Nanoparticle Drug Delivery Systems for Cancer Therapy

NANOPARTICLE DRUG DELIVERY SYSTEMS FOR CANCER THERAPY

DISSERTATION

Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Bryant Chinung Yung, B.A.

Graduate Program in Pharmacy

The Ohio State University

2014

Dissertation Committee:

Dr. Robert J. Lee, Advisor

Dr. L. James Lee

Dr. Mitch A. Phelps

Dr. Thomas D. Schmittgen

Bryant Chinung Yung

2014

ABSTRACT

The design of lipid nanoparticles (LNs) for the delivery of oligonucleotides (ONs) remains a critical challenge in the clinical translation of RNA interference (RNAi) based therapeutics for cancer. Despite facilitating a protective function and passive targeting to ONs, optimization of lipid composition is necessary to promote intracellular delivery. The objective of this dissertation is to provide models for the design, manufacture, and characterization of LN drug delivery systems. Key innovative approaches include the application of lipid coated albumin nanoparticle (LCAN), quaternary-tertiary lipoamine liposome (QTsome), and small peptide lipid nanoparticle (SPLN) for ON delivery.

LCAN is formed by the conjugation of cationic polymers to albumin to form a dense cationic core for electrostatic interaction with ON. This electrostatically stabilized complex is then surrounded by a lipid coat to protect the ON from opsonization. LCAN are utilized for the delivery of LOR-2501, an ON against ribonuclease reductase subunit 1 (RNR1).

QTsome include a combination of permanently ionized quaternary and conditionally ionizable tertiary amine. Upon acidification within the endosome compartment following uptake, QTsome become highly cationized, facilitating interaction with negatively charged lipids of the endosome, thus promoting intracellular delivery. QTsome are applied towards the delivery of anti-miR-21

ii (AM-21) combination therapy with paclitaxel (PTX) in a non-small cell lung carcinoma (NSCLC) model.

SPLN are composed of a combination of lipids and antibiotic peptide, gramicidin.

SPLN employs the fusogenic nature of gramicidin to destabilize lipids in the endosome to promote delivery of ON. A microfluidic hydrodynamic focusing

(MHF) system based on ethanol dilution method is outlined to demonstrate the advantages of microfluidic based manufacture of LNs. SPLN are combined with

AM-221 and AM-21 in a triple negative breast cancer (TNBC) model to examine the potential of combination therapy with tamoxifen (TMX) to induce sensitivity in resistant cells.

iii

DEDICATION

Dedicated to my family.

iv ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. Robert J. Lee for his guidance and support throughout my graduate study. I would also like to thank the members of my committee, Drs. Thomas D. Schmittgen, Mitch A. Phelps, and L. James Lee for their valuable input during the writing of this dissertation. Moreover, I would like to recognize the members of the Robert Lee lab: Hong Li, YoungAh Cho,

Chenguang Zhou, Xiaojuan Yang, Mengzi Zhang, Jilong Li, Yang Liu, Lauren

Cosby, Xinwei Cheng, Oraphan Paecharoenchai, Josimar de Oliveria Eloy,

Raquel Petrilli, Songlin Xu, Yue Zhang, and Xiaoju Zhou, Ying Liu, Lesheng

Teng, Jing Xie, and Jing Zhu, for their friendship and assistance in the laboratory.

Furthermore, none of this work would have been possible without the assistance of our industrial collaborators at Microlin Bio, Rexhan Pharmaceuticals, Inc., and

EMD Millipore and support from the National Science Foundation (NSF)

Nanoscale Science and Engineering Center (NSEC), the National Institutes of

Health (NIH), the Comprehensive Cancer Center (CCC), and the College of

Pharmacy at The Ohio State University. Additionally, I would like to thank the

Pharmacy and Researchers of America (PhRMA) Foundation for awarding the

Pre Doctoral Fellowship in Pharmaceutics, which has supported my studies.

v VITA

December 1st, 1986 ……………………………………….Born – Red Bank, New Jersey, USA

2006 – 2009 ……………………………...... B.A. Chemistry, University of Cincinnati, Cincinnati, Ohio, USA

2009 – 2011 ………………………………………………..Graduate Teaching Associate, College of Pharmacy, The Ohio State University, Columbus, Ohio, USA

2011 – 2013 ………………………………………………..NSEC Fellowship, The Ohio State University, Columbus, Ohio, USA

2013 – Present……………………………………………..PhRMA Pre-doctoral Fellowship, The Ohio State University, Columbus, Ohio, USA

PUBLICATIONS

1. Yang Z, Yu B, Zhu J, Huang X, Xie J, Xu S, Yang X, Wang X, Yung BC, Lee LJ, Lee RJ, Teng L. A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia. Nanoscale. 21;6(16):9742-51, 2014. 2. Kim DC, Cho YA, Li H, Yung BC, Lee RJ. Proteinase K-containing lipid nanoparticles for therapeutic delivery of siRNA LOR-1284. Anticancer Res. 34(7):3531-5, 2014. 3. Paecharoenchai O, Teng L, Yung BC, Teng L, Opanasopit P, Lee RJ. Nonionic surfactant vesicles for delivery of RNAi therapeutics. Nanomedicine. 8(11): 1-7, 2013. 4. Xie J, Teng L, Yang Z, Zhou C, Liu Y, Yung BC, Lee RJ. A polyethylenimine- linoleic acid conjugate for antisense oligonucleotide delivery. BioMed Res. Int. (e-pub ahead of print) 5. Zhang M, Zhou X, Wang B, Yung BC, Lee LJ, Ghoshal K, Lee RJ. Lactosylated gramicidin-based lipid nanoparticles (Lac-GLN) for targeted

vi delivery of anti-miR-155 to hepatocellular carcinoma. J Control Release. 168(3):251-61, 2013. 6. Zhou X, Zhang M, Yung B, Li H, Zhou C, Lee LJ, Lee RJ. Lactosylated liposomes for targeted delivery of doxorubicin to hepatocellular carcinoma. Int. J. Nanomedicine. 7:5465-74, 2012. 7. Zhou X, Yung B, Huang Y, Li H, Hu X, Xiang G, Lee RJ. Novel Liposomal Gefitinib (L-GEF) Formulations. Anticancer Res. 32(7):2919-23, 2012. 8. Piao L, Li H, Teng L, Yung BC, Sugimoto Y, Brueggemeier RW, Lee RJ. Human serum albumin-coated lipid nanoparticles for delivery of siRNA to breast cancer. Nanomedicine 2012 (e-pub ahead of print). 9. Zhang Y, Zhou C, Kwak KJ, Wang X, Yung B, Lee LJ, Wang Y, Wang PG, Lee RJ. Efficient siRNA Delivery Using a Polyamidoamine Dendrimer with a Modified Pentaerythritol Core. Pharm Res. 29(6):1627-36, 2012. 10. Li H, Piao L, Yu B, Yung BC, Zhang W, Wang PG, Lee JL, Lee RJ. Delivery of calf thymus DNA to tumor by folate receptor targeted cationic liposomes. Biomaterials 32(27):6614-20, 2011. 11. Liu Y, Xu S, Teng L, Yung B, Zhu J, Ding H, Lee RJ. Synthesis and evaluation of a novel lipophilic folate receptor targeting ligand. Anticancer Res. 31(5):1521-5, 2011.

FIELDS OF STUDY

Major Field: Pharmacy

vii TABLE OF CONTENTS

Title Page

ABSTRACT ...... ii

DEDICATION ...... iv

ACKNOWLEDGMENTS ...... v

VITA ...... vi

PUBLICATIONS ...... vi

FIELDS OF STUDY ...... vii

LIST OF FIGURES ...... xiii

LIST OF TABLES ...... xxiv

CHAPTER 1 ...... 1

INTRODUCTION ...... 1

1.1 Cancer and conventional therapeutic strategies ...... 1

1.2 The role of microRNA in cancer ...... 3

1.3 Anti-miRs and barriers to delivery ...... 5

1.4 Conjugates and delivery systems for anti-miR delivery ...... 6

1.5 Design of LN delivery systems...... 7

1.6 Manufacture of LNs ...... 9

1.7 Characterization of LNs...... 12

viii 1.8 Pharmacokinetics, Pharmacodynamics, and Nanotoxicity...... 13

1.9 Objectives of the dissertation...... 16

CHAPTER 2 ...... 30

LIPID COATED ALBUMIN NANOPARTICLES FOR THE DELIVERY OF LOR-

2501 ...... 30

2.1 Introduction ...... 30

2.2 Materials and methods ...... 32

2.2.1 Materials...... 32 2.2.2 Synthesis and characterization of APC ...... 33 2.2.3 Synthesis of LCAN...... 34 2.2.4 Physical characterization of LCAN...... 34 2.2.5 Gel retardation assay...... 34 2.2.6 Antisense oligonucleotide transfection...... 35 2.2.7 Cell proliferation assay...... 35 2.2.8 Quantitative qRT-PCR of mRNA...... 36 2.2.9 Establishment of murine KB xenograft model...... 36 2.2.10 Tumor gene regulation analysis...... 37 2.2.11 Statistical analysis...... 37 2.3. Results...... 37

2.3.1. Synthesis and characterization of APC...... 37 2.3.2 Synthesis and characterization of LCAN...... 38 2.3.3 Analysis of LCAN/LOR-2501 in KB cells...... 39 2.3.4 Evaluation of LCAN/LOR-2501 in a KB xenograft murine model...... 39 2.3.5 In vivo target downregulation...... 40 2.4. Discussion...... 40

2.5 Conclusion ...... 42

CHAPTER 3 ...... 53

ix QUATERNARY-TERTIARY LIPOAMINE SYSTEMS FOR THE DELIVERY OF

ANTI-MIR-21 ...... 53

3.1 Introduction ...... 53

3.2 Materials and Methods ...... 55

3.2.1 Materials...... 56 3.2.2 Synthesis of QT...... 56 3.2.3 Mean particle diameter and surface charge...... 57 3.2.4 Drug loading and stability...... 57 3.2.5 Cell culture...... 58 3.2.6 In vivo gene regulation...... 58 3.2.7 Cell viability assay...... 58 3.2.8 Migration assay...... 59 3.2.9 Invasion assay...... 59 3.2.10 Tumor regression analysis...... 60 3.2.11 Combination therapy analysis...... 60 3.2.12 In vivo gene regulation...... 61 3.2.13 Statistical analysis...... 61 3.3 Results...... 61

3.3.1 Particle size and surface charge...... 61 3.3.2 Drug loading and colloidal stability...... 62 3.3.3 Determination of optimal lipid combination...... 62 3.3.4 miR-21 and target regulation...... 63 3.3.5 Dose dependency...... 63 3.3.6 Cell viability...... 64 3.3.7 Invasion and Migration...... 64 3.3.8 In vivo dose response...... 65 3.3.9 In vivo combination therapy...... 65 3.4 Discussion...... 66

3.5 Conclusion ...... 70

CHAPTER 4 ...... 86

x SMALL PEPTIDE LIPID NANOPARTICLES FOR THE DELIVERY OF ANTI-MIR

...... 86

4.1 Introduction ...... 86

4.2 Materials and Methods ...... 89

4.2.1 Materials ...... 89 4.2.2 Preparation of SPLN by bulk mixing ...... 90 4.2.3 Preparation of SPLN by microfluidics ...... 90 4.2.4. Physical characterization ...... 91 4.2.5 Cell culture ...... 91 4.2.6 Formulation optimization ...... 92 4.2.7 Cell viability ...... 92 4.2.8 Gene regulation ...... 93 4.2.9 Migration assay ...... 93 4.2.10 Invasion assay ...... 94 4.2.11 Tamoxifen sensitivity ...... 94 4.2.12 Statistical analysis...... 95 4.3 Results...... 95

4.3.1 Particle size and surface charge ...... 95 4.3.2 Drug loading and colloidal stability ...... 96 4.3.3 Formulation optimization ...... 97 4.3.4 Formulation toxicity ...... 97 4.3.5 miR regulation ...... 98 4.3.6 Target gene regulation ...... 98 4.3.7 Migration and invasion ...... 99 4.3.8 Tamoxifen sensitivity...... 99 4.4 Discussion...... 100

4.5 Conclusion ...... 104

CHAPTER 5 ...... 119

SUMMARY AND PERSPECTIVES ...... 119

xi 5.1 Summary ...... 119

5.2 Perspectives...... 121

5.3 Future Work ...... 122

5.4 Concluding Remarks ...... 123

REFERENCES ...... 124

xii LIST OF FIGURES

Figures Page

Figure 1.1. Oligonucleotide modifications. Structural modifications to oligonucleotides improve drug form resistance against nuclease mediated degradation and increase specificity of binding. (A) natural phosphodiester backbone, (B) phosphorothioate backbone, (C) 2’-O-

Me base, (D) 2’-O-MOE base, (D) LNA, (E) PNA, and (F) morpholino backbone...... 18

Figure 1.2. Selected examples of lipid and polymers in LN synthesis.

Cationic lipids and polymers form electrostatic interactions with negatively charged ONs. Bilayer forming lipids help to stabilize the

LN. Fusogenic lipids promote disruption of endosomal lipid bilayers.

PEG-modified lipids reduce opsonization and decrease off-target uptake...... 19

Figure 1.3. Mechanism of cellular delivery. LN with moderate positive charge bind to the negative charges on the cell membrane and are internalized as endocytic vesicles. Vesicles fuse with the endosome, which becomes increasingly acidic, eventually depositing contents within the lysosome for degradation. LNs must escape the acidic

xiii conditions of the endosome and deliver ON to the cytosol for interaction with miRs or mRNA...... 20

Figure 1.4. Pathways of cellular entry. LN may enter the cell through a variety of pathways including (A) phagocytosis, (B) macropinocytosis, (C) clathrin-mediated endocytosis, (D) caveolin/lipid raft-mediated endocytosis, (E) clathrin and caveolin independent endocytosis. The physical characteristics of the particles influences the mechanism of uptake...... 21

Figure 1.5. LCAN formulation. LCAN consist of a hypercationized

APC-ON complex core (A) surrounded and stabilized by a lipid layer

(B). LCAN are employed to deliver antisense ON, GTI-2501, directed against ribonucleotide reductase subunit 1 (RNR1) in a cervical cancer model...... 22

Figure 1.6. QTsome include a combination of permanently ionized and conditionally ionizable lipoamines at specific ratio. (A) Under acidic conditions within the endosome, conditionally ionized lipoamines become cationized, thereby facilitating endosomal disruption through interaction with negatively charged membrane lipids. (B) QT are used to deliver AM-21 in a NSCLC model. QT/AM-

21 is further co-administered with PTX to evaluate efficacy of combination therapy ...... 23

Figure 1.7. (A) SPLN include a fusogenic peptide, gramicidin, to promote fusogenic potential within the endosomal compartment for

xiv cytosolic delivery of AM. (B) SPLN is applied for the delivery of AM-

221 and AM-21 in a TNBC model to determine if modulation of miRs can induce sensitivity towards hormone therapy (TMX) ...... 24

Figure 2.1. Synthesis of APC. HSA was combined with an excess of low MW PEI (titrated to pH 8) and conjugated using EDC. The resultant conjugate was eluted through a PD-10 column to remove free PEI...... 44

Figure 2.2. BCA and TNBSA assay. BCA protein assay (A) was used to determine reaction yield for the formation of APC. Absorbance data from TNBSA assay (B) was used to calculate the conjugation efficiency of APC. Approximately 37 PEI molecules were bound to each molecule of albumin...... 45

Figure 2.3. Particle size and surface charge. LCAN with varying

APC:ON ratio were prepared. (A) Mean particle diameter was determined by DLS. (B) Surface charge was determined by zeta potential measurement. Data is presented as the mean±SD of three separately prepared samples...... 46

Figure 2.4. Gel retardation assay. LCAN of varying APC:ON ratio were prepared and analyzed for ability to condense ON against an electrophoretic gradient. Red arrows represent complete retardation of ON...... 47

Figure 2.5. Colloidal stability. LCAN/LOR-2501 (3:1) were stored under varying conditions to determine the effect of temperature on

xv particle size over time. Data are presented as the mean±SD of three separate LCAN preparations...... 48

Figure 2.6. Cell viability. Control formulations or LCAN/LOR-2501

(200 nM) at varying APC:ON ratio were transfected in KB cells for 4 h.

MTS assay was conducted to measure relative cell proliferation 48 h after the start of transfection. Data represent the mean±SD of three separate transfections...... 49

Figure 2.7. In vitro regulation of R1. KB cells were treated with control formulations or LCAN/LOR-2501 (200 nM) at varying APC:ON for 4 h in (A) serum-free or (B) 20% serum containing media. R1 expression was measured relative to β-actin. Data is presented as the mean±SD of three separate treatments...... 50

Figure 2.8. Antitumor activity. KB cell xenograft models were initiated in mice (n=5). To determine the in vivo efficacy of LCAN, mice were treated i.v. with saline control, free LOR-2501, LN/LOR-2501,

LCAN/SC, or LCAN/LOR-2501 (3mg/kg). Tumor progression was monitored over a 3 week period. (Mice with tumors over 1.5 cm in diameter were removed from the study per institution policy.) Results are reported as the mean±SE volume...... 51

Figure 2.9. In vivo R1 regulation. Harvested tumors were analyzed for differences in R1 mRNA expression. Data is presented as the mean±SE of QRT-PCR analysis for three separate tumor sections...... 52

xvi Figure 3.1. Particle size and surface charge. QT of varying mol% quaternary and tertiary amine content were prepared and analyzed by

DLS for the effect on nanoparticle size (A). QT and LN composed of only quaternary or tertiary amine were placed in buffer at physiological pH (7.4) and acidic pH (4.0) to determine the pH responsive effect on surface charge (B). Data is presented as the mean±SD of three independent samples (n=3)...... 72

Figure 3.2. Encapsulation efficiency and colloidal stability. (A) Column separation of QT was completed on a CL 4B column, collecting 1 mL fractions. Absorbance at 280nm was measured to detect the presence of ON. (B) To study the relationship between temperature and particle size, QT/AM-21 was stored at varying temperature and the particle size was monitored over 4 weeks...... 73

Figure 3.3. Formulation optimization. QT of varying lipoamine composition were prepared and evaluated for relative transfection efficiency. Data represent the mean±SD of three separate transfections...... 74

Figure 3.4. In vitro gene regulation. (A-B) A549 cells were treated for

4 h with SPLN/NC or SPLN/AM-21 (100 nM). (C) To demonstrate dose dependency of gene regulation, cells were treated with 1.56,

6.25, 25, and 100nM SPLN/AM-221. Relative gene expression to

GAPDH or RNU44 was evaluated 24 h following the start of

xvii transfection. Results are reported as the mean±SD of three independent transfections...... 75

Figure 3.5. Cell viability. (A) A549 cells were treated with QT/AM-21 at varying concentrations with and without paclitaxel. 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. (B) Cells were imaged under light microscope to observe changes in cell morphology following treatment...... 76

Figure 3.6. Migration and invasion assay. (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...... 77

Figure 3.7. Tumor regression analysis. A549 xenograft models (n=10) were created in nude mice and treatment began when tumors reached 180mm3. 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...... 78

Figure 3.8. Weight data. Following the final treatment, mice were sacrificed and body, liver, spleen, and tumor weight data was

xviii collected. Saline treated mice are compared to mice treated with 1 mg/kg QT/AM-21. Weights are expressed as mean±SE, n=10...... 79

Figure 3.9. Kaplan-Meier survival analysis. 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 (p=0.0037) respectively...... 80

Figure 3.10. Combination therapy. 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 24h following the last treatment and evaluated for modulation of targets. Data represent the results (mean±SE) from three separate tumor sections...... 81

Figure 3.11. In vivo gene regulation. Tumors were harvested from mice and analyzed for modulation of miR-21 targets DDAH1 and

PTEN. Results are presented as the mean±SE of QRT-PCR results for three independent tumor sections...... 82

Figure 3.12. Various AM-21 configurations were evaluated using

RNAimax transfection reagent 24 h following the initiation of treatment. AM were screened in the order of priority ranking. Data represent the mean±SD of quadruplicate transfections...... 84

Figure 4.1. Microfluidic method for the synthesis of SPLN. (A)

Photograph of MHF system. A prototype MHF device was constructed for the preparation of SPLN containing AM-221. (B) Photograph of

MHF chip. Sterile syringes containing lipids dissolved in ethanol or ON

xix in PBS were loaded onto syringe pumps and connected to a microfluidic chip. Lipid streams were connected to inlets A and C while AM-221 stream was connected it inlet B. The microfluidic chip was submerged in a water bath to maintain a 25°C temperature during processing. Approximately 2 mL of SPLN was eluted from the outlet port before sample collection to ensure collection of sample within the steady state range of processing...... 106

Figure 4.2. Particle size and zeta potential. SPLN of varying mol% gramicidin content were prepared and analyzed by DLS for differences in particle size (A) and zeta potential measurement for differences in surface charge (B). (C) Average particle size and PDI of

SPLN prepared by bulk mixing and MHF method are compared. Data are presented as mean±SD of three independently prepared SPLNs...... 107

Figure 4.3. Effect of MHF processing parameters on particle size. (A)

Flow rate, (B) lipid concentration, and (C) lipid: ON (w/w) were modulated to determine the relative effect on particle formation.

