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siRNA-loaded Cationic Liposomes for Cancer
Therapy: Development, Characterization and
Efficacy Evaluation
Thesis Presented
By
Bo Ying
To
The Bouvé Graduate School of Health Sciences
in Partial Fulfillment of the Requirements for the Degree of Doctor of
Philosophy in Pharmaceutical Sciences with specialization in
Pharmaceutics and Drug Delivery System
NORTHEASTERN UNIVERSITY
BOSTON, MASSACHUSETTS
April 2010
ii
ABSTRACT
Cancer is a major health problem in the United States and many other
parts of the world. However, cancer treatment is severely limited by the lack of
highly effective cytotoxic agents and selective delivery methods which can serve
as the “magic bullet” (first raised by Dr. Paul Ehrlich, the goal of targeting a
specific location without causing harm to surrounding tissues or to more distant
regions in the body).
The revolutionary finding that tumors cannot grow beyond a microscopic
size without dedicated blood supply provided a highly effective alternative for the
treatment of cancer. Currently, anti-angiogenic therapy and the discovery of RNA
interference makes it possible to treat some conditions by silencing disorder-
causing genes of targeting cells which are otherwise difficult to eradicate with
more conventional therapies. However, before siRNA technology could be widely
used as a therapeutic approach, the construct must be efficiently and safely
delivered to target cells. Strategies used for siRNA delivery should minimize
uptake by phagocytes, enzymatic degradation by nucleases and should be taken
up preferentially, if not specifically, by the intended cell population.
Kinesin spindle proteins (KSP) are the motor proteins which play critical
roles during mitosis. Different from tubulins which are also present in post-mitotic
cells, such as axons, KSP is exclusively expressed in mitotic cells, which makes
them the ideal target for anti-mitotics.
iii
In the present study, we intend to develop, characterize and evaluate a
liposome-based delivery system which can deliver KSP siRNA selectively to the
tumor vasculature (thus inhibiting angiogenesis, destroying tumor vasculature
and eventually, eradicating tumor growth).
We first developed ten different liposome preparation types with different
compositions of lipids. Next, the capacity for loading siRNA and efficiency of
targeting the tumor vascular supply was evaluated using relevant cellular and
tumor models. Pegylated cationic liposomes (PCLs) were selected as carriers for
siRNA. Based on the silencing efficiency of siRNA formulated with different
PCLs, DOPC based cationic liposomes, over DOPE based nanosystems, with a
modest amount of polyetheleneglycol was selected to deliver KSP siRNA to
tumor-bearing mice. Efficacy studies revealed that tumor suppression was
observed when KSP siRNA was delivered using PCLs, but not in mice that
received naked KSP siRNA or KSP siRNA in commercially available transfecting
agents. The results were further supported by MRI (magnetic resonance
imaging) analysis.
To evaluate the role that vasculature supply plays in the development of
the tumor, we also performed tumor response studies using a tumor model
consisting of tumor cells which are resistant to KSP siRNA. The results showed
that a prolonged suppression of tumor growth was achieved only when a large
dose (5mg/kg) KSP siRNA was administered, but not with the administration of a
relatively low dose (2mg/kg) of siRNA, suggesting that a combined treatment
iv
approach containing both anti-vasculature and anti-cancer agents should be
considered to achieve the best treatment outcome.
Finally, it was confirmed by qRT-PCR that the tumor growth inhibition was
due to the successful knock-down of KSP mRNA.
v
ACKNOWLEDGEMENTS
First and foremost, I express my sincerest gratitude to my advisor, Dr.
Robert Campbell, who has supported me throughout my thesis with his patience
and knowledge whilst allowing me the room to work in my own way. One simply
could not wish for a better or friendlier advisor.
It is such a pleasure for me to thank all of my committee members for their
valuable suggestions and precise time.
It is a great honor to have Dr. Dinah Sah in my committee who has
supported me throughout my thesis. Without her and the generous support from
Alnalym Pharmaceutics, this project would be completely impossible.
I would also like to express my special thanks to Dr. John Gatley for the
educative and intriguing conversations about this project.
I can never thank my colleagues enough for the consistent help and
support throughout my thesis work.
I will also like to thank my parents for their support. My mom, as the first
teacher in my life, directed me to the path of intellectual pursuit ever since I was
a child. My Dad never talked a lot, instead, showed me the deepest love that one
can live on for the entire life.
Finally, I would like to thank everybody who was involved in the successful
completion of this thesis, such as Dr. Akio Ohta and Dr Dmitry, as well as to
express my gratitude for everyone who is not mentioned here.
vi
Table of Contents
1. ABSTRACT ..................................................................................................... ii
2. ACKNOWLEDGEMENTS ................................................................................ v
3. LIST OF FIGURES ......................................................................................... ix
4. LIST OF TABLES ............................................................................................ x
5. ABBREVIATIONS: .......................................................................................... xi
6. BACKGROUND AND SIGNIFICANCE ............................................................ 1
7. STATEMENT OF HYPOTHESIS ................................................................... 14
8. SPECIFIC AIMS ............................................................................................ 15
9. MATERIALS AND METHODS ....................................................................... 16
Materials ................................................................................................... 16
Cell culture ............................................................................................... 17
Liposome preparation .............................................................................. 17
Liposome toxicity study ............................................................................ 18
Liposome uptake study ............................................................................ 19
Efficiency of siRNA loading in liposomes ................................................. 19
Stability of siRNA in serum ....................................................................... 19
Liposomal siRNA uptake study ................................................................ 20
Locating liposomal siRNA inside cells ...................................................... 20
vii
Doubling time study .................................................................................. 21
Growth inhibition by KSP siRNA .............................................................. 21
Cell cycle analysis using flow cytometry .................................................. 21
RNA isolation and real-time RT–PCR ...................................................... 22
Animal protocol ........................................................................................ 22
Tumor models and treatment ................................................................... 23
Body weight measurement ....................................................................... 24
Animal survival study ............................................................................... 24
Magnetic Resonance Imaging and analysis ............................................. 25
Immunohistochemistry ............................................................................. 25
Statistics ................................................................................................... 26
10. RESULTS ...................................................................................................... 27
Characterization of liposomes .................................................................. 27
Liposome toxicity study ............................................................................ 28
Cellular uptake study: ............................................................................... 34
siRNA encapsulation efficiency study and stability in serum .................... 34
siRNA uptake study .................................................................................. 42
Growth inhibition by KSP siRNA .............................................................. 47
Cell cycle arrest by KSP siRNA ................................................................ 51
Tumor response study ............................................................................. 53
viii
Magnetic Resonance Imaging analysis .................................................... 56
Quantitative analysis of KSP mRNA on tumor tissues ............................. 58
Animal survival study ............................................................................... 65
11. DISCUSSION ................................................................................................ 67
12. SUMMARY .................................................................................................... 73
13. CONCLUSIONS ............................................................................................ 74
14. REFERENCES .............................................................................................. 76
15. APPENDIX .................................................................................................... 86
ix
LIST OF FIGURES
Figure 1 Mechanism of RNA interference. ......................................................... 5
Figure 2 Tumor angiogenesis and anti-vasculature therapy using cationic
liposomes .......................................................................................... 13
Figure 3 Cell viability as a function of liposome preparation type. ................... 31
Figure 4 DIC microscopic images of MS1-VEGF cells exposed to different
DOPC- or DOPE-based cationic liposome preparations. .................. 33
Figure 5 Cellular uptake as a function of liposome preparation type. .............. 37
Figure 6 siRNA loading efficiency as functions of liposome composition,
concentration and preparations. ........................................................ 40
Figure 7 Influence of liposomes on siRNA stability in serum containing
medium.. ............................................................................................ 41
Figure 8 siRNA uptake by MS1-VEGF cells evaluated using FACS analysis. . 45
Figure 9 siRNA uptake by MS1-VEGF cells using fluorescence-enhanced DIC
microscopy.. ...................................................................................... 46
Figure 10 Doubling time of different tumor and endothelial cell lines. ................ 47
Figure 11 Cell growth inhibition by KSP siRNA-loaded Pegylated cationic
liposomes. ......................................................................................... 50
Figure 12 Cell cycle arrest by inhibition of kinesin spindle protein.. ................... 52
Figure 13 Tumor response as a function of time-KSP silencing effect. ............. 54
Figure 14 Percent body weight change during the treatment period. ................ 55
Figure 15 Tumor response evaluated using magnetic resonance imaging.. ...... 57
Figure 16 Quantitative analysis of KSP mRNA using qRT-PCR. . ..................... 59
x
Figure 17 Tumor response as a function of anti-vasculature therapy. ............... 62
Figure 18 Percent body weight change during the treatment period.. ............... 63
Figure 19 Tumor response evaluated using magnetic resonance imaging.. ...... 64
Figure 20 Animal survival study.. ....................................................................... 66
LIST OF TABLES
Table 1 Characterization of liposomes ................................................................ 27
xi
ABBREVIATIONS:
bEND.3: Brain Endothelial cells;
DAPI: 4, 6-Diamidino-2-Phenylindole;
DMEM: Dulbecco's Modified Eagle Medium;
DOPC: 1, 2-Dioleoyl-sn-Glycero-3-Phosphocholine;
DOPE: 1, 2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine;
DOTAP: 1, 2-Dioleoyl-3-Trimethylammonium-Propane;
EBM-2: Endothelial Cell Basal Medium-2;
FACS: Fluorescence Activated Cell Sorting;
FITC: Fluorescein Isothiocyanate;
HMEC-1: Human Dermal Microvascular Endothelial Cells;
HUVEC: Human Primary Umbilical Vein Endothelial Cells;
KSP: Kinesin Spindle Protein;
MS1: Mile Sven 1 (Murine endothelial cells);
MRI: Magnetic Resonance Imaging;
PCL: Pegylated Cationic Liposome;
RPMI: Roswell Park Memorial Institute medium;
RISC: RNA-Induced Silencing Complex;
SCID: Severe Combined Immunodeficiency;
siRNA: Small Interfering RNA;
SRB: SulfoRhodamine B;
VEGF: Vascular Endothelial Growth Factor;
1
BACKGROUND AND SIGNIFICANCE
Current status of cancer
Cancer is a major public health problem in the United States and many
other parts of the world. Currently, one in four deaths in the United States is due
to cancer 1. It is estimated that about 1.5 million cases of cancer in 2009 and
562,340 Americans will die from cancer, corresponding to more than 1,500
deaths per day 1. All these numbers collectively suggest that the current
treatment for cancer is far away from satisfaction; and that an effective treatment
approach against cancer is urgently needed.
