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Cell penetrating peptide-based polyplexes for sirRNA delivery

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Cell penetrating peptide-based polyplexes for sirRNA delivery

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CELL PENETRATING PEPTIDE-BASED POLYPLEXES FOR SIRNA DELIVERY

by

Robert H. Mo

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(PHARMACEUTICAL SCIENCES)

May 2012

Copyright 2012 Robert H. Mo
ii
DEDICATION

To my parents, Y. Joseph and Jennifer Mo,
thank you for all your love and support.
iii
ACKNOWLEDGEMENTS

My advisor, Dr. Wei-Chiang Shen, has been an excellent role model and mentor for me.
He is full of wisdom and kindness, and he has greatly influenced my growth as a scientist
and a person. I will always be grateful for all the wonderful guidance and opportunities
provided by him. I also thank Mrs. Daisy Shen for all her sweetness and thoughtfulness.
She has been such a positive figure throughout my Ph.D. studies, and I will always
appreciate her assistance and support. Dr. Shen and Daisy are both simply amazing
individuals, and I am very fortunate to have been under their care.

Dr. Jennica L. Zaro has also been an important influence during graduate school. Since
her return to USC, she has provided me with so much help and guidance with my Ph.D.
studies and career development. She is very kind and selfless, and I sincerely appreciate
all of her great advice and support.

My committee members – Dr. J. Andrew MacKay, Dr. Rizwan Masood, Dr. Clay Wang
and Dr. Shao-Yao Ying – have all been instrumental to my academic growth as a
graduate student. I thank them for all their shared knowledge, discussion and guidance.

I also want to acknowledge labmates, friends, professors and staff that I have gotten to
know during my time at USC. I have always appreciated all your help and support during
iv
my many endeavors in lab, the classroom and my extracurricular activities. I would not
be who or where I am without your influences during my graduate studies.

My parents, Dr. Y. Joseph and Jennifer Mo, have always been there for me. I have
always been inspired by their love and dedication for my sister, Stephanie, and me. All of
my accomplishments are due to the great support provided by them throughout my life.
Stephanie H. Mo has also been a dear sister and friend to me. She is incredibly
trustworthy and kind, and she is everything a brother could want in a sister. I love them
all so dearly.

I also thank Stephanie for providing all the schematic figures appearing in this
dissertation.

Finally, I want to acknowledge Yan Wang and all of her unconditional love and support.
Being fortunate enough to work with her over the past few years has made each day all
the more better. Thank you for being you.

v
TABLE OF CONTENTS

DEDICATION ii

ACKNOWLEDGEMENTS iii

LIST OF TABLES ix

LIST OF FIGURES x

LIST OF CALCULATIONS xii

ABBREVIATIONS xiii

ABSTRACT xvii

PREFACE
1. Significance xix
2. Scope of Project xx
3. Overview of Field xxi

CHAPTER 1: INTRODUCTION
1.1. RNA Interference 1
1.2. SiRNA Delivery
1.2.1. Background 4
1.2.2. Cationic Lipids 5
1.2.3. Cationic Polymers 6
1.3. Cell Penetrating Peptides
1.3.1. Background 8
1.3.2. Cell Penetrating Peptides for siRNA Delivery 9

vi
CHAPTER 2: DEVELOPMENT AND CHARACTERIZATION
OF K21-BASED SIRNA POLYPLEX
2.1. Introduction
2.1.1. Background 12
2.1.2. Rationale 13
2.2. Experiments
2.2.1. Quantitative Measurement of Amines in 21mer Oligo-L-lysine 15
2.2.2. Production, Purification and Quantification of N-succinimidyl-
3-(2-pyridyldithio) propionate-modified K21 15
2.2.3. Polyplex Formation between K21-PDP and siGFP 16
2.2.4. Measurement of Amine and Phosphate Interaction for Polyplex 18
2.2.5. Stability of 2:1 N/P Polyplex 18
2.2.6. SiRNA Release from Polyplex
2.2.6.a. Trypsin Digestion 18
2.2.6.b. Polyplex Dilution 19
2.3. Results
2.3.1. Quantitative Measurement of Amines in 21mer Oligo-L-lysine 20
2.3.2. Production, Purification and Quantification of N-succinimidyl-
3-(2-pyridyldithio) propionate-modified K21 21
2.3.3. Polyplex Formation between K21-PDP and siGFP 23
2.3.4. Measurement of Amine and Phosphate Interaction for Polyplex 24
2.3.5. Stability of 2:1 N/P Polyplex 25
2.3.6. SiRNA Release from Polyplex
2.3.6.a. Trypsin Digestion 26
2.3.6.b. Polyplex Dilution 26
2.4. Discussion 28
2.5. Summary 31

CHAPTER 3: COMPARISON OF CATIONIC AND AMPHIPATHIC
CELL PENETRATING PEPTIDES FOR SIRNA DELIVERY
3.1. Introduction
3.1.1. Background 32
3.1.2. Rationale 32
3.2. Experiments
3.2.1. Selection of Oligoarginine for Conjugation 35
3.2.2. Synthesis of R6-polyplex and MAP-polyplex 36
3.2.3. Effect of Carrier Conjugation on Polyplex Stability 37
3.2.4. Cellular uptake 37
3.2.5. GFP Silencing 40
3.2.6. Cell Viability 41
3.2.7. Lack of K21-PDP 41
3.3. Results
3.3.1. Selection of Oligoarginine for Conjugation 43
vii
3.3.2. Synthesis of R6-polyplex and MAP-polyplex 44
3.3.3. Effect of Carrier Conjugation on Polyplex Stability 45
3.3.4. Cellular uptake 46
3.3.5. GFP Silencing 48
3.3.6. Cell Viability 49
3.3.7. Lack of K21-PDP 49
3.4. Discussion 51
3.5. Summary 55

CHAPTER 4: EVALUATION OF MAP-POLYPLEX DESIGN
4.1. Introduction
4.1.1. Background 56
4.1.2. Rationale 57
4.2. Experiments
4.2.1. Effect of Serum on Transfection 58
4.2.2. Different Amounts of MAP Conjugation
4.2.2.a. Uptake 58
4.2.2.b. Activity 58
4.2.3. Uptake Mechanism 59
4.2.4. Model of Cytosolic-like Environment 60
4.2.5. Endogenous Gene Silencing in an Alternative Cell Line 60
4.3. Results
4.3.1. Effect of Serum on Transfection 62
4.3.2. Different Amounts of MAP Conjugation
4.3.2.a. Uptake 63
4.3.2.b. Activity 63
4.3.3. Uptake Mechanism 65
4.3.4. Model of Cytosolic-like Environment 66
4.3.5. Endogenous Gene Silencing in an Alternative Cell Line 66
4.4. Discussion 68
4.5. Summary 72

CHAPTER 5: EFFECTS OF SIRNA DELIVERY ON AUTOPHAGY
IN HEPATOMA CELLS
5.1. Introduction
5.1.1. Background 73
5.1.2. Rationale 74
5.2. Experiments
5.2.1. Cellular uptake of Lipo2000 Lipoplexes and MAP-polyplexes
at Escalating Doses 76
5.2.2. LC3 Conversion in Hepatoma Cells
5.2.2.a. MG-132 Treatment 76
viii
5.2.2.b. SiRNA Transfection in Hepatoma Cells 76
5.2.3. Evaluation of LC3 Conversion in H4IIE 77
5.2.4. Quantification of GFP-LC3 Fluorescence in Huh7.5 Cells
5.2.4.a. MG-132 and Glucosamine Treatment 77
5.2.4.b. Lipo2000 Lipoplex 78
5.2.4.c. MAP-polyplex 78
5.2.5. Detection of Autophagosomes in Huh7.5 Cells using
Confocal Microscopy 78
5.2.6. Effects of Alternative Negative Control siRNAs for siRNA
Delivery on Autophagy in Huh7.5 Cells
5.2.6.a. LC3 Conversion 79
5.2.6.b. Quantification of GFP-LC3 Fluorescence 80
5.3. Results
5.3.1. Cellular Uptake of siDY547 at Different Concentrations using
Lipo2000 Lipoplex and MAP-polyplex in Huh7.5 cells 81
5.3.2. Evaluation of LC3 Conversion in Huh7.5 Cells
5.3.2.a. Positive Control – MG-132 82
5.3.2.b. Lipo2000 Lipoplex 82
5.3.2.c. MAP-polyplex 82
5.3.3 Evaluation of LC3 Conversion in H4IIE Cells 84
5.3.4. Quantification of GFP-LC3 Fluorescence in Huh7.5 Cells
5.3.4.a. Positive controls – MG-132 and Glucosamine 85
5.3.4.b. Lipo2000 Lipoplex 85
5.3.4.c. MAP-polyplex 86
5.3.5. Detection of Autophagosomes in Huh7.5 Cells using Confocal
Microscopy 88
5.3.6. Effect of Alternative Negative Control siRNAs for siRNA
Delivery on Autophagy in Huh7.5 Cells
5.3.6.a. LC3 Conversion 90
5.3.6.b. Quantification of GFP-LC3 Fluorescence 90
5.4. Discussion 92
5.5. Summary 97

CHAPTER 6: SUMMARY AND FUTURE PERSPECTIVES
6.1. Summary 98
6.2. Future Perspectives 101

REFERENCES 104

ALPHABETIZED REFERENCES 115

ix
LIST OF TABLES

Table 1: PDP concentration of K21-PDP using PDP assay. 23

Table 2: Reaction efficiency for R6-polyplex and MAP-polyplex. 45

x
LIST OF FIGURES

Figure 1: RNA interference for post-transcriptional gene silencing. 3

Figure 2: Standard curve of ε-amines for quantification of K21 amines
using TNBSA Assay. 20

Figure 3: Purification and quantification of K21-PDP. 22

Figure 4: Polyplex formation at different N/P ratios between K21-PDP
and siGFP. 24

Figure 5: Determination of N/P interaction at 2:1 N/P polyplex. 25

Figure 6: Polyplex stability based on N/P interaction. 26

Figure 7: SiRNA release from polyplexes. 27

Figure 8: Development of R6-polyplex and MAP-polyplex for
comparison of cationic and amphipathic CPPs for siRNA
delivery and efficacy. 34

Figure 9: Displacement of K21 by N
α
-acetylated oligoarginine (Ac-R4,
Ac-R6 and Ac-R8) at different G/N ratios in 2:1 N/P polyplex
after 48 h. 44

Figure 10: Evaluation of dilution effects on R6-polyplex and MAP-polyplex. 45

Figure 11: Cellular uptake and extracellular surface binding of
R6-polyplex and MAP-polyplex in Huh7.5 cells, stably
transfected to express GFP-LC3. 47

Figure 12: Gene silencing by R6-polyplex and MAP-polyplex in Huh7.5
cells, stably transfected to express GFP-LC3. 48

Figure 13: Cell viability. 49

Figure 14: Role of K21-PDP in R6-polyplex and MAP-polyplex for
gene silencing effects. 50

Figure 15: Effect of 10% FBS on MAP-polyplex gene silencing efficacy. 62

Figure 16: Effect of MAP conjugation for MAP-polyplex cellular uptake
xi
and gene silencing efficacy. 64

Figure 17: Mechanism of MAP-polyplex cellular uptake. 65

Figure 18: Model of siRNA release from MAP-polyplex. 66

Figure 19: Endogenous gene silencing of PTEN in HeLa cells using
MAP-polyplex. 67

Figure 20: Autophagy pathway. 74

Figure 21: Cellular uptake of siDY547 at different concentrations
using Lipo2000 lipoplex and MAP-polyplex in stably
transfected Huh7.5 cells. 81

Figure 22: Western blot of LC3 conversion in stably transfected
Huh7.5 cells. 83

Figure 23: Western blot of LC3 conversion in H4IIE cells. 84

Figure 24: Detection of GFP fluorescence of stably transfected Huh7.5 cells. 87

Figure 25: Visualization of autophagosome formation in Huh7.5 cells, stably
transfected to express GFP-LC3. 89

Figure 26: Comparison of different negative control siRNAs for LC3
conversion and GFP-LC3 fluorescence in Huh7.5 cells, stably
transfected to express GFP-LC3. 91

xii
LIST OF CALCULATIONS

Calculation 1: Comparison of K21 concentration based on weight and amino
groups, as determined by TNBSA assay. 21

Calculation 2: Determination of K21-PDP amine concentration using TNBSA
assay. 22

Calculation 3: Degree of PDP modification per K21-PDP peptide. 23

xiii
ABBREVIATIONS

Ac-K N
α
-acetyl-L-lysine methyl ester HCl
Ac-R N
α
-acetyl-L-arginine
Ac-R4 N
α
-acetyl-tetra-L-arginine
Ac-R6 N
α
-acetyl-hexa-L-arginine
Ac-R8 N
α
-acetyl-octa-L-arginine
Ago2 Argonaute 2
AKT Ak strain of thymoma
BCA Bicinchoninic acid
CMAP L-cysteinyl model amphipathic peptide
CPPs Cell penetrating peptides
CR6 L-cysteinyl-hexa-L-arginine
DAPI 4’,6-diamidino-2-phenylindole
DDH
2
O Double deionized water
DEPC Diethylpyrocarbonate
DMEM Dulbecco’s modified Eagle’s medium
DMF Dimethylformamide
DODAG N’,N’-dioctadecyl-N-4,8-diaza-10-aminodecanoylglycine amide
DOPE Dioleoyl-L-α-phosphatidylethanolamine
DTT Dithiothreitol
EDTA Ethylenediaminetetraacetic acid
xiv
FBS Fetal bovine serum
GFP Green fluorescent protein
GFP-LC3 Green fluorescent protein – light chain 3 fusion protein
G/N Guanidine-to-amine
HBV Hepatitis B virus
HRP Horseradish peroxidase
K21 21mer oligo-L-lysine
K21-PDP Pyridyldithiol-activated K21
LC3 Light chain 3
Lipo2000 Lipofectamine 2000
MAP Model amphipathic peptide
miRNA MicroRNA
mRNA Messenger RNA
mTOR Mammalian target of rapamycin
MTP Membrane transduction peptide
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
N/P Amine-to-phosphate
PBS Phosphate-buffered saline
PDP N-succinimidyl-3-(2-pyridyldithio) propionate-modified
PE Phosphotidylethanolamine
PEI Polyethylenimine
PI3K Phosphoinositide 3-kinase
xv
PQ 9,10 phenanthrenequinone
pre-miRNA Precursor miRNA
pri-miRNA Primary miRNA
PTD Protein transduction domain
PTEN Phosphatase and tensin homologue deleted on chromosome 10
R6 Hexa-L-arginine
R9 Nonaarginine
RFU Relative fluorescence units
RISC RNA-induced silencing complex
RNA Ribonucleic acid
RNAi RNA interference
siDY547 Fluorophore-labeled negative control siRNA
siGFP Green fluorescent protein-targeting siRNA
siNC Negative control siRNA
siNCambion Negative control siRNA from Ambion
siNCqiagen Negative control siRNA from Qiagen
siPTEN PTEN-targeting siRNA
siRNA Small interfering RNA
SPDP N-succinimidyl-3-(2-pyridyldithio) propionate
TAE Tris-acetate-EDTA
TAT Transcriptional trans-activator
TBS-T Tris-buffered saline with Tween 20
xvi
Tf Transferrin
TFA Trifluoroacetic acid
TNBSA 2,4,6-Trinitrobenzene sulfonic acid
VEGF Vascular endothelial growth factor

xvii
ABSTRACT

In this dissertation, a novel cell penetrating peptide (CPP)-based polyplex was
developed and evaluated for small interfering RNA (siRNA) delivery and efficacy. The
design resolved the CPP carrier neutralization issue, where CPP cellular uptake is
compromised by electrostatic interactions between the CPP and siRNA, by using a
chemically modified 21mer oligolysine (K21-PDP) to form a stable and neutralized
polyplex with siRNA. This neutralized polyplex served as a suitable platform to
conjugate distinct CPP carrier moieties – cationic (hexaarginine, R6) and amphipathic
(model amphipathic peptide, MAP) – to determine which CPP property was more
suitable for siRNA delivery. Since K21-PDP already effectively neutralized the siRNA,
the CPP carriers on the conjugated CPP-polyplexes, R6-polyplex and MAP-polyplex,
were able to function without electrostatic interference. Amphipathic MAP was found to
be a better CPP carrier than R6 because MAP-polyplex exhibited greater siRNA
intracellular uptake and gene silencing than R6-polyplex, while still maintaining
comparable cytotoxicity effects.
The design of MAP-polyplex was also further evaluated. MAP-polyplex showed
comparable effects with the commonly used lipid-based siRNA transfection reagent
Lipofectamine 2000. MAP-polyplex entered the cell through a vesicle-based mechanism,
and its transfection efficiency was not affected by the co-incubation of 10% fetal bovine
serum. In addition, the roles of each component of MAP-polyplex – K21-PDP, siRNA,
MAP and disulfide bond linkage – in promoting intracellular siRNA delivery and
xviii
efficacy were elucidated. Therefore, MAP-polyplex is a promising CPP-based platform
that can be used for siRNA delivery.
The effects of siRNA transfection on autophagy in hepatoma cells were also
presented in this dissertation. A method was developed to evaluate the presence of
intracellular autophagosomes using fluorescence spectroscopy. An increased signal of a
stably expressed fluorescent fusion protein as an autophagy marker was detected in
transfected cells relative to non-treated cells. The results corresponded with increased
LC3 conversion and autophagosomal punctate via immunoblotting and confocal
microscopy, respectively, which are also markers commonly used in autophagy studies.
Using these methods, lipid-based (Lipofectamine 2000) and CPP-based (MAP-polyplex)
siRNA transfections elicited an increase of autophagosomes in hepatoma cells in dose-
and time-dependent manners. These effects should be considered for the application of
siRNA delivery systems. Therefore, the findings of this dissertation present relevant
considerations for siRNA delivery and provide important insights for the future
development of siRNA as a therapeutic agent.

xix
PREFACE

1. Significance
This research project is focused on the application of cell penetrating peptides
(CPPs) for small interfering RNA (siRNA) delivery and efficacy. CPPs are short strands
of arginine- and/or lysine-rich peptides, typically of thirty or less amino acids. They rely
on their cationic nature to efficiently accumulate intracellularly. Covalent conjugation of
macromolecules, such as proteins, oligonucleotides and nanoparticles, to CPPs has been
shown to enhance their cellular uptake. CPPs have been primarily used for siRNA uptake
by mixing the two moieties together to form a polyplex. However, electrostatic
interactions needed for polyplex formation also leads to the neutralization of the cationic
carrier by the anionic cargo, which may inhibit the intracellular delivery efficiency.
Taking this into consideration, a novel CPP-based polyplex is designed not only to retain
the cationic properties of CPPs, but also permit the comparison of different CPP carriers
for siRNA delivery and efficacy. In addition, studies are performed to reveal the effects
of siRNA uptake on autophagy in hepatoma cells. The results from these studies will
provide insight for the development of a CPP-based siRNA delivery system and also
raise awareness for autophagic cellular responses to siRNA transfection.

