2), white blood cell count (WBC), hematocrit (Hct), hemoglobin (Hg), red blood cell count
(RBC), platelet count (Plt), mean corpuscular volume in femtoliters (MCV fL), mean corpuscular hemoglobin concentration (MCHC), red blood cell distribution width-
coefficient of variation (RDW-CV), prothrombin time (PT), international normalized ratio
(INR), and, activated partial thromboplastin time (APTT) compared between blood of
saline-treated, unmodified particle-treated and SynthoPlateTM-treated pigs. Furthermore,
platelet aggregometry was conducted on PRP isolated from pig blood samples, using
methods described previously. In addition, effect of the various treatment groups on clot
viscoelastic properties was assessed by Rotational Thromboelastometry (ROTEM) via
analysis of drawn blood samples.
At the 1-hour time point pigs were euthanized and major organs (lungs, heart, liver, kidney,
spleen) were harvested, weighed and fixed in formalin. Three random samples 200-350mg
were removed from each pig organ and placed into pre-weighed homogenizing tubes, and the wet weight of each organ sample was recorded. The samples were then vacuum dried and their dry weights recorded. The dry organs were homogenized with a BeadBug
238 Microtube Homogenizer using a Mammalian Cell Lysis kit and then centrifuged (20
minutes, 12,000 x g) to separate the organ tissue from the supernatant. The supernatant was
added to 1:1 methanol:chloroform to dis-assemble the Rhodamine B labeled lipids of
SynthoPlateTM in the samples. These samples were analyzed with Ultra Performance
Liquid Chromatography (UPLC) using a fluorescence detector (λex = 560nm, λem = 580nm)
to assess Rhodamine B fluorescence. Biodistribution of the SynthoPlateTM particles was
determined by calculating the nanogram (ng) of particles per milliliter (ml) of supernatant
utilizing an appropriate calibration curve that correlates RhB fluorescence intensity to
particle concentration. Samples from each organ were also processed for histological
analysis. For this, samples were embedded in paraffin and sectioned and stained with
hematoxylin and eosin (H & E stain). The resultant slides where imaged on a Zeiss
Axiophot microscope and images were obtained with Zeiss Axiovision camera and
software.
6.4.7 Femoral Artery Bleeding Model in Pigs
Figure 5 shows a representative experimental set-up of the model for these studies. Pigs
(total n=12; Saline: n=4, unmodified particles: n=4, SynthoPlateTM: n=4) were prepared as
described previously. The skin at the inguinal area of the thigh was incised ~10cm, the
femoral artery was exposed and injured (near transection) using a 3.5mm aortic punch
(Scanlan International, St Paul, Minnesota, USA). Saline or particle treatments were administered 1 minute after injury via the jugular at a rate of 50ml/min. Blood loss from femoral artery injury was measured by carefully suctioning the shed blood without disturbing the injury site, into a container that was weighed every 30 seconds for the first
239 10 minutes, every minute for the next 20 minutes, and every 5 minutes thereafter until
bleeding stopped due to death or hemostasis. Blood mass was correlated to blood volume
by approximating blood density to be 1000 kg/m3. With saline administration, which is a
standard volume resuscitation strategy for treating hemorrhage in pre-hospital scenarios, this injury resulted in an average of 31.5 mL/kg blood loss in 24 minutes, and showed only
25% survival within the first 60 min (i.e. within the ‘golden hour’ period). At the end of
experiments, pigs were euthanized with an I.V. overdose of pentobarbital (0.22ml/kg).
6.4.8 Hemostatic Efficacy Evaluation in Femoral Artery Bleeding Model
The femoral artery bleeding model was performed as described above. Induction of the
injury was designated as time-point zero (0). Saline or particle (unmodified control or
SynthoPlateTM) treatments were administered 1 minute after injury via the jugular, (50 ml
particle concentration 1 x 1011 particles/ml followed by 450 ml saline) at 50 ml per minute.
Blood loss was measured as described previously. Arterial blood was drawn via the carotid artery angiocatheter at baseline, and 15, 30, 60 and 120 minutes, to run ex vivo analysis.
At the end of experiments, pigs were euthanized, major organs (lungs, heart, liver, kidney, spleen) and clot were harvested, weighed and fixed in formalin. Organ samples were prepared and analyzed for SynthoPlateTM (or control unmodified particle) biodistribution
at time of death as described previously for uninjured animals. Sections of the femoral
artery injury site from SynthoPlateTM-treated animals were deparaffinized and rehydrated
by washing with xylene 2 times for 2 minutes, 1:1 xylene: ethanol for 3 minutes, ethanol 2
times for 3 minutes, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes and finally 50%
ethanol for 3 minutes. Antigen retrieval was performed by incubating in Tris-EDTA buffer
240 in a water bath at 60oC overnight. Slides were washed, blocked in 10% serum with 1%BSA
in TBS for 2 hours, incubated with the FITC-labeled (green fluorescent) CD42b antibody
(for staining platelet glycoprotein GPIbα), washed, protected with a cover-slip and then imaged (using a Zeiss AxioObserver inverted fluorescence microscope). Brightfield,
SynthoPlateTM fluorescence (red, Rhodamine B) and platelet fluorescence (green, FITC)
were captured for the same field of view.
6.4.9 Statistical Analysis
Statistical analysis of aggregometry, biodistribution, blood loss data, and complement data
was done using two-way ANOVA with a Bonferroni post-test. Vitals data, blood analysis data and ROTEM data were analyzed using one-way ANOVA with a Tukey post-test.
Survival data was analyzed using a Log-rank test. In all analyses, significance was considered to be p < 0.05.
6.5 Acknowledgements
This research was supported in part by funding from the National Institutes of Health
(NIH), specifically National Heart Lung and Blood Institute (NHLBI Grant R01
HL121212, PI: Sen Gupta) and National Institute of General Medical Sciences (NIGMS,
Grant R35 GM119526, PI: Neal; Co-I: Sen Gupta), as well as, funding from Case Coulter
Translational Research Partnership (Grant 419, PI: Sen Gupta), National Center for
Accelerated Innovation – Cleveland Clinic (NIH Grant U54HL119810, PI: Sen Gupta),
and Ohio Third Frontier Technology Validation and Start-up Funds (TECG20160125, PI:
Sen Gupta). The authors also acknowledge NIH National Center for Research Resources
241 (Grant1 C06 RR12463-01, PI: Kutina) for supporting the laboratory facilities for Sen Gupta in the Wickenden Building at Case Western Reserve University. The authors acknowledge
Steve Schomisch and Cassie Cipriano for their assistance with animal studies. The content of this publication is solely the responsibility of the authors and does not represent the official views of the NIH and other funding entities.
6.6 Author Contributions
A. Sen Gupta, V. Kashyap and M. Neal directed research. E. Niedoba, C. L. Pawlowski, and D. A. Hickman prepared all particles, and dynamic light scattering characterizations.
D. A. Hickman, C. L. Pawlowski, N. F. Luc, U. D. S. Sekhon, J. Marks, and M. Sun carried out ex vivo aggregometry and ROTEM studies. A. Girish carried out in vitro cytotoxicity studies with SynthoPlateTM. S. Ganjoo and S. Huang carried out in vivo complement studies. D. A. Hickman and C. L. Pawlowski carried out histology and immunohistochemistry studies. D. A. Hickman, C. L. Pawlowski, A. Kim, A. Shevitz, S.
Ganjoo, S. Huang, E. Niedoba, N. F. Luc, U. D. S. Sekhon, J. Marks, and M. Sun carried out in vivo pig studies. D.A. Hickman, C.L. Pawlowski, M. Dyer, M. Neal and A. Sen
Gupta carried out data analysis, interpretation and wrote the paper.
6.7 Competing Financial Interests
A. Sen Gupta is a co-inventor on patents related to SynthoPlateTM technology: US 9107845 and US 9,6363,383. The SynthoPlateTM trademark is currently recorded with USPTO (U.S.
Serial Number: 86-829,160). The other authors state that they have no conflict of interest.
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254 Chapter 7: Trauma-targeted Delivery of Tranexamic Acid for Augmenting
Hemostasis
7.1 Abstract
Trauma-associated non-compressible hemorrhage remains a leading cause of global mortality. Rapid stoppage of bleeding (hemostasis) is critical to save lives in trauma.
However, traumatic hemorrhage and coagulopathy often lead to a hyperfibrinolytic phenotype where hemostatic clots are incapable of stabilizing due to upregulated tPA and plasmin activity. Tranexamic Acid (TXA), a synthetic lysine analog that can inhibit tPA, plasminogen and plasmin, has emerged as a promising drug to mitigate fibrinolysis in hemorrhage. Tranexamic Acid is currently FDA-approved for treating heavy menstrual and post-partum bleeding, and has shown clinical promise for trauma treatment. However,
TXA at high or repeat dose causes thrombo-embolic complications, neuropathy, systemic coagulopathy etc., possibly due to off-target action. We hypothesized that encapsulation of
TXA within injury site-targeted nanoparticles can enable its delivery and clot-stabilizing action selectively at the trauma site, to improve hemostasis and survival while avoiding systemic off-target effects. To test this hypothesis, we used liposomes as a model delivery vehicle, decorated its surface with a fibrinogen-mimetic peptide (FMP) for anchorage to activated platelets within trauma-associated developing clot, and encapsulated TXA within them at clinically effective doses. These TXA-loaded trauma-targeted nanovesicles
(TTNVs), ~170 nm in diameter, were evaluated in vitro for their clot stabilizing effect in rat blood, and then in vivo for systemic safety, hemostatic efficacy and survival benefit in a liver hemorrhage injury model in rats. Treatment with TXA-loaded control (untargeted)
255 nanovesicles (TCNVs), free TXA or saline only (no drug) were used as comparison. Our studies show that in vitro, the TTNVs were capable of resisting lysis in tPA-spiked rat blood, at levels comparable to free TXA. In vivo, I.V.-administration of TTNVs did not cause any systemic negative effects in un-injured rats. Furthermore, in the rat liver hemorrhage model, post-injury administration of TTNVs showed substantial reduction in blood loss and improvement of short (1 hr) and long term (72 hr) survival. Histopathologic and immunofluorescence evaluation of excised tissue from euthanized rats confirmed systemic safety and trauma site-targeted action of the TTNVs. Overall, these studies establish the potential of the TTNV system for safe injury site-selective enhancement of hemostasis to improve survival outcomes in trauma.
Key words: Trauma, Hemorrhage, Hyperfibrinolysis, Tranexamic Acid, Targeted
Delivery, Rat Model
256 7.2 Introduction
Trauma-associated non-compressible hemorrhage, shock and coagulopathy remain among
the leading causes of mortality in both civilian and military populations, especially in the
age group 1-501–3. Recent clinical studies, e.g. the seminal Pragmatic, Randomized
Optimal Platelet and Plasma Ratios (PROPPR) trial and the Pre-Hospital Air Medical
Plasma (PAMPer) trial, have established that in such trauma hemorrhage scenarios,
transfusion of whole blood or blood components (e.g. 1:1:1 RBC: plasma: platelets) can
significantly improve mortality outcomes4–7. While the role of RBCs is to restore tissue oxygenation, it is the platelets and the coagulation components in plasma that work together to form a fibrin enmeshed hemostatic clot at the injury site that seals the vascular breach and stops the bleeding.
