Development of a QTsome Lipid Nanoparticle Delivery Platform for Oligonucleotide
Therapeutics
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University
By
Jilong Li
Graduate Program in Pharmaceutical Sciences
The Ohio State University
2018
Dissertation Committee:
Robert J. Lee Ph.D. Advisor
Sashwati Roy Ph.D. Co-Advisor
Mitch A. Phelps Ph.D.
Yizhou Dong Ph.D.
Copyright by
Jilong Li
2018
Abstract
The objective of this dissertation thesis is to develop a novel lipid nanoparticle oligonucleotide delivery platform with pH responsive lipid combination and enhancement ligands to achieve therapeutic goals in cancer and wound management.
Favored by numerus scientists and pharmaceutical industries, gene therapy has received a lot of compliments and has been extensively studied over many decades. A series of pre-clinical and clinical trials have revealed superior efficacy over conventional chemo-drugs on many genetic disorder diseases including cancer, inflammation, neural degenerative disease, hereditary disease. However, successful commercialization of gene therapy has been compromised due to several unfavorable properties. Naked oligonucleotides are considerably unstable due to chemical and enzymatic cleavage during manufacture and administration. Additionally, incapability of penetrating cell membranes due to high polarity and charge density has forbidden its bench-side application via conventional formulations. Moreover, highly-potent and long-last efficacy of gene therapeutics has compelled the demands for target-specific delivery to avoid off- target side effects or cytotoxicity. Hence, carefully engineered and delicate development of novel delivery system specifically designed for gene therapy has been called upon by research scientists and medical practionors.
ii In Chapter 2, we have proposed a well-designed pH responsive lipid nanoparticle delivery platform (QTsome) with enhanced in vitro &in vivo gene trafficking efficacy to treat non-small cell lung cancer (NSCLC). The novelty of such pH responsive platform is substantially improved endosomal escape facilitated by the combination of quaternary and tertiary amine based cationic lipids. Three lead advantages can be acquired based on quaternary/tertiary cationic combination: firstly, near neutral or weak positive surface charges can be micro-adjusted to achieve optimal interactions between delivery system and targeting cells, while avoiding drawbacks including cytotoxicity and RES clearance associated with traditional cationic lipid nanoparticles; secondly, higher oligo encapsulation efficiency can be achieved by mixing the oligo cargos with lipid compositions at pH~4.0 to introduce stronger electrostatic interactions; thirdly, enhanced release of gene cargos into cytoplasm is introduced by presence of highly positive charges in late endosome stage. In our study, encapsulated anti-miR-21 has shown excellent stability and loading capacity within the QTsome, along with more than 50% of miR-21 suppression. Additionally, combinational therapy facilitated by incorporation of
PTX (Paclitaxel) into QTsome/ anti-miR-21 has demonstrated greater capacity in cell proliferation inhibition and metastasis reduction.
In Chapter 3, QTsome platform has been further adjusted to accommodate chronic wounds. To further improve in vivo transfection efficiency, ionophore reagent
Gramicidin A was incorporated into the formulation. Gramicidin A has been proposed to enhanced cell penetration capacity as well as endosomal escape via promoting hexagonal
II (HII) phases. QTsome surface properties have been carefully studied to in adaption to
iii the presence of phagocytes and macrophages on the wound bed. Anti-miR-210 has been successfully inserted into keratinocytes to reverse ischemia memory of proliferation cease. Results have suggested restoration of basal keratinocyte cell hyper-proliferation and migration in mice ischemic wound models.
In Chapter 4, a keratinocytes-specific targeting ligand has been conjugated onto the surface of QTsome to enhance cell-specific recognition. Studies have pointed out complications and side effects involved in non-specific targeting. Deep-burn wound bed environment has recruited a variety of cell types including bacteria, macrophage, phagocyte, fibroblast, keratinocyte. Random delivery of high potent oligo cargos has great potentials in causing unknown therapeutic outcomes and severe toxicities.
Therefore, target delivery of miR cargos into specific cells without detection from surrounding tissues have become the major barrier to break. In this study, we have proposed a sophisticated designed targeting delivery system loaded with anti-miR-107 that is capable of keratinocytes recognition and accelerating wound closure and restoration of barrier function. In addition, lyophilization has been incorporated to accommodate a non-injection route of application with additional long-term storage capacity.
iv Dedication
This document is dedicated to my beloved family.
v Acknowledgments
Firstly, I would like to give my most sincere appreciation to my primary advisor,
Dr. Robert J. Lee, for all the support, tutoring, and encouragement throughout all these years. I have been greatly inspired by his depth of knowledge, his way of carrying out an independent research and his creative thought of problem shooting. I would not be able to properly and successfully complete my doctoral degree without his inspiration and support. Additionally, I would also like to thank my co-advisor Dr. Sashwati Roy for providing such precious opportunity to work as a team member in Sen lab and for her patient mentorship and expertise on wound healing project.
I am very grateful to faculty members from College of Pharmacy including Dr.
Yizhou Dong and Dr. Mitch A. Phelps, who kindly accept my request to serve committee members for candidacy examination and final defense examination. I deeply appreciate their generosity for their valuable time and helpful advice on my candidacy proposal and dissertation thesis. I would also like to thank Dr. Chandan K Sen for his support on my graduate study and wound healing projects.
Secondly, I would like to thank my lab mates and colleagues: Dr. Bryant C. Yung,
Dr. Hong Li, Dr. Mengzi Zhang, Dr. Subhadip Ghatak, Dr. Surya C. Gnyawali, Dr.
Mohamed S El Masry, Dr. Das Amitava for their valuable insights and inspiring discussions on my projects. I truly appreciate Dr. Xin Xin, Dr. Dongzhu Wu, Mr. Xinwei
vi Cheng, Mr. Yang Liu, for their friendly help throughout my study at OSU. Plus, I would like to thank all the friends and loved ones for being a part of my memorable journey at the Ohio State University.
Lastly, and most importantly, I would like to give my sincere thanks to my parents for their unconditional support on my good times and bad times through all these years. They have been continuously encouraging me and give me all their understandings for me to carry on. I wouldn’t stand a single chance to have accomplished this much on my journey without their love.
vii Vita
1988…………………………...... Born--Chengdu, P.R. China
2006-2010…………………………...... B.S. Pharmacy, Southern Medical University,
Guangzhou, China
2010-2012…………………………...... M.S. of Pharmaceutical Science, University of
Pittsburgh
2012-present…………………………...Graduate Research Associate, Department of
Pharmaceutics and Pharmaceutical Chemistry,
The Ohio State University
Publications
Ghatak S, Li J, Chan YC, Gnyawali SC, Steen E, Yung BC, Khanna S, Roy S, Lee RJ, Sen CK. AntihypoxamiR functionalized gramicidin lipid nanoparticles rescue against ischemic memory improving cutaneous wound healing. Nanomedicine. 2016 Mar 29.
Yung BC*, Li J*, Zhang M, Cheng X, Li H, Yung EM, Kang C, Cosby LE, Liu Y, Teng L, Lee RJ. Lipid Nanoparticles Composed of Quaternary Amine-Tertiary Amine Cationic Lipid Combination (QTsome) for Therapeutic Delivery of Anti-miR-21. Mol Pharm. 2016 Jan 19. (* These authors contribute equally to this paper)
viii Jaime-Ramirez AC, McMichael EL, Kondadasula S, Skinner CC, Mundy-Bosse BL, Luedke E, Jones NB6, Mani A, Roda J, Karpa V, Li H, Li J, Elavazhagan S, La Perle KM, Schmitt AC, Lu Y, Zhang X, Pan X, Mao H, Davis M, Jarjoura D, Butchar JP, Poi M, Phelps M, Tridandapani S, Byrd JC, Caligiuri MA, Lee RJ, Carson WE. The NK Cell- mediated Anti-tumor Effects of a Folate-conjugated Immunoglobulin are enhanced by Cytokines. Cancer Immunol Res. 2016 Feb 10.
Li J, Ghazwani M, Zhang Y, Lu J, Li J, Fan J, Gandhi CR, Li S. miR-122 regulates collagen production via targeting hepatic stellate cells and suppressing P4HA1 expression. J Hepatol. 2013 Mar; 58(3):522-8.
Zhang X, Sun X, Li J, Zhang X, Gong T, Zhang Z. Lipid emulsions loaded with doxorubicin-oleic acid ionic complex: characterization, in vitro and in vivo studies. Pharmazie. 2011 Jul; 66(7):496-505.
Field of Study
Major field: Pharmaceutical sciences
ix Table of Contents
Abstract ...... ii
Dedication ...... v
Acknowledgments...... vi
Vita ...... viii
Table of Contents ...... x
List of Tables ...... xvi
List of Figures ...... xvii
CHAPTER 1: Introduction and Background ...... 1
1.1 Overview of novel nano-sized therapeutic delivery (carrier) system ...... 1
1.2 Lipid nanoparticle (LNP) based gene delivery system ...... 4
1.3 Engineered cell transfection modifications ...... 7
1.4 Cell-tissue specific targeting ...... 12
1.4.1 Passive targeting delivery ...... 12
1.4.2 Active targeting delivery ...... 14
x CHAPTER 2: Lipid Nanoparticles Composed of Quaternary Amine–Tertiary Amine
Cationic Lipid Combination (QTsome) for Therapeutic Delivery of AntimiR-21 for Lung
Cancer ...... 21
2.1 Introduction ...... 21
2.2 Materials and Methods ...... 24
2.2.1 Materials ...... 24
2.2.2 Synthesis method of QTsome ...... 25
2.2.3 Mean Particle Diameter and Surface Charge ...... 25
2.2.4 Drug Loading and Stability ...... 26
2.2.5 Cell Culture ...... 26
2.2.6 In Vitro Gene Regulation by QTsome-Encapsulated AM-21 ...... 27
2.2.8 In Vitro Tumor Cell Migration Assay...... 28
2.2.9 In Vitro Tumor Cell Invasion Assay ...... 29
2.2.10 In Vivo Therapeutic Activity of QT/AM-21 ...... 29
2.2.11 Combination Therapy Analysis ...... 30
2.2.12 Vivo Gene Regulation by QTsome-Encapsulated AM-21 ...... 30
2.2.13 Statistical Analysis ...... 31
2.3 Results ...... 31
2.3.1Particle Size and Surface Charge ...... 31
xi 2.3.3 Determination of Optimal Lipid Combination...... 32
2.3.4 Regulation of miR-21 and miR-21 Targets ...... 33
2.3.5 Dose and pH Dependency ...... 33
2.3.6 Cell Viability ...... 34
2.3.7 Invasion and Migration ...... 35
2.3.8 In Vivo Dose Response ...... 35
2.3.9 In Vivo Combination Therapy ...... 36
2.4 Discussion ...... 36
2.5 Conclusion ...... 40
CHAPTER 3: AntihypoxamiR Functionalized Gramicidin Lipid Nanoparticles Rescue
Against Ischemic Memory Improving Cutaneous Wound Healing ...... 60
3.1 Introduction ...... 60
3.2 Materials and Methods ...... 61
3.2.1 General study method ...... 61
3.2.2 Preparation of antihypoxamir functionalized gramicidin lipid nanoparticles. . 62
3.2.3 Particle size and surface charge characterization of AM-210 encapsulated in
AFGLN...... 62
3.2.4 Study of encapsulation efficiency & colloidal stability ...... 63
3.2.5 Human subject ...... 64
xii 3.2.6 Animals and wound models ...... 64
3.2.7 In vivo imaging ...... 65
3.2.8 Laser capture microdissection (LCM) of epidermis ...... 66
3.2.9 RNA extraction and quantitative real-time PCR...... 67
3.2.10 Immunohistochemistry and microscopy...... 67
3.2.11 Data collection and statistical analyses...... 68
3.3 Results ...... 68
3.3.1 Negative correlation between expression of miR-210 and keratinocyte
proliferation in ischemic wound...... 68
3.3.2 Development and characterization of AFGLN lipid nanoparticles for miRNA
delivery ...... 69
3.3.3 In vivo evaluation of AFGLNantimiR-210 efficacy in ischemic wound re-
epithelialization...... 70
3.3.4 In vivo evaluation of AFGLNantimiR-210 efficacy in restoring oxidative
metabolism and proliferation...... 71
3.4 Discussion ...... 71
CHAPTER 4: Topical Lyophilized Targeted Lipid-Nanoparticles in the Restoration of
Skin Barrier Function Following Burn Wound ...... 94
4.1 Introduction ...... 94
xiii 4.2 Materials and methods ...... 97
4.2.1 Materials ...... 97
4.2.2 Lipid-Peptide Conjugation...... 98
4.2.3 Preparation of TLNκ...... 98
4.2.4 Lyophilization procedure...... 99
4.2.5 Size and zeta potential measurements...... 99
4.2.6 Encapsulation efficiency...... 100
4.2.7 Cell and cell culture ...... 101
4.2.8 Transfection of miRNA mimic and inhibitors...... 102
4.2.9 Determination of cell proliferation...... 102
4.2.10 Cell migration assay...... 103
4.2.11 Measurement of targeting efficiency by Flow cytometry ...... 103
4.2.12 Animal model...... 103
4.2.10 Trans-epidermal water loss (TEWL)...... 105
4.2.13 Laser capture microdissection (LCM) of epidermis...... 105
4.2.14 RNA extraction and quantitative real-time PCR...... 105
4.2.15 Histology, Immunohistochemistry and microscopy...... 106
4.2.16 Data collection and statistical analyses...... 106
4.3 Results ...... 107
xiv 4.3.1 Preparation and characterization of lyophilized TLN: ...... 107
4.3.2 In vitro targeting efficiency of TLN: ...... 108
4.3.3 In vivo targeting efficiency of TLN: ...... 108
4.3.4. Functional wound closure ...... 110
4.3.5 Epidermal junctional proteins ...... 110
4.4 Discussion ...... 111
CHAPTER 5: Summary and Future Direction ...... 136
Bibliography ...... 141
xv List of Tables
Table 1.1 List of liposomal based therapeutic currently in clinical trials ...... 18
xvi List of Figures
Figure 1. 1 Schematic diagram of liposomal nano-carriers cytoplasm cargo release via endosome escape and the pH drop from early endosome to lysosome...... 19
Figure 1. 2 Schematic diagram illustrating enhanced permeability and retention
(EPR) effect, showing the physiology foundation as hyper-permeable tumor vasculature. Nano-sized particles and micelles are therefore retained at tumor interstitial spaces...... 20
Figure 2. 1 Schematic diagram of QTsome and pH responsive mechanism ...... 42
Figure 2. 2 Particle size of QTsomes prepared under various mol%...... 43
Figure 2. 3 Surface charge of QTsomes suggesting pH responsive...... 44
Figure 2. 4 Encapsulation efficiency of oligo cargos in QTsomes...... 45
Figure 2. 5 Colloidal stability of QTsomes...... 46
Figure 2. 6 Optimization of transfection efficiency on various quaternary–tertiary lipid ratio...... 47
Figure 2. 7 Upregulation of miR-21 and its target genes by QT/AM-21 in A549 cells.
...... 48
Figure 2. 8 Upregulation of mRNA expression in dose dependent pattern in A549 cells...... 49
Figure 2. 9 Upregulation of protein expression in dose dependent pattern in A549 cells...... 50
Figure 2. 10 Inhibited endosome release suggested by the presence of NH4Cl...... 51
Figure 2. 11 Cell viability in response to treatment by QT/AM-21...... 52 xvii Figure 2. 12 Changes in cell morphology following treatment with QT/AM-21 alone or in combination with PTX were imaged under light microscope...... 53
Figure 2. 13 Migration and invasion in response to treatment by QT/AM-21...... 54
Figure 2. 14 Tumor regression by QT/AM-21 in A549 xenograft mice...... 55
Figure 2. 15 Weight data on tumor-bearing mice treated with QT/AM-21...... 56
Figure 2. 16 Kaplan–Meier survival analysis of A549 xenograft mice treated with
QT/AM-21...... 57
Figure 2. 17 Efficacy of QT/AM-21-PTX combination therapy in A549 xenograft model...... 58
Figure 2. 18 In vivo miR-21 target gene regulation by QT/AM-21 in A549 murine xenografts...... 59
Figure 3. 1 Schematic diagram showing the different proteins involved in cell cycle regulation that are targeted by miR-210. The number in the parenthesis indicates the references...... 75
Figure 3. 2 Schematic diagram illustrating AFGLNmiR-210 rescuing ischemic wound hypoxia memory to facilitate efficient wound closure...... 76
Figure 3. 3 miR-210 expression from laser microdissected epidermis of human wound-edge tissue...... 77
Figure 3. 4 Serial human wound cross-sections stained with anti-Ki67 and keratin-
14 antibody, counter stained with DAPI...... 78
Figure 3. 5 Regression plot of miR-210 expression from the human wound edge biopsies against number of Ki67 positive cells/field (20X). (n = 8) ...... 79
xviii Figure 3. 6 miR expression level after LNA treatment from wound edge tissue...... 80
Figure 3. 7 Schematic representation of the AntihypoxiamiR Functionalized
Gramicidin Lipid Nanoparticles (AFGLN) and the zeta potential of the AFGLN at different pH. Shaded region represents the pH range in chronic wounds...... 81
Figure 3. 8 Representation of nanoparticle size and counts...... 82
Figure 3. 9 miR-210 expression from murine non-ischemic and ischemic wound- edge tissue 24 h after intradermal delivery of AFGLNmiR-210. (n = 4). * P < 0.01 compared to AFGLN, ANOVA...... 83
Figure 3. 10 Percentage of wound closure after delivery of empty GLN,
AFGLNscramble and AFGLNmiR-210. (n = 5), * P < 0.001 compared to Day 0; ANOVA.
