Extracellular Matrix-Derived Nanoparticles for Imaging and Immunomodulation

EXTRACELLULAR MATRIX-DERIVED NANOPARTICLES FOR IMAGING AND IMMUNOMODULATION

BY: JOHN KRILL

A THESIS SUBMITTED TO JOHNS HOPKINS UNIVERSITY IN CONFORMITY WITH THE REQUIREMENTS FOR THE DEGREE OF MASTER IN SCIENCE AND ENGINEERING

BALTIMORE, MARYLAND

MAY, 2016

The extracellular matrix (ECM) is a complex component of tissue that includes collagens, glycoproteins, proteoglycans, and elastic fibers. These proteins serve both as a structural foundation upon which cells organize and communicate, and to inherently direct many physiological phenomena within cells, such as migration, proliferation, and differentiation. Due to these unique properties of ECM, laboratory generated matrix derived from the decellularization of native tissue, has become a preferred scaffolding material for use in tissue engineering and regenerative medicine. ECM particulate forms have been of increasing interest, as they provide several advantages over macro-scale patches. Particles enable minimally invasive delivery alternatives, including injections. In addition, by reducing ECM particle size to the nanoscale,

ECM can be directly taken up by cells, such as macrophages, through phagocytosis or other engulfment methods. This internalization of ECM may lead to enhanced potency of directing cell behavior or differentiation versus simple material contact. This is highly relevant in the development of immune therapies that aim to modulate the host immune response for more positive outcomes in cancer treatment, implant integration, and vaccines.

This thesis has two major parts. First, the methodology and characterization of successfully produced ECM nanoparticles derived from several porcine tissue sources are provided. This encompasses the tissue decellularization process and nanoparticle generation procedure. The second part focuses on modification of ECM nanoparticles with a variety of molecules, including fluorescent dyes, polyethylene glycol and functional peptides to alter their properties. Preliminary data regarding the ability of non-modified ECM nanoparticles to influence macrophage polarization in vitro is offered, with changes in cytokine expression suggesting immunomodulatory effects. Overall, ECM nanoparticles are a promising biomaterial for medical imaging, cancer research and immunology, and thus deserve further exploration.

Thesis Readers: Dr. Jennifer H. Elisseeff, Dr. Kevin Yarema, and Dr. Hai-Quan Mao

SPECIAL THANKS TO: Dr. Matthew Wolf Tony Wang Christopher Anderson

And the rest of the Elisseeff Lab for their assistance and guidance throughout this entire project, as well as members from the Green and Yarema Labs who facilitated many of these studies.

iii

TABLE OF CONTENTS

List of Tables ...... v List of Figures ...... vi 1. Introduction 1.1 Overview of ECM ...... 1 1.2 ECM as a Biomaterial in Tissue Repair ...... 4 1.3 ECM in Immunology ...... 8 1.4 ECM Particulates: Unlocking New Applications ...... 12 2. Decellularization Process 2.1 Tissue Decellularization Methodology ...... 14 2.2 ECM Characterization ...... 15 3. ECM Nanoparticle Preparation 3.1 Cryomilling to Generate Micron-Scale Powder ...... 17 3.2 Processing of ECM Powder Into Nanoparticles ...... 20 3.3 ECM Nanoparticle Characterization ...... 22 3.4 ECM Nanoparticle Cytotoxicity Studies ...... 25 4. ECM Nanoparticle Modification and Functionalization 4.1 Fluorescent Marker Conjugation ...... 29 4.2 PEGylation ...... 39 5. Effect of ECM Nanoparticles on Macrophage Polarization 5.1 Intrinsic ability of ECM Nanoparticles to Influence Macrophage Polarization ...... 44 5.2 Functionalization of ECM Nanoparticles for Immune Therapy: Future Works ...... 47 6. Conclusion ...... 50 References ...... 51 Curriculum Vitae ...... 55

iv LIST OF TABLES

Table 1: List of common ECM components, their functions and where they can be found within the body ...... 3

Table 2: Examples of commercially available scaffolds composed of ECM ...... 8

Table 3: Abbreviated list of associated factors for M1 and M2 macrophage phenotypes ...... 11

Table 4: Overview of PAA/Triton decellularization procedure ...... 14

Table 5: Overview of SDS Decell Procedure ...... 15

Table 6: Settings used for SPEX sampleprep 6870 freezer/mill ...... 18

Table 7: Emulsiflex operating pressures for ECM nanoparticle generation ...... 21

Table 8: List of NHS dyes successfully conjugated to ECM nanoparticles...... 38

v LIST OF FIGURES

Figure 1: H&E comparison of decellularized ECM batches with native tissue counterparts ...... 16

Figure 2: Masson’s trichrome comparison of decellularized ECM batches with native tissue counterparts ...... 17

Figure 3: Size comparisons of multiple cryomilled ECM batches ...... 18

Figure 4: SEM images highlighting morphology differences between ECM powders ...... 20

Figure 5: Representative size profile for ECM nanoparticle batches ...... 23

Figure 6: Cumulative Z-averages and PDIs of ECM nanoparticle batches ...... 23

Figure 7: Effects of filtering on ECM nanoparticle batches ...... 24

Figure 8: Cell counts for polystyrene beads added to hASCs ...... 26

Figure 9: Cell counts for lower concentrations of polystyrene beads added to hASCs ...... 27

Figure 10: hASC viability images under addition of various nanoparticles ...... 28

Figure 11: Cell counts for hASC viability images under addition of various nanoparticles ...... 29

Figure 12: NHS-ester chemistry overview...... 30

Figure 13: Fluorescence microscope image of ECM particles ...... 31

Figure 14: Centrifuge method to wash synthetic particles ...... 32

Figure 15: Sizing data of ECM nanoparticle batch before and after centrifugation ...... 33

Figure 16: Overview of centrifuge filter unit method to wash nanoparticles...... 33

Figure 17: Size profile changes of FITC-ECM nanoparticles after centrifuge washing ...... 34

Figure 18: Flow-through and fluorescence data of free dye and FITC-ECM nanoparticle batches ...... 34

Figure 19: Comparison of FITC-NHS, fluorescein and glycine quenched ECM nanoparticle batches ...... 35

vi Figure 20: Addition of FITC-tagged ECM nanoparticles to bone marrow-derived macrophages 37

Figure 21: Signal-to-noise ratios of several dyes conjugated to ECM nanoparticles ...... 38

Figure 22: Distribution of Licor-tagged ECM nanoparticles introduced by tail vein injection ..... 39

Figure 23: Size profiles of ECM nanoparticle batches conjugated with NHS-PEG ...... 40

Figure 24: Zeta potentials of ECM nanoparticle batches conjugated with NHS-PEG ...... 41

Figure 25: Illustration of QCM-D principles of operation ...... 42

Figure 26: Real-time adsorption layer thickness of ECM nanoparticles onto QCM-D disks ...... 43

Figure 27: Timeline of in vitro macrophage polarization study ...... 44

Figure 28: Gene expression changes for several M1 and M2 associated factors produced by bone marrow-derived macrophage after exposure to cardiac ECM nanoparticles ...... 46

Figure 29: Simplified T cell activation pathway demonstrating antigen presentation to trigger stimulation of immature T cells into cytotoxic CD8+ T cells...... 48

vii 1. INTRODUCTION

1.1 OVERVIEW OF ECM

The tissue microenvironment is composed of two major components: cells and a surrounding extracellular matrix (ECM). This ECM consists of a complex combination of proteins and polysaccharides secreted by resident cells that influence and direct many physiological phenomenon. These molecules include collagens, elastins and proteoglycans, the ratios of which differ depending on the tissue source. By considering the components of ECM and analyzing their functions within the body, we can appreciate the myriad of applications that

ECM-derived materials have in tissue engineering, wound healing and immunology.

The ECM was first credited for providing a structural foundation on which cells can organize and communicate. Two of the major structural contributors to ECM are collagens and elastic fibers.[1] Collagens are comprised of three polypeptide -chains that wrap around each other to form highly stiff triple-helices, called tropocollagens. Based on the -chain peptide composition, tropocollagen molecules assemble together to form a wide variety of larger architectures, including fibrillar structures, beaded fibrillar strings and hexagonal networks. This supramolecular structure dictates the overall properties of the final collagen product.[2][3] For example, fibrillar type I collagen found in tendons grants outstanding tensile strength and slight elastic properties that enables contraction and stretching without tearing.[4] Over 28 classes of collagen have been identified and can be grouped into fibrillar, nonfibrillar, association, transmembrane and multiplexin classifications.[5][6] While each type of collagen has a unique purpose and location within the body, they generally serve to provide tensile strength to tissues and form a framework on which cells can adhere to and organize on. Complementary to collagens, elastic fibers are composed of a highly cross-linked network of flexible elastin protein bundles. Found in the lungs, blood vessels, dermis and many other tissues, elastin networks provide elastic properties to tissues, enabling their recoil to original dimensions upon the removal

1 of a deforming force. Elastic fibers are heavily associated with the microfibrillar proteins fibrillin and fibulin, both of which mediate elastic fiber formation and also confer inherent mechanical stability in non-elastic tissues.[7][8]

Collagens, elastic networks and various other proteins are highly incorporated in a viscous milieu comprising mainly of proteoglycans.[9] These structures consist of a core protein with many covalently attached glycosaminoglycan (GAG) chains branching off. These polysacharride chains tend to be extremely hydrophilic, causing them to spread out and occupy large volumes in aqueous environments. Because of the sulfate and carboxyl groups located on the sugars of GAGs, these chains are typically highly negative. This attracts osmotically active cations such as Na+, which absorb water and leads to the formation of gels that provide compressive resistance to surrounding tissues.[9]

