surface engineered pei-modified liposomes ...cj...surface engineered pei-modified liposomes for...

SURFACE ENGINEERED PEI-MODIFIED LIPOSOMES FOR COMBINATION

TREATMENT OF DRUG-RESISTANT CANCERS BY CO-DELIVERY OF siRNA AND

PACLITAXEL

Doctoral Dissertation Presented

By

Lívia Palmerston Mendes

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 Systems

NORTHEASTERN UNIVERSITY

BOSTON, MASSACHUSETTS

July 2018

Department of Pharmaceutical Sciences Investigator: Lívia Palmerston Mendes

i

Northeastern University

Bouvé College of Health Sciences

Doctoral Dissertation Approval

Dissertation title: Surface engineered pei-modified liposomes for combination treatment of

drug-resistant cancers by co-delivery of siRNA and paclitaxel

Author: Lívia Palmerston Mendes

Program: Ph.D. in Pharmaceutical Sciences with Specialization in Pharmaceutics and Drug

Delivery Systems

Approval for dissertation requirements for the Doctor of Philosophy in: Pharmaceutical Science

Dissertation Committee (Chairman):

Dr. Vladimir P. Torchilin Date: 7/17/2018

Other committee members:

Dr. Ban-An Khaw Date: 7/17/2018

Dr. Rebecca Carrier Date: 7/17/2018

Dr. Tania (Tali) Konry Date: 7/17/2018

Dr. Tatyana Levchenko Date: 7/17/2018


ii

ABSTRACT

Surface-modification of nanoparticles with PEG to confer stealth properties has been

considered the most common practice for many years. However, controversial and serious

challenges related to PEGylation have been identified, including lack of functionality, hindered

cellular interaction, allergic reactions, and stimulation of IgM production after repetitive dosing

that accelerates blood clearance of nanoparticles. The development of alternative nanoparticle

modifications has been encouraged to overcome these obstacles and improve therapeutic

outcomes.

The general aim of this study was to develop, characterize and test surface-modified liposomes

as an anticancer nanopreparation based on the combination of cytotoxic drug and siRNA for

enhanced activity against drug-resistant cell lines.

In the first approach, polyethyleneimine (PEI)-modified liposomes (PEIPOS) containing

paclitaxel (PTX) were prepared and characterized as an alternative to PEGylated ones. Various in

vitro studies were performed to thoroughly characterize the formulation. The coating of liposomes

with PEI improved their stability over conventional non-PEGylated liposomes in ion- and protein-

rich mediums. Coating also increased their interaction with various cancer cell lines despite

obvious protein adsorption on the surface of liposomes, as shown from the investigation of their

ability to associate with different cancer cell lines and penetrate cells of 3D tumor spheroids. Cell

viability was also evaluated to show the correlation of the drug encapsulation and surface-

modification with the cytotoxic effects.

In the second approach, we evaluated the capability of the liposomes to complex and deliver

siRNA that downregulates P-glycoprotein, a multidrug resistant (MDR) protein. PEIPOS


iii

complexed siRNA and protected it from RNase degradation. Additionally, PEIPOS delivered

siRNA intracellularly and downregulated resistance-associated proteins.

In the final approach, the in vivo efficacy of the optimized formulation was investigated.

Tumor growth inhibition was observed in a mouse ovarian xenograft tumor model, without

signs of toxicity, in animals treated with the siRNA/PTX co-loaded formulation.

These complex-in-nature but simple-in-design novel liposomal formulations constitute a

viable and promising alternative to their PEGylated counterparts because of their added

functionality and the possibility of combination therapy using drug and oligonucleotide co-

delivery.


iv

ACKNOWLEDGEMENTS

First, I would like to express my sincere gratitude to my advisor Dr. Vladimir P. Torchilin for his

continuous support, motivation, and inspiration. Thank you for accepting me as part of your

research group and for giving me enough guidance and freedom to develop my research. For your

concern not only for my scientific but also personal success. Your confidence in me over the past

four years helped me improve as a scientist and be confident that I can overcome even greater

challenges.

To Dr. Can Sarisozen, for all the patience, attention, mentoring and discussion during my Ph.D.

path. You played a big role in my development as a scientist, taught me numerous techniques and

fomented even more of the scientific curiosity I already had. You are a great example of the

professional that I hope one day to be.

Thanks to my committee members: Dr. Khaw, Dr. Carrier, Dr. Konry and Dr. Levchenko for their

time dedicated to serve on this committee and for comments that helped build the thesis on a

stronger base. I also want to thank Dr. Hartner for providing helpful comments that improved my

manuscripts and consequently my English.

To Ed Luther, who took long hours explaining to me in detail some of the techniques we used for

this thesis that contributed greatly to this work.

To the Holographic Imaging Cytometry Program of Excellence, which through a partnership

between Northeastern University and Phase Holographic Imaging that provided the resources for

the development of the experiments involving holographic imaging cytometry.

I would also like to thank the many past and current lab members, who guided me and helped me

through a difficult day, an unsuccessful experiment, or a personal frustration. Many came and left


v

during this four years, but all of them contributed somehow to help me analyze from different

perspectives, both in life and in science.

A special thanks to Daniel Costa who started this journey with me before we met in person. I’m

glad we had each other to face so many new things, overcome the innumerous challenges in the

Ph.D. life, and try to (re)discover ourselves in this scientific world.

To the Brazilian government, whose inclusive policies from the past administration provided the

opportunity for me and approximately other 4,500 students to pursue our Ph.D. degree abroad in

high level institutions. Unfortunately, in the past few years there have been substantial cuts in

budgets for research in Brazil, threatening the advancement of science due to political and

economic turmoil. I hope one day I can contribute to improve this scenario in appreciation of the

opportunities that were given to me and inspire others to follow this hard, but rewarding path.

I must not forget to thank Dr. Eliana Lima, my first mentor in science and my constant inspiration.

I had the happy opportunity to have her literally only miles away in the first months of my

transition to Boston, which I believe made it indescribably more comforting. Thank you for still

being so present and supportive.

Last, but not least, I would like to immensely thank my family for all the support they have given

me my whole life, which ultimately allowed me to follow this path, but mainly during this graduate

period. You will always be my haven. To my friends, during all the times that I doubted myself,

who were there to remind me who I am and evoke the strength I often tended to forget I have. A

special shout out to my husband André, a real partner, who was immediately ready to get on board

for this adventure with me. Thanks for being so patient and supportive, cheering me up during the

hard times and always being my best example of perseverance, love and kindness.