Particle sizes are reported as the mean±SD of three independent experiments...... 108

Figure 4.4. Encapsulation efficiency. The relationship between loading efficiency and gramicidin content was evaluated by measurement of relative fluorescent intensity between intact particles and those lysed by Triton X-100. Data are presented as mean±SD of three independently prepared SPLNs...... 109

xx Figure 4.5. Colloidal stability. SPLN were stored at varying conditions to evaluate the relationship between temperature and stability of the formulation. Particle size was monitored for 4 weeks. Particle sizes are reported as the mean±SD of three samples...... 110

Figure 4.6. Formulation optimization. SPLN of varying mol% gramicidin were combined with siLuc and transfected in SK-HEP-1 cells stably expressing luciferase in serum-free and 20% serum- containing media. Cell viability (A) was assessed by MTS assay and luciferase assay was used to quantify relative luciferase suppression

(B). Bars represent the mean±SD of three independent transfections...... 111

Figure 4.7. Cytotoxicity of SPLN. (A) MCF-7 and (B) MDA-MB-231 cells were treated with varying levels of SPLN/AM-221. Cytotoxicity was assessed by MTS assay 24 h following removal of transfection medium. Data is presented as the mean±SD of three independent transfections...... 112

Figure 4.8. Gene regulation. SPLN/AM-221 and SPLN/AM-21 were transfected in MCF-7 and MDA-MB-231 cells. miR regulation in (A)

MCF-7 and (B) MDA-MB-231 cells. (C) Effect on downstream gene targets: PTEN, PDCD4, RECK, TIMP3, ERα, p27/kip1, CAD, VIM in

MDA-MB-231 cells. Data are expressed as the mean±SD of three separate transfections...... 113

Figure 4.9. Migration and invasion assays. (A) Wound healing assay was used to determine relative migration of cells following treatment.

xxi (B) Matrigel invasion assay was used to determine the invasive potential of cells following treatment. Data is reported as the mean±SD of three independent transfections...... 114

Figure 4.10. SPLN/C and TMX Combination therapy. (A) MCF-7 and

(B) MDA-MB-231 cells were treated with a combination of SPLN/C

(100 nM) and TMX (27 µM) to measure increased sensitivity towards

TMX after 5 days. Cell viability was determined by MTS assay as the mean±SD of three independent treatments...... 115

Figure 4.11. Figure 4.11. Antibiotic peptides gramicidin (G), polymyxin

B (PMB), and colistin (polymyxin E, PME) were combined with lipids and siLuc to evaluate for relative transfection activity for the downregulation of luciferase in SK-HEP-1 cells. Formulations are labeled SPLN-antibiotic (mol% antibiotic). Values are reported as the mean±SD of three independent experiments...... 116

Figure 4.12. Transfection activity of CD3/CD3ac peptide. Derivatives of gramicidin, CD3 and acetylated CD3ac were evaluated for enhanced transfection activity when combined with lipids for Bcl-2 downregulation mediated by antisense ON G3139. Formulations are labeled SPLN- peptide-molar percent composition peptide.

Experiments were done in triplicate in 20% serum-containing media...... 117

Figure 4.13. SPLN/AM-210 therapy. SPLN was combined with AM-

210 for evaluation of wound healing potential in diabetes using an ischemic wound mouse model through topical treatment. Wounds

xxii were stained with nuclear DAPI stain (blue) and monoclonal antibody macrophage marker Ki-67 (purple) and cytokeratin stain K14 (green).

(Image courtesy of Ghatak et al.) ...... 118

Figure 4.14. SPLN/AM-200b therapy. SPLN was combined with AM-

200b for evaluation of wound healing potential in diabetes using an ischemic wound mouse model. Photographs of the wound site were taken during the time course of topical treatment. (Image courtesy of

Ghatak et al.) ...... 119

xxiii LIST OF TABLES

Tables Page

Table 1.1. Selection of miRs involved in cancer...... 25

Table 1.2. Methods of LN preparation. Adapted from (81-82)...... 26

Table 1.3. Selection of commercially available LN formulations.

Adapted from reference (83) and (84). (i.v. = intravenous, i.m. = intramuscular) ...... 27

Table 1.4. Selection of LNs undergoing clinical trial. Adapted from references (83) and (85). (i.v. = intravenous, ipl. = intrapleural) ...... 28

Table 3.1. Optimization of AM-21 structural modifications...... 83

xxiv CHAPTER 1

INTRODUCTION

1.1 Cancer and conventional therapeutic strategies

Cancer ranks among the leading causes of death worldwide; placing second, just behind heart disease. There are an estimated 1,665,540 new cases and 585,720 cancer deaths in the United States every year (1). Leading cancers include lung, colon, breast, pancreas, and prostate cancer (2). Cancer may occur in virtually any part of the body and is characterized by unregulated and undifferentiated cell growth. The uncontrolled proliferation of cancer cells comes as the result of DNA damage to genes coding for cell differentiation or apoptosis (3-4). While errors in

DNA replication are routinely targeted for repair in normal cells, the DNA in cancer cells remains uncorrected, leading to dysregulation of the cell cycle and propagation of the coding error with every successive generation. Tumors are characterized as benign or malignant, depending on their origin, which in turn determines their propensity to grow and migrate (5). Malignant tumors that invade surrounding tissues and blood vessels are termed metastatic and are especially hard to treat due to the aggressive nature of the cancer and de- centralized sites of proliferation (6).

1 The complex and varied nature of the disease makes cancer very difficult to treat. Risk factors associated with cancer include exposure to carcinogens and radiation, infection, obesity, and a hereditary history of cancer (7). Conventional modes of treatment for cancer therapy may involve surgical resection of the tumor, chemotherapy, radiation therapy, or combinations thereof. The aggressive nature of cancer stresses the importance of early detection of the disease as conventional modes of therapy greatly diminish in efficacy during late or advanced stages (8). Surgical resection of tumors is particularly effective for isolated tumors and involves physical removal of the tumor mass and surrounding lymphatic tissue. Removal of small sections of tumor tissue may also be conducted for biopsy to determine the stage of the tumor (9). Chemotherapy involves the administration of cytotoxic anti-neoplastic drugs. These types of therapeutic agents may be broadly classified as alkylating agents or antimetabolites. Alkylating agents damage DNA of cancer cells by adding an alkyl group to guanine bases. Cancer cells are more sensitive to alkylating agents compared to normal cells due to their rapid growth and reduced mechanisms for DNA error detection and repair (10). Antimetabolites target pathways necessary for cell growth or division by inhibiting use of metabolites.

Radiation therapy exposes tumor tissue to ionizing radiation as a means of causing DNA damage to cancer cells, consequently leading to cell death. In the treatment of cancer, often more than one therapeutic strategy is employed.

Resistance to monotherapy is often experienced in response to continuous treatment with a single chemotherapeutic agent. A combination of two or more

2 drugs at lower dose is usually preferred as it reduces toxicity, diminishes fatalities, and is less prone to drug resistance relative to monotherapies (11-12).

1.2 The role of microRNA in cancer

A growing body of evidence supports the importance of understanding the role of microRNA (miR) in carcinogenesis and its development (13-14). Over 2,500 unique miRs have been discovered and they are capable of regulating hundreds of pathways directly or indirectly. Indeed, miRs are thought to regulate at least two thirds of the human genome (15-16). miRs are small, non-protein coding

RNA measuring 20-25 nucleotides in length. Although most miRs perform some type of endogenous activity, a certain number of miRs are found to be differentially expressed in cancer (Table 1.1). In fact, the level of miR expression can often be used as an indicator of disease stage (17). When miRs are upregulated in cancer, they typically operate as oncogenes. Conversely, when miRs are downregulated, they function as tumor suppressors. The correlation between miR expression levels and tumor progression enable miR to serve as both a prognostic biomarker and target for therapy. Therapy with miRs comes as a contrast to conventional modes of therapy where a chemotherapeutic may target only a single disease pathway or cellular function (i.e. DNA repair, inhibition of microtubule disassembly, etc.). Targeting of miR treats cancer at the epigenetic level. miRs are synthesized in the nucleus by polymerase II as primary-miR (pri-miR). pri-miR are then processed by proteins Drosha and Pasha which cleave the polyadenosine tail, forming precursor-miR (pre-miR). pre-miR is then exported out of the nucleus and into the cytoplasm by exportin 5, where it is

3 then cleaved by Dicer, an enzyme which cleaves the stem-loop structure and leaves behind two nucleotide overhangs on the 5’ sense strand and the 3’ passenger strand of the miR duplex. miR is recruited by argonaute 2 protein and incorporated into the RNA-induced silencing complex (RISC). RISC degrades the passenger strand and helps the mature miR find its complementary target messenger RNA (mRNA). miR binds to its target in the 3’-untranslated region (3’-

UTR) of mRNA. This matching may vary in degrees of complementarity. Greater binding specificity usually leads to degradation of the mRNA strand while incomplete matching may simply result in translational repression of the mRNA target without degradation (18). The inhibition of oncogenic miRs may be accomplished by the delivery of complementary oligonucleotide (ON) sequences termed anti-miRs (AMs). AMs hybridize to miR targets, thereby inhibiting translational repression on downstream genes. miRs acting as tumor suppressors may be administered exogenously in the form of miR mimics to increase anticancer activity as well. In addition to therapeutic miRs, miRs may be used as prognostic markers. The systemic distribution of miR throughout the body has recently become a topic of increasing interest in the propagation of cancer. The transport and spread of miRs between cells occurs through packaging into highly stable lipid-based structures such as exosomes and microvesicles which are released into the bloodstream (19). The differential expression levels of miRs then can be used to determine the severity of the disease and propose the best course of treatment. Collection of blood samples for miR analysis further carries the advantage of being far less invasive than

4 traditional biopsies (20). Thus, the study of miRs offers promise as a powerful tool for the identification of disease progression and specific regulation of oncogenic regulatory pathways.

1.3 Anti-miRs and barriers to delivery

AMs are administered as single-stranded ONs, measuring 15-20 nucleotides in length. While techniques such as electroporation may work well in vitro, AMs face several critical barriers in their in vivo implementation (21). A number of these barriers stem from the physiological constraints set by the biological system of the host. AM are typically delivered by parental routes of administration. Oral delivery is not possible due to the harsh conditions of the digestive system and limitations in permeability of the gastrointestinal mucosa

(22). AM are on average 5000-8000 Da. Further, the phosphodiester backbone of ONs is highly anionic in nature. Dedicated cellular membrane trafficking mechanisms for the transport of naked ONs do not exist and the high molecular weight and anionic charge present formidable barriers to delivery. The high anionic charge also leads to clearance by the reticuloendothelial system (RES).

Instability in serum and degradation by endo- and exonucleases further hamper the prospects of delivery. Non-specific interaction with endogenous miR or off- target uptake may also cause unwanted toxicity (23-24). Since the development of AM technology, a number of chemical modifications to ONs have been implemented to improve functionality of the drug form (Figure 1.1). The first generation of modifications included phosphorothioate linkages, which resist nuclease degradation. This was succeeded by the second generation of

5 modifications including 2’-O-methoxyethyl bases, 2’-O-methyl bases, and locked nucleic acids (LNA), which further prevent degradation and improve the specificity of binding. Other modifications include morpholino and peptide nucleic acid modifications (25).

1.4 Conjugates and delivery systems for anti-miR delivery

Despite modifications to the ON structure, tissue specific delivery of AMs remain a challenge. AMs may be conjugated to a variety of molecules to improve delivery or targeting properties. For instance, conjugation to a peptide can facilitate better binding specificity and carries natural resistance against DNA- and RNA-specific nucleases (26). Attachment of an antibody may increase circulation time and further provide a means for targeted delivery (27). Alternative strategies for gene regulation include aptamers, ribozymes, and small interfering

RNA (siRNA). Aptamers may be derived from peptide or RNA and bind to small molecules, nucleotide sequences, proteins, and/or tissues with high affinity and specificity (28). Ribozymes are RNA sequences with catalytic sequences which are similar to that of enzymes. When bound to RNA, ribozymes induce a splice in the RNA sequence, inhibiting translation (29). siRNA are typically administered as a short hairpin RNA (shRNA) or long double stranded RNA and are activated by the Dicer enzyme, which cleaves the sequence and leaves two nucleotide overhangs. The cleaved sequence is then loaded onto the RISC complex and cleaves its mRNA target. siRNA performs similarly to miR, but is better able to cleave the target sequence due to specific binding with the mRNA. siRNA may

6 also function non-specifically as a miR if the binding is not completely complementary (30).

The nanoscale formulation of drugs is another area rapidly gaining attention in the scientific community. The rheological properties, distribution, and biological activity of drugs can vary greatly on the microscale and nanoscale compared to macroscopic counterparts (31). Nanocarriers for gene-based delivery are broadly classified as viral or nonviral vectors. Viral vectors are high efficient vehicles for gene delivery. However, viral vectors often trigger immunogenic response in the patient, which prevents repeated dosing (32). Nonviral vectors are less efficient in terms of delivery, but offer less toxicity and allow for greater customization in delivery approach. Nonviral vectors are principally composed of a cationic lipid

(i.e. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)) or cationic polymer

(i.e. polyethylenimine (PEI)), components which are able to form electrostatic interactions with negatively charged ONs (Figure 1.2). Lipids, polymers, and ON may also be combined to form ternary structures called lipopolyplex.

Nanoparticles are overall more stable than conjugates and naturally can carry larger quantities of drug (33). Furthermore, little to no modifications are required for encapsulation of the ON, thus allowing ONs to retain maximal therapeutic activity (34).

1.5 Design of LN delivery systems

Liposomes are vesicles composed of a lipid bilayer, encapsulating an aqueous core containing hydrophilic components. Hydrophobic molecules may partition within the nonpolar region of the lipid bilayer. Liposomes are thus high versatile

7 structures for the delivery of thousands of already existing active pharmaceutical ingredients (APIs). LNs are similar to liposomes in structure and at times the terms liposome and LN are used interchangeably. Just as there are barriers for delivery of AM, special considerations must be made to promote delivery of AM.

Mean particle diameter and surface charge are key determinants of LN potential in vitro and in vivo. Particles need to fall below 150 nm to take advantage of the enhanced permeation and retention (EPR) effect and allow for post-processing procedures such as sterile filtration (<220 nm) (35). Further, surface charge, as indicated by zeta potential is required to fall within a moderate range of ±10-

30mV. Particles that carry a high positive charge are taken up nonspecifically by macrophages. Particles exhibiting a highly negative charge are removed by the

RES and moreover are unsuitable for electrostatic interaction with anionic ONs.

Finally, particles with neutral or low charge exhibit poor stability and tend to aggregate over time (36). Formulation naturally requires precise optimization of component concentrations to achieve small particle size, moderate zeta potential, and maximal delivery efficiency. Additional considerations such as stability and providing a mechanism for endosomal escape are important as well.

LNs are typically taken up by the process of endocytosis (Figure 1.3). LNs enter the cell packed inside endocytic vesicles. These vesicles then fuse with the endosome. A critical factor in ON delivery is escape from the endosome. Proton- pumps on the surface of the endosome create increasingly acidic conditions within the endosome leading to degradation of the LN and electrostatically complexed ON. Further degradation occurs in the caustic conditions of the late

8 endosome and lysosome compartments of the cell (37). Endosomal escape is facilitated by lipids that are capable of promoting lamellar or bilayer to hexagonal

II (HII) or non-bilayer phase transitions. Some lipids capable of this activity are the class of lipids known as phosphatidylethanolamines and cholesterol (38).

Addition of polyethylene glycol (PEG) is often required to produce small and stable particles. Lipid-modified PEG is able partition within the lipid bilayer and the long PEG chains adsorb water, creating a barrier against opsonization. PEG- modified liposomes are sometimes referred to as stealth liposomes, which are able evade macrophage uptake (39). Lastly, the addition of targeting agent aids the specificity of uptake for LNs. Several researchers have demonstrated a several-fold increase in delivery following conjugation of targeting moieties to

LNs (40-41). Targeting is usually directed towards the overexpressed receptors on the surface of cancer cells. Examples of such receptors include folate, transferrin, and epidermal growth factor receptors (EGFR). Further targeting may be accomplished by the addition of either a full antibody or antibody fragment to the surface of LNs (i.e. CD44) (42).