There are many other notable molecules that contribute to the ECM’s composition, including laminins and fibronectin, which are listed in Table 1. It is important to note that all of these components play a far more complex and dynamic role in the cellular environment than simply providing a structural foundation. Cells secrete many signaling molecules into the extracellular space that interact with the matrix, including growth factors, cytokines and proteases. Collagen networks help sequester these molecules until they are needed.[6]

Proteoglycans often interact with these secreted factors, mediating a variety of cell behaviors. For example, heparan sulfate chains in GAGs bind to and regulate fibroblast growth factors (FGFs), which have implications in angiogenesis, development, proliferation and wound healing.[9][11]

Certain members of the transforming growth factor- (TGF-) family have demonstrated binding to decorin, a proteoglycan core protein found in bone matrix, to enhance its bioactivity.[12] TGF- is secreted by white blood cell lineages and is highly important in immunology. Proteoglycans also bind to proteases and protease inhibitors that control the production and degradation of the

ECM material, including matrix metalloproteases and serine proteases. These proteases help detach cells from the matrix and enable cell migration by creating pathways through which they

2 can travel.[9] Hyaluronan, a GAG not bound to a core protein, serves as an important matrix component during embryonic development and in adult tissues, forms complexes with proteoglycans, and also generates empty spaces in which cells can migrate during wound healing.[9] The elastic fiber components fibrillin and fibulin also mediate all sorts of cellular activity, including normal lung and kidney development, matrix deposition, and activation of

TGF- and bone morphogenetic protein-7 in mice.[8]

Table 1: List of common ECM components, their functions and where they can be found within the body.[5]

Function Example Locations Fibrillar Collagens Structural, cell adhesion and Bone, dermis, heart (I); cartilage (II); I, II, III, V, XI proliferation, mediates proteoglycan granulation tissue (III); basement membrane interactions (V); articular cartilage, ear (XI) Nonfibrillar collagens Structural, angiogenesis, Kidney (IV); sclera and vasculature (VIII); IV, VIII, X compartmentalizes ECM hypertrophic cartilage (X) components Association collagens Structural, interaction with other Liver (VI); basement membrane (VII); VI, VII, IX, XII, XIV, XIX ECM components, cartilage (IX), skin (XII); blood vessels (XIV); muscle cells (XIX) Transmembrane collagens Structural, interacts with ECM Skin (XIII); cutaneous basement membrane XIII, XVII components, cell-matrix adhesion (XVII) Multiplexins Organizes ECM, inhibits Basement membrane XV, XVIII angiogenesis and tumor growth Elastin Structural, provides elasticity Lungs, blood vessels, dermis Fibrillins Structural, tissue homeostasis Microfibrils 1, 2 Fibulins Structural, interacts with ECM Basement membrane, blood vessels 1, 2, 3, 4, 5, 6, 7 components, modulates platelet adhesion, angiogenesis, formation of elastic fibers Hyaluronan Angiogenesis, cell motility, wound Most tissues; notably skin, cartilage, vitreous healing, cell adhesion humour, joints Hyalectans Structural, interacts with ECM Articular cartilage (aggrecan), brain components, cell adhesion and (brevican, neurocan), blood vessels (versican) migration Fibronectin Cellular adhesion, ECM assembly, Most tissues tissue injury and inflammation, angiogenesis Laminins Cell adhesion, migration and Basement membrane differentiation

3 These are just several examples in which extracellular matrix components regulate cellular activity, though there are many others. However, these brief examples illustrate the effectiveness of ECM components to influence cell organization, proliferation, differentiation and migration. The effect goes both ways. While the ECM provides physical and chemical cues that direct cell behavior, the cells also produce cytokines that remodel the ECM. This dynamic reciprocity is important in homeostasis and tissue repair, as will be discussed in following sections.[29-31] With the ECM playing such a critical role in cell behavior, it makes sense to consider this material in biomedical applications.

1.2 ECM AS A BIOMATERIAL IN TISSUE REPAIR

With the extracellular matrix directing so many physiological phenomena, there has been an increasing interest in isolating ECM material for use as a biomaterial in tissue repair. Upon a destructive stimulus, the wound repair process is triggered. Regardless of the type or location of injury, this process is highly directed by chemical signals, including numerous cytokines and growth factors, and generally involves three major overlapping steps: inflammation, cell proliferation and remodeling.[13][14] Immediately after injury, circulating platelets adhere to exposed collagen in the tissue. These platelets produce clotting factors that trigger the formation of a provisional fibrin matrix, as well as platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-) that signal the chemotaxis of neutrophils, macrophages, smooth muscle cells and fibroblasts to the damaged region. TGF- also plays a role in stimulating macrophages to produce pro-inflammatory cytokines, including tumor-necrosis factor-alpha

(TNF-) and interleukin-1 (IL-1). Recruited neutrophils, as well as macrophages differentiated from monocytes, actively remove dead tissue and foreign material through phagocytosis. Upon transition to the proliferation phase, the fibrin matrix is replaced with granulation tissue, and angiogenesis facilitated by vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (bFGF) occurs. Macrophages can release soluble factors that trigger the differentiation of

4 some fibroblasts into myofibroblasts, which work towards closing the wound and depositing new

ECM. In the last stage, the newly deposited matrix is remodeled by matrix metalloproteinases, which typically involves the conversion of type-III collagen to type-I, and the tissue achieves homeostasis.

This describes the typical wound-healing cascade in many tissues. However, injuries involving high volumetric damage or non-regenerative tissues (i.e. cardiac) may result in alternative responses, including excessive or deficient healing.[14] Excessive healing, commonly known as fibrosis, leads to the deposition of too much matrix material that interferes with proper tissue re-growth, resulting in an overall loss in tissue functionality. The build-up of this non- functional tissue is thought to lead to many diseases, including congestive heart failure, cirrhosis of the liver, transmission blockage following nerve injury and hypertrophic scarring. Deficient healing can occur when infiltrating neutrophils degrade deposited matrix, for example through collagenase or elastase production, faster than it can accumulate. This is the main cause of chronic ulcers that affect debilitated and elderly patients.[14]

Treatment for both overhealing and underhealing in patients focuses on the administration of key growth factors or healthy cells to the defective area in attempts to regenerate functional tissue. However, single-agent therapies have seen limited success.[13]

Delivery of individual growth factors shows little impact due to the redundant nature of the wound healing response and short half-life of the agent at the wound site. Cells delivered by themselves are difficult to isolate and deliver while maintaining viability, and they lack the physical and chemical cues that help them organize and proliferate. Thus, there has been a recent trend in integrating both growth factors and cells into carefully engineered, three-dimensional scaffolds that help promote proper incorporated cell organization and mimic specific biological signals. Synthetic scaffolds derived from polymers have been used in this manner. For example, surgical implantation of porous poly(lactide-co-glycolide) scaffolds, loaded with autologous mesenchymal stem cells, demonstrated cartilage regeneration in damaged sheep joints.[15] Aligned

5 electrospun poly-caprolactone fiber meshes displayed improved cell proliferation, migration and orientation in neural regeneration.[16] These polymer scaffolds, extending but not limited to polystyrene, poly-l-lactic acid, polyglycolic acid and poly (acrylonitrile-co-methacrylate), have also shown some success in repairing bone defects, nerves, liver, skin and blood vessels.[15-17]

However, while these synthetic scaffolds are highly customizable, easily reproduced, and have shown positive tissue regeneration effects, their structures are often too simplistic to properly mimic the in vivo cell environment. The extracellular matrix is a highly complex three- dimensional combination of proteins and secreted molecules, each with a unique structure specifically suited to their function. A simple porous or mesh scaffold cannot replicate the intricate features of numerous matrix proteins, such as the triple helix of collagen or the carefully cross-linked network of elastin. As a result, synthetic scaffolds lack the true bioactivity of native matrix that promotes proper cell differentiation and behavior. Synthetic scaffolds have also been shown to trigger the host immune response, typically resulting in fibrous encapsulation of the scaffold.[18][19]

ECM-derived scaffolds show much more promise in tissue repair, as they address many of the problems found in their synthetic counterparts. As discussed previously, the in vivo cellular environment consists of a highly dynamic and complex extracellular matrix. The structure and composition of this matrix is optimized to promote cell adhesion, organization, proliferation, differentiation and migration through a combination of physical and chemical cues. Thus, by deriving the scaffold material from tissue ECM itself, we can inherit the native structural and functional molecules, as well as the exact three-dimensional environment, that provide bioactivity.[13] ECM-derived scaffolds have been successfully produced and demonstrate improved tissue regeneration in a wide variety of applications. A decellularized equine cartilage matrix scaffold was introduced in a horse knee containing a critically sized osteochondral defect.