xx
2. Scope of Project
The development of a distinct neutralized polyplex is described in Chapter 2. The
polyplex is made of a chemically modified 21mer oligolysine and siRNA. Not only does
the oligolysine neutralize the siRNA, but also it is able to release the oligonucleotide
under cytosolic-like conditions. The polyplex serves as a platform to conjugate functional
delivery moieties, such as cationic and amphipathic CPPs. In Chapter 3, cationic and
amphipathic CPP carriers are covalently conjugated to the polyplex (via a reducible
disulfide bond) to determine which CPP is more suitable for siRNA delivery. The design
allows for comparison of CPP carriers, where the CPP is free to facilitate cellular uptake
and not inhibited by siRNA neutralization. Once the appropriate CPP carrier is selected,
the development of a CPP-based siRNA delivery system is further evaluated and
optimized in Chapter 4.
Furthermore, in Chapter 5, a new method is established to quantify the
intracellular fluorescence of stably expressed green fluorescence protein fusion protein as
an indicator of intracellular autophagosome presence. The results gathered from this
method are confirmed by additional standard autophagy studies. The results from these
assays indicate the effects of siRNA delivery on autophagy in hepatoma cells.

xxi
3. Overview of Field
Development of efficient intracellular siRNA delivery system is an important
consideration for the realization of siRNA as a therapeutic agent. Unfortunately, cells do
not readily take in siRNA due to its anionic nature and relatively large size. One non-viral
method proposed to enhance siRNA cellular uptake is to use CPPs. However, CPPs are
neutralized by anionic siRNAs through electrostatic interactions, and loss of cationic
charges results in reduced CPP uptake efficiency. In order to overcome this obstacle, a
novel chemically modified 21mer oligolysine-based siRNA polyplex is designed and
developed to neutralize siRNA. The polyplex serves as a platform to conjugate different
functional moieties to facilitate siRNA uptake and efficacy. Cationic and amphipathic
CPPs are compared to establish which CPP property is more suitable for siRNA delivery.
The novelty of this comparison is that each CPP is conjugated to a neutralized polyplex,
so that the CPP carrier functions without being inhibited by siRNA neutralization effects.
Therefore, one of the significant contributions of this work is the finding that amphipathic
CPPs are more suitable for intracellular siRNA delivery than cationic CPPs. The
amphipathic CPP-polyplex is shown to enhance siRNA uptake and knockdown reporter
and endogenous gene expression. In addition, lipid- and peptide-based siRNA
transfections are found to increase the presence of intracellular autophagosomes. This
important discovery indicates that autophagy may have an important role in the
intracellular processing and efficacy of siRNA. The findings of this research work should
be taken into consideration for the future design and development of siRNA delivery
systems.
1
CHAPTER 1: INTRODUCTION

1.1. RNA Interference
RNA interference (RNAi), discovered by Fire and Mello in 1998, has been
commonly defined as an endogenous cellular process where short strands of RNA direct
the degradation of target messenger RNA (mRNA) [2]. The loss of the targeted mRNA
has resulted in reduced protein expression that has been classified as a gene silencing
effect. The post-transcriptional gene silencing process has been evolutionary conserved
and required precise oligonucleotide sequence specificity based upon Watson-Crick base
interactions. The biological functions of RNAi have been an on-going area of interest.
Responsibilities currently attributed to RNAi have been immunity and gene regulation
[3,4]. Viral infection has occurred by transferring the viral genetic material via double-
stranded RNA into the host cells. Double-stranded RNA produced from transgenes in
plants has been shown to suppress this viral transfer and avoid infection [5]. In addition,
plants with mutated RNAi genes have been shown to be more susceptible to viral
infection [6]. RNAi components have also been demonstrated to regulate gene expression
through transcriptional silencing in the nucleus [7,8] and post-transcriptional silencing in
the cytoplasm [4,9-11]. The contents of this dissertation focus on RNAi for post-
transcriptional silencing.
The endogenous pathway for RNAi post-transcriptional silencing initiates in the
nucleus where primary microRNAs (pri-miRNAs) are transcribed and then cleaved to
~70 nucleotide-sized microRNA precursors (pre-miRNAs) by the microprocessor
2
complex (Fig. 1). Pre-miRNAs are then exported out of the nucleus via a complex of
Exportin-5 and Ran-GTP. Once in the cytoplasm, a protein called Dicer processes pre-
miRNAs into microRNAs (miRNAs), 21-23 base pairs RNA duplexes with symmetrical
2-nucleotides 3’ overhangs. MiRNA is then loaded onto the multi-protein RNA-induced
silencing complex (RISC), which contains a key cleaving protein called Argonaute 2
(Ago2). Ago2 slices the center of the miRNA sense (passenger) strand, and the cut strand
is then released from RISC. The remaining antisense (guide) strand of miRNA in RISC
directs the sequence-specific gene silencing of target mRNA based on Watson-Crick base
pairing and subsequent cleavage by Ago2 [9-11]. Since RNAi functions by targeting
specific gene expression, RNAi has been receiving high expectations as an innovative
form of therapy for human diseases and ailments (reviewed in [10]).
3

Figure 1. RNA interference for post-transcriptional gene silencing. RNA interference can
be initiated by endogenous miRNA or exogenous siRNA. Gene silencing by miRNA
begins in the nucleus, where pre-miRNAs are produced and then exported out via a
complex of Exportin-5 and Ran-GTP. Dicer then cleaves pre-miRNAs into 21-23 base
pair miRNAs with symmetrical 2-nucleotides 3’ overhangs. The resulting miRNA
product is loaded onto the RISC, and Ago2 removes the passenger strand. The remaining
guide strand then directs the RISC to degrade target mRNA and induce a gene silencing
effect. Alternatively, exogenous siRNA can also be delivered into the cell, where it
directly interacts with the RISC (without requiring Dicer processing) to reduce gene
expression via the same mechanism.

4
1.2. SiRNA Delivery
1.2.1. Background
A pioneering finding in 2001 has indicated that exogenous small interfering RNA
(siRNA), 21-23 base pair RNA duplexes with 2-nucleotides 3’ overhangs, could also
initiate RNAi similar to miRNA (Fig. 1). However, siRNA has been reported to directly
interact with RISC to induce a specific gene silencing effect, without requiring Dicer
processing [12]. Since this discovery, siRNA has been considered as a promising new
therapeutic molecule. RNAi therapy using siRNA delivery has been evaluated for many
diseases [9,13]. In addition to its prospects as a pharmaceutical drug, siRNA has also
been used in the pharmaceutical industry for drug target screening and genomic pathway
elucidation [14].
Although siRNA has many promising features, it has several limitations
concerning its potential therapeutic application. First, its primary shortcoming has
regarded its inability to enter the cell. The size of siRNA molecules (~13 kd) prevents it
from passively diffusing through the cell membrane, because its strong anionic charges of
phosphate backbone repel it from the negatively charged cell surface [15]. A common
method of applying siRNA for in vitro studies has been to use polycationic reagents, such
as Lipofectamine 2000 [16]. However, these reagents have not been appropriate for in
vivo therapies, so developing novel delivery methods is necessary. Moreover, once the
siRNA has been delivered into the cell, it has to localize in the cytosol and be properly
released from its delivery vesicle so that it can interact uninhibitedly with the RNAi
mechanical components. Finally, siRNA also has to be well protected because it is
5
sensitive to nuclease degradation and remains instable in serum [17]. Therefore, in
addition to the efficient intracellular delivery, the latter two concerns have to be
addressed and resolved in order for RNAi therapy to fulfill its promise.
There have been many studies regarding the development of siRNA delivery.
Recombinant viral vectors have been used to achieve prolonged gene silencing in
mammalian cells [18,19]. Viral vectors encoding siRNA have also been shown to obtain
RNAi in vivo [20,21]. However, safety concerns regarding the use of viral vectors due to
potential mutagenesis and immunogenicity have limited its clinical application [22].
Another promising approach for siRNA delivery has been through non-viral vehicles,
such as cell penetrating peptides (CPPs), cationic lipids and cationic polymers. The
introduction to siRNA delivery using cationic lipids and polymers will be introduced in
this section (1.2). An overview of CPPs and its application for siRNA delivery, which is
the primary focus of this dissertation, will be discussed more elaborately in the following
section (1.3).

1.2.2. Cationic Lipids
Cationic lipids interact with anionic siRNAs through electrostatic interactions to
form lipoplexes, which protect oligonucleotides from degradation and prevent
electrostatic repulsion from the negatively charged extracellular membrane [23].
Lipofectamine 2000 has been a well-recognized cationic lipid reagent for siRNA delivery
[16]. However, it has been predominantly used for in vitro cell cultures. Other lipoplexes
have been developed for siRNA delivery both in vitro and in vivo. Novel N
4
,N
9
-
6
diacylated asymmetrical spermine derivatives with two different fatty acid chains,
varying in length (from C18 to C24) or their oxidation state, have been evaluated for
structure activity relationships and siRNA delivery in vitro. It has been established that
N
4
,N
9
-oleoyl-1,12-diamino-4,9-diazadodecane was the most suitable non-toxic cationic
lipid carrier for siRNA efficacy [24]. Another cationic lipid, N’,N’-dioctadecyl-N-4,8-
diaza-10-aminodecanoylglycine amide (DODAG), mixed together a neutral helper lipid
dioleoyl-L-α-phosphatidylethanolamine (DOPE) for siRNA delivery, has exhibited
efficient luciferase knockdown and minimal cytotoxicity in three different cell lines. In
addition, DODAG has delivered anti-hepatitis B virus (HBV) siRNAs to murine livers in
vivo and produced a specific reduction of HBV markers [25]. There have been many
additional examples of cationic lipids used successfully for siRNA delivery (reviewed in
[26,27]).

1.2.3. Cationic Polymers
Nanoparticles are formed when cationic polymers are mixed with siRNA, and its
application for siRNA delivery has been demonstrated in various models. Chitosan is a
natural linear polysaccharide with low toxicity and immunogenicity, and excellent
biodegradability and biocompatibility. Different forms of chitosan have been evaluated,
and an optimial chitosan-based nanoparticle has been characterized and established to be
effective for siRNA delivery in vitro [28]. Another chitosan-based nanoparticle has
produced siRNA efficacy in vivo in transgenic mice via nasal and pulmonary routes of
administration [29-31]. Polyethylenimine (PEI) is a synthetic cationic polymer used for
7
siRNA delivery. The properties of PEI vary based on structure (linear or branched) and
size. Studies have evaluated different PEI and determined that linear 22 kd JetPEI [32,33]
and purified branched ~10 kd PEI F25-LMW [34] were suitable for siRNA delivery and
efficacy in vitro and in vivo. Dendrimer is a synthetic spherical branched polymer that
has different designs based on terminal groups (acidic or basic) and sizes. Various
cationic polyamidoamine dendrimers have been used effectively for silencing activity
with minimal cytotoxicity [35,36]. Gold nanorods are nanoparticles that can be modified
to exhibit cationic charges on its surface to form complexes with siRNA. Gold
nanocomplexes, loaded with a sphingosine kinase-1 siRNA, have been shown to improve
the efficiency of radiation therapy and reduce tumor growth in mice after subcutaneous
injection [37]. Additional reports regarding successful developments of nanoparticles for
siRNA delivery have been extensively summarized (reviewed in [38]).

8
1.3. Cell Penetrating Peptides
1.3.1. Background
CPPs are arginine- and/or lysine-rich peptides consisting of 30 or less amino acids
that depend on their cationic nature for efficient intracellular uptake. CPPs also have been
known as membrane transduction peptides (MTPs) and protein transduction domains
(PTDs). They have been classified as either cationic or amphipathic CPPs. Cationic CPPs
have included TAT (GRKKRRQRRR), derived from the HIV-1 nuclear transcription
activator [39], penetratin (RQIKWFQNRRMKWKK), derived from the third alpha helix
of the Drosophila Antennapedia homeodomain protein [40], and synthetic oligoarginine
(Rn) and oligolysine (Kn) [41,42]. On the other hand, examples of amphipathic CPPs
have been transportan (GWTLNSAGYLLGKINLKALAALAKKIL), derived from a
fusion of a wasp venom peptide and a neuropeptide [43], MPG
(GALFLGWLGAAGSTMGAPKKKRKV), derived from a fusion of HIV-1 gp41 protein
and SV40 large T antigen [44], and the model amphipathic peptide (MAP,
KLALKLALKALKAALKLA), designed for optimal cellular uptake [45].
CPPs have been based on or derived from sequences of endogenous proteins that
are essential for their intracellular delivery. Two important factors for efficient cellular
uptake of CPP have included the peptide sequence and the basic amino acids featured in
all CPPs. There have been many theories regarding the mechanism of CPP cell entry.
CPPs have been proposed to transported into the cell through vesicle- and non-vesicle-
based pathways [46]. Vesicle-based internalization occurs through energy-dependent
endocytosis where the plasma membrane either extends outward or invaginates inward to
9
form a vesicle containing the CPP and extracellular cargo. Non-vesicle-based cellular
uptake is through energy-independent membrane transduction, where the CPP can
directly penetrate across the cell membrane and enter the cytosol. Moreover, attachment
of cargo to the CPP may also alter the transport properties [46]. Therefore, it is important
to evaluate the mechanism of the CPP-cargo as a single moiety.
Although the exact mechanism of CPP uptake remains controversial, CPP has
demonstrated enhanced delivery of a variety of conjugated cargo. Intratracheal delivery
of oligoarginine (R9)-insulin conjugates has reduced blood glucose levels significantly
for a longer period of time than insulin in diabetic rats [47]. R9 also has increased cellular
uptake of cytochrome c, which has resulted in increased apoptosis in vitro [48]. R9 also
has showed increased cytosolic delivery of p16 peptide, which has corresponded with
inhibition of cell proliferation [49]. Conjugation of CPPs to small molecule
chemotherapies, such as cyclosporine A, doxorubicin, methotrexate and paclitaxel, also
has improved the drugs’ delivery and activity [50-53]. Thus, the ability of CPPs to readily
enter cells has been very promising for delivery of therapeutic drugs. There have been
many additional examples of CPP for enhanced delivery of small molecules, proteins,
peptides, oligonucleotides and imaging agents (reviewed in [46,54,55]).

1.3.2. Cell Penetrating Peptides for siRNA Delivery
The primary approaches for using CPPs for siRNA delivery have included 1.)
CPP conjugating directly to siRNA, 2.) CPP conjugating to a cargo containing siRNA, 3.)
CPP forming a polyplex, and 4.) CPP acting as a platform for conjugating functional
10
moieties. The CPPs TAT, penetratin and transportan have all been directly conjugated to
siRNA and demonstrated intracellular uptake and knockdown of target gene [56-59].
CPPs have also been conjugated to cargos carrying siRNA. A fusion protein consisting of
TAT and a double stranded RNA binding domain has showed efficient siRNA delivery in
primary and difficult-to-transfect cells [60]. Oligoarginine R8 has been conjugated to a
multi-functional lipid-based nanoparticle to stimulate cellular uptake, and the developed
nanoparticle has demonstrated positive gene effects in vitro and in vivo [61]. Penetratin
has been conjugated along with the targeting moiety folate to a PLGA nanoparticle for
siRNA delivery. The combination of the two functional ligands has shown a synergistic
effect, and the multi-functional nanoparticle has exhibited significant reduction of
luciferase expression in tumor bearing mice [62].
The positive charges from the basic amino acids of these CPPs have been used to
form polyplexes with anionic siRNA via electrostatic interactions. The polyplex
formation has depended on the amine-to-phosphate (N/P) ratio between CPP and siRNA.
Each CPP and siRNA pairing has a different minimal N/P ratio needed to form a
polyplex. Polyplexes typically have been made of higher N/P ratios, and the excess
positive charges of the polyplex have been responsible for the cellular uptake. Polyplexes
consisting of MPG, penetratin or transportan have demonstrated target gene silencing in
vitro [44,63,64]. Oligoarginine, such as R9 and R12, has also been used to polyplex with
siRNA. R12 has been packaged with siRNA to induce RNAi of target genes in tobacco
suspension cells [65]. A polyplex based on a novel secondary amphipathic CPP called
CADY has been shown to knockdown target gene expression in vitro with minimal
11
cytotoxicity [66]. Therefore, both cationic and amphipathic CPPs have been used to form
polyplexes for siRNA delivery and activity.
CPP-based polyplexes have also been conjugated with functional moieties to
enhance siRNA cellular uptake and efficacy in vitro and in vivo. Systemic administration
of an oligoarginine siRNA polyplex targeted for neuronal cells has been able to cross the
blood brain barrier by fusing the oligoarginine to a short peptide that specifically binds to
acetylcholine receptors of neuronal cells [67]. Another oligoarginine-based polyplex,
loaded with VEGF-targeting siRNA, has been conjugated to a hydrophobic lipid,
cholesterol chloroformate, and locally administered to tumor-bearing mice, which
resulted in tumor regression [68]. Polylysine-based siRNA polyplex has been covalently
conjugated to a pH responsive endosomolytic peptide that facilitated endosomal escape
of siRNA into the cytosol for siRNA efficacy [69,70].

12
CHAPTER 2: DEVELOPMENT AND CHARACTERIZATION OF K21-BASED
SIRNA POLYPLEX

2.1. Introduction
2.1.1. Background
SiRNA requires a delivery vehicle in order to be transported intracellularly for
RNAi. It has been susceptible to nuclease degradation, and it has not been readily taken
into cells due to its negatively charged phosphate backbone [71]. Many non-viral
methods have been established to overcome these barriers by using different types of
delivery vehicles. Cationic materials have been commonly used for siRNA delivery
because the positive charges have allowed the formation of a complex with the anionic
oligonucleotides (discussed in 1.2.2, 1.2.3 and 1.3.2). The complex has provided not only
a layer of protection from siRNA degradation, but it has also allowed the siRNA to be
delivered into the cells. In addition, once the siRNA delivery vehicle has entered the cell,
it has to localize in the cytosol and release siRNA to interact with RISC for RNAi [22].
These have been the primary design considerations for siRNA delivery.
Cationic and amphipathic CPPs have exhibited efficient intracellular
internalization and been used to form polyplexes for siRNA delivery [44,63,64,66].
However, these studies have not fully exploited the CPP carriers’ ability for cellular
uptake since siRNA neutralizes the basic amino acids of CPPs, and the loss of
cationic/amphipathic nature of the CPP have been shown to compromise its uptake
efficiency [72,73]. Therefore, a more effective way to use CPP for siRNA delivery has
13
been to conjugate it to a siRNA complex cargo. This design has permitted the
neutralization of siRNA in a complex and the opportunity to release the siRNA
intracellularly, while also allowing for the conjugated CPP carrier to efficiently deliver
the cargo into the cell. Thus, the focus of this chapter will regard the development of
siRNA as a cargo for intracellular delivery and efficacy.

2.1.2. Rationale
CPP-based polyplexes have been reported to achieve siRNA intracellular delivery
and RNAi activity [44,63,64,66]. These results have indicated CPPs’ ability to form a
stable polyplex, which can enter the cell and then be able to release siRNA in the cytosol.
Prior to investigating the capability of entering cells, the ability to form a reversible
polyplex has to be first appreciated, since this has also been an essential requisite for
siRNA delivery. A pH responsive modified oligolysine has been used to make polyplexes
and exhibited a strong gene silencing effect [69]. It has also shown that low molecular
weight PEI-based complex produced a better knockdown than high molecular weight
PEI-based complex [34]. Based on the insights of these previous reports, a low molecular
weight oligolysine peptide has been considered to form a neutralized polyplex with
siRNA. Without conversing about its intracellular delivery efficiency, the focus of this
chapter is to design and develop a chemically modified low molecular weight 21mer
oligo-L-lysine (K21)-based siRNA polyplex and evaluate its stability and capacity to
release siRNA.
14
K21 has been selected to form a polyplex with siRNA based on its following
advantages. First, siRNA duplexes usually has contained 21 base pairs in length [74].
K21 sequence has been chosen since the number of charges would make a suitable
pairing with the siRNA backbone. The formed polyplex should permit strong electrostatic
interactions between the two moieties and neutralize anionic siRNA. Second, K21 has
included ε-amino groups of the lysyl residues, which can be chemically modified to
conjugate the polyplex to functional moieties. Third, K21 is biodegradable [69] and thus
the potential digestion of K21 by cytosolic lysine specific proteases [75,76] could allow
siRNA to be released from the polyplex. Therefore, a unique siRNA polyplex has been
developed using a chemically modified K21 peptide, and the properties of this polyplex
are evaluated in this chapter. The success of this design can subsequently serve as a
unique platform to conjugate functional moieties that can enhance siRNA delivery and
efficacy.