Characteristics of tumor vasculature
Cancer is generally induced by uncontrolled cell proliferation 2. However, a
tumor cannot grow beyond ~ 1 mm3 in size without a dedicated blood supply, and
these in situ cancers are harmless to the host 3-7. Therefore, the formation of a
lethal malignancy requires tumor cell proliferation plus angiogenesis 7. The
revolutionary finding that angiogenesis is critical for tumor growth and
development has created new opportunities to fight cancer 8. For example,
disrupting the structure and function of tumor blood vessels, and suppressing
cancer associated neovessel formations, are two extensively studied areas in
basic and clinical cancer research 9-11. Compared to targeting tumor cells directly,
interfering with tumor blood vessels has some special advantages. First, because
of the direct access to their targets, if administered intravenously, therapeutic
2
agents used for tumor vascular destruction avoid some critical physiological
barriers compared to tumor interstitial targeting, such as relatively high interstitial
fluid pressure, disorganized tumor vessels and long interstitial transport
distances 12. Second, drug resistance is a major problem in cancer treatment,
since after lone term exposure to chemotherapeutic agent, cancer cells may no
longer respond to the treatment. Or more too often, cancer cells, due to the
intrinsic instability of their genome, may develop resistance to several completely
different chemotherapeutic agents simultaneously, also known as multidrug
resistance (MDR). However, endothelial cells are genetically more stable
compared to cancer cells, which minimizes the potential to acquire drug
resistance 13. Finally, compared to normal endothelial cells in quiescent tissues,
endothelial cells lining the tumor vasculature proliferate at an accelerated rate, as
high as 1000-fold has been reported 10, 13-14.
The tumor vascular supply is highly disorganized and newly formed blood
vessels may possess very different properties based on anatomic location of
tumors or exogenous growth factors 15. But all angiogenic blood vessels exhibit
the overexpression of negatively charged functional groups such as
proteoglycans and glycosaminoglycans on the luminal side of the tumor vessel
wall 10-11, 16-17, making it possible to target therapeutic agents more selectively to
angiogenic vessels 13. Studies have also shown that tumor endothelial cells
preferentially take up cationic liposomes rather than electroneutral or anionic
components. Although the exact mechanism(s) involved is not clear, studies
3
suggest that the over abundance of anionic functional groups on the surface of
tumor endothelia might play a critical role 13, 18. In further support of the overall
importance of charge in tumor vascular targeting, other studies have reported
that the overall cationic charge content, resulting from the inclusion of 10 to 50
mol% of a cationic lipid of liposomes, can be used to control the distribution of
liposomes between the vascular and extravascular tumor compartment 19.
Kinesin Spindle Protein and its important roles in mitosis
Since the fundamental difference between endothelial cells lining the
tumor vasculature and normal vasculature is the division of cells, and mitosis is
the critical step during cell division, attacking mitotic endothelial cells is a highly
effective therapeutic approach. Microtubules have been long employed as
targets for treat cancer and microtubule targeting agents belong to the most
successful anti-cancer drugs (i.e. paclitaxel and vinca alkaloids). Disrupting the
dynamics of microtubule by microtubule-targeted drugs will lead to cell cycle
arrest at mitosis, and ultimately cell death 20-21. However, microtubules are also
present in post-mitotic cells and have been widely involved in many physiological
processes. They are a component of the cytoskeleton and regulate motility,
transport of proteins and vesicles along axon fibers, which is major reason for the
neurotoxicity observed on cancer patients with microtubule targeting drugs 22-25.
Therefore, there is an increasing need for developing new antimitotics which can
specifically target dividing cells so as to reduce the side effect associated with
the use of tubulin-targeting agents.
4
Kinesin spindle protein (KSP), a member of kinesin superfamily, plays a
critical role in mitosis as it mediates the separation of centrosomes and bipolar
spindle assembly 22, 25. Kinesins are characterized by a ~340 amino acid motor
domain, which contains an ATP binding pocket and the microtubule binding
interface 25. Next to the motor domain or ‘head’, kinesins contain a ‘stalk’ region,
followed by the ‘tail’ domain. The highly conserved motor domain of KSP
provides binding sites for microtubules and nucleotides, and the mechanical
force required for the plus-end directed movement of KSP is generated through
the hydrolysis of ATP 22, 25. At the onset of mitosis, the duplicated centrosomes
separate and generate two star-like structures (microtubule asters). KSP motors
are moving to the plus-ends of microtubules, and promote bipolarity. During
metaphase, a stable bipolar spindle is formed and KSP generate a poleward flux
so that duplicated centrosomes can be separated equally to two daughter cells 25.
Inhibition of KSP function will lead to the failure of centrosome segregation,
prolonged cell cycle arrest at mitosis and eventually, cell death. In contrast to
microtubules which are also present in post-mitotic neurons, KSPs are
exclusively expressed in mitotic cells, which make them the ideal targets for anti-
cancer agents 25. Therefore, since tumor endothelial cells are dividing quickly, if
selective delivery of KSP inhibitors to the tumor vasculature can be achieved,
apoptosis of endothelial cells will be induced hence tumor growth will be inhibited
due to nutrient and oxygen deprivation.
5
Figure 1 Mechanism of RNA interference. Ref 30
RNA interference
RNA interference (RNAi) is a mechanism for RNA-guided regulation of
gene expression in which double-stranded ribonucleic acid inhibits the
expression of genes with complementary nucleotide sequences 26. RNAi was first
observed on worms
where a complete
silencing of
targeting gene was
achieved after the
injection of long
dsRNA to the
embryos of
Caenorhabditis
elegans 27. Subsequently, it was found RNAi could also be induced in cultured
mammalian cells using duplexes of 21-nucleotide RNAs called small interfering
RNAs (siRNA) 28. These findings sparked the explosion of research to uncover
new mechanisms of gene silencing, and provided powerful new tools for both
biological research and drug discovery. RNA interference, among the most
conservative pathways during the long history of evolution, is employed as a
defending mechanism for eukaryotes and prokaryotes against virus infection.
Intrinsically, RNAi is guided by microRNAs (miRNA) which are naturally
transcribed from genome. In the endogenous pathway, stem loops or hairpin
structures containing RNAs, usually encoded in the untranslated regions or
6
introns, are processed in nucleus and exported to the cytoplasm by exportin 5 as
~70-nt-long precursor strands (pre-miRNA) 29-31. In the cytoplasm, pre-
microRNAs will be further processed by dicer, another RNA enzyme III, into a
transient ~22- nucleotide miRNA:miRNA duplex. This duplex is then loaded into
the miRNA-associated multiprotein RNA-induced silencing complex (miRISC),
which includes the Argonaute proteins, and the mature single-stranded miRNA is
preferentially retained in this complex. Then, the single-stranded miRNA will be
employed by RISC as a template for searching and binding to mRNAs which
share complementary sequences and regulate gene expression in one the the
two ways depending on the complementarity between the miRNA and targeting
mRNA. If the complementarity is perfect, the mRNA will be cleaved and
degraded; however, if the complementarity is less than perfect, protein
translation will be halted 29-30.
RNA interference can also be induced using exogenous siRNA which is a
class of ~20-25-nt-long double-stranded RNA molecules. siRNA enter RNA
interfering pathway in the late stage since they are directly mimicking the
products after dicer cleavage. Since siRNA are double-stranded RNA molecules
the designed functional strand is called antisense and the other one, sense or
passenger strand. However, how does RISC know which strand is the one to
incorporate and which strand is to be degraded? Analysis of siRNA duplex
sequences shows that the thermodynamic stability of their two RNA ends
determines which strand is incorporated into RISC 32-33. A duplex RNA that is
less thermodynamically stable in the 5′ region than in the 3′ region of the
7
guide strand would efficiently initiate the directional unwinding activity of RISC
from the 5′ end and would incorporate the guide strand into a functional RISC.
During the siRNA-RISC assembly process, the passenger strand is destroyed
and the removal of this strand facilitates RISC formation, but destruction of the
sense strand is not crucial for miRISC assembly 34-36. Different from miRNA,
siRNA preferentially bind to mRNA which share perfect complement sequences,
which is the advantage of using siRNA over miRNA since the binding is
predictable and easy to manipulate.
Since the Nobel prize-winning discovery of RNA interference 27, billions of
dollars have been invested in both basic and clinical research of silencing
disease-causing genes which are otherwise difficult to treat by conventional
approaches 37-41. Despite the early successes gained from ongoing clinical trials
with topical administration of siRNA 41-42, the unfavorable pharmacokinetic profile
and the lack of organ-specific uptake limit the use of RNAi therapeutics for
disease prevention and treatment 43-45. For example, siRNA are very potent in
the cytosol and the silencing effect of one i.v. injection can last for weeks 46,
however, siRNA are extremely unstable in serum when unprotected (with a half
life of about 1.5 minutes) 47. SiRNA are highly specific to their targeting mRNA
molecules in the cytosol but have no preference on the targeting organs or
eventual accumulation sites 26, 45, 48. Even though a small part of administered
siRNA may reach the target cells, the high molecular weight (~13 kDa) and
negative charges make it unfavorable for them to diffuse across cell membranes
8
43-44. Lastly, even though a small quantity of naked siRNA can be randomly taken
up by cells through endocytosis, without an efficient way of escaping, siRNA will
be degraded in lysosomes before the silencing function can be achieved in the
cytosol 26, 43-45, 48. Therefore, the development of effective drug delivery vehicles
is in high demand.