After 8 weeks, significant bone and cartilage regeneration was observed.[20] Implantation of porcine urinary bladder matrix (UBM) facilitated constructive healing of the esophageal wall in a

6 dog model, evidenced by growth of functional and innervated neotissue.[21] Porcine UBM was also capable of inducing reconstruction of the temporomandibular disk in vivo.[22] Tube-shaped

ECM scaffolds from porcine small intestine were implanted into patients suffering from high- grade dysplasia of the esophagus, showing rapid remodeling in the form of new epithelium and submucosal layer, and porcine small intestine submucosa (SIS) scaffolds implanted in a canine volumetric muscle loss model showed formation of vascularized, functionally innervated skeletal muscle.[21][23][24]

Several FDA approved, commercially available ECM products are highlighted in Table

2.[21][25] The production of these scaffolds requires careful washing of whole organs or tissue sections with a combination of detergents, acids, enzymatic solutions and other solvents. These solutions work towards removing unwanted cellular material and solulizable proteins while maintaining the physical structure and key functional components of the organ or tissue. Ideally, decellularized ECM scaffolds retain all of the structural and functional proteins and polysaccharides of native tissue, including collagens, GAGs, fibronectin and laminin ligands that promote cell adhesion and growth in a natural, organized three-dimensional space. These ECM components are largely conserved across and within species and are relatively non-immunogenic.

By removing the cellular content susceptible to immune recognition, decellularized ECM scaffolds can circumvent immunological complications witnessed by synthetic scaffolds.

Tissues within the body vary greatly in morphology, cellular density, protein ratios and composition. The source of the ECM also dictates physical properties such as rigidity, porosity and topography. As a result, the optimal decellularization process may differ between each tissue type. Most processes utilize a combination of the chemical decellularization agents mentioned above, and physical disruption of the tissues to achieve acceptable decellularization. However, it is important to consider the impact of these treatments on the mechanical and physiological properties of tissues. The physical architecture of proteoglycans, collagen fibers and other proteins within tissues contributes greatly to its mechanical properties, and they also provide the

7 matrix with functionality. Destruction of any component through too harsh a decellularization process can lead to diminishing performance of the decellularized material. Badylak et al. provides a comprehensive summary of potential effects of many decellularization agents and processes.[26] Striking a balance between optimal cell removal and preservation of critical ECM components is necessary for producing functional ECM materials.

Table 2: Examples of commercially available scaffolds composed of ECM [21][25]

Product Material Form Use Company AlloDerm Human skin Dry sheet Abdominal wall, breast, head and LifeCell neck reconstruction, grafting Collamend Porcine dermis Dry sheet Soft tissue repair Bard CuffPatch Porcine SIS Hydrated sheet Soft tissue reinforcement Arthrotek Matristem Porcine UBM Dry sheet; Soft tissue repair ACell powder Oasis Porcine SIS Dry sheet Partial and full-thickness burns Healthpoint Permacol Porcine skin Hydrated sheet Soft connective tissue repair Tissue Science Laboratories Veritas Bovine Hydrated sheet Soft tissue repair Synovis Surgical pericardium Xenform Fetal bovine skin Dry sheet Colon, rectal, urethral repair TEI Biosciences Zimmer Porcine dermis Dry sheet Orthopedic applications Tissue Science Collagen Patch Laboratories

This section served to highlight one of the prominent uses of extracellular matrix-derived biomaterials. Widely considered for tissue regeneration applications, ECM scaffolds show promise in their ability to organize cells and produce functional tissue patches for wound repair.

However, the extracellular matrix contributes to many more physiological phenomena within the body, with host immunological response of particular interest.

1.3 ECM IN IMMUNOLOGY

Hinted at earlier, immune cells play a critical role in every step of the wound healing process, with major players including neutrophils, eosinophils, basophils, dendritic cells, monocytes and macrophages, T cells and B cells. The recruitment of these cells and their

8 behavior at the injury site are heavily influenced by local and systemic cascades of complex immune events.[27] These events are often triggered and controlled by secreted cytokines or other soluble mediators within the local tissue microenvironment, among which include numerous extracellular matrix components. For example, mindin is a secreted ECM protein that serves as an opsonin for macrophage phagocytosis of bacteria, and biglycan found commonly in bone, cartilage and tendon matrix can stimulate production of pro-inflammatory cytokines in macrophages.[28][29]

ECM remodeling plays a particularly important role in regulating the immune response.

The breakdown of ECM components, either through interaction with adhered pathogens or by specific proteases such as matrix metalloproteinases (MMPs), exposes cryptic binding sites that facilitate the activation of the innate immune response.[29] The detection of these sites by pattern recognition receptors on immune cells leads to the initiation of an inflammatory response that enables infiltration of immune cells to the injured region. Among the major contributors are low molecular weight hyaluronan (LMWHA) fragments, which induce the release of pro- inflammatory cytokines TNF- and IL-1 by macrophages.[29] Fibronectin fragments expressing an extra domain A have demonstrated activation of receptors that induce responses similar to those triggered by bacteria.[30] In addition to initiating the inflammatory response, ECM fragments also act as chemoattractants that further promote cell infiltration.[31] For example, specific cleavage of collagen I by MMPs generates fragments that contribute to neutrophil recruitment in the early stages of inflammation. Elastin fragments demonstrate similar chemotactic properties for monocytes. The release of cytokines by infiltrating cells also helps to tailor the production of

MMPs and subsequent generation of ECM fragments, re-illustrating the dynamic reciprocity between matrix components and the surrounding cells. The ECM even provides physical functionality, acting as a barrier to filter certain molecules and as a scaffold to mediate cell infiltration.[31]

9 On top of these functions, ECM proteins have been shown to directly modulate the differentiation and polarization of certain immune cell types. Macrophages are particularly involved in both the inflammation and regenerative steps of the healing process. Found in nearly all tissues, macrophages play a huge role in an organism’s development, homeostasis, and wound healing response.[32][33] Macrophages change phenotype depending on signaling provided by the environment. This polarization has been described as a complex continuum, with pro- inflammatory M1 and pro-regenerative M2 classifications representing two extremes.[34] Upon initial signaling, inflammatory monocytes differentiate into M1 macrophages as they travel to the affected tissue. These macrophages express inducible nitric oxide synthase (iNOS) and secrete inflammatory mediators such as TNF- and IL-1 that help to direct cellular activities including differentiation, proliferation and apoptosis.[35] M1 macrophages also work to remove cellular debris and foreign substances through phagocytosis. After this initial phase, macrophages switch to a pro-regenerative M2 phenotype that promotes collagen deposition and tissue repair.

Associated with this phase include IL-4 and IL-10 (Table 3).[34] The polarization of macrophages often dictates the wound healing response, with M2 phenotypes correlating to better tissue repair.[36] ECM proteins, such as LMWHA mentioned previously, have been shown to heavily influence this polarization, and thus by manipulating M1/M2 polarization through ECM biomaterials, it may be possible to control the wound-healing environment and generate better regenerative outcomes.[37]

Macrophage polarization also contributes greatly to the success of biomedical implants.

Biomaterials elicit a foreign body response upon implantation.[18][19] Non-specific blood plasma proteins adsorb to the implant surface and serve to recruit inflammatory cells to the site. Upon arrival, these cells secrete cytokines, chemotactic factors and reactive oxygen species in attempt to degrade the implant. Macrophages are the driving force of this response. They generate a fibrous capsule that surrounds the implant and can fuse together to form foreign body giant cells that attempt to engulf the foreign material. Macrophage polarization often determines whether the

10 scaffold is rejected or remodeled. M1 inflammatory macrophage populations indicate poor scaffold integration, while M2 suppresses inflammation and helps to repair damaged tissue.

Therefore, manipulation of macrophage polarization can also lead to control over the outcome of an implant.[37]

Table 3: Abbreviated list of associated factors for M1 and M2 macrophage phenotypes[37]

Induced by Marker Expression Associated cytokines Functions

M1 IFN-, LPS CD86, CD80, CD68, TNF-; Pro-inflammatory; destruction MHC II, IL-1R, TLR2, of pathogens via phagocytosis TLR4, iNOS IL - 1, 6, 12, 15, 18, 23 and release of oxidative species IL-4, IL-13 CD163, MHC II, IL - 10, 1ra; Anti-inflammatory Fizz1*, Arg-1* TGF-

M2 LPS, ICs, IL- CD86, MHC II IL - 1, 6, 10; Immunoregulatory; interaction 1 with B cells TNF- IL-10, TGF-, CD163, TLR1, TLR8 IL-10; Matrix deposition, tissue GCs remodeling and repair TGF-

IFN- – interferon-gamma; LPS – liopolysaccharide; IL – interleukin; IC – immune complex, TGF – transforming growth factor; GC – gluco-corticoids; CD – cluster of differentiation; MHC – major histocompatibility complex; TLR – toll-like receptor; iNOS – inductible nitric oxide synthase; TNF – tumor necrosis factor. * Mouse only

Several groups have successfully demonstrated the ability of ECM-based materials to modulate macrophage polarization. Urinary bladder matrix (UBM) coatings of polypropylene meshes demonstrated an increased M2/M1 ratio compared to uncoated meshes.[38] Another study showed that decellularized small intestine submucosa (SIS) contains TGF- and VEGF, two growth factors highly important in tissue regeneration and the M2 macrophage phenotype.[24] It is important to mention the issues associated with pure M2 polarization. If left unmediated, excess collagen deposition due to M2 macrophages can greatly hinder the development of new tissue.

This can lead to scarring and an overall loss in tissue function, and thus it is imperative to strike a balance between M1 and M2 responses when modulating macrophage polarization.