1.6 Manufacture of LNs

The method of preparation for LNs is perhaps as important as the composition of the formulation itself. The synthetic method plays a major role in determining physical characteristics such as size and encapsulation efficiency, which in turn determine biodistribution and pharmacologic activity (43) (Table 1.2). The first generation of LNs, termed “lipoplexes”, performed poorly in vivo due to heterogeneous ON encapsulation and poor stability (44, 81). Passive ON loading

9 is based on encapsulation methods used for small molecule drugs. However, active loading methods utilizing gradient (i.e. pH) loading are not possible for ON due to their high molecular weight and hydrophilic nature, which prevent efficient

ON transport between lipid bilayers. Passive loading involves rehydration of a dried lipid film with ON solution in buffer. This is then followed by 5 to 10 cycles of freeze-thaw to facilitate encapsulation and 10 cycles of extrusion through a polycarbonate or similar membrane to reduce particle size. To prevent excessive charge, free ON must be removed by dialysis or column filtration. Encapsulation efficiency by passive loading ranges from 3 to 45% (45, 81). Ethanol dilution is another method for the production of ON-loaded LNs. Lipids are rapidly injected into ON solution and are extruded to form LN. Up to 70% encapsulation efficiency is achieved by this method (46, 81). Often, ionizable lipoamines are used in combination with ethanol dilution method, where ionizable lipoamines are injected into acidic buffer, thereby protonating the lipoamines to enhance ON entrapment efficiency. In a related strategy, preformed cationic lipid vesicles may be added to 40% ethanol solution to destabilize lipid bilayers and facilitate encapsulation. Following encapsulation, free ON and ethanol must be removed by dialysis or column filtration to stabilize the formulation (47, 81).

Extrusion is difficult to accomplish on the commercial scale so additional methods are necessary to streamline production of stable particles with low batch-to-batch variation. Stepwise ethanol dilution is an alternative to traditional ethanol dilution method, which does not require extrusion of LNs. Lipids are dissolved in ethanol and combined with ON solution in buffer. Vesicles are

10 formed spontaneously in solution and stepwise dilution with buffer solution results in stabilized LN. Further ethanol is removed by dialysis or column filtration. Encapsulation efficiency by this method ranges from 80-95% with particle diameters falling below 150nm.

While bulk mixing is a scalable method for the rapid production of LNs, it is not provide any mechanisms for controlled mixing. This leads to a heterogeneous product with inconsistent therapeutic activity. Microfluidic hydrodynamic focusing

(MHF) provides several advantages in the area of LN production. Microfluidics involves the manipulation of nanoliter volumes using fine channels measuring only a few microns in diameter (48). Microfluidic technology has found application in a variety of biological and chemical analyses and synthetic methods (49).

Mixing occurs at the interface of adjacent streams of laminar flow in a microfluidic channel. Defined mixing conditions improve homogeneity of the sample, reduce batch-to-batch variability, and increase the drug loading. The system is furthermore able to rapidly synthesize LNs and is easily scalable for clinical and commercial production of LNs. Microfluidic chips are usually designed by lithography and can take on a number of designs, however commercially available microfluidic chips are starting to become available. In addition to controlling mixing geometry, processing specifications such as temperature, flow rate, and sonication may have further application in modulating particle formation and drug loading (50).

Prior to in vitro or in vivo administration, LNs must be sterile filtered with 0.22 µm filters to remove accumulated contaminants acquired during synthesis. Sterile

11 filtration is not cost effective for preparation of small batch samples due to inherent loss of the sample during the filtration process. Over time, liposomes tend to degrade and release drug into the surrounding buffer. For long term storage of LNs, lyophilization is recommended to maintain integrity of the formulation. Inclusion of a cryoprotectant is needed to reduce damage to LNs, especially during the thawing process (51). Typical cryoprotectants include carbohydrates and polyalcohols, such as sucrose or mannitol, which reduce the glass transition temperature and form hydrogen bonds with LNs, thereby displacing water molecules and reducing the incidence of freezing (52).

Lyophilization of LNs is conducted using a two-phase drying process. The first stage is conducted under low temperature (-25-40°C) under moderate vacuum

(0.1-0.12 bar). This stage removes the frozen water in the sample through sublimation. The second stage is conducted at room temperature and under high vacuum (0.01-0.012 bar) and removes any remaining unfrozen water. The resultant liposome cake is then vacuum sealed and stored at room temperature or under refrigeration for best results (53).

1.7 Characterization of LNs

LNs are typically characterized in terms of particle size, surface charge, loading efficiency, and colloidal stability. Particle size may be determined by dynamic light scattering (DLS) or atomic force microscopy (AFM). DLS measures the time- dependent fluctuations of light scattering of particles moving in Brownian motion.

Fluctuations are processed through correlation algorithms and the rate of molecule diffusion through the aqueous solvent is derived. This diffusion value is

12 then used to determine the hydrodynamic radius of the particle through the

Stokes-Einstein equation. AFM measures the deflections of a laser beam off a cantilever moving across the sample surface. Measured deflections are then converted and reported as heights of particles. Particle size may be reported by volume, weight, or area-based methods. Particle sizes in this dissertation are documented by volume-based method, which assumes that spherical particles of the same volume have the same diameter (54). Surface charge is measured in terms of zeta potential and measures the potential between the disperse phase and the interface between the particle and bulk phase. Surface charge is calculated from the mobility of particles following application of an electrical field

(55). Zeta potential is used as an indirect indicator of particle stability and plays a role in determining transfection ability. Loading or encapsulation efficiency are usually calculated to determine the in vivo dosing parameters and commercial feasibility. This is calculated by measuring the relative absorbance drug before and after addition of lysis buffer. Lastly, colloidal stability measures the ability of particles to retain physical and biological integrity following long term storage or dialysis against serum or buffer.

1.8 Pharmacokinetics, Pharmacodynamics, and Nanotoxicity

The extension of nanotechnology towards medicine has spawned a broad range of drugs broadly classified as nanodrugs. Examples of nanodrugs include dendrimers, engineered nanoparticles, emulsions, liposomes, solid LNs, micelles, and polymeric nanoparticles. Nanodrugs often feature advantages in terms of safety and efficacy over their macromolecular counterparts. Despite

13 these advantages, concerns over potential cytotoxicity due to oxidative stress, inflammation, and genetic damage, have led to the investigation of nanotoxicity

(56).

The pharmacokinetic (PK) and pharmacodynamics (PD) profile of nanodrugs may differ profoundly from drugs delivered in the bulk form. Nanodrugs are often more stable, feature extended circulation time and activity, reach a higher peak concentration (Cmax) in a shorter amount of time (Tmax), and are better able to permeate membranes (57). Encapsulation of the drug form protects the drug from metabolism or degradation by proteases or nucleases. Nanoscale drugs are able to take advantage of the EPR effect to achieve potentially higher local doses within the tumor vasculature (58). With longer circulation time and duration of activity, doses of usually toxic chemotherapeutics may also be lowered to improve safety and patient compliance (59). Moreover, nanodrugs may be formulated with targeting agents to specify deliver to a specific organ or cellular subtype to further boost efficacy and safety. Large molecules are opsonized by macrophages associated with the RES. LNs likewise may be removed by the

RES within a few hours unless they are surface-treated. Hydrophilic surface modifications to nanoparticles such as PEG-coating may minimize opsonization of nanoparticle drugs. Alternative coatings including polyethylene oxide, poloxamers, poloxamines, and polysorbates are also possible (60). Many of the advantages in delivery with nanodrugs stem from the inherent properties of the formulation itself, including the size, surface charge, and hydrophobicity of the formulation. The small size of nanoparticles does not require a dissolution phase

14 as seen with large macromolecular drugs and therefore system drug release is nearly instantaneous with intravenous delivery. Several liposomal drugs are already on the market with hundreds more undergoing clinical trial (Table 1.3-

1.4). Doxil, or liposomal doxorubicin, is perhaps the most well-known liposome- based drug. Relative to the free drug at a 50mg/m2 dose, Doxil is able to achieve a 300-fold greater plasma concentration with a 250-fold reduction in clearance and 60-fold reduction in volume of distribution (61). Cellular PK/PD to understand uptake of nanoparticles at the cellular level is another growing area of clinical investigation. Nanoparticles typically enter the cell through clathrin-mediated endocytosis, but may also be taken up by caveolin-mediated endocytosis, potocytosis, pinocytosis, or patocytosis (Figure 1.4) (62). Cellular uptake may be particularly effective for nanoparticles carrying a moderate positive charge, as interaction with negative charges of mucin found on the surface of epithelial cells may increase the likelihood of uptake (63). Once taken up into the cell, LNs must escape an increasingly acidic endosomal compartment and deliver drug to the intracellular target.

Nanoparticles are able to decrease the toxicity of a number of chemotherapy drugs by lowering the effective dose. However, several in vitro and in vivo studies have suggested that nanoparticles are able to trigger cytotoxicity or inflammatory responses. Nanoparticles may induce the production of reactive oxygen species and free radicals, which in turn cause oxidative stress and trigger inflammatory responses, mutagenesis, and fibrosis (64). Repeated dosing with nanoparticles may also be of concern as accumulation of nanoparticles or degraded materials

15 within the cell has been known to cause cytotoxicity (65). Just as physical characteristics play a role in describing drug stability and efficacy, size, shape, and surface charge contribute to nanotoxicity. For instance, the small size of nanoparticles makes them susceptible to internalization by virtually any cell via pinocytosis. This creates a high risk of cytotoxicity (66). In another example, cationic polymers are known to have an effect on cell proliferation and differentiation in epithelial tissue. Nanoparticles are typically taken up by the RES where they tend to accumulate in the liver and spleen, which both house a large endogenous population of macrophages (67). While many toxicity studies have considered the acute-toxicity to determine the 50% lethal dose (LD50), much fewer studies by comparison have explored the potential organ damage due to nanoparticle therapy. Generally speaking, lipid based carriers are less toxic than synthetic formulations because they are mostly composed of naturally occurring organic molecules. Any synthetic compounds used in delivery should be biodegradable to avoid toxicity (68).

1.9 Objectives of the dissertation

The development of efficient vehicles for ON delivery is paramount to the clinical translation of therapeutic ONs. The overarching goal of this dissertation is to provide a guide for the rational design and implementation of LNs for the delivery of ON-based drugs. Several novel lipid-based delivery technologies are explored in the present work with emphasis on physical characterization of the carrier along with validation of in vitro and in vivo efficacies.

16 Chapter 2 focuses on development of lipid coated albumin nanoparticles (LCAN).

The core of LCAN consists of a hypercationized albumin molecule conjugated to several short chain PEI molecules (Figure 1.5). This core is then surrounded by lipids to protect the ON from nuclease mediated degradation. An antisense ON,

GTI-2050, is delivered to demonstrate efficacy in vitro and in a mouse xenograft model.

Chapter 3 focuses on development of quaternary-tertiary amine liposomes

(QTsome). QTsome include quaternary and tertiary lipoamines combined at a specific ratio to potentiate delivery efficiency by activating a pH-sensitive destabilization mechanism under acidic conditions of the endosome (Figure 1.6).

AM-21 is delivered by QTsome in a non-small cell lung cancer (NSCLC) model.

Chapter 4 focuses on development of small peptide lipid nanoparticles (SPLN).

SPLN include a fusogenic peptide, gramicidin to promote endosomal lysis

(Figure 1.7). SPLN are prepared by traditional bulk mixing and a prototype MHF method. AM-221 and AM-21 encapsulated SPLN are co-administered and evaluated for therapeutic efficacy in a triple negative breast cancer model.

Chapter 5 provides a summary and perspective on the key findings from the dissertation and includes suggestions as to further development of nanoparticle systems for the delivery of nucleic acid-based drugs.

Figure 1.2. Selected examples of lipid and polymers in LN synthesis. Cationic lipids and polymers form electrostatic interactions with negatively charged ONs.

Bilayer forming lipids help to stabilize the LN. Fusogenic lipids promote disruption of endosomal lipid bilayers. PEG-modified lipids reduce opsonization and decrease off-target uptake.

ON to the cytosol for interaction with miRs or mRNA.

A B C D E

21 A

Figure 1.5. LCAN formulation. LCAN consist of a hypercationized APC-ON complex core (A) surrounded and stabilized by a lipid layer (B). LCAN are employed to deliver antisense ON, GTI-2501, directed against ribonucleotide reductase subunit 1 (RNR1) in a cervical cancer model.

22 A

QT/AM-21 is further co-administered with PTX to evaluate efficacy of combination therapy.

Figure 1.7. (A) SPLN include a fusogenic peptide, gramicidin, to promote fusogenic potential within the endosomal compartment for cytosolic delivery of

AM. (B) SPLN is applied for the delivery of AM-221 and AM-21 in a TNBC model to determine if modulation of miRs can induce sensitivity towards hormone therapy (TMX).

24 Table 1.1. Selection of miRs involved in cancer.

Cancer Upregulated Downregulated miRs Reference miRs Breast miR-221, 222, 181, miR-26b, 27b, let-7, 69 21, 200c, 28, 106a, 15b, 342, 451, 126, 206, 155, 301 127 Chronic Lymphocytic miR-150, 21, 106b, miR-155, 181b, 70 Leukemia 650 29a/b/c, 223, 34a, 17- 5p, 15a, 16-1 Colon miR-802, 194, 27a miR-107, 145 71 Head and neck miR-374, 340, 22, miR-27a, 34a/b/c, 72 10a, 140, 181a, 203, 302c, 23a, 27b, 146a, 126, 31, 9 215, 28, 330, 337, 107, 133b Liver miR-25, 92a, 35, 122, miR-29a, 197-3p, 73 34, 885-5p, 21, 106b, 505-3p, 223-3p, 181b let7f Lung miR-17-92, 21, 31, miR-126, 34, 145, 74 221, 222, 214, 155, 200, let-7 210, 98 Oral miR-221, 222, 203, miR-125b, 100, 138, 75 31, 210, 24, 21, 181, 101, 7, 124, 200, 15a, 345, 211, 184, 133, 1, 195a, 205 Ovary miR-20a, 92, 93, miR-let-7a/b/d/f, 22, 76 106a, 146b, 182, 34a/b/c, 12b, 127-3p, 200, 205, 223 152, 155 Pancreas miR-101, 143, 145, miR-34a, 96, 141, 77 146a, 150, 155, 148a/b, 217, 29c, 181a/b/d, 196a/b 30a-3p, 375 Prostate miR-let-7i, 21, 26, 93, miR-let7c/e, 24, 30c 78 106a, 106, 141, 195, 221, 375, 378, 622 Renal miR-9, 7, 156, 23a/b, miR-16, 141, 122 79 222, 200b/c, 17-5p, 429, 106, 20 Stomach miR-155 miR-124, 512-5p, 80 129-2, 181c, 375, 34b/c, 212, 137, 10b, 9, 129, 155, let-7, 195, 378, 34c-5p

25 Table 1.2. Methods of LN preparation. Adapted from (81-82).

Formulation Size* Encapsulation* Advantages Disadvantages Method (nm) (%) Passive 100- 3-45  Well-  Low Loading 500 characterized encapsulation  Requires extrusion and post- encapsulation filtration  Difficult to scale up Ethanol drop 100- 65  Stabilization of  Requires 200 ionizable lipids extrusion using buffer  Difficult to scale exchange up Reverse- 100- 85  Prevents  Requires phase 200 aggregation of extrusion and evaporation charged post- liposomes encapsulation filtration  Difficult to scale up Ethanol 70- 90  Rapid formation  Heavily dilution 130 of LNs dependent on  Low cost ionic strength,  Scalable method cationic lipid content, and PEG lipid content. Microfluidics 50- 90  Rapid formation  Heavily 150 of LNs dependent on  Automated ionic strength, process cationic lipid  Scalable method content, and PEG lipid content.  Potential high start-up cost

*Size and encapsulation will vary depending on lipid composition.

26 Table 1.3. Selection of commercially available LN formulations. Adapted from reference (83) and (84). (i.v. = intravenous, i.m. = intramuscular)

Route Brand of Name Delivery Drug Indication Amphotericin Abelect i.v. B severe fungal infections Abraxane i.v. Paclitaxel breast cancer Amphotericin Ambisome i.v. B severe fungal infections Amphotericin Amphotec i.v. B severe fungal infections DaunoXome i.v. Dauorubicin blood tumors neoplastic meningitis, Depocyt Spinal Cytarabine lymphomatous meningitis Morphine DepoDur Epidural sulfate pain management Karposi's sarcoma, ovarian Doxil i.v. Doxorubicin cancer, breast cancer Inactivated hepatitis A virus (strain Epaxal i.m. RG-SB) Hepatitis A Inactivated hemaglutinine of influenza virus strains A Inflexal V i.m. and B Influenza Karposi's sarcoma, ovarian Lipo-dox i.v. Doxorubicin cancer, breast cancer Myocet i.v. Doxorubicin breast cancer Vincristine Onco TCS i.v. sulfate non-Hodgkin's lymphoma age-related molecular degeneration, pathologic myopia, ocular Visudyne i.v. Verteporfin histoplamosis

27 Table 1.4. Selection of LNs undergoing clinical trial. Adapted from references

(83) and (85). (i.v. = intravenous, ipl. = intrapleural)

Product Route Drug Indication Phase siRNA targeting transthyretin ALN-TTR i.v. (TTR) TTR amyloidosis I Newly diagnosed or relapsed solid Alocrest i.v. Vinorelbine tumors I portable aerosol Arikace delivery Amikacin Lung infection III Cisplatin analog (L- Aroplatin ipl. NDDP) Metastatic colorectal cancer II Acute promyelocytic leukemia, Atragen i.v. Tretinoin hormone-refractory prostate cancer II Brakiva i.v. Topotecan Relapsed solid tumors I Irinotecan HCl: CPX-1 i.v. floxuridine Colorectal cancer II Cytarabine: CPX-351 i.v. daunorubicin Acute myeloid leukemia II EndoTAG-1 (powder/24 Anti-angiogenic properties, breast months) i.v. Paclitaxel cancer, pancreatic cancer II Exparel i.v. Bupivacaine Nerve block II INX-0076 i.v. Topotecan Advanced solid tumors I INX-125 i.v. Vinorelbine Advanced solid tumors I Leukemia, breast, stomach, liver, and LEM-ETU i.v. Mitroxantrone ovarian cancers I LEP-ETU (powder 12/months) i.v. Paclitaxel Ovarian, breast, and lung cancers I/II SN-38, active metabolite of LE-SN38 i.v. irinotecan Metastatic colorectal cancer I/II Lipoplatin Pancreatic, head and neck, (suspension/36 mseothelioma breast, gastric, and non- months) i.v. Cisplatin squamous non-small cell lung cancer III Prostaglandin Liporstin i.v. E1 Peripheral vascular disease II/III Grb2 Liposomal antisense Acut myeloid leukemia, acute Grb-2 i.v. oigonucleotide lymphoblastic leukemia I Liposome- annamycin (powser) i.v. Annamycin Acute lymphocytic leukemia I/II Marqibo i.v. Vincristine Metastatic malignant uveal melanoma III continued 28 Table 1.4 continued.