15
2.2. Experiments
2.2.1. Quantitative Measurement of Amines in 21mer Oligo-L-lysine
21mer oligo-L-lysine (K21, KKKKKKKKKKKKKKKKKKKKK) was
synthesized by Genemed Synthesis, Inc. (San Antonio, TX). 40 mg of K21 was dissolved
in phosphate buffer saline (PBS) (pH 7.4), and 4 mg aliquots of the peptide were stored at
–80ºC. 2,4,6-Trinitrobenzene sulfonic acid (TNBSA) and N
α
-acetyl-L-lysine methyl ester
HCl (Ac-K) (Sigma-Aldrich, St. Louis, MO) were used to quantify the amino groups of
K21 [77]. 1 mg of Ac-K was dissolved in 1 ml of 0.1 M sodium bicarbonate buffer (pH
8.5) and then 0.5 ml of 4, 8, 12 and 16 μg/ml Ac-K were prepared as standards for ε-
amine groups. K21 was diluted in 0.1 M sodium bicarbonate buffer to a final volume of
0.5 ml (concentration 10 μg/ml). 0.25 ml of 0.01% TNBSA solution (prepared in 0.1 M
sodium bicarbonate buffer) was added to each 0.5 ml solution and vortexed. The samples
were sealed with parafilm and then incubated at 37ºC for 2 h. Thereafter, 0.25 ml of 10%
SDS and 0.125 ml of 1 N HCl were added to each sample. The samples were vortexed
and evaluated at 335 nm using a UV spectrophotometer (Shimadzu, Columbia, MD).

2.2.2. Production, Purification and Quantification of N-succinimidyl-3-(2-pyridyldithio)
propionate-modified K21
K21 (100 μl of 40 mg/ml in PBS) was reacted with 110 μl of N-succinimidyl-3-
(2-pyridyldithio) propionate (SPDP, Pierce, Rockford, IL) (10 mg/ml
dimethylformamide, DMF) for 30 min at room temperature and then the N-succinimidyl-
3-(2-pyridyldithio) propionate-modified (PDP) peptide product (K21-PDP) was purified
16
using Sephadex G-25 (GE Healthcare, Uppsala, Sweden) size exclusion chromatography
with PBS as the mobile phase (1 x 25 cm column dimensions). Prior to reaction, the
column was washed with 50 ml PBS (pH 7.4). A micro fractionator (Gilson, Middleton,
WI) collected 25 1 ml fractions (22 drops/fraction), and each sample was detected for
absorbance at a wavelength of 220 nm. Samples containing K21-PDP were combined and
filtered through a 0.22-μm filter (VWR, Radnor, PA) to sterilize the peptide solution.
Aliquots (10 and 20 μl) of sterilized K21-PDP were collected to determine the
degree of PDP modification on K21-PDP. The K21-PDP aliquots were diluted in 1 ml of
0.1 M sodium bicarbonate buffer, and their amine concentrations were determined using
the TNBSA assay as previously described (2.2.1). Meanwhile, a PDP assay was
performed where 10 and 20 μl of K21-PDP aliquots first added to 970 and 980 μl PBS,
respectively, and the samples were detected for absorbance at a wavelength of 343 nm.
Then, 10 μl of 1 M dithiothreitol (DTT, Sigma-Aldrich) was added to the K21-PDP
samples, and the reaction proceeded for 30 min at room temperature. After the reaction,
the reduced samples were read again at a wavelength of 343 nm.

2.2.3. Polyplex Formation between K21-PDP and siGFP
PBS was treated with 0.1% v/v diethylpyrocarbonate (DEPC, Sigma-Aldrich) and
then autoclaved. The sterilized solution, PBS+DEPC, (1 ml) was used to dissolve 40
nmole of green fluorescent protein (GFP)-targeting siRNA (siGFP, sense: 5’-
CAAGCUGACCCUGAAGUUdTdT-3’; antisense: 5’-GAACUUCAGGGUCAGC
UUdTdT-3’; Dharmacon, Lafayette, CO) [78] in a sterile environment. SiGFP was mixed
17
at 4ºC for 2 h, and then the concentration was confirmed by measuring absorbance at a
wavelength of 260 nm.
Increasing amounts of K21-PDP (20.1, 100.6, 201.2, 402.4, 804.8 and 1000.6
nmole amine groups) were mixed with siGFP (2.4 nmole siRNA, 100.6 nmole phosphate
groups) to form polyplexes at different N/P ratios (0.2:1, 1:1, 2:1, 4:1, 8:1 and 10:1 N/P).
These 1 ml polyplex solutions (prepared in PBS) were vortexed and then incubated at
room temperature for 30 min. Prior to preparing the polyplexes, a 2% agarose gel was
prepared by heating 1.6 g of agarose (Invitrogen, Carlsbad, CA) in 80 ml tris-acetate-
EDTA (TAE) buffer for 45 sec (divided into intervals of 30, 10 and 5 sec heating where
the agarose solution is swirled between each heating) using a microwave (Panasonic,
Seacaucus, NJ). After the heated agarose gel cooled to a warm temperature, 8 μl of
ethidium bromide (Sigma-Aldrich) was added. The 2% gel was poured onto a 12-lane
frame and left to polymerize for 1 h. After the gel solidified, it was transferred to an
electrophoresis unit (VWR) and covered with a mixture of 200 ml TAE buffer and 200
ml double deionized water (DDH
2
O). Polyplex samples (30 μl) (along with controls of
siRNA, K21-PDP and PBS only) were mixed with 6 μl of 6x loading buffer (Thermo
Scientific – Fermentas, Glen Burnie, MD) and then loaded on the submerged 2% agarose
gel. The gel ran for 30 min at 125 V and then observed using ChemiDoc XRS (Bio-Rad,
Hercules, CA).
18
2.2.4. Measurement of Amine and Phosphate Interaction for Polyplex
The number of K21-PDP amines interacting with siGFP phosphates was
determined using TNBSA assay. 2:1 N/P polyplex (2.4 μM siRNA) was made as
previously described (2.2.3), while K21-PDP and siGFP only were also prepared at equal
concentrations with 2:1 N/P polyplex. Samples (0.25 ml) of 2:1 N/P polyplex, K21-PDP
and siGFP were used for TNBSA assay as previously described (2.2.1).

2.2.5. Stability of 2:1 N/P Polyplex
The stability of 2:1 N/P polyplex in room temperature was assessed using TNBSA
assay. The setup of the assay was exactly the same as 2.2.4, except that TNBSA assay for
2:1 N/P polyplex was performed four times at 12, 24, 48 and 72 hours after the start of
polyplex formation. Polyplex (2:1 N/P), K21-PDP and siGFP remained at room
temperature before being assayed.

2.2.6. SiRNA Release from Polyplex
2.2.6.a. Trypsin Digestion
2.4 μM polyplexes (1 ml) were prepared at 1:1, 2:1, 3:1 and 4:1 N/P as previously
described (2.2.3). Aliquots of the polyplexes, each containing equal amounts of K21-PDP
(90, 45, 30 and 22.5 μl for 1:1, 2:1, 3:1 and 4:1 N/P, respectively) were then mixed with
10 μl of 0.97 U/μl trypsin, and PBS was also added so that the final volume was 100 μl.
The trypsin-treated samples were incubated at 37ºC for 15 min. Afterwards, 30 μl
19
aliquots of treated and untreated polyplexes were mixed with 6 μl of 6x loading buffer
and loaded onto a 2% agarose gel under the settings as previously described (2.2.3).

2.2.6.b. Polyplex Dilution
2:1 N/P polyplexes (1 ml) was prepared as previously described (2.2.3) and then
diluted in PBS 1:25, 1:10, 1:5, 1:4 and 1:2 to a final volume of 100 μl. Then 30 μl
aliquots of the diluted polyplexes, along with undiluted polyplex and siRNA only, were
mixed with 6 μl of 6x loading buffer and loaded onto a 2% agarose gel under the settings
as previously described (2.2.3).

20
2.3. Results
2.3.1. Quantitative Measurement of K21 Amines
The product of TNBSA and Ac-K standards (4, 8, 12 and 16 μg/ml) produced a
linear curve (y = 0.0027x + 0.0123, R
2
=0.9957, Fig. 2) used to calculate the K21 amine
concentration. Absorbance at a wavelength of 335 nm for 10 μg/ml K21 was measured as
0.114, which was equal to 37.67 μM amino groups (Calc. 1). Therefore, the concentration
of 10 μg/ml K21 was determined to be 1.712 μM using the TNBSA assay. On the other
hand, the concentration of K21 was calculated to be 1.888 μM based on weight
(molecular weight, including counterion trifluoroacetic acid (TFA): 5104 g/mole; peptide
purity 96.36%). Therefore, the TNBSA assay produced a 9.3% error for quantifying the
K21 amines, and this assay was confirmed as a suitable method to quantify amine
concentrations (Calc. 1).

Figure 2. Standard curve of ε-amines for quantification of K21 amines using TNBSA
Assay. Ac-K (4, 8, 12 and 16 μg/ml) and TNBSA assay were used to produce a standard
curve to quantify K21 amines.

21
y = 0.0027x + 0.0123 (Standard curve from Fig. 2)
Absorbance at 335 nm for 10 μg/ml K21: 0.114
0.114 = 0.0027x + 0.0123
x = 37.67 μM NH
2

37.67 μM NH
2
x (1 K21 / 22 NH
2
) = 1.712 μM K21
[K21] = 1.712 μM K21 (based on amino concentration)
[K21] = 1.888 μM K21 (based on weight)

| 1.712 – 1.888 |
% Difference = ---------------------- x 100% = 9.3%
1.888
Calculation 1. Comparison of K21 concentration based on weight and amino groups, as
determined by TNBSA assay.

2.3.2. Production, Purification and Quantification of K21-PDP
K21-PDP was detected in the first peak (fractions 9-12), while the latter broad
peak corresponded to excess, unreacted SPDP (Fig. 3.A). TNBSA and PDP assays were
conducted to confirm the presence of K21-PDP. TNBSA assay indicated that the
concentration of amines was 1.45 mM (Fig. 3.B, Calc. 2), while PDP assay determined
that the concentration of PDP was 440 μM (Table 1). Thus, there was 1 PDP
modification for every 4.15 amine groups, which resulted in an average of 4.25 PDP
modifications per K21-PDP peptide (Calc. 3).
22
A.
B.
Figure 3. A. Purification of K21-PDP. Elution profile of K21-PDP using Sephadex G-25
size exclusion chromatography with PBS (pH 7.4) as the mobile phase. B. Standard curve
of ε-amines for quantification of K21-PDP amines using TNBSA Assay. Ac-K (4, 8, 12
and 16 μg/ml) and TNBSA assay were used to produce a standard curve to quantify K21-
PDP amines.

y = 0.003x - 0.0133
Absorbance at 335 nm for 10 μl K21-PDP: 0.026
Absorbance at 335 nm for 20 μl K21-PDP: 0.057
Calculation for K21-PDP [NH
2
] (x dilution factor)
10 μl K21-PDP: 0.026 = 0.003x - 0.0133 x = 18.61 μM NH
2
1.86 mM NH
2
20 μl K21-PDP: 0.057 = 0.003x - 0.0133 x = 35.83 μM NH
2
1.79 mM NH
2

Average K21-PDP [NH
2
]: 1.83 mM
Calculation 2. Determination of K21-PDP amine concentration using TNBSA assay.

23

K21-PDP (μl) Abs. at 343 nm (+) 10 mM DTT Adj. Abs. [PDP] (μM)
0 0 0.007 N/A N/A
10 0 0.044 0.037 445.6
20 0.001 0.078 0.07 433.2
Average 439.4

Table 1. PDP Concentration of K21-PDP using PDP Assay.

K21-PDP (μl) [NH
2
]/[PDP] NH
2
/K21-PDP PDP/K21-PDP
10 1.86 mM / 445.6 μM = 4.18 17.8 4.3
20 1.79 mM / 433.2 μM = 4.14 17.7 4.3
Average: 4.16 17.75 4.3
Calculation 3. Calculation for degree of PDP modification per K21-PDP peptide.

2.3.3. Polyplex Formation for K21-PDP and siRNA
Polyplexes began to form at 2:1 N/P ratio based on the loss of signal observed
from the 2% agarose gel (Fig. 4). Polyplexes at 0.2:1 and 1:1 N/P did not contain enough
K21-PDP to form a polyplex, since free siRNA was readily noticeable (Fig. 4, Lanes 1,
2). On the other hand, increasing N/P ratios above 2:1 resulted in positively charged
polyplexes, as evidenced by detectable faint signals above the loading wells (Fig. 4,
Lanes 4-6). Therefore, conditions for 2:1 N/P polyplex formation were selected, since it
was the minimal amount of K21-PDP needed to form a neutralized polyplex (Fig. 4, Lane
3).

24

Figure 4. Polyplex formation at different N/P ratios between K21-PDP and siGFP.
Different N/P ratios of K21-PDP and siGFP were mixed together at room temperature for
30 min to form polyplexes. Each N/P polyplex was then loaded onto 2% agarose gel
(containing ethidium bromide) and run for 30 min at 125 V (Reproduced from [79] with
permission from American Chemical Society, Copyright 2011).

2.3.4. Measurement of Amine and Phosphate Interaction for Polyplex
The N/P interaction between K21-PDP amines and siGFP phosphates was
calculated as ~1:1 for 2:1 N/P polyplex (Fig. 5). SiGFP alone did not produce a signal
because it did not react with TNBSA. Thus, the 50% loss of absorbance for polyplex
prepared at 2:1 N/P ratio compared with K21-PDP alone corresponded with the K21-PDP
amines that were electrostatically interacting with the siRNA phosphates.

25

Figure 5. Determination of N/P interaction at 2:1 N/P polyplex. Polyplex (2:1 N/P) was
reacted with TNBSA, and the samples were measured for absorbance at a wavelength of
335 nm. Error bars indicate standard deviation (n = 3). Asterisk (*) represents p < 0.05
based on analysis of variance and the Bonferroni test (Reproduced from [79] with
permission from American Chemical Society, Copyright 2011).

2.3.5. Stability of 2:1 N/P Polyplex
The N/P interaction between K21-PDP amines and siGFP phosphates was ~1:1
for 2:1 N/P polyplex at 12, 24, 48 and 72 h post-polyplex formation (Fig. 6). There was
no significant difference in the N/P interaction at any detected time point. The lack of
changes for N/P interaction indicated that the 2:1 N/P polyplex remained stable when
stored at room temperature for up to 72 h.

26

Figure 6. Polyplex stability based on N/P interaction. The number of K21-PDP amine
groups electrostatically interacting with siRNA phosphate groups was measured by
taking the difference of absorbance values (at a wavelength of 335 nm) for K21-PDP and
2:1 N/P polyplex at 12, 24, 48 and 72 post-formation using TNBSA assay.

2.3.6. SiRNA Release from Polyplex
2.3.6.a. Trypsin Digestion
2.3.6.b. Polyplex Dilution
Two methods were demonstrated to release the siRNA from the polyplexes:
trypsin treatment and polyplex dilution. Trypsin treatment of different N/P polyplexes
resulted in the appearance of unbound siRNA in the agarose gel (Fig. 7.A). Therefore,
degradation of the K21-PDP peptide in the polyplexes (0.2-4:1 N/P) could release the
siRNA. Furthermore, greater dilution of 2:1 N/P polyplex in PBS led to lower stability of
the polyplex and thus increased release of siRNA (Fig. 7.B).

27
A.
B.

Figure 7. SiRNA release from polyplexes. (A) Trypsin treatment of different N/P
polyplexes for 15 min at 37°C. (B) Different dilutions of 2:1 N/P polyplex in PBS.
Samples were then loaded onto a 2% agarose gel and run for 30 min, 125 V (Reproduced
from [79] with permission from American Chemical Society, Copyright 2011).

28
2.4. Discussion
Quantification of K21 was necessary to confirm the amount of peptide provided
by the vendor Genemed. K21 was prepared with the counterion TFA that contributes to
its amount based on weight. After dissolving the peptide with sterile PBS (pH 7.4) in a
sterile environment, TNBSA assay was used to quantify the amine groups of K21 [77].
K21 quantification using TNBSA assay was performed to validate the method and verify
the amount of peptide received (Fig. 2, Calc. 1). After determining ~10% difference
between the K21 amount based on weight and amine groups (Calc. 1), TNBSA assay was
deemed sufficient to use for K21 quantification.
It was important to control the degree of PDP modification on K21. Too much
modification would result in loss of cationic charges on K21 and inhibition of polyplex
formation, whereas too little would minimize the number of conjugation sites for
functional moieties. A suitable reaction parameter was determined to produce ~4 PDP
groups per K21-PDP (Calc. 3). This value was calculated by performing TNBSA (Fig. 3,
Calc. 2) [77] and PDP assay (Table 1) [48]. It should be noted that Ac-K was freshly
prepared each time when producing a standard curve for TNBSA assay (Fig. 3.B). K21-
PDP was then mixed with siGFP at different N/P ratios to demonstrate that the modified
K21 could still form a polyplex (Fig. 4). Although oligolysine had been previously used
for siRNA polyplex formation [69], a peptide with the size of K21 had not been reported
before for this application. A mixture of 2:1 N/P ratio showed a fully neutralized
polyplex because no free siRNA was detected (Fig. 4, Lane 3). The neutralization of
siRNA was further assured by calculating a ~1:1 interaction between K21-PDP and
29
siRNA for polyplexes when prepared at 2:1 N/P ratio (Fig. 5). Interestingly, increasing
the N/P ratio corresponded with increased positive charges of the polyplexes since the
band migrated above the loading well (Fig. 5, Lanes 4-6). Polyplexes with higher N/P
ratios were previously shown to exhibit cellular uptake and gene silencing due to excess
cationic charges [44,63,64]. Thus, K21-PDP was capable of forming a polyplex with
siRNA, and polyplex formed at 2:1 N/P ratio (2:1 N/P polyplex) was selected as the
platform for conjugation of functional moieties.
Another important consideration for siRNA delivery is that siRNA can be freed
from the polyplex. Even if a delivery vehicle is able to carry siRNA to the cytosol, if it is
unable to release to the siRNA intracellularly, siRNA cannot interact with the RISC and
thus, RNAi cannot occur. Two methods were proposed to demonstrate siRNA release
from the polyplex. First, lysine is trypsin-sensitive, so trypsin can be used to digest K21-
PDP peptide and release siRNA. This was evidenced by the appearance of a single
siRNA band on the agarose gel after treating 0.2:1 to 4:1 N/P polyplexes with trypsin
(Fig. 7.A). A similar occurrence should happen inside the cell, where intracellular lysine-
specific proteases could degrade K21-PDP and release siRNA from the polyplex [75,76].
On the other hand, polyplex dilution, through weakening the electrostatic interactions
between the cationic amino acids and anionic phosphates, allows the dissociation of
siRNA from K21-PDP. Results from Fig. 7.B clearly demonstrated the siRNA release
from the polyplex in a dilution dependent manner. During intracellular delivery process
through a vesicle-based mechanism, internalized polyplex usually escapes from the
endosome (a smaller volume) to cytosol (a larger volume). This dramatic change in
30
volume would drive a dilution force that would cause the breakup of siRNA from the
polyplex in the cytosol. It is possible that siRNA can be separated from the polyplex prior
to entering the cells due to dilution effects. However, this can be resolved by conjugating
functional moieties that can provide steric stability [80]. This approach is reported in
Chapter 3.
31
2.5. Summary
In Chapter 2, a novel K21-PDP-based siRNA polyplex was developed as a
platform for conjugation of functional moieties. The 2:1 N/P polyplex exhibited a ~1:1
N/P interaction between K21-PDP and siRNA and remained intact for 72 h at room
temperature. In addition, the polyplex also showed the ability to release the siRNA cargo
after trypsin digestion and polyplex dilution, both of which represent part of cytosolic-
like environment. Therefore, the polyplex is a promising siRNA delivery platform
because it can neutralize the siRNA, provide conjugation sites for functional ligands and
allow the release of siRNA under certain conditions.