vi

TABLE OF CONTENTS

Approval Sheet ………………………………………………………………………….. i

Abstract ………………………………………………………………………………….. ii

Acknowledgements ……………………………………………………………………... iv

Table of Contents ……………………………………………………………………….. vi

List of Tables ……………………………………………………………………………. ix

List of Figures …………………………………………………………………………… x

1. SPECIFIC AIMS ………………………………………………………………….… 1

2. BACKGROUND AND SIGNIFICANCE ………………………………………….. 3

2.1. Cancer: hallmarks and challenges ………………………………………. 3

2.2. Nanotechnology and drug delivery systems for cancer therapy ………… 4

2.3. Paclitaxel ………………………………………………………………... 9

2.4. RNA interference ………………………………………………………... 10

2.5. Rationale and overview of the approach …………………………………. 13

3. MATERIALS AND METHODS ……………………………………………………. 15

3.1. Materials ………………………………………………………………… 15

3.2. Synthesis of polyethyleneimine-phospholipid conjugate (bPEI-PE) ……. 16

3.3. Preparation and characterization of liposomes …………………………... 16

3.4. In vitro drug release ……………………………………………………… 17

3.5. Stability of PEIPOS in fetal bovine serum ………………………………. 18

3.6. Analysis of PEIPOS-associated proteins ………………………………… 18

3.7. Cell culture and spheroid formation …………………………………….. 19


vii

3.8. Cell association with monolayers ……………………………………….. 20

3.9. 3-D spheroid association and penetration ………………………………... 21

3.10. In vitro cytotoxicity on monolayers ……………………………………... 22

3.11. β-tubulin immunostaining ……………………………………………….. 22

3.12. Laser scanning cytometry ………………………………………………. 23

3.13. Phase Holographic Imaging ……………………………………………... 24

3.14. Complexation of PEIPOS with siRNA, gel retardation, size and zeta

potential changes ………………………………………………………… 25

3.15. Protection of siRNA from RNases by PEIPOS …………………………. 26

3.16. Characterization of P-glycoprotein overexpression of cells …………….. 26

3.17. P-gp downregulation in MDR cells …………………………………….. 27

3.18. Hemolysis ………………………………………………………………. 27

3.19. In vivo animal model studies …………………………………………….. 28

3.20. Preliminary in vivo evaluation of the efficacy of nanopreparations ……… 28

3.21. Toxicity evaluation of PEIPOS in mice ………………………………….. 29

3.22. Statistical analysis ………………………………………………………. 30

4. RESULTS …………………………………………………………………………… 31

4.1. Preparation and characterization of PEIPOS …………………………….. 31

4.2. In vitro drug release from bPEI-coated liposomes ……………………….. 32

4.3. Stability of PEIPOS in FBS and analysis of PEIPOS-associated proteins 33

4.4. bPEI-coated PEIPOS improve uptake in monolayers and penetration in

3D spheroid models ……………………………………………………… 36

4.5. bPEI-coating enhances the in vitro cytotoxicity of liposomes …………… 41


viii

4.6. bPEI-modified liposomal PTX induces β-tubulin polymerization ………. 43

4.7. bPEI-modified liposomal PTX induces apoptosis ………………………. 44

4.8. Phase Holographic Imaging ……………………………………………... 49

4.9. bPEI-modified liposomal formulations can complex siRNA

simultaneously with anticancer drugs ……………………………………. 51

4.10. PEIPOS can successfully downregulate resistance-related target proteins 54

4.11. In vitro hemolysis evaluation ……………………………………………. 55

4.12. In vivo efficacy and toxicity ……………………………………………... 56

5. DISCUSSION ……………………………………………………………………….. 62

5.1. Preparation and characterization of PEIPOS ……………………………. 62

5.2. In vitro drug release ……………………………………………………... 63

5.3. PEIPOS-protein association and cell interactions ……………………….. 64

5.4. bPEI-coating enhances the in vitro cytotoxicity of liposomes by inducing

β-tubulin polymerization and subsequent apoptosis……………………... 68

5.5. bPEI-modified liposomes can simultaneously complex siRNA with

chemotherapeutics for combination treatment of MDR cancer ………….. 70

5.6. Investigation of the efficacy of the formulation in vivo ………………….. 71

6. CONCLUSION ……………………………………………………………………… 73

REFERENCES ………………………………………………………………………….. 75


ix

LIST OF TABLES

Table 1: Clinically approved liposomes for cancer treatment in the United States ……. 7

Table 2: Physicochemical characteristics of non-coated and coated liposomes ……….. 32

Table 3: Changes in 0.5% PEIPOS characterization parameters after complexation with

siSCR at various N/P ratios …..………………………………………………... 51


x

LIST OF FIGURES

Figure 1: Schematic representation of the passive targeting approach ………………… 5

Figure 2: Chemical structure of a fragment of branched polyethyleneimine (bPEI) …... 9

Figure 3: Chemical structure of paclitaxel ……………………………………………... 10

Figure 4: Mechanism of action of siRNA in gene silencing …………………………… 12

Figure 5: Holomonitor® system ………………………………………………………... 25

Figure 6: Schematic representation of the synthesis of bPEI-PE and coated liposomes 31

Figure 7: Cumulative in vitro release of PTX ………………………………………….. 33

Figure 8: Stability of PEIPOS upon incubation in 10% (v/v) FBS in PBS pH 7.4 at

37°C for over 24h …………………………………………………………….. 34

Figure 9: Protein adsorption of the surface of liposomes ……………………………… 35

Figure 10: Cellular association of coated and non-coated PEIPOS with monolayers in

three different cancer cell lines …………………………………………….. 37

Figure 11: Kinetics of cellular association and penetration of formulations in 3D HeLa

spheroids …………………………………………………………………..... 40

Figure 12: Viability of HeLa and A2780-ADR cells …………………………………... 42

Figure 13: Immunofluorescent detection of liposomal PTX-mediated β-tubulin

polymerization on HeLa cells ……………………………………………... 43

Figure 14: iCyte laser scanning cytometer images …………………………………….. 45

Figure 15: . Quantification of events using laser scanning cytometry analysis ………... 46

Figure 16: Laser scanning cytometry analysis of HeLa cells treated with different

formulations ……………………………………………………………….. 48


xi

Figure 17: Images and graphs generated based on analysis with Holomoniter® M4 ….. 50

Figure 18: siRNA complexation of surface-modified PEIPOS liposomal formulations 53

Figure 19: P-gp overexpression and downregulation on the cell membrane …………... 55

Figure 20: Hemolytic potential of the developed formulations in mouse erythrocytes ... 56

Figure 21: Fluorescence intensity of rhodamine-labeled liposomes (red) in A2780-

ADR xenograft tumors after injections administered intravenously every

other day via the tail vein ...………………………………………………… 57

Figure 22: Fluorescence intensity of rhodamine-labeled liposomes (red) in the liver of

A2780-ADR tumor-bearing animals after injections were administered

intravenous every other day via the tail vein ………………………………. 58

Figure 23: In vivo evaluation of the formulation efficacy in nude athymic mice bearing

an A780-ADR resistant human ovarian tumor xenograft ...………………… 60

Figure 24: Evaluation of toxicity of the treatment of nude athymic mice bearing

A780-ADR resistant human ovarian tumor xenograft with different

formulations ………………………………………………………………... 61


1

1. SPECIFIC AIMS

The general aim of this study was to develop, characterize and test surface-modified liposomes

as an anti-cancer nanopreparations based on a combination of a cytotoxic drug and a siRNA for

enhanced activity against drug-resistant cell lines. We hypothesized that the coating of liposomes

with polyethyleneimine (PEI) would improve their stability over conventional non-PEGylated

liposomes and increase their interaction with various cancer cell lines. Moreover, this surface

modification would allow the possibility of co-delivering siRNA along with an anti-cancer drug

in vitro and in vivo. With this in mind, the following approaches and specific aims were pursued:

1.1. Specific Aim I. To develop, characterize and optimize PEI-modified liposomes with as

an anticancer formulation and study their effect against various tumor cell lines, including

multidrug resistant (MDR). To achieve this, the following steps were undertaken:

a) Preparation of PEI-modified liposomes. PEI-coated and non-coated liposomes were loaded

with paclitaxel and characterized for their size, surface charge, drug loading efficiency and

stability in a serum containing environment.

b) Investigation and comparison of the in vitro efficacy of PEI-coated and non-coated

liposomes in cervical cancer cells, HeLa, and in ovarian carcinoma cells, A2780-ADR and

SKOV3-TR. Cell association with monolayers was assessed, as well as cell viability.

Penetration in 3D tumor spheroid models was also evaluated.

c) Evaluation of the cell cycle distribution and apoptotic events using quantitative imaging

cytometry analysis (iCyte), and quantitative analysis of living cells using time-lapse long-

term imaging analysis (Phase Holographic Imaging).


2

1.2. Specific Aim II. To combine drug-loaded liposomes developed in Aim I with siRNA and

investigate their ability to downregulate resistance mechanisms in MDR cells, along with the

synergistic effects of the co-delivery of the two loads. To achieve this, the following steps were

undertaken:

a) Evaluation of the ability of the developed liposomes to complex siRNA for co-delivery

along with paclitaxel and the stability of these nanopreparations.

b) Characterization of the overexpression of P-glycoprotein (P-gp) in the resistant ovarian

carcinoma cell line A2780-ADR.

c) Evaluation of the ability of PEI-modified liposomes complexed with siRNA against P-gp

to downregulate this well-known resistance mechanism.

1.3. Specific Aim III. To evaluate the in vivo antitumor activity of the nanopreparations

selected from Aim II in mouse models of human ovarian cancers and to identify the most

promising formulation and treatment strategies. To achieve this, the following steps were

undertaken:

a) Investigation of the effectiveness of the formulation developed in Specific Aim II in

experiments in vivo in mouse xenograft tumor models of A2780-ADR cell.

b) Evaluation of toxicity in the tumor-bearing mice treated for the tumor inhibition efficacy

study.


3

2. BACKGROUND AND SIGNIFICANCE

2.1.Cancer: hallmarks and challenges

Cancer is the second leading cause of death in the United States [1] and one of the major health

concerns worldwide. It is a disease characterized by a rapid and disorderly cell growth. These

cells tend to present an aggressive and uncontrolled division. They can invade tissues and organs,

and thus are able to spread to other parts of the body (metastasis). There are more than 100 types

of cancer, classified based mainly on the tissue or organ of the disease’s origin [2].

Cancer cells present some differences in relation to the normal cells, including the inability to

undergo differentiation, uncontrolled proliferation, great potential for invasion, an ability to

undergo metastasis, faulty DNA repair, and apoptosis evasion. The uncontrolled cell proliferation

occurs due to alterations of growth factors and/or receptors, in pathways of intracellular signaling,

especially those that control the cell cycle, in telomerase expression (regulatory enzyme), and in

tumor-related angiogenesis [3].

Conventional treatment for cancer most commonly involves surgery, radiotherapy or

chemotherapy, but also can involve immunotherapy, hormone and targeted therapies. A

combination of more than one type of these treatments is usually utilized, depending on the specific

disease and its stage. Chemotherapy involves the administration of drugs with proven therapeutic

efficacy and different mechanisms of action. The choice of the drugs to be used is also closely

related to the type of cancer [4]. However, conventional chemotherapy is frequently associated

with adverse effects resulting from lack of specificity of the drugs [5].