Transferrin- targeted Gastric cancer and gastro-esophageal MBP-436 i.v. oxaliplatin junction II ErbB2/ErbB3- targeted MM-302 i.v. doxorubicin ErbB2-positive breast cancer I MM-398 i.v. CPT-11 Gastric and pancreatic cancer II Nyotran i.v. Nystatin System fungal infections I/II OSI-211 i.v. Lurtotecan Ovarian and head and neck cancer II Camptothecin Recurrent or progressive carcinoma of S-CKD602 i.v. analog the uterine cervix I/II SPI-077 i.v. Cisplatin Lung and head and neck cancer I/II BLP25 lipopeptide (MUC1-targted Cancer vaccine for multiple myeloma Stimuvax i.v. peptide) developed encephalitis III T4N5 Bacteriophage liposome T4 lotion topical endonuclease 5 Xeroderma pigmenosum III ThermoDox i.v. Doxorubicin Non-resectable hepatocellular carcinoma III RNAi targeting polo-like kinase TKM-PLK1 i.v. 1 (POLO) Liver tumors I

29 CHAPTER 2

LIPID COATED ALBUMIN NANOPARTICLES FOR THE DELIVERY OF LOR-2501

2.1 Introduction

Antisense oligonucleotides (ONs) have been developed to treat a variety of diseases including cancer. By suppressing sequences responsible for tumor development, progression, or drug resistance, ON have become an invaluable tool for the treatment of cancer (1-3). Recent clinical trials and publications have indicated the therapeutic advantage of a 20-mer ON, LOR-2501 (previously GTI-

2501) developed by Lorus Therapeutics (4-5). LOR-2501 targets R1, the large subunit of ribonucleotide reductase. R1 plays a critical role in the cell cycle and catalyzes the reduction of ribonucleotides (ADP, GDP, UDP, CDP) to deoxyribonucleotides (dADP, dGDP, dUDP, dCDP). Implementation of LOR-

2501 into clinical practice however has been limited by the lack of efficient vectors for delivery. The high charge density and large molecular weight of ON render it impermeable to cell membranes and the presence of serum nucleases make it susceptible to degradation. Previous studies have evaluated several viral and nonviral nanocarriers for gene delivery, neither of which have demonstrated satisfactory efficacy in vivo. Often, these agents cause off-target toxicity, or fail to facilitate endolytic action necessary for cytoplasmic delivery (6-7). Principle

30 amongst nonviral polymer-based transfection agents is high molecular weight polyethylenimine (PEI, 25kDa) (8-9). The high charge density of this polymer facilitates strong electrostatic interaction with the negatively charged phosphate backbone of ONs. The use of high molecular weight polymer for transfection however, has largely been limited to in vitro studies as accumulation of the polymer inside cells following delivery confers cytotoxicity (10). Low molecular weight PEI (600 Da) causes substantially less cytotoxicity, however the low charge density of the polymer is not conducive for the rigorous conditions of in vivo delivery, which require formation of a stable electrostatic complex. In order to mitigate the cytotoxicity of high molecular weight PEI and to amplify the transfection activity of low molecular weight PEI, a new class of transfection agents, albumin polycation conjugates (APCs), was developed (Figure 2.1). In previous studies, the coating of albumin on cationic lipid nanoparticles boosted the transfection efficacy of small interfering RNA (siRNA) in several breast cancer models (11-12). The increased cellular response due to albumin is believed to stem from its ability to deter serum proteins from binding to the nanoparticle (11). Conjugation of albumin with low molecular weight polymers such as PEI (600 Da) may greatly enhance the transfection activity of plasmids, siRNA, and ON. Although APCs may be used as alone for gene delivery, APCs combined with lipids may further potentiate transfection activity. In the current study, APCs were combined with neutral and cationic lipids and PEGylated lipid to generate a long circulating lipid nanoparticles for ON delivery. The rationale of this model arises from the concept of lipopolyplexes, which include both lipid and

31 polymer components. Previous research has demonstrated the benefit of using a combination of these classes of molecules which exhibit greater transfection efficiency than either component alone (13-14). The inclusion of pre-formed

APCs with lipids has been termed lipid albumin coated nanoparticles (LCANs). In the present work, LCANs are designed and optimized for ON delivery and combined with LOR-2501 to demonstrate anticancer activity in vitro and in a xenograft mouse model.

2.2 Materials and methods

2.2.1 Materials. Human serum albumin (HSA) was obtained from Octapharma Plasma, Inc.

(Charlotte, NC, USA). PEI (MW 600 Da) and ethidium bromide (EtBr) were obtained from Sigma Aldrich (St. Louis, MO, USA). 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) was obtained from Thermo Scientific

(Rockford, IL, USA). LOR-2501 (5′-

C*T*C*T*A*G*C*G*T*C*T*T*A*A*A*G*C*C*G*A-3′, where * represent phosphorothioate linkages) and scrambled control and primers for R1 and β-actin were purchased from Alpha DNA (Montreal, Quebec, Canada). PD-10 desalting columns were obtained from GE Healthcare Life Sciences (Pittsburgh, PA, USA).

Bicinchoninic acid (BCA) protein assay and 2,4,6-Trinitrobenzene sulfonic acid

(TNBSA) assay solution were purchased from Thermo Scientific. Agarose and

5X gel loading dye were obtained from Fisher Scientific (Pittsburgh, PA, USA).

CellTiter 96 AQeous One Solution Cell proliferation was purchased from 32 Promega (Madison, WI, USA). TRIzol® reagent, SuperScript® III First-Strand

Synthesis System, and SYBR Green PCR Master Mix were purchased from Life

Technologies (Carlsbad, CA, USA). All reagents were used without further purification.

2.2.2 Synthesis and characterization of APC

APC were prepared by combining 41.9 mg HSA dissolved in water (titrated to pH

8.0) with 18.7 mg PEI dissolved in water (titrated to pH 8.0) to a total volume of

2.0 mL in HEPES buffered saline (HBS, 50mM, pH 8.0). 9.6 mg EDC was allowed to equilibrate to room temperature before dissolution in a small volume of water and dropwise addition to a stirring solution of HSA and PEI. The molar ratio of compounds used was set at 1:50:80, HSA:PEI:EDC. The reaction was allowed to proceed for 1 h at room temperature. pH was maintained at 8.0 throughout the course of the reaction with 1 μM HCl used to adjust the pH. An aliquot of purified

APC for further incorporation into liposomes was taken and eluted through a PD-

10 column to remove unreacted reagents using phosphate buffered saline (PBS, pH 7.4) as an eluent. BCA assay was then used to determine the concentration of the conjugate in the collected fractions. The filtered product was stored at 4°C.

In order to evaluate conjugation yield, TNBSA assay was conducted to determine relative primary amine content of the purified sample relative to unreacted albumin. APCs were combined with 100 μM LOR-2501 at varying nitrogen to phosphate ratio (N:P) to form stable electrostatic complexes.

33 2.2.3 Synthesis of LCAN. DDAB, CHOL, and TPGS lipid stocks dissolved in pure ethanol were combined at a molar ratio of 60:35:5. 100 μL lipid mixture in ethanol was added to 900μL

HBS buffer to form empty lipid nanoparticles (LNs) at 2 mg/mL concentration in

10% ethanol. LNs were allowed to stand at room temperature for 10 min before combining with varying amounts of APC/LOR-2501 to form LCANs. LCANs were briefly vortexed and allowed to stand for 15 min at room temperature prior to further analysis. LCAN without APC, designated LN/LOR-2501, were prepared as a control for in vitro and in vivo study.

2.2.4 Physical characterization of LCAN.

Particle sizes of LCAN/LOR-2501 complexes were determined by dynamic light scattering on a NICOMP 370 Submicrometer Particle Sizer (Santa Barbara, CA,

USA) under the volume-weighted setting. Zeta potential measurement was conducted on a ZetaPALS (Brookhaven Instruments Corp., Worcestershire, NY,

USA) by diluting the complexes to a total volume of 1.4 mL in 0.1X PBS.

Samples of LCAN were stored at -20, 4 and 25°C with 10% sucrose as a cryoprotectant to evaluate colloidal stability. Particles were periodically monitored over a 30 day period for changes in particle size.

2.2.5 Gel retardation assay.

A 0.8% agarose gel in 1X TBE buffer containing 0.5 μg/mL EtBr was prepared for gel electrophoresis. 10 μL aliquots of LCANs containing varying amounts of APC were combined with 2 μL 5x gel loading buffer and loaded onto a gel.

Electrophoresis was conducted at 100V for 45 min at room temperature with 1X 34 TBE as the running buffer. The resultant gel was imaged under a UV transilluminator (Ultra-Lum, Claremont, CA, USA) and documented using a

Kodak DC 290 digital camera (Eastman Kodak, Rochester, NY, USA).

2.2.6 Antisense oligonucleotide transfection. KB (a subline of HeLa cervical cancer) cells were grown in complete media consisting of RPMI 1640 medium supplemented with 10% fetal bovine serum

(FBS), 2 mM L-glutamine, penicillin (100 U/mL), and streptomycin (100 μg/mL) at

4 2 37°C and 5% CO2 atmosphere. Cells were plated at a density of 2×10 cells/cm in 6-well plates 24 h prior to transfection and allowed to reach 70% confluency.

At the time of transfection, media was removed and replaced with appropriate controls and formulations containing varying amounts of LCAN (1:1, 3:1, 6:1

APC:ON, w/w) and 200 nM LOR-2501 in 1 mL serum-free or 20% serum containing media. Transfection was allowed to proceed for 4 h at 37°C before replacing the transfection media with fresh complete media and incubating the cells for an additional 44 h at 37°C. Following the period of incubation, cells were evaluated for cell proliferation and target gene downregulation.

2.2.7 Cell proliferation assay. Cell viability 48 h following LCAN/LOR-2501 treatment was assessed by MTS assay. 20 µL MTS solution was added to each well of a 96-well plate and incubated for 45 min. Following the incubation period, the absorbance was measured at 490nm on a Dynatech MR-600 microplate reader (Bio-Rad,

Hercules, CA, USA). Cell proliferation was reported as a percentage relative to the untreated cell sample, set at 100% viability.

35 2.2.8 Quantitative qRT-PCR of mRNA. The mRNA of R1 was quantified by quantitative real-time PCR (qRT-PCR). RNA was isolated from cells using TRIzol® reagent and purified according to the manufacturer’s instruction. cDNA was generated using SuperScript® III First

Strand Synthesis System kit per manufacturer’s protocol. β-actin was used as a reference gene to determine relative R1 gene regulation. SYBR Green® was used to conduct qRT-PCR according to the manufacturer’s instruction on an

Applied Biosystems StepOnePlusTM RT-PCR system (Life Technolgies). mRNA levels of R1 were computed under the comparative ΔΔCT method (16-17) and reported as levels of RNA relative to β-actin.

2.2.9 Establishment of murine KB xenograft model. Athymic nude-foxn1nu mice, obtained from NCI/Fredericks, were inoculated with

KB cells via subcutaneous injection of 1×106 cells per mouse. Tumors were allowed to reach a size of 100 mm3 before starting treatment. Groups of five mice each were randomly selected for treatment with saline control, free LOR-2501,

LCAN with scrambled LOR-2501 control (LCAN/SC), LN/LOR-2501, or

LCAN/LOR-2501. Mice were treated every four days at a dose of 2.5 mg/kg (50

μg/20g mouse) for a total of seven doses. Tumor volume was monitored prior to

퐿푊퐻 each dosing and was calculated by: 푉 = , where V = volume, L = length, W = 2 width, and H = height. Mice were then euthanized according to university guidelines by CO2 inhalation. Tumor samples were harvested and immediately frozen prior to analysis.

36 2.2.10 Tumor gene regulation analysis. A small section of frozen harvested tissue sample was processed in TRIzol® reagent and homogenized with a Tissue-Tearor homogenizer (BioSpec Products

Inc, model 985370, Bartlesville, OK, USA). qRT-PCR as described previously was conducted on the homogenized tissue to determine relative gene regulation in vivo.

2.2.11 Statistical analysis. All experiments were done in triplicate and results were reported as mean ± standard deviation (S.D.). Significance between two groups was analyzed by

Student’s t-test and multiple groups were compared by analysis of variance

(ANOVA) on Microsoft Excel 2007 software (Microsoft, Redmond, WA). Results were considered statistically significant at the p < 0.05 level. Animal group sizes were derived from power analysis based on previous data in the literature (18).

With 5 mice per group, differences as small as 100 mm3 will be detectable between groups. *, **, and *** represent p<0.05, 0.01, and 0.001 respectively for treatment group relative to untreated control.

2.3. Results

2.3.1. Synthesis and characterization of APC. HSA was conjugated to low molecular weight PEI to form APCs. APCs were eluted by PD-10 column filtration to purify the conjugate. As shown in Figure 2.2, nearly 90% PEI was retained within the conjugated fractions (4-6) signifying high reaction yield. Fractions 13-15 contain the free PEI. Analysis by TNBSA similarly showed high primary amine content within the APC product fractions. 37 Comparison with the absorbance spectra of HSA revealed that approximately 37 molecules of PEI were attached to each molecule of HSA.

2.3.2 Synthesis and characterization of LCAN. LCANs composed of varying amounts of APCs were evaluated for the effect on particle size and zeta potential. Differences in APC concentration between samples did not significantly alter the resultant particle size as only slight decreases were observed for increasing APC concentration (Figure 2.3a). LNs were consistently between 100 to 200 nm, meeting size criteria for passive targeting to the tumor vasculature. Zeta potential (Figure 2.3b) demonstrated a moderate charge, between 11 and 26 mV, suggesting the formation of stable

LNs for transfection. As a point of comparison, commercial transfection reagent,

Lipofectamine 2000 (LF2K), had a zeta potential of 65.8 mV. Increasing APC content increased the overall surface charge, suggesting that the net charge on

APC/ON complexes was positive, most likely due to the high density of cationic

PEI. Gel retardation analysis revealed stable formation of LCAN/LOR-2501 complexes at w/w above 3:1 (APC:ON) (Figure 2.4). Below 3:1, complexes displayed a sharp reduction in ON retention. Encapsulation efficiency studies showed that LCAN have a high encapsulation rate of 72.4%. Further studies evaluated the colloidal stability of LCAN (Figure 2.5). LCAN were most stable at

4 and -20 °C. At room temperature (25°C), LCAN increased in size after a few days.

38 2.3.3 Analysis of LCAN/LOR-2501 in KB cells. The vehicle related toxicity of LCAN/LOR-2501 formulations was evaluated in KB cells at varying concentrations. Toxicity appeared to be dose dependent at the tested levels. As shown from the data (Figure 2.6), LCANs did not confer significant cytotoxicity below the 3:1 (APC:ON) level, however moderate signs of toxicity appeared for LCAN (5:1). The high density of charge in the LCAN (5:1) formulation may have attributed to the observed cytotoxicity. For animal studies,

LCAN (3:1) was used to minimize cytotoxicity and to avoid confounding effects in the measurement of gene regulation. No significant toxicity was conferred by

LCAN lipids or LOR-2501 alone. Downregulation of R1 mRNA was evaluated by qRT-PCR (Figure 2.7). The greatest decrease in R1 levels was observed for

LCAN (3:1) under serum-free conditions. Transfection under 20% serum however did not significantly inhibit transfection activity of LCAN, though the

LCAN (1:1) formulation performed slightly better than the LCAN (3:1) formulation.

Minimal downregulation activity was observed for LCAN lipids or free LOR-2501.

2.3.4 Evaluation of LCAN/LOR-2501 in a KB xenograft murine model. Tumor growth (Figure 2.8) was used as a therapeutic marker to gauge the potential of LCAN/LOR-2501 for tumor suppression. Mice treated with free LOR-

2501 or LCAN with scrambled control showed limited anticancer activity.

Although LN/LOR-2501 demonstrated initial signs of tumor suppression, tumor size increased substantially during the second and third week of study. Mice treated with LCAN/LOR-2501 demonstrated significant decreases in tumor size and displayed fewer signs of morbidity relative to the control groups. These results comes a contrast to the previously published literature on LOR-2501, 39 which observed considerable tumor suppression activity and target gene and protein downregulation with free LOR-2501 (4). The cancer types and models used for these studies were different and may account for the difference in observed transfection activity for free LOR-2501.

2.3.5 In vivo target downregulation. qRT-PCR was conducted to verify whether the observed decrease in tumor size was due to inhibition of R1 expression (Figure 2.9). Free LOR-2501 only accomplished minor downregulation of R1 levels. LN/LOR-2501 exhibited moderate downregulation activity (31%) while LCAN/SC accomplished only marginal R1 suppression. Treatment with LCAN/LOR-2501 demonstrated strong downregulation of R1 levels at 69%. Thus, APC plays a vital role in mediating enhanced transfection efficiency.

2.4. Discussion

LOR-2501 is a broad spectrum chemotherapeutic, with tumor suppressive activity in colon, pancreatic, lung, breast, ovarian, skin, brain, prostate, and renal cancers (4). The antisense targets the R1 subunit of RNR, a potent regulator of

DNA synthesis during cell proliferation and metastasis (19-21). As cancer cells divide at a much faster rate relative to normal cells, cancer cells are more sensitive to inhibition of RNR activity (22). Additional studies have suggested anticancer activity through the involvement of p53R2-mediated DNA repair mechanisms through downregulation of R1 (23-24). During DNA repair processing, R1 provides the dNTPs necessary for p53R2 to correct transcription 40 errors. Therefore, LOR-2501 may further be applied towards increasing cancer sensitivity towards chemotherapeutics or radiotherapy (25). LOR-2501 has demonstrated superior activity over chemotherapeutics 5-fluorouracil (5-FU), gemcitabine, and vinblastine, which also target RNR as part of their mechanism of action (4). Relative to LOR-2501, these chemotherapeutics carry greater risks of side effects because of off-target toxicity (26).

Clinical trials have previous evaluated LOR-2501 in prostate and renal cancer patients (27-28). Encapsulation of LOR-2501 within LNs such as LCAN may further increase the safety and specificity of delivery of LOR-2501 by passive targeting of tumor vasculature through the enhanced permeation and retention

(EPR) effect (29-30). LNs 150 nm and below are able to passively traffic to the tumor site and remain in circulation due to reduced lymphatic drainage. The APC used in this study is but one of several possible cationic low molecular weight polymers that may be conjugated with albumin. Pentaethylenehexamine (PEHA) or diethylaminoethyl (DEAE) are low molecular weight cationic polymers may similarly be conjugated to albumin to form an APC for oligonucleotide delivery

(31-32).

ONs are inherently unstable in serum and are prone to degradation by nucleases

(33). Furthermore, ONs may bind with serum proteins such as immunoglobulins and complement proteins which may change its biological properties (34). The delivery of antisense oligonucleotides and similar materials remains a critical challenge for pharmaceutical formulation. LCANs protect LOR-2501 from nuclease degradation by securing it electrostatically within the APC core. The ON

41 is further protected by a layer of lipids and PEGylating agent which reduces opsonization and removal by macrophages. Our studies demonstrated the benefit of using a hypercationized albumin core to promote transfection activity.

Several other studies have suggested he beneficial effect of including albumin in formulations designed for in vivo transfection to tumors. Interestingly, albumin tends to preferentially accumulate within tumor and inflamed tissues. Moreover, pre-coating liposomes with albumin has been found to decrease hepatic disposition and immunostimulation, improve drug circulation time, and increased uptake at the tumor site. (35-36).