The cellular and molecular mechanisms of this hemostatic clot formation is shown in
Figure 7-1. In this process, as per the current consensus on the cell-based model of hemostasis8,9, vascular injury exposes tissue factor (TF) bearing cells (e.g. vascular smooth
muscle cells, adventitial fibroblasts etc.) to blood plasma such that the interaction of
coagulation factor FVII with TF and subsequent formation of the activated FVIIa-TF
complex leads to the generation of an initial amount of thrombin (FIIa). This thrombin then
activates other coagulation factors, e.g. FIX to FIXa and FX to FXa, as well as activates
blood platelets in the locality of the vascular damage. The vascular injury also exposes sub-
endothelial collagen, as well as induces secretion and deposition of von Willebrand Factor
(vWF) from the vascular endothelium and activated platelets10–15. Platelets can adhere to
these exposed proteins (e.g. to vWF by platelet GPIbα and to collagen by platelet GPIa/IIa)
257 and this adhesion further activates platelets which aggregate at the injury site principally
by bridging of platelet surface integrin GPIIb-IIIa by fibrinogen (Fg)
Figure 7-1. Cellular and molecular components of hemostasis regulation mediated by multifactorial mechanisms involving Tissue Factor (TF) bearing cells, adherent active platelets expressing negatively charged phosphatidylserine (PS), co-localized coagulation factors, thrombin generation, thrombin-induced conversion of fibrinogen to fibrin, crosslinking of fibrin to stabilize clot and degradation of crosslinked fibrin over time; in trauma-associated hyperfibrinolysis the fibrin degradation processes are upregulated while inhibitors for the processes are downregulated.
to form the primary hemostatic plug16,17. The surface of these aggregated active platelets
expose a high extent of negatively charged phospholipids like phosphatidylserine (PS)18,19
which allows for co-localization of the various coagulation factors, especially forming the
‘tenase’ complex (FVIIIa-FIXa-FX) in presence of co-factor calcium (Ca++) to generate
activated FXa. This in turn forms the ‘prothrombinase’ complex (FXa-Fva-FII) on the PS- rich platelet surface in presence of Ca++, leading to amplified generation of thrombin (FIIa).
The thrombin converts local Fg into fibrin, which via self-assembly as well as
transglutaminase FXIIIa-induced reaction forms a highly crosslinked biopolymeric
mesh20,21 to secure the platelet plug and other blood components (e.g. RBCs) to stop
bleeding (i.e. hemostasis).
258 Therefore, crosslinked fibrin formation and stabilization is central to the establishment of
hemostatic clots. As the injury environment transitions from the ‘hemostatic’ phase to
‘healing’ phase, natural well-regulated mechanisms contribute to clot (fibrin) degradation.
The most pertinent process is the secretion of tissue plasminogen activator (tPA) by
vascular endothelium which converts plasminogen (Plg) into plasmin, predominantly
amplified when Plg is bound to fibrin, and this plasmin degrades fibrin (e.g. into fibrin D- dimers)22,23. This process remains well-regulated by plasminogen activator inhibitor (PAI-
1), also secreted by the endothelium, which inhibits tPA locally24 to optimize the balancing
kinetics of fibrin formation (by thrombin) and fibrin degradation (by plasmin). Other
processes, e.g. the thrombin-thrombomodulin mediated activated Protein C (aPC) pathway
to deactivate FVa and FVIIIa also regulate thrombin (and hence fibrin) generation
kinetics25. However, in traumatic hemorrhage these mechanisms are severely dysregulated, often resulting in a unique coagulopathic scenario termed ‘hyperfibrinolysis’26–29. In this
hemorrhagic scenario, the lethal combination of plasma depletion and dilution,
hypothermia and hypoxic tissue acidosis has been reported to amplify the aPC pathway,
reduce fibrin deposition and crosslinking, upregulate tPA production and downregulate
PAI-1 activity. As a result, fibrinolytic activity offsets fibrin generation activity and the
clot becomes unstable, leading to impaired hemostasis and subsequent mortalities. Clinical
observation of this pathophysiology in trauma patients has prompted robust research into
the use of anti-fibrinolytic agents for clot stabilization and hemostatic benefit. Such agents
include, tranexamic acid or trans-4-(aminomethyl) cyclohexanecarboxylic acid
(abbreviated as TXA) and ε-aminocaproic acid (abbreviated as EACA), synthetic Lysine
analogs that inhibit tPA activation of Plg, Plg conversion to plasmin as well as action of
259 plasmin directly30–32. We have decided to utilize TXA over EACA due to it being 10 times
more potent in vitro and binding more strongly to both the weak and strong receptor sites
on the plasminogen molecule than EACA33. Additionally, a summary of the cost associated
with TXA compared to EACA show that the cost per course of treatment of TXA is slightly
cheaper than EACA ($3.70 vs $3.80)34. Several recent clinical trials have established a beneficial role of TXA in mitigating hemorrhagic shock in trauma35–37, and TXA is now
listed as an ‘essential medicine’ by the World Health Organization (WHO) for use in
hemostatic management of heavy menstrual and post-partum hemorrhage, traumatic
bleeding and surgery (pre- and post-operative). TXA is available in both oral and
intravenous form, and in traumatic hemorrhage the intravenous form is used
predominantly. While these studies have advanced the intravenous use of TXA in trauma,
several parallel reports have also emphasized potential systemic side-effect risks of TXA
use, especially at high dose or repeat dose, in the context of systemic off-target thrombotic,
thrombo-embolic and seizure risks38–40. Such findings have prompted recent research into
packaging of TXA within various delivery vehicles to explore site-localized controlled
release potential41–45. However, these research reports are majorly in the context of topical
application of TXA-loaded particles directly at the injury site. A formulation for
intravenous administration of TXA for injury site-targeted delivery and action in a setting
of traumatic hemorrhage has not been studied. In this framework, we hypothesized that
encapsulation of TXA within injury site-targeted nanoparticles can enable its delivery and
clot-stabilizing action selectively at the trauma site, to improve hemostasis and survival
while avoiding systemic off-target effects. Here we report our findings from testing this
260 hypothesis using injury site-targeted liposomal nanovesicles as the TXA carrier system in
a traumatic liver hemorrhage model in rats.
7.3 Materials and Methods
7.3.1 Materials
The lipid components, namely, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]
(DSPE-mPEG1000), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-
[azido(polyethylene glycol)-2000] (DSPE-PEG2000-Azide) were purchased from Avanti
Polar Lipids (Alabaster, AL, USA). Rhodamine B-dihexadecanoyl-sn-glycero-3-
phosphoethanolamine (DHPE-RhB) was purchased from Invitrogen (Carlsbad, CA, USA).
The peptide cyclo-{Pra}CNPRGD{Tyr(OEt)}RC (FMP) was custom-synthesized by
Genscript (Piscataway, NJ, USA). Fibrinogen mimetic peptide was conjugated to DSPE-
46 PEG2000-azide via copper-catalyzed alkyne-azide cycloaddition (CuCAAC) and was
purified by dialysis and characterized by MALDI-TOF mass spectrometry. Sterile normal
saline solution (0.9% NaCl) was purchased from Baxter (Deerfield, IL, USA). Cellulose
dialysis tubing (MWCO 2k and 3k), phosphate buffered saline (PBS), Calcium chloride
(CaCl2), chloroform, methanol, and Dimethyl sulfoxide (DMSO) were purchased from
Fisher Scientific (Pittsburgh, PA, USA). L-Ascorbic acid 99% (C6H8O6) for the 2%
Ascorbic Acid solution was purchased from Sigma-Aldrich (Saint Louis, MO, USA). Tris
Hydrochloride (tris(hydroxymethyl)aminomethane hydrochloride) was purchased from
Promega Corporation (Madison, WI, USA). For rotational thromboelastometry (ROTEM)
analysis, all reagents were purchased from ROTEM (Munich, Germany). For in vivo
261 studies on pigs, SAS Sprague Dawley Rats were purchased from Charles River. Isoflurane,
buprenorphine, pentobarbital, lidocaine, ketamine, and xylazine were obtained from
Patterson Veterinary (Greely, CO, USA). The Beadbug Mircotube Homogenizer was
purchased from Benchmark Scientific (Edison, NJ, USA). The Rat D-dimer (D2D) ELISA
Kit was purchased from Biomatik (Wilmington, DE, USA). CD41 antibody FITC for
immunohistochemistry was purchased from Biorbyt (San Francisco, CA, USA).
7.3.2 TXA-loaded targeted nanovesicle (TTNV) development and characterization
For targeted delivery of TXA to hemorrhagic injury site, we utilized a liposomal vehicle
decorated on the surface by a cyclic RGD peptide, namely cyclo-CNPRGDY(OEt)RC that
has high affinity for the activated form of platelet surface integrin GPIIb-IIIa47,48. The
binding of this peptide to integrin GPIIb-IIIa mimic the binding of fibrinogen alpha chain
to that integrin, and thus we designate this peptide as fibrinogen mimetic peptide (FMP).
Compared to various integrin-binding ubiquitous RGD sequences (e.g. GRGDS), the FMP
sequence has high specificity towards the stimulated form of platelet integrin GPIIb-IIIa47,
and thus provides an ideal way to direct nanoparticle platforms to sites of active platelet aggregation and clot formation in hemorrhagic injury, as demonstrated in our previous
49,50 studies . This peptide was conjugated to the lipid DSPE-PEG2k-Azide by reacting with
Propargylglycine-terminated FMP via copper-catalyzed cycloaddition (i.e. ‘click’ chemistry) as shown in the schematic of Figure 7-5A. The resultant DSPE-PEG2K-FMP
conjugate was combined at 2.5 mol% with DSPC (46.5 mol %), cholesterol (45 mol%),
DSPE-mPEG(2k)(2.5 mol%), DSPE-mPEG(1k)(2.5 mol%), and DHPE-Rhodamine (1
mol%) to form FMP-decorated liposomal nanovesicles by film rehydration and extrusion
262 technique. To confirm the ability of the FMP-decorated nanovesicles to bind to clot-
associated activated platelets, the FMP-decorated nanovesicles were incubated with
platelet-rich plasma (PRP) in well plates in presence of thrombin to induce clot formation.
In these incubation samples, platelets were pre-stained with calcein and the plasma was spiked with AlexaFluor 647-labeled fibrinogen. Post 30 min incubation, the clots were harvested and analyzed by an Olympus FV 1000 confocal microscope, to detect platelet, fibrin and nanovesicle fluorescence in the clot.