4

Among the challenges related to current therapy are the non-specific bodily distribution of

antineoplastic agents, inadequate drug concentration at the site of action and a limited therapeutic

response. Since current anti-cancer agents cannot differentiate normal cells from cancer cells, a

series of adverse effects and high toxicity are common characteristics of the conventional therapy

against the cancer [6]. Additionally, some tumors recur and acquire resistance to the widely used

and generally efficacious drugs [7].

2.2.Nanotechnology and drug delivery systems for cancer therapy

To increase the specificity, and consequently the efficacy of the treatment, extensive research

is being done to develop drug delivery systems that, most of the time, carry already established

and widely used anti-cancer drugs [5, 8, 9]. This approach overcomes some obstacles of

conventional therapy, such as increased drug solubility so they can be administered in a safer

formulation that allows a decrease in the amount of drug administered as a result of the specificity

of the treatment. The main advantage of the use of drug delivery systems the higher delivery to the

tumor, which reduces off-target effects (fewer side-effects, an increase in safety and efficacy, and

an optimization of treatment) [10-12].

The use of nanoparticles often takes advantage of the intrinsic characteristics of the tumors.

The most widely known and explored advantage is passive targeting utilizing the neo-vasculature

that develops around solid tumors (Figure 1). Tumor cells need a supply of oxygen and nutrients

to maintain their metabolism and growth. It is necessary, therefore, that the tumor promotes

angiogenesis or development of vascular networks that will provide the necessary supplies for its

continued development and spread. In this process, hypoxic tumor cells begin to secrete factors


5

that induce the formation of new vessels. These factors are signaling molecules that stimulate the

migration of vascular endothelial cells from vessels near the tumor and, consequently, result in the

formation of new capillaries essential for tumor growth and dissemination. This new vasculature

formed around the tumor has specific abnormal characteristics. The vessels are highly

disorganized, tortuous and dilated, with multiple branches. Endothelial junctions have wider

openings and a discontinuous or absent basement membrane that provides high capillary

permeability [13, 14]. This enhanced vascular permeability is important for tumor growth and

survival and also facilitates the entry of proteins and macromolecules, as well as drug-loaded

nanoparticles. This feature is known as the enhanced permeability and retention effect (EPR) and

allows nanocarriers to pass through the fenestrated endothelial tissue of the tumor vasculature and

accumulate in the tumor. Nanoparticle-based drugs, on the other hand, are much less able to

passively enter normal tissues, unlike what occurs with the free drug molecules [15, 16].

Figure 1. Schematic representation of the passive targeting approach. Passive targeting is a

strategy widely used in the field of nanoparticles for anti-cancer therapy, which allows the

accumulation of the nanosystems in the tumor microenvironment (adapted from Oliveira, Mendes

[17])


6

Liposomes are the most commonly used lipid-based nanoparticle for delivery of anticancer

drugs [17, 18]. They were first described in the 1960’s by Alec D. Bangham but their application

as drug delivery systems were explored only later in the early 1970’s [19, 20]. Liposomes are

spherical vesicles ranging in size from a few nanometers to hundreds of micrometers. These

vesicles consist of lipid bilayers and resemble biological membranes that encapsulate an aqueous

interior. Liposomes are prepared using biodegradable, biocompatible and non-toxic materials. The

lipids used in their preparation can be either synthetic or come from natural sources. Due to the

innocuous nature of the liposomal components and the versatility of their structure, combined with

an ability to entrap hydrophobic agents in their bilayers or encapsulate aqueous molecules such as

hydrophilic drugs and nucleic acids in their core, these lipid vesicles became widely studied

candidates for use as drug carriers [21, 22]. In 1995, Doxil® was the first FDA liposomal

formulation approved for cancer treatment. It has since been used for recurrent ovarian cancer,

AIDS-related Kaposi’s sarcoma, multiple myeloma and metastatic breast cancer [23]. Following

Doxil®, other liposomal formulations were approved and introduced into the clinic in the United

States (Table 1). All of them rely on the passive targeting EPR effect for better tumor accumulation

and improved therapeutic outcome.

The design of liposomal formulations depends on many factors, including the intended cargo,

the targeted site of action, and desired pharmacokinetic properties. For nucleic acid delivery,

liposomes are usually designed with an overall positive surface charge to improve the

encapsulation of the oligonucleotides, including siRNA, which have a negative charge [24, 25].


7

Table 1. Clinically approved liposomes for cancer treatment in the United States.

Trade name Year of approval Active agent Surface modification Reference

Doxil® 1995 Doxorubicin PEG [23]

DaunoXome® 1996 Daunorubicin None [26]

DepoCyt® 1999 Cytarabine None [27]

Marqibo® 2012 Vincristine Sulfate None [28]

Onivyde™ 2015 Irinotecan PEG [29]

Vyxeos™ 2017 Daunorubicin and

cytarabine None [30]

PEG = Polyethylene glycol

Most of the time, micro- and nanoparticulate carriers are easily recognized by the mononuclear

phagocytic system (MPS) and rapidly removed from the blood circulation via opsonization [31].

Certain hydrophilic polymers can be used to modify the liposomal membrane and increase surface

hydrophilicity. Moreover, this modification hides or repels the opsonizing blood proteins and

components to increase the residence time of liposomes in the blood circulation, giving them so

called “stealth” characteristics [32]. Polyethylene glycol (PEG) is the current gold standard for

molecules used for surface modification of liposomes and other nanoparticles used to improve

circulation time and enhance their passive localization in the tumors due to the EPR effect [33].

On the other hand, the PEG-coating, which hinders the interaction with cells, can diminish

cellular internalization. In the case of gene delivery, it jeopardizes the success of transfection [34].

Furthermore, thorough studies have shown that repeated administration of PEG can generate IgM

antibodies against it. The presence of these antibodies may lead to a rapid elimination of PEGylated


8

formulations from the circulation, a phenomenon termed “accelerated blood clearance (ABC)”.

PEG can also activate the complement system and provoke serious side effects such as anaphylaxis

[35, 36]. In approaches alternative to PEG, long-circulation can be provided by other polymers

such as polyvinyl, poloxamers, chitosan and dextran. The composition of the polymers, molecular

weight, and the flexibility of their chains are properties affecting in the stealth aspect of liposomes

[32].

Polyethyleneimine (PEI) is a hydrophilic polymer (Figure 2) well-known as a vector for

nucleic acid delivery. Its high positive charge density enables effective condensation of negatively

charged siRNA by electrostatic interactions. The relatively high transfection efficacy of PEI can

be attributed in part to the intrinsic capacity of PEIs to buffer the endosomal acidic pH and disrupt

the endosomal vesicles via a proton sponge effect [37]. The major drawback of PEI is its high

toxicity, especially of the high molecular weight PEIs. Lower MW PEIs have better toxicity

profiles, but they are far less efficient. Branched PEIs of low molecular weight, on the other hand,

present low toxicity and are still efficient as a transfection agents [38]. Lipidation of PEI through

covalent association with dioleoyl phosphatidylethanolamine (DOPE) has been shown to further

improve its gene silencing ability when used as a siRNA carrier [39, 40].


9

Figure 2. Chemical structure of a fragment of branched polyethyleneimine (bPEI).

2.3.Paclitaxel

Paclitaxel (Figure 3) is a widely-studied drug used in the field of nanotechnology to treat

several solid tumors such as breast [41], ovarian [42] and lung [43] carcinomas. Its mechanism of

action involves the inhibition of cell growth by stabilization of microtubules due to the drug’s

covalent conjugation with tubulin, preventing its depolarization and thus blocks the process of cell

division. The conventional market formulation of paclitaxel, approved by the Food and Drug

Administration (FDA) in 1992 for the treatment of ovarian cancer, is Taxol®, and utilizes

Cremophor EL (polyoxyethylated castor oil) and ethanol (50:50 v/v) as a vehicle [44]. Despite the

toxic effects and hypersensitivity reactions presented by this vehicle, it is currently used to

overcome the poor water solubility of the paclitaxel itself [45]. Because of these problems, a great

effort has been made to develop Cremophor EL-free formulations that are more effective and less

toxic than the conventional one.


10

OO

O

OHCH3

OO

OH

O

O CH3

CH3

CH3

OO

O

CH3

OH

CH3

NH

O

Figure 3. Chemical structure of paclitaxel.

In 2005, FDA approved the use of a formulation of paclitaxel without Cremophor for the

treatment of breast cancer, Abraxane. This preparation contains 130 nm albumin nanoparticles

conjugated with paclitaxel, which increases its solubility, allowing its administration in an aqueous

solvent. This Cremophor-free formulation prevents hypersensitivity reactions and side effects

related to this vehicle, in addition to allowing the possibility of administering higher amounts of

the drug [12, 46]. Genexol®PM is another approved paclitaxel-loaded nanomedicine that is free

from toxic surfactants [47]. No significant overall survival benefits have been observed to date,

however.