11 1.4 ECM PARTICULATES: UNLOCKING NEW APPLICATIONS

The previous examples highlighted several uses of ECM scaffolds as macroscale materials. Already extensively used in tissue engineering and regenerative medicine applications, decellularized ECM sheets display desirable properties, including features that promote cell adhesion, organize and enable proliferation of these cells in functional three-dimensional structures, and generate positive immunological effects. More recently, ECM particulate forms have been of extreme interest, because they hold several inherent advantages to their macroscale counterparts. Most notably, powder formulations allow for alternative delivery mechanisms of matrix material, such as hypodermic injection or aerosol inhalation.[25][39] This unlocks more minimally invasive methods of introducing ECM into the body while simultaneously maintaining highly targeted therapy. In addition, as particle size decreases, high surface area to volume ratio may begin to play a role in the potency of ECM materials. At smaller size scales, the surface area greatly outweighs the volume of the particle. This translates to having more of the functional proteins available on the surface of the material for interaction with the surrounding environment, which may lead to stronger effects. Compaction of ECM powders also opens the possibility of constructing highly complex three-dimensional scaffolds for use in tissue repair.

Several extracellular matrix powders have been produced, including ones derived from

UBM and adipose tissue.[39][40] These powders were generated via mechanical milling and yielded particles around 20-500um in diameter. These materials have been successfully injected through

22-gauge needles, a typical size for intramuscular injection, and they maintain the ultrastructure and three-dimensional surface characteristics of parent ECM sheets that promote tissue repair.[25]

However, while micron-scale powders have been successfully produced and well characterized,

ECM nanoparticles have yet to be considered.

Nanoparticles interact much differently within the body compared to macro-scale or even micro-scale materials. Research has shown that synthetic nanoparticle biomaterials, including polymer nanoparticles, iron oxide particles, liposomes and carbon nanotubes, exhibit

12 opsonization of blood proteins on their surfaces when introduced in vivo.[41][42] The composition of these adsorbed proteins, which include albumin, immunoglobulins and complement, determines how these particles are distributed and cleared throughout the body. For particles smaller than 0.5um in diameter (500nm), opsonization can trigger particle phagocytosis by macrophages through one of four receptor-mediated pathways: mannose, complement, Fc and scavenger.[43] Nanoparticles can be engineered to target any of these pathways through surface modifications, though the specific mechanisms are not well understood. However, particle phagocytosis via mannose receptor-, complement receptor- and Fc receptor-mediated pathways all resulted in inflammatory cytokine production in macrophages.[43] Thus, through internalization of ECM nanoparticles, it may be possible to trigger macrophage cytokine production and drive the polarization and related behaviors of immune cells to effects beyond that of macroscale interactions. Coupled with chemical conjugation to functional proteins, ECM nanoparticles may serve as an effective delivery vehicle for active agents to immune cells, helping to elicit specific responses. Functionalization of these nanoparticles with fluorescent dyes also enables medical imaging applications.

We believe that extracellular matrix-based particles can drive the same behavioral changes in cells compared to macroscale ECM scaffolds, while also providing an alternative form that enables different delivery methods and possibilities. This thesis provides an overview of successful methods used to produce ECM micro- and nanoparticles of consistent size, shape and functionality from various tissue sources. We cover post-modification of these particles using fluorescent markers and functional molecules and demonstrate the ability of these particles to influence macrophage polarization in several polarization conditions.

13 2. DECELLULARIZATION PROCESS

2.1 TISSUE DECELLULARIZATION METHODOLOGY

Whole organs (heart, liver, lung, etc.) were harvested from pigs (Wagner Meats,

Yorkshire breed, 5-6 months old, 250-260 lbs) and individually diced into fine pieces approximately 1-2 mm in size. Tissues were washed with deionized water and subsequently treated with 3% peracetic acid (PAA), 1% Triton X-100/20mM disodium ethylenediaminetetraacetic acid (EDTA), and 600 U/mL DNAse I in 10mM magnesium chloride and 10% antifungal-antimycotic solution, with water washes in between. The finished product was re-equilibrated in water and lyophilized for further processing (Table 4).

Table 4: Overview of PAA/Triton decellularization procedure

TIME TEMP Dice tissues - - Freeze until decell - - Deionized water Until blood is removed RT 3% PAA – 1 1 hr 37C 3% PAA – 2 3 hrs 37C Deionized water Until PAA is removed RT 1% Triton X-100 + 20mM EDTA – 1 24 hrs RT 1% Triton X-100 + 20mM EDTA – 2 24 hrs RT Deionized water Until Triton solution is removed RT 600 U/ml DNAse in 10mM MgCl + 10% anti/anti 24 hrs 37C Deionized water Until DNAse solution is removed RT

RT – room temperature

Depending on the end application of the ECM material, the user may require a gentler or more thorough decellularization method. As mentioned previously, different decellularization agents will remove different tissue components.[26] The PAA/Triton X-100 protocol discussed here is designed to maintain as much of the original ECM protein composition and structure as possible. This approach is relatively gentle and thus can be susceptible to cellular residue.

Alternative approaches aim to completely eliminate cellular components, though often at the expense of altering the ECM composition away from that of native matrix. A popular alternative

14 method involves using 1% sodium dodecyl sulfate (SDS) (Table 5). This detergent is much stronger than Triton X-100 and provides much more efficient disruption of cell membranes and removal of cellular debris, though at the expense of potentially damaging collagen, removing

GAGs and disrupting the ECM ultrastructure.

Table 5: Overview of SDS Decell Procedure

TIME TEMP Dice tissue - - Freeze until decell - - Deionized water Until blood is removed RT 1% SDS – 1 24 hrs RT 1% SDS – 2 24 hrs RT Deionized water Until SDS is removed RT Triton 24 hrs RT Deionized water Until Triton is removed RT

2.2 ECM CHARACTERIZATION

By the end of the decellularization process, the tissue sample should ideally contain no cellular content. However, decellularization efficiency rarely achieves 100%, and the sample may contain some residual cellular material. Though a standard has yet to be set in determining acceptable decellularization, routine histology of nuclear content through DAPI or H&E staining can provide qualitative verification of decreased cellular content.[26] Decellularized ECM samples from several tissue sources were characterized via H&E staining. As shown in Figure 1, decellularized samples contained fewer nuclei compared to native tissue samples, indicating the successful removal of cells. It is important to note the various degree to which decellularization is effective across tissue types. This is heavily dependent on the relative cellular content of the starting tissue, with more densely cellular tissues (i.e. liver) being more susceptible to residual nuclei after processing.

Figure 1: H&E comparison of decellularized ECM batches with native tissue counterparts. Images shows qualitative decrease of the presence of nuclei (purple). Histology can stain for other interesting components. For example, Masson’s trichrome stain utilizes three separate dyes that identify the presence of connective tissue (blue) and cytoplasm (red), in addition to nuclei (dark purple). The images in Figure 2 demonstrate the abundance of connective tissue remaining in cardiac, liver and bladder samples post- decellularization. These histology images aim to verify the ability of the selected decellularization protocol to remove cellular content while maintaining certain critical components found in native

ECM. Other stains exist that can reveal the presence of elastin (Verhoeff-Van Gieson), collagens

(PicroSirius Red), GAGs (Alcian Blue) and lipids (Oil Red O), enabling many avenues of characterization for these materials.

Figure 2: Masson’s trichrome comparison between decellularized ECM samples and their native tissue counterparts. Images show the presence of connective tissue (blue) alongside cytoplasm (red).

Upon confirmation of the successful removal of cellular content, the discussion shifts towards generating powders and particles out of this decellularized ECM material.

3. ECM NANOPARTICLE PREPARATION

3.1 CRYOMILLING TO GENERATE MICRON-SCALE POWDER

Creating nanoparticles out of decellularized ECM is a two-step process. The first step is cryomilling, also known as cryogenic grinding, which is a method that turns samples into powders. It involves the use of a solenoid magnetic coil to induce rapid oscillations of a rod- shaped magnet. The impact of the magnet against the sample container crushes the enclosed sample into fine particulates. The entire process is done within a bath of liquid nitrogen to prevent heat from altering the sample.

17 A SPEX sampleprep 6870 freezer/mill was used to process decellularized ECM batches into fine powders. Briefly, lyophilized ECM was manually broken up with a razorblade and inserted into polycarbonate tubes containing stainless steel impactors. Tubes were sealed with special plugs and inserted into the freezer/mill chamber. The body of the freezer/mill was filled with liquid nitrogen and run at the settings shown in Table 6.

Table 6: Settings used for SPEX sampleprep 6870 freezer/mill

CYCLES 10 PRECOOL 3 RUN TIME 1 COOL TIME 3 RATE 10

Scanning electron microscopy (SEM) images of various ECM powders produced by cryomilling show very high consistency among particle sizes, both within and between tissue types. Figure 3 compares three cardiac ECM powders to that of native heart tissue powder (left), as well as ECM powders from seven different tissue sources (right). There was no statistical difference in average ECM powder size between all batches excluding cartilage, which maintained a slightly larger size.

Figure 3: Size comparisons of multiple cryomilled cardiac ECM batches (left) and cryomilled ECM batches from a variety of tissue sources (right).

18 Though size remained relatively constant, several different particle morphologies were identified between ECM particles of different tissue origins (Figure 4). For example, cardiac, liver, skeletal muscle and lung-derived ECM particles had rounded shapes. Particles derived from small intestine submucosa (SIS) had flatter, sheet-like morphologies. Collagen ECM particles were highly elongated and fibrous. Different particle morphologies may lead to different interactions between these particles and the cells they come in contact with.[42] Flatter, elongated shapes increase the particle surface area and allow for more points of contact with cells. Rounder and more compact particles may be easier for cells to engulf, for example through phagocytosis.

Thus, particle morphology is expected to play a vital role in the behavior of cells and must be considered. In addition, features resembling intact collagen fibers were detected on bladder-based

ECM samples, indicating the preservation of such structures through both the decellularization and cryomilling processes (Figure 4, Image I). Coupled with histology results, this helps to confirm that decellularized ECM samples contain certain functional proteins found in native

ECM.