Liposome as drug carriers
Since the first observation by Alec Bangham roughly 40 years ago that
phospholipids in aqueous systems can form closed bilayer structures, liposomes
have become a pharmaceutical carrier of choice for many drugs and the
enthusiasm of developing liposomal drug is only increasing. Compared to many
other drug carriers, liposomes have their unique advantages. First, because of
the structure of liposome is an aqueous core enclosed by lipid bilayers,
hydrophilic drugs can be encapsulated into the aqueous core, while for
hydrophobic drugs, lipid bilayers. Second, most phospholipids used in preparing
liposomes, such as phosphatidylcholine and phosphatidylethanolamine, are the
natural component in cell membranes, which make liposomes the ideal candidate
for biologically compatible and degradable carriers 49-50. Third, drugs
incorporated in liposomes will be protected from direct contact with serum
proteins thus to show prolonged half-life in serum 49, 51. For instance, doxorubicin
has a half life in blood of ~5 minutes, indicating rapid tissue uptake of the drug.
However, after incorporation into liposomes, most of the drug is cleared with an
elimination half-life of 20–30 hours, and the area under the concentration-time
9
curve (AUC) is increased at least 60-fold compared with free doxorubicin 52.
Conventional doxorubicin is administered at doses of 60–90 mg/m2 every 3
weeks, while the current FDA-recommended dose of PEGylated liposomal
doxorubicin is 50 mg/m2 every 4 weeks 53. Fourth, to utilize the enhanced
permeability and retention (EPR) effect in tumor, liposomes have been employed
to improve the specific delivery of cytotoxic drugs to solid tumors. As a
consequence, a decreased toxicity because of less accumulation of cytotoxic
drugs in the normal tissues was observed in clinic 49, 54. Last but not the least, the
surface characterizations of liposomes can be easily modified based on different
delivering requirements. For example, polyetheleneglycol (PEG) has been used
to enhance the circulating time of conventional liposomes in blood 55-56.
Moreover, different antibodies have been attached to the surface of liposomes or
to the distal end of PEG chains to enhance the uptake of liposomes by target
cells overexpressing specific ligands under certain pathological conditions 49, 57-
60.
Cationic liposomes in tumor vascular targeting and gene therapy
As relatively safe alternatives to virus vectors, liposomes have not only
been used for delivering chemotherapeutic agents to the tumor site, but also for
the successful delivery of genes and proteins to target cells 49, 61. To employ the
acidic environment in the endosome, liposomes are constructed from pH-
sensitive components. Phosphatidylethanolamine (PE)-containing liposomes are
stable in the blood, but undergo a phase transition at acidic endosomal pH. This
10
destabilizes the endosome membrane which facilitates the release of
oligonucleotide into the cytosol 61-62. Although cationic lipid-based carriers have
been used quite successfully to deliver genes to cells 49, 63-66, toxicity is
associated with the use of cationic lipids 61, 67-69, which severely limits its potential
for clinical applications. Another drawback for cationic lipid-based gene delivery
system is the relatively short circulating time in bloodstream and massive uptake
by the organs from reticulo-endothelial system (RES), especially liver, which may
result in an insufficient exposure of targeting cells to the liposomes 49, 51, 70.
Therefore, polymers such as polyetheleneglycol (PEG) have been used to shield
liposome from opsonization so as to minimize the RES uptake 49, 51-52, 54, 70-71.
However, decreased transfection efficiency was also observed after the inclusion
of PEG into liposomes, which is believed due to the prevention of transition from
lamellar phase to hexagonal phase 72. . Interestingly, different effects on DNA
transfection and siRNA-induced gene targeting efficacy were also observed on
PEG-grafting polyethylenimine (PEI) 73. So whether or not to use PEG and the
proper amount of PEG to be used is still under investigation.
Another fundamental difference between gene delivery and siRNA
delivery is in terms of schedule and dose administration. As we know, protein
expression from a successful gene transfection can last for a long time, however,
silencing effect from siRNA is relatively shorter, which means a more frequent
administration of siRNA will be required for clinic use. In order to secure the gene
silencing-potential of siRNA, the carrier molecule must be capable of delivering
11
siRNA to target cells with minimum-associated cellular toxicity. Given the level of
cellular toxicity reported for the use of many cationic liposomes, at the moment it
is thus difficult to simultaneously archieve both safe delivery and efficient gene-
silencing function of siRNA to a given population of cells 67-68.
12
13
Figure 2 Tumor angiogenesis and anti-vasculature therapy using cationic
liposomes (Reference 13). Left: Tumor angiogenesis is a step by step
process. An angiogenic stimulus is secreted by a developing tumor and a
vessel sprouts in the direction of the stimulus (A), proteases begin to degrade
the basement membrane (B), while endothelial cells migrate in the direction of
the stimulus formed through the newly formed openings in the basement
membrane (C), and a new vessel sprout forms (D). Right: Vascular targeting
with cationic liposomal therapeutics: The tumor vasculature is lined with an
overexpression of anionic functional groups (A), cationic liposomal
therapeutics interact with tumor vessels (B), injury to the tumor
microvasculature results in damage to the endothelial cells (C), and eventual
loss of tumor vessel function results in the death of thousands of cancer cells
owing to severe oxygen and nutrient deprivation (D). Ref. 13
14
STATEMENT OF HYPOTHESIS
1. The inclusion of polyethylene glycol (PEG) will efficiently reduce the toxicity of
liposomes associated with the use of cationic lipids.
2. Both the cationic charge and the inclusion of PEG are critical for the most
efficient cellular uptake of liposomes.
3. Inclusion of cationic lipids and PEG will improve the efficiency of loading
siRNA in liposomes.
4. PEG modified cationic liposomes (PCLs) will provide greater protection to
siRNA in serum compared to the unmodified liposomes.
5. PCLs will enhance silencing efficiency of siRNA (KSP) in vitro and in vivo.
6. Tumor growth will be suppressed through silencing KSP in vivo.
7. Tumor growth can be inhibited using anti-vasculature therapy.
8. MRI can be used to evaluate tumor response.
15
SPECIFIC AIMS
1. To develop and characterize different siRNA liposome formulations.
2. To compare the targeting efficiency of different liposome carriers using
cellular models of the tumor vasculature.
3. To evaluate the loading efficiency for siRNA in different liposome carriers.
4. To evaluate the protective role of different liposome carriers for siRNA in
serum.
5. To evaluate the delivery efficiency of siRNA to tumor endothelial cells by
different liposome carriers using FACS analysis.
6. To evaluate the intra-cytosol delivery of siRNA by different liposome carriers
using fluorescence enhanced DIC microscopy.
7. To evaluate the growth inhibition efficiency of siRNA (KSP) using various cell
lines.
8. To evaluate the potential of tumor growth inhibition by silencing kinesin
spindle proteins using KSP siRNA-loaded PCLs.
9. To evaluate the potential of tumor growth inhibition by destroying tumor
vasculature using KSP siRNA-loaded PCLs.
10. To use MRI to evaluate tumor response to siRNA-PCLs therapy compared to
other formulation varieties.
16
MATERIALS AND METHODS
Materials
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE), 1,2-Dioleoyl-sn-
Glycero-3-Phosphocholine (DOPC), 1, 2-Dioleoyl-3-Trimethylammonium-
Propane (DOTAP), 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-
[Methoxy(Polyethylene glycol)-2000 (DOPE-PEG2000), 1,2-dipalmitoyl-sn-glycero-
3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (rhodamine-DPPE),
1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(Carboxyfluorescein) (FITC-
DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL). Sulforhodamine
B (SRB) was purchased from Sigma-Aldrich (St Louis, MO), TCA (trichloroacetic
acid) and 1% acetic acid were purchased from Fisher Scientific Company (Fair
Lawn, NJ). Cy3 labeled random sequence siRNA (MW ~13kDa) was purchased
from Applied Biosystems (Austin, TX). KSP siRNA and negative control siRNA
were generously provided by Alnylam Pharmaceuticals (Cambridge, MA).
Dialysis membranes were purchased from Spectrum Laboratories (Rancho
Dominguez, CA). E-Gel® (4% agarose gel) was purchased from Invitrogen
(Carlsbad, CA). Primers for KSP and beta-actin mRNA were purchased from
Applied Biosystems (Austin, TX). Male SCID (severe combined immunodeficient)
mice were purchase from Charles River Laboratories International, Inc.
(Wilmington, MA)
17
Cell culture
Murine endothelial cell line MS1-VEGF and bEND-3, human primary
umbilical vein endothelial cell line (HUVEC), murine melanoma cell line B16-F10
and murine breast cancer cell line 4T1 were purchased from ATCC (American
Type Culture Collection, Manassas, VA). Human dermal microvascular
endothelial cell (HMEC-1) line was obtained by Centers for Disease Control and
Prevention (Atlanta, GA). Cell culture growth media DMEM, EBM and RPMI were
purchased from ATCC (Manassas, VA). Heat-inactivated FBS (fetal bovine
serum) was purchased from Hyclone (Logan, UT). MS1-VEGF, bEND-3 and
B16-F10 cells were maintained in DMEM with 10% FBS, HMEC-1 and HUVEC
were maintained in EBM with additional endothelial cell growth kit-BBE (ATCC
PCS-100-040). 4T1 cells were maintained in RPMI with 10% FBS. All cell line
cultures were grown in a humidified atmosphere of 5% CO2 at 37 °C.