2.4.RNA interference

Proteins, in general, are among the main pharmacological targets of many therapies, including

cancer, but since the discovery of RNA interference (RNAi) therapy in the late 1990’s, alternative


11

approaches have been explored to indirectly target these proteins [48]. RNAi is a post-

transcriptional gene silencing therapy that utilizes oligonucleotides to silence gene expression or

inhibit translation. The main oligonucleotides involved in RNAi are micro RNA (miRNA) and

small interfering RNA (siRNA). They can bind to the targeted messenger RNA (mRNA) and

induce or inhibit its activity, which may lead for example, to the downregulation of a protein that

causes some specific disease. One of the greatest differences between these two RNA’s is that

siRNA binds specifically to a complementary sequence in the mRNA, whereas miRNA can bind

to various mRNAs that have similar sequences [49].

siRNAs are double-stranded RNA molecules usually comprised of 19 to 25 base pairs. Once

introduced into the cell cytoplasm, siRNA couples with a RNA-induced silencing complex (RISC).

The double strand is unwound and only the antisense strand remains coupled with RISC. This

antisense/RISC complex then binds specifically to a complementary sequence in the target mRNA,

which induces mRNA cleavage (Figure 4). The degradation of the mRNA prevents the translation

of the protein [50, 51].

For delivery into the cytoplasm, however, siRNA encounters several obstacles that constitute

the biggest challenges to the use of RNAi therapy. The stability of naked siRNA in the blood is

very low due to degradation by serum nucleases, with a siRNA half-life


12

Figure 4. Mechanism of action of siRNA in gene silencing (adapted with permission from

Navarro, Pan [57]. Copyright (2015) American Chemical Society.

Among the clinical approaches using siRNA are cancer therapy [58]. Therapy can be based on

the downregulation of proteins involved in the disease and its resistance mechanisms. One example

related to multi-drug resistance (MDR) is the overexpression of ATP-binding cassette transporters

that reduce intracellular drug concentration through efflux pumps. The most extensively studied

ABC transporter related to MDR is the P-glycoprotein (P-gp), also known as MDR1, responsible

for transporting antitumor agents such as vincristine, doxorubicin and paclitaxel out of the cells.

To bypass MDR and restore chemotherapy sensitivity in resistant cells, oligonucleotides are being

used to silence genes related to this and other resistance mechanisms [39]. Many other proteins are

related to resistance mechanisms and can also be targeted using RNAi. One example is survivin,

an anti-apoptotic member of the inhibitor of apoptosis (IAP) genes. It is overexpressed in many


13

tumors including breast [59], pancreatic [60] and ovarian [61] cancers, is related to resistance to

chemotherapy and is a marker for a poor prognosis. For these reasons, therapeutic targeting of

survivin is a promising approach in cancer therapy.

2.5.Rationale and overview of the approach

Nanostructured carriers have been extensively studied as an approach for cancer treatment to

address common drawbacks of conventional therapies, such as low efficacy and high non-

specificity associated with toxicity and severe adverse effects. Despite these improvements,

chemoresistance remains one of the greatest obstacles to improving patient prognosis and survival.

MDR involves various mechanisms that are nonrelated to each other, thus complex and “smart”

strategies are required to overcome this MDR bottleneck in usage of nanomedicine. One of the

strategies to bypass MDR and restore chemotherapy sensitivity in resistant cells is the use of

oligonucleotides to silence genes related to resistance mechanisms.

Combination therapy is the approach that has recently shown great potential. The co-delivery

of two anticancer drugs is the most common combinatorial strategy. Recent studies have been

invested in the use of nanopreparations co-delivering chemotherapeutic agents and siRNA to

promote a synergistic effect by enhancing intracellular drug accumulation and the cytotoxicity

effect of the drug once the resistance gene is silenced. Based on this scenario, this research project

intended to investigate the potential of a liposomal formulation modified with PEI as an alternative

to PEG to impart increased cell association with the liposomes. Branched PEI has the desirable

characteristics of hydrophilic polymers used to stabilize formulations, including flexible chains,

and a hydrophilic nature. It may also provide steric stabilization to avoid opsonization. In addition,


14

PEI is expected to complex siRNA and enable the co-delivery of an oligonucleotide against a

resistance mechanism to improve the cytotoxic effect of paclitaxel.


15

3. MATERIALS AND METHODS

3.1. Materials

Egg phosphatidylcholine (ePC), cholesterol (Chol), 1,2-dioleoyl-sn-glycero-3-

phosphoethanolamine-N-(glutaryl) (NGPE) and 1,2-dipalmitoyl-sn-glycero-3-

phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE) were purchased from

Avanti Polar Lipids (Alabaster, AL, USA). 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-

[methoxy (polyethylene glycol)-2000] (PEG2000-PE) was acquired from Corden Pharma

International (Plankstadt, Germany). Branched polyethyleneimine (bPEI) with a molecular weight

of 600 Da (confirmed with MALDI-TOF) was purchased from Polysciences, Inc. (PA, USA) and

paclitaxel (PTX) from LC Laboratories (Woburn, MA). Human cancer cell lines of HeLa (cervical)

was purchased from ATCC (Manassas, VA) and A2780-ADR (adriamycin-resistant ovarian

carcinoma) was purchased from Sigma (ECACC, UK). The paclitaxel-resistant human ovarian

cancer cell line SKOV-3TR was a kind gift from Dr. Duan Zhenfeng (MGH, Boston, MA).

CellTiter-Blue® cell viability assay was obtained from Promega Corp. (Madison, WI).

Hochest33342, Yo-Pro-1-iodide (YoPro), and propidium iodide (PI) were purchased from Life

Technologies (Carlsbad, CA). siRNA targeting MDR1 (siMDR1): 5′-

GGAAAAGAAACCAACUGUCdTdT-3′ (sense) and siRNA non-targeting (siSCR) duplex: 5´-

AGUACUGCUUACGAUACGGdTdT-3´ (sense) were purchased from Dharmacon (CO, USA).

Phycoerythrin-conjugated anti-P-glycoprotein antibody [UIC2] was purchased from Abcam

(Cambridge, MA). All other reagents were of analytical grade.


16

3.2. Synthesis of polyethyleneimine-phospholipid conjugate (bPEI-PE)

The bPEI-PE conjugate used to coat the liposomes was synthesized following the method in

Sawant et. al [62] with slight modifications. Briefly, N-Hydroxysuccinimide (NHS) (88 µmol) and

ethyl(dimethyl aminopropyl) carbodiimide (EDC) (86 µmol) were solubilized in methanol and

added to a chloroform solution containing NGPE (8 µmol) under magnetic stirring, keeping

agitation for 1 hour at room temperature. BPEI (27 µmol) was solubilized in chloroform, and 5 µL

of triethylamine was added to this solution. bPEI solution was added dropwise to the activated

NGPE under magnetic stirring, and agitation was maintained overnight at room temperature. The

organic solvent was removed under nitrogen gas; conjugate was resuspended in deionized water

and purified from free bPEI by dialysis (MWCO 3.5 kDa) against excess deionized water for 24

hours at 4°C. The purified solution was lyophilized, resuspended in chloroform and stored at -

80°C. The synthesized conjugate was characterized by thin layer chromatography (TLC).

3.3. Preparation and characterization of liposomes

PTX loaded liposomes (1.5% to 4% w/w) were prepared using ePC and Chol (90:10 mol%)

by the thin-film formation method followed by extrusion. Briefly, ePC, Chol and PTX were

solubilized in chloroform/methanol mixture, and the organic solvents were removed by rotary

evaporation to form the thin lipid film. The film was rehydrated with phosphate buffered saline

(PBS) pH 7.4 at 10 mg/mL concentration of lipids, heated up to 45°C, and extruded 15 times

through 100 nm polycarbonate membranes. bPEI-PE films were also prepared by solubilizing the

conjugate in chloroform, which was evaporated under nitrogen flow to form a thin film. After

extrusion, liposomes were added to bPEI-PE films at the mol ratios of 0.1% and 0.5% (mol% of


17

bPEI-PE:liposomal lipids). Liposome formulations were incubated at 37°C under mild agitation

for 4 hours for stable post-insertion of bPEI-PE into the lipid bilayer. Non-coated (plain) and

coated liposomes were filtered through 0.22 µm PES membranes for sterilization purposes as well

as separation of non-encapsulated drug. To prepare fluorescently labeled liposomes, Rhod-PE was

added in the preparation of the lipid film at a final concentration of 0.1% (%mol of total lipids).