Figure 4: SEM images highlighting morphology differences between ECM powders: cardiac (A), collagen (B), liver (C), bladder (D), skeletal muscle (E), cartilage (F), lung (G), and SIS (H). Evidence of collagen in bladder samples (I) and zoomed in morphology of sheet-like SIS (J).

3.2 PROCESSING OF ECM POWDER INTO NANOPARTICLES

While cryomilling produces a fine powder, the individual particles measure on the scale of tens of microns (Figure 3). This size is still too large for many applications of interest, and thus further processing must be done to bring these particles to the nanoscale. There exist many techniques to generate metallic and polymeric nanoparticles. For example, gold nanoparticles are commonly produced by a citrate reaction at boiling temperatures, yielding distinct particles ranging from 1-50nm in diameter.[44] Unfortunately, methods conducted at such high temperatures would destroy the functional proteins of ECM samples and thus cannot be used.

Polymer nanoparticles are typically made using an emulsion approach called solvent evaporation.

20 This involves mechanical agitation of a dissolved polymer solution within an immiscible aqueous solution. The polymer forms individual spherical droplets in solution, corresponding to the lowest surface energy, and particles between 60-200nm in diameter are generated.[45] The main issue with this strategy is that an organic solvent is required to dissolve the polymer. These solvents can denature proteins by disrupting non-covalent interactions, such as hydrogen bonds, that help stabilize the protein’s structure.[46] However, the relatively soft properties of ECM allow forgoing the use of organic solvent. Instead, the ECM material can be directly subjected to mechanical agitation that aims to physically break apart the powder particulates into nanoparticles. Many high-pressure homogenizers are commercially available that provide this mechanical agitation.

These machines work by a principle similar to a French press, where a high-pressure region is generated against a low-pressure region separated by a pinhole. The sample, suspended in solution, passes through the pinhole, and the drastic pressure change generates extremely high shear forces that mechanically break apart the sample. This method is typically used to lyse cell walls for extraction of nucleic material, but it also proves highly effective at generating nanoparticles. An Avestin C5 model Emulsiflex was used to produce nanoparticles from ECM cryomilled powder. Upon equilibration in water or PBS, ECM powder samples were suspended in solution and run through the machine at increasing pressures (Table 7).

Table 7: Emulsiflex operating pressures for ECM nanoparticle generation

CYCLE NUMBER EMULSIFLEX PEAK PRESSURE (PSI) 1-2 10000-15000 3-4 15000-20000 5-7 25000-30000

This gradual increase in pressure aims to first disperse groups of particles clumped together by weak physical interactions. Afterwards, higher pressures can begin to break apart

21 individual particles into smaller nanometer sizes. The effectiveness of this homogenization procedure is illustrated in the following section.

3.3 ECM NANOPARTICLE CHARACTERIZATION

To quantify the size distribution of nanoparticles generated by the Avestin C5

Emulsiflex, a type of dynamic light scattering (DLS) was used. DLS is a technique where a laser shines through a dispersed colloidal solution. As light travels through the solution, suspended particles interfere with the light’s path by scattering it in different directions. Traditional DLS machines have detectors that collect this scatter to mathematically calculate particle size, though this method only works on relatively stationary particles.[47] As described by the Stokes-Einstein equation (Equation 1), the diffusion coefficient of low Reynolds number particles is inversely proportional to the particle radius.[48] This means that smaller particles, such as those on the nanometer scale, have high movement in solution due to Brownian motion.

Equation 1: Stokes-Einstein equation for diffusivity of spherical particles in fluids of low Reynolds number.

D – diffusion constant; kB – Boltzmann’s constant; T – absolute temperature;  - dynamic viscosity; r – particle radius

The Zetasizer, developed by Malvern Instruments, takes advantage of this principle to determine size distributions for nanoscale particles that would otherwise be too small for traditional DLS. It operates by measuring the fluctuations of reflected light intensity due to particle movement within a sample, where more rapid fluctuations link to smaller particles.

Figure 5 shows a sample Zetasizer size distribution of a representative cardiac ECM nanoparticle batch produced by the Emulsiflex.

Figure 5: Representative size profile for ECM nanoparticle batches. Note three prominent peaks near 100nm, 1um and 5um.

Cumulative data of seventy ECM nanoparticle batches across seven tissue sources show highly consistent Z-averages between tissue types, with values falling between 300-1000nm

(Figure 6, left). Of interest is the difference in Z-averages between tissues, indicating that certain tissue sources homogenize better than others. The protein composition of each tissue source will vary, and ECM materials high in tough proteins like collagens provide the greatest resistance to breaking apart. For example, urinary bladder matrix (UBM) has been shown to contain an intact basement membrane complex consisting of laminin, collagen IV and collagen VII.[49] These features may contribute to the larger overall Z-average seen in certain ECM nanoparticle batches.

Figure 6: Cumulative Z-averages (left) and PDIs (right) of ECM nanoparticle batches.

23 It is important to note the relatively high polydispersity index (PDI) of ECM nanoparticle batches, as indicated by three prominent peaks in the size distribution (Figure 5). The PDI serves as a measurement of how narrow or wide the size ranges of particles are within a sample, with a lower value indicating a more uniform particle population. Because of the irregular features of cryomilled ECM powder, each micron-scale particle breaks apart differently when homogenized.

This may be due to the starting size and shape of the particles or their ratio of tougher ECM components. As a result, a single processed batch may contain particles ranging between 10nm and 10um. There are methods by which this high PDI can be circumvented, which is critical for ensuring more predictable and controllable particle behavior in vivo. A quick look at the size distribution data identifies a prominent peak around 100nm. Recall that particles under 0.5um

(500nm) in diameter are susceptible to opsonization and subsequent phagocytosis by macrophages.[43] This internalization can lead to the production of cytokines that alter the cellular environment and may play critical roles in generating or modulating immune responses. Thus, particles in this size range are of great interest in immunology. By isolating this portion of the sample, nanoparticle batches of small size and high uniformity can be achieved. One simple approach is to selectively filter for particles of a certain size cutoff. A 0.22um polyethersulfone syringe filter was used to separate the smaller particles from the rest of the nanoparticle solution.

Figure 7 demonstrates the effectiveness of this method to remove large particles and decrease sample PDI.

Figure 7: Effects of filtering on ECM nanoparticle batches. Note drastic decrease in Z-average and PDI.

24 Along with size distribution, particle surface characterization must be considered. Zeta potential is one important measurement used to describe the overall stability of particles in dispersions. In solution, particles experience two major competing forces as Brownian motion brings them together: attractive van der Waals interactions and repulsive electrostatic forces.[50] If the attractive forces outweigh the repulsive ones, particles will flocculate or aggregate. This is critical when considering the injection of ECM nanoparticles in vivo and their circulation throughout the body. Particle aggregation can lead to the blockage of blood vessels or other harmful affects and is best to be avoided. Zeta potential describes the strength of the repulsive forces that keep particles apart. Thus, a higher magnitude, positive or negative, indicates a more stable particle solution. In addition, zeta potential has been correlated with the phagocytosis of nanoparticles by macrophages, with more negatively charged particles exhibiting higher uptake efficiency compared to neutral ones.[42][43]

The average zeta potential across nine ECM nanoparticles of four different tissue sources

(cardiac, bladder, small intestine, skeletal muscle) was -26.0  13.3, characteristic of moderate stability. A negative value is expected, likely due to an abundance of negatively charged functional groups commonly present in proteoglycans or residual DNA content. This result is encouraging, as ECM nanoparticles may be able to resist aggregation without the need for chemical modification and show promise in being phagocytosed by macrophages in vivo.

3.4 ECM NANOPARTICLE CYTOTOXICITY STUDIES

Determining the cytotoxicity of ECM nanoparticles is a top priority when evaluating the potential of this material to be used in biomedical applications. To generate a comparison group, two batches of commercially available polystyrene beads (0.2um average size) were obtained, one containing a stabilizing surfactant (SFPS) and one without (PS). Beads were diluted from a

4.1 mg/mL stock solution and washed various times with deionized water to remove residual storage buffer. Washed beads were subsequently added to human adipose stem cells (hASCs) and

25 incubated at 37C for three days. Cell counts were compared to live and dead control wells to determine overall viability. At these high concentrations of polystyrene beads, there was a clear inverse relationship between the amount of beads added and the final cell count (Figure 8).

Samples containing the highest concentrations of beads (0.41 mg/mL) showed very different morphologies compared to control wells. These cells were much larger and contained features representative of vacuoles throughout the cell body (Figure 8, Image H). While displaying poor vibility, this suggests internalization of the polystyrene beads, supporting the idea that nanoparticles can interact with cells and deliver conjugated molecules through cellular uptake.

Figure 8: Polystyrene beads (diluted from 4.1 mg/mL stock) added to hASCs. Imaged after three days: 1:10 PS (A), 1:50 PS (B), 1:200 PS (C), 1:10 SFPS (D), 1:50 SFPS (E), 1:200 SFPS (F), live control (G), and zoomed in image of 1:10 PS highlighting vacuoles (H).

The cytotoxicity study was repeated at lower PS concentrations to identify the threshold at which the beads affect cell viability. Concentrations of 10 ug/mL and 100 ug/mL were

26 investigated, with both surfactant and surfactant-free samples showing very little effect on the hASC count at these levels (Figure 9). Thus, ECM nanoparticles were not expected to be cytotoxic at concentrations around 100 ug/mL.