LCAN/LOR-2501 prepared in this study exhibited lower surface charge than

Lipofectamine, while maintaining greater transfection ability. Lipofectamine’s high cationic charge makes its unusable for in vivo purposes because the high amount of charge is toxic to cells and attracts uptake by macrophages. In vitro studies demonstrated low cytotoxicity conferred by LCANs, emphasizing the safety of the formulation over high molecular PEI. LCAN further demonstrated optimal transfection efficiency at an APC:ON ratio of 3:1. Testing of LCAN (3:1) in a xenograft mouse model demonstrated promising results. LCAN also performed better than LOR-2501 alone, providing further evidence of the benefit of using LN delivery systems.

2.5 Conclusion

The present study evaluated LCAN for the delivery of LOR-2501. LCAN include a highly cationized albumin-PEI core, which facilities strong interaction with ONs 42 and deters serum protein binding. LCAN were prepared by stepwise addition of

APC to LOR-2501, followed by addition of lipids. Electrostatically stabilized LCAN demonstrated small particle size, moderate zeta potential, high drug loading, and long term colloidal stability. LCAN was well tolerated by KB cells at moderate concentrations of APC and further suppressed R1 expression in the presence of serum. Xenograft models in mice demonstrated the ability of LCAN/LOR-2501 to successfully suppress tumor growth through inhibition of R1 expression.

Figure 2.1. Synthesis of APC. HSA was combined with an excess of low MW

PEI (titrated to pH 8) and conjugated using EDC. The resultant conjugate was eluted through a PD-10 column to remove free PEI.

44 A 300

250

200

150 APC

100 HSA

Protein concentration (ug/mL) concentration Protein 50

0 0 5 10 15 20 Fraction (mL)

B 0.9 0.8 0.7 0.6 0.5 0.4 APC 0.3 HSA

Absorbance (490 nm) (490 Absorbance 0.2 0.1 0 0 5 10 15 20 Fraction (mL)

37 PEI molecules were bound to each molecule of albumin.

45 A 160

140

120

100

60 Particle Size (nm) Size Particle 40

0 LCAN (without APC) LCAN (1:1) LCAN (3:1) LCAN (5:1)

B 80

Zeta potential (mV) potential Zeta 20

0 LF2K LCAN (without LCAN (1:1) LCAN (3:1) LCAN (5:1) APC)

Figure 2.3. Particle size and surface charge. LCAN with varying APC:ON ratio were prepared. (A) Mean particle diameter was determined by DLS. (B) Surface charge was determined by zeta potential measurement. Data is presented as the mean±SD of three separately prepared samples.

46 0:1 0.5:1 1:1 3:1 6:1

Figure 2.4. Gel retardation assay. LCAN of varying APC:ON ratio were prepared and analyzed for ability to condense ON against an electrophoretic gradient.

Red arrows represent complete retardation of ON.

47 170

160

150

140

130 -20C

120 4C

110 25C

Mean Diameter (nm) Diameter Mean 100

80 0 5 10 15 20 25 30 Days

Figure 2.5. Colloidal stability. LCAN/LOR-2501 (3:1) were stored under varying conditions to determine the effect of temperature on particle size over time. Data are presented as the mean±SD of three separate LCAN preparations.

48 120

100 p=0.038 * 80

40 20

Cell Cell viability (%of control) 0

Figure 2.6. Cell viability. Control formulations or LCAN/LOR-2501 (200 nM) at varying APC:ON ratio were transfected in KB cells for 4 h. MTS assay was conducted to measure relative cell proliferation 48 h after the start of transfection.

Data represent the mean±SD of three separate transfections.

49 A 1.2

actin) 1 - β *** ** 0.8 ** ***

0.6 *** 0.4

0.2

0 Fold expression R1 (rel.R1expression Fold

1.2 B

actin) 1

β p=0.015 0.8 * ** 0.6 **

0.4

0.2

0 Fold expression R1 (rel.R1expression Fold

50 1800

1600 ) 3 1400 1200 Untreated 1000 *** LOR-2501 800 LCAN/SC 600 LN/LOR-2501

400 Tumor Volume (mm Volume Tumor 200 LCAN/LOR-2501 0 0 5 10 15 20 25 Days

Figure 2.8. Antitumor activity. KB cell xenograft models were initiated in mice

(n=5). To determine the in vivo efficacy of LCAN, mice were treated i.v. with saline control, free LOR-2501, LN/LOR-2501, LCAN/SC, or LCAN/LOR-2501

(3mg/kg). Tumor progression was monitored over a 3 week period. (Mice with tumors over 1.5 cm in diameter were removed from the study per institution policy.) Results are reported as the mean±SE volume.

1.4

1.2 actin) - 1 β p=0.033 * p=0.019 * 0.8

0.6

0.4 ***

Fold R1 expression (rel.expressionR1 Fold 0.2

0 Untreated LOR-2501 LCAN/SC LN/LOR-2501 LCAN/LOR-2501

Figure 2.9. In vivo R1 regulation. Harvested tumors were analyzed for differences in R1 mRNA expression. Data is presented as the mean±SE of QRT-

PCR analysis for three separate tumor sections.

52 CHAPTER 3

QUATERNARY-TERTIARY LIPOAMINE SYSTEMS FOR THE DELIVERY OF ANTI-MIR-21

3.1 Introduction

Lung cancer is currently the leading cancer and accounts for over 160,000 deaths in the United States each year (1). 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 (2).

Moreover, NSCLC is highly metastatic and rapidly spreads to other parts of the body including the adrenal glands, liver, brain, and bone (3). The destructive nature of NSCLC and the lack of effective means of treatment outline the critical need for new modes of therapy.

Several researchers have associated the progression of NSCLC with overexpression of microRNA-21 (miR-21) (4-5). miR-21 operates at the epigenetic level of cancer, directly and indirectly impacting the gene pathways associated with cell apoptosis, DNA repair, tumor suppression, cell invasion, metastatsis, and drug resistance (6). Successful inhibition of miR-21 is expected to provide better outcomes for patients due to its broad spectrum of activity in modulating cancer progression. Administration of a miR inhibitor oligonucleotide

(ON), anti-miR-21 (AM-21), has demonstrated much success in vitro for the

53 modulation of miR-21 targets (i.e. ANKRD46, DDAH1, PTEN, RECK, PDCD4,

TIMP3) in addition to decreasing relative cell migratory and invasion potential (7-

8). Moreover, studies of miR-21 in ovarian cancer have linked overexpression of miR-21 to resistance against chemotherapeutics such as paclitaxel (PTX) (9).

Despite initial promising results with AM-21, progress in vivo has been limited due to poor pharmacokinetic (PK) properties and stability. ONs are often subject to degradation by serum nucleases, which reduces bioavailability. Further non- specific uptake by macrophages and other cells leads to unwanted toxicity, limiting therapeutic application (10-11).

Quaternary-tertiary lipoamine systems, termed “QTsome” (QT), have been developed to overcome these barriers in delivery. Based on cationic lipid nanoparticle technology, QT when combined with AM are able to improve the PK profile of AM and further protect it from nuclease-mediated degradation. Cationic lipids in QT form electrostatic interactions with the negatively charged phosphate backbone of AM, resulting in stabilized lipid nanoparticles (LNs). LNs smaller than 150nm are able to take advantage of the enhanced permeability and retention (EPR) effect, leading to passive targeting of tumor vasculature and accumulation within the tumor site due to reduced lymphatic drainage (12).

Escape from the endosome and release of AM into the cytosol is a critical step in the efficacious delivery of AMs. A number of studies have proposed the inclusion of helper lipids to facilitate escape from the endosome. Helper lipids such as phosphatidylethanolamines and cholesterol promote endosomal disruption by destabilizing lipid bilayer phases within the endosome (13-15). QT further

54 enhance this endolytic activity by the inclusion of a conditionally ionizable tertiary amine. While a moderate positive surface charge on LNs is necessary for cell surface binding, excessive charge leads to non-specific uptake and clearance by macrophages. Under normal physiological pH (7.4), tertiary amines carry a neutral charge, however within the acidic (pH <5) conditions with the endosome, tertiary amines become protonated, facilitating interaction with negatively charged lipids of the endosome, thus leading to membrane disruption and delivery of the AM. Application of QT technology towards the delivery of AM-21 is expected to increase the potency of AM-21 both in vitro and in vivo by increasing cellular uptake and delivery to the cytosol.

The present study will investigate the therapeutic potential of QT/AM-21 in

NSCLC. Physical characterization studies will determine the particle size, pH- dependent surface charge, encapsulation efficiency, and relative stability of the formulation. In vitro studies in A549 cells will evaluate the ability of QT/AM-21 to upregulate pathway targets of miR-21 and to reduce metastatic potential.

Additional studies will be completed to evaluate whether QT/AM-21 administration is able to increase sensitivity of A549 cells towards PTX. Finally, a xenograft mouse model will evaluate the in vivo potential of QT/AM-21 for modulating tumor progression and pathway targets of miR-21.

3.2 Materials and Methods

55 3.2.1 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

Cremephor EL were obtained from Sigma Aldrich (St. Louis, MO, USA). N-

(carbonyl-methoxypolyethyleneglycol 2000)-1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine (DPPE-PEG) was purchased from NOF America Corp.

(White Plains, NY, USA). The AM-21 sequence, u*c*a*acaucagucugauaag*c*u*a, where lower case letters represent 2’-O-methyl bases and asterisks represent phosphorothioate linkages, was obtained from

Alpha DNA (Montreal, Quebec, CA) at desalted purity. PrimeTime qPCR assay primer probes and kits for DDAH1, PTEN, RECK, PDCD4, TIMP3 target genes and GAPDH housekeeping gene were purchased from Integrated DNA

Technologies (Coralville, IA, USA).

3.2.2 Synthesis of QT. QT were prepared by serial ethanol dilution method. Briefly, all lipids

(X/X/36/20/4 mol/mol, DODMA/DOTAP/DOPC/CHOL/DPPE-PEG) were dissolved in ethanol and combined with an equal volume of AM-21 dissolved in citric acid buffer (20 mM, pH 4.5), maintaining a 10:1, lipid:AM weight ratio.

DODMA and DOTAP content were varied with the total molar percent of tertiary and quaternary amine maintained at 40 molar percent composition. This solution was further diluted by equivalent volumes (1:1) of citric acid buffer, NaCl/NaOH buffer (300 mM NaOH, 20 mM NaOH, pH 7.4), and PBS (10 mM, pH 7.4). The 56 resultant LN solution was concentrated by tangential diafiltration to remove excess ethanol and to reach the appropriate final concentration. Samples were stored at 4°C prior to characterization.

3.2.3 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 complexes containing only tertiary or quaternary amine were diluted in citric acid buffer or

PBS to demonstrate the pH dependency of surface charge. Zeta potential measurement was conducted on a Zeta PALS Analyzer (Brookhaven

Instruments Corp., Worcestershire, NY, USA).

3.2.4 Drug loading and stability. Encapsulation efficiency 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 (480nm λex, 520nm λem). Relative encapsulation efficiency was determined with the formula:

퐸푛푐푎푝푠푢푙푎푡𝑖표푛 퐸푓푓𝑖푐𝑖푒푛푐푦

퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ표푢푡 푇푟𝑖푡표푛 푋 − 100 = (1 − ) × 100% 퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ 푇푟𝑖푡표푛 푋 − 100

Formulation stability was evaluated at -20, 4, and 25°C for a period of 30 days.

The particle size was periodically monitored by DLS. 10% sucrose was added as a cryoprotectant prior to storage.

57 3.2.5 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.

3.2.6 In vivo gene regulation. Cells were grown in 24-well plates at a density of 7.0×105 cells/well 24 h prior to transfection. QT/AM-21 of vary lipid composition or QT/negative control (NC) were administered at 50 nM in the presence of 20% serum containing media to determine the optimal QT composition. QT/AM-21 was also tested at 1.56, 6.25,

25, and 100 nM to demonstrate dose dependency of treatment. Cells were incubated at 37°C with transfection media for 4 h and then washed three times with PBS. Fresh complete cell culture media 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 StepOnePlusTM RT-PCR system (Life Technolgies). The relative amount of DNA was calculated and compared according to the 2-ΔΔCt method (16-17).

3.2.7 Cell viability assay. 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 58 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 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 cytotoxicity relative to the untreated control.

3.2.8 Migration assay. A 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 33mm 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 media was removed and replaced with transfection media containing QT/AM-21 or appropriate controls diluted in complete media. 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) and SPOT

Advanced Imaging Software (v5.0, Diagnostic Instruments Inc., Sterling Heights,

MI, USA).

3.2.9 Invasion assay. Matrigel (BD Biosciences, San Jose, CA, USA) was combined with serum-free

RPMI 1640 culture media 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 insert. Transfection media containing various formulations or controls at 2X

59 concentration in a 100 μL volume were added to the top of the well inserts. 500

μL 10% fetal bovine supplemented media 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 in the top of the well inserts were removed with a cotton swab. Well inserts were rinsed with PBS and placed in 500uL 0.25% trypsin solution for 1 h at 37°C. Detached cells were counted on a hemocytometer.

3.2.10 Tumor regression analysis. A549 mouse xenograft models were generated by inoculating female athymic nude mice with 1.0×106 cells/mouse. Tumors were allowed to reach a size of

≥100 mm3 before treatment began (~2 weeks). 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 three 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).

3.2.11 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

60 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. 48 h following the last dose, mice were euthanized and tumors were collected for gene regulation study.

3.2.12 In vivo gene regulation. Tumors were harvested and placed in TRIzol reagent (Life Technologies) following treatment and homogenized. mRNA was isolated per the manufacturer’s protocol. QRT-PCR was then completed according to the same procedure as outlined in the in vitro section.

3.2.13 Statistical analysis. All studies were done in triplicate unless otherwise mentioned. Student’s t-test was used to determine statistical significance between two or more groups. p≤0.05 was selected as the cutoff for statistical significance. *, **, and *** represent p<0.05, 0.01, and 0.001 respectively for treatment group relative to untreated control.

3.3 Results

3.3.1 Particle size and surface charge. Previous studies have demonstrated the need for small particles (<150 nm) and moderate zeta potential (+10-30 mV) to potentiate delivery of ONs (18-19).

Furthermore, LNs must exhibit high stability and slow release to maximize clinical application and therapeutic efficacy (20). Particle size measurement by DLS indicated particles of approximately 80-170 nm in diameter (Figure 3.1a).

61 Particles with greater amount of quaternary amine (15-40 mol%) achieved particles of smaller size (<120 nm) suggesting the ability to facilitate the EPR effect. Zeta potential measurement (Figure 3.1b) revealed QT 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 demonstrating the pH responsive behavior of the conditionally ionizable formulation and potential application in promoting endosomal lysis. The pH responsive behavior of QT fell between the range of charges for LNs containing only tertiary or quaternary amine at similar pH values. The charge on tertiary amines changed from 3 to 15 mV with decreasing pH. Conversely, quaternary amine containing LNs did not vary much with changes in buffer solution (27 and

29 mV).

3.3.2 Drug loading and colloidal stability. Encapsulation efficiency studies demonstrated high drug loading of 83.3 ± 4.17% by fluorescent intensity measurement. Further studies by CL 4B column filtration analysis (Figure 3.2a) showed approximately 90% encapsulation of ON within the encapsulated drug fractions (4-6) with very little ON remaining in the free drug fractions (10-12). The formulation further demonstrated high stability under storage at -20 (with cryoprotectant), 4, and 25°C over a period of 30 days, maintaining a size of ~110 nm (Figure 3.2b).

3.3.3 Determination of optimal lipid combination. A series of QT formulations were evaluated to determine the best ratio of quaternary and tertiary lipoamine for transfection. Formulations are identified as

QT(mol% quaternary amine)-(mol% tertiary amine) in Figure 3.3. Treatment with

62 50 nM AM-21 revealed QT with 15 mol% DOTAP and 25 mol% DODMA to perform best in upregulation of DDAH1 (1.33-fold) and this combination of lipids was 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 respectively. Formulations containing only quaternary or tertiary lipoamine did not perform well relative to combinations.

3.3.4 miR-21 and target regulation. Treatment with 100 nM QT/AM-21 resulted in moderate to strong upregulation of miR-21 and its downstream targets (Figure 3.4a-3.4b). Relative to the untreated control, miR-21 decreased by 50.3 ± 2.1% following administration of QT/AM-21.

Little to no effect on target gene regulation was observed for the scrambled negative control 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 1.5-fold. Migration inhibitors ANKRD46 and DDAH1 were upregulated 1.22 and 2.95-fold respectively.

3.3.5 Dose dependency. Varying dosages of QT/AM-21 between 1.56 to 100 nM were administered and qPCR was conducted to evaluate the relationship between AM concentration and gene upregulation (Figure 3.4c). DDAH1, PTEN, and RECK all demonstrated a direct correlation between dose and response. DDAH1 and PTEN were relatively more sensitive to changes in concentration relative to RECK.

63 3.3.6 Cell viability. Treatment with free AM-21 or QT lipids did not result in significant cytotoxicity as analyzed by MTS assay (Figure 3.5a). Likewise, the combination of QT/AM-21 at

50 nM did not demonstrate much cytotoxicity. However, moderate 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 QT lipids to PTX did not result in significant decreases in cell viability.

However, substantial gains in cytotoxicity were attributed to the addition of

QT/AM-21 at increasing doses. Cell viability was reduced to 51.2%, 41.0%, and

31.8% for 50, 100, and 200 nM doses respectively. The difference between untreated and QT/AM-21 was not significant, but differences between the

QT/AM-21 and PTX monotherapies and the combination therapy had p<<0.05.

Changes in cell morphology were also noted by microscopy. Little changes in cellular 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 3.5b).

3.3.7 Invasion and Migration. Wound healing assay was completed to monitor the relative mobility of A549 following treatment with AM-21. QT lipids or AM-21 alone did not confer significant decreases in cell migration in the wound region. With 100 and 200 nM treatment, mobility was reduced to 43.0 and 22.2% relative to the untreated control (Figure 3.6a). Matrigel is often used to simulate biological conditions of the basement membrane. The ability of cancer cells to migrate plays a role in 64 determining metastatic potential and increases with cancer progression.

Treatment 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 QT lipids or AM-21 did not significantly retard cell invasion (Figure 3.6b).

3.3.8 In vivo dose response. Treatment with QT/AM-21 demonstrated strong anti-tumor activity (Figure 3.7) at

1 mg/kg, but diminished activity at 0.5 mg/kg. Treatment of tumors initiated at

~180 mm3 and ended at 816.75, 618.125, and 172.5 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 groups, with a difference of about 1.5 g. Liver and spleen weights remained fairly consistent between the two groups, suggesting little to no toxicity for these organs. Tumor weight was over 16-fold lower for the treated group compared to the untreated group (Figure 3.8). 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 3.9).

3.3.9 In vivo combination therapy. In a following study, PTX and QT/AM-21 combination therapy was evaluated for therapeutic efficacy. Treatment began when tumors reached ~80mm3 in volume.

Tumors progressed to 380, 246.4, 201.1, and 138.1 mm3 for the untreated, PTX,

QT/AM-21, and combination treatment groups respectively (Figure 3.10).