Plain liposomes were named 0% PEIPOS and coated ones were named as 0.1% PEIPOS or 0.5%

PEIPOS depending on the mol% of bPEI-PE used in the coating step. After filtration, PEIPOS

were characterized by size, polydispersity index (PdI) and zeta potential (ZP) by dynamic light

scattering (DLS) in a Zetasizer Nano ZS 90 (Malvern Instruments, UK), with the samples

previously diluted with ultrapure water. Encapsulation efficiency was determined by a validated

reversed-phase HPLC method using a Hitachi Elite LaChrome HPLC system equipped with an

auto-sampler (Pleasanton, CA) and diode array detector [63]. Chromatographic separation was

achieved in a Xbridge C18 (4.6 mm250 mm) column (Waters Corporation, Milford, MA) with a

mobile phase containing 60:40 (v/v) acetonitrile:water at a flow rate of 1 ml/min and wavelength

of 227 nm.

3.4. In vitro drug release

For the in vitro release, the acceptor media were prepared with PBS containing 1% (w/v)

sodium dodecyl sulfate (SDS) at pH 5.0 and 7.4. A volume of 500 μl of either PEIPOS containing

PTX or free PTX solubilized in the release media was added to a Float-A-Lyzer® G2 ready-to-use

dialysis device (MWCO 50 kD). The devices were immersed in 13 mL of each acceptor media,

and maintained in an orbital shaker at 37°C at 125 rpm for 48h. Samples were withdrawn from the

release media and replaced with fresh media at predetermined time points over 48h. The amount


18

of drug released in the acceptor compartment was analyzed by HPLC after dilution with mobile

phase. The assay was performed in triplicate, and sink conditions were maintained during the

experiment. Samples were then filtered through a 0.45 μm membrane, diluted in mobile phase and

analyzed by HPLC. To ensure sink conditions, solubility of the drug was assessed in PBS

containing 1% SDS for 24 h at 37°C under agitation. Solubility of PTX in these media was

determined as 200 μg/mL.

3.5. Stability of PEIPOS in fetal bovine serum

Non-coated, 0.1% and 0.5% coated PEIPOS were incubated in 10% (v/v) fetal bovine serum

(FBS) containing PBS at 37°C under mild agitation (150 rpm on a horizontal shaker). At

predetermined time points of 1, 4 and 24h the changes in size and ZP were measured as described

earlier. All samples were incubated at a concentration of 500 µg/mL of lipids and diluted with

ultrapure water for the measurements.

3.6. Analysis of PEIPOS-associated proteins

To evaluate protein adsorption onto the surface of the liposomes, non-coated (0%) and 0.5%

PEIPOS at 1 mg/mL of lipid concentration were incubated in 10% (v/v) FBS containing PBS pH

7.4 overnight at 37°C under mild agitation. Liposomes of ePC and Chol (90:10 mol%) coated with

PEG2000-PE (2 mol%) were also prepared in the same way as PEIPOS for this assay. After

incubation, samples were submitted to ultrafiltration in 300 kDa MWCO filters centrifuged at

4,000 g for 10 minutes to separate free and PEIPOS-associated proteins. The proteins were

analyzed by SDS-PAGE under non-reducing conditions after lysing the liposomes with RIPA®


19

buffer (Thermo Scientific, Waltham, MA). The run was performed at 225V for 40 minutes in

polyacrylamide/bis-acrylamide gradient gel (4-20%) using prestained SDS-PAGE molecular

weight standards (Thermo Scientific Spectra Multicolor Broad Range Protein Ladder) to estimate

the molecular weight of the proteins adsorbed on to the PEIPOS. After the run was complete, the

gel was stained with GelCode® Blue Stain Reagent (Thermo Scientific, Waltham, MA) to allow

visualization of the protein bands.

3.7. Cell culture and spheroid formation

The human cervical cancer cell line HeLa, PTX-resistant human ovarian adenocarcinoma cell

line SKOV3-TR and adriamycin-resistant human ovarian carcinoma cell line A2780-ADR were

allowed to grow in complete RPMI cell culture medium supplemented with 10% FBS (v/v), plus

50 U/mL penicillin, and 50 µg/mL streptomycin. A2780-ADR cell line was exposed to 100 nM

doxorubicin twice a week for 48h, as recommended by the supplier. All cell lines were incubated

at 37°C, in a 5% CO2 atmosphere at high humidity.

Multicellular tumor spheroids of HeLa cells were prepared by the liquid overlay method as

described previously [64]. Briefly, 1.5% (w/v) of high-gelling temperature agar was dissolved in

serum-free RPMI and sterilized by autoclaving. Each well of a 96-well plate was coated with 50

µL of agar solution to prevent cells from adhering to the surface of the wells. After cooling down

for 45 min, 10,000 cells/well in 100 µl complete medium were seeded into the wells. The plates

were then centrifuged for 15 min at 1,500 rcf at 20°C. Spheroids were formed after 3-5 days after

seeding under the same conditions as monolayers and used in studies.


20

3.8. Cell association with monolayers

HeLa, SKOV3-TR and A2780-ADR cells were used to evaluate the association of the

formulations with these cell lines in monolayers. Briefly, the cells were seeded at a density of

100,000 cells/well in 12-well plates 24h prior to the experiment and then treated with Rhod-PE

labeled 0%, 0.1%, and 0.5% PEIPOS in serum-complete medium. The final lipid concentration for

the treatments was 100 µg/mL for all cell lines. HeLa cells were also treated with a low lipid

concentration of 10 µg/mL of lipids to investigate the liposome count:cell number effect. After 4h

of incubation at 37ºC, the cells were harvested, washed and immediately analyzed by a

FACSCalibur flow cytometer (Beckton Dickinson, Franklin Lakes, NJ) using 488 nm blue laser

for excitation and FL2 channel (585/42 nm wavelength filter) for recording the rhodamine

fluorescence intensity. Cells were gated to exclude debris and dead cells, and 10,000 events were

collected for each sample (n=3). Mean fluorescent intensity of the cells was compared to a control

group treated with non-labeled PEIPOS.

In another approach, A2780-ADR cells were seeded in 12-well plates at 80,000 cells/well 24h

prior to the assay. One plate was incubated at 4°C to inhibit any energy-dependent uptake and the

other at 37°C. A pre-treatment group was included where cells were pre-incubated with bPEI at

10 µg/mL for 15 min at both temperatures. This was done to saturate the cell surface with PEI and

verify that the signal obtained from the labeled liposomes was not originating from the electrostatic

interaction of the naturally negative charge of cell membranes with the positively charged PEIPOS.

After the pre-incubation period with free bPEI, cells were washed thrice and rhodamine-labeled

liposomes were added to each well at 200 µg/mL lipid concentration and incubated for an

additional 90 min. At the end of the treatment, the cells were washed with PBS once, detached,

centrifuged, washed again twice and analyzed by flow cytometry following the previously


21

described protocol. PEGylated liposomes were also used in this assay to compare the difference in

endocytosis of bPEI- and PEG-coated formulations.

3.9. 3D spheroid association and penetration

3D HeLa spheroids were treated for 4h with Rhod-PE labeled 0%, 0.1% and 0.5% PEIPOS

formulations 5 days after seeding. Lipid concentration was kept at 100 µg/mL, and five spheroids

were used as one replicate and treatments were done in triplicates (total of 15

spheroids/formulation). For flow cytometry analysis, spheroids were washed twice with PBS and

dispersed into single cells by 10 min incubation with AccuMax® cell detachment/disassociation

solution at 37°C [65]. The cell detachment solution was inactivated by FBS addition and cells were

centrifuged, washed and redispersed in PBS, and immediately analyzed by flow cytometer using

the same settings described earlier.

For optical sectioning with confocal laser scanning microscopy (CLSM), the spheroids were

treated the same way as in flow cytometry studies without disassociation. Following treatments,

the spheroids were washed with PBS and fixed in 4% paraformaldehyde overnight at 4°C. After

washing with PBS, spheroids placed in 16-well glass chamber slides (Nunc™ Lab-Tek™ Chamber

Slide System, USA) and the analysis was performed with a Zeiss LSM 700 confocal microscope.

The spheroids were imaged using 10 µm Z-stack intervals starting from the apex of the spheroids

using 555 nm laser and 10x objective. The images were obtained and analyzed using Image-J

software 1.51n (NIH, Bethesda, MD) with Fiji package (version 1.0) [66] to evaluate the

penetration profiles. A circular area with the same diameter and location indicating the core of the

spheroids were selected in every optical section. The relative area of selection was kept constant


22

between different spheroids. The corrected integrated pixel densities in the selection area were

plotted against the section distance from the apex to create the profiles. Z-projections using

maximum pixel intensity were also created to better reflect the differences between the overall

penetration profiles.

3.10. In vitro cytotoxicity on monolayers

To evaluate in vitro cytotoxicity, HeLa and A2780-ADR cells were seeded in 96-well plates

at a density of 3,000 cells/well 24h before treatment. Different formulations were added to the

cells at various concentrations and for various treatment times. For HeLa cells, the drug treatment

was continuous for 48h at 37ºC in complete media, whereas for A2780-ADR the cells were washed

after 4h of treatment, followed by 44h of incubation in fresh complete medium at same conditions.