...... 84
Figure 3. 11 The percentage of re-epithelialization at day 6 post wounding was plotted graphically...... 85
Figure 3. 12 Representation of wound closure and re-epithelialization...... 86
Figure 3. 13 Wound closure suggested by contrast digital photographs and immune- staining...... 87
Figure 3. 14 Laser speckle image showing perfusion level in the bi-pedicle flap at day 0 and day 7 after delivery of only GLN, AFGLNscramble and AFGLNmiR-210...... 89
Figure 3. 15 IVIS image from repTOP™mitoIRE showing cell proliferation in animal treated with lipid nanoparticles with AFGLNmiR-210.The images from quantification of the mean luminescence have been presented. (n = 3) ...... 90
xix Figure 3. 16 Serial wound cross-sections stained with anti-Ki67 antibody counter stained with DAPI (blue). (n = 3)...... 91
Figure 3. 17 31P NMR spectra of the ischemic wound-edge tissue at day 7 post wounding showing iP, and the three subunits α-, β- and γ- subunit of ATP normalized with phosphocreatinine ...... 92
Figure 3. 18 Schematic diagram showing interventions available for reoxygenation of ischemic tissue...... 93
Figure 4. 1 Schematic representation of Keratinocytes Targeting Lipid Nanoparticle
(TLNκ)...... 114
Figure 4. 2 Change of TLNκ size due to aqueous storage condition under 4 degree over a period of 7 days...... 115
Figure 4. 3 Representative photograph of the lyophilized Keratinocytes Targeting
Lipid Nanoparticle...... 116
Figure 4. 4 Representation of nanoparticle size and concentration...... 117
Figure 4. 5 The zeta potential of the TLNκ at varying pH suggesting pH responsive behavior...... 118
Figure 4. 6 miR-210 expression level followed by TLNκ treatment...... 119
Figure 4. 7 Overlay of phase contrast and fluorescence microscopic images...... 120
Figure 4. 8 Study of in vitro uptake...... 121
Figure 4. 9 Representative mosaic image of murine dorsal skin after application of the burner for 5 secs (top) and 15 secs (bottom) for developing the full thickness burn wound. Scale bar = 500μm...... 122
xx Figure 4. 10 Digital photos of TLNκ application...... 123
Figure 4. 11 miR-107 expression level from Laser Capture Microdissection...... 124
Figure 4. 12 Co-localization on normal skin by confocal microscopy...... 125
Figure 4. 13 Co-localizations on burn edge skin by confocal microscopy...... 126
Figure 4. 14 Cell proliferation and migration ...... 127
Figure 4. 15 Quantitative PCR analysis of miR-107 effectiveness...... 128
Figure 4. 16 miR-107 downstream gene expression level followed by treatment. .. 129
Figure 4. 17 Dicer expression level followed by TLNκ treatment...... 130
Figure 4. 18 p21 waf1/Cip1 expression level followed by TLNκ treatment...... 131
Figure 4. 19 Representation of wound closure...... 132
Figure 4. 20 Representation of re-epithelization and restoration of barrier function.
...... 133
Figure 4. 21 upregulated epidermal junction proteins suggesting restoration of barrier function...... 134
xxi CHAPTER 1: Introduction and Background
1.1 Overview of novel nano-sized therapeutic delivery (carrier) system
Since the emerging of medicinal nanotechnology, numerous patients and physicians have benefited from better medicines as well as diagnostic tools. Such innovation has revolutionized several traditional chemical drugs, such as Doxorubicin and Paclitaxel, into bigger commercial success [1, 2]. The initial intention of developing drug carriers was to facilitate controlled or triggered release with preferred pharmacokinetic/pharmacodynamics profiles [3]. Significantly reduced toxicity and side effects were therefore achieved with better patient compliance and less dosing frequency.
It has also benefited pharmaceutical industries to reconsider some incompetent small molecule drugs which are suffered from poor water-solubility or poor membrane permeability. Significantly improved bioavailability is thus endorsed to these compounds to better help our patients [4]. The first concept of nano-carrier delivery system was introduced to public in 1960s, many years of endeavor have contributed to the success development of first FDA approved liposomal drug named AmBisome followed by
Doxil [5]. In the following years, emerging formulations such as lipid nanoparticles, biodegradable polymer nanoparticles, micelles, and dendrimers have proven their
1 tremendous advantages over conventional drug delivery systems in many clinical trials
[6]. Development of nano-sized medicine has become more favorable due to reduced renal toxicity along with improved stability and circulation time [7]. In recent years, along with Abraxane, which has been approved by FDA not too long ago, many irinotecan nanomedicines have been reported in the late stage of clinical trials [8]. This has indicated a successful transition from bench-top production into fully developed industrial manufacturing process. A summarize of information about recent clinical trials on lipid nanoparticles based therapeutics are presented in Table 1.1. While early developed nanoparticles are mainly focused on low molecular weight chemical entities, the era of gene therapy has already begun. Due to the premium properties of nanomedicine, it has become desirable for scientists to transform this technology to adapt the needs of gene therapy. In the past few years, dedicated researches and investigations have paved the road to successful development of gene delivery system on similar basis
[9].
Newly developed nano delivery systems have shown great potential in successful encapsulating and delivering oligonucleotide cargos efficiently. Oligonucleotide therapeutics are majorly suffered from low cell membrane permeability due to its high polarity and charge density. Its fragile genetic structure has also yielded research scientists with problems including instability and high serum clearance [10]. Other than systemic barrier, cellular barrier including cell membrane and nucleus have also prevented gene therapeutics to reach their target sites [11]. Therefore, manufacture and administration of naked oligonucleotide therapeutics has become a dead-end, which
2 demands a significantly amount of work to develop a suitable delivery system. Careful considerations including physiochemical properties, loading capacity, surface modification, immunogenicity and overall toxicity are all essential works to ensure successful development of gene delivery system. In the beginning, viral vectors are considered as one of the most promising delivery method for genetic cargos. However, extremely unsafety profiles were observed from pre-clinical and clinical trials. Retroviral vector has been studied for the therapeutic of X-SCID, despite showing of therapeutic effectiveness, incidence of leukemia has been observed in many patients due to inappropriate insertion of gene cargos in host genome [12-14]. Similar clinical results have suggested similar outcome from adenoviral vectors as well, where serious side effects and even incidents of patient’s death have sounded the alarm for using viral vectors as gene delivery systems with enough safety profiles [15-17]. Follow-up studies have shown a serial of drawbacks using viral vectors as gene delivery systems: triggering severe immunogenic events and lethal inflammation, lack of recognition devices resulted in high risk of insertional oncogenesis and uncontrollable cell targeting, high cost of production in upstream and downstream purification, and lack of toolbox for loading detection and separation in industrial productions [18].
Thus, fully customizable synthetic nano drug delivery platforms have indisputable advantages over conventional viral vectors due to its amendable properties in overcoming above critical drawbacks. Among these delivery platforms, loads of work has been done to accommodate their capacities as superior gene delivery platforms serving various purposes. Other than traditional lipophilic or pH gradient encapsulation strategies that are
3 designed for small molecule compounds, electrostatic interactions and chemical conjugations methodology was introduced to satisfy the needs of high drug loading capacity [19]. Sophisticated designed and evaluated carrier composition has protected genetic cargos from environmental instability, enzymatic degradation, and systemic clearance. Multiple transfection enhancement methods were designed and studied including stimuli-responsive components and cell penetration ligands to help with cell membrane penetration. Both passive and active targeting strategies have been included to further improve therapeutic index while reducing unwanted side effects. Among all, polymer and lipid based nanoparticles (LNP) are the most successful platforms in preparing gene therapeutic carriers with huge potentials in future development.
1.2 Lipid nanoparticle (LNP) based gene delivery system
Composed by multi-functional phospholipid components, LNP delivery platform is considered as one of the most successfully developed carrier systems. A smooth translation from delivering low molecular weight drugs to genetic cargos can be achieved thanks to many years of hard work by both academic and industry. It is now almost fully customizable to achieve various purposes including variable particle size, variable surface charges, suitable of multiple surface modifications, suitable of controlling release profiles. In addition, a large pool of composition components can be selected from in order to prepare the most desirable LNPs that serves our goals, and most of the formulation gradients have already been approved by FDA [20-22]. Its relatively simple
4 manufacturing process has also contributed to its many advantages as a desired genetic carrier: by mixing lipid compositions and lipophilic components into aqueous solution, thermodynamic favorable reaction will lead to spontaneous formation of a bilayer membrane-like spherical structure automatically [10]. Nevertheless, substantial difference between genetic cargos and chemical drugs have represented the first challenge in successful development of LNP gene delivery platform [23]. The general goals in design and development of a robust LNP gene delivery platform are considered to [24]: 1. Maintain a colloidal stable hydrodynamic particle size; 2. Highly efficient encapsulation of genetic therapeutics via electrostatic or conjugation; 3. Robust and scalable manufacturing process to satisfy industrial production needs; 4. Considerable stability of both nanoparticles and loaded genetic materials in manufacturing and delivery; 5. Sophisticated design of transfecting mechanism.
Conventional strategies of loading small molecule drug into LNPs via hydrophobicity or pH transmembrane gradient cannot be directly translated into oligo drugs. Therefore, electrostatic binding of oligos to cationic lipids were first introduced to achieve high loading capacity [25]. A scalable development of manufacture process by combine ionizable cationic lipids with oligo cargos were manufactured by ethanol- dilution method [26, 27]. Such loading strategy involves rapid mixing lipid ethanol mixture with aqueous buffer containing oligos at lower pH. Rapid mixing process is extremely critical in encapsulation of oligo cargos. Upon mixing, a quick formation of inverted micelle structure was constructed by electrostatic reaction between cationic lipids and negatively charge RNA/DNA [28, 29]. A properly designed mixture process
5 with high Reynolds number is considered a critical aspect to uniformity. As the polarity of micelles increases, backbone lipids will start to cover the micelles into a bilayer membrane structure before aggregation [29]. Subsequently, microfluid mixing technology was adopted to refine the mixing process resulted in a well-defined production of uniform high gene cargo loading LNP [30]. Residual ethanol can be removed by dialysis method or size-exclusion chromatography. Such step is considered to further decrease the size and stabilize the spherical structure [26].
To fully take advantage of cationic LNP encapsulation method, multiple cationic lipids have been studied and utilized to load gene cargos. DOTAP (1,2-dioleoyl-3- trimethylammonium-propane), DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), DC-Chol (3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol), and multivalent cationic lipids (MLVs) composed liposomes have been extensively studied and successfully commercialized [31-33]. A rational design and selecting strategy of cationic lipids are largely based on physiochemical properties. Degree of saturation, ester bonds, chain length, charge density and flexibility are addressed as predominant factors to be considered [34]. Three distinctive structures have been discovered within the self- assembly lipid/nucleic acid complexes: lamellar structure indicates where gene cargoes enveloped in between cationic membranes; inverted hexagonal suggests genes loaded within inverted lipids with a formation of hexagonal lattice; intercalated hexagonal shows a micelle-like lipids covering gene drugs with sub-structure arranged as honeycomb symmetry [35]. Conical structured helper lipids including DOPE (1,2-dioleoyl-sn- glycero-3-phosphoethanolamine) and Chol (Cholesterol) have been proven to
6 thermodynamically favor the formation of inverted hexagonal shape. Such formation has enhanced transfection efficiency by inducing fusogenic membrane fusion and endosomal escaping [36]. To better design and select proper lipid compositions, additional study has been carried out to suggest the presence of ester bond is beneficial in improving biodegradability and reducing cytotoxicity lipids [37]. The combination of
DOPC/DOTAP has shown significant improvement over commercial transfection reagents giving high serum content [38].
1.3 Engineered cell transfection modifications
Genetic therapeutics encapsulated in LNP delivery systems demand successful delivery into the cell cytoplasm. Therefore, it is rather critical to develop sophisticated cell membrane penetration mechanisms to achieve effective delivery. Enhanced endosomal escape and engineered cell membrane penetration methods have represented the two most common ways to that goal [39]. Due to the native structure of oligo cargos, failure of escaping from endosomes in the endosome stage will result in the fate of degradation in the presence of acidic lysosomes [40]. Therefore, by taking advantage of
LNP composition and cell penetration peptides, these bio-barriers could be potentially overcome.
Four major categories of cationic liposome designs have been employed to enhance in vitro/in vivo cell transfection efficiency: monovalent cationic LNPs, pH- sensitive cationic LNPs, lipidoids, and multivalent cationic LNPs [41]. Most
7 representative monovalent cationic LNPs are composed by either or combination of
DOTAP, DC-Chol, DOTMA. Single incorporation of monovalent cationic lipids forms lipoplexes often yield with a high gene transfection efficiency in vitro. High degree of cellular uptake via ionic interactions and endosomal escape by the ion-pair mechanism is often achieved [42]. Monovalent cationic lipids are capable of forming electrostatic interaction with cell surface anionic membrane lipids thus induce the cellular uptake events. Afterwards, ion-pairs were assembled to release of gene cargos while promoting the inverted hexagonal structure and disrupt endosomal membrane [43]. At the same time, the use of neutral helper lipids in monovalent cationic LNPs have been demonstrated to be extremely beneficial. Some commonly employed neutral lipids are
DOPE, DOPC (dioleoyl phosphatidyl choline), DSPC (1,2-distearoyl-sn-glycero-3- phosphocholine) and Chol. The utilization of neutral helper lipids and cationic lipids have yielded with improved transfection efficiency and stabilized lipid structure in many researches. DOPE has been known for its function of destabilizing lipid bilayers which results in endosomal disruption [44]. By using DC-Chol/DOPE complexes, significantly increased gene expression level was observed when compare to DC-Chol/DOPC complexes in the delivery of CAT (chloramphenicolacetyl-transferase) plasmid gene
[45]. DOTMA composed LNPs have shown similar transfection efficiency compare to commercial Lipofectamine in vitro siRNA knock down study [46].
The underlying mechanism for the pH-sensitive cationic LNPs is enhanced escaping from early endosome by endocytic pathway. After binding to cell membrane, liposomes are retained in early endosomes. As shown in Figure 1.1, early endosomes
8 (pH=6.5) will mature into relative lower pH late endosomes (pH=5.0-6.0), then become lysosomes (pH=4.0) [47]. By taking advantage of such pH environment gradient, pH sensitive LNPs have been designed to break down endosome membranes and release cargos into cytoplasm at late endosome stage [48, 49]. Two theoretical mechanisms have been proposed as in pore formation and diffusion through endosomal membrane.
Quaternary amine based cationic lipids such as DOTAP possess one permanently positive charge in spite of pH environment. On the other hand, tertiary amine based cationic lipids such as DODMA have a slightly lower pKa, meaning it is uncharged at biological environment but become ionized at acidic pH [50]. Therefore, stronger positive charges will interact with endosome membrane and lead to breakage. Early developed
DOPE/CHEMS (cholesteryl hemisuccinate) lipid complexes have shown significantly higher level of cell association compare to conventional cationic LNP designs [51].
Current strategies of formulating pH sensitive LNPs are composed by the combination of monovalent cationic lipid and a protonated helper lipid [38]. Therefore, such composition could result in neutral or weakly charge at physiological pH and strongly positive charged at acidic environment. Studies have discovered that pH responsive liposomes tend to form a SNALP structure other than lipoplexes [52]. By combination of DOTAP,
DOPE and DODAP (1,2-dioleoyl-3-dimethylammonium-propane), SNLAP has shown more siRNA silencing effectiveness by that of traditional designed liposomes [53]. Other than monovalent cationic LNPs, novel designed lipidoid nanoparticles and multivalent cationic LNPs can also be formulated with pH sensitive property [41]. Several research
9 studies have suggested improved transfection efficiency associated with reduce cytotoxicity [54, 55].
Other than modifications of lipid components, the incorporation of PEGylation has shown tremendous effects on circulation extension and structural stability enhancement that eventually have an indirectly impact on transfection efficiency.
PEGylation is often acquired by including PEG-modified structural lipids during the preparation or post-insertion method. The inclusion of PEG motif does certainly improved blood circulation time by avoiding RES (Reticuloendothelial System) recognition, however, one study has suggested that PEG is also responsible for reducing gene transfection efficiency as well as endosomal escape capacity [41]. PEG length and covering percentage have both been playing important factors in regulating LNPs properties. PEG1000 or shorter chain length is found to insufficiently protect the particles from protein adsorption and RES recognition, while PEG5000 or longer would significantly reduce cellular uptake [56, 57]. Therefore, PEG2000 has become the most commonly used PEGylation for LNP surface engineering. Also, PEG covering density has been shown to have a major impact on avoid macrophage detection. 10% PEG2000 have been shown to significantly induce tumor accumulation [58, 59]. Only 2% of
PEG2000 is adequate to reduce protein adsorption. Interestingly, if to increase PEG2000 to
5%, increased protein adsorption is observed with transforming of mushroom-like surface structure into a brush structure with inhibited macrophage uptake [60]. Therefore, fine modification of PEG content on the surface of LNP is also essential characteristics to evaluate.
10 Moreover, helper ligands such as cell penetration peptide (CPP) represents another outstanding way of penetrating cell membranes. Upon the discovery from viral vectors, CPPs have become highly favorable due to their biocompatibility plus easy route of conjugation. Relatively small molecular weight and simply structure allow scientists to modify their properties including charge density, hydrophilicity and binding affinity.
CPPs are able to translocate themselves into cytoplasm via endocytic pathways [61].
Typical example of successfully developed CPPs are HIV-TAT, R8, MPG8 and BP16
[62]. Optimized peptide sequence with highest transfection efficiency has been proved to suited LNPs in delivering siRNA to dendritic cells [63]. Similar work has been done with selection of multiple CPPs to facilitate synergistic effects both in vitro and in vivo [64]. A family of prototypical channel formers have been extensively studied for understanding of dynamics and functionality of membrane-spanning channels. Antibiotics like
Gramicidin A are found to form cationic ion channels in membranes [65]. Gramicidin A has been considered an excellent transmembrane channel former with a channel pore size
~4 Å [66]. The properties of accommodation for monovalent cations passage has suggested promising potentials as ionophore in which helps with endosomal escaping.
Permeation assays have been conducted to examine its pore formation capacity. Results have suggested disordered lipid bilayer matrix after incubation of Gramicidin A along with significantly increased membrane permeability.[65-67].
11 1.4 Cell-tissue specific targeting
One very important advantage of nano-sized LNPs gene delivery system is the capability of specific cell targeting modulation. It has become a predominate function in the consideration of improved efficacy and reduced toxicity. As described above, some attributes have been discovered to prevent RES/renal clearance and to enhance specific tissue/cell accumulation with adequate quantity. Two major mechanisms have been described to achieve targeting effects. By taking advantage of nanoparticle surface technology in coordinate with physical micro-environment, passive targeting has shown satisfying accumulation of nanoparticles at solid tumor site. On the other hand, advanced technologies have also helped with surface representation of active targeting chemical motifs to achieve cell-specific recognition.
1.4.1 Passive targeting delivery
In the recent years of discovery and research, passive delivery has fulfilled its full potentials in solid tumor targeting and immune cell targeting. As the first and foremost acknowledged the tumor micro-environment targeting strategy, enhanced permeability and retention (EPR) effect was introduced. As suggested in Figure 1.2, the foundation of
EPR physiology is based on hyper-permeable tumor vasculature [68]. Therefore, larger particles such as proteins, nano-sized particles and micelles that cannot be cleared by renal system are accumulated at tumor interstitial spaces due to impaired lymphatic drainage [69]. A synergistic effect with PEGylated nanoparticles have further improved accumulation effects. Enhanced tumor uptake was achieved by avoiding mononuclear
12 phagocyte system (MPS), improved colloidal stability and homogenized size distribution
[70]. In addition, surface charges are also considered to play an important role in the overall performance of passive targeting effect. Studies have claimed that negatively charged or neutral surface could reduce renal clearance [71], while positively charged nano-carriers tends to have stronger interaction with negatively charged proteins and trigger RES clearance [72].