Figure 9: Cell counts for lower concentrations of polystyrene beads added to hASCs: live control (A), dead control (B), 10ug/mL PS (C), 100ug/mL PS (D), 10ug/mL SFPS (E), and 100ug/mL SFPS (F).

27 Following PS studies, cardiac ECM nanoparticles were added to hASCs for three days, and a live/dead assay was used to determine cell viability (Figure 10). In these images, calcein-

AM (2mg/mL) marks live cells as green, while ethidium bromide (1mg/mL) tags the nuclei of dead cells red. At first glance, neither non-filtered nor 0.22um-filtered cardiac ECM batches appear to affect cell viability, as illustrated by similar cell morphologies and densities compared to controls. However, nuclei counts of the red channel indicate that samples containing ECM had overall slightly higher cell death. This death appears to be higher at larger concentrations

(500ug/mL vs 100ug/mL), as well as in non-filtered samples that contain larger particles (Figure

11, left).

Figure 10: hASC viability images under addition of various nanoparticles: live control (A), dead control (B), 100ug/mL PS (C), 100ug/mL SFPS (D), 500ug/mL cardiac ECM (E), 500 ug/mL filtered cardiac ECM (F).

28 Because of this, the ECM nanoparticle concentration was further lowered to 10ug/mL and 1ug/mL, and new cell counts were taken. There was very little difference between hASCs cultured in 10ug/mL or 1ug/mL of ECM compared to a live control, suggesting that this is the threshold at which ECM nanoparticles do not affect cell viability. With this concentration identified, the applications for ECM nanoparticles can be investigated.

Figure 11: Cell counts for hASC viability in: 500ug/mL and 100ug/mL nanoparticles (left); 10ug/mL and 1ug/mL cardiac ECM nanoparticles (right).

4. ECM NANOPARTICLE MODIFICATION AND FUNCTIONALIZATION

4.1 FLUORESCENT MARKER CONJUGATION

One of the main motivations behind an ECM nanoparticle formulation is using this material in theranostics. Native ECM has been widely established to play an important role in cell signaling, behavior and differentiation. Shown to have certain immunomodulatory properties,

ECM as a standalone material could provide therapeutic effects in immunological diseases. A major area of interest is to combine ECM nanoparticles with a visual marker that helps track the location and movement of these particles within the body. This would enable greater understanding of the mechanisms behind which ECM nanoparticles are providing their effect.

29 As mentioned previously, ECM nanoparticles have a very high surface area to volume ratio, translating to a highly exposed surface available for interaction with cells and the environment. The protein content varies greatly depending on the tissue source and the exact portion of that tissue, but it contains a highly diverse set of reactive groups, including amines, thiols, phenols and carboxylic acids. Because of this diversity, there are many types of chemistries that can be employed to functionalize ECM nanoparticle surfaces. Of the reactive groups mentioned, amines are among the most abundant. Usually present in all proteins, amines such as lysine serve as strong nucleophiles above pH 8.0 and can react cleanly with many reagents. Because of their abundance and ease of reaction, amines are the most widely targeted group for conjugation.[51]

There exist many reactive agents for amines, each of which using a different method to achieve conjugation. Reactive esters, such as N-hydroxysuccinimide (NHS), are the most commonly used and directly target lysines and -amino groups.[52] Forming amide bonds under slightly basic conditions, these reagents are highly selective towards aliphatic amines and form very stable products (Figure 12). Virtually any molecule containing a carboxylic acid can be converted into its NHS ester, providing extreme utility amongst this family of reactive agents.

Isothiocyanates, aldehydes and sulfonyl halides provide alternatives to reactive esters and may be necessary to react aromatic amines. However, these reagents require harsher conditions that may not be suitable for more sensitive proteins.[51] Because of the ease and flexibility provided by

NHS-esters, this chemistry was primarily used to functionalize ECM nanoparticles.

NHS Ester Primary Amine Stable Conjugate NHS

Figure 12: NHS-ester chemistry overview

30 ECM particle batches were dispersed in a sodium bicarbonate solution to create a slightly basic environment (pH 8). Various NHS-dyes were dissolved in separate solvents, which were then added to the particle solutions. Reactions were allowed to take place under gentle shaking in the dark. Figure 13A shows the results of a typical fluorescein isothiocyanate-NHS (FITC-NHS) conjugation of ECM particles using this procedure. This tagging demonstrates not only the effectiveness of NHS chemistry, but also highlights the unique morphology of these particles.

Synthetic particles such as PLGA or polystyrene beads tend to exhibit uniform spherical shapes, but ECM particles feature much more irregular structures and sizes. Additionally, there are similarities of these particles to the features found in SEM images of ECM powders, specifically fibrous strands that resemble collagen. These characteristics may contribute to the cellular responses generated by this material.

Figure 13: Fluorescence microscope image highlighting irregular, fibrous morphology of ECM particles (A). Subsequent addition of these particles to hASC cultures at 0% (B), 1% (C), and 10% (D) volume ratios.

31 These FITC-particles were subsequently added to hASCs in several concentrations to determine cell-particle affinity. Figure 13B-D demonstrates a clear difference in FITC signal between hASCs cultured in 1% FITC-particles (1% of the solution volume) and 10% FITC- particles. FITC-particles accumulated almost exclusively in regions containing hASCs, indicating that the ECM material preferentially attaches to cellular material. This contact is encouraging, because the close proximity of ECM material to cells enables ECM surface proteins to interact with cells and ultimately influence behavior, differentiation and the immune response.

For sufficient conjugation of dye to ECM particles, the modification process must be done in high excess of dye to account for a tagging efficiency less than 100%. Upon reaction completion, the remaining non-conjugated free dye needs to be removed from the sample to avoid any background signal that may compete with the true signal of the particles. Centrifugation is a standard method used to wash synthetic particles.[53] The procedure involves spinning particles under centrifugal force to separate the solution into a particle pellet and a particle-free supernatant

(Figure 14). The supernatant is carefully removed, and the particle pellet is resuspended in clean solution. This process is repeated until the supernatant appears clear, indicating the removal of all non-conjugated excess.

Figure 14: Centrifuge method to wash synthetic particles The main limitation with this approach is that small enough particles (roughly <220nm) do not form a pellet under reasonable centrifuge speeds. Even after a 10 minute 21130G (rcf) spin, sizing data of an ECM nanoparticle batch clearly shows the presence of a high number of

32 nanoparticles remaining within the supernatant (Figure 15). Thus, this method is not suitable for processing the smaller (< 220nm) portion of nanoparticles in solution that is of high interest in immune modulation.

Figure 15: Sizing data (number %) of ECM nanoparticle batch before and after centrifugation at 21130G, the maximum speed of available equipment. Clear presence of ~100nm particles remain in suspension. An alternative approach to ECM particle purification uses centrifuge filter units. This process involves passing the nanoparticle solution through a fine filter membrane using centrifugation as the driving force (Figure 16). By discarding the flow through and replacing the sample with fresh solution, enough wash cycles will theoretically remove all excess dye. Batches of 0.22um-filtered FITC-ECM nanoparticles were washed using 15mL cellulose-based centrifuge filter units with a 100k molecular weight cutoff (MWCO). Preliminary results show a slight increase in the size profile post-washing, though particles maintained an acceptable Z-average

(<500nm) for use in immunological applications (Figure 17).

Figure 16: Overview of centrifuge filter unit method to wash nanoparticles.

Figure 17: Size profile changes of 0.22um-filtered FITC-ECM nanoparticles after centrifuge washing. Size increase observed, but particles remain within target size of <500nm.

Analysis of the flow-throughs for each washing step show a much different washing profile versus a free dye sample of the same concentration. The observed higher retention of dye can be attributed to the successful conjugation of FITC molecules to ECM nanoparticles (Figure

18, left). In addition, fluorescence measurements of the samples show an average signal of 1097.5 relative fluorescence units (RFU) for the final product versus 57 RFU for the final flow through

(Figure 18, right). This ratio, which is defined as the signal-to-noise ratio, should be as high as possible to indicate a statistical difference in the fluorescence of the sample versus background signal. The high signal-to-noise in our sample, combined with evidence suggesting minimal particle size change and different flow through patterns versus free-dye samples suggests successful production of FITC-conjugated cardiac ECM nanoparticles.

Figure 18: Flow-through washing profiles of free dye and FITC-ECM nanoparticle batches (left). Fluorescence data of the final products versus final flow throughs of the same batches (right).

34 While this initial data looks promising, it is necessary to consider the FITC-NHS dye adsorbing to the nanoparticle surface, rather than actually chemically conjugating to it. To test for this, the FITC conjugation procedure was repeated, substituting FITC-NHS dye for a fluorescein dye. This dye is identical to FITC-NHS but lacks the functional NHS group that enables chemical conjugation to the amine groups on the protein nanoparticle surface. As a result, all signal detected in fluorescein nanoparticle batches is expected to be from adsorption. Sure enough, when comparing the fluorescein batch to a standard FITC-NHS batch, there is a clear difference in the signal-to-noise ratio (Figure 19). The FITC-NHS batch displays a signal-to-noise ratio of

8.9, while the fluorescein batch is almost exactly 1:1 to the background signal of the final flow through. This indicates that the FITC-NHS dye is likely not adsorbing to the particle surface, but instead is properly conjugated to it.