Furthermore, qPCR conducted on tumor sections revealed moderate to strong upregulation of DDAH1 and PTEN (Figure 3.11). DDAH1 and PTEN were only

65 modulated slightly by PTX, 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 combination therapy.

3.4 Discussion miR-21 is involved in a number of gene pathways regulating tumor progression and resistance to chemotherapy. Therefore it is a key prognostic and diagnostic biomarker for NSCLC. miR-21 is also found to have similar roles in other cancers as well, including ovarian, breast, and prostate cancer (21). AM-21 therapy to inhibit miR-21 is therefore an important therapeutic tool for the treatment of

NSCLC and other cancers. The application of AM-21 and related ONs for therapy is hindered by several physical and biological barriers to delivery. Many of these obstacles are overcome by the use of nanoparticle carriers. LNs are comonly employed to improve delivery of ONs. Quaternary amines are the most commonly used class of lipids and form strong electrostatic interactions with negatively charged ONs. Quaternary amines are found in a number of commercial transfection agent products such as Lipofectamine 2000, which includes quaternary amine 2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N- dimethyl-1-propanaminium pentahydrochloride (DOSPA) and helper lipid 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (22). Tertiary amines are used commercially for delivery as well. 1,2-dilinoleyloxy-3-dimethylaminopropane

66 (DLinDMA) is a major component of the stable nucleic acid lipid particles

(SNALP) delivery system developed by Tekmira Pharmaceuticals (23-24).

In the present study, a pH-sensitive carrier, QT, was evaluated for the delivery of

AM-21 to NSCLC. Tertiary lipoamines form the pH-sensitive component of QT and upon exposure to acidic conditions as in the endosome, the tertiary lipoamine becomes cationized, enabling interaction with negatively charged lipids forming the endosomal bilayer and consequently leading to endosomal lysis.

Release of drug from the endosomal compartment is a critical step in determining drug efficacy (25). The inclusion quaternary amine is necessary to electrostatically stabilize the charge of LNs under physiological pH conditions. A higher amount of quaternary amine was demonstrated to better interact with the negative charge of the ONs, thereby resulting in particles of smaller average particle size relative to particles containing mostly tertiary amine content. In terms of surface charge, QT15-25 displayed an intermediary response to changing buffer condition relative to QT40-0 or QT0-40. LNs furthermore demonstrated excellent colloidal stability and efficient drug loading, suggesting practicality for clinical use and commercialization. QT may be further stabilized by the addition of cryoprotectant and processing by lyophilization to retain the drug form’s integrity over long term storage (26).

QT/AM-21 was able to strongly 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%

67 response rate, but many of these patients develop resistance to PTX over time

(27-28). The combination of QT/AM-21 and PTX demonstrated greater reductions in cell proliferation over use of 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, the 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) (29). HIF-1α and HIF-1β are distinct subunits of the transcription factor HIF-1. HIF-1α is upregulated in response to oncogene activity, hypoxia, and the presence of growth factors. HIF-1β is comparatively benign and constitutively expressed in the body. P-gp is a member of the ATP- binding cassette (ABC) transporter family, which has been found to play a role in the development of multidrug resistance in cancer (30-31). Decreased migratory and invasion potential was observed in a dose dependent manner in response to

QT/AM-21 treatment. Lung cancer is often diagnosed in late in its development, when metastasis has begun (32). Therefore, QT/AM-21 addresses a critical need that is currently unmet by chemotherapy administered in the late stage.

Treatment with QT/AM-21 in a xenograft mouse model was able to significantly suppress tumor growth at 1 mg/kg. Targets of miR-21 were also found to be upregulated, supporting trends observed in vitro. Relative increase in body weight indicates mice with better health and body condition (33). 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

68 populations of macrophages, and LNs may accumulate in these organs due to their similarities with tumor vasculature (34-35). The combination of PTX and

QT/AM-21 demonstrated greater therapeutic activity than either agent administered alone. Interestingly, while PTX displayed greater therapeutic activity in vitro than QT/AM-21, the trend in tumor suppression was opposite and QT/AM-

21 was slightly more effective than PTX. The relative increase in QT/AM-21 activity may be attributed to increased retention of the LNs in the tumor vasculature following injection, leading to a greater duration of therapeutic effect.

Additional studies will be required to validate the safety and efficacy of QT/AM-

21. Studies by Western blot would confirm whether the increases in mRNA for target genes correlates with an increase in the associated protein levels (36). It would also be interesting to see if miR-21 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 mechanism behind uptake of QTs, specifically to determine if QT are taken up in the same clathrin-mediated endocytosis pathway as in the case of quaternary or tertiary-based LNs (37-38). A major advantage of LN formulations is the broad spectrum of potential application for drug loading and treating other diseases. The large majority ONs are roughly 20-25 nucleotides in length and encapsulation is sequence independent, dependent instead on the relative anionic charge of the sequence. Therefore, QT is not limited to administration for AM-21 and may be used to deliver virtually any type of AM, siRNA, or miR mimic, with little optimization required (39). (Refer to Table 3.1

69 and Figure 3.12 for AM-21 optimization studies.) Addition of a targeting agent may also improve the specificity of delivery of QT to NSCLC cells. Previous studies has suggested targeting the epidermal growth factor receptor with chitosan or cetuximab (40). Similarly, bevacizumab may be included to target the vascular epidermal growth factor receptor (41). Further toxicity, pharmacokinetic, and pharmacodynamics studies are also necessary to better characterize

QT/AM-21 and aid its translation to the clinic.

3.5 Conclusion

QT were prepared by a modified ethanolic dilution method. QT particles 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 amines alone. The combination of quaternary and tertiary amine form a pH sensitive system to enhance the fusogenic activity of the formulation.

Moreover, increased sensitivity to PTX and reduced migration and invasion were demonstrated with varying levels of QT/AM-21 treatment. In vivo analyses reveal tumor regression, improved body condition, upregulation of target genes, enhanced anticancer activity with combination therapy, and prolonged survival.

QT is thus an effective therapeutic option for the treatment of NSCLC, warranting further study. QT may further be applied for the formulation of other AMs and miR mimics to treat NSCLC and other diseases.

70 A 250

200

150

100

50 Mean Particle Diameter (nm) Diameter Particle Mean

0 40-00 35-05 30-10 25-15 20-20 15-25 10-30 05-35 00-40 Quaternary:Tertiary mol% Lipid Nanoparticle

40 B 35 30 25 pH 4 20 pH 7 15

10 Zeta potential (mV) potential Zeta 5 0 Tertiary QT Quaternary

Figure 3.1. Particle size and surface charge. QT of varying mol% quaternary and tertiary amine content were prepared and analyzed by DLS for the effect on nanoparticle size (A). QT and LN composed of only quaternary or tertiary amine were placed in buffer at physiological pH (7.4) and acidic pH (4.0) to determine the pH responsive effect on surface charge (B). Data is presented as the mean±SD of three independent samples (n=3).

71 1.2 A 1

0.8

0.6

0.4

Absorbancenm) (280 0.2

0 0 5 10 15 20 Fraction (mL)

B 140 130 120 110 100 -20C 90 4C 80 25C 70

Mean particle diameter (nm) diameter particle Mean 60 0 5 10 15 20 25 30 Days

Figure 3.2. Encapsulation efficiency and colloidal stability. (A) Column separation of QT was completed on a CL 4B column, collecting 1 mL fractions.

Absorbance at 280nm was measured to detect the presence of ON. (B) To study the relationship between temperature and particle size, QT/AM-21 was stored at varying temperature and the particle size was monitored over 4 weeks.

72 160

140

120

100

Fold DDAH1 expression (rel. GAPDH) (rel. expression DDAH1 Fold 0

3.5 ***

A 1.2 B

) ) 1 3 *** 0.8 2.5 DDAH1 0.6 *** p=0.013 PTEN (rel.GAPDH 2 * 0.4 RECK 1.5 0.2 PDCD4 expression (rel. RNU44 expression(rel. 1 0 TIMP3

Fold 0.5 Fold Expression Fold

0 Untreated Neg. Ctrl. QT/AM-21

C 3.5 3

(rel. 2.5 100 nM ) 2 25 nM 1.5 GAPDH 6.25 nM 1 1.56 nM Fold Expression Expression Fold 0.5 0 DDAH1 PTEN RECK

Figure 3.4. In vitro gene regulation. (A-B) A549 cells were treated for 4 h with

SPLN/NC or SPLN/AM-21 (100 nM). (C) To demonstrate dose dependency of gene regulation, cells were treated with 1.56, 6.25, 25, and 100nM SPLN/AM-

221. Relative gene expression to GAPDH or RNU44 was evaluated 24 h following the start of transfection. Results are reported as the mean±SD of three independent transfections.

74 120A n.s.

100 *** 80 **

Figure 3.5. Cell viability. (A) A549 cells were treated with QT/AM-21 at varying concentrations with and without paclitaxel. 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. (B) Cells were imaged under light microscope to observe changes in cell morphology following treatment.

75 A 120

100 p=0.023 * 80

60 ***

40 ***

Percent Percent migration 20

0 Untreated AM-21 QT lipids QT/AM-21 QT/AM-21 QT/AM-21 (200nM) (50nM) (100nM) (200nM)

120 B p=0.025 100 * *** 80 *** 60

Number Number of invaded cells 0 Untreated AM-21 QT lipids QT/AM QT/AM QT/AM (200nM) (50nM) (100nM) (200nM)

76 1000 900

800 ) 3 700 600 500 *** Untreated 400 0.5 mg/kg

300 1 mg/kg Tumor Volume Tumor (mm 200 100 0 0 5 10 15 20 25 30 35 Days

(mean±SE) was monitored to determine relative tumor progression.

77 Body weight Liver weight

30 1 p=0.047 0.9 25 * 0.8 20 0.7 0.6 15 0.5

mass(g) 0.4 10 mass(g) 0.3 5 0.2 0.1 0 0 Untreated Treated Untreated Treated

Spleen weight Tumor weight

0.2 0.7 0.195 0.6 0.19 0.185 0.5 0.18 0.4 0.175 0.3

0.17 mass(g) mass (g) mass 0.165 0.2 0.16 0.1 *** 0.155 0.15 0 Untreated Treated Untreated Treated

Figure 3.8. Weight data. Following the final treatment, mice were sacrificed and body, liver, spleen, and tumor weight data 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.

78 120

100

80 Untreated 60 0.5 mg/kg

40 1.0 mg/kg Percent Survival Percent 20

0 0 10 20 30 40 50 60 Days

Figure 3.9. Kaplan-Meier survival analysis. 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 (p=0.0037) days respectively.

79 500 450

400

) 3 350 300 p=0.032 Untreated 250 * PTX 200 QT/AM-21 150

Tumor Volume (mm Volume Tumor Combination 100 50 0 0 5 10 15 20 25 30 35 Days

Figure 3.10. Combination therapy. 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 24h following the last treatment and evaluated for modulation of targets. Data represent the results (mean±SE) from three separate tumor sections.

80 *** A 6 ** 5

4 ***

GAPDH) ** 2

1 Fold DDAH1 expression (rel. (rel. expression DDAH1 Fold

0 Untreated PTX QT/AM-21 Combination *** 4.5 B *** 4 3.5 3 *** 2.5

2 *** GAPDH) 1.5

1 Fold PTEN expression (rel. (rel. expression PTEN Fold 0.5 0 Untreated PTX QT/AM-21 Combination

Figure 3.11. In vivo gene regulation. Tumors were harvested from mice and analyzed for modulation of miR-21 targets DDAH1 and PTEN. Results are presented as the mean±SE of QRT-PCR results for three independent tumor sections.

Table 3.1. Optimization of AM-21 structural modifications.

MLB# Color Notation 101 T*C*A*A*C*A*T*C*A*G*T*C*T*G*A*T*A*A*G*C*T*A 102 C*A*G*T*C*T*G*A*T*A*A*G*C*T*A 103 G*A*T*A*A*G*C*T 104 T*C*A*G*T*C*T*G*A*T*A*A*G*C*T*A 105 T*C*A*G*T*C-T*G*A*T-A*A-G-C*T*A 106 T*C*A*G*T*C*T*G*A*T*A*A*G*C*T 107 T*C*A*G*T*C*T*G*A*T-A*A-G*C*T 108 T*C*A*G*T*C*T*G*A*T*A*A*G*C*T*A 109 T*C*A*G*T*C*T*G*A*T-A*A*G-C*T*A 110 T*C*A*G*T*C*T*G*A*T-A*A*G-C*T*A 111 T*C*A*G*T*C*T*G*A*T*A*A*G*C*T*A 112 T*C*A*G*T*C*T*G*A*T*A*A*G*C*T 113 T*C*A*G*U*C*U*G*A*T*A*A*G*C*T*A 114 T*C*A*G*T*C*T*G*A*T*A*A*G*C*T 115 T*C*A*G*T*C-T*G*A*T-A*A-G-C*T*A 116 T*C*A*G*T*C-T-G*A*T*A-A-G*C*T*A 117 T*C*A*G*T*C-T-G*A*T*A-A-G*C*T 118 T*C*A*G*T*C-T-G*A*T-A-A-G*C*T*A 119 T*C*A*G*T*C*T*G*A*T-A*A*G-C*T 120 T*C*A*A*C*A*T*C*A-G-T-C-T-G-A-T-A-A-G*C*T*A 121 T*C*A*A*C*A*T-C-A-G-T-C-T-G-A-T-A*A*G*C*T*A 122 C*A*T*C-A-G-T-C-T-G-A-T-A-A-G*C*T*A 123 T*C*A*G-T-C-T-G-A-T-A-A-G*C*T*A 124 T*G*A*T*A*A*G*C*T 125 T-G-A-T-A-A-G-C-T Key LNA, 2'OMe, "-"PO linkage "*" PS linkage

82 100nM 25nM 4 6.25nM 1.56nM 3.5

2.5

1.5

Fold DDAH1 expression (rel. GAPDH) DDAH1(rel.expression Fold 0.5

0 116 118 117 112 113 114 108 109 110 NC AM-21

100 nM 3 25nM 6.25nM 2.5 1.56nM

1.5

0.5 Fold PDCD4 expression (rel. GAPDH) (rel.expression PDCD4 Fold 0 116 118 117 112 113 114 108 109 110 NC AM-21

continued

Figure 3.12. Various AM-21 configurations were evaluated using RNAimax transfection reagent 24 h following the initiation of treatment. AM were screened in the order of priority ranking. Data represent the mean±SD of quadruplicate transfections. 83 Figure 3.12. (continued)

100 nM

3.5 25 nM 6.25 nM

3 1.56 nM

2.5

1.5

Fold DDAH1 expression (rel. GAPDH) (rel.expressionDDAH1 Fold 0.5

0 104 103 102 101 120 121 122 123 124 125

100 nM 3 25 nM 6.25 nM 2.5 1.56 nM

1.5

Fold PDCD4 expression (rel. GAPDH) (rel.expressionPDCD4 Fold 0.5

0 104 103 102 101 120 121 122 123 124 125

continued 84 Table 3.12. (continued)

1.8

1.6

1.4

1.2 100 nM 1 25 nM 0.8 6.25 nM 0.6 1.56 nM

0.4

Fold DDAH1 expression (rel. GAPDH) (rel. expression DDAH1 Fold 0.2

0 111 115 119 107 106 105

1.4

1.2

100 nM 0.8 25 nM 0.6 6.25 nM 1.56 nM 0.4

0.2 Fold PDCD4 expression (rel. GAPDH) (rel. expression PDCD4 Fold

0 111 115 119 107 106 105

85 CHAPTER 4

SMALL PEPTIDE LIPID NANOPARTICLES FOR THE DELIVERY OF ANTI-MIR

4.1 Introduction

Triple negative breast cancer (TNBC) accounts for roughly 20% of breast cancer cases worldwide (1). It is a heterogeneous group of cancer characterized by the absence of estrogen receptor α (ERα), progesterone receptor, and human epidermal growth factor receptor 2 (HER2) expression. Prognosis of this cancer is typically poor due to the aggressive nature of the disease and resistance against traditional modes of therapy (2-3). Small molecule drugs, especially kinase inhibitors, have been applied to target specific transduction pathways in cancer cells promoting extravasation, evading apoptosis, or facilitating drug efflux

(4-5). Furthermore, antibody based treatments, aimed at triggering the innate immune system against TNBC cells have been tested (6-7). Unfortunately, many of these approaches have generated varied success in the clinic (8). RNA interference (RNAi) stands as a frontier approach in the treatment of TNBC.

Several microRNA (miR) have been associated with the apparent drug resistance and progression of TNBC (9-10). However, much research is necessary to identify a suitable vector for efficacious delivery of anti-miR (AM)

(11).

86 Lipid nanoparticles (LNs) are often employed for the delivery of oligonucleotides

(ONs). The development of nanocarriers for gene delivery has largely focused on cationic lipids and polymers, which are able to form electrostatic interactions with oligonucleotides (11-12). However, very few studies have been devoted to the exploration of peptide based transfection agents. Peptides, though not necessarily significant contributors of charge density, may facilitate oligonucleotide delivery by promoting endosomal escape (13-14). Previous studies have evaluated an antibiotic, gramicidin S, in combination with lipids for the transfection of plasmid DNA (15). Other variants of gramicidin (A, B, C, D) have not been previously investigated as transfection agents. Though these gramicidin subtypes share a conserved sequence of peptides, gramicidin A, B, C, and D form a beta-helix structure, while gramicidin S forms a cyclic structure

(16). Therefore, natural gramicidins (A-D) are fundamentally different from synthetic gramicidin S in terms of activity. Gramicidin dimerizes and forms an ion channel that promotes membrane fusion, which is necessary for the destabilization of lipid bilayers of the endosome and of the LN (17).

Consequently, gramicidin is an ideal candidate for formulation in gene-based therapies. Incorporation of gramicidin (A, B, C, and D) into LNs may significantly increase the cellular transfection efficiency of antisense oligonucleotides (ONs) and siRNA. Formulations of this nature have been termed small peptide lipid nanoparticle (SPLN). SPLN in this study will be prepared by bulk mixing as well as a novel microfluidic hydrodynamic focusing (MHF) method to determine if there are improvements in manufacture. Several studies have cited the benefit of

87 using MHF technology to reduce polydispersity and minimize particle size. Mixing geometries are well-defined in MHF and give rise more consistent sample preparation (18-19).

Advancements in the understanding of miRNAs have led to novel diagnostic and prognostic tools in the treatment of cancer (20). Recent studies have reported overexpression of miR-221 and miR-21 in aggressive basal-like breast cancer

(21-22). This aberrant expression has been linked to promotion of the epithelial- to-mesenchymal transition (EMT) in breast cancer (23). miR-221, an effector in the RAS-RAF-MEK signaling pathway (Fig. 3), is involved at several levels of tumor progression. miR-221 inhibits TRPS1, which in turn increases ZEB2, a protein directly acting upon the inhibition of EMT suppressing e-cadherin (CAD) and upregulation of EMT promoting vimentin (VIM). miR-221 also plays a role in the inhibition of p27/Kip1, a key regulator in cell cycle termination (24). miR-21 similarly plays a role in the promotion of EMT and angiogenesis by upregulation of metalloproteinase inhibitors RECK and TIMP3 (25). miR-21 is also involved in the suppression of anti-apoptotic factors PTEN and PDCD4 which act on the Akt pathway and translation initiation factors eIF4A/G respectively (26-27).