On the other hand, above strategies could be used against to improve uptake by innate immune cells. Some studies were carried out to discover the principal of using
LNPs platform to target innate immune cells including phagocytes and macrophages. It was concluded that small sized liposome (~85nm) with negatively charge surface behave the best in internalization by MPS cells [73, 74]. At the same time, other studies have also reported improved uptake by increasing the LNP size to 1000nm [75-77]. It is very likely the optimal size should be studied case-by-case as targeting cell and LNP formulation varies. Negatively charged LNP surface are preferentially recognized by macrophages when comparing neutral charge phosphotidylcholine (PC) with negatively charge phosphatidylserine (PS) and phosphatidylglycerol (PG) [57]. The PEGylating was removed from design to ensure better recognition and internalization for innate immune cells. Additionally, short PEG surface mods is capable of triggering complement reaction and induce macrophage recognition, which serves as a promising targeting tool for macrophages [78].
13 1.4.2 Active targeting delivery
The fundamental of receptor mediated cell targeting has been established based on antibody binding mechanisms [79]. In order to enhance nano-carriers with enhanced therapeutic effects and minimized off-target cytotoxicity. The concept of active targeting has been introduced and developed into nanoparticle delivery systems. Targeting ligands are chemically conjugated onto the surface of nano-carriers where they are more likely to be captured by cells with over-expressed complementary receptors. Small chemical entity, peptide, antibody, aptamer and protein are among the most popular targeting ligands that has been show promising potentials.
After years of development of antibody drug therapeutics, they have been recognized it as a superior strategy with the highest targeting efficiency. However, due to the large molecular weight and complex structure, the actual use of antibody as targeting ligand has been compromised. In cancer treatment, most antibodies that can diffuse into tissue are countered by antigens presented on perivascular tumor cells [80, 81]. Great effort has to be made to successfully conjugate antibody onto the surface of nano-carriers without compromising its structural integrity [82]. Study has suggested fast clearance of antibody-liposome conjugates due to intense immunogenicity [83]. Therefore, peptide and small chemical entity have been considered to better serve the goal.
Small molecule ligands are considered not to be constrained by those factors.
They are easily amenable to chemical modifications and conjugations, while maintaining their structural stability [84]. As one of the most extensively studied targeting mechanisms, FR targeting induce receptor-mediated endocytosis of cancer cells that
14 expresses high affinity for folic acid (Kd=1-10nM) [85]. Study has suggested folate conjugates can leak from blood vessel and saturate folate receptors (FR) within 5 mins of i.v. injection [86]. Folic acid is generally non-immunogenic with high efficiency of tissue permeation. It has been suggested a significantly higher affinity to over-expressed cells upon incorporation of folate to the surface of LNPs via short PEG spacer [87, 88]. Biotin receptor is another promising target for cancer specific delivery or imaging. Biotin major transporter, sodium-dependent multivitamin transporter (SMVT), has been correlated with several over-expression cancer cells: renal (RENCA, RD0995), leukemia
(L1210FR), mastocytoma (P815), ovarian (OV 2008, ID8), lung (M109), colon (Colo-
26), and breast (4T1, JC, MMT06056) [84, 89]. Research has showed preferred internalization into targeted cancer cells, while reduced cytotoxicity over normal cells has been observed [90].
Integrin receptors are heterodimeric transmembrane glycoproteins express on cell membrane surface. V3 and V5 sub-class receptors are found to express much more on many carcinoma endothelial cell types [91]. Integrin receptors have been frequently found to mediate cell adhesiveness to extracellular matrix (ECM) therefore in direct correlation with cancer tissue invasion, proliferation and migration [84]. Also, by manipulation of ECM, integrin receptors are capable of regulating tumor angiogenesis and metastasis. A tri-peptide chemical motif RGD (Arg-Gly-Asp) has been discovered to express high affinity for V3 and V5 sub-class receptors. Cyclic RGD peptide have been linked to PEGylated LNPs to enhance targeting efficiency in many reports [92].
Most commonly used cyclic RGD peptides are characterized in c(RGDfK), c(RGDfC)
15 and RGD10 (DGARYCRGDCFDG) subgroups [93-95]. Results from pre-clinical study has already shown inhibition of metastases with targeted nanoparticles loaded with doxorubicin [96].
Besides integrin receptor targeting peptides, transferrin receptor targeting peptides have also gain considerable attentions in cancer management. Transferrin receptors
(TFRs) are transmembrane glycoproteins. It has been found that a significantly increase of TFRs expression associated with malignant cancer cells such as glioma, chronic lymphocytic leukemia, and bladder-transitional cell carcinoma, while the expression level of TFR1 is considerably low in normal tissues [97-99]. Peptide T7 (HAIYPRH) was selected using standard phage display method with relatively higher binding affinity than
Transferrin itself [100]. T7 modified nanoparticles loaded with PTX have shown a significantly enhanced efficacy against A2780 human ovarian cancer cells [101].
Additionally, extensive studies have been conducted over HER-2 receptors among EGF family. It is overexpressing over several cancers including pancreatic, prostate ovarian and breast carcinomas [102]. HER-2 overexpression has been found among 20-30% of all breast cancer types, and has been acknowledged as an important biomarker indicating poor prognosis [103]. Other than using a recombinant monoclonal antibody
(Tratuzumab), low cost HER-2 binding peptide ligands were produced by standard phage display method [104]. The binding affinity has been evaluated over HER-2 positive breast and prostate cancer cells: peptide conjugated liposomes have suggested cell growth inhibition on HER-2 positive cells and mice models [105]. A5G33 peptide
(ASKAIQVFLLAG) were developed from library of cell-adhesive peptide. Such peptide
16 has shown strong cell binding capacity for keratinocyte cells via heparin-like receptors
[106].
17 Trial Product name Drug Lipid composition Approved indication Ref phase LEP-ETU (powder/12 DOPC, cholesterol, and cardiolipin (90:5:5 Paclitaxel Ovarian, breast, and lung cancers Phase I/II [89, 90] months) molar ratio) DOPC, cholesterol, and cardiolipin (90:5:5 LEM-ETU Mitoxantrone Leukemia, breast, stomach, liver, ovarian cancers Phase I [89, 91] molar ratio) EndoTAG-1 DOTAP, DOPC, and paclitaxel (50:47:3 Anti-angiogenic properties, breast cancer, pancreatic Paclitaxel Phase II [89, 92, 93] (powder/24 months) molar ratio) caner Arikace Amikacin DPPC and cholesterol Lung infection Phase III [94, 95] Cholesterol and egg sphingomyelin (45:55 Marqibo Vincristine Metastatic malignant uveal melanoma Phase III [89, 96, 97] molar ratio) DPPC, MSPC, and PEG 2000-DSPE (90:10:4 ThermoDox Doxorubicin Non-resectable hepatocellular carcinoma Phase III [98, 99] molar ratio) Acute promyelocytic leukemia, hormone-refractory Atragen Tretinoin DMPC and soybean oil Phase II [89] prostate cancer Acute myeloid leukemia, chronic myelogenous Liposomal Grb-2 Grb2 ASO Unknown Phase I [100] leukemia, acute lymphoblastic leukemia Nyotran Nystatin DMPC, DMPG, and cholesterol Systemic fungal infections Phase I/II [89] BLP25 Monophosphoryl lipid A, cholesterol, Cancer vaccine for multiple myeloma developed Stimuvax Phase III [101, 102] lipopeptide DMPG, and DPPC encephalitis SPI-077 Cisplatin SHPC, cholesterol, and DSPE-PEG Head and neck cancer, lung cancer Phase I/II [89] Lipoplatin Pancreatic cancer, head and neck cancer, SPC, DPPG, cholesterol, and mPEG 2000- (suspension/36 Cisplatin mesothelioma, breast and gastric cancer, and non- Phase III [89, 103] DSPE months) squamous non-small-cell lung cancer Camptothecin Recurrent or progressive carcinoma of the uterine [89, 104, S-CKD602 DPSC and DSPE-PEG (95:5 molar ratio) Phase I/II analog cervix 105] OSI-211 Lurtotecan HSPC and cholesterol (2:1 molar ratio) Ovarian cancer, head, and neck cancer Phase II [106, 107] Cholesterol and egg sphingomyelin (45:55 INX-0125 Vinorelbine Advanced solid tumors Phase I [89, 108] molar ratio) Table 1.1 List of liposomal based therapeutic currently in clinical trials
18
Figure 1. 1 Schematic diagram of liposomal nano-carriers cytoplasm cargo release via endosome escape and the pH drop from early endosome to lysosome.
19
Figure 1. 2 Schematic diagram illustrating enhanced permeability and retention
(EPR) effect, showing the physiology foundation as hyper-permeable tumor vasculature. Nano-sized particles and micelles are therefore retained at tumor interstitial spaces.
20 CHAPTER 2: Lipid Nanoparticles Composed of Quaternary Amine–Tertiary
Amine Cationic Lipid Combination (QTsome) for Therapeutic Delivery of AntimiR-
21 for Lung Cancer
2.1 Introduction
Lung cancer accounts for over 160,000 deaths in the United States each year
[107]. Non-small-cell lung cancer (NSCLC) accounts for over 80% of lung cancers and relative to small-cell lung cancer (SCLC) is less responsive to surgery, radiotherapy, and chemotherapy [108, 109]. The destructive nature of NSCLC and the lack of effective means of treatment outline the critical need for new modes of therapy. microRNAs are noncoding RNAs that regulate gene expression via RNA interference. Progression of
NSCLC has been linked to overexpression of microRNA-21 (miR-21) [110, 111]. miR-
21 is an oncomiR that operates at the epigenetic level, directly and indirectly impacting pathways associated with cellular apoptosis, DNA repair, proliferation, invasion, metastasis, and drug resistance [112]. Inhibition of miR-21 should result in upregulation of tumor suppressor genes that are miR-21 targets. Indeed, antimiR-21 (AM-21) has been shown to upregulate miR-21 targets (i.e., ANKRD46, DDAH1, PTEN, RECK, PDCD4,
TIMP3) while decreasing relative cell migratory and invasion potential [113, 114].
21 Moreover, miR-21 in ovarian cancer has been linked to resistance to chemotherapeutic paclitaxel (PTX) [115].
As potential therapeutic agents, antimiRs are sensitive to nucleases, are rapidly cleared from blood circulation, and cannot penetrate the cellular membrane due to their high molecular weight and charge density [116, 117].Incorporation of oligonucleotides into lipid nanoparticles (LNPs) combined with chemical modification of their backbone is a commonly used strategy to address these issues. A common chemical modification scheme is to introduce 2′-O-methylation on the ribose throughout, with phosphorothioate
(PS) linkages at the two ends of an antimiR, which improves target affinity and nuclease stability relative to unmodified RNA. In designing LNPs, a cationic lipid is typically used to enable electrostatic complexation with the oligonucleotide, which is a polyanion due to the negative charges contributed by phosphates and/or PS in its structure.
Quaternary amine based cationic lipids, such as DOTAP and DOTMA, are permanently charged. They are used extensively in cationic LNPs for plasmid delivery for gene therapy. In contrast, tertiary amine based cationic lipids, such as DODAP and
DODMA, are mostly uncharged at neutral pH and become fully ionized only at acidic pH, such as that found in endosomes. It has been shown that high zeta potential for a
LNP is detrimental to its in vivo activity while introducing toxicity due to undesired interaction with blood components [118]. Weakly charged LNPs can be prepared by combining an oligonucleotide with a tertiary amine based cationic lipid at low pH and in
40% ethanol to facilitate electrostatic interaction and oligo loading, followed by raising the pH and ethanol removal [119-121].At the cellular level tertiary amine based cationic
22 lipids facilitate endosomal escape of the oligonucleotide by going back to positive charge in response to the low pH environment in the endosome following internalization of the
LNPs [119-121].
QTsome is a novel formulation of LNP based on a combination of a quaternary amine based cationic lipid and a tertiary amine based cationic lipid. As shown in Figure
3.1, this design leverages the advantage of high charge density provided by the quaternary amine cationic lipid and the pH responsiveness of tertiary amine cationic lipid to achieve a balanced charge-vs-pH profile optimal for delivery of oligonucleotides
[119].In addition to superior activity, an advantage of QTsome is that it can be produced using lipid excipients (DOTAP, DODMA) that have previously been used in clinical trials and are readily available, providing a straight path toward rapid clinical translation.
Application of the QTsome formulation toward the delivery of AM-21 is expected to increase its efficacy in vivo. The present study investigates the therapeutic potential of
QTsome/AM-21 (QT/AM-21) in NSCLC using a cell line and a xenograft model. In vitro studies in A549 cells were carried out to evaluate the ability of QT/AM-21 to upregulate tumor suppressor targets of miR-21 and to reduce metastatic potential of tumor cells.
Additional studies were completed to evaluate whether QT/AM-21 was able to increase sensitivity of A549 cells toward PTX. Finally, a xenograft mouse model was used to evaluate the in vivo efficacy of QT/AM-21 for inhibiting tumor progression and modulating targets of miR-21.
23 2.2 Materials and Methods
2.2.1 Materials
1,2-Dioleyloxy-N,N-dimethyl-3-aminopropane (DODMA) was obtained from
Corden Pharma (Boulder, CO, USA). 1,2-Dioleoyl-3-trimethylammonium-propane
(DOTAP) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were purchased from
Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol (CHOL) and Cremophor EL were obtained from Sigma-Aldrich (St. Louis, MO, USA). N-(Carbonyl-methoxy-polyethylene glycol 2000)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG) was purchased from NOF America Corp. (White Plains, NY, USA). A fully 2′-O-methyl modified AM-21 oligonucleotide, u*c*a*acaucagucugauaag*c*u*a, where asterisks represent phosphorothioate linkages, was obtained from Alpha DNA (Montreal, Quebec,
CA). PrimeTime qPCR assay primer probes and kits for DDAH1, PTEN, RECK,
PDCD4, and TIMP3 target genes, with GUSB as a non-target control and GAPDH as a housekeeping gene control, were purchased from Integrated DNA Technologies
(Coralville, IA, USA). Goat IgG directed against DDAH1 was obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Rabbit IgG directed against GAPDH was purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibody directed against goat or rat IgG conjugated with HRP was purchased from Jackson
ImmunoResearch Laboratories (West Grove, PA, USA). Ammonium chloride was purchased from Fisher Scientific (Pittsburgh, PA, USA).
24 2.2.2 Synthesis method of QTsome
QTsomes were prepared by a serial ethanol dilution method [27, 122]. Initial dissolution of lipids under low pH and high ethanol resulted in a disperse lipid phase that was able to form electrostatic complexes with oligonucleotides. By increasing pH and reducing ethanol, LNPs became stabilized. Briefly, all lipids (X/Y/36/20/4 mol/mol,
DODMA/DOTAP/DOPC/CHOL/DPPE-PEG) were dissolved in ethanol and then combined with an equal volume of AM-21 dissolved in citric acid buffer (20 mM, pH
4.5), maintaining a 10:1, lipid:AM-21 weight ratio. DODMA and DOTAP contents were varied with the sum of molar percentages of tertiary and quaternary amine maintained at
40 (X + Y = 40). This mixture was further diluted by equivalent volumes (1:1) of citric acid buffer, 300 mM NaCl, and then PBS (10 mM phosphate, 135 mM NaCl, pH 7.4).
The resultant QTsome was concentrated by tangential diafiltration to remove unencapsulated AM-21 and ethanol, and to reach the appropriate final concentration.
Samples were stored at 4 °C prior to characterization. For long-term stability, 10% sucrose was added as a cryoprotectant and the QTsome formulation was either frozen or lyophilized.
2.2.3 Mean Particle Diameter and Surface Charge
Aliquots of QT/AM-21 were diluted in PBS. Particle size was measured by dynamic light scattering (DLS) on a NICOMP 370 submicron particle sizer (NICOMP,
Santa Barbara, CA, USA). Aliquots of QT/AM-21 or LNPs containing only tertiary or quaternary amine cationic lipid were diluted in either citric acid buffer or PBS to
25 determine the pH dependency of surface charge. Zeta potential measurement was conducted on a Zeta PALS Analyzer (Brookhaven Instruments Corp., Worcestershire,
NY, USA).
2.2.4 Drug Loading and Stability
Encapsulation efficiency for AM-21 was determined by Quant-iT Ribogreen
RNA assay kit (Life Technologies, Carlsbad, CA, USA). Briefly, QT/AM-21 complexes were lysed with Triton X-100, and mean fluorescent intensity was compared with intact
QT/AM-21 at (480 nm λex, 520 nm λem). The encapsulation efficiency was determined with the formula
Formulation stability was evaluated at −20, 4, and 25 °C over a period of 30 days. The particle size was periodically monitored by DLS; 10% sucrose was added as a cryo- protectant prior to storage.
2.2.5 Cell Culture
A549 cells were purchased from the American Type Culture Collection
(Rockville, MD, USA) and grown in RPMI 1640 (Corning, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA) and 100 units/mL penicillin and 100 mg/mL streptomycin. Cells were maintained at
37 °C and grown under a humidified atmosphere containing 5% CO2.
26
2.2.6 In Vitro Gene Regulation by QTsome-Encapsulated AM-21
A549 cells were grown in 24-well plates at a density of 7.0 × 105 cells/well 24 h prior to transfection. QT/AM-21 or QT/negative control (NC) of varying lipid composition was added at 50 nM oligonucleotide concentration in the presence of 20% serum containing medium to determine the optimal QTsome lipid composition. QT/AM-
21 was also tested at concentrations of 1.56, 6.25, 25, and 100 nM to determine dose dependency of treatment. Cells were incubated at 37 °C with transfection medium for 4 h and then washed three times with PBS. Fresh complete cell culture medium was added, and the cells were incubated at 37 °C for an additional 44 h. RNA was isolated from cells by RNeasy 96 kit (Qiagen, Valencia, CA, USA). qRT-PCR was conducted with Taqman
MicroRNA Assay (Life Technologies) or EXPRESS One-Step Superscript qRT-PCR kit
(Life Technologies) on an Applied Biosystems StepOnePlus RT-PCR system (Life
Technologies). The relative amount of DNA product was calculated and compared according to the 2–ΔΔCtmethod [123, 124].Western blot was completed to assess changes in protein expression following QT/AM-21 treatment. Cell lysates were denatured in sample buffer. Equal amounts of proteins were loaded and electrophoresed on 10% SDS– polyacrylamide gels and transferred onto nitrocellulose membranes. The transferred blots were blocked with 5% nonfat milk in Tris-buffered saline (TBS, 150 mM NaCl, 20 mM
Tris–HCl, pH 7.4) and incubated for 2 h at room temperature or overnight at 4 °C with primary antibodies in TBS, 0.05% Tween 20. After washing, the blots were reacted with
HRP-conjugated secondary antibodies for 45 min and developed using an enhanced
27 chemiluminescence (ECL) detection system. Quantitative analysis of Western blot bands was performed with ImageJ software (National Institutes of Health, Bethesda, MD,
USA).