Liposome preparation
The liposomes employed for this study were classified as either PC
(DOPC/DOTAP/PEG2000) or PE (DOPE/DOTAP/PEG2000) based liposome
preparations, and were used at different molar ratios (100/0/0, 50/50/0, 48/50/2,
45/50/5 and 40/50/10). Fluorescein isothiocyanate (FITC) or rhodamine was
added into liposome formulations at the ratio of 5 mol% if fluorescence indicators
were needed. Liposomal formulations were prepared by thin film and hydration
method as previously reported 74. Briefly, a rotary evaporator was employed to
remove solvent from a pyrex tube containing lipid mixed at the appropriate ratios
18
and the purex tube (placed inside a round bottom flask) rotated continuously in
the water bath at 42 °C for 30 minutes or until a thin film was deposited on the
inside wall of pyrex tube. The lipid film was hydrated with 1X PBS, or PBS
containing siRNA in the water bath at 37 °C, and then placed in an ice bucket at
15-minute intervals for at least 8 cycles. To reduce particle size, liposomes were
passed through a 0.1μm filter for 11 times by extrusion (Avanti Polar Lipids,
Alabaster, AL). Particle size and zeta (ζ) potential of liposomes after extrusion
were determined by 90 Plus Particle/Zeta Potential Analyzer (Brookhaven
Instruments, Holtsville, NY).
Liposome toxicity study
Cells were seeded at 1*104 cells/ml in 48-well plates. Following a 24-hour
incubation period cells were exposed to various amounts of liposomes for an
additional 24 hours. Percent of cell viability was determined using
sulforhodamine B (SRB) cytotoxicity assay 75, and the percent of viable cells
remaining was calculated as follows:
Percent of cell viability= fluorescence intensity of treated cells / fluorescence
intensity of untreated cells (control) *100
19
Liposome uptake study
Cells were seeded at 1*104 cells/ml in 48-well plates. Following a 24-hour
incubation period, cells were exposed to various amount of rhodamine labeled
liposomes for an additional 24 hours. Fluorescence intensity was next analyzed
by a fluorescence spectrophotometer (Bio-Tek® Instruments Inc., VT) at the
excitation/emission wavelength of 540/590 nm.
Efficiency of siRNA loading in liposomes
FITC labeled lipid films were hydrated with siRNA-containing PBS. The
final concentration of liposomes and siRNA in stock was 2µmol/ml and 100nM,
respectively. Next, liposomes were transferred to a dialysis membrane with a
pore size of 30 kDa and then dialyzed overnight. Liposomes in control groups
were dialyzed under similar experimental conditions but in membranes with pore
sizes of 500 Da to prevent free siRNA from diffusing through the membrane.
Fluorescence signals from FITC and Cy3 were used as indicators for presence of
liposomes and siRNA, respectively. Fluorescence intensity values were
determined with a use of a fluorescence spectrophotometer.
Stability of siRNA in serum
Fetal bovine serum (FBS) was used at a concentration of 50% (v/v) to
simulate the serum levels in vivo. Following a 3-hour incubation period with FBS,
samples from both experimental and control groups were loaded onto 4%
agarose gel followed by electrophoresis for 30 minutes. Pictures were acquired
20
using Molecular Imager ChemiDoc XRS+System (Bio-Rad Laboratories,
Hercules, CA).
Liposomal siRNA uptake study
Cells were seeded at 5*105 cells/1ml in 6-well plates. Following a 24-hour
incubation, cells were exposed to various amount of FITC-labeled liposomes
containing Cy3-labeled siRNA for an additional 6 hours. Next, cells were
trypsinized and cell suspensions were centrifuged at 1000 rpm for 5 minutes.
Pellets were then resuspended in sheath solution and fluorescence signals from
FITC and Cy3 were analyze using FACS analysis (BD Biosciences, Franklin
Lakes, NJ).
Locating liposomal siRNA inside cells
Cells were seeded at 5*105 cells/1ml on a covering slip in 6-well plates.
Following a 24-hour incubation, cells were exposed to FITC-labeled liposomes
(200 nmole) containing Cy3-labeled siRNA (100pmole) for an additional 6 hours.
Cells on the covering slips were mounted on slides with slow fade (Invitrogen,
Carlsbad, CA). Images were captured using a fluorescence enhanced differential
interference contrast microscope (Olympus BX61WI, Melville, NY) under different
fluorescence channels at 40X magnification. Fluorescence pictures were further
processed and analyzed using IPLAB software (BD Biosciences, Franklin Lakes,
NJ).
21
Doubling time study
Cells were seeded at equal numbers into T25 flasks. An additional 24
hours was given to allow complete attachment of cells to the flasks. Then cells
were harvested at different time points and counted under a conventional light
microscope using a hemocytometer. The doubling time was calculated based on
the cell counts at each time point using GraphPad Prism 5.0 (GraphPad
Software, Inc., La Jolla, CA).
Growth inhibition by KSP siRNA
Cells were seeded at 1*104/ml into 48-well plates and incubated for 24
hours to allow complete attachment. KSP siRNA or control siRNA formulated by
liposomes were added to cells at increased concentrations. Sulforhodamine B
assay was employed to detect the growth inhibition after 48 hours of incubation.
Percent of growth inhibition= [1 - fluorescence intensity of treated cells /
fluorescence intensity of untreated cells (control)] *100
Cell cycle analysis using flow cytometry
MS1-VEGF cells were seeded at 5*105 cells/1ml in 6-well plates.
Following a 24-hour incubation period, cells were then exposed to KSP siRNA or
control siRNA (100 pmole) formulated by different liposomes. After 24 hours,
cells were detached from the surface using trypsin and cell suspensions were
centrifuged at 1000 rpm for 5 minutes. Pellets were then resuspended in 1X
PBS, centrifuged at 1000 rpm for another 5 minutes before cells were fixed using
22
70% ice cold ethanol. Fixed cells were centrifuged for 5 min at 3000 rpm. The
ethanol was then discarded and the pellet was resuspended. Cells were stained
with propidium iodide for 30 min at room temperature before being analyzed
using flow cytometry.
RNA isolation and real-time RT–PCR
Tissue samples collected from tumors in different groups were subjected
to RNA-STAT-60 (TEL-TEST, Friendswood, TX) according to the manufacturer's
instructions. The total RNA extracted from cells was reverse transcribed to
synthesize cDNA using SuperScript first-strand synthesis kit (Invitrogen Life
Technologies, Carlsbad, CA). The total RNA was reverse transcribed in a final
reaction volume of 20μl, using random hexamers for 10min at room temperature,
1h at 42°C and 15min at 70°C. Real-time PCR was performed using Taqman
master mix (Applied Biosystems, Foster City, CA). Beta-actin mRNA was
employed as the internal standard. The real-time PCR was performed using
Applied Biosystems 7300 Real-Time PCR system (Foster City, CA).
Animal protocol
For studies involving the use of mice, the animal protocol was approved
by the Institutional Animal Care and Use Committee at Northeastern University
(NEU), Boston, MA, USA. All experiments were performed in accordance with
the institutional guidelines at approved NEU facilities.
23
Tumor models and treatment
Melanoma tumor model was established on male SCID mice. In brief,
B16-F10 cells (1*106) were suspended in 0.1cc growth medium and injected
subcutaneously into the right flank of mice. Mice were randomly divided into
different treatment groups. Tumor volume was measured daily from day 12
following the injection of cells, and the treatment started when the tumor volume
reached ~400mm3 (considered as day 0). KSP siRNA or control siRNA in PCL
formulation were administered at 2mg/kg to mice on day 0, 3 and 7. Mice in the
liposome control group received empty liposome carriers (DOPC/DOTAP/DOPE-
PEG2000) at the concentration of 40µmol/ml.
Breast cancer model was established on male SCID mice. In brief, 4T1
cells (1*106) were suspended in 0.1cc growth medium and injected
subcutaneously into the flank of mice. Mice were randomly divided into different
treatment groups. Tumor volume was measured everyday from day 7 after
inoculation, and treatment started when tumor volume reached ~200mm3
(considered as day 0). KSP siRNA were administered at 2mg/kg (low dose
group) or 5mg/kg (high dose group) to mice on day 0, 4 and 8. Mice in control
siRNA group received 5mg/kg control siRNA at the same time interval. Mice in
liposome control group received empty liposome carriers (DOPC/DOTAP/DOPE-
PEG2000) at 40µmol/ml.
24
Tumor volume was measured daily using digital caliper and calculated using the
formula below
V = 0.52 * (Length) * (Width) 2
Body weight measurement
Body weight was monitored daily, and was used as an indicator of the
toxicity caused to the mice during the entire treatment period. Euthanasia was
applied whenever mice lost more than 20 percent of the initial body weight.
Animal survival study
4T1 cells (1*106) were suspended in 0.1cc growth medium and injected
subcutaneously into the right flank of mice. Mice were randomly divided into
different groups and treatment started on day 7 following tumor cell injection. In
PCL alone group, mice received liposomes at 40µmol/ml. Mice in PCL/control
siRNA group received 5mg/kg control siRNA. KSP siRNA in 2mg/kg and 5mg/kg
were administered to mice in low dose and high dose PCL/KSP siRNA treatment
groups, respectively. The criteria for animal survival were one or more of the
following: 1) natural death; 2) more than 20% of body weight loss observed; 3)
tumor volume of 1000 mm3 observed. Mice received euthanasia when the last
two situations applied.