The TXA was loaded within the liposomal vesicles during the lipid hydration step of the process, since TXA is highly soluble in water and allows for lipid hydration with an aqueous solution of TXA (167mg/ml). The TXA dose to be encapsulated was chosen as
93mg/kg in relevance to reported clinical doses37. The scaling of the dose from human to
rat was carried out per allometric scaling51. The dose for trauma associated hemorrhage in
humans is ~15mg/kg. This is converted to a rat dose using the allometric scaling factor of
6.2 to obtain the rat dose of 93mg/kg. Resultant TXA-loaded FMP-decorated trauma- targeted liposomal nanovesicles (TTNVs, schematic shown in Figure 7-5A) were characterized for size by dynamic light scattering (DLS). The encapsulation efficiency and release kinetics of TXA from were characterized by utilizing a colorimetric assay by conjugating TXA to ascorbic acid and analyzing with absorbance spectrometry (reaction schematic and calibration curve shown in Figure 7-2). FMP particles encapsulating TXA were synthesized, sealed in a 3k MWCO dialysis tube and placed in a beaker of 250 mL
5mM Tris-HCl 1mM CaCl2 buffer (pH 8.0) and incubated at 37°C for 3 hours. At each time
point (0 and every 10 minutes for 3 hours) 450uL of the buffer solution,
263
Figure 7-2. Ascorbic Acid-based reaction for assaying TXA, and representative calibration curve using this assay, based on which TXA release kinetics from nanovesicles was estimated.
which contained the released TXA, was removed from the 250mL reaction vessel and
replaced with 450uL of fresh 5mM Tris-HCl 1mM CaCl2 buffer (pH 8.0). After 3 hours, a
colorimetric assay was performed by conjugating TXA to ascorbic acid. 2mL of Ascorbic
acid stock solution (2% w/v in DMSO) was added to each collected sample and heated at
100°C for 45 minutes. The optical density of each sample was quantified by measuring the
absorbance at 390nm and the amount of TXA released at each time point and thus TXA
release kinetics was determined by utilizing an appropriate calibration curve that correlates
TXA-ascorbic acid absorbance intensity to TXA concentration.
7.3.3 In vitro analysis of the effect of free TXA and TTNVs on tPA-induced fibrinolysis
Rotational thromboelastometry (ROTEM) of rat whole blood was used to characterize and confirm the effect of TXA as well as TXA-loaded liposomal vesicles on improving clot stability in fibrinolytic conditions. ROTEM profiles provide effective characterization of
264
Figure 7-3. Representative instrument set-up, characteristic profile, actual instrument and relevant measured parameters in Rotational Thromboelastometry (ROTEM) analysis of whole blood (WB). For ROTEM analysis, whole blood sample is placed into a cuvette warmed at 37oC and a cylindrical pin is immersed in the blood. A gap of 1 mm remains between the pin and the cup wall, with blood in that space. The pin is rotated by a spring to the right and the left at 4.75o. When the blood is liquid, this movement is unrestricted. As the blood starts clotting, the developing clot increasingly restricts the rotation of the pin with rising clot firmness. Over time the clot can naturally lyse or undergo lysis due to action of extraneously added agents (e.g. tPA). The kinetics of this clot development, growth, stabilization and lysis processes are detected mechanically and processed digitally provide typical ROTEM profiles (TEMogram) from which various numerical parameters are calculated as shown above. In the specific studies described in the current manuscript, the parameter of interest were the lysis parameters (LI 30, LI 60 and ML) since the focus was to analyze the effect of TXA (free or nanovesicle-loaded) on clot stability.
clotting and clot formation time (CT and CFT), maximum clot firmness (MCF) and clot lysis parameters in whole blood52. The relevance of various parameters to clot characteristics is described in the schematic in Figure 7-3. As evident in comparing A1 and A2 in Figure 7-6A, a highly fibrinolytic effect could be created by adding 0.75ug/ml tPA to human blood in ROTEM assay, signified by the rapid onset of lysis (Lysis Index
265 after 30 minutes, LI30) and maximum lysis (ML) compared to that for healthy human
blood. Saline (control), or TXA at 4.9mg/ml as free TXA or loaded within liposomal
vesicles were added in these tPA-treated human blood assays and studied by ROTEM to
characterize clot properties, with the rationale that the TXA will inhibit the tPA effect and
thereby ‘rescue’ some of the clot stability (evidenced by improvement in MCF, LI30
profiles and delay in lysis). The TXA-loaded vesicles used in these studies did not have
FMP decorations since these are in vitro studies with blood in pooled conditions and thus an effect of ‘targeting’ the injury site is not relevant. The studies were mainly focused on establishing that TXA loaded within vesicles had comparable anti-fibrinolytic activity to free TXA, such that in subsequent in vivo studies the clot-targeted delivery of TXA-loaded vesicles is rationalized.
7.3.4 In vivo safety of FMP-decorated empty nanovesicles (TNVs) and TXA-loaded
TTNVs
In these studies, we designate FMP-decorated empty nanovesicles as ‘targeted nanovesicles’ (TNVs) and TXA-loaded versions of these targeted nanovesicles as TTNVs.
All in vivo rat studies were carried out in accordance with relevant guidelines and regulations approved by Case Western Reserve University Institutional Animal Care and
Use Committee (IACUC, Protocol Number 2017-0102). SAS Sprague Dawley Rats 200-
320g were acclimated to the laboratory space for 48 hours. Rats were anesthetized with isoflurane (1-5% to effect). Following induction of anesthesia, ophthalmic ointment is applied, a rectal thermometer, pulse oximeter, blood pressure cuff and tail vein catheter are placed. For studying in vivo safety of TNVs as well as TTNVs, rats were administered with
266 nanovesicle dose of 8.8mg/kg. For evaluating TXA-loaded nanovesicles the TXA dose was kept at 93mg/kg. All injections were given via tail vein catheter at a 0.5ml volume. The vitals were monitored every 1 minute for the first 30 minutes, every 5 minutes for the next
30 minutes, then every 8 hours for 24 hours for anomalies that could be indicative of systemic effects. 24 hours after treatment administration, rats were euthanized with an
overdose of pentobarbital. Blood was drawn at baseline and pre-euthanasia endpoints to
analyze systemic effects. Post-euthanasia, clearance organs (heart, lungs, liver, spleen, kidney) were harvested for histology.
7.3.5 Development of rat liver hemorrhage traumatic injury model
TXA-loaded targeted nanovesicles (TTNVs) were evaluated in a rat liver injury model adapted from Morgan et al53. Expected 1hr mortality is 30-40% in untreated (saline only,
no drug) animals. Figure 7-8A shows a representative experimental set-up of the model
for these studies. Rats were prepared as previously described. The abdomen was shaved, a
subcutaneous buprenorphine administration and intramuscular injection of Lidocaine at the
incision site were administered. The abdomen was scrubbed, the peritoneal cavity opened
and the liver exposed. The peritoneal cavity was packed with pre-weighed absorbent gauze
and the liver was cut sharply 1.3 cm from the superior vena cava. The abdomen was
immediately closed, and treatment was administered 1 minute after injury. The vitals were
monitored every 1 minute for the first 30 minutes, every 5 minutes for the next 30 minutes,
then every 8 hours for 72 hours. One hour after injury, the gauze was removed from the
abdomen and weighed to determine blood loss, the abdomen was sutured closed and the
wound area was injected with lidocaine. 72 hours post-injury, rats were euthanized with an
267 overdose of pentobarbital. Blood was drawn at baseline and pre-euthanasia endpoints to analyze systemic effects. After euthanasia, the major organs (heart, lungs, liver, spleen, kidneys, brain, and blood) were harvested for histological and microscopic evaluation of off-target effects (if any).
7.3.6 Evaluation of TTNVs in rat liver hemorrhage model
Rats (total: n=48; saline: n=12, Free TXA n:=12, TXA-loaded control (unmodified) nanovesicles (TCNV): n=12, and TXA-loaded targeted (FMP-decorated) nanovesicles
(TTNV): n=12) were prepared and injured as previously described. TXA at a dose of
93mg/kg was administered 1 minute after injury via tail vein injection in rats at 0.5ml volume as free TXA, or loaded within nanoparticles (control or targeted). The vitals were monitored as previously described for anomalies that could be indicative of systemic effects. One hour after injury, blood loss was determined. 72 hours post-injury, rats were euthanized with an overdose of pentobarbital. Blood was drawn at baseline and pre- euthanasia endpoints to analyze systemic effects. After euthanasia, the major clearance organs (heart, lungs, liver, spleen, kidney) were harvested for histological and microscopic evaluation of off-target effects (if any). Organs were fixed in formalin, processed and slides were made for H&E and immunostaining. Sections of the liver traumatic injury site were deparaffinized and rehydrated by washing with xylene 2 times for 2 minutes, 1:1 xylene: ethanol for 3 minutes, ethanol 2 times for 3 minutes, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes and finally 50% ethanol for 3 minutes. Antigen retrieval was performed by incubating in Tris-EDTA buffer in a water bath at 60 °C overnight. Slides were washed, blocked in 10% serum with 1%BSA in TBS for 2 hours, incubated with the
268 FITC-labeled (green fluorescent) CD41a antibody (for staining platelet integrin GPIIb),
washed, protected with a cover-slip and then imaged (using an Olympus FV 1000
fluorescence confocal microscope). Cell Nucleus (blue, DAPI), nanovesicle fluorescence
(red, Rhodamine B) and platelet fluorescence (green, FITC) were captured for the same
field of view.
7.3.7 Statistical analysis
Vitals data, blood loss data and ROTEM data were analyzed using one-way ANOVA with
a Tukey post-test. Survival data was analyzed using a Log-rank test. In all analyses,
significance was considered to be p < 0.05.
Figure 7-4. Representative confocal images showing red fluorescent FMP-decorated nanovesicles (designated in the article as trauma-targeted nanovesicle or TNV) can bind and localize with activated platelets (pseudocolored blue) within crosslinked fibrin-rich (pseudocolored green) clots formed from platelet-rich plasma (PRP) in presence of thrombin; these simulated studies confirm the ability of TNVs to incorporate within developing clots at the trauma site upon injury.