To investigate the role of endosomal pH in QTsome-mediated delivery, the above studies were repeated with addition of 100 mM NH4Cl, a lysosomotrophic agent, into the medium during QT/AM-21 treatment to prevent endosome acidification.
2.2.7 Cell Viability Assay
A549 cells were grown in 96-well plates at a density of 2.0 × 104 cells/well. Cells were treated with controls or QT/AM-21 at 50, 100, or 200 nM, with and without PTX
(4.1 nM) dissolved in a small volume of 1:1 Cremophor EL:ethanol solution. Relative cell viability was quantified by CellTiter 96 AQueous One Solution Cell Proliferation
Assay (Promega, Madison, WI, USA) according to the manufacturer’s protocol 72 h following the start of treatment. Briefly, 20 μL of MTS assay solution was added to each well, and the plates were incubated for 1 h. The absorbance at 490 nm was recorded to determine cell viability.
2.2.8 In Vitro Tumor Cell Migration Assay
A scratch wound healing model was conducted to examine the migratory ability of A549 cells following treatment. A549 cells were plated at a density of 6.0 ×
105 cells/well in a 33 mm Petri dish 24 h prior to transfection. A scratch wound across the dish was made using a 10 μL pipet tip immediately before treatment. Culture medium
28 was removed and replaced with transfection medium containing QT/AM-21 or appropriate controls diluted in complete medium. Cells were allowed to proliferate at
37 °C for 48 h. Distances between edges of the wound were measured on a Nikon E800 microscope (Nikon, Tokyo, Japan) with SPOT Advanced Imaging Software (v5.0,
Diagnostic Instruments Inc., Sterling Heights, MI, USA).
2.2.9 In Vitro Tumor Cell Invasion Assay
Matrigel (BD Biosciences, San Jose, CA, USA) was combined with serum-free
RPMI 1640 culture medium in a 1:1 ratio; 70 μL of gel was added to each well insert of a
24-well plate. The gel was allowed to set for 1 h at 37 °C. A549 cells were seeded at 7.5
× 105 cells/well in a volume of 100 μL/well on top of the gel in the inset. Transfection medium containing various formulations or controls at 2× concentration in a 100 μL volume were added to the top of the well inserts. Then, 500 μL of 10% fetal bovine serum supplemented medium was added as a chemoattractant below the Transwell insert.
The plate was incubated at 37 °C for 48 h. Following the incubation period, cells remaining on the top of the well inserts were removed with a cotton swab. Well inserts were rinsed with PBS and placed in 500 μL of 0.25% trypsin solution for 1 h at 37 °C.
Detached cells were counted on a hemocytometer.
2.2.10 In Vivo Therapeutic Activity of QT/AM-21
An A549 mouse xenograft model was generated by inoculating female athymic nude mice with 1.0 × 106 cells/mouse. Tumors were allowed to reach a size of ∼80
29 mm3 before treatment initiation (∼2 weeks after inoculation). Mice (n = 10 per group) were dosed by tail vein injection with saline control, 0.5, or 1 mg/kg QT/AM-21. Tumor progression was routinely monitored through the course of the study. Tumor volume was calculated according to the formula V = (L·W2)/2. Mice were dosed every 3 days for the first three treatments and then once a week following the first dose for a total of seven doses. All mice were treated according to the guidelines deemed appropriate by the
Institutional Animal Care and Use Committee (IACUC) of the Ohio State University
(OSU).
2.2.11 Combination Therapy Analysis
Female athymic nude mice (n = 5 per group) were implanted with 1.0 × 106 A549 cells/mouse, and treatment began when tumors reached a size of ≥100 mm3. Mice were treated by tail vein injection with saline control or 1 mg/kg QT/AM-21. Mice receiving
PTX treatment as monotherapy or combination therapy received PTX dissolved in 1:1
Cremephor EL: ethanol solution at a dose of 3 mg/kg via intraperitoneal injection. Mice were dosed on days 1, 3, 5, 8, 15, 22, 29 and were monitored over a 4-week period. At 48 h following the last dose, mice were euthanized and tumors were collected and analyzed for miR-21 target gene expression.
2.2.12 Vivo Gene Regulation by QTsome-Encapsulated AM-21
Tumors were harvested and placed in TRIzol reagent (Life Technologies) following treatment and were homogenized. mRNA was isolated per the manufacturer’s
30 protocol. qRT-PCR was then completed according to the same procedure as outlined above in section 2.2.6.
2.2.13 Statistical Analysis
All studies were done in triplicate unless otherwise indicated. Statistical analysis was conducted using Microsoft Excel Software (2013, Redmond, WA, USA).
Student’s t test was used to determine statistically significant difference(s) between two or more groups. A p value of 0.05 was selected as the cutoff for statistical significance.
2.3 Results
2.3.1Particle Size and Surface Charge
Previous studies have demonstrated the need for small particle size (<150 nm) and moderate zeta potential (+10–30 mV) to potentiate delivery of oligonucleotides[125,
126]. QTsome particle size measurement by DLS indicated particles of approximately
80–170 nm in diameter (Figure 2.2). Particles with greater amounts of quaternary amine
(15–40 mol %) achieved particles of smaller size (<120 nm). The minimization of particle size was obtained at equal molar percentages of quaternary and tertiary amine, which may signify the point at which the QTsomes adopted a condensed structure[127,
128]. Zeta potential measurement (Figure 2.3) revealed formation of QTsome particles of
12.49 ± 1.45 mV in PBS (pH 7.4) and 29.89 ± 8.16 mV in citric acid buffer (pH 4.0), thus
31 demonstrating the pH responsive behavior of a conditionally ionizable LNP formulation.
As expected, the pH responsiveness of QTsomes fell between that of those LNPs containing only tertiary amine cationic lipid and those with only quaternary amine cationic lipid. The charge on tertiary amine only LNPs changed from 3 to 15 mV with increasingly acidic pH. Conversely, the charge on quaternary amine only LNPs did not vary much with changes in buffer pH (Figure 2.3).
2.3.2 Drug Loading and Colloidal Stability
Encapsulation efficiency obtained during QTsome synthesis was 83.3 ± 4.17%.
Further studies by size exclusion chromatography analysis on a Sepharose CL-4B column
(Figure 2.4) showed approximately 90% of oligonucleotide within the encapsulated drug fractions [110-112], with very little oligonucleotide remaining in the free drug fractions
[116-118].The formulation (with 10% sucrose as cryoprotectant) further demonstrated high stability under storage at −20, 4, and 25 °C over a period of 30 days, maintaining a size of ∼110 nm (Figure 2.5).
2.3.3 Determination of Optimal Lipid Combination
A series of QTsome formulations were evaluated to determine the best ratio of quaternary and tertiary cationic lipid for transfection. Formulations are identified as QT
(mol % quaternary amine)-(mol % tertiary amine) in Figure 2.6. Treatment with 50 nM
AM-21 revealed QTsome with 15 mol % DOTAP and 25 mol % DODMA to perform the best in upregulation of DDAH1 (1.33-fold), and this combination of lipids was therefore
32 chosen for further in vitro and in vivo investigation. Formulations QT10-30 and QT5-35 also performed well, with 1.32- and 1.27-fold upregulation of the target genes, respectively. Formulations containing only quaternary or tertiary cationic lipid did not perform as well relative to the quaternary–tertiary combinations.
2.3.4 Regulation of miR-21 and miR-21 Targets
Treatment with 100 nM QT/AM-21 resulted in significant downregulation of miR-21 and upregulation of its downstream targets (Figure 2.7). Relative to the untreated control, miR-21 level was decreased by 50.3 ± 2.1% following administration of
QT/AM-21. In contrast, little to no effect on target gene regulation was observed for the scrambled negative control (NC) group. Tumor suppressors PTEN and PDCD4 were upregulated 2.7- and 1.3-fold, respectively. Matrix metalloprotease inhibitors RECK and
TIMP3 were both upregulated by approximately 1.5-fold. Migration inhibitors
ANKRD46 and DDAH1 were upregulated 1.2- and 3.0-fold, respectively. Meanwhile, little to no change in expression was seen for GUSB control across treatment groups.
2.3.5 Dose and pH Dependency
Varying dosages of QT/AM-21 between 1.56 and 100 nM were administered and qRT-PCR was conducted to evaluate the relationship between AM-21 concentration and miR-21 target gene upregulation (Figure 2.8). miR-21 targets DDAH1, PTEN, and
RECK all demonstrated a direct correlation between AM-21 dose and mRNA levels.
DDAH1 and PTEN were relatively more sensitive to changes in concentration relative to
33 RECK. In addition, concentration-dependent upregulation of DDAH1 in response to
QT/AM-21 was shown at the protein level by Western blot (Figure 2.9). Furthermore,
QT/AM-21 treatment of A549 cells in the presence of 100 mM NH4Cl demonstrated a significant reduction in DDAH1 upregulation. This might be due to the inhibition of endosomal release of the oligonucleotide (Figure 2.10). By preventing the acidification of the endosome, overall cationic charge of QTsome would have been diminished as the tertiary amines remained neutral and thus unable to interact with negatively charged lipids of the endosomal membrane. The results of this finding suggest a pH-dependent mechanism of QTsome delivery.
2.3.6 Cell Viability
Treatment with free AM-21 or empty QTsome did not result in significant cytotoxicity as analyzed by MTS assay (Figure 2.11). Likewise, the combination of
QT/AM-21 at 50 nM did not demonstrate much cytotoxicity. However, increases in cytotoxicity were observed at increased concentrations of QT/AM-21 (100, 200 nM).
Addition of PTX alone diminished cell viability by 40%. Addition of free AM-21 or
QTsome lipids to PTX did not result in significant decreases in cell viability. However, substantial gains in cytotoxicity were observed with QT/AM-21 treatment in a dose- dependent manner. Cell viability was reduced to 51.2%, 41.0%, and 31.8% at 50, 100, and 200 nM doses, respectively. The differences between the QT/AM-21 and PTX monotherapies and the combination therapy were both significant at p ≪ 0.05. Changes in cell morphology were also noted by microscopy. Minor changes in cellular
34 morphology were observed for control treatment groups and for low doses of QT/AM-21.
Major alterations in morphology were observed corresponding to increasing cytotoxicity for increasing doses of QT/AM-21 and especially QT/AM-21 with PTX (Figure 2.12).
2.3.7 Invasion and Migration
Wound healing assay was completed to monitor the relative mobility of A549 following treatment with AM-21. QTsome lipids or AM-21 alone did not confer significant decreases in cell migration in the wound region. With 100 and 200 nM AM-21 treatment, mobility was reduced to 43.0 and 22.2% relative to the untreated control
(Figure 2.13 A). Matrigel was used to simulate biological conditions of the basement membrane. The ability of cancer cells to migrate plays a role in determining metastatic potential and increases with cancer progression. Treatments with QT/AM-21 at 50, 100, and 200 nM were able to reduce migration to 87.7, 76.7, and 62.6% respectively, while
QTsome lipids alone or free AM-21 did not significantly retard cell invasion (Figure 2.13
B).
2.3.8 In Vivo Dose Response
Treatment with QT/AM-21 demonstrated strong antitumor activity (Figure 2.14) at 1 mg/kg, but diminished activity at 0.5 mg/kg, suggesting a strong dose-dependence in therapeutic response. Treatment of tumors initiated at ∼80 mm3 and ended at 816, 618, and 172 mm3 for the untreated, 0.5 mg/kg, and 1 mg/kg groups, respectively. Moderate differences in terms of body weight were observed between the treated and untreated
35 groups. Liver and spleen weights remained fairly consistent between the two groups, suggesting little to no significant toxicity for these organs. Tumor weight was over 16- fold lower for the treated group compared to the untreated group (Figure 2.15). In terms of median survival time, the untreated group was 21 days, the 0.5 mg/kg treated group was 24 days, and the 1 mg/kg treated group was significantly prolonged, at 33 days
(Figure 2.16).
2.3.9 In Vivo Combination Therapy
PTX and QT/AM-21 combination therapy was evaluated for therapeutic efficacy.
Treatment began when tumors reached ∼80 mm3 in volume. Tumors progressed to 380,
246, 201, and 138 mm3 for the untreated, PTX, QT/AM-21, and combination treatment groups, respectively (Figure 2.17). Furthermore, qPCR conducted on tumor sections revealed moderate to strong upregulation of DDAH1 and PTEN (Figure 2.18). DDAH1 and PTEN were only modulated slightly by PTX, by 1.7- and 1.5-fold, respectively.
DDAH1 was upregulated 3.4-fold while PTEN was upregulated 2.5-fold with QT/AM-
21. DDAH1 was strongly upregulated by 5-fold and PTEN was upregulated 4.1-fold with the QT/AM-21/PTX combination therapy.
2.4 Discussion
miR-21 is involved in a number of pathways regulating tumor progression and resistance to chemotherapy. Therefore, it can serve as a prognostic and diagnostic
36 biomarker for NSCLC. miR-21 is also found to have similar roles in other cancers as well, including ovarian, breast, and prostate cancer [129]. AM-21 therapy to inhibit miR-
21 is a potential strategy for the treatment of NSCLC and other cancers. The in vivo application of AM-21 and related oligonucleotides for therapy is hindered by several physical and biological barriers to their in vivo delivery. LNPs are employed to improve delivery of oligonucleotides. Quaternary amines are the most commonly used class of lipids for gene therapy delivery and form strong electrostatic interactions with negatively charged oligonucleotides. Meanwhile, tertiary amines are used for delivery of oligonucleotides, such as siRNA. For example, the tertiary amine lipid 1,2-dilinoleyloxy-
3-dimethylaminopropane (D-Lin-DMA) is a major component of a version of the stable nucleic acid lipid particles (SNALP) delivery system developed by Tekmira
Pharmaceuticals [130, 131].Tertiary amine based lipids are pH responsive, which has been shown to be critical to oligonucleotide delivery [119-121].
In the present study, a pH-sensitive carrier, QTsome, was evaluated for the delivery of AM-21 to NSCLC. Tertiary amine cationic lipids form the pH-sensitive component of QTsome. Upon exposure to acidic conditions as found in the endosome, the tertiary amine cationic lipid becomes cationized, enabling interaction with negatively charged lipids in the endosomal membrane and facilitating endosomal release. Release of drug from the endosomal compartment is a critical step in determining antimiR efficacy
[32, 132]. Meanwhile, inclusion of quaternary amine lipids can contribute to structural stability of LNPs under physiological pH. A positively charged quaternary amine can better interact with the negative charge of the oligonucleotides, thereby resulting in
37 particles of smaller average particle size relative to particles containing only tertiary amine lipids. In terms of pH-dependence of surface charge, QT15-25 displayed an intermediary response to pH relative to QT40-0 or QT0-40. QT/AM-21 furthermore demonstrated excellent colloidal stability and efficient drug loading, which are important factors for clinical translation. It is worth noting that LNP formulations also based on a combination of two pH-sensitive lipids, one cationic and one anionic, called Smarticles, have recently been used in clinical studies for therapeutic delivery of miR-based oligonucleotides with promising initial data. QTsomes may be further stabilized by the addition of cryoprotectant and by lyophilization for long-term stability [133].
QT/AM-21 was able to downregulate miR-21 and upregulate several key targets of miR-21 including tumor suppressors, matrix metalloprotease inhibitors, and migration inhibitors. PTX kills cancer cells by stabilization of microtubules, which prevents mitosis. Patients normally respond to PTX with a 40–80% response rate, but many of these patients develop resistance to PTX over time[134, 135]. The combination of
QT/AM-21 and PTX demonstrated greater ability to reduce cell proliferation than the combination of free AM-21 and PTX, suggesting greater uptake and/or efficacy of AM-
21 when delivered via QT. In an ovarian cancer model, resistance against PTX is suggested to be regulated by miR-21’s effect on hypoxia-inducible factor-1α (HIF-1α) and P-glycoprotein (P-gp) [136-138]. Decreased migratory and invasion potential was observed in a dose-dependent manner in response to QT/AM-21 treatment. Lung cancer is often diagnosed late in its development, when metastasis has already
38 begun[139]. Therefore, QT/AM-21 addresses a critical need that is currently unmet by chemotherapy administered in the late stage of the disease.
Treatment with QT/AM-21 in a xenograft mouse model could significantly suppress tumor growth at 1 mg/kg. Targets of miR-21 were also found to be upregulated at this dose. Relative increase in body weight indicated mice treated with QT/AM-21 as having better health and body condition [140]. Liver and spleen weight did not differ much between the two groups, suggesting that the formulation was not toxic to those organs. Liver and spleen are heavily fenestrated organs involved in the reticuloendothelial system with large local populations of macrophages, and LNPs may accumulate in these organs due to their similarities with tumor vasculature [141,
142]. The combination of PTX and QT/AM-21 demonstrated greater therapeutic activity than either agent administered alone. Interestingly, PTX appeared to have effects on miR-
21 targets. Actually, this is consistent with previous findings, showing effects of PTX on
PTEN and DDAH1 expression levels [143, 144]. Within certain cytotoxic thresholds,
PTX has been found to increase the expression as well as the activity of PTEN. It is therefore plausible that upregulation of DDAH1 can occur by a similar mechanism.
Additional studies will be required to validate the safety and efficacy of QT/AM-
21. Studies by Western blot would confirm whether the increase in mRNA for other target genes correlates with an increase in the associated protein levels [145]. It would also be interesting to see if miR-21 in NSCLC also regulates HIF-1α as in ovarian cancer to verify the mechanism by which miR-21 promotes resistance against PTX. Cellular uptake studies with pathway inhibitors would be likewise helpful to understand the
39 mechanism behind uptake of QTs, specifically to determine if QTsomes are taken up in the same clathrin-mediated endocytosis pathway as in the case of quaternary- or tertiary- based LNPs [146]. Incorporation of antimiR oligonucleotides into lipid nanoparticles is generally based on their electrical charge rather than base composition. Therefore, application of QTsome is not limited to delivery of AM-21. QTsomes may be used to deliver virtually any type of antimiRs, siRNAs, or miR mimics [147].Addition of a targeting agent may improve the specificity of delivery of QTsomes to NSCLC cells
[148]. Further toxicity, pharmacokinetic, and pharmacodynamics studies are needed to better characterize QT/AM-21 and aid its translation to the clinic.