Figure 19: Comparison of FITC-NHS ECM nanoparticle batches to ECM nanoparticle batches containing fluorescein dye and batches containing FITC-NHS quenched with glycine. Note a difference in the signal-to- noise ratios between all three samples. As an extra confirmation, the FITC-NHS dye was quenched with glycine prior to adding it to ECM nanoparticles. Glycine serves to inactivate the FITC molecules by reacting with its

35 NHS group. This serves as a separate check to make sure that the NHS version of the dye does not have any inherent adsorption over fluorescein. The dye was quenched in 100 molar excess glycine, 158.6mg to 10mg of dye, and allowed to react at room temperature overnight. The dye was then added to cardiac ECM nanoparticles following the normal conjugation procedure.

Resulting plate reader data helps to verify that the dye is not attaching by adsorption, as indicated by a significantly reduced signal-to-noise ratio of 1.87 versus 8.9 for the non-quenched batch

(Figure 19). It is likely that the slightly higher signal-to-noise ratio, relative to the fluorescein batch, is due to incomplete quenching of the dye.

Upon confirmation of successful FITC conjugation and the encouraging results of ECM nanoparticle localization to hASCs, FITC-tagged ECM nanoparticles were added to bone marrow-derived macrophages from C57BL/6 mice in preparation for immune modulation studies.

Similar to the hASC culture, ECM nanoparticles appeared to selectively adhere to macrophages after 4 hours (Figure 20, top). Flow cytometry of the culture calculated that nearly 100% of the cell population expressed a strong FITC signal, regardless of the induced polarization condition

(Figure 20, bottom right). This experiment serves to directly demonstrate the capability of ECM nanoparticles to interact with macrophages. This is highly promising when considering the major role macrophages play in regulating the host wound healing and immune responses. Having fluorescently tagged, potentially immunomodulatory particles that reliably adhere to macrophages sets up many possibilities for future studies.

Figure 20: Addition of FITC-tagged ECM nanoparticles to C57BL/6 mouse bone marrow-derived macrophages after 4 hours (top). Flow cytometry data indicating expression of FITC in nearly 100% of analyzed cells (bottom). All of the work thus far has focused on FITC-NHS modification of ECM nanoparticles.

However, there exist many other NHS-ester dyes that can be conjugated to particles for different applications. Table 8 provides a list of dyes that have been successfully conjugated to ECM nanoparticles, including a summary of the dye ratios, solvents and reaction times used during the conjugation procedure. Data illustrating the signal-to-noise ratio of these tagged nanoparticle batches can be found in Figure 21.

37 Table 8: List of NHS dyes successfully conjugated to ECM nanoparticles

Dye Name Visible Color Dye Amount (w/w) Solvent Reaction Time (min) FITC Green 1:20 DMSO 60 Licor IRDye 800CW Near Infrared 1:48 DI-Water 120 AF-546 Yellow 1:20 DMSO 90 Rhodamine Yellow-Orange 1:20 DMSO 120

FITC – Fluorescein isothiocyanate; AF - Alexa Fluor; DMSO – Dimethyl sulfoxide

Figure 21: Signal-to-noise ratios of several dyes conjugated to ECM nanoparticles. A batch of Licor-tagged ECM nanoparticles, which provided the highest overall signal out of all samples, was later injected in the tail vein of C57BL/6 mice. Particles were imaged over a span of one week to visualize how they travel within the body and how long they are retained.

Starting at day one and persisting past day three, there was clear migration and accumulation of

Licor-tagged ECM nanoparticles in the inguinal lymph nodes (Figure 22). Lymph nodes are an integral part of the body’s immune system, functioning as a filter that traps bacteria, viruses and other foreign substances. These nodes are highly concentrated with immune cells, including B and T lymphocytes, dendritic cells and macrophages.[54]

Figure 22: Distribution of Licor-tagged ECM nanoparticles introduced by tail vein injection after 3 days.

Accumulation of ECM nanoparticles in the lymph nodes is very encouraging. The close proximity of these nanoparticles to a highly dense population of immune cells enables the presentation of nanoparticle surface markers to lymphocytes. Through further surface modification, ECM nanoparticles can be engineered to elicit specific immune responses within the lymphatic system. In addition, ECM nanoparticles can potentially take advantage of the lymphatics system to travel throughout the body when attached to circulating antigen-presenting cells. This experiment demonstrates the power of fluorescent imaging to track ECM nanoparticles in vivo.

4.2 PEGYLATION

ECM nanoparticle modification is not limited to fluorescent dyes. Particles can be functionalized with any NHS-based molecule to alter its properties and behavior. NHS-

PEGylation is one such modification. Polyethylene glycol (PEG) has been commonly used as a functional coating for nanoparticles, providing “stealth-like” characteristics that prevent particle detection within the body and consequently increase particle retention time.[55] Secondary effects

39 of PEGylation include aggregation resistance and improved pharmacokinetics of immunomodulatory effects.[43] By PEGylating ECM nanoparticles, we can reduce the occurrence of nonspecific interactions that may lead to particle agglomeration and clearance from the reticular-endothelial system. In addition, nonspecific uptake by macrophages is reduced, leading to longer circulation times essential for imaging applications.[42]

Several ECM nanoparticle batches were functionalized with NHS-PEG. The protocol closely follows that of fluorescent dye conjugation. Briefly, ECM nanoparticles are suspended in a 0.1M sodium bicarbonate solution and passed through a 0.22um filter. NHS-PEG is dissolved in water and added to the nanoparticle solution at a concentration of 2mg/mL, and the solution is incubated for 1 hour at room temperature. Sizing data before and after the PEGylation procedure show slight size increases that appear directly proportional to the PEG concentration (Figure 23).

This result is expected, as individual PEG molecules commonly vary between 2000 and 20000 daltons and would contribute to an increased particle diameter upon successful conjugation. This trend continued through the washing step. While all PEG batches maintained acceptable sizes, higher z-averages corresponded to batches with more PEG.

Figure 23: Size profiles of ECM nanoparticle batches conjugated with various amounts of NHS-PEG. A slight increase in particle size can be detected, with increased PEG amounts translating to larger size increases.

40 Since NHS-PEG is not fluorescent, alternative methods are needed to quantify the

PEGylation efficiency of particles. One approach is to look at the zeta potential. As previously discussed, the zeta potential is related to the electrostatic repulsion of particles in solution, related to stability. A zeta potential of higher magnitude correlates to a more stable solution, translating to particles that prefer interacting with the surrounding solution rather than each other. ECM nanoparticles have been demonstrated to feature negative zeta potentials, likely due to negatively charged GAGs or residual DNA. Conjugation to PEG is expected to yield a more neutral value, because non-charged PEG molecules cover these negatively charged features on the ECM particle surface. This can theoretically be detected by a shift towards a more neutral zeta potential, which should be directly proportional to the degree of PEGylation.

PEGylation of several ECM nanoparticle batches highlight this decrease in zeta potential with increasing PEG concentration. Batches were PEGylated under four different concentrations, and excess PEG was removed through washing via centrifuge filter unit. The zeta potentials of each batch were measured by Zetasizer, and the results provide a good indication that PEG contributes to the sample’s zeta potential (Figure 24). However, the method lacks quantification, as the PEGylation efficiency cannot be directly extrapolated from this data.

Figure 24: Zeta potentials of ECM nanoparticles conjugated in a variety of NHS-PEG concentrations.

41 To supplement zeta potential measurements, quartz crystal microbalance with dissipation

(QCM-D) was used to help quantify the degree of PEGylation. QCM-D involves passing a highly controlled, oscillatory voltage through a thin quartz crystal disk. This voltage matches the resonance frequency of the crystal, causing cyclic deformation. By flowing a solution over this crystal, certain molecules can adsorb to the quartz crystal surface. As these molecules accumulate, the resonance frequency of the crystal changes, and this change can be detected as

“overtones”, which is used to calculate the adsorption layer thickness (Figure 25).[56] Therefore,

QCM-D should be able to identify different adsorption layer thicknesses between PEGylated and non-PEGylated samples, with greater thickness corresponding to higher PEGylation.

Figure 25: Illustration of QCM-D principles of operation. Particles adsorb onto quartz crystal disk, affecting its vibration in response to an applied cyclic voltage. Particles containing long PEG chains will affect oscillation differently by providing more of a dampening effect.

Figure 26 shows a graph of the real-time average thickness layer accumulation for several batches of PEGylated ECM nanoparticles. Two concentrations of PEG were analyzed, 0.3 mg/mL and 2 mg/mL, as well as washed versus non-washed batches. All nanoparticle batches adsorbed at similar times, as illustrated by a sharp increase in layer thickness around 13 minutes.

However, there is a clear trend between higher concentrations of PEG yielding thicker adsorption layers (green, gold and black lines). This suggests that ECM nanoparticles that were allowed to

42 react with NHS-PEG are larger than their non-PEGylated counterparts, indicating successful conjugation. There was also a notable difference in washed versus non-washed PEG-ECM layer thickness. As demonstrated in previous sections, the washing process tends to increase the average particle size, and thus this result is unsurprising. The quartz crystal used to measure the no-PEG sample was slightly damaged, which could account for unnaturally steady increase in that sample’s thickness after other samples had equilibrated.

Figure 26: Real-time adsorption layer thickness of ECM nanoparticles onto QCM-D disks. The end point of each line represents the final layer thickness.

This PEGylation experiment serves as a proof-of-concept that ECM nanoparticle functionalization extends beyond fluorescent dyes. NHS chemistry, as well as other methods, can ultimately serve to modify ECM nanoparticles for all sorts of biomedical uses. In the next section, we discuss some of these potential modifications and outline their clinical significance.