269 7.4 Results
7.4.1 Characterization of TTNVs and TXA release kinetics
Figure 7-4 shows representative confocal microscopy images of clots where FMP- decorated nanovesicles were incubated with PRP in presence of thrombin. As evident from the images, RhB-labeled nanovesicles (red) were capable of specifically binding to calcein- stained activated platelets (pseudocolored blue) within AlexaFluor 647 stained fibrin-rich
clots (pseudocolored green). Figure 7-5B shows a schematic for envisioned clot-localized delivery of TXA from injury site-targeted liposomal nanovesicles. Figure 7-5C shows representative results of DLS based analysis of size-distribution of TXA-loaded liposomal vesicles, demonstrating that the nanovesicle formulations can be consistently prepared at an average diameter of ~ 170 nm. Figure 7-5D shows representative results of encapsulation efficiency (EE) of TXA in such vesicles, demonstrating a consistent encapsulation efficiency of ~ 65%. These TXA-loaded nanovesicles were put in 3,000
MWCO dialysis tubing and incubated in a beaker of 250 mL 5mM Tris-HCl 1mM CaCl2
buffer (pH 8.0) at 37°C for 3 hours. Samples (450ul) were removed every 10 minutes,
replaced with fresh buffer, and underwent an ascorbic acid detection assay to determine
TXA concentration. As shown in Figure 7-5E, the release of TXA from these vesicles
followed a first order kinetic profile. It is important to note here that such release kinetics
are in agreement with profiles shown for other water-soluble drugs like streptokinase (SK)
from similar clot-targeted nanovesicles in fibrinolysis studies54. Furthermore, in those
studies the release of the drug was demonstrated to be a combination of diffusional release
as well as release due to degradation of DSPC-enriched vesicles by phospholipase enzymes
(e.g. sPLA2) that are reportedly upregulated at the site of clot formation (secreted from
270 stimulated platelets and macrophages)55,56. Since the lipid composition of the TXA- encapsulating nanovesicles incorporate the same mole% of DSPC, a similar release mechanism for the encapsulated payload can be rationalized for these systems.
Figure 7-5. [A] Schematic of the manufacturing process of TTNVs using DSPE-PEG2K-FMP, DSPC, DHPE- RhB lipids and incorporating TXA in the nanovesicle core; [B] Envisioned targeted delivery of TXA at the trauma site from TTNVs anchored to active platelets in the developing clot, to allow local inhibition of tPA, plasminogen and plasmin to site-specifically reduce hyperfibrinolysis; [C] Representative DLS characterization results from multiple batches of TTNVs manufactured for the studies show an approximate diameter of ~170 nm; [D] Representative encapsulation efficiency (EE) of TXA in TTNVs for multiple batches show an approximate encapsulation of ~ 60%; [E] Representative release profile of TXA from TTNVs at 37oC in PBS shows a first order release kinetics.
7.4.2 ROTEM analysis of the effect of TXA-loaded liposomes in human blood
In order to confirm that the TXA delivered via such nanovesicles is capable of resisting hyperfibrinolysis in rat blood clots (correlation to subsequent in vivo studies), ROTEM studies were carried out with rat whole blood spiked with tPA. Figure 7-6A shows representative
271
Figure 7-6. ROTEM analysis of clot characteristics of whole blood spiked with tPA and treated with saline (no TXA), free TXA or TXA loaded within nanovesicles; [A1] Normal ROTEM profile of rat blood; [A2] ROTEM profile of rat blood spiked with 0.75 mg/ml tPA showing rapid onset of lysis; Treatment with [A3] free TXA as well as [A4] nanovesicle-loaded TXA is capable of conserving clot characteristics and delay lysis onset possibly by inhibiting tPA, plasminogen and plasmin in the milieu; [B] shows ROTEM parameters from these studies, demonstrating that treatment with saline only (no TXA) is incapable of inhibiting lysis as the LI30 goes down (i.e. more lysis compared to MCF) and maximum lysis (ML) significantly increases compared to free TXA and TXA-loaded nanovesicle (TNV) groups; [C] shows Lysis 60 values, corroborating that treatment of tPA-spiked blood with free TXA or TNV allows for conservation of clot mechanics and reduce lysis to keep clot stability comparable to normal whole blood, while treatment with saline is incapable of providing such resistance to lysis.
272 ROTEM profiles of normal (A1), tPA-spiked (A2), tPA-spiked free TXA treated (A3) and tPA-spiked TXA-loaded nanovesicle treated (A4) rat whole blood. Spiking with 0.75 ug/ml of tPA resulted in a significant hyperfibrinolytic effect in rat whole blood clots, as evident from the ROTEM profile comparison between Figure 7-6A, A1 and A2, where MCF in
A2 was significantly reduced and clot lysis set in rapidly. Treatment of this tPA-spiked blood with free TXA enabled resistance to this ‘lytic’ state, as evident from Figure 7-6A3 where the MCF was demonstrably conserved and lysis onset was significantly delayed.
Treatment of tPA-spiked blood with TXA-loaded nanovesicles (TNV) allowed for similar resistance to rapid lysis, as evident from Figure 7-6A4. The table in Figure 7-6B shows
ROTEM parameters for the three treatment groups, namely saline (no TXA), free TXA and
TXA-loaded nanovesicles (TNVs), demonstrating that treatment with TNVs is capable of conserving MCF, resisting LI30 and reducing ML, at levels comparable to treatment with free TXA. Figure 7-6C shows quantitative analysis of lysis at 60 minutes (Lysis 60) in these studies, confirming that the treatment of tPA-spiked blood (i.e. induced hyperfibrinolytic state) with TXA-loaded nanovesicles can render substantial resistance to lysis at levels comparable to free TXA. Altogether these results suggest that if TXA is encapsulated within nanovesicles that allow targeted delivery selectively at a clot forming site post-injury, it can render localized inhibition of tPA (and plasmin) to resist clot lysis, while avoiding potential off-target effects associated with systemic delivery of free TXA.
Therefore, this strategy was further evaluated in vivo, in the rat liver hemorrhage model.
273 7.4.3 In vivo safety evaluation of various doses of FMP-decorated liposomes
To enable delivery of TXA-loaded nanovesicles specifically to the injury-associated clot
forming site, the nanovesicle surface was decorated with fibrinogen-mimetic peptides
(FMP) that bind to
Figure 7-7. In vivo safety evaluation with TNVs as well as TTNVs show that intravenous administration of these vesicles at dose of 8.8 mg/kg (TTNVs incorporate TXA dose of 93 mg/Kg) does not affect vitals over 24-hour period (A and B); Representative heart rate and SpO2 traces over the first 60 min period are shown in C and D. Total 6 animals (3 per group) were used for these studies.
the stimulated conformation of integrin GPIIb-IIIa on active platelets (a major component of clots). To ensure that these FMP-decorated nanovesicles themselves (TNVs) as well as loaded with TXA (TTNVs) do not pose any systemic negative effect in rats, safety studies were first conducted by dosing a pilot group of un-injured rats with these nanovesicles and monitoring vitals and reactions for 24 hours. Figure 7-7, A and B shows results from these studies, where administration of TNVs (empty trauma-targeted nanovesicles) or TTNVs
(TXA-loaded trauma-targeted nanovesicles) showed no negative effects on heart rate (HR),
274 oxygen saturation (SpO2) and body temperature over the 24-hour period following injection. Figure 7-7, C and D show representative traces of heart rate and SpO2 respectively over 60 min periods for rats administered with TNVs and TTNVs, showing stable vitals and such vitals were conserved over the 24-hour observation period.
Altogether, these results confirmed that intravenous administration of the TNVs and
TTNVs did not cause any drastic systemic effects in rats and all rats survived with stable vitals, until euthanized. Therefore, in subsequent studies TTNVs were administered to injured (liver hemorrhage) rats and evaluated for blood loss and survival, compared against administration of saline (no TXA), free TXA and TXA-loaded control (untargeted) nanovesicles (TCNVs).
Figure 7-8. [A] Schematic of rat liver injury hemorrhage model set-up (details described in Methods section); [B] Post-injury vitals of animals administered intravenously with saline, free TXA, TCNV or TTNV show that hemorrhagic injury causes higher heart rate while SpO2 stays somewhat consistent due to efficient use of ventilator; [C] Blood loss analysis post-injury and treatment administration shows that rats treated with TCNVs and TTNVs undergo substantially lower blood loss compared to rats treated with saline or free TXA and TTNV-treated rats exhibit lower blood loss than TCNV-treated rats; [D] 1-hr survival analysis post- injury and treatment administration shows that TTNV-treated rats exhibit substantially improved survival from the other treatment groups, and [E] this trend is conserved over 72 hours although mortality in all groups increase to some extent during that period. Total 48 animals (12 per group) were used for these studies.
275 7.4.4 Effect of clot-targeted delivery of TXA-loaded nanovesicles in rat liver injury model
The rat liver injury hemorrhage model was described in detail in the Methods section. In
this model, there is significant bleeding in the peritoneal cavity from the injured liver,
resulting in ~ 30 % fatality within 1 hour if no treatment is given (i.e. saline group in Figure
7-8D). It is important to note here that in the ‘saline’ group, saline was not transfused at
high volumes as a resuscitative transfusion strategy to treat hypovolemia from blood loss,
but administered only at volumes of 0.5ml that corresponds to the injection volume of other
treatments (free TXA and TXA-loaded untargeted and targeted nanovesicles). As shown in Figure 7-8B, post-injury during the first hour, rats without any TXA treatment (saline group) as well as with TXA treatment (as free drug as well as loaded within TCNVs and
TTNVs) showed higher heart rate compared to un-injured rats (Figure 7-7) since the hemorrhagic loss forces the heart to beat faster to compensate and maintain blood pressure.
The SpO2 in un-injured and injured rats remained comparable since they are all under
proper ventilation. More importantly, administration of free TXA was incapable of
reducing blood loss in the injured rats, whereas TXA delivery in TCNVs and TTNVs both
reduced blood loss substantially, with TTNV-treated rats showing reduced blood loss compared to TCNV-treated rats (Figure 7-8C). Furthermore, administration of free TXA as well as TXA in TCNVs were incapable of substantially improving 1-hr survival in rats
(survival level was comparable to ‘saline’ group) as shown in Figure 7-8D. In contrast, administration of TXA in TTNVs substantially improved survival during the 1-hr period.
This is very promising, considering that fact that most preventable mortalities resulting from traumatic hemorrhage occurs within 1-2 hours of injury (termed the ‘golden hour’).
Therefore, the results suggest that TXA nanoformulation in TTNVs can be a promising
276 technology for point-of-injury rapid administration in the hemorrhaging patient to improve
survival. Additionally, monitoring the survival over 72 hrs, TTNV-administered rats
continued to maintain improved survival compared to those administered with saline, free
TXA or TCNV (Figure 7-8E). Interestingly, administration of free TXA performed worse
than saline in maintaining survival over the 72-hr period, suggesting possible off-target
effects or reduced bioavailability at the injury site so as to be incapable of augmenting (or
maintaining) clot stability.
Figure 7-9. Representative images from histopathologic analysis (H & E staining) of excised tissue sections from liver injured rats administered with various treatments show that TTNVs do not have any off-target effects on clearance organs and free TXA can have some off-target effects in lungs.
277 At the end of the 72-hr observation period, rats were euthanized and clearance organs as
well as injured liver tissue were excised for histologic and immunofluorescence evaluation.