2.5 Conclusion
QTsomes were prepared by a modified ethanol dilution method. QTsome nanoparticles exhibit small particle size, moderate zeta potential, high drug loading capacity, and long-term stability. In vitro analyses indicate a strong, dose-dependent upregulation of miR-21 targets with greater activity than formulations with either quaternary or tertiary amine cationic lipids alone. The combination of quaternary and tertiary amine cationic lipids forms a pH-sensitive system that is stable and possesses fusogenic activity in the endosome. Moreover, increased sensitivity to PTX and reduced migration and invasion were demonstrated with QT/AM-21 treatment. In vivo analyses in tumor-bearing mice reveal QT/AM-21 induced tumor regression, upregulated miR-21 target genes, enhanced anticancer activity with combination therapy, and prolonged
40 survival. Thus, these studies suggest that QTsome warrants further evaluation for therapeutic applications in NSCLC and other types of cancer.
41
Figure 2. 1 Schematic diagram of QTsome and pH responsive mechanism
Representation of lipid bilayer spherical structure of QTsome composed by neutral lipid, tertiary amine-cationic lipid and quaternary amine-cationic lipid, and the mechanism of enhanced endosome membrane disruption due to pH response.
42
Figure 2. 2 Particle size of QTsomes prepared under various mol%.
QTsome of varying mol % quaternary and tertiary amine content were prepared and analyzed by DLS for the effect on nanoparticle size (A). Data is presented as the mean ±
SD of three independent samples (n = 3).
43
Figure 2. 3 Surface charge of QTsomes suggesting pH responsive.
QTsome and LNP composed of only quaternary or tertiary amine cationic lipids were placed in buffer at physiological pH (7.4) and acidic pH (4.0) to determine the pH responsive effect on surface charge. Data is presented as the mean ± SD of three independent samples (n = 3).
44
Figure 2. 4 Encapsulation efficiency of oligo cargos in QTsomes.
Column separation of QTsome was completed on a Sepharose CL-4B column, collecting
1 mL fractions. Absorbance at 280 nm was measured to detect the presence of oligonucleotide.
45
Figure 2. 5 Colloidal stability of QTsomes.
To study the relationship between temperature and particle size, QT/AM-21 was stored at varying temperatures and the particle size was monitored over 4 weeks.
46
Figure 2. 6 Optimization of transfection efficiency on various quaternary–tertiary lipid ratio.
QTsomes of varying cationic lipid composition were prepared and evaluated for relative efficiency for modulating miR-21 target DDAH1. Data represent the mean ± SD of three separate transfections.
47
Figure 2. 7 Upregulation of miR-21 and its target genes by QT/AM-21 in A549 cells.
A549 cells were treated for 4 h with 100 nM QT/NC or QT/AM-21. (A) Expression of miR-21 by qRT-PCR, relative to RNU44. (B) Upregulation of miR-21 target genes, relative to GAPDH. Results are reported as the mean ± SE of three independent plates.
48
Figure 2. 8 Upregulation of mRNA expression in dose dependent pattern in A549 cells.
A549 cells were treated for 4 h with 1.56, 6.25, 25, or 100 nM QT/AM-21 to demonstrate the effects of dose dependency on target mRNA. Relative gene expression to GAPDH was evaluated 24 h following the start of treatment. Results are reported as the mean ±
SE of three independent plates.
.
49
Figure 2. 9 Upregulation of protein expression in dose dependent pattern in A549 cells.
A549 cells were treated for 4 h with 1.56, 6.25, 25, or 100 nM QT/AM-21 to demonstrate the effects of dose dependency on protein expression levels. Results are reported as the mean ± SE of three independent plates.
50
Figure 2. 10 Inhibited endosome release suggested by the presence of NH4Cl.
Relative gene expression of DDAH1 following QT/AM-21 treatment in the presence of
100 mM NH4Cl. Results are reported as the mean ± SE of three independent plates.
51
Figure 2. 11 Cell viability in response to treatment by QT/AM-21.
A549 cells were treated with QT/AM-21 at varying concentrations with and without PTX
(4.1 nM). MTS assay after 5 days’ treatment was used to assess cell proliferation.
Combination of QT/AM-21 with PTX significantly inhibited cell proliferation. Results are reported as the mean ± SD of three separate experiments.
52 QTsome/AM-21 QTsome/AM-21 QTsome/AM-21 Untreated AM-21 (200nM) QTsome (lipids) (50nM) (100nM) (200nM)
Without PTX
With PTX
Figure 2. 12 Changes in cell morphology following treatment with QT/AM-21 alone or in combination with PTX were imaged under light microscope.
While high concentrations of QT/AM-21 were evidenced by moderate morphological change, combinations of QT/AM-21 and PTX resulted in the greatest percentage of cell death.
53
Figure 2. 13 Migration and invasion in response to treatment by QT/AM-21.
(A) The percentage of A549 cells migrated into the wound region was evaluated 48 h following generation of a scratch wound across confluent cells. (B) Relative invasion capability of A549 cells following treatment was assessed using Matrigel invasion assay.
Results are reported as the mean ± SD of three separate treatments.
54
Figure 2. 14 Tumor regression by QT/AM-21 in A549 xenograft mice.
A549 xenograft models (n = 10) were created in nude mice and treatment began when tumors reached 180 mm3. Mice were treated with saline control, 0.5, or 1 mg/kg QT/AM-
21. Tumor size (mean ± SE) was monitored to determine relative tumor progression.
55
Figure 2. 15 Weight data on tumor-bearing mice treated with QT/AM-21.
Following the final treatment, mice were sacrificed and data on body, liver, spleen, and tumor weight was collected. Saline treated mice are compared to mice treated with 1 mg/kg QT/AM-21. Weights are expressed as mean ± SE, n = 10.
56
Figure 2. 16 Kaplan–Meier survival analysis of A549 xenograft mice treated with
QT/AM-21.
Mice (n = 10) treated with saline control, 0.5 mg/kg, and 1 mg/kg QT/AM-21 dose had a median survival time of 21, 24, and 33 days, respectively.
57
Figure 2. 17 Efficacy of QT/AM-21-PTX combination therapy in A549 xenograft model.
Mice (n = 5) were treated with saline control, PTX (3 mg/kg), QT/AM-21 (1 mg/kg), or combination of PTX and QT/AM-21 at the same doses. Tumor growth was monitored over 4 weeks. Tumors from mice were harvested 24 h following the last treatment and evaluated for modulation of targets. Data represent the results (mean ± SE) from three separate tumor sections.
58
Figure 2. 18 In vivo miR-21 target gene regulation by QT/AM-21 in A549 murine xenografts.
Tumors were harvested from mice and analyzed for modulation of miR-21 targets
DDAH1 (A) and PTEN (B). Results are presented as the mean ± SE of qRT-PCR results for three independent tumor sections.
59 CHAPTER 3: AntihypoxamiR Functionalized Gramicidin Lipid Nanoparticles
Rescue Against Ischemic Memory Improving Cutaneous Wound Healing
3.1 Introduction
Peripheral vasculopathies are primarily responsible for wound ischemia and hypoxia [149]. The biological response to ischemia depends largely on the state of hypoxia. If the hypoxia is moderate, allowing for sufficient residual oxygen to support tissue survival, cells minimize oxygen cost by economizing metabolism and makes an effort to mount adaptive solutions. On the other hand, if the hypoxia is severe or near- anoxic, then adaptive rescue is no longer an option. Cells bring down oxygen cost to the threshold that separates survival and death. This is sustainable for a limited time within which the tissue may be rescued by intervention or it yields to necrotic death [149].
Our previous work has recognized the induction of master hypoxamiR miR-210 in keratinocytes of ischemic wound-edge tissue[150-152]. Elevated miR-210 lowers oxygen cost of survival by repressing mitochondrial metabolism and attenuating keratinocyte proliferation. miR-210 also silences cell cycle proteins [150, 151] (Figure
3.1). Such measures defend survival but oppose healing because tissue repair requires oxidative metabolism and cell proliferation. Although the state of oxygenation of the
60 wound tissue may be corrected by intervention[149, 153, 154], growth of wound tissue is limited by an abundance of miR-210 repressing mitochondrial metabolism and cell proliferation. Thus, miR-210 may be viewed as an “ischemic memory” at the wound- edge tissue that is in direct conflict with wound healing. Healing outcomes of such ischemic wound tissue may be maximized by sequestering miR-210 (Figure 3.2). In the present work we report the development of a novel antihypoxamiR functionalized lipid nanoparticles (AFGLN) that is able to rescue against the ischemic memory of miR-210 improving cutaneous wound healing.
3.2 Materials and Methods
3.2.1 General study method
1,2-Dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), soy phosphatidylcholine (SPC), gramicidin
(GRAM), and d-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) were dissolved in ethanol and combined at the molar ratio of 40/5/30/20/5
(DODAP/DOTAP/SPC/GRAM/TPGS). The lipid mixture was then combined with an appropriate amount of LNA based miR-210 power inhibitor in 40% ethanol followed by serial dilution. Ischemic wound in C57BL/6 and repTOP™mitoIRE mice were induced by creating a bi-pedicle flap [150]. The repTOP™mitoIRE mice express the luciferase reporter gene under the control of an artificial minimal promoter derived from the Cyclin
61 B2 gene, specifically induced during cell proliferation. LNA based anti-miR-210 and negative control were purchased from Exiqon. miRNA isolation, Western blot, immunohistochemistry was performed as describe previously [155].
3.2.2 Preparation of antihypoxamir functionalized gramicidin lipid nanoparticles.
AntihypoxamiR Functionalized Gramicidin Lipid Nanoparticles (AFGLN) were prepared by an ethanol serial dilution method [27, 122]. First, all lipid components
(DOTAP/DODAP/GRAM/DOPC/TPGS, 25/15/20/30/10 mol/mol) were dissolved in ethanol. An LNA conjugate power anti-miR-210 inhibitor (Exiqon) solution (AM-210) in
1.5-volume of HEPES buffer was prepared. Rapid injection of the lipid solution into AM-
210 HEPES buffer maintaining at 1:10 AM-210: total lipids weight ratio was performed.
The mixture was further diluted with equivalent volumes (1:1) of HEPES buffer (20mM,
100mM NaCl, pH 7.4) twice. The resulting AFGLNmiR-210 were concentrated by tangential flow diafiltration method to remove free AM-210 and ethanol and also to achieve the desired concentration. The final product was stored at 4°C prior to characterization. For long-term stability and storage, 10% sucrose was added as a cryoprotectant and the AFGLN were either frozen under -20°C or lyophilized.
3.2.3 Particle size and surface charge characterization of AM-210 encapsulated in
AFGLN.
For mean particle diameter and surface charge measurement, aliquotes of
AFGLNantimiR-210 were diluted in PBS (20mM, pH 7.4). The particle size was determined
62 by dynamic light scattering method using a Zetasizer Nano ZS90 (Malvern, Worcestershire,
UK). SPLN-G were dispersed in double-distilled water and tested in volume-weighted size distribution mode. AFGLNantimiR-210 morphology and size distribution were further analyzed by NanoSight NS300 (Malvern, Worcestershire, UK). Aliquots of AFGLNantimiR-
210 or AFGLNscramble containing only lipid components were diluted in PBS with a series of pHs (50mM, from 2-11) to determine the pH dependency of surface charge. Zeta potential measurement was carried out on a Zetasizer Nano ZS90 (Malvern, Worcestershire, UK).
3.2.4 Study of encapsulation efficiency & colloidal stability
For encapsulation efficiency and stability, AFGLNantimiR-210 were firstly diluted into
1ml solution by 20mM HEPES buffer, and injected into Slide-A-Lyzer Dialysis Cassette
(Thermo Scientific, Rockford, USA). A one-liter beaker was filled with 20mM HEPES buffer. Dialysis cassette was then dropped into the beaker and incubated at room temperature for 4 hrs. Encapsulation efficiency was then performed by Quant-iT™
RiboGreen RNA Kit (Invitrogen, Grand Island, NY). AFGLNantimiR-210 was lysed with
Triton X-100 and the fluorescence intensity (FI) was determined using a luminescence spectrometer (KS 54B, Perkin Elmer, UK) at (480nm λex, 520nm λem). Loading efficiency was calculated with the following formula:
퐸푛푐푎푝푠푢푙푎푡𝑖표푛 퐸푓푓𝑖푐𝑖푒푛푐푦
퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ표푢푡 푇푟𝑖푡표푛 푋 − 100 = (1 − ) × 100% 퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ 푇푟𝑖푡표푛 푋 − 100
63 Formulation stability was evaluated at 4 and 25°C over a period of 21 days. The particle size was measured by Zetasizer Nano ZS90. Ten percent sucrose was added to serve as cryoprotectant.
3.2.5 Human subject
Human wound biopsy samples were obtained from chronic wound patients seen at
OSU Comprehensive Wound Center (CWC) clinic. All human studies were approved by
The Ohio State University’s (OSU) Institutional Review Board (IRB). Declaration of
Helsinki protocols was followed and patients gave their written informed consent.
3.2.6 Animals and wound models
Male C57BL/6 mice were obtained from Harlan Laboratory. Male repTOPTMmitoIRE mice were obtained from Charles River laboratory. Briefly, these mice have a luciferase reporter tagged with the cyclin B2 promoter. The repTOPTMmitoIRE mouse produces bioluminescence from any proliferating cell of the body, allowing the use of in vivo imaging to visualize areas of high activity with greater sensitivity of immunohistochemistry. All animals were 8-10 weeks old at the time of experiment. All animal studies were performed in accordance with protocols approved by the Laboratory
Animal Care and Use Committee of the Ohio State University. No statistical methods were used to predetermine sample size. Power analysis were not necessary for this study. The animals were tagged and grouped randomly using a computer based algorithm
64 (www.random.org). None of the mice with the appropriate genotype were excluded from this study.
Ischemic wounds were developed on the back of the Mice using a bipedical flap model as described previously [150]. Digital images of the wounds were taken at different time points post wounding as indicated. Wound area measurement was done by digita planimetry using Image-J software (NIH), as described previously [156].
3.2.7 In vivo imaging
Potassium salt of Beetle luciferin was administered intraperitoneally to the repTOPTMmitoIRE mice 15 mins prior to IVIS imaging. The animals were imaged with anesthesia using IVIS Lumina II optical imaging system. Overlay images with luminescence images were made using Living Image software.
Perfusion imaging was performed using Laser Speckle Perfusion imaging system
(Perimed Inc., Sweden). Color coded perfusion maps were acquired at all time points and average perfusion was calculated using PimSoft v1.4 software (Perimed Inc., Sweden). The wound edge and wound bed tissue regions were chosen as region of interests (ROI). From the real-time graphs obtained, time-of-interest (TOI) was chosen to include lower peak regions and to exclude motion related artifacts. Mean and standard deviation of perfusion data were obtained from the selected TOI perfusion data.
High-resolution 31P NMR spectra were obtained at 200 MHz using a Bruker
Advance 500 WB spectrometer (Bruker Bio-spin; Ellington, Germany). Proton-decoupled
31P spectra were collected using a B_31PSpectroscopy pulse sequence with a predelay of
65 1.35s for an acquisition time of 2.25min. An average of 19K 31P free induction decays
(FIDs) were collected in 16K data points with a spectral width of 20kHz, then Fourier transformed with line broadening and manually phase corrected using Bruker ParaVision
5.1 and TopSpin software package. Phosphorus resonances obtained and spectra were acquired. As the molecular weight of each standard phospholipid was unknown due to heterogeneous fatty acid chains, quantization was determined by integrating each phospholipids peak. The spectrum so obtained contains peaks of inorganic phosphorus (Pi),
Phosphocreatine (PCr), and Adenosine Triphosphate (ATP) group including, γ-ATP,α-
ATP,and β-ATP.
3.2.8 Laser capture microdissection (LCM) of epidermis
Epidermal Laser Capture Microdissection (LCM) was performed using the laser microdissection system from PALM Technologies (Bernreid, Germany) as described previously by our group [150, 157]. Briefly, sections were stained with hematoxylin for
30s, subsequently washed with DEPC-H2O and dehydrated in ethanol as described [158].
Epidermal fractions were identified based on morphology, cut and captured under a 20× ocular lens. The samples were catapulted into 25 μl of cell direct lysis extraction buffer
(Invitrogen). Approximately 10,00,000 μm2 of tissue area was captured into each cap and the lysate was then stored at −80°C for further processing [150, 157].
66 3.2.9 RNA extraction and quantitative real-time PCR.
RNA from murine wound edge tissue sample was extracted using miRVana miRNA isolation kit (Ambion) according to the manufacture’s protocol [159]. For determination of miR expression, specific TaqMan assays for miRs and the TaqMan miRNA reverse transcription kit were used, followed by real time PCR using the Universal
PCR Master Mix (Applied Biosystems, Foster City, CA). mRNA was quantified by real- time or quantitative (Q) PCR assay using the double-stranded DNA binding dye SYBR
Green-I [159].
3.2.10 Immunohistochemistry and microscopy.
Immunostaining of Keratin14 (Covance; PRB-155P) and Ki67 (Covance; PRB-
145P) was performed on cryosections of wound edge sample using specific antibodies as described previously [160]. Briefly, OCT embedded tissue were cryosectioned at 10μm thick, fixed with cold acetone, blocked with 10% normal goat serum and incubated with specific antibodies against, Keratin14 (1:400) and Ki67 (Covance; PRB-155P). Signal was visualized by subsequent incubation with fluorescence-tagged appropriate secondary antibodies (Alexa 488-tagged α-rabbit, 1:200; Alexa 568-tagged α-rabbit, 1:200) and counter stained with DAPI. Images were captured by microscope and quantification of fluorescent intensity of image was achieved by software AxioVision Rel 4.6 (Carl Zeiss
Microimaging).
67 3.2.11 Data collection and statistical analyses.
Samples were coded and data analysis was performed in a blinded fashion. For animal studies, data are reported as mean ± SD of at least 4-8 animals as indicated. In vitro data are reported as mean ± SD of 3-6 experiments as indicated in respective figure legends.
Student's t test (two-tailed) was used to determine significant differences. Comparisons among multiple groups were tested using analysis of variance (ANOVA). p<0.05 was considered statistically significant.