25
Magnetic Resonance Imaging and analysis
Magnetic resonance imaging was performed on a 7 T preclinical MRI
system (BioSpec 70/20USR, Bruker BioSpin Corp., Billerica, MA) at the Center
for Translational NeuroImaging at Northeastern University. A whole body
quadrature coil was used for reception and transmission. Imaging sequence used
was a T1 RARE spin-echo (1738/10 [repetition time /echo time msec], 90º flip
angle) with coronal slices of 0.75 mm in thickness covering whole animal. MR
images were processed using MATLAB and MIVA (Medical Image Visualization
and Analysis Software) to calculate tumor volumes. Tumor volumes were
calculated by manually selecting the tumor areas in each slice and counting the
total number of voxels in the entire tumor area.
Immunohistochemistry
Mice were sacrificed; and the liver, spleen, lung, and tumors were
collected. The tissues were fixed with paraformaldehyde, embedded in paraffin
and cut into thin sections. H&E staining was performed on these thin sections by
standard procedure. To visualize blood vessels, CD31 staining was performed on
the sections using conventional staining methods. Briefly, thin tumor sections
were incubated with rat anti-mouse CD31 antibody (dilution, 1:3) (catalog no.
553370; BD-Pharmingen, Sandiego, CA, USA). A goat anti-rat antibody with
horseradish peroxidase was used as the secondary antibody. Next, chromogen
3, 3'-diaminobenzidine tetrahydrochloride was added to the sections followed by
26
counterstaining with hematoxylin, and sections were mounted on slides for
analysis.
Statistics
Unless mentioned specifically, all data were analyzed using SPSS 16.0
with One-Way ANOVA, post-hoc Tukey, and plotted using Sigmaplot 11.0 (Systat
Software, Inc., San Jose, CA). In the present study, the following were used as a
measure of statistical significance, *, p<0.05; **, p<0.01; *** p<0.001.
27
RESULTS
Characterization of liposomes
Particle size and zeta (ζ) potential of liposomes were determined by 90
Plus Particle/Zeta Potential Analyzer after extrusion (Table 1). In the DOPC
based formulation group, the particle size decreased significantly from 178 nm to
149 nm following the inclusion of DOTAP. The inclusion of 2 mol% of PEG2000
decreased the particle size to 113 nm. However, an additional increase in the
amount of PEG did not alter liposome size. Liposomes formed by DOPC alone
showed a zeta potential of 2 mv. The incorporation of 50 mol% of DOTAP
significantly increased the zeta potential to 45 mv. When 2, 5 or 10 mol% of PEG
was incorporated into DOPC/DOTAP liposomes, the zeta potential decreased to
34 mv, 23 mv and 5.0 mv, respectively. A similar trend for particle size and zeta
Composition Ratio Particle size (nm)
Zeta potential(mv)
DOPC
DOPC 100 178 ± 17.6 2 ± 4.1 DOPC/DOTAP 50/50 149 ± 14.3 45 ± 3.7
DOPC/DOTAP/PEG2000
48/50/2 113 ± 7.2 30 ± 3.9 45/50/5 105 ± 4.5 23 ± 2.6 40/50/10 102 ± 6.9 5 ± 2.4
DOPE
DOPE 100 341 ± 41.3 - 3 ± 5.3 DOPE/DOTAP 50/50 217 ± 21.8 43 ± 7.7
DOPE/DOTAP/PEG2000
48/50/2 118 ± 10.5 31 ± 4.9 45/50/5 113 ± 8.3 21 ± 4.6 40/50/10 104 ± 2.3 -1.1 ± 4.7
Table 1 Characterization of liposomes
28
potential was observed for the DOPE containing formulation group, however,
relatively large liposome diameters were reported for these preparations.
Liposome toxicity study
Liposome toxicity experiments were performed in vitro using a
sulforhodamine B cytotoxicity assay. In the DOPC based formulation group,
electroneutral liposomes, at concentrations up to 1mM, were not toxic to MS1-
VEGF (95±6.9%), HMEC (109 ±15.6%) or HUVEC (111±5.2%) cells (Figure 3, A,
B, C). However, non-Pegylated cationic liposomes (CLs) were toxic at
concentrations as low as 10µM. A sharp decrease in cell viability was observed
for both HMEC (88±10.9%) and HUVEC (87±11.9%) cells. The similar toxic effect
was also observed with MS1-VEGF cells at 50 µM (82±4.3%). The inclusion of
PEG for the PC and PE based preparations significantly diminished cellular
toxicity regardless of the molar ratio of PEG used.
Quantitative analysis revealed that CLs are quite toxic to cells, and that
the level of toxicity can be reduced by including PEG in the preparation of
cationic liposomes. However, it was still unknown as to what effect the different
liposome preparation types have on the morphology of cells. Following the
incubation of cells with liposomes for 24 hours, DIC microscopy was used to
determine the effect of liposome type on cell morphology. Cells exposed to ELs
were similar in morphology to control cells, however, healthy looking cells were
less apparent following incubation with the different cationic liposomes (Figure 4
29
C, I). In general, regardless of the PC or PE lipid employed, an increase in PEG
content limited (or prevented) unfavorable changes in cellular morphology
caused by the cationic lipids.
30
31
Figure 3 Cell viability as a function of liposome preparation type. Cells were
seeded at 1×104 cells/well in 48 well plates 24 hours before exposed to
liposomes and cell viability was measured 24 hours after exposure to various
concentrations of liposomes. Solid circles (●) indicate liposomes formed by
electroneutral lipids alone; open circles (◯) indicate liposomes formed by either
DOPC or DOPE with DOTAP. Solid triangles (▲), open triangles (△) and solid
squares (■) represent liposomes formed by electroneutral lipids and cationic lipid
DOTAP with 2, 5 or 10 percent of PEG2000, respectively. Dotted lines (┄) indicate
90% of cell viability. Results were presented as mean ± s.d. (n=6) and analyzed
using One-way ANOVA.* indicates significant difference compared to self-
control. (See appendix for pictures with higher resolution)
32
33
Figure 4 DIC microscopic images of MS1-VEGF cells exposed to different
DOPC- or DOPE-based cationic liposome preparations.
34
Cellular uptake study:
Rhodamine labeled electroneutral and cationic liposomes incorporated
with 2, 5, 10 mol% PEG were employed for cellular uptake studies. Non-
Pegylated cationic liposomes were not evaluated in this study due to the
relatively toxic nature of the preparations in the absence of PEG. Electroneutral
liposomes containing only DOPC (zeta potential: 2 ± 4.1 mv) or DOPE (zeta
potential: -3 ± 5.3 mv) did not show significant uptake by endothelial cells even
when used at concentrations as high as 1 mM (Figure 5). However, when
DOTAP and a modest amount of PEG (2 mol% and 5 mol%) were incorporated
into liposome preparations, a significant amount of cellular uptake was observed.
Although cationic liposomes with a relatively high content of PEG (10 mol%)
were non-toxic to the cells at the concentration of liposomes used, the amount of
PEG proved to be excessive, significantly limiting cellular uptake.
siRNA encapsulation efficiency study and stability in serum
A critical challenge that siRNA must overcome in vivo is the degradation
by nucleases 26, 45. So an ideal carrier should be able to protect siRNA from
nuclease activity. The aqueous core of liposomes has been explored for loading
a variety of agents (i.e., DNA, cytotoxic agents, proteins etc.); however, the
loading efficiency may vary depending on many factors, such as methodologies
used for loading siRNA, ratio between liposome and siRNA, and the lipids
employed for compositing liposomes. For this reason, two commonly used
methods for loading siRNA into liposomes were evaluated. It was revealed that
35
when liposomes were formed before siRNA was added into the suspension
(external addition), a significantly lower encapsulation efficiency of siRNA was
observed compared to the loading efficiency when dry lipid films were hydrated
using siRNA containing PBS (internal addition) (Figure 6A). Next, we determine
the optimal molar ratio of liposomes to siRNA. Using the DOPC-based
nanosystems, it was found that a minimum molar ratio of 2000 was required to
achieve 100 percent encapsulation of siRNA (Figure 6B). Therefore, using the
same ratio of liposome to siRNA, we determined the loading efficiency of siRNA
in liposomes with various compositions. In the electroneutral liposome groups
(prepared with the use of DOPC or DOPE), the Cy3 intensity of siRNA was
significantly lower compared to the control group following dialysis overnight
(FIGURE 6C). This suggests that a poor efficiency of siRNA loading was
achieved using the electroneutral-based preparation types. Once DOTAP was
included in the DOPC liposome preparation, most of the siRNA loaded was
retained, suggesting that a charge interaction between negatively charged siRNA
and positively charged lipids assisted in the efficiency of loading. The inclusion of
varying amounts of PEG further enhanced the loading of siRNA significantly.
36
37
Figure 5 Cellular uptake as a function of liposome preparation type. Cells were
seeded at 1×104 cells/well in 48-well plates for 24 hours. Cellular uptake was
measured 24 hours after cells were exposed to liposomes. Solid circles (●)
indicate liposomes formed by DOPC or DOPE alone; open circles (◯), solid
triangles (▲) and open triangles (△) represent liposomes formed by
electroneutral lipids (DOPC or DOPE) and cationic lipid DOTAP with 2, 5 or 10
percent of DOPE-PEG2000, respectively. Results were presented as mean ± s.d.
(n=6) and analyzed using One-way ANOVA. * indicates significant difference
compared to eletroneutral liposomes. (See appendix for pictures with higher
resolution)
38
B
A
39
C
40
Figure 6 siRNA loading efficiency as functions of liposome composition,
concentration and preparations. A, lipid films were hydrated to yield a final
liposome concentration of 200 µM. siRNA was added at final concentration of
100nM either internally or externally. B, loading efficiency of siRNA at a fixed
concentration (100nM) was determined using DOPC based PCLs at various
concentrations. C, Lipid films were hydrated with 100 nM siRNA in 1X PBS to
yield a final liposome concentration of 200 µM. Samples were dialyzed
overnight in membranes with pore size of 30 kDa. In the control group, samples
were dialyzed in membranes with pore size of 500 Da. Results were presented
as mean ± s.d. (n=8) and analyzed using One-way ANOVA.