Figure 7-9 shows representative images of histologic evaluation (H & E staining) of tissue
sections from the various organs excised from rats administered with the various treatment
groups. The images show that all animals underwent liver injury as noted by the hydropic
changes seen in the hepatocytes near the blood vessels. Of particular note is the prominent
pulmonary congestion observed in the rats treated with free TXA signified by the red blood
cells and exudate in the alveolar space. This could be indicative of pulmonary emboli. In
fact, the lungs in the rats treated with TTNVs showed the least presence of such effects
compared to all other treatment groups (saline, free TXA and TCNV). There were drastic
effects seen in the heart, spleen and kidney for the TCNV and TTNV treated animals,
compared to saline and free TXA treated animals. Overall, these results indicate that
TTNVs do not cause any major systemic effects in injured animals and that free TXA may
cause off-target unwanted effects systemically (e.g. in the lung).
Figure 7-10 shows representative immunofluorescence images of the liver hemorrhage injury site tissue, showing nucleated cells stained by DAPI (blue), platelets in the
hemostatic clot stained by FITC-antiCD41a (green) and TXA-loaded nanovesicles stained
by Rhodamine B (red). As evident from the figure, administration of saline (essentially no
treatment) was incapable of augmenting formation of an effective hemostatic clot (low
levels of green platelet fluorescence). Administration of free TXA improved the hemostatic
clot formation and stability, as demonstrated by the enhanced presence of green fluorescent
platelets in the clot. Administration of TXA in control nanovesicles (TCNV) showed partial
278 incorporation of the vesicles into hemostatic clots, as evident from the overlay of green platelets and red nanovesicles. However, the overlay suggests only low levels of incorporation, possibly because the vesicles did not have surface decorations with a clot site-targeting ligand (i.e. FMP motifs). In contrast, administration of TXA in targeted nanovesicles (TTNV) showed significant incorporation of vesicles (red) into the clot (green platelets) as evident from the overlay, indicating that the vesicle surface-decoration with
FMP motifs enables active anchorage of the vesicles within the clots by virtue of binding to active platelet surface integrin GPIIb-IIIa. Furthermore, the clot structure in TTNV- treated rat liver injury site seemed larger in size and more compact, as evident from the green fluorescence of the platelets. This is possibly due to the fact that localized release of
TXA from clot-incorporated TTNVs inhibits clot lysis and stabilizes the fibrin network of the clots which results in retention of the activated platelets within the clots, thereby enhancing the overall clot composition and stability. Additionally, by virtue of bridging with the active platelets in the developing clot, the TTNVs may amplify the overall aggregation of platelets in an injury site-selective manner. These results corroborate the findings from the in vitro ROTEM analysis shown previously in Figure 7-6 in the context of clot stability (i.e. conserved MCF and reduced lysis). The results further suggest that hemostatic clot stabilization by injury site-targeted delivery of TXA using TTNVs can result in improved short term and long-term survival in hemorrhagic trauma.
279
Figure 7-10. Representative confocal microscopy images of immunostained tissue sections from injured liver site of rats administered with the various treatment groups show that rats treated with saline, free TXA and TCNVs form sub-optimal clot structure in liver vasculature, while rats treated with TTNVs exhibit substantial co-localization of TTNVs with platelets in the hemostasized liver and the clot structure for TTNV-treated rats appeared more compact.
7.5 Discussion
Robust efforts are being directed at improving the survival outcome in traumatic hemorrhage and coagulopathy, especially in resource-limited pre-hospital an in-hospital
280 scenarios. This is evident in continued research and development in the areas of making
whole blood as well blood components more portable, storage stable and available outside
of large trauma centers and blood banks via improving pathogen reduction technologies,
reducing contamination risks, enhancing storage through reduced temperature processing
(e.g. cold-stored as well as cryo-preserved platelets and plasma), engineering donor-
independent biologic and synthetic blood cell mimics (e.g. stem cell derived as well as
synthetic RBCs and platelets) etc.57. Along with such strategic improvements in making
blood-derived products available and accessible for transfusion-based hemorrhage control, significant research continues to be directed at developing materials and technologies that allow augmentation of hemostasis in conjunction as well as absence of blood products, including recombinant coagulation factors, tourniquet systems, biomaterials based topical, intracavitary and injectable hemostat technologies, and pharmaceutical agents that may improve platelet production, fibrin generation and clot stability58. One such agent, TXA,
has generated significant clinical interest due to its ability to inhibit plasminogen activator,
plasminogen itself as well as plasmin, thus allowing a robust reduction in fibrinolytic
processes that are often associated in destabilizing hemostatic clots in trauma. Due to this
capability, an oral formulation of TXA (brand name Lysteda) was approved by the Food
and Drug Administration (FDA) in 2009 to treat heavy menstrual bleeding, and the drug
has been clinically evaluated more extensively in recent years for the hemostatic
management of bleeding complications in trauma, surgery and post-partum hemorrhage.
Tranexamic Acid is currently clinically available at 100 mg/ml injectable formulation
(Cyklokapron) for intravenous administration in the treatment of traumatic hemorrhage and
is currently listed in the ‘Essential Medicine’ list of World Health Organization (WHO).
281 To date, the CRASH 2 trial has been the largest clinical evaluation of TXA in trauma
(20,211 patients over 40 countries) and a detailed look at the trial outcomes show that TXA given within 3 hrs of injury reduced mortality but given after 3 hrs of injury increased bleeding and mortality. This can possibly be due to systemic off-target antiplasmin effects where body’s natural regulation of clot formation and clot dissolution may be affected by
TXA. Furthermore, the optimal dosing of TXA remains a topic of continued research, since
TXA use especially at high doses have been associated with complications like seizures, vascular thrombo-embolism etc. as well as mortality in patients with physiological fibrinolysis. Such effects can be rationalized to be possibly stemming from off-target non- specific action of TXA, especially when administered at high doses. Furthermore, such observations emphasize the continued research on optimizing TXA-based anti-fibrinolytic strategies for broad spectrum application towards hemorrhage control.
In this study, we investigated the potential of encapsulating and delivering TXA in a clot- targeted nanovesicle formulation, that ensures injury site-selective delivery and release of the drug for localized action. For this purpose, we utilized unilamellar liposomes as a model drug delivery system and decorated the liposome surface with peptide motifs that allow active anchorage to the stimulated conformation of integrin GPIIb-IIIa on activated platelets involved in clot formation at the injury site. Our in vitro studies demonstrate that the nanovesicles can effectively bind to platelets within clots and TXA released from the nanovesicles can effectively stabilize the clot and inhibit clot lysis. The TXA dose in these formulations were kept at 93mg/kg to maintain relevance to the clinical dosing. One should note here that loading of TXA is not limited to liposomal vesicles, since a few recent studies
282 have demonstrated the ability to load and deliver TXA in other particulate systems as stated
previously. For intravenously administrable nanoformulation of TXA, we chose liposomes
as a model vehicle since it is a clinically well-studied drug delivery platform59.
Furthermore, developing such injury site-targeted nanovesicle systems provide the
possibility of intravenously delivering multiple types of hemostasis augmenting agents in
combination with TXA as well as stand-alone, to maintain high availability of the agents at therapeutically active concentration at the target site, while minimizing off-target
distribution and effects. Our in vivo studies indicate that the injury site-targeted
nanovesicles are systemically safe at doses of 8.8mg/kg in rats, and delivery of TXA using
these targeted nanovesicles (TTNV) substantially reduced blood loss and improved
survival. We show here that all in vivo studies described here were carried out at pilot scale, where 6 animals were used for in vivo safety studies and 12 animals per treatment group (4 groups = 48 animals) were used for in vivo hemostatic efficacy studies, totaling
54 animals. Histology based evaluation of excised tissue from clearance organs of animals indicate that animals treated with TTNV after liver injury experienced less lung injury complications compared to control groups. Immunofluorescence-based evaluation of injury site tissue sections from the liver indicate that TXA-loaded TTNV could efficiently incorporate within clots at high extent and possibly contributed to robust clot formation and stability for improving hemostasis, reducing blood loss and enhancing survival in the rat liver hemorrhage model.
The current studies are indeed the first ‘proof-of-concept’ experiments and analyses of injury site-targeted intravenous delivery of TXA in a model nanoparticle system for post- injury administration in an established traumatic hemorrhage model in rats. Based on the
283 promise of the current studies, we anticipate future studies from our group and others
directed at evaluating such TXA delivery in the context of varying the ‘time of
administration’ of the TXA nanoformulation post-injury to explore whether such encapsulation and targeted delivery can improve the survival outcome in traumatic hemorrhage when administered at longer time-points post injury. It is also important to note here that the mechanistic elucidation of whether TXA can maintain its anti-fibrinolytic capabilities upon long time periods in vivo, can be efficiently studied by developing specific animal models that present persistent levels of trauma-associated hyperfibrinolysis. While in the current studies we have used an acute liver injury model in normal wild-type rats, future studies can be directed at developing genetically engineered murine and rodent models that inherently present hyperfibrinolytic phenotype. Associated studies should also be directed towards analyzing pharmacology/toxicology profiles and potential immune response attributes in utilizing such TXA-loaded nanoformulation intravenously, especially to find the maximum tolerated dose (MTD) level and the potential of repeat dosing in severely injured exsanguinating patients.
7.6 Conclusion
In this study, we present proof-of-concept studies of an injury site-targeted TXA nanoformulation to treat traumatic hemorrhage and improve hemostasis and survival. For this, we utilized active platelet integrin GPIIb-IIIa-anchoring liposomes as the injury site- targeted delivery system and encapsulated TXA in the aqueous core. The TXA could be released via first order kinetics to potentially allow local inhibitory action on tPA and plasmin to reduce fibrinolysis. Our in vitro ROTEM studies demonstrated the ability of
284 this TXA-nanoformulation in inhibiting lysis and maintaining clot stability. Our in vivo
studies demonstrated the systemic safety of the nanovesicles, as well as, the ability of the
TXA-nanoformulation in reducing blood loss and improving survival over 1 hr to 3-day
observation period in a rat liver injury traumatic hemorrhage model. Post-euthanasia evaluation of excised tissues indicated the enhanced ability of the targeted TXA nanovesicles (TTNV) to get incorporated within developing clots at the injury site and improve clot size and quality. Histopathological evaluation of the TTNVs did not demonstrate systemic off-target thrombotic risks in clearance organs (liver, lung, kidney
and spleen).
7.7 Acknowledgement
This research was supported in part by funding from the National Institutes of Health
(NIH), specifically National Heart Lung and Blood Institute (NHLBI Grant R01
HL121212, PI: A. Sen Gupta). D.A. Hickman was supported by AHA Pre-doctoral
Fellowship 17CPRE33670016, NIH grant T32 GM007250 and NIH grant TL1 TR000441.
The authors also acknowledge NIH National Center for Research Resources (Grant1 C06
RR12463-01, PI: Kutina) for supporting the laboratory facilities for Sen Gupta in the
Wickenden Building at Case Western Reserve University. The content of this publication
is solely the responsibility of the authors and does not represent the official views of the
NIH and other funding entities.