Several studies have shown that miR-221 activity is involved in promoting the

ERα-negative phenotype and hormone therapy resistance associated with

TNBC. A combination of inhibition of apoptotic proteins and ERα-negative phenotype is believed to be the primary factor conferring resistance to hormone therapy in TNBC cells (28). Restoration of miR-221 regulated p27/Kip1 and ERα expression could open up new avenues of treatment for TNBC. Tamoxifen (TMX)

88 has long been prescribed as an agent for treating ERα-positive breast cancer.

However, tumor cells develop a resistance to TMX over prolonged treatment (29-

30). Decreases in protein levels of p27/Kip1, PTEN, and TIMP3 have been correlated with this resistance (31-32). Increased levels of miR-221 and miR-21 have been found in TMX resistant cells including both ERα-positive and TNBC cell lines (33-34). Therefore, administration of AM targeting miR-221 and miR-21 would be a rational approach for restoring sensitivity to TMX. If successful, this therapy would provide significant opportunities to both patients who have developed TMX resistance as well as those with TNBC.

4.2 Materials and Methods

4.2.1 Materials 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). N-(carbonyl-methoxypolyethyleneglycol 2000)-1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-PEG) was purchased from NOF America Corp. (White Plains, NY, USA). Gramicidin from Bacillus aneurinolyticus and TMX were purchased from Sigma Aldrich (St. Louis, MO).

Luciferase Assay System and CellTiter 96® AQueous One Solution Cell

Proliferation (MTS) Assay kit were purchased from Promega (Madison, WI).

Stealth RNAi™ siRNA Luciferase Reporter Control was purchased from was purchased from Life Technologies (Carlsbad, CA, USA). AM-221 (5'- g*a*aacccagcagacaaugu*a*g*c*u-3', lower case letters represent 2’O-methyl bases 89 and asterisks represent phosphorothioate linkages), AM-21

(u*c*a*acaucagucugauaag*c*u*a), and scrambled control were purchased from

AlphaDNA (Montreal, Quebec, Canada). Primers for p27/kip1, PTEN, PDCD4,

ERα, RECK, TIMP3, CAD, VIM, and GAPDH were custom synthesized by

Integrated DNA Technologies (Coralville, IA, USA). All reagents were of analytic grade or higher and used without further purification.

4.2.2 Preparation of SPLN by bulk mixing SPLN formulations were prepared by a modified ethanol dilution method. Briefly,

DOTAP, DOPC, DPPE-PEG, and gramicidin were dissolved in ethanol and combined with a 9-fold excess volume of AM-221 or AM-21 dissolved in phosphate buffered saline (10 mM, pH 7.4), maintaining a 10:1, lipid:AM weight ratio. The concentration of gramicidin was varied between 0-50 molar percent molar composition, with DOPC varied between 6-56 percent. DOTAP and DPPE-

PEG were maintained at 40 and 4 molar percent for all formulations. The resultant solution was concentrated by tangential diafiltration to remove excess ethanol and to reach the appropriate final concentration. Samples were stored at

4°C prior to analysis.

4.2.3 Preparation of SPLN by microfluidics SPLN were prepared by MHF as depicted in Figure 4.1. Two syringe pumps

(Model 78-0100I, Fisher Scientific, Pittsburg, PA, USA) were set up with three 10 or 5 mL syringes and appropriate tubing and luer fittings connected to a microfluidic chip (µ-Slide III3in1 uncoated, Ibidi, Martinsried, Germany). Two syringes were filled with SPLN in 30% ethanol and connected to the lateral ports

90 of the microfluidic chip. AM solution was loaded into the central microfluidic channel. Pumps were activated simultaneous to allow for consistent processing.

An approximate 1-2 mL volume was eluted through the system before sample collection to ensure measurement within the steady state phase of mixing. The effect of flow rate, lipid concentration, and lipid:ON ratio were evaluated in the

MHF system to determine the effect on particle size.

4.2.4. Physical characterization Mean particle diameter of SPLN and SPLN/AM complexes were determined by dynamic light scattering on a NICOMP Submicron Particle Sizer Model 370

(Santa Barbara, CA, USA) under volume-weighted setting. Zeta potential measurement of complexes was completed on a ZetaPALS instrument

(Brookhaven Instruments Corp., Worcestershire, NY, USA). Encapsulation efficiency of SPLN/AM complexes was analyzed by Quant-iT™ OliGreen® ssDNA Kit (Life Technologies, Grand Island, NY, USA) per the manufacturer’s instructions, using Triton X-100 to lyse SPLN/AM complexes. Encapsulation efficiency was calculated according to the following equation:

퐸푛푐푎푝푠푢푙푎푡𝑖표푛 퐸푓푓𝑖푐𝑖푒푛푐푦

퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ표푢푡 푇푟𝑖푡표푛 푋 − 100 = (1 − ) × 100% 퐹푙푢표푟푒푠푐푒푛푐푒 푤𝑖푡ℎ 푇푟𝑖푡표푛 푋 − 100

4.2.5 Cell culture TNBC cell line MDA-MB-231 and ERα-positive breast cancer cell line MCF-7 were grown in DMEM/F12 media (with 20mM L-glutamine and without phenol red) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin cultured under a 5% CO2 atmosphere at 37°C. SK- 91 HEP-1 cells stably expressing luciferase were grown in DMEM media supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 0.1 mg/ml streptomycin cultured under a 5% CO2 atmosphere at 37°C.

4.2.6 Formulation optimization SK-HEP-1 cells were grown in 96-well plates at a density of 2.0×104 cells/well 24 h prior to transfection. SPLN with 50 nM siRNA against luciferase (siLuc) or

Lipofectamine 2000 (LF2K) with siLuc were transfected in the presence of serum free or 20% serum containing media. SPLN are labeled as SPLN followed by the corresponding molar percentage of gramicidin in the formulation. Cells were incubated at 37°C with transfection media for 4h and then washed three times with PBS. Fresh complete cell culture media was added and the cells were incubated at 37°C for an additional 44h. Cells were then analyzed by luciferase assay according to the manufacturer’s protocol to determine relative decreases in luciferase expression.

4.2.7 Cell viability The vehicle-related cytotoxicity of SPLN formulations was evaluated by MTS assay in MDA-MB-231 and MCF-7 cell lines. Cells were seeded at a density of

2×104 cells/cm2 in a 96-well plate 24 h prior to treatment. Cells were washed three times with PBS and replaced with serum-free media 30 min prior to transfection. 50-250 nM SPLN/AM-221 was then added, followed by incubation for 4 h at 37°C. Transfection medium was then removed and the cells were washed three times with PBS and placed in fresh serum-containing media. Cells were allowed to proliferate for an addition 44 h, at which time 20 μL MTS solution

92 was added to each well and the plate was further incubated for 1 h. The optical density (OD) was observed at 490 nm on a standard plate reader. Cell viability was calculated relative to the OD of the untreated control for each cell line.

4.2.8 Gene regulation MDA-MB-231 and MCF-7 cells were grown in 24-well plates at a density of

8.0×105 cells/well 24 h prior to transfection. Free AM-221, free AM-21, SPLN lipids, SPLN/AM-221, SPLN/AM-21, or combination of SPLN/AM-21 and

SPLN/AM-221 (SPLN/C) were administered at 200 nM total AM concentration in the presence of 20% serum containing media. Cells were incubated at 37°C with transfection media for 4 h and then washed three times with PBS. Fresh complete cell culture media 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). Real-time quantitative polymerase chain reaction (QRT-

PCR) was conducted with EXPRESS One-Step Superscript® qRT-PCR kit (Life

Technologies) on an Applied Biosystems StepOnePlusTM RT-PCR system (Life

Technolgies). The relative amount of DNA was calculated and compared according to the 2-ΔΔCt method (35-36).

4.2.9 Migration assay A wound healing model was conducted to examine the migratory ability of TNBC cells following treatment. MDA-MB-231 cells were plated at a density of 6.0 ×105 cells/well in a 33 mm petri dish 24h prior to transfection. A scratch wound across the dish was initiated using a 10 μL pipet tip immediately preceding treatment.

Culture media was removed and replaced with transfection media containing

93 SPLN/C (50-200 nM) or appropriate controls diluted in complete media. 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) using

SPOT Advance Imaging Software (v5.0, Diagnostic Instruments, Inc., Sterling

Heights, Michigan, USA).

4.2.10 Invasion assay Matrigel (BD Biosciences, San Jose, CA, USA) was combined with serum-free

DMEM culture media 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. MDA-MB-231 cells were seeded at 7.5×105 cells/well in a volume of 100 μL/well on top of the gel in the insert. Transfection media containing various SPLN/C (50-200 nM) formulations or controls at 2X concentration in a 100 μL volume were added to the top of the well inserts. 500 μL 10% fetal bovine supplemented media 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 in the top of the well inserts were removed with a cotton swab. Well inserts were rinsed with PBS and placed in 500 μL 0.25% trypsin solution for 1 h at 37°C. Detached cells were counted on a hemocytometer.

4.2.11 Tamoxifen sensitivity TMX sensitivity was evaluated in MCF-7 and MDA-MB-231 cells following treatment with SPLN/AM-221, SPLN/AM-21, or SPLN/C at 100 nM. Cells were plated in a 96-well plate at a density of 2.0×104 cell/well 24 h prior to treatment.

TMX was administered at 27 µM, a dose equivalent to the IC50 for MCF-7 cells

94 (37). Following treatment for five days, cells were analyzed by MTS assay as indicated previously for relative cell viability.

4.2.12 Statistical analysis Statistical significance between two groups was analyzed by Student’s t-test and between multiple groups was compared by analysis of variance (ANOVA) on

Microsoft Excel 2013 software (Microsoft, Redmond, WA). Results were considered statistically significant at the p < 0.05 level. *, **, and *** represent p<0.05, 0.01, and 0.001 respectively for treatment group relative to untreated control.

4.3 Results

4.3.1 Particle size and surface charge Various SPLN formulations were prepared and analyzed for differences in terms of particle size and zeta potential. The addition of gramicidin had a noticeable effect on particle size (Figure 4.2). Particles without gramicidin were 66.5 nm and increased to 300 nm in diameter with the inclusion of 50% molar composition gramicidin. SPLN were also prepared by MHF method. Several processing conditions including lipid concentration, flow rate, and lipid:ON ratio were modulated to determine the effect on particle size. Lipid concentrations under 2 mg/mL were able to produce particles under 150 nm, but at higher concentrations, the particle size rapidly increased to above 200 nm. In terms of flow rate, SPLN requires at least 0.2 mL/min to retain a small particle size under

150 nm. For lipid:ON ratio, a combination of 15:1 resulted in the smallest

95 particles. Taken together, SPLN prepared by MHF requires dilute and rapid mixing conditions with 15-fold excess lipid to maximize electrostatic interaction and reduce aggregation of the formulation. The faster flow rates may be necessary to simulate the rapid mixing that occurs in the traditional bulk mixing method. However, combination of fluid streams within the defined mixing geometries may offer a greater amount of control over resultant particle size.

Indeed, the batch-to-batch variation and polydispersity (0.115 (MHF) versus

0.224 (bulk mixing)) of SPLN prepared by MHF was less than that of SPLN prepared by bulk mixing. Modulation of gramicidin content did not have a profound effect on observed zeta potential as gramicidin carries a neutral charge

(Figure 4.3). Surface charge was maintained between 8 to 13.5 mV when SPLN was dissolved in PBS containing varying amounts of gramicidin from 0 to 50 molar percent content.

4.3.2 Drug loading and colloidal stability Encapsulation efficiency experiments were conducted to calculate loading efficiency and to determine whether gramicidin concentration had any effect on loading capacity (Figure 4.4). Encapsulation efficiency was maintained around

83% for all formulations, regardless of gramicidin concentration. Further analysis evaluated the colloidal stability (Figure 4.5) of the formulation. When stored at room temperature, SPLN gradually aggregated, leading to increased particle size. However when stored at 4°C or -20°C with the addition of cryoprotectant,

SPLN remained stable for at least 30 days.

96 4.3.3 Formulation optimization SPLN/siLuc containing varying amounts of gramicidin were administered in SK-

HEP-1 cells stably expressing luciferase to determine the optimal percentage of gramicidin for transfection (Figure 4.6). Formulations with 10 and 20 molar percent gramicidin were relatively non-toxic, but moderate toxicity was observed at 35 molar percent gramicidin. In regards to downregulation of luciferase, the

SPLN-35 formulation had the greatest reduction in luciferase expression at 80%, while 40% was observed for SPLN-20 and little to no downregulation activity was observed for SPLN-10. The SPLN-20 formulation was used in the following studies as it offered the best compromise between low vehicle toxicity and high transfection activity. SPLN formulations also performed better than commercial transfection reagent, LF2K, which is largely inhibited by serum binding, limiting its application to in vitro study. Additional studies involving similar fusogenic antibiotic peptides, polymyxin B and collistin, were completed to determine the relative effect on transfection efficiency in the SK-HEP-1 luciferase model.

Though both of these peptides facilitated greater suppression of luciferase than

LF2K formulated siRNA, neither compound performed as well as gramicidin. A summary of these results may be found in the Figure 4.11.

4.3.4 Formulation toxicity MCF-7 and MDA-MB-231 cells were treated with varying levels of SPLN/AM-221 to determine the effect on cell viability. A concentration dependent relationship on cell viability was observed (Figure 4.7) after 48 h treatment. SPLN/AM-221 was well tolerated at concentrations below 100nM, but began to show moderate toxicity at 250 nM for both cell lines. In following studies, evaluating gene 97 regulation, AM-221 concentration was maintained below 250 nM to avoid confounding effects due to cell death. The cell death observed at high concentrations of SPLN, may be due to vehicle related toxicity, specifically from the cationic component DOTAP and the fusgogenic component, gramicidin.

Toxicity resulting from administration of the AM alone is unlikely as cells maintained a high level of viability following treatment with 250 nM AM-221.

4.3.5 miR regulation MCF-7 and MDA-MB-231 cells were treated with free AM-221, free AM-21,

SPLN/AM-221, SPLN/AM-221, or SPLN/C to assess the effects on miR levels

(Figure 4.8). miR-21 levels were suppressed by 29.3% with SPLN/AM-21 and

34.1% with SPLN/C in MCF-7 cells. Less than 10% miR-21 suppression was observed for SPLN/AM-221 MCF-7 cells. Similar results were observed in MDA-

MB-231 cells. miR-21 was reduced by 8.3, 31.2, and 42.7% by SPLN/AM-221,

SPLN/AM-21, and SPLN/C respectively. miR-221 levels were strongly downregulated by SPLN/AM-221, 34.2% for MCF-7 cells and 41.3% for MDA-

MB-231 cells. Administration of SPLN/AM-21 did not significantly contribute to downregulation of miR-221 in either cell line. Results of SPLN/C modulation of miR-221 were relatively close to treatment by SPLN/AM-221 alone, with approximately 34% and 42% reductions in miR-221 expression for MCF-7 cells and MDA-MB-231 cells respectively.

4.3.6 Target gene regulation The combination of AM-21 and AM-221 was assessed in MDA-MB-231 cells

(Figure 4.8). Tumor suppressors, PTEN, PDCD4, and p27/kip1, were

98 upregulated by 3.4-fold, 2.8-fold, and 2.5-fold respectively. Metalloproteinase inhibitors RECK and TIMP3 were upregulated 2.3-fold and 2.1-fold respectively.

ERα, the target of competitive inhibitor, TMX, was upregulated by 1.8-fold. CAD, a marker for EMT was upregulated 3.1-fold. Conversely VIM, a gene promoting

EMT, was downregulated by 0.4-fold.

4.3.7 Migration and invasion Both miR-21 and miR-221 affect genes downstream regulating cell migratory potential (Figure 4.9). MDA-MB-231 cells were treated with SPLN/C at 50, 100, and 200 nM. In the wound healing assay, 40.8, 33.5, 21.9% of cells migrated into the scratch region for 50, 100, and 200 nM treatments respectively. Moderate reductions in invasive potential were observed as well. Treatment with SPLN/C resulted in dose dependent reductions in invasion from 28.9 to 46.3%. No significant decreases in cellular migration and invasion were observed for

SPLN/NC.

4.3.8 Tamoxifen sensitivity Cell viability of MDA-MB-231 and MCF-7 to TMX was assessed by MTS assay

(Figure 4.10). TMX-sensitive MCF-7 cells showed a much greater decrease in cell proliferation compared to TMX-resistant cell line MDA-MB-231. miR-21 and miR-221 modulate TMX resistance and downregulation of these targets is therefore expected to influence sensitivity to TNBC cells as well as those who have become refractory towards treatment. Administration of 100 nM SPLN/C was able to both increase sensitivity of MCF-7 cells towards TMX as well as sensitize MDA-MB-231 to chemotherapy.

4.4 Discussion

TNBC is a metastatic and aggressive cancer that is generally responsive to initial treatment with chemotherapeutics but quickly relapses (especially within the first

3-5 years), leading to poor survival outcomes (38). TNBC is characteristically non-responsive to hormone-based therapies due to an absence of targetable receptor expression. miR-221 and miR-21 are upregulated in TNBC and were investigated as therapeutic targets to modulate response to TMX and decrease metastatic potential. Both miR-221 and miR-21 are upregulated in a number of cancers including lung, liver, pancreatic, prostate, and breast cancers (39-40). miR-221 expression is higher in TNBC relative to other subtypes of breast cancer. Interestingly the expression levels of miR-21, though elevated relative to normal tissue are lower in TNBC compared to other types of breast cancer (41). miR-221 is thought to regulate ERα expression and mediate anti-estrogen response through regulation of TIMP3 and p27/kip1 (42). miR-21 similarly influences the expression of TIMP3 and a number of tumor suppressors including

PTEN, which regulates apoptosis through the PI3K/Akt pathway. miR-21 has been associated with the development of trastuzumab resistance in HER2- positive breast cancers (43). By targeting these two regulatory pathways mediating drug resistance simultaneously, we were able to demonstrate increased sensitivity towards TMX. It is furthermore likely that treatment with other anti-estrogens such as raloxifene or toremifene will experience enhanced

100 therapeutic effect in combination with SPLN/C, thus opening even more opportunities for TNBC therapy (44-45).

In addition to miR-221 and miR-21, a number of other miRs are involved in chemotherapeutic resistance. miR-34a is a tumor suppressor typically downregulated in TNBC. It acts as a regulator in the p53 family of genes which regulates Bcl-2, CD44, Rac1, and Notch-1 expression. Bcl-2 has been indicated in a number of studies as an important regulator of apoptosis. Deng et al. demonstrated that by treating TNBC cells with miR-34a, increased sensitivity towards doxorubicin (DOX) was induced (46). let-7 is similarly downregulated in

TNBC and plays a role in the regulation of ER-α36 expression, which in controls

MAPK and Akt pathways involved in cellular signaling and apoptosis. High levels of ER-α36 are correlated with poor response to TMX. Treatment with let-7 miR mimics was shown to increase the sensitivity of TNBC cells towards TMX (47).