Liposomes can be used as carriers for siRNA, however, it is well
established that cationic liposomes bind extensively to insoluble blood proteins 76-
77. It is not known, however whether the inclusion of PEG, and how much of it, is
required to protect against nuclease degradation. To investigate the protective
function of the different liposome formulations, PBS with 50% FBS was employed
to simulate the serum levels in vivo. Naked or liposomal siRNA wase first
incubated in 1X PBS or PBS/FBS for 3 hours before loaded onto 4% agarose gel
for electrophoresis. A clear band was observed on lane 1 but not on lane 2
(Figure 7), which indicated that all naked siRNA were degraded within 3 hours
following exposure to serum enzymes. A similar result was also observed in the
DOPE-siRNA group (lane 3 and lane 4), which further confirmed that DOPE by
itself could not efficiently load siRNA. Following the inclusion of DOTAP, clear
41
bands were observed in the loading wells on lane 5 and lane 6. Encapsulated
siRNA was immobilized during electrophoresis, indicating that siRNA was
retained in the well; however, the band in lane 6 was less prominent when
compared to lane 5. Following the inclusion of PEG into liposome formulations,
clear bands were observed in lanes 7 through 10, and the intensity of siRNA
bands in both control groups and FBS groups was similar, suggesting a better
protection of siRNA against degradation by nucleases. In the DOPC-siRNA
group, a band was observed in the well and another band was found on the gel
(lane 11), suggesting that siRNA was only partially protected by the DOPC
liposome preparation type. Finally, a single band in the well of lane 12 suggests
that all non-encapsulated siRNA was degraded.
Figure 7 Influence of liposomes on siRNA stability in serum containing
medium. Samples from each group were incubated either in 1X PBS (-) or
PBS/FBS (+) at 37 ⁰C for 3 hours before subjected to electrophoresis. Lane
1&2, naked siRNA; Lane 3&4, DOPE + siRNA; Lane 5&6, DOPE / DOTAP +
siRNA; Lane 7&8, DOPE / DOTAP / PEG (2%) + siRNA; Lane 9&10, DOPE /
DOTAP / PEG (10%) + siRNA; Lane 11&12, DOPC + siRNA. Arrows indicate
siRNA bands on the gel.
42
siRNA uptake study
Small interfering RNAs exert their silencing function against
complimentary messenger RNA in the cytoplasm. Liposomes can encapsulate,
and therefore protect, siRNA from being degraded. It is still unclear, however,
whether the encapsulated siRNA interacts with cells more extensively when
compared to naked siRNA. So, FACS analysis was employed to investigate the
uptake of siRNA by cells. Compared to the baseline control, no uptake of siRNA
could be observed in the DOPE-siRNA group and only a modest amount of
uptake was observed in the DOPC-siRNA group (Figure 8) when compared to
the naked siRNA group. The figure suggests that ELs are not an efficient
nanosystem for loading siRNA. Following the inclusion of DOTAP in the
liposomes a dramatic shift was observed when compared to the naked siRNA
group, suggesting an increase in siRNA uptake by cells compared to naked
siRNA. The enhanced cellular uptake was not abolished following the inclusion of
a modest amount of PEG (2% or 5%). However, when 10 mol% PEG
formulations were used, the uptake of siRNA by cells was similar to that of the
naked siRNA group, suggesting ≥10 mol% PEG is not beneficial.
One question FACS analysis was not able to address is whether siRNA
was taken up by cells or just superficially associated with cell membranes. For
this reason, fluorescence microscopy was employed for more visible
observations. Results from fluorescence images revealed that the Cy3 signal
intensity from siRNA was barely detectable for naked siRNA, DOPE-siRNA or
43
DOPE/DOTAP/PEG (10 mol%) groups (Figure 9), which is consistent with results
from FACS analysis. Although a significant amount of siRNA and liposome
uptake was observed in DOPE/DOTAP-siRNA group, no co-localization of the
two fluorescence signals, or perinuclear localization, was observed. This
suggests that the signal was a result of the background noise due to cellular
debris. However, a clear co-localization of Cy3 labeled siRNA and FITC labeled
liposomes was observed inside cells when siRNA was formulated using
DOPE/DOTAP/PEG (2 mol% or 5 mol%).
44
45
Figure 8 siRNA uptake by MS1-VEGF cells evaluated using FACS analysis.
FL1-H and FL2-H indicate FITC and Cy3 channels which were employed to
monitor the uptake of liposomes or siRNA by MS1-VEGF cells, respectively.
Black dotted lines indicate MS1-VEGF cells alone as baseline. Dark blue lines
represent siRNA alone. Light blue lines indicate the uptake of electroneutral
liposomes. Cationic liposomes without polyetheleneglycol are indicated in red
lines. Green, yellow and orange lines indicate PCLs with 2, 5, 10 percent of
DOPE-PEG2000, respectively. (See appendix for pictures with high resolution)
46
Figure 9 siRNA uptake by MS1-VEGF cells using fluorescence-enhanced DIC
microscopy. Nucleus were stained in DAPI (blue), liposomes and siRNA were
labeled using FITC (green) and Cy3 (red), respectively. Orange indicates the
merging of green and red.
47
Growth inhibition by KSP siRNA
Doubling time of all cell lines employed was determined. According to the
proliferating rate, 4T1 is the fastest growing cell line, followed by B16-F10, then
followed by three endothelial cell lines: MS1-VEGF, HMEC-1 and bEND-3,
respectively (Figure 10). To maintain the consistency, the above five cell lines
were exposed to KSP siRNA or control siRNA formulated by liposomes with
different compositions for 48 hours before the SRB cytotoxicity assay was
performed. Liposomes with control siRNA showed a marginal inhibition effect on
cell proliferation (Figure 11, upper panel). However, liposomes with KSP siRNA
Figure 10 Doubling time of different tumor and endothelial cell lines.
48
showed a concentration dependent growth inhibition on all endothelial cells and
tumor cells except 4T1, and the greatest inhibitory effect was observed on B16-
F10 cells when KSP siRNA was formulated using DOPC based liposome carriers
containing 5 mol% PEG (Figure 11, lower panel).
49
50
Figure 11 Cell growth inhibition by KSP siRNA-loaded Pegylated cationic
liposomes. Cells were seeded at 1×104 cells/well in 48-well plates 24 hours
before exposed to PCLs with KSP siRNA or control siRNA. Cell viability was
measured using SRB assay after 48-hour exposure to liposomes. Brown
represents naked siRNA without any liposome formulation. All liposomes used
above contain 50 mol% DOTAP and variable mol% of electroneutral lipids
(DOPC or DOPE, as indicated in the legend) and PEG2000 (as indicated by
numbers in the legend). Results were presented as mean ± STD. (See
appendix for pictures with high resolution)
51
Cell cycle arrest by KSP siRNA
The kinesin spindle protein plays an important role in mitosis as it
mediates the segregation of centrosomes during cell division. Therefore, the
knock-down of KSP will result in cell cycle arrest at metaphase when cells have
double the amount of genetic material in terms of total DNA content. After a 24-
hour incubation of cells with naked KSP siRNA or KSP siRNA formulated using
DOPC-based PCLs, MS1-VEGF cells were fixed and stained using propidium
iodide (PI) before FACS analysis. Untreated cells showed a ratio of ~1:5 between
cells undergoing G2 phase and G1 phase, respectively. Cells treated with naked
KSP siRNA exhibited a ratio of 1:4 between the two populations (G2/G1).
However, after the cells were exposed to PCL/KSP siRNA for 24 hours, the ratio
of cells that were arrested at G2/G phase was 1:2. The experimental findings thus
support the growth inhibitory effect of KSP siRNA against MS1-VEGF cells in
vitro.
52
Figure 12 Cell cycle arrest by
inhibition of kinesin spindle protein.
After 24-hour incubation with KSP
siRNA, MS1-VEGF cells were fixed
and stained using propidium iodide.
The criteria for selecting cells in G1
and G2 cycle were as shown on the
graph. In the control group (upper
panel), 25% of gated cells contained
one set of chromosome (2N), 5% of
gated cells showed doubled
chromosome contents (4N), a ratio
of ~1:5 was observed between cells
in G2 and G1. In cells treated with
naked KSP siRNA group (middle
panel), a ratio of ~1:4 (G2/G1) was
observed. In the PCL/KSP siRNA
treated group (lower panel), a ratio of
~1:2 was observed between cells in
G2 and G1 phase.
53
Tumor response study
B16-F10 cells were injected subcutaneously on SCID mice at number of
1*106 per mouse. All tumor-bearing mice were randomly divided into six groups.
Tumor length and width was measured daily using digital caliper and treatment
started only when the tumor volume reached ~400mm3 (Figure 13, day 0). Mice
received injections with different agents on day 0, 3 and 7 (indicated by arrows).
Compared to the mice in untreated group, a significant decrease in tumor growth
was observed in the KSP siRNA-loaded PCL-treatment group from day 4 after
injection, but not observed in mice from other groups. At the end of the treatment
(day 10), only the size of the tumors in the PCL/KSP-siRNA treatment group was
reduced to a significant extent (1562 ± 209 mm3), while tumors in all other groups
were approaching 4000 mm3 in size (Figure 13). During the entire treatment
period, no body weight decrease was observed in the tumor-bearing mice (Figure
14).
54
Figure 13 Tumor response as a function of time-KSP silencing effect. B16-F10
cells were inoculated on the right flank of SCID mice at number of 1×106 per
mouse. The treatment started when the tumor volume reached 400mm3 (day 0).