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295 Chapter 8: Design and Administration of Nanoparticles to Prevent Pseudoallergic
Responses in Intravenous Hemostatic Management of Bleeding
8.1 Introduction
Traumatic injury is the leading cause of mortality globally for individuals 5-44 years of
age and accounts for 10% of all deaths in the US1. Blood loss is the primary cause of death
at acute time points post injury2,3. Research has demonstrated that immediately halting blood loss is one of the most effective means of minimizing mortality associated with severe trauma. The current standard of care for treating internal bleeding, blood products, has a number of limitations such as low in-hospital and no pre-hospital availability, short shelf life, and risk of bacterial contamination. Our lab has created an I.V.-administrable platelet-inspired nanomedicine, that can mimic and amplify body’s natural hemostatic mechanisms specifically at the bleeding site while overcoming these limitations and maintaining systemic safety.
The hemostatic capability of these platelet-inspired nanomedicines in decreasing blood loss and increasing survival have previously been described in a murine and porcine model of acute hemorrhagic shock and in correcting tail-bleeding time in thrombocytopenic mice4–
6. When these nanomedicines were first used in a porcine femoral artery injury model, a
model that better represents the human cardiovascular system, it was important to explore
methods to eliminate hypersensitivity reactions such as complement activation-related
pseudoallergy (CARPA), which pigs are highly susceptible and has been found to be
present in 23% of humans receiving nanomedicine7. CARPA is a hypersensitivity reaction
296 to nanoparticles and causes over-activation of the complement system and can lead to
symptoms such as arrhythmias, systemic hypotension, skin flushing, bronchospasm,
increased adenosine and is responsible for over 20,000 deaths per year in the US7,8.
Hypersensitivity reactions to nanoparticles have been linked to the over activation of the
complement system9. The complement system acts to distinguish between healthy cells, compromised cells, and foreign bodies and alters its response accordingly10. Often,
nanoparticles are designed, unintentionally, to have the same size and charge as pathogens
that the complement system is primed to combat7. Infusion of billions of these
nanoparticles can lead to a massive response that is not only detrimental to the
nanoparticles but also to the patient. Currently, this pseudoallergy is mitigated clinically
with FDA approved nanoparticle formulations such as Doxil, by infusing the treatment
slowly over many hours thereby avoiding high plasma concentrations of particles11.
Unfortunately, this is not a practical method of nanomedicine administration for someone
with fatal internal hemorrhage.
Alternatively, we could seek to modify the properties of our nanomedicine to eliminate
CARPA. Various characteristics of nanoparticles known to impact complement system
activation, include administration protocol, size, PEGylation, and zeta potential12,13.
Previously, I have contributed to research published by Onwukwe et al., where we were
able to mitigate the complement response by engineering a particle with a more neutral
zeta potential14. In the following studies, we observed the impact of PEGylation and administration protocol on CARPA response.
297 8.2 Materials and Methods
8.2.1 Materials
The lipid components, namely, 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-
Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-1000]
(DSPE-mPEG1000), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Distearoyl- sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE- mPEG2000), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-
[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Mal) and 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[azido(polyethylene glycol)-2000] (DSPE-PEG2000-
Azide) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Rhodamine B- dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE-RhB) was purchased from
Invitrogen (Carlsbad, CA, USA). The peptides CTRYLRIHPQSWVHQI (VBP), C[GPO]7
(CBP) and cyclo-{Pra}CNPRGD{Tyr(OEt)}RC (FMP) were custom-synthesized by
Genscript (Piscataway, NJ, USA). VBP and CBP were conjugated to DSPE-PEG2000-Mal
15 via thiol-maleimide coupling and FMP was conjugated to DSPE-PEG2000-azide via
copper-catalyzed alkyne-azide cycloaddition (CuCAAC)16, conjugates were purified by
dialysis and characterized by MALDI-TOF mass spectrometry. Sterile normal saline
solution (0.9% NaCl) was purchased from Baxter (Deerfield, IL, USA). Cholesterol, rat
tail type I collagen, copper(II) sulfate (CuSO4), Tris(3-
hydroxypropyltriazolylmethyl)amine (THPTA), sodium ascorbate, decaethylene glycol
monododecyl (C12E10) and the Mammalian Cell Lysis Kit were from Sigma-Aldrich (Saint
Louis, MO, USA). Ethylenediamine Tetraacetic Acid (EDTA), cellulose dialysis tubing
(MWCO 2k and 3.5k), phosphate buffered saline (PBS), chloroform, methanol, For
298 rotational thromboelastometry (ROTEM) analysis, all reagents were purchased from
ROTEM (Munich, Germany). For in vivo studies on pigs, Yorkshire Farm Pigs were
purchased from Shoup Investments Ltd. Telazol, isoflurane and pentobarbital were
obtained from Patterson Veterinary (Greely, CO, USA). The Guinea Pig Complement C3
ELISA kit and Human Complement C3a des Arg ELISA kit were from Abcam
(Cambridge, MA, USA).
Table 8-1. Characteristics of SynthoPlate PEG variants
8.2.2 Manufacture of SynthoPlateTM 1.0
SynthoPlateTM 1.0 was manufactured using ‘film rehydration and extrusion’ technique as
17,18 described previously . Briefly, DSPC, cholesterol, DSPE-PEG2000-VBP, DSPE-
PEG2000-CBP, and DSPE-PEG2000-FMP were homogeneously mixed at 0.475, 0.45,
0.0125, 0.0125, and 0.025 and mole fractions, respectively, in 1:1 chloroform:methanol.
Solvent was removed via rotary evaporation, and the thin lipid film was rehydrated with
normal saline solution (0.9% NaCl) at a concentration of 1 x 105 moles lipid per mL. This
lipid suspension was subjected to 10 freeze/thaw cycles and subsequent extrusion through
200nm pore diameter polycarbonate membrane using a pneumatic extruder (Northern
Lipids, Burnaby, Canada) to create heteromultivalently decorated SynthoPlateTM vesicles.
299 Dynamic light scattering (DLS) and electron microscopy characterization indicated fresh- made vesicles were ~200nm in diameter.
8.2.3 Manufacture of SynthoPlateTM 1.5
SynthoPlateTM 1.5 was manufactured using ‘film rehydration and extrusion’ technique as described above except DSPC, cholesterol, DSPE-PEG2000-VBP, DSPE-PEG2000-CBP,
DSPE-PEG2000-FMP and DSPE-mPEG2000 were homogeneously mixed at 0.475, 0.45,
0.0125, 0.0125, 0.025 and 0.025 mole fractions, respectively, in 1:1 composition of
chloroform : methanol.
8.2.4 Manufacture of SynthoPlateTM 2.0
SynthoPlateTM 1.5 was manufactured using ‘film rehydration and extrusion’ technique as described above except DSPC, cholesterol, DSPE-PEG2000-VBP, DSPE-PEG2000-CBP,
DSPE-PEG2000-FMP and DSPE-mPEG1000 were homogeneously mixed at 0.475, 0.45,
0.0125, 0.0125, 0.025 and 0.025 mole fractions, respectively, in 1:1 chloroform:methanol.
8.2.5 In vivo Safety Studies in Pigs
All in vivo porcine studies were carried out in accordance with relevant guidelines and
regulations approved by Case Western Reserve University Institutional Animal Care and
Use Committee (IACUC, Protocol Number 2015-0135). Yorkshire Farm Pigs 25-36kg
were acclimated to the laboratory space for 48 hours. Pigs were fasted for 12 hours before
experiments but had free access to water. Pigs were sedated with Telazol (6-8mg/kg)
injected intramuscularly, intubated with an endotracheal tube and anesthetized with
300 isoflurane (1-5% to effect). A CO2 sensor was placed at the end of the endotracheal tube.
Mechanical ventilation was provided to keep end-tidal CO2 and respiration rate initially at
normal values. Electrocardiogram electrodes were placed on the pig’s limbs. A pulse-
oximeter probe was placed on the pig’s mouth (cheek or tongue). An esophageal
temperature probe was placed to measure core temperature, which was maintained between
36oC-38oC with a water-filled warming blanket. An angiocatheter was placed in the carotid
artery to acquire invasive blood pressure and withdraw blood samples for ex vivo testing.
Another angiocatheter was placed in the internal jugular vein to deliver saline or particle
treatments. The 500ml dose of SynthoPlateTM to be administered in the pigs was 1X1010
particles/ml, while the 50ml dose of SynthoPlateTM was 1X1011 particles/ml followed by a
450ml saline infusion. Invasive arterial blood pressure, CO2, SpO2, temperature, and heart
rate were recorded every 30 seconds for the first 10 minutes, every minute for the next 20
minutes, and every 5 minutes thereafter for a total of 60 minutes. Arterial blood was drawn
via the carotid artery angiocatheter at baseline, 30 minutes, and 60 minutes. At the end of
experiments, pigs were euthanized with an I.V. overdose of pentobarbital (0.22ml/kg).
In order to evaluate the CARPA response of SynthoPlateTM PEG variants within the
‘golden hour’ time-frame, without any confounding effects from injury, treatments were administered (at the 500ml volume) in a pilot group of non-injured animals (total n=5;
SynthoPlateTM 1.0: n=2, SynthoPlateTM 1.5: n=2, SynthoPlateTM 2.0: n=1), and the animals
were observed for 1 hour. To evaluate the CARPA response of SynthoPlateTM volume
adminstration within the ‘golden hour’ time-frame, without any confounding effects from
injury, SynthoPlate 2.0 was administered at the 500ml volume or the 50ml volume
301 followed by 450ml saline in a pilot group of non-injured animals (total n=3; 50ml: n=2,
500ml: n=1), and the animals were observed for 1 hour. The surgeons were blinded to the
administered treatments, and blinding was ensured by a researcher who assigned a
randomly generated code to each treatment. The effects of the administered treatments on
vitals, skin and blood chemistry were examined. The risk for CARPA reactions was also
assessed ex vivo. For this, specific complement activation marker (C3 to C3a) was
measured in the blood drawn from the pig at baseline as well as 30 and 60 minutes post
administration treatment. In complement activation, C3 is cleaved into C3a and C3b
leading to an increase in plasma C3a levels, and thus the plasma C3a concentration can
indicate such risks. For these studies, platelet poor plasma was isolated from pig blood
samples by centrifuging for 25 minutes at 2500 x g and the measurement of C3a was carried
out using Human Complement C3a des Arg ELISA kit respectively (Abcam, Cambridge,
MA, USA).
302
Figure 8-1 Schematic representation of pig femoral artery hemorrhage model setup. A CO2 sensor was placed at the end of the endotracheal tube and mechanical ventilation was provided, EKG electrodes were placed on the pig’s limbs, a pulse-oximeter probe was placed on the pig’s mouth, an esophageal temperature probe was placed to measure core temperature, an angiocatheter was placed in the carotid artery to acquire invasive blood pressure and also withdraw blood samples for ex vivo analysis, an angiocatheter was placed in the internal jugular vein to deliver saline (or nanoparticle treatments) via an infusion pump.