Many additional combinations of miR mimics and inhibitors are possible and the

SPLN platform may facilitate the rapid screening and clinical translation of these combinations. (Refer to Figure 4.12-4.14 for additional examples).

The synthesis of LNs on the laboratory scale is well documented and routine practice. However, preparation of liter volumes of LNs for clinical implementation or commercialization requires a scalable and robust method. MHF processing is both a scalable and reproducible method for the synthesis of LNs. Other commonly used methods such as thin film hydration and detergent dialysis result in LNs with asymmetric physical and biological properties in addition to poor reproducibility. As microfluidics is gaining popularity as a platform for both the

101 synthesis of nanomaterials and biological analysis, microfluidic chips with a variety of designs are becoming increasingly available. The design of channels including parameters such as channel width, number of channels, and angle of flow all play a role in the determination of mixing geometry (48). The MHF system detailed in this study is fully scalable, capable of producing anywhere from a few milliliters of LNs to several liters. The current studies used syringe pumps to manufacture milliliter quantities of LNs. These could easily be replaced by peristaltic pumps drawing from large reservoirs of lipid and oligonucleotide solutions to increase yield. Increased throughput may be accomplished by adding multiple chips in parallel or by increasing the number of pumps. The formation of LNs relies heavily upon the mixing geometries established by the microfluidic chip, but as the present studies have demonstrated, lipid concentration, processing flow rate, and lipid:ODN ratio play a critical role as well in LN formation. Related studies have shown the influence of processing parameters such as sonication on particle formation. Sonication appears to decrease particle size through the introduction of cavitation events, which promote dispersion of lipids within a fluid stream (49).

The use of gramicidin as a promoter of transfection still requires much validation before it finds practical application in clinical studies. Pathway studies to understand the kinetics of ON internalization with SPLN would be beneficial to determine if gramicidin operates intracellularly in a manner similar to its activity as an antibiotic. Comparative studies would also be beneficial to determine gramicidin’s fusogenic activity relative to other fusogenic molecules such as

102 phosphatidlyethanolamines (50). It is currently unknown how gramicidin will perform under in vivo conditions. Moreover, while gramicidin is an excellent fusogenic peptide, it may cause unwanted hemolytic action. Triggering of hemolytic response has largely limited the use of many antibiotics to topical application. Previous studies have shown that a dose of gramicidin S exceeding

300 µg/mL is capable of inducing hemolytic activity (51). Though xenograft models have not been completed for the evaluation of SPLN, it is unlikely that the gramicidin content for the therapeutic dose of SPLN will reach this concentration.

The inclusion of gramicidin itself may also pose a problem for other reasons as its fusogenic activity is not particularly directed towards the endosomal membrane. Over time, especially at physiological temperature, the action of gramicidin may destabilize the lipid bilayer of SPLN well before it has a chance to reach the tumor site. Use of a pH-sensitive system as described in Chapter 3 may be advantageous. JTS-1 is a mimic of INF7, a peptide derived from HA2 protein expressed on the influenza virus envelope. Upon entering acidic conditions as in the endosome, JTS-1 is activated, promoting fusogenic activity

(52).

The results of the current studies warrant further investigation of SPLN and its application in the treatment of TNBC. In following, it is necessary to test SPLN in a xenograft TNBC model to demonstrate safety of the delivery vehicle and to define appropriate dosing for effective tumor regression and increased sensitivity towards TMX or related hormone-based chemotherapeutics. Characterization of the formulation in vivo will best gauge SPLN’s suitability for clinical application.

103

4.5 Conclusion

SPLN is a novel formulation that includes gramicidin to potentiate fusogenic activity within the endosome and enhance overall drug release. SPLN were prepared by modified ethanolic dilution processing through traditional bulk mixing and MHF methods. Particles formed by MHF were similar in size to those formed by bulk mixing, but exhibited less variability and polydispersity across batches.

The synthesized SPLN demonstrated small particle size, moderate zeta potential, high oligonucleotide encapsulation efficiency, and excellent colloidal stability. Treatment with SPLN/C was well tolerated and able to modulate miR levels as well as downstream gene targets. Gene regulation further translated into significant reductions in migration and invasion potential. Most importantly, treatment with SPLN/C was able to increase sensitivity towards TMX in estrogen- positive and estrogen-negative cell lines. Taken together, SPLN/C represents a promising new strategy for the treatment of TNBC and breast cancers that have become refractory towards chemotherapy.

104

Figure 4.1. Microfluidic method for the synthesis of SPLN. (A) Photograph of

MHF system. A prototype MHF device was constructed for the preparation of

SPLN containing AM-221. (B) Photograph of MHF chip. Sterile syringes containing lipids dissolved in ethanol or ON in PBS were loaded onto syringe pumps and connected to a microfluidic chip. Lipid streams were connected to inlets A and C while AM-221 stream was connected it inlet B. The microfluidic chip was submerged in a water bath to maintain a 25°C temperature during processing. Approximately 2 mL of SPLN was eluted from the outlet port before sample collection to ensure collection of sample within the steady state range of processing.

105

A 16 400 B 14 350 300 12 250 10 200 8 150 6

100 4 Zeta Potential (mV)Potential Zeta

50 2 Mean Particle Diameter (nm) Diameter Particle Mean 0 0 0 10 20 30 40 50 0 10 20 30 40 50 Percent Gramicidin Percent Gramicidin

C Mean Diameter (nm) PDI Bulk Mixing 122.76 ± 24.21 0.224 MHF 102.9 ± 11.02 0.115

Average particle size and PDI of SPLN prepared by bulk mixing and MHF method are compared. Data are presented as mean±SD of three independently prepared SPLNs.

106 A 400 350 300 250 200 150 100

Particle Size (nm) Size Particle 50 0 0.05 0.1 0.2 0.5 1 Flow Rate (mL/min) B 250 200 150 100

50 Particle Size (nm) Size Particle 0 0.5 1 2 5 10 Lipid Concentration

C 500 400 300 200

100 Particle Size (nm) Size Particle 0 1 5 10 15 30 Lipid:ON (w/w)

Figure 4.3. Effect of MHF processing parameters on particle size. (A) Flow rate,

(B) lipid concentration, and (C) lipid: ON (w/w) were modulated to determine the relative effect on particle formation. Particle sizes are reported as the mean±SD of three independent experiments.

107 90 88 86 84 82 80 78 76 74

72 Encapsulation Efficiency (Percent) Efficiency Encapsulation 70 0 10 20 30 40 50 Percent gramicidin

108 250

200

150 -20C 100 4C 25C

50 Mean Particle Diameter (nm) Diameter Particle Mean

0 0 5 10 15 20 25 30 Days

Figure 4.5. Colloidal stability. SPLN were stored at varying conditions to evaluate the relationship between temperature and stability of the formulation. Particle size was monitored for 4 weeks. Particle sizes are reported as the mean±SD of three samples.

109 A 160

140

120

100

80 ** Serum Free 20% FBS

60 Percent Viability Percent 40

0 Untreated LF2K SPLNG-0 SPLN-10 SPLN-20 SPLN-35

140 B 120

100

80 *** Serum Free 60 20% FBS *** 40 *** ***

Percent Luciferase Activity Luciferase Percent **** ** 20

0 Untreated LF2K SPLN-0 SPLN-10 SPLN-20 SPLN-35

Figure 4.6. Formulation optimization. SPLN of varying mol% gramicidin were combined with siLuc and transfected in SK-HEP-1 cells stably expressing luciferase in serum-free and 20% serum-containing media. Cell viability (A) was assessed by MTS assay and luciferase assay was used to quantify relative luciferase suppression (B). Bars represent the mean±SD of three independent transfections.

110

A 120 p=0.041 p=0.019 100 * p=0.015 * * 80

40 Percent Cell viabilityCell Percent 20

0 Untreated AM-221 & SPLN Lipids SPLN/C (50 SPLN/C (100 SPLN/C (250 AM-21 (250 nM) nM) nM) nM)

B 120

100 ** ** 80 **

40 Percent Cell ViabilityCell Percent

0 Untreated AM-221 & SPLN Lipids SPLN/C (50 SPLN/C (100 SPLN/C (250 AM-21 nM) nM) nM)

111 A 1.2 B 1.2

1 1

***

*** 0.8 *** 0.8

*** *** *** 0.6 0.6

0.4 miR-221 0.4 miR-221

(rel. RNU44) (rel. (rel. RNU44) (rel.

0.2 miR-21 0.2 miR-21

Fold miR expression expression miR Fold Fold miR expression expression miR Fold 0 0

C 4

***

3.5

*** PTEN

3 ***

*** PDCD4

2.5 ***

*** RECK

2 *** TIMP3 ERa 1.5 1 1.14 2.54

1 CAD

** Fold expression (rel. GAPDH) (rel. expression Fold 0.5 VIM

0 Untreated SPLN/NC SPLN/C

Figure 4.8. Gene regulation. SPLN/AM-221 and SPLN/AM-21 were transfected in MCF-7 and MDA-MB-231 cells. miR regulation in (A) MCF-7 and (B) MDA-MB-

231 cells. (C) Effect on downstream gene targets: PTEN, PDCD4, RECK, TIMP3,

ERα, p27/kip1, CAD, VIM in MDA-MB-231 cells. Data are expressed as the mean±SD of three separate transfections.

112 120% A p=0.031 100% *

80%

60% *** *** 40%

Percent Migration Percent ***

20%

0% Untreated AM-221 & SPLN Lipids SPLN/C (50 SPLN/C (100 SPLN/C (200 AM-21 nM) nM) nM)

B 300

250

200 ** *** *** 150 ***

100 Number of Invaded cells Invaded of Number 50

0 Untreated AM-221 & SPLN Lipids SPLN/C (50 SPLN/C (100 SPLN/C (200 AM-21 nM) nM) nM)

Figure 4.9. Migration and invasion assays. (A) Wound healing assay was used to determine relative migration of cells following treatment. (B) Matrigel invasion assay was used to determine the invasive potential of cells following treatment.

Data is reported as the mean±SD of three independent transfections.

113 A 120 ** 100 *** 80 ** 60

Percent Cell Viability Cell Percent 20

*** 120 *** B 100 ***

Percent Cell Viability Cell Percent 20

Figure 4.10. SPLN/C and TMX Combination therapy. (A) MCF-7 and (B) MDA-

MB-231 cells were treated with a combination of SPLN/C (100 nM) and TMX (27

µM) to measure increased sensitivity towards TMX after 5 days. Cell viability was determined by MTS assay as the mean±SD of three independent treatments.

114 120

100

20 Percent luciferase expression luciferase Percent 0

Figure 4.11. Antibiotic peptides gramicidin (G), polymyxin B (PMB), and colistin

(polymyxin E, PME) were combined with lipids and siLuc to evaluate for relative transfection activity for the downregulation of luciferase in SK-HEP-1 cells.

Formulations are labeled SPLN-antibiotic (mol% antibiotic). Values are reported as the mean±SD of three independent experiments.

115

120 p=0.017 * 100 p=0.036 80 *

116

K14/DAPI Ki67/DAPI SPLN

d6 d6

K14/DAPI Ki67/DAPI SPLN/NC

d6 d6

K14/DAPI K14/DAPI

210 SPLN/AM d6 d6 Scale bar = 1000µm Figure 4.13. SPLN/AM-210 therapy. SPLN was combined with AM-210 for evaluation of wound healing potential in diabetes using an ischemic wound mouse model through topical treatment. Wounds were stained with nuclear DAPI stain (blue) and monoclonal antibody macrophage marker Ki-67 (purple) and cytokeratin stain K14 (green). (Image courtesy of Ghatak et al.)

117

day 0 day 2 day 4 day 6

SPLN

SPLN/NC

200b

- SPLN/AM Scale bar = 50µm Scale bar = 1 cm

Figure 4.14. SPLN/AM-200b therapy. SPLN was combined with AM-200b for evaluation of wound healing potential in diabetes using an ischemic wound mouse model. Photographs of the wound site were taken during the time course of topical treatment. (Image courtesy of Ghatak et al.)

118 CHAPTER 5

SUMMARY AND PERSPECTIVES

5.1 Summary

The objective of this dissertation was to design, synthesize, and characterize several novel lipid nanoparticle (LN) formulations for the delivery of oligonucleotides (ONs) to cancer. LNs confer several advantages to ON-based therapies by extending circulation time and promoting delivery to the tumor site

(1-3). Careful selection of lipids and optimization of design is essential to the preparation of LNs with good physical properties and consistent therapeutic activity (4-5).

In Chapter 2, Lipid-coated albumin nanoparticle (LCAN) was developed for the delivery of LOR-2501. LCAN are formed by conjugation of human serum albumin

(HSA) to low molecular weight polyethylenimine (PEI) (600 Da) to form a dense albumin polycation core (APC) for electrostatic interaction with ONs. The cationic core is then surrounded by a shell of lipids and PEGylating agent to prevent opsonization. LCAN when combined with LOR-2501 further exhibited strong downregulation activity of R1 relative to LN without an APC. Moreover, LCAN demonstrated enhanced transfection activity in the presence of FBS relative to commercial transfection agent, suggesting potential utility in vivo. When

119 evaluated in vivo, LCAN/LOR-2501 demonstrated strong tumor suppressive activity and the ability to downregulate R1.

In Chapter 3, QTsome (QT) was developed for the delivery of anti-miR-21 (AM-

21) to non-small cell lung carcinoma (NSCLC). QT combine quaternary and tertiary amines at specific ratios to form a pH-sensitive drug delivery system for the delivery of AM within the acidic conditions of the endosome (6). QT/AM-21 demonstrated strong upregulation of miR-21 targets DDAH1, PTEN, PDCD4,

RECK, and TIMP3 with low vehicle related toxicity. Furthermore, treatment with

QT/AM-21 was able to reduce migration and invasion in vitro. When combined with paclitaxel (PTX), QT/AM-21 greatly increased the sensitivity of A549 cells towards PTX. Delivery of QT/AM-21 in a xenograft model of A549 cells was able to induce strong tumor suppressive activity and treatment in combination was

PTX was able to further suppress tumor growth over either agent administered as monotherapy.

In Chapter 4, small peptide lipid nanoparticle (SPLN) was developed for the delivery of AM-221 to triple negative breast cancer (TNBC). SPLN, similar to QT, includes a fusogenic component, namely an antibiotic peptide, gramicidin.

Gramicidin is able to promote delivery by promoting destabilization of lipid bilayers, such as those in the endosome (7). A prototype microfluidic hydrodynamic focusing (MHF) system was designed to prepare SPLN.

Microfluidic processing provides defined mixing geometries which result in better sample reproducibility. Analysis of processing conditions determined that flow rate, lipid concentration, and lipid:ON ratio were critical determinants of particle

120 size. LN prepared by MHF demonstrated less polydispersity relative to samples prepared by bulk mixing. SPLN/C was able to effectively deliver AM-221 and AM-

21 to MCF-7 and MDA-MB-231 cells as indicated by upregulation of PTEN, estrogen receptor α (ERα), and e-cadhein and downregulation of vinmentin. The combination of TMX and SPLN/C resulted in sensitization towards TMX for normally resistant MDA-MB-231 cells and increased sensitivity for MCF-7 cells thus, opening new possibilities for the treatment of breast cancer. Following treatment with SPLN/C, decreases in cell motility were also observed.

5.2 Perspectives

The potent regulatory ability of RNA interference based therapeutics in cancer will undoubtedly be the focus of study for many years to come. Equally important to this study will be determination of efficient vectors for mediating delivery.

Successful development of ON delivery systems depends on a complex interplay between physiochemical properties and biological response. Preparing particles of correct size and surface charge may not necessarily guarantee therapeutic response if ON is not correctly released into the cytosol. The studies provided in this dissertation stress the importance of a fusogenic or helper lipid to enhance delivery. Furthermore, greater understanding of the drug itself is needed to fully potentate therapy. The degree of ON complementarity as well as the modifications to base chemistry all play a role in how siRNA, AM, and miR- mimics bind to and interact with their targets in vitro and in vivo (8-9). Screening of hundreds of ONs is often not feasible due to limitations inherent in assay size and high cost of materials. Therefore, establishment of a set of guidelines for the

121 design of ONs would be most beneficial to the continuation of the science. With the growing interest in ON and LNs, answering questions regarding the safety of the formulations will be a top priority. Better understanding of the cellular pharmacokinetics (PK) and pharmacodynamics (PD) and how it relates to overall

PK/PD will lead these studies and guide the transition of LN to the clinic.

Moreover, producing LN under current good manufacturing practice (cGMP) conditions remains a challenge for the commercialization and industrial application of LNs. A collaboration between pharmaceutical scientists and material science engineers will guide the development of standardized equipment for LN manufacture.

5.3 Future Work

The formulations outlined in this dissertation are continuing development to improve upon LN design and to expand in new clinical directions. A folate targeted LCAN is currently being investigated for the delivery of Archexin, an antisense directed against the Akt pathway. Preliminary animal data shows significant tumor regression activity over docetaxel treated mice and prolonged survival time. QT/AM-21 formulation is continuing testing to determine appropriate dosing regimen and to evaluate cytokine response. These studies will aid the translation of QT/AM-21 into clinical study for the treatment of

NSCLC. QT/miR-484 mimic, QT/miR-34a mimic, and QT/AM-17-5p are also being evaluated for the treatment of ovarian, prostate, and pancreatic cancers respectively. Furthermore, combinations of AM and miR mimics will be investigated to determine whether any synergetic interaction occurs between

122 agents. SPLN is similarly being explored to discover new therapeutic opportunities. Recently SPLN was used to deliver AM-220b in an ischemic diabetic wound model, resulting in greatly accelerated wound closure. Moreover, while in vivo studies of SPLN are beginning, addition in vitro studies are being completed to better elucidate the mechanistic pathways mediating intracellular delivery. Additionally, the MHF used to prepare SPLN is being adapted to produce liter volumes of liposomes to determine the scalability of the manufacturing process.

5.4 Concluding Remarks

The current work presents several novel formulations for the purpose of ON delivery. The high degree of customization and versatility make LNs a highly valuable platform technology for both the screening of ONs and for use in the clinic. LNs greatly enhance the potential of small interfering RNA (siRNA), AM, and miR mimics, by providing protection against nucleases, limiting off-target uptake, and promoting delivery to the site of therapeutic action. Rational design of LNs allow for efficient uptake and delivery both in vitro and in vivo. Such systems thus facilitate translational research in cancer and accelerate the screening and approval of ON-based therapies. A greater understanding of the connection between physical properties such as particle size and surface charge and the resultant therapeutic activity is still needed to fully take advantage of LN technology. It is my hope that the work included in this dissertation will be used as a stepping stone for others in the rapidly expanding field of LN for the delivery of ON in cancer.

123

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