43 5. EFFECT OF ECM NANOPARTICLES ON MACROPHAGE POLARIZATION

5.1 INTRINSIC ABILITY OF ECM NANOPARTICLES TO INFLUENCE

MACROPHAGE POLARIZATION

As previously discussed, the extracellular matrix consists of many components known to interact with and affect immune cells.[29-33] Among these cells are macrophages, which play critical roles in both the host immune response to biomaterials and wound healing. Macrophage polarization into M1 or M2 phenotypes contributes greatly towards determining whether the response mediates damaging inflammation or regeneration, respectively.[35] Cytokines secreted by macrophages vary between these response types, with a list of several associated factors provided in Table 3.[34] By influencing macrophage polarization towards M1 or M2 phenotypes using

ECM particles, we can theoretically increase or decrease the production of these secreted factors and consequently change the immune environment.

The first step is to determine whether or not non-modified ECM-derived nanoparticles have an intrinsic ability to modulate macrophage polarization. Macrophages were isolated from the bone marrow of C57BL/6 mice and allowed to mature for one week. Cardiac ECM-derived nanoparticles were added to these macrophages at concentrations of 2ug/mL, 20ug/mL,

200ug/mL (straight from homogenizer, serial diluted) and 2000ug/mL (lyophilized and resuspended) under M0 (standard media), M1 (+ 1:5000 LPS and 1:10000 IFN-) and M2 (+

1:5000 IL-4) polarization conditions. After 24 hours, macrophage RNA was extracted, and qPCR was run for several M1 and M2 associated factors (Figure 27).

Figure 27: Timeline of in vitro macrophage polarization study

44 PCR results for three M1 factors (TNF-, iNos, and IL1) and three M2 factors (IL10, resistin like alpha also known as Fizz-1, and Arg1) were analyzed using Livak’s 2^-CT method.[57] Comparisons of the fold changes for several of these factors reveal notable differences in gene expression before and after the introduction of cardiac-derived ECM nanoparticles

(Figure 28). Of particular interest are M1 factor iNos and M2 factor Arg1, which displayed similar trends. Both of these factors show dose-response upregulation in M0 conditions, with iNos also exhibiting this behavior in the M2 condition. This hints at the intrinsic ability of ECM nanoparticles to alter the immune environment. Several other changes were observed, including large fold changes in nearly every gene for 2000ug/mL nanoparticle concentrations across all polarization conditions. This may be due to either the increased concentration or the different processing method (lyophilization) used to generate these batches. ECM nanoparticles also appear to contribute to the slight downregulation of iNos and IL1 expression in M1 conditions.

ECM nanoparticles appear to have some inherent effect on macrophage polarization in vitro. Fold change patterns across all genes suggests a complex polarization that can be attributed to a dynamic spectrum of M1 and M2 macrophage phenotypes within the population. ECM nanoparticles likely do not promote one specific polarization, though certain trends such as general up or downregulation of M1 and M2 factors can be established through more trials. ECM nanoparticles derived from other tissue sources (liver, lung, SIS, etc.) may trigger different macrophage phenotypes and thus need to be investigated. In addition, modulation of other immune cell types, including T cells and B cells, ought to be considered in future studies.

Figure 28: Gene expression fold changes for several M1 (left column) and M2 (right column) associated factors produced by bone marrow-derived macrophages of C57BL/6 mice after 24-hour exposure to cardiac ECM nanoparticles (2, 20, 200 and 2000 ug/mL concentrations). M0, M1 and M2 are polarization controls. Homogenized DMEM group represented by “Homo DMEM”. Samples marked with “X” were not run.

46 5.2 FUNCTIONALIZATION OF ECM NANOPARTICLES FOR IMMUNE THERAPY:

FUTURE WORKS

After more thorough characterization of the intrinsic ability of ECM nanoparticles to modulate immune cell behavior, the next step is to consider functionalization of these particles to induce enhanced or altered cellular responses. We have already demonstrated the effectiveness of

NHS-chemistry to conjugate fluorescent dyes and PEG to ECM nanoparticles. We can further extend this chemistry to the conjugation of functional peptides that are known to trigger certain responses within the body. Using ECM nanoparticles as a vehicle, delivery of these peptides to specific cell types within the body can prove useful for developing vaccines for immune therapy.[58] In this section, we touch upon development of a method to produce peptide- conjugated ECM nanoparticles to trigger killer T cell activation.

Conjugation of nanoparticles with functional peptides is a common approach for diagnosis and therapy in biomedicine. PEG-PLA polymer nanoparticles functionalized with K237 peptide have been used to facilitate targeted delivery of the anti-cancer drug paclitaxel.[59] Iron oxide nanoparticles used in magnetic resonance imaging have been functionalized with urokinase plasminogen activator receptor antagonist to achieve higher specificity towards specific cell types.[60] Amyloid growth inhibitor peptide and sweet arrow peptide conjugated to gold nanoparticles enabled recognition by macrophages and induced an increased production of pro- inflammatory cytokines.[61] The possibilities are endless, as peptide sequences can be engineered for any application, but these examples provide a good overview of the applications of peptide- conjugated nanoparticles in cancer therapy, medical imaging and immune modulation.

Development of these peptide-functionalized nanoparticles often starts with the use of a model antigen to better understand the mechanisms behind certain cellular responses. Ovalbumin

(OVA), derived from chicken egg whites, is an extremely well characterized biologically active agent that has been widely used in the development of general immune therapies, including the immunization of patients for allogenic transplants, cancer immunotherapy against tumor antigens,

47 and induction of tolerance against self-antigens for autoimmune diseases.[62][63] Upon breakdown in the body, OVA generates many peptide fragments, including the SIINFEKL peptide known to stimulate CD8+ cytotoxic T cell activation. These CD8+ cytotoxic T cells, better known as killer T cells, are a critical part of the adaptive immune system. As SIINFEKL is internalized by antigen- presenting cells (APCs), typically dendritic cells or macrophages, the peptide selectively attaches to major histocompatibility complex (MHC) class-I found within the cell. This complex then migrates to the surface of the APC, where it is detected by immature CD8+ T cells. This detection activates CD8+ cytotoxic T cells, which go on to target and destroy other cells expressing the

SIINFEKL peptide on their surface (Figure 29).

Figure 29: Simplified T cell activation pathway demonstrating antigen presentation to trigger stimulation of immature T cells into cytotoxic CD8+ T cells.

Typically, the response time of CD8+ T cell activation is very slow. The presence of an antigen alone is not enough to trigger the cascade of responses that lead to killer T cell activation.

This is likely designed to avoid false positive detection of antigen and subsequent unnecessary destruction of host cells. Therefore, an inflammatory environment is required during antigen presentation. Current synthetic nanoparticle vaccines attempt to conjugate inflammatory adjuvants, such as alum, alongside the antigens in order to generate this inflammatory

48 environment.[64] However, these particle systems are limited in the responses they can generate, mainly due to the low number of cytokines that can be co-conjugated onto these particles.

ECM nanoparticles may serve as a more effective antigen delivery system than its synthetic counterparts. Recall that the extracellular matrix contains highly functional proteins that demonstrate immunomodulatory capabilities. This inherent property of ECM may serve as the inflammatory adjuvant needed to trigger CD8+ T cell activation. This would eliminate the need for complex co-conjugations of adjuvants required by synthetic particle systems. In addition,

ECM nanoparticles contain a much wider range of functional groups that can be targeted in conjugation, allowing for more complex co-conjugation of supplementary adjuvants, if required.

ECM nanoparticles have already been shown to accumulate in lymph nodes, demonstrating their ability to present antigens to APCs and T cells (Figure 22).

The SIINFEKL peptide serves as a well-characterized model antigen to help in method development. Upon method finalization, SIINFEKL can be substituted with different antigens to trigger many immune pathways. We focus specifically on SIINFEKL because of its well- documented characterization and the existence of the OT-1 mice strain, whose T cells exhibit only the SIINFEKL epitope. By coupling antigens to ECM nanoparticles and presenting them to immune cells, we hope to generate immune therapies revolving around the presentation of peptides and subsequent training of immune cells to recognize antigens.

49 6. CONCLUSION

The extracellular matrix is a complex material consisting of many components, each of which plays important structural and biologically functional roles within tissues. ECM-derived biomaterials take advantage of these unique properties to generate positive outcomes in wound healing and tissue repair. Particle formulations enable many new uses for ECM materials, with immune modulation of particular interest. We have provided methods for consistent production of

ECM micro- and nanoparticles, with characterization that illustrates the conservation of critical functional molecules after processing. Successful particle conjugation with fluorescent dyes and

PEG was confirmed through several quantitative techniques. We have demonstrated the ability of fluorescently tagged ECM nanoparticles to selectively adhere to macrophages in vitro, and in vivo imaging of these particles shows accumulation within the lymph nodes of mice. Finally, we provided preliminary data suggesting modulation of macrophage polarization by cardiac ECM nanoparticles and offered several avenues for future studies. ECM nanoparticles show promise as a novel immunological tool to modify immune environments and thus deserves further investigation.

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54 CURRICULUM VITAE

John Krill received his degree in Biomedical Engineering from Worcester Polytechnic

Institute in May, 2014. With emphasis on tissue engineering and a minor in biomaterials, he was involved in several cardiovascular research labs that aimed to understand cardiac development and treat myocardial infarction. Post-graduation, John shifted his research focus towards nanoparticle formulation, which lead him to this project provided by the Elisseeff lab.