8.2.6 Femoral Artery Bleeding Model in Pigs
Figure 8-1 shows a representative experimental set-up of the model for these studies. Pigs
(total n=9; 50ml: n=6, 500ml: n=3) were prepared as described previously. The skin at the
inguinal area of the thigh was incised ~10cm, the femoral artery was exposed and injured
(near transection) using a 3.5mm aortic punch (Scanlan International, St Paul, Minnesota,
USA). SynthoPlate 2.0 at the 500ml volume or the 50ml volume followed by 450ml saline
were administered 1 minute after injury via the jugular at a rate of 60ml/min. Blood loss
from femoral artery injury was measured by carefully suctioning the shed blood without
303 disturbing the injury site, into a container that was weighed every 30 seconds for the first
10 minutes, every minute for the next 20 minutes, and every 5 minutes thereafter until
bleeding stopped due to death or hemostasis. Blood mass was correlated to blood volume
by approximating blood density to be 1000 kg/m3. With saline administration, which is a
standard volume resuscitation strategy for treating hemorrhage in pre-hospital scenarios, this injury resulted in an average of 31.5 mL/kg blood loss in 24 minutes, and showed only
25% survival within the first 60 min (i.e. within the ‘golden hour’ period). At the end of
experiments, pigs were euthanized with an I.V. overdose of pentobarbital (0.22ml/kg).
8.2.7 Hemostatic Efficacy Evaluation in Femoral Artery Bleeding Model
The femoral artery bleeding model was performed as described above. Induction of the
injury was designated as time-point zero (0). SynthoPlate 2.0 at the 500ml volume or the
50ml volume followed by 450ml saline treatments were administered 1 minute after injury
via the jugular, (at the concentrations previously described) at 60 ml per minute. Blood loss
was measured as described previously. Arterial blood was drawn via the carotid artery
angiocatheter at baseline, and 15, 30, 60 and 120 minutes, to run ex vivo analysis. At the
end of experiments, pigs were euthanized.
8.2.8 Statistical Analysis
Statistical analysis of blood loss data and complement data was done using two-way
ANOVA with a Bonferroni post-test. Vitals data were analyzed using one-way ANOVA
with a Tukey post-test. Survival data was analyzed using a Log-rank test. In all analyses,
significance was considered to be p < 0.05.
304 8.3 Results
8.3.1 Synthesis of SynthoPlate PEG variants
PEGylation of a nanoparticles has been reported to effect the complement activation- related pseudoallergy response19. This led us to develop and evaluate three iterations of
SynthoPlate (1.0, 1.5, 2.0) with different PEGylation compositions (Table 8-1).
SynthoPlate 1.0 was designed with 5 mol percent 2,000 MW PEG that was all conjugated between DSPE and functional peptide (VBP, CBP or FMP). The 2,000 MW PEG mol percentage was increased to 7.5 for SynthoPlate 1.5 by adding an additional 2.5 mol percent of DSPE conjugated (peptide free) PEG. In the SynthoPlate 2.0 formulation, the additional
2.5 mol percent of DSPE conjugated (peptide free) PEG was replaced with 1,000 MW PEG to help reduce steric interference of the PEG chains. The SynthoPlate PEG variants had a hydrodynamic diameter that was roughly 200nm and zeta potential of about -6mV (Table
8-1). Varying PEG content did not significantly affect other characteristics of these nanoparticles.
8.3.2 Evaluation of SynthoPlate PEG variants
The SynthoPlate PEG variants were evaluated in our in vivo porcine safety model to assess their effect on complement activation-related pseudoallergy (total n=5; SynthoPlateTM 1.0:
n=2, SynthoPlateTM 1.5: n=2, SynthoPlateTM 2.0: n=1). As previously mentioned CARPA
can lead to changes in heart rate, blood pressure and breathing, so these parameters were
assessed by monitoring heart rate, mean arterial pressure and CO2 respectively. After
injection in non-injured pigs, SynthoPlate 1.0 caused a drastic drop in pigs’ heart rate, mean
arterial pressure and CO2 (Figure 8-2A, B, and C respectively). This ultimately led to the
305 pigs administered SynthoPlate 1.0 dying within 30 minutes of particle administration
(Figure 8-2D). This was indicative of a drug induced hypersensitivity reaction. Pigs
administered, SynthoPlate 1.5 had spikes in their heart rate, mean arterial pressure, and
CO2 within 10 minutes of particles administration but stabilized within 40 minutes without
intervention (Figure 8-2A, B, and C respectively). All animals treated with SynthoPlate
1.5 survived 60 minutes (Figure 8-2D). These observations are indicative of a mild drug hypersensitivity reaction. The Pig administered SynthoPlate 2.0 had stable heart rate and
CO2 and a slight spike in mean arterial pressure at 10 minutes that stabilized within 10
minutes without intervention (Figure 8-2A, C, and B respectively). This animal survived
the 60 minutes experiment time period (Figure 8-2D). These observations are likely an
indication of no reaction to the drug. The increase in mean arterial pressure alone is likely
a compensatory mechanism due to the increase in total blood volume from the 500ml
treatment.
306
Figure 8-2 Vital analysis in safety pigs administered SynthoPlate PEG variants show that [A] treatment with SynthoPlate 2.0 had no effect on heart rate while SynthoPlate 1.5 did show minimal effect and SynthoPlate 1.0 demonstrated more pronounced effects. There were similar results seen for [B] Mean Arterial Pressure and [C] CO2. [D] Animals administered SynthoPlate 2.0 and 1.5 had 100% survival rates while those administered SynthoPlate 1.0 had a 0% survival rate.
CARPA can also lead to a skin response. For this reason, the skin was monitored for
changes after SynthoPlate administration. SynthoPlate 1.0 administration lead to a
generalized erythematous rash (Figure 8-3A) that appeared roughly 1 minute after injection and resolved at the end of the injection (5 minutes). Administration of
SynthoPlate 1.5, lead to a marbled erythematous rash (Figure 8-3B) that also appeared roughly 1 minute after injection and resolved at 5 minutes. A milder skin reaction was observed with SynthoPlate 2.0 (Figure 8-3C). We observed generalized small erythematous patches with the same time course as SynthoPlate 1.0 and 1.5. These results support the vitals data.
307
Figure 8-3 Images of pig skin responses after SynthoPlate PEG variant administration. [A] A Generalized erythematous rash is seen after SynthoPlate 1.0 administration. [B] After SynthoPlate 1.5 administration a marbled erythematous rash is observed. [C] A generalized rash with small erythematous patches is observed after 500ml administration of SynthoPlate 2.0. [D] Little to no reaction is observed after 50ml bolus administration of SynthoPlate 2.0.
One of the main risk indicators for drastic activation of complement system and deleterious reactions like CARPA is a more than 4 -fold increase in complement protein20–22. We measured the product of C3 cleavage when the complement system is activated, complement C3a. We did not measure a significant increase in complement system activation in our SynthoPlate PEG variants (Figure 8-4A). There was a slight increase of
308 C3a plasma concentration in animals treated with our SynthoPlate 1.0 variant (Figure
8-4A). Animals treated with SynthoPlate 1.5 had relatively stable C3a plasma
concentrations while animals treated while animals treated with our SynthoPlate 2.0 variant
had a decrease in C3a plasma concentration (Figure 8-4A).
Figure 8-4 Analysis of C3a plasma concentration in blood drawn from pigs administered with [A] SynthoPlate PEG variants showed no significant alterations and [B] 50ml or 500ml SynthoPlate showed no significant alterations.
8.3.3 Evaluation of SynthoPlate Administration Protocol Safety
SynthoPlate 2.0 elicited the mildest reaction in pigs after administration and was therefore chosen to evaluate the effect of administration volume in our porcine safety model. Pigs were evaluated with an infusion speed of 60ml/min with either 50ml (bolus injection- <1
min) followed by 450ml saline infusion or 500ml (infusion- 8.3min) (total n=3; 50ml: n=2,
500ml: n=1). SynthoPlate 2.0. Pigs treated with bolus injections had peaks in heart rate and
CO2 at 20min that began to stabilize shortly after, while their mean arterial pressure was relatively stable (Figure 8-5A, C and B respectively). Pigs infused with SynthoPlate 2.0 had stable heart rate and CO2 but a peak in mean arterial pressure at 10 minutes, likely due
to an increase in intravascular volume (Figure 8-5A, C and B respectively). Both
treatments had a 100% 60-minute survival rate (Figure 8-5D). Pigs treated with bolus
309 injection had little to no skin changes (Figure 8-3D) while pigs infused with SynthoPlate
2.0 experienced generalized small erythematous patches (Figure 8-3C).
Figure 8-5 Vital analysis in safety pigs administered SynthoPlate 2.0 at 50ml (bolus) or 500ml (infusion) show that [A] Heart rate remains stable after infusion while there is a spike at 20min with bolus injection [B] Mean Arterial Pressure remains relatively stable with bolus injection but has a spike with infusion and [C] CO2 remains stable with infusion but has slight variations with bolus injection. [D] All pigs administered SynthoPlate 2.0 via bolus or infusion survived the experiment time (60 minutes).
8.3.4 Evaluation of SynthoPlate Administration Protocol Efficacy
These administration protocols where then evaluated in our porcine femoral artery injury
model (Figure 8-1). SynthoPlate 2.0 was injected by bolus or infusion, 1 minute after injury (total n=9; 50ml: n=6, 500ml: n=3). Pigs receiving a bolus injection had an increased
yet stable heart rate; decreased, yet stable mean arterial pressure; and stable CO2 (Figure
8-6A, B and C respectively). Pigs treated with SynthoPlate 2.0 infusion had a decreased
310
Figure 8-6 Vital analysis in injury pigs administered SynthoPlate 2.0 at 50ml (bolus) or 500ml (infusion) show that [A] Heart rate increases with bolus injection and decreases with infusion [B] Mean Arterial Pressure decreases slightly and stabilized with bolus injection but, decreases significantly and rebounds (in animals that survive) and [C] CO2 remains stable with bolus injection but drops significantly and rebounds with an infusion. [D] Analysis of C3a plasma concentration in blood drawn showed no significant alterations.
yet stable heart rate; a large decrease, then increase, then stabilization of mean arterial pressure; and a large decrease in CO2, before stabilizing around 50 minutes after injury
(Figure 8-6A, B and C respectively). There is a slight increase in blood loss rate and total blood loss in bolus injected animals compared to infusion animals (Figure 8-7A and B), but this is likely a result of bolus injected animals maintaining a higher heart rate and blood
311 pressure during the experiment. Bolus treated animals have an 83%
Figure 8-7 Hemostatic efficacy analysis in injured pigs shows that administration of SynthoPlate 2.0 [A] with bolus injection leads to a slightly higher blood loss rate compared to infusion injection [B] and pigs administered with bolus injection have increased total blood loss rate compared to pigs that received infusion injection, however [C] bolus injection in pigs lead to an increase in survival compared to animals that received infusion injection (83% survival vs. 33% survival).
survival rate compared to a 33% survival rate in animals treated with an infusion of
SynthoPlate 2.0 (Figure 8-7C). Like the safety studies, bolus injected animals had little to
no skin response (Figure 8-3D) while animals receiving an infusion had generalized small
erythematous patches (Figure 8-3C). Neither administration method had a more than 4-
fold increase in complement C3a plasma concentration levels (Figure 8-6D).