32
CHAPTER 3: COMPARISON OF CATIONIC AND AMPHIPATHIC CELL
PENETRATING PEPTIDES FOR SIRNA DELIVERY

3.1. Introduction
3.1.1. Background
Not only does siRNA need to be taken into the targeted cell, but it also needs to
reach the cytosol to interact with RISC and induce RNAi. Thus, various functional
moieties have been proposed and used for siRNA delivery. They have been either
conjugated directly to siRNA [56,58,81,82] or to a complex carrying the siRNA as cargo
[60,69,79]. Functional moieties have been applied to achieve different purposes for
siRNA delivery, such as facilitating its cellular uptake [71,79], permitting its cytosolic
localization via endosomal escape [83,84], delivering it to the desired site of action
[67,80,84] and extending its plasma half-life [69,82,84]. One of the widely studied
functional moieties used for siRNA delivery is CPPs due to their ability to enhance
cellular internalization of conjugated cargos [22,46,54,55]. The focus of this chapter
regards the use and comparison of two types of CPPs for siRNA intracellular delivery
and efficacy.

3.1.2. Rationale
CPPs have been utilized for siRNA delivery by conjugating or forming
polyplexes with siRNA. However, it has been shown that the loss of positive charges on
basic amino acids of CPPs through electrostatic interactions inhibit the amount of cellular
33
uptake [72]. As a result, studies comparing CPPs for siRNA delivery may not provide
accurate evaluation of the peptides’ delivery properties, since the internalization
capabilities of CPPs are partially compromised due to the neutralization effects. In order
to resolve this problem, the neutralized K21-PDP-based siRNA polyplex developed in
Chapter 2 can serve as a suitable platform for conjugation of different CPPs without
concerns about loss of cationic charges. This design would allow the direct
characterization and comparison of the delivery capability for different CPPs (Fig. 8).
In this chapter, two different types of CPP, a cationic oligoarginine (R6) and an
amphipathic MAP, have been each conjugated to the K21-PDP polyplex to form R6-
polyplex and MAP-polyplex, respectively. Cationic oligoarginine is considered because
its ability for membrane transduction during internalization allows the majority of the
peptide to be localized in the cytosol where RNAi initiates [42]. On the other hand,
amphipathic MAP has exhibited significantly greater levels of cellular uptake than other
CPPs, including oligoarginine, but subcellular fractionation studies have suggested that
MAP localizes predominantly in the vesicular fraction and not noticeable in the cytosol
region [85]. Therefore, in this chapter, two different types of CPP carriers, each
possessing a distinct internalization property, will be compared for their ability to deliver
siRNA and induce gene silencing effects. The results from these studies will indicate
which feature is most important for siRNA delivery and efficacy.
34

Figure 8. Development of R6-polyplex and MAP-polyplex for comparison of cationic
and amphipathic CPPs for siRNA delivery and efficacy. K21 was reacted with SPDP to
form K21-PDP. The chemically modified product was purified using size exclusion
chromatography, and the degree of modification was calculated as an average of 4 PDP
per K21-PDP. K21-PDP was mixed with siRNA at 2:1 N/P ratio for 30 min at room
temperature to form a polyplex, and then R6 or MAP was added to the polyplex solution
to make R6-polyplex and MAP-polyplex, respectively. The reaction proceeded overnight,
and the carrier CPP was conjugated to the polyplex by a reducible disulfide bond. The
conjugate products were then ready for studies (Reproduced from [79] with permission
from American Chemical Society, Copyright 2011).
35
3.2. Experiments
3.2.1. Selection of Oligoarginine for Conjugation
N
α
-acetyl-tetra-L-arginine (Ac-R4, Ac-RRRR), N
α
-acetyl-hexa-L-arginine (Ac-
R6, Ac-RRRRRR), N
α
-acetyl-octa-L-arginine (Ac-R8, Ac-RRRRRRRR) (Genemed
Synthesis) (40 mg each) were dissolved in 1 ml sterile PBS, and 125 μl aliquots were
stored at -80ºC. The concentrations of Ac-R4, Ac-R6 and Ac-R8 were calculated based
on their guanidine groups using 9,10 phenanthrenequinone (PQ, Sigma-Aldrich) [86]. PQ
assay samples were prepared by first diluting Ac-R4, Ac-R6 and Ac-R8 (25 μl) 40-fold in
1 ml PBS and then further (20 μl) diluting another 50-fold in 1 ml PBS. N
α
-acetyl-L-
arginine (Ac-R, Sigma-Aldrich) (0.5 ml of 5, 8, 10, 12, 16 and 20 μg/ml) was used to
produce a standard curve to quantify the guanidine groups. The 2000-fold diluted
samples and standards (100 μl) were mixed with 300 μl of 500 μM PQ (1 mg in 10 ml
ethanol) and 50 μl of 2 N NaOH. After incubating for 1 h at 60ºC, 450 μl 1.2 N HCl was
added to stop the reaction, and the fluorescence was measured at excitation 312 nm and
emission 395 nm using a fluorescence spectrophotometer (Hitachi, Tokyo, Japan).
2:1 N/P polyplexes (2.5 μM siRNA) were prepared using K21 and siGFP. SiGFP
(25 μl of 25 μM) was first mixed with 165 μl of PBS, and then K21 (10 μl of 212 μM)
was added. After 30 min incubation at room temperature for polyplex formation, Ac-R4,
Ac-R6 and Ac-R8 were added to the polyplexes at different guanidine-to-amines (G/N)
ratios. Each N
α
-acetylated-oligoarginine peptide was first diluted in 2 ml PBS so that the
final concentration of guanidine was 2.32 mM. Thereafter, 6, 20 and 60 μl of each N
α
-
acetylated-oligoarginine, along with 244, 230 and 190 μl PBS, respectively, were added
36
to 2:1 N/P polyplex so that the final volume was 0.5 ml. The oligoarginine and polyplex
mixtures were stored for 48 h at room temperature, and then 100 μl aliquots were used to
perform TNBSA assay (2.2.1).

3.2.2. Synthesis of R6-polyplex and MAP-polyplex
The synthesis of R6-polyplex and MAP-polyplex were conducted in a sterile
environment. R6-polyplex and MAP-polyplex were prepared with different siRNAs:
siGFP, negative control siRNA (“siNC,” Ambion, Foster City, CA) and fluorophore-
labeled (DY547) negative control siRNA (“siDY547,” Dharmacon). K21-PDP (120 μl of
1.83 mM) was mixed with siGFP (62.5 μl of 40 nM), siNC (62.5 μl of 40 nM) and
siDY547 (35.7 μl of 70 nM) and PBS to form 2:1 N/P polyplex as previously described
(2.2.3). The volume of the polyplex was adjusted so that after the addition of the carrier,
the final volume of the conjugate was 1 ml. The carriers, L-cysteinyl model amphipathic
peptide (CMAP, CKLALKLALKALKAALKLA) and L-cysteinyl-hexa-L-arginine
(CR6, CRRRRRR) (Genemed Synthesis), were synthesized with a cysteine group at the
N-terminus, so that the thiol group could react with K21-PDP for conjugation. After 30
min incubation for polyplex formation, each peptide (1 mg) was dissolved in 1 ml of 0.2
M sodium acetate buffer (pH 4.5) and added to the polyplex at a 1:1 CPP/PDP ratio (128
and 156 μl for 1 mg/ml CMAP and CR6, respectively) in a sterile setting. The reaction
proceeded overnight at room temperature, and then the conjugates, R6-polyplex and
MAP-polyplex were ready for evaluation (the final stock concentrations of K21-PDP,
siRNA and carrier CPP were 12.5, 2.5 and 60 μM, respectively). The polyplex
37
nomenclature will include the carrier name followed by the kind of siRNA used in the
polyplex (i.e.: MAP-polyplex with siNC will be designated as “MAP-siNC-polyplex”). In
addition, any delivery vehicle containing siRNA will be indicated by the siRNA
concentration (i.e.: MAP-siNC-polyplex containing 50 nM siNC will be referred to as
“50 nM MAP-siNC-polyplex”).
The reaction efficiency between 2:1 N/P polyplex and carrier peptide was
evaluated by measuring the absorbance for release of pyridine-2-thione at a wavelength
of 343 nm after the disulfide bond formation. The absorbance of 2:1 N/P polyplex was
first measured at a wavelength of 343 nm before the addition of a carrier. After the
overnight reaction, the carrier-polyplex conjugate was detected again for absorbance at a
wavelength of 343 nm. The difference of absorbance between before and after carrier
addition indicated the reaction efficiency.

3.2.3. Effect of Carrier Conjugation on Polyplex Stability
2.5 μM R6-siGFP-polyplex and MAP-siGFP-polyplex were diluted 10-fold in
PBS (100 μl in 1 ml). Aliquots of each conjugate (30 μl) were mixed with 6 μl of 6x
loading buffer and subsequently examined on a 2% agarose gel as previously described
(2.2.3).

3.2.4. Cellular Uptake
Huh7.5 human hepatoma cells, stably transfected to express GFP-light chain 3
(LC3) (GFP-LC3), were used to evaluate R6-polyplex and MAP-polyplex. Stable
38
expression of GFP-LC3 was achieved by pGFP-LC3 transfection followed by G418
(Invitrogen, Carlsbad, CA) selection [87]. Cells were maintained at 37°C, 5% CO
2
in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 1% nonessential amino acids, and 2 mM L-glutamine (“Huh7.5 growth
medium”). The day before siRNA transfection, Huh7.5 cells were seeded in 6-well plates
at a density of 0.2x10
6
cells/ml in 2 ml of Huh7.5 growth medium.
SiDY547 was used to evaluate the siRNA cellular uptake of the developed
delivery vehicles. Lipofectamine 2000 (“Lipo2000”) (Invitrogen), a cationic lipid-based
transfection reagent, was used as a control. Lipo2000/siRNA lipoplexes were prepared
according to manufacturer’s instructions. SiDY547 was diluted in OptiMEM (Invitrogen)
to a concentration four-times (200 nM) the final dosing concentration (50 nM). Lipo2000
was also diluted in OptiMEM to a concentration corresponding to 20 pmole siRNA / 1 μl
Lipo2000. Equal volumes of Lipo2000 and siDY547 solutions were mixed together to
form Lipo2000/siDY547 lipoplexes for 20 min at room temperature. During the
incubation, Huh7.5 growth medium was aspirated, and 0.5 ml of OptiMEM was added to
each well. Thereafter, 0.5 ml Lipo2000/siDY547 lipoplex was added to the wells.
SiDY547-polyplex, R6-siDY547-polyplex and MAP-siDY547-polyplex (2.5 μM,
prepared as previously described 3.2.2) were diluted in OptiMEM to 50 nM (63 μl in 3.15
ml OptiMEM), and then 1 ml of each dosing solution was added per well. Additional
control dosing solutions (50 nM siDY547, 1.2 μM R6 and 1.2 μM MAP) were prepared
by directly diluting the siRNA or peptide in OptiMEM and then adding 1 ml per well.
39
Cells were treated for 1 and 6 h at 37°C, 5% CO
2
. At the designated time points,
treatment medium was aspirated, and cells were washed with 1 ml of 4°C PBS. Trypsin-
EDTA (300 μl, Gibco) was added per well and incubated at 37°C, 5% CO
2
for 5 min.
PBS (700μl, 4°C) was added to each well, and the cells were collected and transferred to
1.5 ml microcentrifuge tubes. Cell pellets were obtained by centrifuging at 1000 g, 4°C
for 7 min. The supernatant was collected for evaluation of trypsin removable supernatant
and stored at 4°C. Cell pellets were resuspended in 4°C PBS by gentle vortexing and then
cells were pelleted again as previously described. Cell extraction buffer (Invitrogen),
supplemented with 10x protease inhibitor cocktail and 0.3 M phenylmethylsulfonyl
fluoride (Sigma-Aldrich), was prepared according to manufacturer’s instructions (Ratio
of 300:1.5:1, respectively). The supernatant was discarded, and then 75 μl of
supplemented cell extraction buffer was added to each tube. After vigorous vortexing, the
tubes were stored on ice for 30 min, while vortexing again every 10 min. After incubation
on ice, the cell lysates were centrifuged at 13,000 g, 4°C for 10 min. The cell lysates (65
μl/well) were transferred, along with the trypsin removable supernatant (250 μl/well), to a
96-well fluorescence flat-bottom microplate (BD Biosciences, Sparks, MD). SiDY547
fluorescence was detected using a plate reader (Molecular Devices, Sunnyvale, CA). The
microplate was read from the “bottom” setting and the excitation and emission were set at
544 nm and 590 nm, respectively.
Thereafter, cell lysates were quantified for protein content using bicinchoninic
acid (BCA) assay (Pierce). Protein concentration standards (0.25, 0.5, 1 and 2 mg/ml
bovine serum albumin (Pierce)) were prepared in PBS. Protein standards and cell lysates
40
(25 μl/well) were transferred to a 96-well plate (Greiner Bio-one, Monroe, NC). BCA
reagent solution was prepared at a ratio (50 parts Component A mixed with 1 part
Component B) provided by manufacturer’s instruction, and 200 μl of the BCA reagent
solution was added per well. The 96-well plate was sealed in a plastic bag and incubated
for 30 min at 37°C. After incubation, the plate was scanned for absorbance using a plate
reader (Tecan, Männedorf, Switzerland) at a wavelength of 570 nm. SiDY547 uptake
fluorescence was defined as relative fluorescence units (RFU) per mg of cell protein for
cell lysates and RFU/ml for trypsin removable supernatant.

3.2.5. GFP Silencing
Huh7.5 cells, stably transfected to express GFP-LC3 fusion protein, were used to
evaluate the gene silencing effect of R6-polyplex and MAP-polyplex. Each was loaded
with siGFP to knockdown the GFP fusion protein and reduce intracellular fluorescence.
SiNC-loaded R6- and MAP-polyplexes were also prepared as negative controls, while
Lipo2000 was used as a positive control. Huh7.5 cell seeding and treatment dosing
solutions (50 nM) were prepared as previously described (3.2.4). Cells were treated for 6
h at 37°C, 5% CO
2
, and then replaced with 1 ml Huh7.5 growth medium for an additional
42 h at 37°C, 5% CO
2
. Cell lysates were collected as previously described (3.2.4), and 65
μl of each sample was transferred to a 96-well microplate. Intracellular GFP fluorescence
was measured at wavelengths of 485 nm and 518 nm for excitation and emission,
respectively, using a plate reader (Tecan). BCA assay was performed to determine the
protein content of each sample. Intracellular GFP fluorescence was defined as RFU per
41
mg of cell protein for cell lysates. SiGFP-treated cells were normalized to siNC-treated
counterparts when applicable, whereas other control treatments (Lipo2000, R6 and MAP)
were presented relative to non-treated cells.

3.2.6. Cell Viability
Cytotoxicity assay was performed similarly to the GFP silencing assay (3.2.5).
However, after the 48 h assay cycle (6 h treatment followed by 42 h incubation), instead
of collecting the cell lysates, cell viability was evaluated. 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) solution was prepared at 2
mg/ml in Huh7.5 growth medium. MTT solution was then filtered through a 0.22-μm
filter, and 0.25 ml was added to each well. After a 4 h incubation at 37°C, 5% CO
2
, the
medium was removed, and 1 ml of 1:24 1 N HCl:isopropanol solution was added per
well. The plates were thoroughly mixed to dissolve the cells, and cell viability was
measured using a plate reader (Tecan) at a wavelength of 570 nm.

3.2.7. Lack of K21-PDP
Additional polyplexes consisting of just the carrier CPPs and siRNA were
prepared to evaluate the importance of K21-PDP. CMAP and CR6 were each dissolved
as previously described (3.2.2) and mixed with siRNA (siGFP and siNC) in PBS to form
“MAP/siRNA” and “R6/siRNA” polyplexes (the final stock concentrations of siRNA and
carrier CPPs were 2.5 and 60 μM, respectively; preparation was the same as MAP-/R6-
polyplexes, except that no K21-PDP was added (3.2.2)). MAP/siGFP and R6/siGFP
42
polyplexes were diluted to 50 nM in OptiMEM and compared with MAP- and R6-GFP-
polyplexes (50 nM) in gene silencing assays to reduce GFP fluorescence in Huh7.5 cells,
stably transfected to express GFP-LC3. SiNC-loaded counterparts were used as negative
controls. The gene silencing assays were conducted as previously described (3.2.5).

43
3.3. Results
3.3.1. Selection of Oligoarginine for Conjugation
Displacement of K21 was determined using TNBSA assay due to an increased
absorbance signal detected at 335 nm with each unbound lysyl-amine group. The α-
amines on the oligoarginine peptides were N
α
-acetylated so that they would not react with
TNBSA. When incubating 2:1 N/P polyplex with Ac-R4, no significant displacement was
observed between 0.3:1, 1:1 and 3:1 G/N (1, 0, -3% displacement, respectively, p>0.05)
or Ac-R6 (0.7, 5, 8%, respectively, p>0.05) (Fig. 9). However, Ac-R8 showed
significantly more displacement of the 2:1 N/P polyplex at 1:1 G/N (32%, p<0.01) and
3:1 G/N (38%, p<0.001) vs. 0.3:1 G/N (19%) (Fig. 9). Moreover, Ac-R8 significantly
displaced more K21 than Ac-R6 and Ac-R4 at 0.3:1, 1:1 and 3:1 G/N (p<0.001), whereas
Ac-R6 only exhibited significant differences compared to Ac-R4 at 3:1 G/N (p<0.05)
(Fig. 9). These results demonstrated that oligoarginine peptide length and G/N ratio had
significant effects on 2:1 N/P polyplex stability. Therefore, R6 was chosen to conjugate
to 2:1 N/P polyplex because it was the longest oligoarginine CPP with minimal polyplex
displacement (<10%).

44

Figure 9. Displacement of K21 by N
α
-acetylated oligoarginine (Ac-R4, Ac-R6 and Ac-
R8) at different G/N ratios in 2:1 N/P polyplex after 48 h (Reproduced from [79] with
permission from American Chemical Society, Copyright 2011).