3.3 Results
3.3.1 Negative correlation between expression of miR-210 and keratinocyte proliferation in ischemic wound.
Ischemic foot ulcers in patients have the worst outcome of all chronic skin wounds, with high amputation and mortality rates. Although commonly encountered in diabetic patients with ischemic limbs, impaired cell proliferation is a critical determinant of wound closure. Wound-edge biopsies were collected from chronic ischemic and non-ischemic wounds from patients at the comprehensive wound center at the Ohio State University
Wexner Medical Center. Consistent with the previous reports on other models[150, 161], laser capture microdissection of human wound-edge epithelium revealed elevated miR-210 expression compared to the non-ischemic epithelium (Figure 3.3). Expression of Ki-67, a
68 marker of cell proliferation, was also found to be significantly low in the epithelial tongue
[162] of the human ischemic wound (Figure 3.4). The expression of miR-210 was inversely co-related with the number of proliferating keratinocytes in patients with chronic ischemic wounds (Figure 3.5). In order to evaluate an efficacy of anti-miR-210 in ischemic wound closure, locked nucleic acid (LNA) conjugated antimiR-210 power inhibitor was delivered intra-dermally in murine model. However, even at a higher dose of 500nM, the antimiR-
210 was ineffective in bringing down the expression of hypoxia induced miR-210 (Figure
3.6).
3.3.2 Development and characterization of AFGLN lipid nanoparticles for miRNA delivery
Wound closure is the single-most important outcome, as viewed by the United
States Food and Drug Administration, when evaluating wound-related therapeutics.
Clinical application of antagomir requires safe and efficient delivery in vivo. The implementation of therapeutic antisense oligonucleotides or miRNA is limited by several critical barriers to delivery [163]. We sought to develop LNPs optimized for the delivery of antagomir cargo to the cutaneous wound tissue. SPLN-G have been increasingly recognized as one of the most promising delivery systems in RNA interference therapy due to their biocompatibility and the ease of large-scale production [164, 165]. We have designed and optimized the AFGLN composition based on SPLN-G and evaluated the surface charge to accommodate the wound bed microenvironment (Figure 3.7). The formulation had an average diameter of 150 nm and a zeta potential of +10 to -10 mV
69 within the pH of 5-8 respectively. The encapsulation efficiency of these nanoparticles was found to be 80.12%. This composition was selected as the delivery vehicle for the subsequent experiments and termed Antihypoxiamir (antimiR-210) functionalized gramicidin lipid nanoparticles (AFGLN). Size and concentration of AFGLN were further examined by nanosight (Figure 3.8).
3.3.3 In vivo evaluation of AFGLNantimiR-210 efficacy in ischemic wound re- epithelialization.
Intradermal delivery of AFGLN containing LNA conjugate antimiR-210 power inhibitor (AFGLNantimiR-210) at the wound-edge tissue in the murine model of ischemic wound significantly downregulated the hypoxia induced miR-210 expression in C57Bl/6 mice (Figure 3.9). To evaluate the effect of AFGLNantimiR-210 on ischemic wound closure, bi-pedicle flaps were prepared on C57BL/6 mice. Digital planimetry demonstrated significant improvement of closure in AFGLNantimiR-210 group from day 3 post-wounding
(Figure 3.10). To study whether delivery of AFGLNantimiR-210 accelerated wound re- epithelialization, frozen ischemic wound-edge tissue sections at day 7 post-wounding were stained for keratin-14. Wound re-epithelialization was severely compromised in the ischemic wounds that were treated with empty GLN (devoid of any cargo) alone or
AFGLN packed with scrambled oligos (AFGLNscramble) (Figure 3.11 & 3.12). In contrast,
AFGLNantimiR-210 treated ischemic wound showed improved re-epithelialization with the presence of characteristic hyperproliferative epithelium as would be expected in healing wounds (Figure 3.12). Because mouse is a loose skin animal and wound contraction may
70 confound closure, we used a splinted wound model to limit contraction. AFGLNantimiR-210 treatment improved re-epithelialization (Figure 3.13).
3.3.4 In vivo evaluation of AFGLNantimiR-210 efficacy in restoring oxidative metabolism and proliferation.
Consistent with the above-mentioned favorable effects of AFGLNantimiR-210, bi- pedicle flaps were created with ischemic wounds on repTOP™mitoIRE mice. These mice express the luciferase reporter gene under the control of an artificial minimal promoter derived from the Cyclin B2 gene, specifically induced during cell proliferation. As shown in Figure 3.14, in a setting where the perfusion of ischemic wound was equally limited in all groups, delivery of AFGLNantimiR-210 rescued cell proliferation as evident from the luminescent images of the in vivo imaging system (Figure 3.15). This finding was substantiated by immunohistochemical detection of Ki67 (Figure 3.16). 31P NMR studies validated the rescue by demonstrating elevated ATP content at the ischemic wound-edge tissue in AFGLNantimiR-210 treated group compared to GLN alone or AFGLNscramble group
(Figure 3.17), suggesting that the wound-edge keratinocytes are metabolically active in spite of being ischemic. These observations demonstrate that AFGLNantimiR-210 is effective in restoring oxidative metabolism in ischemic wound edge tissue.
3.4 Discussion
One of the common outcome of wound healing in ischemic injury is revascularization [166]. Till date five major factors have been identified that limits wound
71 healing post revascularization. They are A) the site and extent of ischemic insult [167], B) infection [168] B) host immune response post revascularization [169] D) inappropriate revascularization [170] and E) suboptimal therapy [171]. In the present work, we identified yet another potential factor that compromises ischemic wound re-epithelialization.
Clinical intervention to reoxygenate chronic ischemic wounds such as oxygen therapy, debridement, or recanalization is likely to face metabolic barriers such as the miR-
210 ischemic memory which when abundant in the wound tissue resists tissue growth
(Figure 3.18). MicroRNAs are widely recognized as key players in wound healing [172-
175]. The hypoxymiR-210, driven by HIF-1 has been known to impair tissue metabolism by suspending mitochondrial respiration and other important cellular process [150].
Although this may be regarded as a cellular response to enter into a hibernating mode under conditions of ischemic insults, yet it has some drawback. MicroRNAs are extremely stable with an average half-life of 5 days, almost 5 times that of mRNA [176]. Thus, once expressed, they persist within the cells for a considerable period even when the source is removed as in this case hypoxia. Neutralizing such barriers is likely to improve the effectiveness of standard clinical interventions aimed at re-oxygenating the ischemic wound tissue. In this work, we designate the presence of miR-210 within the tissue post hypoxia as “the ischemic memory”.
Anti-miR therapy have potential therapeutic relevance [177]. However proper delivery of the anti-miR to the site action has always been a challenge. Several potential methods to deliver the oligonucleotides has been developed to enhance delivery efficiency such as direct injection, lentivirus, nanoparticles etc [178]. However, there is no evidence
72 of lipid nanoparticle based intervention of miR delivery or inhibition in the skin. In cutaneous injury, an additional challenge is the rapid removal of such nanoparticles by wound associated phagocytes [179].Furthermore, the wound microenvironment features low pH which influences the dynamics of cargo release to the site of injury [52]. Thus, design of lipid nanoparticles for skin health application, including tissue repair, must troubleshoot these challenges. Soy phosphatidylcholine has been utilized for the bilayer formation along with cationic lipid with tertiary (DODAP) and quaternary (DOTAP) amine headgroups. Incorporation of Gramicidin A was aimed at improving endosomal escape and facilitating ion channel formation in the lipid bilayer [180]. Intradermal injection of
AFGLN was effective in cargo-delivery. Observations of this work substantiate the ischemic memory hypothesis by rescuing wound-re-epithelialization in response to inhibition of hypoxamir miR-210 [152]. AFGLN was effective in rescuing the ischemia affected wound tissue and resuming re-epithelialization. The formulation of AFGLN reported in this work would be effective to deliver any cargo of interest to the healing cutaneous wound tissue. A clear translational advantage of AFGLN is that all materials used for its formulation have prior history of FDA approval for human use [20-22]. Our method of antimiR-210 delivery using lipid nanoparticle based approach is effective, safe, stable and most importantly, more translationally relevant.
One of the limitation of this study is that the nanoparticles are not targeted.
However, the presence of the hydrophobic peptide, Gramicidin A, significantly increases the cellular uptake of the nanoparticles. Incorporation Gramicidin A improve endosomal escape and facilitate ion channel formation in the lipid bilayer [180]. Since expression of
73 miR-210 is robust and universal in all cell types in response to hypoxia with similar biological outcome, hence non-specific delivery is not a major concern in this study.
74
Figure 3. 1 Schematic diagram showing the different proteins involved in cell cycle regulation that are targeted by miR-210. The number in the parenthesis indicates the references.
75
Figure 3. 2 Schematic diagram illustrating AFGLNmiR-210 rescuing ischemic wound hypoxia memory to facilitate efficient wound closure.
76
Figure 3. 3 miR-210 expression from laser microdissected epidermis of human wound-edge tissue.
Expression level of miR-210 were analyzed between non-ischemic tissue (NI) and ischemic wound tissue (I). (n = 7). * P < 0.001; ANOVA.
77
Figure 3. 4 Serial human wound cross-sections stained with anti-Ki67 and keratin-
14 antibody, counter stained with DAPI.
The plot represents quantification of the Ki67 positive cells/field (20X). Ki67 fluorescent intensity level were analyzed between non-ischemic tissue (NI) and ischemic wound tissue (I). (n = 4-5).
78
Figure 3. 5 Regression plot of miR-210 expression from the human wound edge biopsies against number of Ki67 positive cells/field (20X). (n = 8)
79
Figure 3. 6 miR expression level after LNA treatment from wound edge tissue. miR-210 expression from murine non-ischemic and ischemic wound-edge tissue 24 h after intradermal delivery of naked LNA-anti-miR-210. (n = 4). * P < 0.01; † P < 0.05 compared to non-ischemic wound, ANOVA.
80
Figure 3. 7 Schematic representation of the AntihypoxiamiR Functionalized
Gramicidin Lipid Nanoparticles (AFGLN) and the zeta potential of the AFGLN at different pH. Shaded region represents the pH range in chronic wounds.
81
Figure 3. 8 Representation of nanoparticle size and counts.
Representative nanoparticle tracking analysis (NanoSight™) showing particles size and concentration of the empty GLN (devoid of any cargo), AFGLN packed with scrambled oligos (AFGLNscramble) and AFGLN packed with anti-miR-210 (AFGLNmiR-210) (n = 4).
82
Figure 3. 9 miR-210 expression from murine non-ischemic and ischemic wound- edge tissue 24 h after intradermal delivery of AFGLNmiR-210. (n = 4). * P < 0.01 compared to AFGLN, ANOVA.
83
Figure 3. 10 Percentage of wound closure after delivery of empty GLN,
AFGLNscramble and AFGLNmiR-210. (n = 5), * P < 0.001 compared to Day 0; ANOVA.
84
Figure 3. 11 The percentage of re-epithelialization at day 6 post wounding was plotted graphically.
Digital photographs of open wounds were taken and calculated using ImageJ on the same scale (n = 3), * P < 0.001; ANOVA.
85
Figure 3. 12 Representation of wound closure and re-epithelialization.
(i) Digital photographs of murine ischemic wound at days 0, 2, 4 and 6 after delivery of empty GLN, AFGLNscramble and AFGLNmiR-210 (ii) H&E images stained sections from ischemic wounds at day 6 post-wounding. (iii) Wound cross sections were stained with anti-keratin 14 antibody and counterstained with DAPI to show re-epithelialization at day
6 following treatment with GLN, AFGLNscramble and AFGLNmiR-210. (n = 4).
86
Figure 3. 13 Wound closure suggested by contrast digital photographs and immune- staining.
(A) High contrast digital photographs of murine ischemic wounds were treated with empty GLN, AFGLNscramble and AFGLNmiR-210. Wound closure has been represented graphically (n=4). Data expressed as mean ± SD (n=4), *p<0.001 compared to d0. Scale
87 bar=3mm (B) Representative serial wound cross-sections stained with anti-Keratin14 antibody counterstained with DAPI (blue). The white arrow head indicates the leading edge of the epithelium. Scale bar=1000μm. (n=4). The percentage of re-epithelialization was plotted graphically. Data expressed as mean ± SD (n=4), *p<0.001; ANOVA.
88
Figure 3. 14 Laser speckle image showing perfusion level in the bi-pedicle flap at day 0 and day 7 after delivery of only GLN, AFGLNscramble and AFGLNmiR-210.
89
Figure 3. 15 IVIS image from repTOP™mitoIRE showing cell proliferation in animal treated with lipid nanoparticles with AFGLNmiR-210.The images from quantification of the mean luminescence have been presented. (n = 3)
90
Figure 3. 16 Serial wound cross-sections stained with anti-Ki67 antibody counter stained with DAPI (blue). (n = 3).
91
Figure 3. 17 31P NMR spectra of the ischemic wound-edge tissue at day 7 post wounding showing iP, and the three subunits α-, β- and γ- subunit of ATP normalized with phosphocreatinine
92
Figure 3. 18 Schematic diagram showing interventions available for reoxygenation of ischemic tissue.
Although effective, healing is often associated with complications due to presence of miR-210.Sequestration of miR-210 prior to intervention can eliminate the complications associated with healing of ischemic wound. Anti-miR therapy may involve direct injection, viral vectors or lipid nanoparticles. Intranslational study, lipid nanoparticle based anti-miR delivery (marked in green) is much safer than direct injection or lentiviral delivery.
93 CHAPTER 4: Topical Lyophilized Targeted Lipid-Nanoparticles in the Restoration
of Skin Barrier Function Following Burn Wound
4.1 Introduction
Lipid nanoparticles (LNPs) are promising as carriers of nucleic acid therapeutics.
They have been used in cancer treatment for delivering targeted agents [181], antisense oligonucleotides [182, 183], small interfering RNA (siRNA) [184-187], mRNA [188] and
DNA inhibitors [189]. Particle size and surface modifications are important parameters for
LNPs [190, 191]. Cell targeting can be achieved by conjugating antibodies to LNP surface
[191]. Although such approach is effective in vitro, in vivo applications are fraught with the risk of immunogenicity following repeated injections [192]. Furthermore, barriers such as high cost, regulatory hurdles and limited shelf life complicate the translational path [191].
Nonetheless, both therapeutic as well as diagnostic nanoparticles are currently undergoing clinical testing [193].
Skin is a promising route of administration for local effects [194]. Small LNPs
(<10nm) may directly penetrate the stratum corneum of viable human skin [195].
Meanwhile, larger LNPs (10-200nm) access the skin via hair follicle openings, especially after massage [195]. The low density of hair follicles (32 follicles/cm2) relative to the whole
94 skin surface represents a limitation of the delivery system [196]. Newer delivery systems rely on chemical enhancers, non-cavitational ultrasound and iontophoresis, as well as additional measures such as microneedles or thermal abrasion to penetrate the stratum corneum [194]. Targeted delivery of therapeutic cargo across intact stratum corneum remains a challenge.
In wounds, although skin barrier function is breached, abundance of inflammatory cells at the site of injury poses a different set of challenges. Phagocytic clearance of nanoparticles poses an issue for delivery [197-199]. In this work, we sought to develop a novel delivery platform based on lyophilized keratinocyte targeting LNPs (TLNκ) to facilitate keratinocytes-specific delivery of oligonucleotides such as LNA anti-microRNAs.
Hydrogel assisted delivery of lyophilized TLNκ was shown to be robust, offering translational advantages and a longer shelf life in wound care.
The understanding of burn wound healing is generally based on the depth of burn
[200]. Wound infections have been thought to complicate wound healing as well as affecting post-trauma skin heath due to the formation of biofilm [201]. Formation of biofilm is considered as the leading cause of death in burns due to sepsis [200]. Although speed of wound closure has not been found to relate to biofilm infected skin, it has been suggested that biofilm can compromise the barrier function of regenerated skin [201].
Loss of barrier function deteriorates recovery of full healthiness skin and results in frequent infection and chronic wound [201]. Skin barrier is the formation of upper epithelium cornification contributed by differentiation of basal keratinocytes which function as a barrier of outside environment [156]. Our previous research has shown that
95 arrest of Dicer would impair regeneration of barrier function [156]. Dicer depletion results in inhibited production of miR-93, miR-106b, and miR-20a, causing elevation of p21 expression thus disrupting the barrier function [156]. It has been elaborated that miR-
107 is found to target Dicer formation, therefore, we considered it an interesting target for barrier function reconstruction [156, 202]. More interestingly, miR-107 has been previously shown to regulate cell proliferation and migration [203-205]. Thus, we hypothesized that upon successful delivery of antimiR-107 into keratinocytes, we could achieve barrier function regeneration as well as accelerated wound closure.
Upon the boosting discovery of RNA/DNA based therapeutics, nanoparticles have become a spotlight on the stage and become favorable as a trustworthy delivery platform.
Several nanoparticles based drug have been approved by FDA, and many of them are in the clinic [193]. However, its function in wound management remains undiscovered, therefore we proposed to explore a topical miRNA delivery platform for the treatment of full thickness burn. Based on our previous research, we have developed a sophisticated nanoparticle delivery platform with pH-stimuli release modules to benefit transfection efficiency [26]. Quaternary and tertiary amine based phospholipids are employed to introduce high delivery efficiency. A previous established design has been employed and reformed to match wound bed scenario [206]. At the wound site with the presence of phagocyte, monocyte and macrophage cell, highly charged nanoparticles are prone to be recognized and captured by immune cells, which results in reduced delivery to keratinocyte cells while cause unwanted uptake and potential adverse effects [207].
Hence, we designed TLNκ with mild zeta potential at environmental pH while equipped
96 with keratinocyte targeting ligand to induce cell uptake. Keratinocyte targeting ligand was first reported to equipped with a strong binding to keratinocyte surface heparin receptors[106]. Higher surface charges are triggered when TLNκ are captured into endosome/lysosome complex to benefit cargo release into cytoplasm [26].
Lyophilized TLNκ are developed due to the consideration of long-term storage and convenient application. In topical delivery, conventional lipid nanoparticles were restricted by intact stratum corneum, and tends to accumulated in hair follicles [208]. Due to the damage of skin barrier function, nano-sized particles can effectively pass through damaged stratum corneum and reach to the keratinocyte basal cells. The lyophilized
TLNκ is reconstituted with commercial available hydrogels as water suppliant and wound dressing. Further Tegadrem film are used to cover the wound site while reducing exposure to infection.
4.2 Materials and methods
4.2.1 Materials
1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane
(DODAP), and 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000-Amine) were obtained from Avanti Polar Lipids (Alabaster,
AL, USA). D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and
97 trimethylamine (TEA) were obtained from Sigma-Aldrich (St. Louis, MO, USA).