8.4 Discussion
Applications of intravenously administered nanoparticles continue to grow for a variety of
therapies. Nanoparticles are small enough that they can be injected into the blood stream
and can be designed to target, treat, and monitor with specificity. The surge of
nanomedicine is being aided by the scientific community’s expanding understanding of the
pathophysiology underlying the diseases we hope to treat and our expertise to engineering
nanoparticles23. With the widespread popularity of nanomedicine comes an increased risk
associated with their use. Adverse drug reactions will occur in 30% of hospitalized patients
312 and 77% of these will be complement mediated. Nanoparticles’ similarities to the pathogens the complement system is primed to combat make them likely to illicit an over activation of the complement system7.
In this study, we examine the effect different components of the drug delivery system can have on a hypersensitivity reaction. We have demonstrated that by just altering the composition (mol percent) of PEGylation of our nanoparticles, we can create a minimal versus a fatal hypersensitivity reaction. Increased PEGylation lead to stabilization of the heart rate and CO2, a mitigated skin response and decreased activation of the complement system. Though there were only slight differences in the hypersensitivity reaction observed when we varied the volume administered, we did see significant differences in efficacy, most importantly in survival. A 50ml injection of our hemostatic nanoparticles was able to increase survival by 50% compared to a 500ml injection. These results demonstrate that mitigating hypersensitivity reactions seen by promising nanomedicines is possible.
A four-fold increase in complement C3a plasma levels, which would be indicative of detrimental complement system activation, was not observed in our study, though the
SynthoPlate 1.0 PEG variant did show an increase in complement C3a levels. Complement
C3a plasma concentration was ideal to monitor for this study because complement C3 is cleaved into C3a in all complement pathways allowing us to study the involvement of the complement system in general. Subsequent studies should be performed to determine which complement pathway is being activated. The classical, alternative and lectin pathways can be studied individually by monitoring plasma levels of complement C1
313 complex, complement Ba (cleaved from C3bB), and the activity of MASP-1 or MASP-2 respectively24,25. It likely that the increases in C3a observed in this study are due to the activation of the alternative pathway, due to the how fast (less than one minute) the reaction to the treatments are observed. Additionally, to determine if the adaptive immune response is playing a role in the responses seen, IgE plasma concentrations should be measured.
Future studies should be targeted towards different characteristics of the nanoparticle design in hopes of understanding not only the pathophysiology of the drug hypersensitivity but also the cellular mechanisms. Our in vivo studies confirm how important each, seemingly minor, aspect of a nanoparticle design can have on hypersensitivity reactions, the success of a technology, and ultimately translating life altering treatments to our patients.
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318 Chapter 9: Conclusion and Future Directions
9.1 Conclusion
Trauma is a ubiquitous condition that could happen to anyone at any time in any place.
Knowing that it is one of the leading causes of deaths worldwide and that we have the
technology to stabilize patients if we could manage their bleeding until they reach medical
services, makes this an urgent and appealing problem to work towards solving1–3. Current hemostatic management of bleeding complications in trauma have limitations such as no pre-hospital availability, short shelf life, and high risk of contamination that lead to the high rate of preventable traumatic hemorrhage related deaths seen today2,3. These risks are
increased in patients with thrombocytopenia. The overall hypothesis for this work is that
the design of nanoscale delivery systems with platelet-inspired heteromultivalent surface- modifications, can enable injury site-specific biointeractions and drug delivery to enhance platelet-mediated hemostatic processes, leading to decreased blood loss and increased
survival.
Chapters 2 and 3, are a detailed review of current materials and technologies for the
management of traumatic bleeding. Though there are a number of approved and ground-
breaking technologies for managing bleeding, treatment of internal non-compressible
hemorrhage still heavily depends on transfusion of whole blood or blood’s hemostatic
components (platelets, fibrinogen and coagulation factors). The recent clinical trial
PROPPR (Pragmatic, Randomized Optimal Platelet and Plasma Ratios) demonstrated the
importance of platelets, in particular, for the management of traumatic bleeding. The study
319 determined that early platelet administration is associated with improved hemostasis and
reduced mortality in traumatic bleeding patients compared to patients who received blood
products without platelets4.
In Chapter 4, our ability to engineer a synthetic nanoparticle based functional mimic of platelets that can leverage and amplify the body’s physiological clotting mechanisms specifically at the bleeding site was reported. This nanoparticle, SynthoPlateTM, was
evaluated in vitro and found to be a ~150nm unilaminar vesicle that does not activate or
aggregate circulating quiescent platelets, but that co-localizes specifically with activated
platelets in clots enhancing their aggregations and in effect, enhancing fibrin generation.
Also described, was that in a thrombocytopenic mice model, the hemostatic efficacy of
these particles was found to be dose-dependent, with doses comparable to normal murine
platelet count (800-1000 particles per nL) improving bleeding time in thrombocytopenic
mice to levels of normal mice.
Building on these results, Chapter 5 outlines the evaluation of SynthoPlate in the
pretreatment and an emergency application of a mouse liver injury. Blood loss was
significantly reduced and 72hr survival was significantly increased for mice treated with
SynthoPlate in both models. After these promising results, we moved to a large animal
injury model, the pig femoral artery injury, to evaluate the efficacy and safety of
SynthoPlate in Chapter 6. Pigs were chosen because of the similarities between their
cardiovascular system and the human cardiovascular system5. In these studies, SynthoPlate
significantly reduces blood loss rate, stabilizes blood pressure and increases survival during
320 the ‘golden hour’ after traumatic arterial injury compared to control groups. It was also found to be systemically safe at the administered dose.
The development of a platelet-inspired nanovehicle for injury-site targeted delivery of
Tranexamic Acid, TXA, was reported in Chapter 7. We were able to successful encapsulate
TXA in our platelet-targeting nanovehicle and demonstrate in vivo efficacy of drug release.
In vivo, the TXA-loaded targeting nanovesicles were able to safely reduce blood loss and increase 1hr and 72hr survival in a rat liver injury model compared to free TXA and controls.
Chapter 8 investigates ways to refine nanomedicine design to improve systemic safety, by altering surface charge and hydrophilicity, as well as modulating the administration protocol. Here we demonstrate that by altering the composition (mol percent) of
PEGylation of our nanoparticles, we can create a minimal versus a fatal hypersensitivity reaction.
Overall, these studies prove the hypothesis that the design of nanoscale delivery systems with platelet-inspired heteromultivalent surface-modifications, can enable injury site- specific biointeractions and drug delivery to enhance platelet-mediated hemostatic processes, leading to decreased blood loss and increased survival.
321 9.2 Future Directions
9.2.1 Nanovesicle Design
In our attempt to manage traumatic bleeding, we studied the bleeding mechanisms and decided to mimic adhesion and aggregation properties of platelets. Now that we have
successfully mimicked these properties, we could possibly increase the efficacy of our
technology by mimicking even more platelet functions. When platelets become activated
they release a number of molecules from their granules which help with propagation of the
clotting cascade and thrombus formation. In Chapter 7, we have demonstrated that we can
load and deliver payloads with our nanovesicles. Selecting a platelet relevant molecule, or
cocktail of molecules such as polyphosphate, ADP, Factor V, vWF, and fibrinogen to
deliver could help enhance the hemostatic environment. Activated platelets also provide a
highly negative surface for the propagation of the coagulation cascade. We could create a
negatively charged surface on our nanovehicles that is masked, to prevent spontaneous
thrombus formation, until the masking molecules are cleaved by a clot relevant enzyme.
This would allow for our nanovehicle to interact directly with and enhance the actions of
clotting factors.
Additionally, we might consider mimicking the shape and size of platelets. Our lab just
published a study reporting that in the presence of red blood cells, micro-scale non-
spherical particles undergo enhanced ‘margination and adhesion’ compared to nano-scale
spherical particles, resulting in their higher binding6. Though our particles do show efficacy
at reducing blood loss and increasing survival demonstrating their availability to marginate
and adhere in vivo, altering their shape and size may further increase their efficacy.
322 9.2.2 Nanoparticle Evaluation
Our nanovesicle design are evaluated in vitro before they are tested in vivo for practical, ethical and cost reasons. It is our goal to have a system that will model in vivo systems as close as possible. Our current in vitro systems do have their own limitations. For example, in the flow studies, we typically run our samples in platelet rich plasma over vWF and collagen coated surfaces with laminar flow. In the future, we could design a system that allows us to test our designs in more biologically relevant condition such as a design that incorporates endothelial cells, pulsatile flow and whole blood would begin to incorporate some of the variables currently lacking in our system.
Our current in vivo models work to recreate trauma scenarios, but here is currently no standard traumatic hemorrhage animal model. This may be in part because there is no single animal model that can answer all questions. We do our best to utilize the appropriate model for the question at hand. A recent study examining experimental models of coagulopathy found 62 relevant publications describing 27 distinct models of traumatic coagulopathy7. Though pig models were the most common species, other model species included rat, rabbit, sheep, mouse and hamster. Although the overall hemostatic processes may be conserved among many mammals, the response to nanoparticle administration may vary widely from species to species and is important to understand before beginning animal studies. Though we have used mice and rats to model traumatic hemorrhage, our pig model is likely the best model because the pig cardiovascular system is similar to the human cardiovascular system. Unfortunately, the pig model is costly, requires special surgery facilities, special housing, and complex handling which all lead to reduce sample sizes. In
323 our studies, we tested one dose one minute after injury. It is unlikely that patients will receive our treatment one minute after trauma. In future studies it will be important to correlate the treatment efficacy with administration time to better simulate real-world scenarios. Additionally, future studies should be conducted to determine the maximum tolerated dose and minimum effective dose.
9.2.3 Nanoparticle Technology Translation
One of the most challenging yet promising future directions for this research will be its development into an FDA approved drug. Patents have been issued on SynthoPlateTM design (composition) and methods of use, and a company (Haima Therapeutics, LLC) has been established to move SynthoPlateTM to the marketplace. The next steps will include adopting good manufacturing practice so that an industry consistent product is being utilized. The therapeutic window will need to be determined by performing maximum tolerated dose and minimum effective dose studies as well as performing toxicity studies to determine a safety profile. Stability studies in both suspension and lyophilized conditions at varying temperatures will also need to be conducted. Hopefully, the results of this future work will lead to an FDA approved drug that can be utilized for the hemostatic management of bleeding complications in thrombocytopenia and trauma.
9.3 References
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