3.3.2. Synthesis of R6-polyplex and MAP-polyplex
The pyridine-2-thione product released after the conjugation of the carrier peptide
and polyplex was detected for absorbance at a wavelength of 343 nm and used to
estimate the reaction efficiency. The change in absorbance before and after the addition
of the carrier peptide was 0.416 and 0.425 for R6-polyplex and MAP-polyplex,
respectively. Thus, the concentration of pyridine-2-thione was calculated as 51.4 and 52.5
μM using the Beer-Lambert law (A=εcl, ε
pyridine-2-thione
at 343 nm is 8080/M·cm) (Table
2). If the reaction proceeded at 100%, then 60 μM pyridine-2-thione should have been
detected in solution. However, the reaction efficiency for R6-polyplex and MAP-
polyplex was only 86% and 87.5%, respectively (Table 2).
45
Δ between before and [Pyridine-2-thione]
after carrier addition (μM) %Efficiency
R6-polyplex 0.416 51.4 86
MAP-polyplex 0.425 52.5 87.5
Table 2. Reaction efficiency for R6-polyplex and MAP-polyplex

3.3.3. Effect of Carrier Conjugation on Polyplex Stability
Conjugation of either CPP carrier, R6 or MAP, to 2:1 N/P polyplex prevented
polyplex dissociation after dilution. When diluting R6-polyplex and MAP-polyplex 10-
folds, neither exhibited siRNA being released (Fig. 10, Lanes 7-8), whereas 10-fold
diluted polyplex did (Fig. 10, Lane 6). Therefore, carrier conjugation stabilized the
polyplex structure and allowed for lower concentration treatments for siRNA
transfections.

Figure 10. Evaluation of dilution effects on R6-polyplex and MAP-polyplex. SiGFP,
siGFP-polyplex, R6-polyplex and MAP-polyplex were diluted 10-fold in PBS. The
samples were loaded onto 2% agarose gel and run for 30 min, 125 V (Reproduced from
[79] with permission from American Chemical Society, Copyright 2011).

46
3.3.4. Cellular Uptake
MAP-siDY547-polyplex (50 nM) had significantly greater siDY547 uptake (4-
and 3-folds) than 50 nM Lipo2000/siDY547 lipoplex at 1 and 6 h, respectively (Fig.
11.A). On the other hand, 50 nM siDY547, siDY547-polyplex and R6-siDY547-polyplex
exhibited minimal uptake at each time point (Fig. 11.A). The amount of siDY547
fluorescence detected in the trypsin removable supernatant corresponded with the
intracellular uptake. MAP-siDY547-polyplex exhibited more surface binding than
Lipo2000/siDY547 lipoplex and R6-polyplex at both time points (Fig. 11.B). Neither
CPP carrier alone demonstrated any fluorescence emission (at 590 nm) for the excitation
wavelength (of 544 nm).
47
A.
B.

Figure 11. (A) Cellular uptake and (B) extracellular surface binding of R6-polyplex and
MAP-polyplex in Huh7.5 cells, stably transfected to express GFP-LC3. Cells were
treated for 1 and 6 h in OptiMEM. After treatment, the (A) cell lysates and (B) trypsin
removable supernatant were collected. They were measured at Ex. 544 nm and Em. 590
nm and normalized with protein concentrations determined using BCA assay. Error bars
indicate standard deviation (n = 3). Asterisk (*) represents p < 0.05 based on analysis of
variance and the Bonferroni test (A – Reproduced from [79] with permission from
American Chemical Society, Copyright 2011).
48
3.3.5. GFP Silencing
MAP-siGFP-polyplex and R6-siGFP-polyplex (50 nM) exhibited 53 and 0%
reduction of GFP fluorescence, respectively, relative to their siNC equivalent
counterparts in Huh7.5 cells, stably transfected to express GFP-LC3 (Fig. 12). The
fluorescence knockdown by MAP-siGFP-polyplex was comparable to Lipo2000/siGFP
lipoplex (60%, p>0.05), whereas there was no significant difference between the
silencing effect of R6-siGFP-polyplex and siGFP-polyplex (9%, p>0.05) (Fig. 12).
Furthermore, 50 nM siGFP, 2.5 μg/ml Lipo2000, 1.2 μM MAP and 1.2 μM R6 alone did
not produce any significant gene silencing effect.

Figure 12. Gene silencing by R6-polyplex and MAP-polyplex in Huh7.5 cells, stably
transfected to express GFP-LC3. Cells were treated for 6 h in OptiMEM and then
replaced with Huh7.5 complete medium for an additional 42 h. The cells were then
washed, and cell lysates were collected. They were measured at Ex. 485 nm and Em. 518
nm and normalized with protein concentrations determined using BCA assay. Data
indicated with a caret (^) represent the value is relative to no treatment; unmarked data
are relative to siNC equivalent counterpart. Error bars indicate standard deviation (n = 3).
Asterisk (*) represents p < 0.05 based on analysis of variance and the Bonferroni test
(Reproduced from [79] with permission from American Chemical Society, Copyright
2011).
49

3.3.6. Cell Viability
MAP-siGFP-polyplex and R6-siGFP-polyplex (50 nM) reduced cell viability by
17 and 7%, respectively, relative to non-treated Huh7.5 cells (Fig. 13). There was no
significant increase of cytotoxicity between the CPP carrier alone and CPP-polyplex (Fig.
13). In addition, neither CPP-siGFP-polyplex exhibited significant difference of relative
cell viability compared with Lipo2000/siGFP lipoplex.

Figure 13. Cell viability. Stably transfected Huh7.5 cells were treated for 6 h in
OptiMEM and then replaced with Huh7.5 complete medium for another 42 h. MTT
solution was then added to the medium for additional 4 h incubation. The cells were
dissolved in a 1:24 N HCl:isopropanol solution, and cell viability was detected at a
wavelength of 570 nm (Reproduced from [79] with permission from American Chemical
Society, Copyright 2011).

3.3.7. Lack of K21-PDP
Neither MAP/siGFP nor R6/siGFP polyplex (no K21-PDP) (50 nM) was able to
produce a significant GFP silencing effect in Huh7.5 cells (Fig. 14). Only 50 nM MAP-
50
siGFP-polyplex was able to significantly reduce GFP fluorescence (Fig. 14). Thus, the
presence of K21-PDP in the MAP-polyplex design was able to significantly facilitate a
gene silencing effect.

Figure 14. Role of K21-PDP in R6-polyplex and MAP-polyplex for gene silencing
effects. Huh7.5 cells, stably transfected to express GFP-LC3, were treated for 6 h in
OptiMEM and then replaced with Huh7.5 complete medium for an additional 42 h. The
cells were then washed, and cell lysates were collected. They were measured at Ex. 485
nm and Em. 518 nm and normalized with protein concentrations determined using BCA
assay. Data are relative to siNC equivalent counterpart. Error bars indicate standard
deviation (n = 3). Asterisk (*) represents p < 0.05 based on analysis of variance and the
Bonferroni test (Reproduced from [79] with permission from American Chemical
Society, Copyright 2011).
51
3.4. Discussion
In this chapter, cationic (R6) and amphipathic (MAP) CPPs have been compared
and evaluated for siRNA delivery and efficacy. A previous report comparing CPP-based
siRNA delivery vehicles did not accurately reflect the CPPs’ abilities to knockdown the
target gene [64] because the neutralization effects could potentially weaken the
internalization abilities of CPPs [72]. However, R6- and MAP-polyplex overcome this
issue because each CPP carrier is conjugated to a neutralized polyplex.
Both CPP-polyplexes had similar conjugation efficiencies between the polyplex
and the CPP carrier (Table 2). Furthermore, both CPP carriers stabilized the polyplex so
that siRNA was not freed after 10-fold dilution (Fig. 10). This observation was attributed
to steric stabilization after the CPP carrier was conjugated to the polyplex [80].
Despite the similarities between the CPP-polyplexes, our results indicated that
MAP was a more suitable CPP than R6 for siRNA delivery. MAP demonstrated
significantly greater intracellular uptake of siDY547 (Fig. 11.A) and a stronger
knockdown of GFP fluorescence (Fig. 12) than R6-polyplex. However, the loss of K21-
PDP for MAP/siGFP resulted in no activity (Fig. 14). This observation supported the role
of K21-PDP as a scaffold necessary to conjugate the CPP carrier and facilitate cellular
uptake. R6-polyplex uptake and silencing effect were limited, where no intracellular
siDY547 was detected (Fig. 11.A) and no GFP fluorescence reduction was observed (Fig.
12). R6-polyplex results were similar with those observed for siRNA polyplex without
CPP conjugation, which suggested that R6 did not enhance siRNA polyplex uptake and
siRNA efficacy. In addition, R6/siRNA, without any K21-PDP, was also unable to
52
knockdown the gene expression at carrier and siRNA concentrations equivalent to R6-
polyplex (Fig. 14). The lack of R6/siGFP activity (and MAP/siGFP) was most likely due
to CPP carrier charge neutralization and subsequent uptake inhibition. On the other hand,
whether given as CPP alone or CPP-polyplex, R6 exhibited less cytotoxicity than MAP,
but the difference was not significant (Fig. 13).
The finding that MAP-polyplex was more effective than R6-polyplex in siRNA
cellular uptake and exhibiting gene silencing effects are very intriguing and informative.
MAP contains one less positive charge than R6 (5 vs. 6, respectively). Thus, the number
of cationic charges did not indicate the delivery efficiency of the CPP. On the other hand,
the order of cationic charges among hydrophobic regions in the amphipathic MAP has
shown the significance of amino acid sequence for efficient cellular uptake and activity
of siRNA polyplex. Another deciding factor could be the CPP size. MAP is 19 amino
acids long, while R6 is only 7 (counting also the cysteine at the N-terminus). The longer
peptide may exhibit better accessibility to the extracellular surface to allow higher
polyplex uptake.
Cationic oligoarginine was selected for conjugation to the polyplex due to its
advantage to directly deliver siRNA to the cytosol, where RNAi initiates, following
cellular uptake [42]. Selection of the appropriate oligoarginine peptide length is crucial
because the length of cationic CPP is an important factor in its internalization efficiency
[41]. In addition, for the design of the K21-based polyplex, there were concerns that the
cationic CPP could displace K21’s interaction with siRNA because the guanidine group
of arginine is a stronger base than the ε-amine group of lysine. R6 was selected as the
53
cationic CPP carrier because it was the longest oligoarginine with minimal displacement
of K21 in 2:1 N/P polyplex (Fig. 9). The displacement assay indicated the G/N ratio and
the oligoarginine peptide length were both significant variables for polyplex
displacement, where higher G/N ratios and longer oligoarginine peptides contributed to a
higher degree of separation between K21 and siRNA. Although R6 was selected as the
optimal oligoarginine for conjugation to the polyplex, R6-polyplex was not as effective
as MAP-polyplex for delivering enough siRNA into the cell to induce a biological
response.
Lipo2000 is a commonly used transfection reagent for siRNA. Our results
indicated that MAP-polyplex transported more siDY547 intracellularly than Lipo2000
lipoplex (Fig. 11.A), but only showed comparable amounts of gene silencing (Fig. 12)
and cytotoxicity (Fig. 13) in stably transfected Huh7.5 cells. There are two reasons to
explain this discrepancy. First, the intracellular amount of RNAi components, such as
miRNA, RISC and Ago2, also affects siRNA potency [88-91]. Both Lipo2000 lipoplex
and MAP-polyplex may have transported enough siRNA to occupy available RNAi
components and reach the limits of gene silencing effects. Meanwhile, any surplus
siRNA delivered intracellularly may have been consumed by cellular defense processes,
such as autophagosomes [1], before they had an opportunity to interact with RNAi
components for gene silencing. Another possible reason can be due to vesicle-based
internalization mechanism of MAP. Previous reports have indicated that MAP enters the
cell primarily through endocytosis and targets to nuclear localization [85,92]. Therefore,
a significant portion of the internalized siRNA may have been directed to compartments,
54
such as the lysosome or the nucleus, where RNAi for post-transcriptional gene silencing
cannot be initiated. Consequently, only comparable inhibitory effects were observed
between MAP-polyplex and Lipo2000 lipoplex. Additional studies evaluating properties
of MAP-polyplex are discussed in Chapter 4.
One additional technical note regards the storage of K21-PDP. Initially, K21-PDP
required fresh preparation before each assay. Storing K21-PDP at 4, -20 or -80°C
produced CPP-polyplexes that did not exhibit any silencing activity. However, it has been
found that snap freezing can resolve these issues [70]. After purifying and quantifying the
modified peptide, K21-PDP was aliquoted and dipped in liquid nitrogen for 3 min.
Subsequently, snap frozen K21-PDP was stored at -80°C. Prior to CPP-polyplex
preparation, K21-PDP was thawed in 37°C water bath and ready for use. This method
provides good consistency among different batches of products. Thereafter, fresh
preparations of the modified peptide were no longer required to produce active CPP-
polyplexes.

55
3.5. Summary
In Chapter 3, cationic R6 and amphipathic MAP was compared for siRNA
delivery and efficacy. A valid comparison was evaluated between the two types of CPPs
because each carrier was conjugated to the polyplex, which contained a neutralized
siRNA. Thus, there were no concerns regarding the compromise of internalization
capabilities caused by electrostatic interactions. MAP-polyplex has exhibited
significantly greater siRNA delivery efficiency and gene silencing effects than R6-
polyplex, and both CPP-polyplexes exhibited comparable changes in cell viability. In
addition, MAP-polyplex delivered more siRNA intracellularly than Lipo2000 lipoplex,
but both managed comparable gene silencing and cytotoxicity effects. Therefore,
amphipathic MAP was determined to be a suitable CPP carrier for siRNA delivery and
efficacy.
56
CHAPTER 4: EVALUATION OF MAP-POLYPLEX DESIGN

4.1. Introduction
4.1.1. Background
The properties and mechanisms for CPP-based siRNA delivery vehicles are
important to study and understand. Each component has a proposed role for facilitating
siRNA delivery and efficacy, and it is necessary to confirm that each component
functions as designed. The mechanism of siRNA delivery vehicles has been a common
area of interest. Cellular uptake of siRNA delivery vehicles has occurred through vesicle-
or non-vesicle-dependent pathways. Amphipathic CPP-based polyplexes have been
reported to enter the cells through different pathways. An amphipathic MPG-derived
polyplex and an amphipathic CADY polyplex have been demonstrated to enter the cell
through a endosomal and non-endosomal pathway, respectively [63,66]. Thus, despite
being classified as the same type of CPP, each polyplex has exhibited a different cellular
uptake pathway. In addition, the presence of serum has also been an important
consideration for siRNA transfections. In vitro transfections may be conducted in
minimal or serum-free conditions because serum may interact with siRNA delivery
vehicles and inhibit the transfection efficiency [16]. It has been preferable to conduct
transfections in the presence of serum because cells require serum for growth, and the
results would give better insight into the in vivo efficacy where the microenvironment is
more complicated [93]. Finally, how the conjugated functional ligands affect the delivery
and efficacy of siRNA polyplex also has to be evaluated. A targeting peptide fused to an
57
oligoarginine CPP-based polyplex exhibited specific binding and uptake to neuronal cells
in a dose-dependent manner [67]. A folate- and penetratin-conjugated nanoparticle has
shown synergistic siRNA uptake and efficacy results relative to only having either the
targeting or intracellular transport moiety [62]. Therefore, characterization of siRNA
delivery vehicle properties will provide insights into its attributes, which can be further
applied for future designs and developments.

4.1.2. Rationale
In the previous chapter, MAP-polyplex has been shown to be a better siRNA
delivery vehicle than R6-polyplex. MAP-polyplex and R6-polyplex have produced
comparable cytotoxic effects, but the former has exhibited significantly greater siRNA
intracellular uptake and stronger gene knockdown. In this chapter, the design of MAP-
polyplex is further evaluated to determine the factors that contribute to the enhanced
siRNA delivery and efficacy. The parameters to be examined include the effect of serum
on MAP-polyplex transfection, the necessary amount of MAP conjugation on MAP-
polyplex, the mechanism of MAP-polyplex cellular uptake, the model of siRNA release
from MAP-polyplex and the evaluation of MAP-polyplex targeting an endogenous gene
in another cell line. These findings are important will offer valuable information to the
further development of CPP-based siRNA delivery vehicles.
58
4.2. Experiments
4.2.1. Effect of Serum on Transfection
The effect of serum on MAP-polyplex efficacy was evaluated by diluting MAP-
siGFP-polyplex in Huh7.5 growth medium (supplemented with 10% FBS), instead of
OptiMEM (serum-free), for treatment. The rest of the GFP silencing assay was performed
as previously described (3.2.5).

4.2.2. Different Amounts of MAP Conjugation
The importance of the MAP:polyplex ratio on siRNA cellular uptake and efficacy
was evaluated by comparing MAP-polyplex formed at different ratios of MAP to PDP
groups. An additional MAP-polyplex was prepared at 0.5:1 CPP/PDP ratio (0.5:1
CPP:PDP MAP-siRNA-polyplex) with a final carrier CPP concentration of 30 μM in the
same manner as previously described (3.2.2).

4.2.2.a. Uptake
Cellular uptake assays were performed as previously described (3.2.4), except that
only 6 h time point was evaluated.

4.2.2.b. Activity
GFP silencing assays were performed as previous described (3.2.5).

59
4.2.3. Uptake Mechanism
Studying transport of MAP-siDY547-polyplex in Huh7.5 cells at different
temperatures elucidated the uptake mechanism of MAP-polyplex. As an internal control,
radiolabeled transferrin (Tf) (Sigma-Aldrich) was co-incubated with MAP-siDY547-
polyplex during treatment. Tf was radiolabeled with Na
125
I (PerkinElmer, Inc., Waltham,
MA) (
125
I-Tf) using the chloramine-T method. Briefly, 100 μl of 10 mg/ml Tf was added
to 150 μl of PBS and then mixed with 50 μl Na
125
I. The oxidizing agent chloramine-T (50
μl of 4 mg/0.5 ml PBS) was added and allowed to react for 5 min on ice. Then, the
reducing agent sodium metabisulfite (2.4 mg/0.5 ml PBS) was added for an additional
reaction of 5 min on ice. The iodination was completed by adding 100 μl potassium
iodide (10 mg/ml DDH
2
O). The reaction mixture was purified using Sephadex G-50 (GE)
size exclusion chromatography. Fifteen fractions (1 ml/fraction) were collected and
detected using a gamma counter (Packard, Downers Grove, IL).
125
I-Tf was aliquoted and
stored at -20ºC. Huh7.5 cells, stably transfected to express GFP-LC3, were seeded and
prepared as previously described (3.2.4). Dosing solutions were prepared with 3 μg/ml
125
I-Tf and 50 nM MAP-siDY547-polyplex in OptiMEM. Huh7.5 cells were then dosed
with treatments prepared in OptiMEM for 1 h at 4, 16 and 37ºC. After the incubation, the
cell lysates was collected and analyzed for siDY547 uptake and protein content, as
previously described (3.2.4). However, before protein quantification, cell lysates were
also evaluated for
125
I-Tf radioactivity using the gamma counter.

60
4.2.4. Model of Cytosolic-like Environment
A cytosolic-like environment was modeled to demonstrate the release of siRNA
from MAP-polyplex. Trypsin and DTT were used to simulate proteolysis and reducing
activities, respectively, typically found in the cytosol. SiGFP polyplex and MAP-siGFP-
polyplex (40 μl of 2.5 μM) was reacted with trypsin (5 μl of 0.97 U/μl) and PBS (5 μl) for
15 min at 37ºC. MAP-siGFP-polyplex was also reacted with DTT (5 μl of 1 M) for 30
min, room temperature and then trypsin (5 μl of 0.97 U/μl) for 15 min at 37ºC. The
treatments (30 μl) were mixed with 6 μl of 6x loading buffer and then loaded onto a 2%
agarose gel. Untreated siGFP polyplex and MAP-siGFP-polyplex were also loaded as
controls. Agarose gel shift assay was performed as previously described (2.2.3).