Cell viability at the end of the treatments was assessed by CellTiter Blue® following the

manufacturer’s protocol. Fluorescence intensity was recorded (excitation 560 nm, emission 590

nm) in a microplate reader (BioTek, Model EL800, Winooski, VT). The viability of the treated

cells was compared to a control of non-treated ones.

3.11. β-tubulin immunostaining

To confirm the main mechanism of action of PTX, being a microtubule-stabilizing effect of

PTX, and whether it was affected by PEIPOS-mediated delivery, immunofluorescent imaging

studies were conducted. Briefly, HeLa cells were seeded on glass coverslips in 12-well plates at

100,000 cells/well one day before treatment. Cells were treated with the free drug, 0% and 0.5%

PEIPOS at 2.5 nM of PTX concentration and incubated for 24h continuously at 37°C in complete


23

media. After that, cells were fixed with 2% paraformaldehyde (PFA) at room temperature for 15

mins, permeabilized for 5 min with cold methanol and incubated with PBS containing 3% bovine

serum albumin (BSA) (w/v) at room temperature for 30 mins to block non-specific protein

interaction. Next, cells were incubated overnight at 4°C with 1:20 dilution of primary anti-β-

tubulin antibody (mouse monoclonal to β-tubulin, G-8, sc-55529, Santa Cruz Biotechnology,

Dallas, TX). The primary antibody solution was removed, coverslips were washed thrice, and the

FITC-labeled secondary goat anti-mouse IgG (Santa Cruz Biotechnology, Dallas, TX) was used

at a 1:30 dilution to incubate the cells for 2h at room temperature. All antibody dilutions and

staining were performed in blocking buffer. Finally, cells were washed with PBS, their nuclei

stained with 5 µM Hoechst 33342, and coverslips were mounted on slides for CLSM analysis.

3.12. Laser scanning cytometry

HeLa cells were seeded at 3,000 cells/well in black-walled optical-bottom 96-well plates one

day before the experiment. Cells were treated with the free drug, 0%, and 0.5% PEIPOS at different

concentrations for 48h. Next, Hoechst 33342, YoPro and PI were diluted with medium and added

directly to the wells at 5 µg/mL, 0.12 µg/mL, and 1 µg/mL final concentrations, respectively.

Incubation was followed at 37°C for 30 minutes, and cells were analyzed in situ by iCyte imaging

cytometer (CompuCyte Corp., Westwood, MA). Hoechst was excited with a 405 nm laser, and the

fluorescent signal was collected through a 440/30 nm bandpass filter. YoPro and PI were excited

by a 488 nm laser and fluorescent signals collected at 515/30 nm bandwidth and from 650 nm long

pass filter, respectively. The nuclei stained by Hoechst allowed the quantification of total nuclear

DNA content. Single live cells were assigned into G1, S, and G2 phases to analyze the cell cycle

distribution. Quantification of apoptotic events was done based on random segmentation, where


24

the area of the images covered by Hoechst, YoPro and PI fluorescence was determined. By doing

so, we discriminate live (Hoechst only) from early apoptosis (YoPro) and late apoptosis/necrosis

(PI).

3.13. Phase Holographic Imaging

Phase Holographic Imaging is a laser based methodology using a microscope that resides

in a standard CO2 incubator, and captures time-lapse images of unlabeled cells (Figure 5). For

holographic microscopy, 20,000 cell/well were seeded in glass-bottom 24-well plates 24h prior to

the experiment. Cells were treated with free PTX and 0% and 0.5% PEIPOS at 2.5 nM of PTX.

The Holomonitor M4 (Phase Holographic Imaging, Lund, Sweden) was used to collect images

every 5 minutes during 48h from each well (3 spots/well) using a 20x objective lens. The data

collected was analyzed using Hstudio analysis software (Phase Holographic Imaging).


25

Figure 5. Holomonitor® system. (A) Schematic representation of the principal of digital

holography. The light that illuminates the sample is split into a reference beam and a sample

beam, which rejoin later creating a hologram. The hologram recorded digitally is processed

using the software to obtain a holographic image of the optical thickness (B) Image of the M4

base unit, which is kept in a CO2 incubator during the analysis to maintain cell-growth optimum

conditions. (Retrieved from https://www.phiab.se/holomonitor/technology/holographic-time-

lapse-cytometry/ on June 28th, 2018)

3.14. Complexation of PEIPOS with siRNA, gel retardation, size and zeta potential changes

The complexation of PEIPOS with siRNA was monitored by gel retardation electrophoresis.

The 0.5% PEIPOS formulation was used to complex siSCR at different ratios of

nitrogen/phosphate (N/P), present in PEI-PE and siRNA, respectively. The complexes were

prepared with a fixed amount of siRNA diluted in 5% glucose (w/v) buffered HEPES pH 7.4

(BHG) and varying amounts of liposomes diluted with the same solution, mixed in equal volumes

and incubated for 15 minutes. The nucleic acid (1 µg) was loaded on a 0.8% agarose gel, and

electrophoresis was performed using an E-Gel electrophoresis system (Life Technologies,

Carlsbad, CA), followed by evaluation of the bands under UV light. Size and zeta potential of the


26

complexes were also verified to check for changes of these parameters upon complexation with

siRNA.

3.15. Protection of siRNA from RNases by PEIPOS

The protection of PEIPOS-complexed siRNA from nuclease digestion was determined by

treatment of the samples with 1 U of RNase III/µg siRNA for 30min at 37°C. EDTA was then

added at 30 mM for 5 min to stop the nuclease reaction. Heparin was then added at 40 U/µg siRNA

to break the complexes and free the siRNA. Samples were analyzed by agarose gel electrophoresis

using the setting described earlier.

3.16. Characterization of P-glycoprotein overexpression of cells

A2780-ADR cells were seeded in 6-well plates at 500,000 cells/well, along with A2780 cells,

their sensitive counterpart, to characterize their P-glycoprotein (P-gp) expression patterns. The

cells were detached on the next day with the aid of Cellstripper™ non-enzymatic cell

disassociation buffer, washed twice with 2% BSA (w/v) in PBS pH 7.4 containing 0.1% (w/v)

sodium azide (staining buffer) and incubated for 45 minutes on ice with phycoerythrin-labeled

anti-P-glycoprotein antibody (UIC2, Abcam). Cells were then washed twice with staining buffer

and resuspended in this same solution for immediate flow cytometer analysis. An isotype matching

control was used to evaluate the non-specific antibody binding and to normalize the results.


27

3.17. P-gp downregulation in MDR cells

Transfection efficiency of PEIPOS/siMDR1 complexes was evaluated in P-gp overexpressing

A2780-ADR cells. Cells were seeded into 6-well plates at 100,000 cells/well. Complexes were

prepared at N/P 13 and cells were treated one day after seeding with 100 nM of siMDR1/well for

4h at 37°C in serum-complete medium. Cells were washed gently and incubated for an additional

44h at same conditions. Then, the cells were detached using Cellstripper™ and processed using

the same protocol described earlier for P-gp staining. bPEI-PE micelles complexed with siMDR1

at N/P 16 were used as a positive control. Samples were analyzed by flow cytometry, and

fluorescence intensity of treated cells was compared to a P-gp stained non-treated control.

3.18. Hemolysis

Blood was collected from a healthy mouse in anticoagulant-containing tube, homogenized and

centrifuged at 500 x g for 5 minutes. The supernatant was discarded and 1 mL of calcium chloride

solution at 10 mM containing 0.85% sodium chloride was added to wash the erythrocytes. The

solution was washed 3 times by centrifuging at 500 x g for 5 minutes. The erythrocytes were re-

suspended at 2% (v/v) in the saline solution and used immediately. Triton X-100 solution at 0.2%

(v/v) in saline was used as positive control and a blank of all concentrations of PEIPOS was

included to null any color interference. The samples analyzed included 0% and 0.5%

PEIPOS/PTX, 0.5% PEIPOS/PTX/siMDR1, free PTX and 0.5% PEIPOS without drug (blank).

The starting concentration for drug, siRNA and lipid were 1.8 mg/mL, 0.25 mg/mL and 8.3

mg/mL, respectively, followed by 2-times serial dilution. Samples were added to a round-bottom

96-well plate, followed by addition of the erythrocyte solution. The plate was incubated for 1 h


28

under mild agitation at 37°C, following plate centrifugation at 500 x g for 10 min. The supernatant

was transferred to another plate and hemoglobin released due to hemolysis was measured by

spectrophotometer at 540 nm. The percentage of hemolysis was calculated considering the

absorbance in the presence of Triton X-100 as 100%.