The days of injection were indicated by arrows. Solid circles (●) indicate
untreated group; open circles (◯) indicated mice treated with naked siRNA, solid
triangles (▲) indicate mice received liposome control; open triangles (△)
represent mice treated with KSP siRNA formulated by DOPE and DOTAP at
equal mole percentage; solid squares (■) and open squares (□) indicated mice
treated with control siRNA or KSP siRNA formulated by PCLs. Results were
presented as mean ± SEM. (n=4) and analyzed using One-way ANOVA.*
indicates significant difference compared to untreated group. (See appendix for
pictures with higher resolution)
55
Figure 14 Percent body weight change during the treatment period. Each
line indicates an individual mouse. Arrows indicate days when mice
received injections.
56
Magnetic Resonance Imaging analysis
Digital calipers have been widely used to measure superficial tumor
growths in tumor-bearing mice. However, there is a severe limitation with this
method given that tumors often grow beneath the skin surface, which is a major
drawback of using digital caliper alone for the evaluation of treatment effects.
Therefore, tumor responses to different treatments were also evaluated using
MRI (magnetic resonance imaging) and analysis. Several mice, one from each
experimental group were selected for MRI and analysis. The mice selected were
the most representative from each group. As shown on the images below, the
entire tumor, not only the part above the skin surface, was observed by MRI
(Figure 14). The entire tumor volume was next measured by counting the voxels
in the tumor area of all the MRI pictures. Mice in the untreated group, naked KSP
siRNA treated group, PCL alone group and PCL/control siRNA group showed
over 2000 voxels in the tumor area. Although the tumor volume of mice from
DOPE/DOTAP/KSP siRNA group did not show any difference from control
tumors when measured using digital calipers, MRI revealed that the tumor border
located beneath the skin was less compared to all the control groups (1700
voxels). However, a much greater decrease of the total number of voxels was
observed in PCL/KSP siRNA treated group (790 voxels). The results further
supported the notion that the suppression of tumor growth was best achieved
using KSP siRNA-loaded PCLs.
57
Figure 15 Tumor response evaluated using magnetic resonance imaging. The
number of voxels is the sum of results from 28 pictures per mouse.
58
Quantitative analysis of KSP mRNA on tumor tissues
Tumor tissues collected from mice in different groups were treated with
STAT-60 to extract the RNA. Quantitative RT-PCR analysis was performed
according to manufacturer’s instructions. β-actin was employed as the loading
control. No obvious changes in the total amount of KSP mRNA were observed in
mice treated with naked siRNA or PCL control (Figure 16). A slight decrease of
KSP mRNA was observed in mice that received PCL with control siRNA.
Although no significant decrease in tumor volume was observed in mice that
received KSP siRNA formulated in DOPE/DOTAP liposomes, the total amount of
KSP mRNA extracted from the tumor was decreased to ~ 50 percent compared
to the untreated tumors. However, a dramatic decrease in KSP mRNA was
observed in the mice that received KSP siRNA formulated by PCLs, only ~ 10
percent of KSP mRNA was recovered from the tumor tissue. This experimental
finding supports the notion that the tumor response to KSP siRNA treatment is
due to the successful knock-down of KSP mRNA (Figure 16).
59
Figure 16 Quantitative analysis of KSP mRNA using qRT-PCR. β-actin was
employed as loading control. All results were presented as percentage of
untreated tumors.
60
From the results above, melanoma from B16-F10 cells responded well to
treatment and PCL formulated KSP siRNA successfully suppressed the tumor
growth in the mice. However, since it has been shown that the growth of both
B16-F10 cells and endothelial cells can be inhibited by KSP siRNA, it was still not
clear if this suppression on tumor growth was due to the growth inhibition of
tumor cells or endothelial cells lining the tumor vasculature, or even both. Since
4T1 cells did not respond to KSP treatment in vitro, the tumors derived from the
same cell line could be used as a good model to evaluate the importance of
tumor vascular disruption during our KSP/PCL treatment approach.
4T1 cells were injected subcutaneously in SCID mice at 1*106 per mouse.
All tumor-bearing mice were randomly divided into five groups. Tumor length and
width was measured daily using digital caliper and treatment started only when
the tumor volume reached ~200mm3 (Figure 17, day 0). Mice received injections
with different agents on day 0, 4 and 8 (indicated by arrows). Compared to the
mice in untreated group, a significant suppression in tumor growth was observed
in mice given both low and relatively high dose of PCL/KSP siRNA treatment
(from day 4 after injection), but not observed in mice that received PCL alone or
PCL with control siRNA. There was no significant difference in tumor volume
observed between mice in untreated group and low dose PCL/KSP siRNA group
from day 7 and thereafter. At the end of the treatment (day 11), when the size of
tumors was approaching 1600 mm3 in the untreated group, PCL treated group
and PCL/control siRNA group, the average tumor volume from mice in the low
61
dose and relatively high dose KSP siRNA –treated groups was 1159 ± 202 mm3
and 815 ± 6 mm3, respectively. However, during the treatment period, loss of
body weight was also observed in the two mice that received relatively high dose
of PCL/KSP siRNA (Figure 18).
Magnetic resonance imaging was again employed to analyze tumor
response. A greater number of voxels in tumor area was observed in the MRI
images from mice in the untreated group (730 voxels), PCL treated group (680
voxels) and PCL with control siRNA treated group (1000 voxels). Mice that
received PCL with low dose KSP siRNA also showed a slightly decreased
number of total voxels (530 voxels), however, a much greater decrease in total
number of voxels was observed in the mice that received relatively high dose of
KSP siRNA (300 voxels) (Figure 19).
62
Figure 17 Tumor response as a function of anti-vasculature therapy. 4T1 cells
were inoculated on the right flank of SCID mice at number of 1×106 per
mouse. Treatment started when tumor volume reached ~200mm3 (day 0).
Days of injection were indicated by arrows. Solid circles (●) indicate untreated
group; open circles (◯) indicated mice treated with PCL alone, solid triangles
(▼) indicate mice received PCL with control siRNA; open triangles (△) and
solid squares (■) represent mice received KSP siRNA formulated using PCLs
at low dose (2mg/kg) or high dose (5mg/kg), respectively. Results were
presented as mean ± s.e.m. (n=5) and analyzed using One-way ANOVA with
post-hoc Tukey. * indicates significant difference compared to mice in
untreated group. (See appendix for pictures with higher resolution)
63
Figure 18 Percent body weight change during the treatment period. Each
line indicates an individual mouse. Arrows above the lines indicate days
when different treatments were administered. Arrows on the bottom indicate
two mice from the high dose KSP siRNA treatment group when they lost
~20% of body weight.
64
Figure 19 Tumor response evaluated using m
agnetic resonance imaging. The num
ber of voxels is the sum of results
from 28 pictures per m
ouse.
65
Animal survival study
4T1 cells were injected subcutaneously in SCID mice at 1*106 per mouse.
All tumor-bearing mice were randomly divided into five groups. Different
treatments were administered through i.v. injection on day 7, 11 and 15.
According to the preset criteria which have been mentioned in materials and
methods, all mice in the untreated group died within 14 days, and within 17 days
for mice in PCL alone, and PCL with control siRNA groups (Figure 20). However,
a significant prolonged survival time was observed in mice that received the
relatively high dose of KSP siRNA (P<0.01), but not with mice that received the
low dose of KSP siRNA.
66
Figure 20 Animal survival study. 4T1 cells were inoculated on the right flank of
SCID mice at number of 1×106 per mouse. Mice received different treatment on
day 7, 11 and 15 (indicated by arrows, red). Solid circles (●) indicate untreated
group; open circles (◯) indicated mice treated with PCL alone, solid triangles
(▼) indicate mice received PCL with control siRNA; open triangles (△) and solid
squares (■) represent mice received PCLs with low dose (2mg/kg) or high dose
(5mg/kg) KSP siRNA, respectively. Results were analyzed using LogRank test
(P<0.01).
67
DISCUSSION
Biocompatible and biodegradable liposomes formed by phospholipids are
considered as promising carrier systems for chemotherapeutic agents through
the enhanced permeability and retention (EPR) effect 49, 78-79. In the present
study, we report limited uptake of conventional electroneutral liposomes making
them difficult to be considered as carriers for targeting the tumor vascular supply.
Cationic liposomes have been used in gene delivery to enhance cellular uptake
and transfection efficiency, and the cationic lipid DOTAP was used specifically for
this purpose. 63-64, 66 However, the toxicity associated with the use of more
conventional cationic liposomes was observed even when relatively low
concentrations of liposomes were used. Similar toxic effects have been reported
for other polycation-based gene transfer systems 48, 67-68, 80-81, suggesting a
problem associated with the use of traditional gene delivery vehicles for multiple
administration of siRNA as therapeutics.
The cationic charge potential of liposomes facilitates their ability to interact
with target cells. However, results from the present cell viability and microscopy
studies offer little support for the use of conventional cationic liposomes to deliver
siRNA to cells, at least not without including some additional liposome
components during the formulation process. For this reason, novel methods are
being developed to modify surface characteristics of cationic liposomes to
preserve the electrostatic-mediated cell targeting characteristics while reducing
68
the unwanted cellular toxicity. In the present study, toxic effect of cationic
liposomes was significantly diminished following the inclusion of only 2% of PEG,
and similar benefits were reported for 5% PEG content as well. Although 10% of
PEG almost completely reversed the toxic effects of DOTAP (Figure 3), the
relatively high level PEG content almost completely abolished cellular uptake
(Figure 5). The findings support a delicate balance between cellular toxicity and
optimal efficiency of targeting when cationic liposomes are employed; this
balance can be altered by regulating the total PEG content.