PEGylated bis(sulfosuccinimidyl)suberate (BS(PEG)5) and Vybrant™ DiD Cell-Labeling
Solution were purchased from ThermoFisher Scientific (Waltham, MA, USA). A5G33 peptide (sequence: ASKAIQVFLLAG) was custom synthesized by Genscript (Township,
NJ, USA). 3MTM TegadermTM hydrogel was obtained from 3M (St. Paul, MN, USA). Anti- miR-107 and anti-miR-210 with Locked-Nucleic-Acid (LNA) modification as well as negative control A LNA were synthesized by Exiqon Inc (Woburn, MA, USA).
4.2.2 Lipid-Peptide Conjugation.
Lipid peptide conjugation was done by dissolving 4mg of DSPE-PEG2000-Amine in 500ul of dimethyl sulfoxide (DMSO) and combined with 5.7µl of 250mM homobifunctional crosslinker (BS(PEG)5) with addition of 4µl of TEA. The reaction proceeded in an oxygen-free environment (nitrogen purge) at room temperature for overnight. Next, 0.5mg of A5G33 peptide was added into the solution and incubated for 2 hours at room temperature. The molar ratio between DSPE-PEG2000, BS(PEG)5 and A5G33 peptide was controlled at 1:1.2:1.4. Thin layer chromatography was used to evaluate the efficiency of conjugation reaction.
4.2.3 Preparation of TLNκ.
TLNκ were prepared by an ethanol serial dilution method, as described previously
[26, 27]. First, lipid components (DOTAP/DODAP/DSPE-PEG2000-A5G33/DOPC/TPGS,
30/24/3/41/2 mol/mol) were dissolved in ethanol. If lipophilic fluorescence dye DiD were
98 chosen to label the LNPs, 0.2% mol/mol amount of dye was added into the above formulation recipe. LNA based antimiR was dissolved in equal volume of citric acid buffer
(5 mM, pH 4.5) as prepared lipid ethanol solution. The lipid solution was rapidly injected into LNA citric acid buffer at 1:10 antimiR-107: total lipids weight ratio. This mixture was further diluted by equivalent volumes (1:1) of HEPES buffer (25 mM, 135 mM NaCl, pH
7.4) twice. Non-targeting LNPs were formulated similarly by substituting DSPE-PEG2000-
A5G33 with DSPE-PEG2000-Amine and used as controls. The final product was stored at
4°C.
4.2.4 Lyophilization procedure.
A primary-secondary drying program was employed[209]. 1ml of LNP samples were transferred into 5ml lyophilization vials (Wheaton, Millville, USA) and diluted with equal volume of 20% sucrose solutions. Slow-freezing process were chosen: sample vials were partially capped and cooled in a sequence of 2 hours each at 4 °C, −20 °C and −80 °C.
Frozen samples were primary dried at −30 °C and 0.12mbar for 24 hours followed with
25°C and 0.08mbar secondary drying for additional 6 hours in a shelf dryer from Labconco
(Kansas City, MO, USA). Lyophilized sample stoppers were seated in vacuum and stored at 4 °C.
4.2.5 Size and zeta potential measurements.
For mean particle diameter and surface charge measurement, aliquotes of TLNκ were diluted in PBS (20mM, pH 7.4). The particle size was determined by dynamic light
99 scattering method using a Zetasizer Nano ZS90 (Malvern, Worcestershire, UK). TLNκ were dispersed in double-distilled water and tested in volume-weighted size distribution mode. TLN morphology and size distribution were further analyzed by NanoSight NS300
(Malvern, Worcestershire, UK). Aliquots of TLNκ containing antimiR-107 were diluted in
PBS with a series of pHs (50mM, from 2-11) to determine the pH dependency of surface charge. Zeta potential measurement was carried out on a Zetasizer Nano ZS90 (Malvern,
Worcestershire, UK).
4.2.6 Encapsulation efficiency.
For encapsulation efficiency and stability, TLNκ/antimiR-107 were firstly diluted into
1ml solution by 20mM HEPES buffer, and injected into Slide-A-Lyzer Dialysis Cassette
(Thermo Scientific, Waltham, MA, USA). A one-liter beaker was filled with 20mM
HEPES buffer. Dialysis cassette was then dropped into the beaker and incubated at room temperature for 4 hrs. Encapsulation efficiency was then performed by Quant-iT™
RiboGreen RNA Kit (Invitrogen, Grand Island, NY, USA). TLN-LNA-107 was lysed with
Triton X-100 and the fluorescence intensity (FI) was determined using a Multi-Mode
Microplate Readers (Biotek, Winooski, VT, USA) at (480nm λex, 520nm λem). Loading efficiency was calculated with the following formula:
퐸푛푐푎푝푠푢푙푎푡𝑖표푛 퐸푓푓𝑖푐𝑖푒푛푐푦
퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ표푢푡 푇푟𝑖푡표푛 푋 − 100 = (1 − ) × 100% 퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ 푇푟𝑖푡표푛 푋 − 100
100 Formulation and lyophilized cake stability was evaluated at -20, 4 and 25°C over a period of 9 days. The particle size was measured by Zetasizer Nano ZS90.
4.2.7 Cell and cell culture
Immortalized human keratinocytes (HaCaT) were grown in Dulbecco’s low- glucose modified Eagle’s medium (Life Technologies) as described previously [210].
Human dermal microvascular endothelial cells (HMECs) were cultured in MCDB-131 medium supplemented 10 mm l-glutamine, and 100 IU/ml of penicillin, 0.1 mg/ml of streptomycin (Invitrogen), as described previously [211]. THP-1 are human (pro-) monocytic cell lines that can be differentiated into either dendritic cells or tissue macrophages [212]. THP-1 cells were differentiated to macrophages using PMA (20 ng/ml,
48hr) as previously described [213]. Differentiated human THP-1 cells were cultured in
RPMI complete medium as described previously [213, 214]. Human skin fibroblast BJ cells (ATCC® CRL-2522™) were obtained from ATCC and were cultured in Eagle's
Minimum Essential Medium, (Catalog No. 30-2003) as per the instruction provided. All cell cultures media were supplemented with 10% FBS and 1% antibiotic-antimycotic (AA)
(Life Technologies) unless stated otherwise. Cells were maintained in a standard culture incubator with humidified air containing 5% CO2 at 37°C.
101 4.2.8 Transfection of miRNA mimic and inhibitors.
HaCaT cells (0.05 x 106 cells / well in 12-well plate) were seeded for 18-24h prior to transfection. HaCaT cells were transfected with miRIDIAN hsa-miR-107 mimic (50 nM), hsa-miR-107 inhibitor (100nM) using DharmaFECTTM 1 transfection reagent
(Thermo Scientific Dharmacon RNA Technologies, Lafayette, CO) as per the manufacturer’s instructions[215-217]. miRIDIAN miR mimic/inhibitor negative controls
(Thermo Scientific Dharmacon RNA Technologies, Lafayette, CO) were used as control.
Cells were collected after 48h of miR mimic or 72h of miR inhibitor transfection for quantification of miRNA or protein expression. In some experiments, the cells were further reseeded in 96-wells plate or 2-well inserts for cell proliferation or cell migration assay.
4.2.9 Determination of cell proliferation.
Cell proliferation was measured using a Vybrant MTT Cell Proliferation Assay
Kit (Thermo Fisher Scientific) per the manufacturer’s instructions as described previously[218]. At 24h after treatment, cells were incubated in fresh low glucose
DMEM culture medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) for 4–6 h at 37°C with 5% CO2. After MTT treatment, medium was removed, and DMSO was added (10–20 min at 37°C with 5% CO2) to solubilize formazan produced as a result of MTT metabolism. DMSO extract from each well (100
μl) was collected in a 96-well plate, and formazan content was determined by reading absorbance at 540 nm.
102 4.2.10 Cell migration assay.
Cell migration assay was performed using culture inserts (IBIDI, Verona, WI) according to the manufacturer’s instructions as discussed previously [219]. Briefly, confluent cellular monolayer was made in the presence of the insert inside a 24-well plate. Removal of the insert generated a gap in the monolayer. Migration of cells across that gap was studied at different time points following withdrawal of the insert. Images were captured using an inverted microscope (Axiovert 200M, Zeiss, Germany) and analyzed using Zen software.
4.2.11 Measurement of targeting efficiency by Flow cytometry
The fluorescence of the cells was determined by using an Accuri C6 flow cytometer
(Accuri Cytometers, Ann Arbor, MI, USA). Signals from cells labeled with DiD fluorescent dye were collected on channel FL4 (650 ± 20 nm) after excitation with a 488- nm solid-state laser on a gated population of cells. Data were collected from at least 10,000 cells at a flow rate 250–300 cells/s. A logarithmic scale was used to measure both background and cell fluorescence. Background fluorescence was then subtracted from cell fluorescence, allowing linear comparisons.
4.2.12 Animal model.
To generate reproducible, full-thickness burn wounds, an electrically heated burn device with controlled pressure delivery was manufactured. A 0.8cm diameter stainless steel burning stylus was brought to temperature by an electrically heated element (150-
103 Watt soldering iron, Great American Inc.) inserted into the middle of the stylus.
Temperature was controlled by an electronic thermostat (eTRON M, JUMO Process
Control Inc., NY) which measured the burn stylus temperature using a thermocouple inserted into the center of the stylus and controlled the amount of current sent to the heating element. Applied pressure was regulated through a compression system comprised of upper and lower aluminum guides connected by spring loaded guide posts. The heating element can be brought into contact with the murine skin and pressure applied. When maximum pressure is reached the springs collapse fully and prevent further depression of the burner stylus into the skin below.
Male C57BL/6 mice were obtained from Harlan Laboratory (Indianapolis, IN).
Experimental thermal injury was caused by 110°C heated stylus for 15 seconds. Animals were randomly divided into groups based on a computer-generated algorithm
(www.random.org). All animal studies were performed in accordance with protocols approved by the Laboratory Animal Care and Use Committee of The Ohio State University.
During the wounding procedure, mice were anesthetized by low-dose isoflurane inhalation as per standard recommendation. Each wound was digitally photographed at the time point indicated. Wound area was calculated using ImageJ software (NIH, Bethesda, MD).
The animals were euthanized at the indicated post-wounding time point, and wound-edge tissues (2 mm away from the wound, snap frozen) or the wound tissues in optimal cutting temperature compound (OCT) were harvested.
104 4.2.10 Trans-epidermal water loss (TEWL).
TEWL serves as a reliable index to evaluate the skin barrier function in vivo [201,
220]. TEWL was measured from the wounds using DermaLab TEWL Probe (cyberDERM,
Broomall, PA)[201]. The data were expressed in gm-2 h-1.
4.2.13 Laser capture microdissection (LCM) of epidermis.
Epidermal Laser Capture Microdissection (LCM) was performed using the laser microdissection system from PALM Technologies (Bernreid, Germany) as described previously by our group[150, 157]. Briefly, sections were stained with hematoxylin for 30s, subsequently washed with DEPC-H2O and dehydrated in ethanol as described previously
[158]. Epidermal fractions were identified based on morphology, cut and captured under a
20× ocular lens. The samples were catapulted into 25 μl of cell direct lysis extraction buffer
(Invitrogen). Approximately 10,00,000 μm2 of tissue area was captured into each cap and the lysate was then stored at −80°C for further processing[150, 157].
4.2.14 RNA extraction and quantitative real-time PCR.
RNA from mouse wound-edge tissue or cells was isolated using miRVana miRNA
Isolation Kit according to the manufactures protocol (Ambion Life Technologies) as described previously[221]. The RNA quality was assessed using Agilent 2100 Bioanalyzer
(Agilent Technologies, Santa Clara, CA). Quantification of mRNA expression was done by quantitative real-time PCR (qRT-PCR) using SYBR Green-I [157, 222]. For
105 determination of miR expression, specific TaqMan assays for miRs and the TaqMan miRNA reverse transcription kit were used, followed by real-time PCR using the Universal
PCR Master Mix (Applied Biosystems, Foster City, CA) [159].
4.2.15 Histology, Immunohistochemistry and microscopy.
Histology of skin was performed from 8µm thick paraffin sections after staining with Hematoxylin and Eosin (H&E). Immunostaining of Dicer (Abcam; ab13502; 1:200), p21Waf1/Cip1 (Santa Cruz; sc-397; 1:200), Keratin 14 (Covance; PRB-155P; 1:400), Claudin
1 (Invitrogen, RB9209P; 1:200), loricrin (Covance; PRB-145P; 1:400), filaggrin (Covance;
PRB-417P; 1:500), ZO-1 (Invitrogen; 61–7,300; 1:200), ZO-2 (Invitrogen; 38–9,100;
1:200) were performed on paraffin and cryosections of skin sample using specific antibodies as indicated [223]. Specificity of the antibodies was validated using rabbit isotype control (Abcam, ab27478; 1:400). Briefly, OCT embedded tissue were cryosectioned (10μm), fixed with cold acetone, blocked with 10% normal goat serum and incubated with specific antibodies overnight at 4°C. Signal was visualized by subsequent incubation with either biotinylated–tagged for DAB staining and fluorescence-tagged appropriate secondary antibodies (Alexa 488-tagged α-rabbit, 1:200; Alexa 568-tagged α- rabbit, 1:200).
4.2.16 Data collection and statistical analyses.
Samples were coded and data analysis was performed in a blinded fashion. For animal studies, data are reported as mean ± SD of at least 4-8 animals as indicated. In vitro
106 data are reported as mean ± SD of 3-6 experiments as indicated in respective figure legends.
Student's t test (two-tailed) was used to determine significant differences. Comparisons among multiple groups were tested using analysis of variance (ANOVA). p<0.05 was considered statistically significant.
4.3 Results
4.3.1 Preparation and characterization of lyophilized TLN:
TLNκ was prepared using a keratinocyte-targeting peptide sequence
ASKAIQVFLLAG (A5G33) [106]. The peptide was conjugated to DSPE-PEG2000 and incorporated into the surface of LNPs (Figure 4.1). Colloidal stability of the LNPs was studied under storage condition. Over a period of 6 days the particle size doubled from
88nm to >200nm (Figure 4.2). To address such limitation, TLNκ was lyophilized with addition of a lyoprotectant as described in Methods (Figure 4.3). The lyophilized keratinocytes-targeted LNPs when stored at 4°C, retained their size when reconstituted after 9 days (Figure 4.4). Zeta potential of these reconstituted TLNκ ranged from +10mV to -10mV in the pH range of 6-8 (Figure 4.5). Encapsulation of antimiR in lyophilized
TLNκ after 9 days at 4°C was found to be 96.54%. In order to assess cargo stability of lyophilized TLNκ, in vitro transfection was performed with TLNκ loaded with anti-miR-
210. miR-210 has been shown as an important target in wound healing previously [150,
206]. Human immortalized keratinocytes cells when subjected to hypoxia showed elevated
107 miR-210 expression. TLNκ loaded with anti-miR-210, stored at 4°C for 9 days, significantly inhibited miR-210 response in cells exposed to hypoxia. Storage of LNPs at
4°C yielded the best result, compared to storage at room temperature or at -20°C (Figure
4.6).
4.3.2 In vitro targeting efficiency of TLN:
To test specificity of targeted cell delivery of TLNκ in vitro experiments were carried out using different cell lines including keratinocytes (HaCaT cells), endothelial cells (HMEC), fibroblasts (BJ-1) and differentiated monocytes (THP-1). DiD, a lipophilic fluorescent dye, was incorporated into the LNPs as described in Method [224]. Cells were exposed to DiD-labelled TLNκ for 4h followed by imaging and flow cytometric analyses.
The uptake of DiD-labelled TLNκ was significantly higher in keratinocytes compared to that of non-targeted LNPs (nTLNκ without the targeting peptide) that served as control
(Figure 4.7). Flow cytometric data showed more than 90% keratinocytes showed uptake of the DiD-labelled TLNκ unlike that in endothelial cells (HMEC) or in fibroblasts (BJ-1)
(Figure 4.8). Differentiated monocytes (THP-1) appeared promiscuous demonstrating 12% uptake of even non-targeted LNPs. Nonetheless, delivery of TLNκ to keratinocytes was over 4-fold higher compared to uptake by monocytic cells.
4.3.3 In vivo targeting efficiency of TLN:
To evaluate targeting efficiency of TLNκ in vivo, full thickness burn wound was developed on the dorsal skin of mice. Full thickness burn wound was confirmed by H&E
108 staining (Figure 4.9). Lyophilized DiD-labeled TLNκ were applied with hydrogel on the burn wound (Figure 4.10). Lyophilized TLNκ was readily dispersed in hydrogel within 30 seconds. The burn wound tissue was harvested after 24h of application. To assess antimiR delivery efficiency of TLNκ in vivo, targeted as well as nTLNκ containing anti-miR-107 was applied topically on murine skin and burn wound-edge tissue. Tissues were collected after
24h of treatment and subjected to laser capture microdissection. Quantitative RT-PCR demonstrated significant down regulation of miR-107 in the epidermis in TLNκ treated group while the dermis remained unaffected (Figure 4.11). Lyophilized TLNκ thus effectively sequestered keratinocyte miR-107 in vivo.
Confocal microscopic images of the normal skin demonstrated that TLNκ was successful in penetrating the wound edge stratum corneum and co-localized in basal keratinocytes (Figure 4.12). Non-targeted LNκ primarily localized in the stratum corneum
(Figure 4.12). Single plane confocal co-localization analysis of wound-edge sections harvested at day 1, day 3 and day 7 was performed. Our data showed that repeated application of TLNκ every third day resulted in cumulative DiD fluorescence in the epidermis unlike nTLNκ (Figure 4.13i). Furthermore, Z-stack maximum intensity projected images also demonstrated that the nTLNκ was primarily localized in the stratum corneum
(Figure 4.13ii) even at day 3. At day 7, the TLNκ were found to be more evenly distributed throughout the epidermis.
109 4.3.4. Functional wound closure
MicroRNA-107 inhibits cell proliferation and migration [225-228]. Delivery of anti-miR-107 using commercially available transfection reagent (Dharmafect1) accelerates keratinocyte proliferation and migration (Figure 4.14). MicroRNA-107 also targets the
Dicer, a key contributor to miR biogenesis [229, 230]. Dicer is an RNAase-III enzyme that plays a critical role in reestablishing the barrier function of the skin [156]. Delivery and sequestration of miR-107 inhibited and upregulated dicer expression in keratinocytes, respectively (Figure 4.15). Dicer is responsible for the biogenesis of key miRs including miR-20a, miR-93 and miR-106b (Figure 4.16), which play pivotal roles in establishing the barrier function of the repairing skin by inhibiting the expression of p21Waf1/Cip1 [156].