3.19. In vivo animal model studies

The experiments were performed in 5 to 6-week-old female athymic nude mice following

protocol 16-1134R approved by Northeastern University Institutional Animal Care and Use

Committee. The animals were obtained from Charles River (Wilmington, MA) and weighed

between 22g and 25g throughout the study. They were provided food and water ad libitum. After

the acclimatization period of 7 days, the animals were inoculated subcutaneously near the right

flank with two million A2780-ADR cells in 100 µL of sterile PBS pH 7.4.

3.20. Preliminary in vivo evaluation of the efficacy of the nanopreparations

Before starting the efficacy study, two animals were injected with either 0.5% PEIPOS/PTX

or 0.5% PEIPOS/PTX/siSCR labeled with 1% rhodamine-PE (%mol). The animals were injected

every other day for a total of 3 times, via intravenous (IV) injection of the tail vein, with the same

drug, lipid and siRNA concentration to be used in the following efficacy study. Four hours after

the third injection, the animals were euthanized and their tumor and liver were harvested for

analysis of the presence of liposomes in these structures. For this analysis, the tumors and liver

were sliced using a Cryostat Microtome HM 550 (MICROM GmbH, Walldorf, Germany) at 10

mm thickness and collected on glass slides. They were fixed with paraformaldehyde solution at


29

2% (g/v) for 30 minutes, washed 3 times with PBS solution pH 7.4 and stained with 5 ug/mL of

Hoechst. An animal injected with PBS only was used as control for comparison of the rhodamine

intensity in the analyzed tissues.

For the tumor inhibition assay, when tumors reached approximately 150 mm3, animals were

randomly separated into 5 groups (n = 4). The different treatments administered IV, via the tail

vein, were: a) PBS Ph 7.4 as control; b) Free paclitaxel; c) 0.5% PEIPOS/PTX; d) 0.5%

PEIPOS/PTX/siMDR1. The treatment regimen consisted of IV injections every other day

containing 5.5 mg/kg of PTX and 0.8 mg/kg of siMDR1 for up to 12 injections or until tumor

reached 1500 mm3, when animals were then euthanized. The tumor sizes were monitored by

measuring with the aid of a Vernier caliper, every other day, beginning from the first day of

injection until the day the animals are euthanized. Tumor volume was calculated based on the

formula: V = 0.5 x W x W x L, where W is the width (smaller dimension of the tumor) and L is

the length (the largest dimension). At the end of the experiment, animals were euthanized, tumors

were collected, weighed, and snap-frozen with liquid nitrogen for further analysis. Blood was also

collected via cardiac puncture method from all the mice and centrifuged at 2,000 g for 30 min at 4

°C to separate the plasma, which was then stored at −80°C for further analysis of toxicity.

3.21. Toxicity evaluation of PEIPOS in mice

The toxicity of the formulations used was evaluated based on change in body weight of the

animals along the treatment. For that, animals were weighed every other day, beginning from the

first day of injection until the day the animals were euthanized. Alanine aminotransferase (ALT)


30

levels were measured in the plasma using a colorimetric assay kit following the manufacturer’s

instructions (Abcam, Cambridge, MA).

3.22. Statistical analysis

Experiments were performed in triplicate and results were expressed as mean ± standard

deviation. Unless otherwise indicated, an analysis of variance test (ANOVA) followed by Tukey’s

multiple comparisons test was used for comparison of differences between groups. Statistical

difference was accepted when P


31

4. RESULTS

4.1. Preparation and characterization of PEIPOS

The bPEI-PE conjugate was obtained by the reaction of a NHS-activated carboxyl group in

NGPE and an amino group in bPEI, resulting in an amphiphilic structure able to form micelle-like

nanoparticles spontaneously in aqueous media (Figure 6A) [62]. This conjugate was used at 0.1%

and 0.5% by mol to coat plain liposomes, represented as a scheme in Figure 6B (referred to as

0.1% PEIPOS and 0.5% PEIPOS, respectively; uncoated liposomes were named 0% PEIPOS).

Figure 6. (A) Schematic representation of the synthesis of bPEI-PE and (B) of bPEI-coated

liposomes complexing siRNA.

B

A


32

All liposomal formulations were monodispersed and presented a mean diameter of

approximately 160 nm, with ZP varying from -4.3 mV to +13.7 mV depending on the mol% of

bPEI-PE coating (Table 2). Drug leakage from the liposomes was evaluated after one-week

incubation at 4°C. Results show that 0.5% PEIPOS retained the drug and significantly avoided

leakage in this period when compared to plain liposomes (P


33

retain their therapeutic cargo when in the circulation, while not preventing the encapsulated drug

release after a longer time in s different pH environment.

Figure 7. Cumulative in vitro release of PTX from bPEI-coated liposomes at different pH under

sink conditions. Free PTX groups were used as controls to evaluate the diffusion rate through

the release membrane. Results express mean±SD.

4.3. Stability of PEIPOS in FBS and analysis of PEIPOS-associated proteins

Liposomes were incubated for 1h, 4h, and 24h in PBS with 10% (v/v) of FBS at 37°C to mimic

the in vivo salt and serum protein concentrations. Their ZP, particle size, and PdI values were

monitored to characterize their stability in physiological conditions [67]. There were small but

significant changes in size of non-coated liposomes (Figure 8B), and their PdI changed drastically

during the incubation period (Figure 8C), although still indicating a monodisperse sample after

24h of incubation. This data suggests a saturation after long-term serum exposure. On the other

hand, the presence of serum did not alter the size of bPEI-coated liposomes, and the changes in


34

their PdI values were only moderate. However, regardless of the surface modification with bPEI

and the ZP values before starting the serum incubation, the ZP of all samples decreased to a value

of approximately -5 mV (Figure 8A).

Figure 8. Stability of PEIPOS upon incubation in 10% (v/v) FBS in PBS pH 7.4 at 37°C for

over 24h. (A) ZP (mV), (B) particle size (nm) and (C) PdI values at given time points. Error bars

represent standard deviation of three independent sample measurements.

The sharp decrease in ZP of 0.5%PEIPOS is consistent with the adsorption of proteins onto

the surface of the liposomes, which was also confirmed by SDS-PAGE (Figure 9A). On the other

hand, 0%PEIPOS did not show changes of ZP in the presence of serum proteins. It is possible to


35

observe by SDS-PAGE bands of proteins adsorbed onto the PEIPOS surface around 50, 70, and

140 kDa, which approximate the molecular weights of mainly albumin (66 kD) and globulins (93–

1193 kDa), the main proteins present in FBS (Figure 6A) [68]. PEGylated liposomes (of the same

composition as 0% PEIPOS with an additional 2 mol% of PEG2000-PE) were also investigated to

compare their protein association with PEIPOS. The results show that the profile of proteins

adsorbed onto PEGylated liposomes (Figure 9A, lane 4) is the same as 0% and 0.5% PEIPOS, but

to a lesser extent. When the band intensities are evaluated by Fiji software (Figure 9B), it was

found that 0.5% PEIPOS and PEGylated liposomes represented approximately 70% and 64% of

the band intensities of 0% PEIPOS.

Figure 9. Protein adsorption of the surface of liposomes. (A) Proteins adsorbed in liposomes

incubated in 10% FBS at 37°C overnight. Lane 1 shows FBS diluted to 2.5% (v/v), lanes 2 and

3 are 0% PEIPOS and 0.5% PEIPOS and lane 4 is a PEGylated liposomal formulation. (B) Band

intensities of the liposome surface adsorbed proteins, calculated by analyzing the SDS-PAGE

gel image by Image-J. Non-coated liposome (0%PEIPOS) surface-adsorbed protein amount was

accepted as 100%. 0.5%PEIPOS and PEGylated liposome-adsorbed protein amounts were

calculated relative to 0%PEIPOS band. bPEI-coated and PEGylated liposomes show 30% and

35% less protein adsorption onto their surface, respectively.

A B


36

4.4. bPEI-coated PEIPOS improve uptake in monolayers and penetration in 3D spheroid

models

Cell association was assessed in HeLa, SKOV3-TR, and A2780-ADR cells. The effect of the

coating at different molar ratios was also evaluated. bPEI-PE coated liposomes presented higher

cell association compared to the non-coated ones in all the tested cell lines (Figure 10). 0.5%

PEIPOS showed the highest cellular association, with a significant difference compared to non-

coated liposomes even when a low concentration of lipids (10 µg/mL) was used. In HeLa cells,

when a low concentration of lipid was used, there was no significant difference between control

and 0% or 0.1% PEIPOS, whereas the association was enhanced by almost 5-fold for 0.5%

PEIPOS. At higher lipid concentration (100 µg/mL), the cell interaction with 0.5% PEIPOS was

27-fold higher than control or non-coated liposomes. In A2780-ADR cells, the difference in

association of 0.5% PEIPOS was of almost 2-fold compared to 0.1% PEIPOS and 26-fold higher

when compared to control. SKOV3-TR, which is another MDR cell line, results indicate a similar

trend. 0.5% PEIPOS showed the highest association with the cells. It is important to note that

surface-engineered liposomes interacted significantly more than with the drug-resistant ovarian

cancer cells in serum-complete medium.