4.2.5. Endogenous Gene Silencing in an Alternative Cell Line
MAP-polyplex efficacy was further evaluated in a different cell line (HeLa),
while targeting an endogenous gene (phosphatase and tensin homologue deleted on
chromosome 10, PTEN). MAP-polyplex (2.5 μM) was prepared with a PTEN-targeting
siRNA (siPTEN) (Cell Signaling Technlogy, Danvers, MA) using the same method
previously described (3.2.2). HeLa cells were maintained in DMEM supplemented with
10% FBS and 2 mM L-glutamine (“HeLa growth medium”). The day before siRNA
transfection, HeLa cells were seeded in 6-well plates at a density of 0.2x10
6
cells/ml in 2
ml of HeLa growth medium. MAP-siPTEN-polyplex and Lipo2000/siPTEN lipoplex
(100 nM) were transfected in OptiMEM for 6 h, before being replaced by HeLa growth
61
medium for an additional 42 h incubation at 37ºC, 5% CO
2
. Cell lysates were collected,
and BCA assay was used to quantify the protein content, as previously described (3.2.4).
Cell lysates (40 μg) were boiled in loading buffer (supplemented with reducing
agent, 2-mercaptoethanol) and loaded onto 13% SDS-PAGE gel for electrophoresis
followed by Western blotting using rabbit anti-PTEN (1:10000 in 1% BSA in TBS-T,
Cell Signaling Technology) and mouse anti-actin (1:8000 in 5% milk in TBS-T, Sigma-
Aldrich) primary antibodies overnight at 4ºC and for 1 h at room temperature,
respectively. Horseradish peroxidase (HRP)-conjugated secondary antibodies were
detected using enhanced chemiluminescence (GE Healthcare), and immunoreactive
bands were quantified using Quantity One software (Bio-Rad, Hercules, CA).

62
4.3. Results
4.3.1. Effect of Serum on Transfection
The effect of serum on MAP-polyplex transfection efficiency was evaluated by
comparing OptiMEM (serum-free) and Huh7.5 complete medium (supplemented with
10% FBS). MAP-siGFP-polyplex (50 nM) significantly reduced GFP fluorescence by 49
and 47% when Huh7.5 cells were transiently transfected in OptiMEM and Huh7.5
complete medium, respectively (Fig. 15). Conversely, siGFP (50 nM) did not exhibit any
gene silencing effect. Thus, MAP-siGFP-polyplex silencing activity exhibited
comparable effects after transfection in either serum-free or 10% FBS supplemented
medium.

Figure 15. Effect of 10% FBS on MAP-polyplex gene silencing efficacy. Huh7.5 cells,
stably transfected to express GFP-LC3, were treated for 6 h in OptiMEM or Huh7.5
complete medium (with 10% FBS) and then replaced with Huh7.5 complete medium for
an additional 42 h. The cells were then washed, and cell lysates were collected. They
were measured at Ex. 485 nm and Em. 518 nm and normalized with protein
concentrations determined using BCA assay. Data are relative to siNC equivalent
counterpart. Error bars indicate standard deviation (n = 3). Asterisk (*) represents p <
0.05 based on analysis of variance and the Bonferroni test (Reproduced from [79] with
permission from American Chemical Society, Copyright 2011).

63
4.3.2. Different Amounts of MAP Conjugation
4.3.2.a. Uptake
The amount of MAP conjugation to polyplex correlated with MAP-siDY547-
polyplex delivery efficiency. MAP-siDY547-polyplex (50 nM, 1:1 CPP:PDP) exhibited a
60% increase of siDY547 cellular delivery than 0.5:1 CPP:PDP MAP-siDY547-polyplex
(Fig. 16.A). Coincubation of MAP with siDY547 polyplex (where no covalent
conjugation occurred between the carrier and polyplex) resulted in an insignificant
amount of siDY547 detected intracellularly, which was comparable with that delivered
by siDY547 polyplex (Fig. 16.A).

4.3.2.b. Activity
The GFP silencing activity of MAP-siGFP-polyplex was dose-dependent on the
MAP:polyplex ratio, where 50 nM 0.5:1 and 1:1 CPP:PDP MAP-siGFP-polyplex reduced
GFP fluorescence by 17 and 50%, respectively (Fig. 16.B). There was an insignificant
fluorescence decrease of 11% when coincubating unconjugated MAP and siDY547
polyplex. SiGFP polyplex exhibited an insignificant silencing effect (Fig. 16.B).

64
A.

B.

Figure 16. Effect of MAP conjugation for MAP-polyplex (A) cellular uptake and (B)
gene silencing efficacy. MAP-polyplexes were made with different amounts of MAP
conjugation. (A) To determine cellular uptake, Huh7.5 cell, stably transfected for GFP-
LC3, were treated for 6 h in OptiMEM. Cell lysates were then collected and detected at
Ex. 544 nm and Em. 590 nm and normalized with protein concentrations calculated using
BCA assay. A, no treatment; B, 1.2 μM MAP; C, 50 nM siDY547; D, 50 nM siDY547-
polyplex; E, 50 nM siDY547-polyplex + 1.2 μM MAP; F, 50 nM 0.5:1 CPP/PDP MAP-
siDY547-polyplex; G, 50 nM 1:1 CPP/PDP MAP-siDY547-polyplex. (B) To evaluate
GFP silencing, after a 6 h transfection and then replacement with Huh7.5 complete
medium for 42 h, cell lysates were collected and measured at Ex. 485 nm and Em. 518
nm and normalized with protein concentrations calculated using BCA assay. A, 1.2 μM
MAP; B, 50 nM siGFP; C, 50 nM siGFP-polyplex; D, 50 nM siGFP-polyplex + 1.2 μM
MAP (unconjugated); E, 50 nM 0.5:1 CPP/PDP MAP-siGFP-polyplex; F, 50 nM 1:1
CPP/PDP MAP-siGFP-polyplex. Data are presented relative control treatments (A,
relative to non-treated cells; B-F, relative to siNC equivalent counterpart). Error bars
indicate standard deviation (n = 3). Asterisk (*) represents p < 0.05 based on analysis of
variance and the Bonferroni test (Reproduced from [79] with permission from American
Chemical Society, Copyright 2011).
65

4.3.3. Uptake Mechanism
MAP-siDY547-polyplex (50 nM) was coincubated with radiolabeled transferrin
(
125
I-Tf) for cellular uptake assays at different temperatures (4, 16 and 37ºC).
125
I-Tf
cellular uptake is through clathrin-mediated endocytosis [94], and thus was used as an
internal control to confirm the loss of vesicle formation and fusion in cells incubated at 4
and 16ºC, respectively. At 4 and 16ºC, MAP-siDY547-polyplex cellular uptake was
reduced by 52 and 35%, respectively, while
125
I-Tf internalization was also diminished by
70 and 54%, respectively (Fig. 17).

Figure 17. Mechanism of MAP-polyplex cellular uptake. Stably transfected Huh7.5 cells
were treated for 1 h at 4, 16 and 37ºC with 50 nM MAP-siDY547-polyplex and 3 μg/ml
of
125
I-Tf in OptiMEM. Cell lysates were collected and detected at Ex. 544 nm and Em.
590 nm for siDY547 uptake and using a gamma counter for
125
I-Tf uptake. Error bars
indicate standard deviation (n = 3). Asterisk (*) represents p < 0.05 based on analysis of
variance and the Bonferroni test (Reproduced from [79] with permission from American
Chemical Society, Copyright 2011).

66
4.3.4. Model of Cytosolic-like Environment
MAP-polyplex was incubated with trypsin and DTT to demonstrate the release of
siRNA from MAP-polyplex. Direct treatment of MAP-polyplex with trypsin did not free
siRNA (Fig. 18, Lane 9), whereas trypsin-treated polyplex did release siRNA (Lane 5).
However, incubating MAP-polyplex with the reducing agent DTT cleaved the disulfide
bonds, which resulted in a pattern similar to the polyplex (Lane 7 vs. Lane 4,
respectively). Subsequent treatment of reduced MAP-polyplex with trypsin did free
siRNA (Lane 8).

Figure 18. Model of siRNA release from MAP-polyplex. Polyplex (2:1 N/P) and MAP-
polyplex were treated with DTT for 30 min, room temperature and/or trypsin 15 min,
37ºC. Samples were loaded onto 2% agarose gel and run for 30 min, 125 V (Reproduced
from [79] with permission from American Chemical Society, Copyright 2011).

4.3.5. Endogenous Gene Silencing in an Alternative Cell Line
MAP-polyplex activity was further studied by targeting an endogenous gene
(PTEN) in another cell line (HeLa). MAP-siPTEN-polyplex (100 nM) reduced PTEN
expression by 50%, which was comparable to the effect of 100 nM Lipo2000 lipoplex
67
(Fig. 19). SiPTEN (100 nM) alone did not exhibit any significant gene silencing effect
(Fig. 19).

Figure 19. Endogenous gene silencing of PTEN in HeLa cells using MAP-polyplex.
HeLa cells were treated in OptiMEM for 6 h and then replaced with HeLa complete
medium for additional 42 h. Cell lysates were collected, quantified using BCA assay, run
on 13% SDS-PAGE, and immunoblotted with anti-PTEN and anti-β-actin. Signal
intensities were quantified using densitometry, and results were expressed as the average
of relative intensities to non-treated HeLa cells. Error bars indicate standard deviation (n
= 3). Asterisk (*) represents p < 0.05 based on analysis of variance and the Bonferroni
test (Reproduced from [79] with permission from American Chemical Society, Copyright
2011).
68
4.4. Discussion
The design of MAP-polyplex was further evaluated in Chapter 4. Different assays
were completed to recognize the role of each component of MAP-polyplex and determine
the efficacy of MAP-polyplex in different conditions. Better understanding of MAP-
polyplex properties can contribute to future developments of siRNA delivery systems.
The presence of serum was reported to dissociate PEI/DNA polyplexes [95] and
affect transfection efficiency [16]. Thus, the effect of serum on MAP-polyplex
transfection was studied. No significant difference of GFP silencing by MAP-polyplex
was observed in the presence of 10% FBS or serum-free conditions (Fig. 15). Thus,
MAP-polyplex appeared to remain stable and unaffected by the presence of 10% FBS.
This bodes well for MAP-polyplex’s prospects for in vivo delivery, since serum is
particularly relevant for siRNA delivery in vivo where its abundance in blood could
potentially disassemble polyplexes [93].
MAP carrier peptide was verified as a potent CPP for enhanced cellular uptake
[85], where MAP conjugated to siRNA polyplex exhibited significant uptake (Fig. 11).
Different amounts of MAP were conjugated to the polyplex to further demonstrate its
role in siRNA uptake and efficacy. The amount of intracellular siRNA detected (Fig.
16.A) correlated well with the degree of gene silencing (Fig. 16.B), where the 1:1
CPP/PDP MAP-polyplex exhibited significantly more uptake and activity than 0.5:1
CPP/PDP MAP-polyplex. Therefore, the amount of MAP conjugation was critical for
siRNA delivery and activity. Furthermore, the conjugation between the carrier and
polyplex was also crucial because when they were incubated together unconjugated, no
69
significant uptake or efficacy was observed (Fig. 16). This also substantiated the
importance of K21-PDP as a necessary scaffold that allows for CPP carrier conjugation
(as discussed in Chapter 3). As a result, both K21-PDP and MAP were necessary
components in this polyplex design to achieve significant reduction in targeted gene
expression. In this design, K21-PDP formed a polyplex with siRNA, while MAP
functioned as an intracellular transport vehicle for siRNA without being inhibited by any
siRNA neutralizing effects.
Temperature-dependent cellular uptake assays revealed that MAP-polyplex
internalization required vesicle formation and fusion events. Cellular uptake was
inhibited when cells were incubated at both 4 and 16ºC, respectively (Fig. 17). These
observations were consistent with the reported characteristics of MAP alone [85].
Radiolabeled
125
I-Tf, which entered cells through temperature-dependent clathrin-
mediated endocytosis [94], also exhibited reduced uptake at 4 and 16ºC (Fig. 17). This
result indicated that the cells were behaving normally at these temperatures. Despite
having vesicle-dependent uptake (Fig. 17), MAP-polyplex still exhibited silencing effects
(Fig. 12, 15, 16, 19), which suggested that MAP-polyplex was still able to escape from
vesicles to deliver siRNA into the cytosol for gene knockdown. The escape properties
may be attributed to MAP because it was previously reported that amphipathic peptides
showed endosomal escape capabilities [70,96,97]. However, inefficient endosomal
escape from intracellular vesicles, such as endosomes, by MAP-polyplex may also
explain why high levels of siRNA internalization did not correlate with a higher degree of
gene silencing.
70
A proposed mechanism of siRNA release from MAP-polyplex in the cytosol after
endosomal escape was tested using an agarose gel shift assay (Fig. 18). Releasing siRNA
from the delivery vehicle was an important design consideration for MAP-polyplex
because if siRNA remained attached to the polyplex, then it would be unable to cooperate
with RISC to induce RNAi. The reducing [98] and proteolytic [75,76] environment of the
cytosol are believed to reduce the disulfide bonds and digest K21-PDP, respectively, of
MAP-polyplex, thereby generating free siRNA to interact with RISC. This proposed
breakdown of MAP-polyplex was examined using DTT and trypsin treatment to simulate
cytosolic reducing and proteolytic activities, respectively. The reducing agent DTT
separated MAP from the K21-PDP polyplex by reducing the disulfide bonds (Lane 7) and
then trypsin treatment of the unconjugated polyplex digested the K21-PDP peptides to
release siRNA (Lane 8). On the other hand, siRNA was not released when MAP-polyplex
was only treated with trypsin (Lane 9). Therefore, disulfide bond reduction between the
carrier and polyplex was demonstrated to be crucial for siRNA separation from MAP-
polyplex. Furthermore, not only did MAP conjugation stabilize the polyplex (Fig. 10),
but it also protected K21-PDP from degradation (Fig. 18, Lane 9).
MAP-polyplex efficacy was also evaluated by targeting an endogenous gene
(PTEN) in an alternative cell line (HeLa cells). PTEN is a lipid phosphatase that
negatively regulates the mitogenic signaling phosphoinositide 3-kinase (PI3K)/AKT
signaling pathway, and thus reduction of PTEN expression induces growth and survival
phenotypes [99,100]. Regulation of PTEN function was recently introduced as a potential
therapeutic treatment for recovery from adult spinal cord injury [101]. Therefore, PTEN
71
silencing was considered a worthwhile target for siRNA knockdown. MAP-polyplex and
Lipo2000 lipoplex showed comparable PTEN silencing effects in HeLa cells (Fig. 19).
These results were similar to targeting the reporter GFP fusion protein in Huh7.5 cells
(Fig. 19 vs. Fig. 12). MAP-polyplex transfection was efficient in two different cells lines
when targeting reporter and endogenous genes.

72
4.5. Summary
In Chapter 4, the properties of MAP-polyplex were further evaluated. The
presence of 10% FBS did not affect MAP-polyplex transfection efficiency. The amount
of MAP conjugation to polyplex was important for siRNA cellular uptake and efficacy.
SiRNA can be released from MAP-polyplex in a cytosolic-like environment. MAP-
polyplex uptake required vesicle formation and fusion. The delivery efficacy of MAP-
polyplex was also validated in an alternative cell line targeting to an endogenous gene.
Therefore, MAP-polyplex functions as a viable delivery vehicle for siRNA delivery and
activity. Further evaluation of MAP-polyplex in a difficult-to-transfect cell line or
primary cells would be an exciting future direction.

73
CHAPTER 5: EFFECTS OF SIRNA DELIVERY ON AUTOPHAGY IN
HEPATOMA CELLS

5.1. Introduction
5.1.1. Background
SiRNA is an important and prominent research technology. Through the RNAi
pathway, exogenously delivered siRNA interacts with other cytosolic proteins to disrupt
protein production by degrading the target mRNA [9]. The ability to knockdown specific
genes has been a useful application to evaluate gene function [102], elucidate molecular
pathways [103], determine prospective drug targets, and discover pharmaceutical
therapeutics [103]. However, due to its negative charge and large size, siRNA has been
limited in its intracellular uptake and required a delivery moiety to aid its transport into
the cells [15]. Lipids and CPPs have been commonly used for siRNA delivery because
they can form complexes (lipoplexes and polyplexes, respectively) with siRNA through
electrostatic interactions and achieve intracellular internalization. However, a limited
number of studies have been completed to evaluate inadvertent cell responses, such as
autophagy, caused by these lipid- and CPP-based transfection systems.
Autophagy is a vital cellular process where intracellular proteins and organelles
are degraded through a lysosomal-dependent process (Fig. 20). It is tightly regulated and
has an essential role in cell growth, cell survival, immune defense and cellular
homeostasis [104]. Macroautophagy is the most commonly referred type of autophagy
and refers to the collection of cytosolic components by double-membrane vesicles called
74
autophagosomes that eventually merge with lysosomes for degradation of cargo [105]. In
this dissertation, the term “autophagy” indicates to macroautophagy.

Figure 20. Autophagy pathway. Upon autophagy induction, an expanding membrane
called phagophore surrounds cytosolic proteins and organelles. Meanwhile, cytosolic
endogenous LC3-I and stably-expressed GFP-LC3-I are converted to lipid-conjugated
LC3-II and GFP-LC3-II, respectively, and inserted into the phagophore’s inner and outer
membranes. The resulting double-membrane autophagosome then fuses with lysosomes,
and the inner compartment is exposed to lysosomal hydrolases, which leads to
degradation of the internalized cargo.

5.1.2. Rationale
Uptake of exogenous nanoparticles has been shown to induce cellular autophagy
[106,107], and the amount of autophagy induction correlated with the particle size [108].
Furthermore, the reported handling of particles may be due to the cellular elimination of
protein aggregates, one of the main responsibilities of autophagy [109-111]. Based on
these findings, it is plausible that autophagy has a role in the processing of siRNA
75
delivery vehicles. The focus of this chapter is to study the cellular autophagic response to
lipid- and CPP-based siRNA transfection systems.
Lipo2000 and MAP-polyplex are the siRNA delivery vehicles used for
evaluation. Lipo2000 is a commonly used lipid-based reagent for siRNA delivery [16],
whereas MAP-polyplex is a recently developed CPP-based siRNA delivery vehicle [79].
Each is loaded with a non-targeting, negative control siRNA without any particular gene
silencing effects and studied for their autophagic responses in hepatoma cells. These
studies will reveal important considerations for the application of siRNA for RNAi.

76
5.2. Experiments
5.2.1. Cellular Uptake of Lipo2000 Lipoplexes and MAP-polyplexes at Escalating Doses
Huh7.5 cells, stably transfected to express GFP-LC3, were used for cellular
uptake assays to evaluate escalating doses (50, 100 and 200 nM) of Lipo2000 lipoplexes
and MAP-polyplexes loaded with siDY547. Cellular uptake assay (6 h incubation) was
performed as previously described (3.2.4).