TLNκ/antimir-107 significantly increased Dicer expression (Figure 4.17) and down-regulated p21Waf1/Cip1 expression (Figure 4.18) compared to lyophilized targeted scramble
(TLNκ/scramble) or lyophilized non-targeted LNκ/antimir-107.
Application of lyophilized TLNκ/antimir-107 on the burn wound every third day accelerated wound closure (Figure 4.19) Transepidermal water loss (TEWL) measures skin barrier function [156, 201]. TLNκ/antimir-107 accelerated re-establishment of successful skin barrier function following burn injury (Figure 4.20).
4.3.5 Epidermal junctional proteins
Junctional proteins help establish the barrier function of the repaired skin. Topical application of lyophilized TLNκ/antimir-107 significantly upregulated the expression of
110 claudin, loricrin, filaggrin, ZO-1 and ZO-2 (Figure 4.20) compared to lyophilized
TLNκ/scramble or lyophilized non-targeted LNκ/antimiR-107.
4.4 Discussion
Lipid nanomaterials with specific biological functions are of therapeutic value. In this work, we report a novel LNP formulation of antimiR that when applied to the skin topically, may penetrate the stratum corneum to specifically target keratinocytes for cargo delivery. The LNPs can be lyophilized to extend the shelf-life. This work constitutes the first report on cell-targeting LNPs in a non-cancer health care application [231]. According to a recent report from The Center for Drug Evaluation and Research (CDER) within the
US Food and Drug Administration (FDA), 234 investigational new drug (IND) applications containing nanomaterials have been submitted, and 34 new drug applications
(NDAs) have been approved by the office [24]. Liposomes account for 61% of nanomaterials targeting cancer therapeutics having size less than 300nm [24]. The following targeted nanoparticles are in clinical trial: MCC-465, MBP-426, SGT-53, SGT-
94, BIND-014, MM-302, TargomiRs, CALAA-01, Cornell Dot and ND-L02-s0201 [232,
233]. Nanoparticles are frequently targeted to cancer cells either through the transferrin receptor or folate receptor [234-236]. In addition, carbohydrates such as lacto bionic acid were used to target hepatic tumor cells by binding to their asialoglycoprotein receptors
[237]. Polysaccharides, such as hyaluronic acid or chitosan have shown promising results in targeting tumor extracellular environment [238]. However the cellular uptake and
111 delivery efficiency are difficult to predict as it depends on the payload-related variables
[238]. Mannose and Galactose derivatives are often used for targeting macrophages through the C-type lectin receptors, which are expressed by all the cells of myeloid origin
[239, 240]. The targeting ability of functionalized nanoparticles may be lost in a biological environment [241]. Even when it does work, efficiency of targeting is highly limited and was estimated to be 30% [242]. Some carbohydrate nanocarriers (mono/oligosaccharides and/or polysaccharides) used in biomedical application are reported to reduce unspecific protein adsorption and increases the circulation time in the blood [243]. The field of targeted nanoparticle therapy is emergent, and at present applications are highly limited.
This work underscores the potential of targeted nanoparticles in wound and skin care and offers a new targeting platform.
For topically applied nanoparticles, non-specific uptake was significantly reduced and is mostly limited to follicular (passive) penetration [244]. Active penetration of nanoparticles with size >45nm is an open and controversial topic of discussion. Although the literature reports variable results on porcine and murine skin showing penetration, there are only two publications that report passive permeation of such particles beyond the stratum corneum [244-246]. Toll et.al reports penetration of stratum corneum via hair follicle conduits. However, Kohli et al attributes penetration of stratum corneum to the strong anionic properties of nanoparticles [245, 246]. If the intent is to target specific cells, high surface charge of nanoparticles is a confounding factor as it would be promiscuous and be readily taken up by most cells in proximity.
112 This work reports a simple and scale-up friendly target nanoparticle platform for skin and wound care applications. The design of TLNκ employed DOTAP/DODAP combination pH-responsive lipid components to improve endosomal escape. Compared to our previously published nanoparticle formulation [206], we replaced gramicidin with keratinocyte targeting ligand to promote cell-specific targeting. To minimize interference of clearance by immune cells surface charge of anti-miR loaded TLNκ was designed to be close to neutral. Additional pH vs zeta-potential analyses were conducted to assess the pH- dependence of surface charge. TLNκ possess near zero surface charges that quickly react to pH drop by boosting surface charges to +15mV for efficient endosomal release. This design does compromise transfection efficiency but maximizes targeting efficiency, which is desirable in a wound microenvironment complicated by inflammation. By virtue of its design properties, at pH 7 TLNκ is near-zero in charge. This poses stability problems due to aggregation, deposition, and sedimentation of the particles [247]. Lyophilization of
TLNκ not only addresses this limitation but also extends storage life.
113
Figure 4. 1 Schematic representation of Keratinocytes Targeting Lipid Nanoparticle
(TLNκ).
114
Figure 4. 2 Change of TLNκ size due to aqueous storage condition under 4 degree over a period of 7 days.
115
Figure 4. 3 Representative photograph of the lyophilized Keratinocytes Targeting
Lipid Nanoparticle.
116
Figure 4. 4 Representation of nanoparticle size and concentration.
Representative nanoparticle tracking analysis (NanoSight™) showing particles size and concentration of freshly prepared TLNκ and reconstituted LTLNκ after 9 days of storage under 4 degree (n = 4).
117
Figure 4. 5 The zeta potential of the TLNκ at varying pH suggesting pH responsive behavior.
118
Figure 4. 6 miR-210 expression level followed by TLNκ treatment. miR-210 expression in HaCaT cells exposed to normoxia and hypoxia 24h after delivery of TLNκ /miR-210. (n=4). * p<0.01 compared to hypoxia, ANOVA.
119
Figure 4. 7 Overlay of phase contrast and fluorescence microscopic images
Showing uptake of DiD- labeled (red) TLNκ in keratinocytes (HaCaT cells), endothelial cells (HMEC), fibroblasts (BJ-1) and differentiated monocytes (TJP-1) over a period of 4 hours. DiD- labeled (red) nTLN served as control. Scale = 50µm.
120
Figure 4. 8 Study of in vitro uptake
Flow cytometric analysis of showing uptake of DiD- labeled (red) TLNκ in keratinocytes (HaCaT cells), endothelial cells (HMEC), fibroblasts (BJ-1) and differentiated monocytes (TJP-1) over a period of 4 hours. The percentage of cells showing red fluorescence of DiD and plotted graphically. Data expressed as mean
±SD. n=4. * p<0.001.
121
Figure 4. 9 Representative mosaic image of murine dorsal skin after application of the burner for 5 secs (top) and 15 secs (bottom) for developing the full thickness burn wound. Scale bar = 500μm.
122
Figure 4. 10 Digital photos of TLNκ application.
Digital photomicrographs showing (I) application of lyophilized TLNκ powder on the dorsal skin of mice post-burn. (II) application of 3M hydrogel to cover and reconstitute the dry powder; (III) application of 3M Tegaderm to cover and strap the dressing from falling off.
123
Figure 4. 11 miR-107 expression level from Laser Capture Microdissection. miR-107 expression from laser microdissected epidermis of murine skin and wound-edge tissue 24h after application of lyophilized TLNk containing anti-miR-107. Data expressed as mean ± SEM. (n=3), § p<0.05; † p<0.01; ANOVA compared to nTLNk/anti-miR-107.
124
Figure 4. 12 Co-localization on normal skin by confocal microscopy. Confocal microscopic images showing localization of the DiD-labelled nanoparticles
(red) in the epidermis 24h after application of nTLNk/anti-miR-107 and TLNk/anti-miR-107. The sections were counter stained with K14 (green) and DAPI (blue). The sections were couter stained with K14 (green) and DAPI (blue). Scale = 50µm.
125
Figure 4. 13 Co-localizations on burn edge skin by confocal microscopy. (i) Single plane confocal microscopic images showing co-localization of the DiD-labelled nanoparticles in the burn wound-edge tissue at day 1, 3 and 7. nTLNk/anti-miR-107 and
TLNk/anti-miR-107 were applied as mentioned in the method. The co-localization signals
(white dots) were merged with DAPI (blue). (ii) z-stack maximum intensity projection of the same frame showing co-localization (yellow) of the DiD-labelled nanoparticles (red) in the burn wound-edge tissue at day 1, 3 and 7. The sections were counter stained with
K14 (green). Scale = 50µm.
126
Figure 4. 14 Cell proliferation and migration HaCaT cells were transfected with either control inhibitor or miR-107 inhibitor for 72h.
(A) Cells were trypsinized and reseeded in 96 wells plate. Cell proliferation was determined after 24h of treatment by using MTT assay. Data expressed as mean ± SD. * p < 0.001; n = 8 (B) Cell migration assay was performed after reseeding in 2-well cell inserts. Migration of cells was observed at 12h following removal of the insert. The black and white dashed line indicated the distance at 0h and 12h respectively. Scale bar = 100 mm. The distance between the two ends are calculated using Zen software (Zeiss) and expressed graphically. Data expressed as mean ± SD. † p < 0.01; n = 3.
127
Figure 4. 15 Quantitative PCR analysis of miR-107 effectiveness Quantitative PCR analysis of miR-107 after delivery of (A) miR-107 mimic and (B) miR-
107 inhibitor in HaCaT cells. Data expressed as mean ± SD (n=4). * p<0.001. (C)
Western blot analysis of dicer expression after transfection of miR-107 mimic and inhibitor.
128
Figure 4. 16 miR-107 downstream gene expression level followed by treatment. Quantitative PCR analysis of miR-20a, miR-93 and miR-106b at day 24 after delivery of
TLNκ/scramble, NTLNκ/anti-miR-107 and TLNκ/anti-miR-107. Data expressed as mean ± SD (n=4).
†, p<0.01.
129
Figure 4. 17 Dicer expression level followed by TLNκ treatment.
TLNκ/anti-miR-107 increased the expression of dicer (red) in murine skin at day 24. Sections were counter stained with DAPI. Dermal-epidermal junction is indicated by dashed white line. Scale bar = 50μm. Abundance of dicer and p21waf1/Cip1 in epidermis were quantified and expressed graphically as mean ± SD. (n=6). †, p<0.01; * p<0.001
130
Figure 4. 18 p21 waf1/Cip1 expression level followed by TLNκ treatment.
TLNκ/anti-miR-107 decreased the expression of p21 (red) in murine skin at day 24. Sections were counter stained with DAPI. Dermal-epidermal junction is indicated by dashed white line. Scale bar = 50μm. Abundance of dicer and p21waf1/Cip1 in epidermis were quantified and expressed graphically as mean ± SD. (n=6). †, p<0.01; * p<0.001
131
Figure 4. 19 Representation of wound closure. (A) Digital photographs of the full thickness burn wound at day 0, 6, 12, 18, 24 days after topical application of TLNκ/scramble, nTLNκ/anti-miR-107 and TLNκ/anti-miR-107. The white dashed lines indicate the wound area. (B) Wound closure after topical application of
TLNκ/scramble, nTLNκ/anti-miR-107 and TLNκ/anti-miR-107 was quantified by digital planimetry.
(n=5), * p<0.001 compared to d0; ANOVA.
132
Figure 4. 20 Representation of re-epithelization and restoration of barrier function.
Transepidermal water loss at day 0 and at day 24 after delivery of TLNκ/scramble, nTLNκ/anti- miR-107 and TLNκ/anti-miR-107 was plotted graphically. (n=5), § p<0.05 compared to day0;
ANOVA.
133 Figure 4. 21 upregulated epidermal junction proteins suggesting restoration of barrier function.
TLNκ/anti-miR-210 upregulates epidermal junctional proteins. TLNκ/anti-miR-107 increased the expression of Claudin (red), loricrrin (green), Filagrrin (red), ZO-1 (green) and ZO-2
(red) in murine skin at day 24. Sections were counter stained with DAPI. Dermal- epidermal junction is indicated by dashed white line. Scale bar = 50μm. Abundance of junctional proteins were quantified and expressed graphically as mean ± SD. (n=6). § p<0.05; * p<0.001.
134
135 CHAPTER 5: Summary and Future Direction
This dissertation thesis has conducted an extensively amount of work focused on the development of pH responsive LNP delivery platform (QTsome) for oligonucleotide therapeutics.
In Chapter 2, we have proposed and demonstrated a successfully developed pH responsive LNP carrier consist of DODMA/DOTAP/DOPC/Chol/DPPE-PEG with a superior encapsulation and transfection strategy. Further identification of loading capacity has suggested 83.3% ± 4.17% encapsulated genetic cargos. Particle size has been reformed with optimum formulation composition ratios to achieve <120nm hydrodynamic diameter. Surface charge of 12.49 ± 1.45mV in PBS buffer (pH=7.4) has been dramatically increased to 29.89 ± 8.16mV in citric buffer (pH=4.0), suggesting sensitive response to pH change. Efficacy of in vitro gene suppression has been revealed to be 50.3 ± 2.1% in miR-21 expression level after administration. 2.7- and 1.3-fold upregulation of PTEN and PDCD4 tumor suppressor gene has been observed in the study. Significantly reduce cytotoxicity has been observed upon delivery of QT/AM-21 and QT/AM-21/PTX with overall 90% cell viability, while naked PTX has resulted in
<40% cell viability. In vivo tumor xenograft mice studies have suggested 16-fold lighter tumor weight after administration and significantly extended survival days compare to
136 controls. Overall results have suggested promising capacity of QTsome platform as oligonucleotide delivery carrier in vivo. Due to the native property gene loading strategy, such platform could serve as a universal template for genetic therapeutic anti-miRs, siRNAs, or miR mimics.
In Chapter 3, modification of Gramicidin A was incorporated in QTsome platform to further improve in vivo transfection efficiency. We have demonstrated successful delivery of anti-miR-210 into ischemic wound to restore cell proliferation functionality.
Challenges were faced with not only the presentation of phagocytes and macrophages, but also the low pH wound microenvironment. Administration of AFGLNmiR-210 has demonstrated improved re-epithelialization in the form of hyper-proliferative epithelium.
In Chapter 4, QTsome platform has been modified to fulfil cell-specific targeting request in deep burn wound bed microenvironment. miR-107 has been known for its regulating functions in cancer carcinoma and macrophages [203, 249, 250]. Therefore, to reduce incidence of unwanted side-effects, cell-specific targeting is demanded to serve the purpose. Via high throughput screening method of phage display, we have located one promising targeting ligand, A5G33 [106]. By conjugation of A5G33 peptide ligand onto the surface of QTsome with space linker, enhanced cell recognition by keratinocytes is proposed. Previous researches have suggested strong interactions between LNPs and innate immune cells in systemic circulation [77]. In chronic wound, the presence of macrophages and phagocytes are more frequent and intense than that in the systemic circulation. Therefore, surface modification of charge density and morphology has become much more critical to facilitate successful cell-specific targeting. The optimal
137 composition of lipids formula has been carefully evaluated that result in a neutral charged
LNP at pH 7.4. However, we have noticed one drawback of such design. Neutral LNPs are suffered from limited electrostatic repulsive force between particles, which leads to aggregation, deposition and sedimentation issues during storage. To solve such problem, frozen and lyophilized formulation have been proposed. It is well known that burn wound bed are presented with large amounts of extracellular wound fluid. Therefore, we proposed a novel administration route via lyophilized LNP powder. Research has found fast reconstitution rate of lyophilization formulation at wound bed with the assistance of hydrogels. One advantage of our proposal is to benefit patients with unpainful and easier route of administration. In vitro cell targeting assays have shown 96.54% uptake by targeted keratinocyte cells, while less than 5% of TLN were found in fibroblast and endothelial cell lines. In vivo targeting efficiency results have suggested co-localization of TLN in epidermal layers suggested by suppressed miR-107 expression. restoration of barrier function was observed on Day24 in the TLNκ/antimir-107 group, while the control groups showed no sign of barrier function reconstruction.
In the aspect of future designs and optimization of QTsome platform, further optimization and validation of surface modifications will accommodate a variety of therapeutic purposes. An increasing number of pH responsive LNP delivery platforms have been patented for distinctive therapeutic purposes [251]. TAT peptide modified
LNPs composed with pH responsive lipids have been examined in animal study to certificate its potential in delivering 6mg/kg cisplatin. 2.1-fold increase in AUC has been observed compare to free cisplatin treatment [252]. A similar research study has reported
138 long-circulation pH responsive LNPs with PEGylation is able to trigger the TAT cell penetration function at tumor microenvironment [253]. It is reported that at biological pH, hydrazine bond conjugated TAT peptide was “hidden” in the long-chain PEG bush to avoid unwanted targeting and release [253]. When LNPs platform reached tumor site, low pH microenvironment would trigger the degradation of TAT-PEG bond and therefore expose the ligands to induce internalization [253].
pH responsive triggered release could be incorporated in QTsome design to benefit the therapeutic effects. The pH sensitivity of QTsome design has a relatively linear relationship to pH variance, which yields limited functionality in acidic microenvironment including some solid tumors and chronic wounds. Therefore, by taking advantage of acid labile chemical bonds, higher sensitivity as well as additional functions can serve as a plus. The utilization of hydrazine bond has been most common and investigated in a variety of polymers and macromolecules in clinical trials [254, 255].
Similarly, imine bond based linker has also suggested sensitive response to acidic environment. Report has suggested benzoic imine and poly (propylene imine) polymer construction showing very sensitive response to hydrolysis at pH (5.0-6.5) [256]. Similar studies using oxime bond and amide bond have suggested tumor microenvironment sensitivity under several circumstances [257-260].
In general, our work on developing QTsome platform has demonstrated great potential in successfully delivering genetic therapeutics effectively. Additional tweaks on surface modifications could further enhance delivery and targeting efficiency to accommodate disease microenvironments. The platform has built a sophisticated base
139 ground for future improvement. Newly synthesized or discovered cationic lipids and pH responsive lipids could serve well as potential candidates in refining the lipid composition. Multi-functional cell penetrating methods can be easily attached onto the surface of QTsome to enhance in vivo transfection efficiency. However, the translation of such technology from bench to bed-side demands further investigated in pre-clinical large animal models. Pharmacokinetic and pharmacodynamics studies are extremely necessary to understand the bio-distribution and bio-availability of QTsome. Surgical induced ischemia and chronic wound porcine models have been constantly providing reliable therapeutic feedbacks before clinical trials. Therefore, the use of porcine models for advanced investigation of therapeutics targeted towards cutaneous wound is highly recommended. Lastly, the lyophilization process of QTsome has not been thoroughly investigated. The possibility of employing spray-dry instead of lyophilization is still under debate. Long-term storage stability, reconstitution quality, and lyophilization process stability will be of next concern in successfully development of QTsome drug product.
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