We performed a cellular association study of the PEIPOS formulations at 37°C and 4°C (Figure

10D) to further characterize and confirm their mode of entry into the cancer cells. The results

showed that PEGylated liposomes had the lowest cellular association at both temperature levels,

highlighting the challenges related to PEGylation. The incubation of PEIPOS with cells at 4°C

lead to a significant decrease in association. However, there was still a 4-fold increase in

association at low temperature when compared to the control. When cells were pre-treated with


37

free bPEI before adding the formulations, there was an overall decrease in association at both

temperatures.

Figure 10. Cellular association of coated and non-coated PEIPOS with monolayers of three

different cancer cell lines; (A) HeLa, (B) A2780-ADR and (C) SKOV-3TR. * indicate difference

from control, whereas # indicate differences within the groups. Mean±SD, n=3, **P


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Following the 2D monolayer experiments, we investigated the interaction of the formulations

with the cancer cells within the 3D spheroid model. Cellular association of the formulations with

the cancer cells in 3D structural organization was investigated by analyzing the individual cells

forming the spheroids by flow cytometry. Results show that, compared to control groups, 0%

PEIPOS had an approximately 5% cellular association increase with HeLa cells in the spheroids,

while 0.1% and 0.5% PEIPOS association increases were 18% and 88%, respectively (Figure

11A). There was a statistically significant increase in association when the liposomes were coated,

confirming the ability of bPEI-PE modification to improve the interaction of the liposomes with

the cells in this tumor mass model.

It should be noted that flow cytometry analysis involves all the cells in the spheroid structure

and give a normalized result. However, it is not possible to understand the distribution of the

formulations in different levels of the spheroids, i.e. their penetration profiles. To better investigate

the penetration ability of liposomes inside a 3D tumor mimicking model with significant

penetration barrier properties, we conducted the CLSM studies. The 5% cellular association of

non-coated liposomes obtained by flow cytometry did not translate to CLSM analysis, and it was

not possible to observe fluorescence signal for 0% PEIPOS in the settings used. It is, however,

possible to see that 0.1% PEIPOS penetrates fairly well into the spheroid structure. The maximum

integrated fluorescent signal was collected at 40 μm depth from the apex of the spheroid (Figure

11B and D). The fluorescent signal obtained with 0.5% PEIPOS was maximum at 50 μm,

suggesting slightly deeper penetration into spheroid layers compared to the 0.1% bPEI-modified

liposome formulation. More importantly, the signal from 0.5% PEIPOS was significantly higher

in layers starting from 30 μm, with the difference of approx. 75% at 50 μm. The representative Z-

section images of the spheroids are given in Figure 3D. The results clearly indicate that the


39

penetration of liposomes in the deeper layers is enhanced with higher concentrations of surface

accessible bPEI on the liposomes. Moreover, coated liposomes are not internalized only by the

cells in the periphery but also by the cells in deeper layers and not subjected to a penetration barrier

effect. Maximum pixel intensity (MPI) Z-projections of the optical stacks were created to better

visualize the penetration differences of the formulations. Taken together, the data confirm that the

0.5% bPEI-modified liposomes penetrated into the 3D spheroids by bypassing several barriers

including physical penetration and decreased cellular interaction in a highly heterogeneously

organized environment. Due to the successful and supportive results obtained with 0.5% PEIPOS

and the promising possibility of this formulation to form siRNA complexes at reasonable N/P

ratios, the 0.1% PEIPOS formulation was not included in further experiments.


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Figure 11. Kinetics of cellular association and penetration of formulations in 3D HeLa spheroids.

(A) Rhodamine intensity increase as the indicator of cellular association of liposomes with HeLa

cells after disassociation of spheroids into single cells, obtained by flow cytometry analysis, n=3,

a total of 15 spheroids, mean±SD, one-way ANOVA with Tukey’s multiple comparison tests,

****P


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4.5. bPEI-coating enhances the in vitro cytotoxicity of liposomes

HeLa cells, a cervical carcinoma cell line and the most studied cancer cell line to date, and

A2780-ADR, a drug-resistant P-gp overexpressing ovarian cancer cell line, were chosen for the in

vitro cytotoxicity studies of the liposomal formulations. On HeLa cells, there was a significant

difference in cytotoxicity between PTX loaded coated and non-coated PEIPOS at most of the

studied drug concentrations (Figure 12A). 0.5% PEIPOS presented similar killing properties to

free PTX, with IC50 of 2.4 nM and 2.5 nM, respectively. The IC50 improvement by bPEI-surface

modification was 50% compared to that for 0% PEIPOS of 3.8 nM. Data confirm and support the

improved cell association promoted by bPEI coating. Moreover, the bPEI-coated empty liposomes

did not induce significant cytotoxicity in HeLa cells, suggesting the safety of the bPEI-modified

liposomes, despite their overall net positive surface charge.

On A2780-ADR cells, however, even though coated liposomes presented better association

(Figure 7B), this result did not translate into improved cytotoxicity (Figure 12B), most probably

due to the MDR characteristic of this cell line. The overexpression of P-gp on the surface of these

cells leads to the removal of the drug through efflux pumps. Despite a lack of differences between

them, coated and uncoated PEIPOS had enhanced cytotoxicity when compared to free PTX in

concentrations varying from 32 µM to 4 µM, underlining the advantages of nanomedicine-based

treatment approaches for treating drug resistant cancers. Empty PEIPOS formulations exerted no

significant cytotoxicity on this cell line either.


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Figure 12. Cell viability of (A) HeLa and (B) A2780-ADR cells after treatment with the

formulations for continuously 48h or 4+44h, respectively. Data shown indicate triplicate

mean±SD. *P


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4.6. bPEI-modified liposomal PTX induces β-tubulin polymerization

Paclitaxel shows its inhibitory effect on cell division, migration and proliferation by binding

to microtubules and stabilizing them, thus leading to eventual mitotic arrest. Immunofluorescent

analysis of β-tubulin showed that cells from untreated control have normal microtubule

organization, with fine and diffuse microtubule structure (Figure 13). Free PTX-treated cells,

however, presented microtubules with thicker structures, an indication of their stabilization. For

the liposomal formulations, especially for 0.5% PEIPOS, it is possible to see a greater effect of

this stabilization, with tubulins polymerizing around the nuclei and causing the cells to round up.

The results of β-tubulin stabilization were consistent with previous reports and indicate that PTX-

loaded bPEI-modified novel liposome formulations did not cause a change of action of cargo drug

and its effects.

Figure 13. Immunofluorescent detection of liposomal PTX-mediated β-tubulin polymerization

on HeLa cells. Nuclei of the cells were stained with Hoechst, displayed in magenta, and β-

tubulin structures are displayed in green, stained by FITC-labeled antibody.

Control Free PTX

0% PEIPOS/PTX 0.5% PEIPOS/PTX


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4.7. bPEI-modified liposomal PTX induces apoptosis

Following the confirmation of the effective β-tubulin stabilization ability of the PEIPOS, we

investigated whether the microtubule polymerizing capacity of PTX caused subsequent apoptosis.

We used different treatment regimens to better evaluate and compare the effects of free and bPEI-

modified liposomal PTX on cells. HeLa cells were treated either for 48h continuously (indicated

as 48) or for 4h, followed by 44 hours of incubation after drug removal (indicated as 4+44). Laser

scanning cytometry (LSC) analysis was used in a single end-point experiment, based on Hoechst-

stained DNA content and also cell morphology. Nuclear segmentation was used to identify the

cells based on DNA staining (analysis of brightness, area, content) and to determine their phase in

the cell cycle, as represented in the histogram in Figure 14A. The whole cell population was then

gated and classified as live, sub-G1 or early apoptotic cells. The sub-G1 population included

apoptotic bodies generated when cells break up during apoptosis. Early apoptotic cells represent

the early effects of the drug in apoptosis. The microtubule polymerization caused by PTX led the

nuclei to start becoming distorted. The cells at this stage fall out of the normal cell cycle, and

present a decrease in area (Gate R9, Figure 14B). At this stage, cell nuclei present a reniform shape

(Figure 14B, lower left).


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Figure 14. iCyte laser scanning cytometer images. Data from cells treated by 0.5% PEIPOS

with PTX at 5 nM for 48h continuous. (A) Cell cycle histogram from cells in gate R11 from

panel B. (B) Cell nuclei stained with Hoechst and clearly demonstrated the different shapes of

nuclei (lower panels). Gate R11 contained live cells; gate R10 included cells in sub-G1

(apoptotic bodies); and gate R9 with cells presenting early effects of PTX. The nuclei of cells in

R9 present a reniform shape, with smaller area when compared to cells in R11 (live cells).

Images were recovered from the indicated areas automatically with the same size and intensity

settings.

It was possible to observe

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