5.2.2. LC3 Conversion in Hepatoma Cells
5.2.2.a. MG-132 Treatment
Huh7.5 cells, stably transfected to express GFP-LC3, were treated with 10 μM
MG-132 (Calbiochem, La Jolla, CA) in OptiMEM for 24 h. Cell lysates were collected
and quantified for Western blotting as previously described (3.2.4). Cell lysates (15 μg)
were subjected to electrophoresis in 15% SDS-PAGE gels followed by immunoblotting
against polyclonal rabbit anti-LC3 (1:1000 in 5% milk in TBS-T, MBL International,
Woburn, MA) and monoclonal mouse anti-β-actin (1:8000 in 5% milk in TBS-T) primary
antibodies with corresponding HRP-conjugated secondary antibodies. Immunoreactive
band analysis was performed as previously described (4.2.5).

5.2.2.b. SiRNA Transfection in Hepatoma Cells
Huh7.5 cells, stably transfected to express GFP-LC3, were transfected with
escalating doses of siNC (50, 100 and 200 nM) delivered using lipid- (Lipo2000 lipoplex)
and peptide-based (MAP-polyplex) transfection methods. After a 6 h transfection in
77
OptiMEM, treatment medium was replaced with Huh7.5 growth medium for an
additional 42 h incubation at 37ºC, 5% CO
2
. Cell lysates were collected and quantified
for LC3 conversion immunoblotting as previously described (5.2.2.a).

5.2.3. Evaluation of LC3 Conversion in H4IIE Cells
H4IIE rat hepatoma cells were maintained in DMEM supplemented with 10%
FBS and 2 mM L-glutamine (“H4IIE growth medium”). The day before siRNA
transfection, H4IIE cells were seeded in 6-well plates at a density of 0.2x10
6
cells/ml in 2
ml of H4IIE growth medium. Lipo2000/siNC lipoplex (100 nM) were transfected in
OptiMEM for 6 h, before being replaced by H4IIE growth medium for an additional 42 h
incubation at 37ºC, 5% CO
2
. Cell lysate processing and immunoblotting for LC3
conversion were conducted as previously described (5.2.2.a).

5.2.4. Quantification of GFP-LC3 Fluorescence in Huh7.5 Cells
5.2.4.a. MG-132 and Glucosamine Treatment
Huh7.5 cells, stably transfected to express GFP-LC3, were treated with 10 μM
MG-132 and 40 mM glucosamine for 24 and 48 h, respectively. Cell lysates were
collected, detected for GFP-LC3 fluorescence and quantified for protein content as
previously described (3.2.5).

78
5.2.4.b. Lipo2000 Lipoplex
Huh7.5 cells, stably transfected to express GFP-LC3, were transfected with
escalating doses of siNC (50, 100 and 200 nM) delivered using Lipo2000 lipoplex. After
a 6 h transfection in OptiMEM, treatment medium was replaced with Huh7.5 growth
medium for an additional 42 h incubation at 37ºC, 5% CO
2
. Cell lysates were collected,
detected for GFP-LC3 fluorescence and quantified for protein content as previously
described (3.2.5).

5.2.4.c. MAP-polyplex
Huh7.5 cells, stably transfected to express GFP-LC3, were transfected with
escalating doses of siNC (50, 100 and 200 nM) delivered using MAP-polyplex. After a 6
h transfection in OptiMEM, treatment medium was replaced with Huh7.5 growth
medium for an additional 42 h incubation at 37ºC, 5% CO
2
. Cell lysates were collected,
detected for GFP-LC3 fluorescence and quantified for protein content as previously
described (3.2.5).

5.2.5. Detection of Autophagosomes in Huh7.5 Cells using Confocal Microscopy
Cover slips (VWR) were sterilized by dipping in 200 proof ethanol and then
passing through a flame. Sterilized cover slips were placed per well in a 6-well plate.
Huh7.5 cells, stably transfected to express GFP-LC3, were seeded at a density of 0.2x10
6

cells/ml in 2 ml of Huh7.5 growth medium the day before treatment. Cells were
transfected with OptiMEM (no treatment), 5 μg/ml Lipo2000, 100 nM siDY547 and 100
79
nM Lipo2000/siDY547 lipoplex for 6 h at 37ºC, 5% CO
2
, and then incubated with
Huh7.5 growth medium for an additional 42 h incubation at 37ºC, 5% CO
2
. Cells were
also treated with 40 mM glucosamine (in OptiMEM) for 48 h as a positive control. Forty-
eight hours after the start of transfection/treatment, medium was removed, and Huh7.5
cells were washed three times using 4ºC PBS. Thereafter, they were fixed with 0.5 ml of
4% para-formaldehyde (Sigma-Aldrich) in PBS for 30 min, washed three times with 4ºC
PBS and then treated with 1 ml of 300 nM 4’,6-diamidino-2-phenylindole (DAPI) for 5
min. The fixed cells were mounted using Prolong Antifade Kit (Invitrogen) according to
manufacturer’s instructions and viewed using Nikon PCM Quantitative Measuring High-
Performance Confocal System (at the USC Research Center for Liver Diseases).

5.2.6. Effects of Alternative Negative Control siRNAs for siRNA Delivery on Autophagy
in Huh7.5 Cells
Alternative negative control siRNAs (“siNCambion” and “siNCqiagen,” from
Ambion and Qiagen, Valencia, CA respectively) were used for evaluation of Lipo2000
transfections. Lipo2000 lipoplexes (Lipo2000/siNC, Lipo2000/siNCambion, and
Lipo2000/siNCqiagen) were formed as previously described (3.2.4).

5.2.6.a. LC3 Conversion
Huh7.5 cells, stably transfected to express GFP-LC3, were seeded as previously
described (3.2.4). They were treated with 100 nM Lipo2000/siNC,
Lipo2000/siNCambion, and Lipo2000/siNCqiagen lipoplexes for 6 h at 37ºC, 5% CO
2
,
80
and then incubated with Huh7.5 growth medium for an additional 18 h. Cell lysates were
collected and quantified for LC3 conversion immunoblotting as previously described
(5.2.2.a).

5.2.6.b. Quantification of GFP-LC3 Fluorescence
Huh7.5 cells, stably transfected to express GFP-LC3, were seeded as previously
described (3.2.4). They were treated with 100 nM Lipo2000/siNC,
Lipo2000/siNCambion, and Lipo2000/siNCqiagen lipoplexes for 6 h at 37ºC, 5% CO
2
,
and then incubated with Huh7.5 growth medium for an additional 66 h. Cell lysates were
collected, detected for GFP-LC3 fluorescence and quantified for protein content as
previously described (3.2.5).

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5.3. Results
5.3.1. Cellular Uptake of siDY547 at Different Concentrations using Lipo2000 Lipoplex
and MAP-polyplex in Huh7.5 Cells
The delivery efficiency of Lipo2000 lipoplex and MAP-polyplex was compared
using siDY547 cellular uptake assay. Compared to 200 nM siDY547 alone, 50, 100 and
200 nM Lipo2000/siDY547 lipoplexes increased siDY547 cellular uptake by 1.5, 4.3 and
9.6-folds, respectively (Fig. 21). Similarly, 50, 100 and 200 nM MAP-siDY547-polyplex
also increased cellular uptake by 4.0, 8.1 and 17.0-folds, respectively, relative to 200 nM
siDY547 alone (Fig. 21). Therefore, cellular uptake of Lipo2000/siDY547 lipoplex and
MAP-siDY547-polyplex was dose-dependent. At the same siRNA concentrations, MAP-
polyplex delivered greater intracellular amounts of siDY547 than Lipo2000 lipoplex.

Figure 21. Cellular uptake of siDY547 at different concentrations using Lipo2000
lipoplex and MAP-polyplex in stably transfected Huh7.5 cells. Huh7.5 cells, stably
transfected to express GFP-LC3, were treated for 6 h in OptiMEM. Cells lysates were
collected and measured at Ex. 544 nm and Em. 590 nm, and normalized with protein
concentrations quantified using BCA assay.

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5.3.2. Evaluation of LC3 Conversion in Huh7.5 Cells
5.3.2.a. Positive Control – MG-132
A known activator of autophagy, the proteasome inhibitor, MG-132, was used as
a positive control to demonstrate LC3 conversion in Huh7.5 cells. Treatment of Huh7.5
cells with 10 μM MG-132 for 24 h led to a significant increase of LC3-II compared with
non-treated cells (Fig. 22.A).

5.3.2.b. Lipo2000 Lipoplex
Increasing concentrations of Lipo2000/siNC lipoplex (50, 100 and 200 nM)
resulted in a dose-dependent response for LC3 conversion. Relative to non-treated
Huh7.5 cells, 50, 100 and 200 nM Lipo2000/siNC lipoplexes increased the amount of
LC3-II by 1.3, 2.1 and 3.4-folds (Fig. 22.B). Lipo2000 (10 μg/ml) also increased LC3
conversion by 2.8-folds, whereas 200 nM siNC showed only 1.1-fold increase (Fig.
22.B). Therefore, treatments with Lipo2000 and escalating doses of Lipo2000/siNC
lipoplex (50, 100 and 200 nM) corresponded with increased amounts of LC3-II relative to
non-treated cells.

5.3.2.c. MAP-polyplex
Increasing concentrations of MAP-siNC-polyplex (50, 100 and 200 nM) resulted
in a dose-dependent response for LC3 conversion. Relative to non-treated Huh7.5 cells,
50, 100 and 200 nM MAP-siNC-polyplex increased the amount of LC3-II by 2.1, 3.0 and
4.1-folds (Fig. 22.C). SiNC (200 nM) showed a 2.0-fold increase. Therefore, treatments
83
escalating doses of MAP-siNC-polyplex (50, 100 and 200 nM) corresponded with
increased amounts of LC3-II relative to non-treated cells.
A.
B.
C.
Figure 22. Western blot of LC3 conversion in stably transfected Huh7.5 cells. (A) Cells
were treated with 10 μM MG-132 for 24 h. Cells were treated for 6 h with different
concentrations of (B) Lipo2000/siNC lipoplexes and (C) MAP-siNC-polyplexes, and then
replaced with Huh7.5 complete medium for additional 42 h. After treatments, cells were
washed, and cell lysates were collected, run on 15% SDS-PAGE and immunoblotted with
anti-LC3 and anti-β-actin (A, B – Reproduced from [1] with permission from Humana
Press, Inc. via Copyright Clearance Center).
84

5.3.3. Evaluation of LC3 Conversion in H4IIE Cells
Treatment of another hepatoma cell line, H4IIE, with Lipo2000/siNC, also led to
an increase amount of LC3-II. Lipo2000/siNC (100 nM) treatment of H4IIE cells resulted
in a 1.7 and 1.6-fold increase of LC3-II relative to non-treated and 100 nM siNC-treated
cells, respectively (Fig. 23). These results verified that the induction of LC3 conversion
by Lipo2000/siNC lipoplex was also exhibited by another hepatoma cell line.

Figure 23. Western blot of LC3 conversion in H4IIE cells. Cells were treated for 6 h with
different concentrations of 100 nM Lipo2000/siNC lipoplexes and then replaced with
H4IIE complete medium for additional 42 h. After treatments, cells were washed, and
cell lysates were collected, run on 15% SDS-PAGE and immunoblotted with anti-LC3
and anti-β-actin (Reproduced from [1] with permission from Humana Press, Inc. via
Copyright Clearance Center).

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5.3.4. Quantification of GFP-LC3 Fluorescence in Huh7.5 Cells
5.3.4.a. Positive Controls – MG-132 and Glucosamine
Autophagy activators, MG-132 and glucosamine, were used to demonstrate
changes to GFP-LC3 fluorescence. Treatment with 10 μM MG-132 and 40 mM
glucosamine resulted in a 5.3 and 2.7-fold increase of GFP-LC3 fluorescence than non-
treated cells (Fig. 24.A). Therefore, treatment of the stably transfected Huh7.5 cells with
the autophagy inducers led to increase amounts of total GFP-LC3 (GFP-LC3-I and GFP-
LC3-II) fluorescence.

5.3.4.b. Lipo2000 Lipoplex
GFP-LC3 fluorescence was measured at different time points in Huh7.5 cells
treated with escalating doses of Lipo2000/siNC lipoplex (Fig. 24.B). At 24 h, 200 nM
Lipo2000/siNC lipoplex significantly enhanced GFP-LC3 fluorescence by 22%. At 48 h,
both 100 and 200 nM Lipo2000/siNC lipoplex significantly increased GFP-LC3
fluorescence by 29 and 54%, respectively. At 72 h, all three siRNA concentrations of 50,
100 and 200 nM Lipo2000/siNC lipoplex led the greatest increase of GFP-LC3
fluorescence (35, 48 and 65%, respectively). No significant changes were observed for 10
μg/ml Lipo2000 or 200 nM siNC alone at any time point. Thus, these results suggested
that Lipo2000/siNC lipoplex treatment increased total GFP-LC3 fluorescence, and these
changes were dose- and time-dependent.

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5.3.4.c. MAP-polyplex
GFP-LC3 fluorescence was also measured at different time points in Huh7.5 cells
treated with escalating doses of MAP-siNC-polyplex (Fig. 24.C). At 24 h, 200 nM MAP-
siNC-polyplex significantly enhanced GFP-LC3 fluorescence by 20%. At 48 h, all three
siRNA concentrations of 50, 100 and 200 nM MAP-siNC-polyplex significantly
increased GFP-LC3 fluorescence by 10, 34 and 62%, respectively. At 72 h, the GFP-LC3
fluorescence continued to rise for 50, 100 and 200 nM MAP-siNC-polyplex-treated cells
(30, 46 and 135%, respectively). No significant changes were observed for 200 nM siNC
alone at any time point. Thus, these results suggested that MAP-siNC-polyplex treatment
also increased total GFP-LC3 fluorescence, and these changes were also dose- and time-
dependent.
87
A.
B.
C.

Figure 24. Detection of GFP
fluorescence of stably transfected
Huh7.5 cells. Huh7.5 cells, stably
transfected to express GFP-LC3,
were treated with (A) 10 μM MG-132
for 24 h and 40 mM glucosamine for
48 h. They were also treated with
different concentrations of (B)
Lipo2000 lipoplexes and (C) MAP-
polyplexes for 6 h and then replaced
with Huh7.5 complete medium. At
designated times of 24, 48, and 72 h
after the start of treatment, cell lysates
were collected and measured at Ex.
485 nm and Em. 518 nm and
normalized with protein
concentrations quantified using BCA
assay. Data is shown as a percentage
of non-treated groups at each time
point. Error bars indicate standard
deviation (n = 3). Asterisk (*)
represents p < 0.05 based on analysis
of variance and the Bonferroni test
(A, B – Reproduced from [1] with
permission from Humana Press, Inc.
via Copyright Clearance Center).
88
5.3.5. Detection of Autophagosomes in Huh7.5 Cells using Confocal Microscopy
Confocal microscopy was used to confirm the presence of autophagosomes after
Huh7.5 cells were treated with Lipo2000/siNC lipoplexes. Cells were treated with
glucosamine, a known autophagy activator, as a positive control. Glucosamine (40 mM)
showed an increased presence of GFP-LC3 puncta compared with non-treated cells (Fig.
25). These GFP-LC3 puncta were previously demonstrated to represent autophagosomes
[87]. A greater number of GFP-LC3 puncta was also detected in cells treated by 100 nM
Lipo2000/siNC lipoplex (Fig. 25). However, treatment of either 5 μg/ml Lipo2000 or 100
nM siNC alone did not produce an increase of GFP-LC3 puncta.

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Figure 25. Visualization of autophagosome formation in Huh7.5 cells, stably transfected
to express GFP-LC3. Cells were treated for 6 h in OptiMEM and then replaced with
Huh7.5 complete medium (except for 40 mM glucosamine treatment which lasted for 48
h). At 48 h from the start of treatment, cells were washed and fixed with 4% para-
formaldehyde. The nuclei of the fixed cells were stained with DAPI and then mounted on
slides for observation using confocal microscopy (Green: GFP-LC3 signal. Blue: DAPI
signal) (Reproduced from [1] with permission from Humana Press, Inc. via Copyright
Clearance Center).
90

5.3.6. Effects of Alternative Negative Control siRNAs for siRNA Delivery on Autophagy
in Huh7.5 Cells
5.3.6.a. LC3 Conversion
Lipo2000 was used to deliver three different negative control siRNAs
intracellularly, and the amount of LC3 conversion was detected. Relative to non-treated
cells, 100 nM Lipo2000/siNC, Lipo2000/siNCambion and Lipo2000/siNCqiagen
lipoplexes increased the amount of LC3-II by 1.6, 2.0 and 1.7-folds (Fig. 26.A). On the
other hand, compared to non-treated cells, 100 nM siNC, siNCambion and siNCqiagen
changes were relatively less (1.2, 1.5 and 1.5-fold, respectively) (Fig. 26.A).

5.3.6.b. Quantification of GFP-LC3 Fluorescence
Lipo2000 was used to deliver three different negative control siRNAs
intracellularly, and the amount of GFP-LC3 fluorescence was also detected. All three
Lipo2000/siRNA lipoplexes led to comparable and significant increases of GFP-LC3
fluorescence relative to non-treated cells (Fig. 26.B). However, the degree of GFP-LC3
fluorescence increase was greater for Lipo2000/siNC lipoplex (40%) than
Lipo2000/siNCambion and Lipo2000/siNCqiagen lipoplexes (23 and 22%, respectively).
The negative control siRNAs alone did not induce any significant changes compared with
non-treated cells.