Ideally, liposomes should not only deliver siRNA, but also offer adequate
protection to siRNA from nuclease activity. Two different methods for
encapsulating siRNA in liposomes were evaluated. Internal addition of siRNA to
lipid films is critical for an efficient loading. To achieve this, the lipid film was first
hydrated with siRNA containing PBS, rather than adding siRNA to pre-formed
liposome preparations. We show that siRNA could be efficiently encapsulated in,
or associated with, cationic (but not electroneutral) liposomes at molar ratio of
1:2000 (siRNA: liposome) regardless of the amount of PEG used. In fact, the
siRNA loading efficiencies reported for siRNA-liposomes (containing 2 and 5
mol% PEG) were significantly higher than that of ELs and CLs (Figure 6C).
However, qualitative results from the siRNA loading studies suggested that only
PCLs offered adequate protection to siRNA from the effects of serum proteins
(Figure 7), which could be explained by either the steric stabilizing effect from
69
PEG against opsonization in serum 71, or the prevention of nucleases from
contacting the siRNA associated on the outer liposome surface 82.
Small interfering RNAs will not exert their silencing function until they are
released to the cytosol where they bind to RISC by an asymmetric assembly 26.
Results from both FACS analysis and fluorescence microscopy studies revealed
that cells showed the most promising uptake of siRNA when 5 mol% of PEG was
included in PCLs. A significant uptake of siRNA as well as cellular toxicity was
also observed using cationic liposomes with 2 mol% of PEG. Our unpublished
experimental observations confirmed that it is still possible to deliver siRNA
efficiently with this formulation, but only when relatively low concentrations of
liposomes are used.
The exact mechanism(s) involved in the role of PEG in protecting human
endothelial cells from the toxic effect of cationic lipids is still unclear. It is widely
accepted however, that PEG forms a repulsive hydration barrier owing to steric
forces when used in the absence of DOTAP 70, 83. In addition to limiting
interaction of liposome membranes with proteins, our data strongly support the
notion that the dynamic changes that occur at the interface of pegylated cationic
liposomes and endothelial cells results in the relatively safe delivery of siRNA to
target cells. This is an important experimental observation, given that the ideal
siRNA carrier molecule should enable adequate gene-silencing without causing
70
unwanted toxic effects caused by the delivery vehicle, which must be achieved in
order to assess and characterize the silencing effect.
Kinesin spindle protein is a promising target for cancer treatment not only
because it mediates the segregation of centrosomes during mitosis, but because
it has little or no expression in non-mitotic cells 22, 24-25. In our study, we showed
tumor growth repression by delivering KSP siRNA in PCLS, an achievement not
observed with the use of cationic liposomes without pegylation. The growth
inhibition of both tumor endothelial cells and tumor cells was observed in vitro
when KSP siRNA was delivered in PCLs (Figure 11). Since targeting tumor
vasculature and targeting the tumor cells directly are both considered as effective
treatment approaches, it was still unclear whether or not the suppression of
tumor growth was due to destroying the tumor vasculature or tumor cells, or both.
Based on this doubt, we decided to employ a tumor model in which the cancer
cells were resistant to KSP treatment. Surprisingly, the tumor responded to both
high dose and low dose KSP siRNA treatment from day 4 after the first
therapeutic injection, and no difference was observed between the two groups
(Figure 17). This suggested that destroying the tumor vasculature played an
important role in preventing the growth of the tumors. However, mice that
received low dose KSP siRNA stopped responding to the treatment from day 7
through the end of the experiment. Tumor vascular disruption has been shown to
suppress tumor growth in orthotopic melanoma models 84-86. What should not be
ignored is that tumor cells can also be affected by vasculature disrupting agents,
71
such as paclitaxel and vincristine, because of the central role that tubulins
playing in the maintenance of cytoskeleton and transportation of proteins and
vesicles along axon fibers. But in our 4T1 tumor model, it has been shown that
this tumor cell line does not respond to KSP siRNA treatment, so any response
from the tumor is likely due to the vascular disruption effect.
Even though a significant suppression of tumor growth and a prolonged
survival were achieved in mice that received high dose KSP siRNA treatment, a
severe loss of body weight was also observed in some animals, indicating a
maximal tolerated dose may have been reached. However, if this toxicity is
associated with the use of KSP siRNA or with the cationic liposome still needs to
be further examined.
We also checked mRNA level in the tumor tissue after KSP siRNA
treatment. Even though the tumor recovered from the mice treated with KSP
siRNA in DOPE/DOTAP liposomes showed ~40 percent decrease of KSP mRNA
compared to untreated controls, this decrease did not translate to a significant
tumor response in vivo (Figure 13, 15). However, a significant suppression of
tumor growth was observed when ~90 percent KSP mRNA was knocked down in
mice that received KSP siRNA in PCLs, indicating a significant level of KSP
mRNA must be silenced to block the function of kinesin spindle proteins during
mitosis. However, this still needs to be further understood using western blot or
ELISA to evaluate protein translation from KSP mRNA.
72
A promising delivery of siRNA to cellular models of tumor endothelia has
been achieved using PCLs. A suppression in the tumor growth has been
observed using KSP siRNA-loaded PCLs using murine models of melanoma and
breast cancer. But the detailed mechanism of KSP-induced cell death (assumed
after prolonged cell cycle arrest) is an area that requires further examination.
73
SUMMARY
1. Cationic liposomes can target the tumor vascular supply more efficiently
compared to electroneutral liposomes.
2. Cationic lipids are critical for the efficient loading of siRNA in liposomes.
3. The inclusion of PEG in cationic liposomes further enhanced the loading of
siRNA and prevented the degradation of siRNA by enzymes in serum.
4. The inclusion of PEG is critical for minimizing the toxicity associated with the
use of cationic liposomes.
5. Cationic liposomes can still reserve the high targeting affinity towards tumor
vasculature after the inclusion of a modest amount of PEG.
6. KSP siRNA-loaded PCLs successfully inhibited the growth of tumor cells and
tumor endothelial cells in vitro.
7. KSP siRNA-loaded PCLs successfully inhibited the tumor growth in vivo.
8. Magnetic resonance imaging can be used to analyze tumor response to
treatments.
9. A relatively high dose of KSP siRNA will be required to suppress tumor
growth and to prolong animal survival, and the tumor vascular supply is the
likely tumor target.
74
CONCLUSIONS
In the present study, we incorporated siRNA in PCLs (PEGylated cationic
liposomes) for targeting the tumor vasculature. The inclusion of PEG in the
cationic liposome preparations significantly reduced the cellular toxicity
associated with the use of cationic lipids, while still preserving its affinity for
targeting endothelial cells. We showed that cationic lipids are critical for high
efficient loading of siRNA in liposomes. We also demonstrated the internal
addition method is more efficient in loading siRNA in liposomes compared to
external addition. The inclusion of PEG in cationic liposomes improved the
loading efficiency of siRNA, and prevented the degradation of siRNA by serum
enzymes, an advantage not achieved by using cationic liposomes alone. We also
evaluated the growth inhibition of different tumor cells and tumor endothelial cells
using KSP siRNA-loaded PCLs. Further, this growth inhibition was confirmed as
a result of the failure in KSP function and prolonged mitosis. The liposome
formulation for KSP siRNA which showed the greatest growth inhibition also
successfully suppressed tumor growth in vivo in tumor-bearing mice. We also
evaluated the possibility of using MRI to evaluate tumor response to therapy and
to probe the tumor environment located beneath the skin. The findings were
consistent with tumor volume measured using the digital calipers. We also
confirmed that the suppression of tumor growth was due to the knock-down of
KSP mRNA using qRT-PCR. We further explored the importance of anti-
angiogenic therapy in the treatment of cancer using a tumor model which itself
doesn’t respond to KSP inhibition. The low dose of KSP siRNA only suppressed
75
tumor growth for a short period of time, however, the relatively high dose of KSP
siRNA showed a significant decrease in tumor growth. Results from MRI analysis
further confirmed this growth suppression of tumor by delivering KSP siRNA in
PCLs to mice. Animal survival was also evaluated to examine if KSP siRNA can
prolong survival of tumor-bearing mice, and results showed a significantly
increased survival rate in mice that received the higher dose of KSP siRNA.
76
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APPENDIX
86
APPENDIX
Figure 3 Cell V
iability as a function of liposome preparations. (H
ME
C)
APPENDIX
87
Figure 3 Cell V
iability as a function of liposome preparations. (H
UV
EC
)
APPENDIX
88
Figure 3 Cell V
iability as a function of liposome preparations. (M
S1-V
EG
F)
APPENDIX
89
Figure 5 Cellular uptake as a function of liposom
e preparation type. (HM
EC
)
APPENDIX
90
Figure 5 Cellular uptake as a function of liposom
e preparation type. (HU
VE
C)
APPENDIX
91
Figure 5 Cellular uptake as a function of liposom
e preparation type. (MS
1-VE
GF)
APPENDIX
92
Figure 6 siRN
A uptake by M
S1-V
EG
F cells evaluated using FAC
S analysis. ()D
OP
C based form
ulation
APPENDIX
93
Figure 6 siRN
A uptake by M
S1-V
EG
F cells evaluated using FAC
S analysis. (D
OP
E based form
ulation)
APPENDIX
94
Figure 11 Cell grow
th inhibition by KS
P siR
NA
-loaded Pegylated cationic liposom
es (4T1).
APPENDIX
95
Figure 15 Tumor response as a function of tim
e-KSP silencing effect
APPENDIX
96
Figure 11 Cell grow
th inhibition by KS
P siR
NA
-loaded Pegylated cationic liposom
es (bEN
D.3).
APPENDIX
97
Figure 11 Cell grow
th inhibition by KS
P siR
NA
-loaded Pegylated cationic liposom
es (B16-F10).
APPENDIX
98
Figure 11 Cell grow
th inhibition by KS
P siR
NA
-loaded Pegylated cationic liposom
es (B16-F10).
APPENDIX
99
Figure 13 Tumor response as a function of tim
e-KS
P silencing effect.
APPENDIX
100
Figure 16 Tumor response as a function of anti-vasculature therapy.