91
A.
B.
Figure 26. Comparison of different negative control siRNAs for (A) LC3 conversion and
(B) GFP-LC3 fluorescence in Huh7.5 cells, stably transfected to express GFP-LC3. Cells
were treated for 6 h and then replaced with Huh7.5 complete medium. (A) At 24 h from
the start of treatment, cell lysates were collected, run on 15% SDS-PAGE and
immunoblotted with anti-LC3 and anti-β-actin. Lane 1: OptiMEM; Lane 2 10 μg/ml
Lipo2000; Lane 3: 100 nM siNC; Lane 4: 100 nM Lipo2000/siNC; Lane 5: 100 nM
siNCambion; Lane 6: 100 nM Lipo2000/siNCambion; Lane 7: 100 nM siNCqiagen; Lane
8: 100 nM Lipo2000/siNCqiagen. (B) At 72 h from the start of treatment, cell lysates
were collected and measured at Ex. 485 nm and Em. 518 nm and normalized with protein
concentrations quantified using BCA assay. Data are shown as a percentage of non-
treated groups at each time point. Error bars indicate standard deviation (n = 3). Asterisk
(*) represents p < 0.05 based on analysis of variance and the Bonferroni test (Reproduced
from [1] with permission from Humana Press, Inc. via Copyright Clearance Center).
92
5.4. Discussion
In this chapter, the autophagic effects of negative control siRNA delivery using
Lipo2000, a commonly used cationic lipid transfection reagent, and MAP-polyplex, a
novel amphipathic CPP-based siRNA delivery vehicle, on Huh7.5 hepatoma cells were
studied. Different assays were performed to evaluate the changes to the basal autophagic
condition of the cells. Negative control siRNAs, reported to have no non-specific
silencing effects, were used as a control to avoid the possibility that silencing a particular
gene would result to inadvertent autophagic effects. It is important to consider the effects
of these siRNA transfection systems during their applications.
Lipo2000 lipoplex and MAP-polyplex were first shown to transport siDY547
intracellularly at different concentrations (Fig. 21). Cellular uptake of Lipo2000/siDY547
lipoplex and MAP-siDY547-polyplex was dose-dependent, where increasing
concentrations of Lipo2000/siDY547 lipoplex or MAP-siDY547-polyplex resulted in
greater internalization of siDY547. At the same siRNA concentrations, MAP-polyplex
delivered greater intracellular amounts of siDY547 than Lipo2000 lipoplex. These results
verified that either transfection reagent was able to deliver siRNA intracellularly.
LC3 conversion is a widely accepted marker of autophagosomes formation [112].
Upon autophagy induction, LC3-I, a soluble cytosolic protein, is conjugated to
phosphotidylethanolamine (PE) to form LC3-II, which subsequently becomes membrane
bound to autophagosomes. Thus, the amount of LC3-II relative to non-treated control
cells is considered as a sign of autophagosomes accumulation and the most direct method
for detection of autophagy induction [112-114]. Despite having a larger molecular weight
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than LC3-I, LC3-II migrates quicker during the separation of these two endogenous
proteins during electrophoresis due to its greater hydrophobicity [112]. LC3 conversion
was used to evaluate the autophagic effects of Lipo2000 lipoplex and MAP-polyplex
transfection in Huh7.5 cells, stably transfected to express GFP-LC3 fusion protein.
Treatment of MG-132, a proteasome inhibitor and a known autophagy activator
[115], on Huh7.5 cells showed that the stably-transfected cells responded as expected
(Fig. 22.A). Increased concentrations of both Lipo2000/siNC lipoplex (Fig. 22.B) and
MAP-siNC-polyplex (Fig. 22.C) resulted in increased amounts of LC3-II. Furthermore,
the amount of intracellular siDY547 after 6 h transfection correlated with the induction of
LC3 conversion by Lipo2000 lipoplex and MAP-siNC-polyplex. These observations
suggested the presence of autophagosomes in treated cells was due to the cellular uptake
of siRNA or the siRNA delivery vehicle. Lipo2000 alone also showed greater LC3-II
levels than non-treated cells, which agreed with a study regarding cationic lipids inducing
autophagy [116]. Nevertheless, the treatment with 200 nM Lipo2000/siNC lipoplex still
resulted in higher expression of LC3-II than the treatment with the same amount of
Lipo2000 or siNC alone. LC3-II amounts were also increased in H4IIE cells treated with
100 nM Lipo2000/siNC lipoplex compared with non-treated cells. Thus, the increased
presence of autophagosomes was observed in human hepatoma cells (Huh7.5) and rat
hepatoma cells (H4IIE).
Changes to the amount of GFP-LC3 fluorescence after lipid- and CPP-based
transfection was evaluated in Huh7.5 cells, stably transfected to express GFP-LC3 fusion
protein. GFP-LC3 was commonly used for the observation of autophagosomes during
94
autophagy [113,117], but it had also been reported for detection using flow cytometry-
based assays to determine autophagic activity [118,119]. A fluorescence-based assay was
developed to quantify total GFP-LC3 (GFP-LC3-I and GFP-LC3-II) expression levels,
which indicate the amount of intracellular autophagosomes. Huh7.5 cells treated with
mTOR-dependent (MG-132 treated) [115] and mTOR-independent (glucosamine treated)
[120] autophagy activators resulted in increased GFP-LC3 fluorescence (Fig. 24.A).
Enhanced GFP-LC3 fluorescence was also observed after treatment with Lipo2000/siNC
lipoplexes (Fig. 24.B) and MAP-siNC-polyplexes (Fig. 24.C). The increase of GFP-LC3
fluorescence depended on the concentration of Lipo2000/siNC lipoplex and MAP-siNC-
polyplex treatment. Although each concentration of treatment led to different amounts of
GFP-LC3, they all shared a similar pattern where the fluorescence values steadily rose at
each time point. The observed dose- and time-dependent trend indicated that the
intracellular processing of internalized Lipo2000 lipoplex and MAP-polyplex may
increase GFP-LC3 levels due to the accumulation of the delivery vehicles over time.
Even lowest concentration of treatment (50 nM) eventually led to significantly greater
fluorescence levels than non-treated cells. Observations using confocal microscopy
confirmed that the detection of relative GFP-LC3 fluorescence corresponded to the
presence of autophagosomes in Huh7.5 cells (Fig. 25). Therefore, the long-term effects of
siRNA uptake should be taken into account, since cellular response may not be
immediately obvious.
A notable difference between GFP-LC3 detection (Fig. 24.B) and LC3 conversion
(Fig. 22.B) assays was the response to Lipo2000 alone. Lipo2000 alone (10 μg/ml) did
95
not increase GFP-LC3 fluorescence relative to control at any time point, whereas the
same treatment showed higher amounts of LC3-II than non-treated cells at 48 h
treatment. The different responses from these two studies may be due to differences in
the sensitivity, or inherent distinctions in the detection method (i.e. measurement of LC3-
II presence in immunoblotting vs. increased total GFP-LC3 in the fluorescence detection
assay).
Additional negative control siRNAs, siNCambion and siNCqiagen, were also
used to alleviate concerns about unintended non-specific effects by siNC and verify that
the Lipo2000/siNC lipoplex produced the observed increases of GFP-LC3 fluorescence
and LC3-II. All three negative control siRNAs, two from Ambion and one from Qiagen,
exhibited similar results, where the Lipo2000 lipoplexes led to greater relative amounts
of LC3-II (Fig. 26.A) and GFP-LC3 (Fig. 26.B), albeit to different amounts. The
differing extents in the studies may be attributed to the distinct complexation structures
formed by different siRNA oligonucleotide sequences and Lipo2000. Thus, two
conclusions can be made based on the treatment of various negative siRNAs using
Lipo2000. First, the siRNA sequence affected the degree of autophagic changes, and
more importantly, second, any siRNA mixed with Lipo2000 will influence basal
autophagy conditions.
The increase of LC3 conversion, GFP-LC3 fluorescence and autophagosomes
presence after siRNA transfection could be attributed to either autophagy induction or
inhibition [112,113]. Autophagy induction involves autophagosomes formation, which
results in GFP-LC3 production and LC3 conversion. Conversely, autophagy inhibition
96
regards accumulation of autophagosomes due to decreased autophagic degradation and
maturation, which results in higher amounts of LC3-II and GFP-LC3 fluorescence. The
cause of increased LC3-II and GFP-LC3 fluorescence may be due to autophagy
induction, rather than inhibition, because of the size of siRNA delivery vehicles.
Intracellular delivery of particles has been reported to induce autophagy [106,107], and
the degree of autophagy activation correlated with the particle size [108]. Thus, Lipo2000
lipoplex and MAP-polyplex are also particles that might be recognized for intracellular
processing through autophagy processing, since one of autophagy’s functions is to clear
out protein aggregates [109-111]. On the other hand, the increased intracellular presence
of siRNA after delivery by Lipo2000 and MAP-polyplex may have contributed to the
increase of autophagosomes. Further studies are required to make more definitive
conclusions the exact cause for the increase of autophagosomes.

97
5.5. Summary
In Chapter 5, lipid- and CPP-based siRNA transfections were shown to increase
the presence of autophagosomes in Huh7.5 and H4IIE hepatoma cells. These conclusions
were demonstrated using detection of LC3 conversion using immunoblotting, observation
of autophagosome formation using confocal microscopy, and measurement of GFP-LC3
fluorescence using a newly established fluorescence spectroscopy-based assay. Thus, it is
necessary to be aware of these autophagic effects when using lipid- and CPP-based
siRNA delivery vehicles for transfections. The results indicated that the effects of these
transfections on autophagy were dose-dependent and also increased with prolonged
incubation time. Therefore, since siRNA dwells intracellular for ~66 h [121,122], the
long term effects need to be considered, especially with regards to the potential
development of siRNA delivery systems as therapeutic agents.

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CHAPTER 6: SUMMARY AND FUTURE PERSPECTIVES

6.1. Summary
Exogenously delivered siRNA has been considered as a potential pharmaceutical
therapeutic agent for various diseases. One of the proposed approaches is to utilize CPPs.
CPPs are short peptide strands (<30 amino acids) that contain a significant number of
basic amino acids (lysine, arginine and histidine). CPPs rely on their cationic nature to
efficiently accumulate intracellularly, which is relevant for drug delivery and
oligonucleotide transfections. However, CPPs can be neutralized by anionic siRNA, and
the loss of cationic charges compromises CPP internalization capabilities. The focus of
this dissertation regarded the development of a new CPP-based polyplex to overcome this
neutralization issue.
First, K21-PDP was demonstrated to form a stable siRNA polyplex that was
capable of releasing the oligonucleotide under cytosolic-like conditions. The polyplex
also exhibited multiple conjugation sites available for functional moieties. Thus, this
polyplex platform was used to compare the delivery efficiencies of two types of CPP –
cationic R6 and amphipathic MAP – by conjugating each CPP carrier to the polyplex to
form R6-polyplex and MAP-polyplex, respectively. Since K21-PDP already neutralized
the siRNA in the polyplex, the conjugated CPP carriers were able to facilitate cellular
uptake without being inhibited by electrostatic interactions with siRNA. Results from
these studies indicated that MAP-polyplex delivered siRNA intracellularly and reduced
protein expression more significantly than R6-polyplex, while both exhibited comparable
99
cytotoxicity effects. MAP-polyplex also demonstrated comparable knockdown activities
to Lipo2000 lipoplex. Therefore, amphipathic MAP was a more suitable CPP carrier for
siRNA delivery than cationic R6.
Evaluation of MAP-polyplex design revealed that each component of the MAP-
polyplex design was shown to have an essential role for facilitating siRNA uptake and
activity. Four elements were involved in this design. First, siRNA was the cargo agent
that induces gene silencing. Second, K21-PDP was the scaffold that neutralized siRNA
and provided conjugation sites for functional moieties, such as CPP carriers for
enhancing cellular uptake. Third, MAP was the CPP carrier that delivered siRNA
intracellularly and also provided steric stability to the polyplex. The cellular uptake of
MAP-polyplex, similar to MAP alone, behaved in vesicle- and temperature-dependent
manners. Fourth, the disulfide bonds connected the carrier with the polyplex, and their
reduction permitted the release of siRNA. The separation of siRNA from the polyplex
was also demonstrated in a cytosolic-like environment that possesses both reducing and
proteolytic conditions. Therefore, MAP-polyplex was a viable agent for siRNA delivery
and efficacy and represents the foundation of a CPP-based siRNA delivery vehicle.
Another advantage of the design of this polyplex/MAP-polyplex was that it allowed
conjugation of any other functional moieties. These studies provided relevant insights for
the further development of CPP-based vehicles for siRNA therapeutic agents.
During the development of MAP-polyplex, lipid- and CPP-based siRNA
transfections were found to enhance the intracellular presence of autophagosomes in
hepatoma cells. Increased LC3 conversion, GFP-LC3 fluorescence and GFP-LC3 puncta
100
indicated changes to basal autophagy conditions due to Lipo2000 lipoplex- and MAP-
polyplex-mediated delivery of siRNA. Not only were these changes dependent of the
treatment concentration, but they were also influenced by the treatment duration. These
findings are particularly relevant for the application of siRNA transfections for autophagy
studies in vitro and the development of siRNA as a therapeutic agent in vivo. However, it
remains to be investigated whether a prolonged, increased presence of autophagosomes is
beneficial or harmful for the treated cells.
The results of this dissertation present relevant considerations for siRNA delivery
and efficacy. Amphipathic MAP has been found to be more suitable for siRNA delivery
than cationic R6, since MAP-polyplex has demonstrated enhanced siRNA cellular uptake
and efficacy compared with R6-polyplex. The properties of MAP-polyplex necessary to
achieve RNAi activity have also been elucidated. Therefore, MAP-polyplex serves as a
promising platform for future siRNA delivery systems. In addition, lipid- and CPP-based
siRNA transfections have also been shown to increase the presence of intracellular
autophagosomes. These effects should be taken into consideration for the application of
siRNA delivery systems. Therefore, the contents of this dissertation provide important
insights for the future development of siRNA as a therapeutic agent.
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6.2. Future Perspectives
MAP-polyplex represents the foundation of a CPP-based siRNA delivery vehicle.
However, for in vivo application in its current state, MAP-polyplex would most likely be
transported to the liver through passive targeting or a tumor through enhanced permeation
and retention effect before being cleared via the reticuloendothelial system or renal
filtration. Therefore, the addition of a targeting ligand could enhance MAP-polyplex
efficacy by directing it to the desired site of interest. One advantage of MAP-polyplex is
the availability of conjugation sites for additional functional moieties, such as a targeting
ligand. Conjugation of a targeting ligand for tumors, such as transferrin, folic acid or an
antibody, would be very relevant for in vivo application of MAP-polyplex because it
would direct MAP-polyplex to the desired location for cancer treatment.
Additional changes to MAP-polyplex would also reveal interesting insights into
its design. In Chapter 4, a model of siRNA release from MAP-polyplex was proposed,
where K21-PDP could be digested by lysine-specific peptidases to release siRNA.
Instead of using a 21mer oligo-L-lysine, 21mer oligo-D-lysine could be used to evaluate
the importance of peptide digestion for in vitro transfections because D-peptides are not
readily degraded. Furthermore, the covalent disulfide bond between the carrier and
polyplex was previously shown to be essential for polyplex stabilization and gene
silencing. A non-reversible covalent linker could be substituted for the disulfide bond to
confirm the significance of a reversible bond. Finally, another design consideration
would be to vary the length of K21-PDP to see whether a shorter or longer oligolysine
would enhance or decrease cellular uptake and RNAi.
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MAP-polyplex can also be used as a model delivery vehicle for in vitro studies
involving siRNA transfections. In particular, certain cells have been shown to exhibit
poor transfection efficiency. MAP-polyplex should be evaluated for siRNA delivery in
difficult-to-transfect cells and primary cells. The design and sequence of MAP is derived
from α-helical peptides that show efficient intracellular uptake without cell specificity, so
MAP-polyplex should also promote high internalization without limitation. On the other
hand, if MAP-polyplex were not successful, then determining the cause of ineffectiveness
would also be intriguing area of study. After all, the reason for lack of sufficient siRNA
uptake and activity in certain cells has not been elucidated yet. Studying the distinctive
properties between cell type and transfection efficiency, such as cellular defense
mechanism, the extracellular expression of proteins and sugars or the intracellular
trafficking of oligonucleotides, may reveal why certain cells are more susceptible to
transfection than others. This knowledge would be useful for enhancing siRNA efficacy
as a therapeutic agent. Therefore, the limiting factor for siRNA transfection in all cell
types remains to be determined.
Another research area of interest is the relationship between siRNA transfection
and autophagy. The increased presence of autophagosomes after lipid- and CPP-based
siRNA transfection was described in Chapter 5. However, it was not clear whether it was
the intracellular presence of siRNA or siRNA and delivery vehicle that increased the
amount of autophagosomes. Particles, such as Lipo2000 lipoplex or MAP-polyplex, may
have induced autophagosome formation due their size and/or charge. Thus, additional
physiochemical characterization of Lipo2000 lipoplex and MAP-polyplex should be
103
performed, so that a correlation may be established between their properties and
autophagy. Furthermore, siRNA itself may also contribute to the effect. The presence of
foreign double-stranded RNA may stimulate the treated cell to eliminate itself through
the autophagy pathway. If this is the case, then autophagy may also play a role in the
extent of gene silencing based on the rate of siRNA clearance from the cytosol.
CPPs are a promising area of research interest due their ability to efficiently
accumulate intracellularly. Their application is particularly relevant for siRNA delivery
and efficacy since cells do not readily take in the oligonucleotide. In this dissertation, a
promising K21-PDP-based polyplex platform was introduced and evaluated to contain
siRNA as cargo. The platform was used to select the optimal type of CPP carrier for
siRNA delivery. Amphipathic MAP was determined to be preferable to cationic R6, and
MAP-polyplex was shown to enhance siRNA uptake and promote gene silencing. In
addition, lipid- and CPP-based transfections were found to alter basal autophagy
conditions, which may affect siRNA efficacy. Therefore, the contents of this dissertation
provide insights into siRNA intracellular delivery properties that can contribute to the
future development of siRNA as a therapeutic agent.
104
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Abstract (if available)

Abstract In this dissertation, a novel cell penetrating peptide (CPP)-based polyplex was developed and evaluated for small interfering RNA (siRNA) delivery and efficacy. The design resolved the CPP carrier neutralization issue, where CPP cellular uptake is compromised by electrostatic interactions between the CPP and siRNA, by using a chemically modified 21mer oligolysine (K21-PDP) to form a stable and neutralized polyplex with siRNA. This neutralized polyplex served as a suitable platform to conjugate distinct CPP carrier moieties – cationic (hexaarginine, R6) and amphipathic (model amphipathic peptide, MAP) – to determine which CPP property was more suitable for siRNA delivery. Since K21-PDP already effectively neutralized the siRNA, the CPP carriers on the conjugated CPP-polyplexes, R6-polyplex and MAP-polyplex, were able to function without electrostatic interference. Amphipathic MAP was found to be a better CPP carrier than R6 because MAP-polyplex exhibited greater siRNA intracellular uptake and gene silencing than R6-polyplex, while still maintaining comparable cytotoxicity effects. ❧ The design of MAP-polyplex was also further evaluated. MAP-polyplex showed comparable effects with the commonly used lipid-based siRNA transfection reagent Lipofectamine 2000. MAP-polyplex entered the cell through a vesicle-based mechanism, and its transfection efficiency was not affected by the co-incubation of 10% fetal bovine serum. In addition, the roles of each component of MAP-polyplex – K21-PDP, siRNA, MAP and disulfide bond linkage – in promoting intracellular siRNA delivery and efficacy were elucidated. Therefore, MAP-polyplex is a promising CPP-based platform that can be used for siRNA delivery. ❧ The effects of siRNA transfection on autophagy in hepatoma cells were also presented in this dissertation. A method was developed to evaluate the presence of intracellular autophagosomes using fluorescence spectroscopy. An increased signal of a stably expressed fluorescent fusion protein as an autophagy marker was detected in transfected cells relative to non-treated cells. The results corresponded with increased LC3 conversion and autophagosomal punctate via immunoblotting and confocal microscopy, respectively, which are also markers commonly used in autophagy studies. Using these methods, lipid-based (Lipofectamine 2000) and CPP-based (MAP-polyplex) siRNA transfections elicited an increase of autophagosomes in hepatoma cells in dose- and time-dependent manners. These effects should be considered for the application of siRNA delivery systems. Therefore, the findings of this dissertation present relevant considerations for siRNA delivery and provide important insights for the future development of siRNA as a therapeutic agent.

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Mo, Robert H. (author)

Core Title Cell penetrating peptide-based polyplexes for sirRNA delivery

School School of Pharmacy

Degree Doctor of Philosophy

Degree Program Pharmaceutical Sciences

Publication Date 05/02/2012

Defense Date 03/02/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag amphipathic,autophagosomes,autophagy,cationic,cell penetrating peptides,membrane transduction domains,model amphipathic peptide,OAI-PMH Harvest,oligoarginine,polyarginine,polyplex,siRNA,siRNA delivery

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Shen, Wei-Chiang (committee chair), Mackay, John Andrew (committee member), Masood, Rizwan (committee member), Wang, Clay C.C. (committee member), Ying, Shao-Yao (committee member)

Creator Email rhm2022@columbia.edu,robert.h.mo@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-22203

Unique identifier UC11288316

Identifier usctheses-c3-22203 (legacy record id)

Legacy Identifier etd-MoRobertH-709.pdf

Dmrecord 22203

Document Type Dissertation

Rights Mo, Robert H.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA