http://informahealthcare.com/drdISSN: 1071-7544 (print), 1521-0464 (electronic)

Drug Deliv, Early Online: 1–14! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/10717544.2014.973082

RESEARCH ARTICLE

EpCAM-targeted liposomal si-RNA delivery for treatment ofepithelial cancer

Dhiraj Bhavsar1, Krishnakumar Subramanian2, Swaminathan Sethuraman1, and Uma Maheswari Krishnan1

1Centre for Nanotechnology & Advanced Biomaterials, School of Chemical & Biotechnology, SASTRA University, Thanjavur, India and2L&T Ophthalmic Pathology Department, Sankara Nethralaya, Vision Research Foundation, Chennai, India

Abstract

Background: RNA interference (RNAi) technology using short interfering RNA (si-RNA) hasshown immense potential in the treatment of cancers through silencing of specific genes.Cationic non-viral vectors employed for gene delivery exhibit toxic effects in normal cellslimiting their widespread use, therefore, site-specific delivery using benign carriers couldaddress this issue.Objective: Design of a non-toxic carrier that enables site-specific delivery of si-RNA into thecancer cells is of prime importance to realize the promise of gene silencing.Methods: In the present study, non-cationic liposomes encapsulating si-RNA against epithelialcell adhesion molecule (EpCAM) were developed and characterized for encapsulationefficiency, colloidal stability, in vitro and in vivo gene silencing efficacy.Results: PEGylated liposomes containing phosphatidyl choline and phosphatidyl ethanolamineexhibited maximum si-RNA encapsulation efficiency of 47%, zeta potential of -21 mV, phasetransition temperature of 51 �C and good colloidal stability in phosphate-buffered saline (PBS)containing bovine serum albumin (BSA) and plasma protein (PP) at 37 �C. Conjugation ofepithelial cell adhesion molecule (EpCAM) antibody to the liposomes resulted in enhanced cellinternalization and superior down-regulation of EpCAM gene in MCF-7 cell lines whencompared with free si-RNA and the non-targeted liposomes. In vivo evaluation ofimmunoliposomes for their efficacy in regressing the tumor volume in Balb/c SCID miceshowed about 35% reduction of tumor volume in comparison with the positive control whenadministered with an extremely low dose of 0.15 mg/kg twice a week for 4 weeks.Conclusion: Our results exhibit that the nanocarrier-mediated silencing of EpCAM gene is apromising strategy to treat epithelial cancers.

Keywords

Colloidal stability, gene delivery, genesilencing, non-cationic liposomes,release kinetics

History

Received 8 August 2014Revised 1 October 2014Accepted 1 October 2014

Introduction

Short interfering RNA (si-RNA) is a powerful tool that has

the potential to down-regulate the expression of the target

genes (Pai, 2005). However, gene silencing through RNA

interference (RNAi) for the treatment of different cancers has

not made significant progress due to several limitations. Most

strategies to improve RNAi efficiency have not been

successful due to the immune response and the additional

barriers presented by the biological system (Whitehead et al.,

2009). The major challenges in the use of RNAi are the poor

stability of si-RNA in circulation, poor targeting to specific

cells, inability to escape endosomal degradation, off-target

effect and production of inflammatory response (Juliano

et al., 2009). Therefore, the prime requisite for successful

implementation of effective gene down-regulation in vivo is to

enable target-specific delivery of si-RNA using an appropriate

carrier.

Numerous approaches for delivery of si-RNA have been

investigated including both viral and non-viral delivery

systems (Gao & Huang, 2008). Viral vectors, such as

adenoviral, lentiviral and retroviral systems exhibit high

transfection efficacy (Raper et al., 2003; Stewart et al.,

2003). But, despite their desirable transfection efficiency, use

of viral vectors for gene delivery is restricted because of their

ability to integrate into the host genome and produce severe

inflammation (Barton & Medzhitov, 2002). Clinical trials

conducted for the treatment of X-linked severe combined

immune deficiency using retrovirus vectors revealed that the

patients exhibited increased T-cell count because of the

integration of viral genome at the first intron of LMO-2

(Hacein-Bey-Abina et al., 2003). These results demonstrate

the risk associated with viral vectors. This has resulted in the

development of non-viral vectors to improve gene therapy

strategies. Majority of the carriers employ cationic moieties

to facilitate better complexation of the negatively charged

si-RNA through electrostatic associations with the carrier.

Address for correspondence: Prof. Uma Maheswari Krishnan, PhD,Centre for Nanotechnology & Advanced Biomaterials, School ofChemical & Biotechnology, SASTRA University, Thanjavur 613 401,India. Tel: +91 4362 264101; Ext 3677. Fax: +91 4362 264120. E-mail:umakrishnan@sastra.edu

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Among the polymers commonly employed as non–viral

si-RNA delivery systems, chitosan (Ma et al., 2014;

Ozpolat et al., 2014; Xie et al., 2014), poly(ethylene imine)

(Urban-Klein et al., 2005; Nabzdyk et al., 2014), poly(ami-

doamine) dendrimers (Patil et al., 2008; Conti et al., 2014)

and poly(L–lysine) (Inoue et al., 2008; Kodama et al., 2014)

have been extensively investigated.

Liposomal carriers, in particular, have received consider-

able attention over the years due to their ease of formation,

tailorable surface functionalities and excellent cell intern-

alization (Immordino et al., 2006; Kong et al., 2012; Nag

& Awasthi, 2013). Cationic liposomes prepared with 1,2–

dioleoyl–3–trimethylammonium propane (DOTAP) and

dioleoyl phosphatidylethanolamine (DOPE) lipids have

been found to be highly efficient in complexing the

si-RNA and have been evaluated for their silencing

efficacy in Many cancer cell lines (Ying & Campbell,

2014). Custom-made cationic lipids (Oh & Park, 2009) and

cationic gemini lipids (Zhao et al., 2014) have also been

explored as gene delivery vehicles. Despite their promise

in in vitro trials, most of these systems are ineffective in

vivo due to the toxicity of the cationic moieties and their

non-specific interactions with non-target cells limit their

widespread use. High-inflammatory responses to these

systems have also restricted their use as gene delivery

systems in vivo (Lonez et al., 2008). Therefore, attempts

to reduce or mask the cationic moieties have been reported

to reduce the adverse effects of these systems. Use of

anionic lipids to form liposomes along with a divalent

cation has also been attempted to overcome the drawbacks

of cationic liposomes (Daniel & Godbey, 2011).

The present study intends to develop si-RNA-encapsu-

lated PEGylated liposomes formed using non-cationic lipids.

We attempt to achieve cancer cell-specific delivery through

surface modification with EpCAM antibody. Epithelial cell

adhesion molecule has been found to be over-expressed in

epithelial cancers, cancer stem cells (Allard et al., 2005) as

well as circulating cancer cells (Simon et al., 2013). The

EpCAM found in normal cells are expressed in the

basolateral side, while it is over-expressed on the apical

side in cancer cells (Slanchev et al., 2009). This character-

istic of EpCAM makes it an attractive target for site-specific

si-RNA delivery. EpCAM-targeting liposomes encapsulating

doxorubicin were found to be more effective in colon

cancers when compared to free drug and the non-targeted

carrier (Allen & Cullis, 2004). Synthetic amphiphiles

(SAINT) were reported to effectively deliver antisense

oligonucleotides to melanoma and colon cancer cells when

conjugated to a monoclonal antibody targeting EpCAM (Van

Zanten et al., 2004). However, significant reduction in

cancer cell numbers was achieved only in colon cancer

cells implying the need for further exploration of this

targeting moiety and its role in different types of cancer.

Delivery of bispecific antisense nucleotides against bcl-2

and bcl-xL has been accomplished using EpCAM antibody

fragment as the targeting moiety (Hussain et al., 2006). The

system was found to sensitize the epithelial cancer cells to

doxorubicin.

EpCAM has also been recognized to possess oncogenic

potential (Osta et al., 2004). The MCF-7 breast cancer cell

line has been extensively studied for EpCAM over-expression

and it has been found that EpCAM over-expression activates

cell proliferation by stimulating gene expression of c–myc and

JNK/AP–1 (Sankpal et al., 2011). The silencing of EpCAM

gene has been demonstrated to down-regulate proliferation

genes and up-regulate the expression of apoptotic genes, such

as DRAM and cytochrome c. It also reduces the expression of

tumor invasive genes like MMP2 and cdc42 (Mitra et al.,

2010). Therefore, EpCAM gene silencing may help in

controlling tumor growth and can be used for the treatment

of epithelial cancers. However, reports on the use of an

appropriate gene delivery system to achieve silencing of

EpCAM gene through RNAi technology are scanty in

literature. The realization of the therapeutic potential of

EpCAM silencing through the use of targeted cationic lipid-

free liposomal system therefore forms the crux of the present

study.

Materials and methods

Materials

Egg phosphatidylcholine (EPC) was purchased from Avanti

Polar Lipids, Alabaster, AL. N–(Carbonyl–methoxypolyethy-

leneglycol 2000)–1,2–distearoyl–sn–glycero–3–phosphoetha-

nolamine sodium salt (PEG chain MW 2000 Da) (DSPE–PEG)

were purchased from NOF, Grobbendonk, Belgium. EpCAM

si–RNA and Alexa Fluor 488 labeled si–RNA were purchased

from Qiagen, Valencia, CA. EpCAM antibody was procured

from Santa Cruz Biotechnology Ltd., Dallas, TX. Bovine

serum albumin (BSA) was purchased from Sigma Aldrich, St.

Louis, MO. All other organic reagents of analytical grade were

purchased from Merck Chemicals, NJ, USA.

Preparation and characterization of liposomes

Liposomes were formed using thin-film hydration. The

solvent in the phospholipid solution was evaporated in a

current of nitrogen to obtain a thin layer of phospholipids. An

aqueous dispersion of the si-RNA complex in phosphate-

buffered saline (PBS) of pH 7.4 was added to the thin film and

stirred constantly for 30 min at 60 �C. The solution was

extruded through polycarbonate membranes with a pore

diameter of 200 nm for 10 cycles to obtain uniform sized

liposomes and were freeze–dried (Christ alpha 2-4 LD Freeze

dryer, Martin Christ, Osterode am Harz, Germany) and stored

until further use.

For preparation of hybrid immunoliposomes, thiolated

EpCAM antibody was used. The antibody was mixed with

Traut’s reagent (2–iminothiolane, sigma Aldrich, St. Louis,

MO) at 1:20 ratio in HEPES-buffered saline (HBS) (25 mM

HEPES, 140 mM NaCl, pH 8) and allowed to stand at room

temperature for 1 h. The reaction buffer contains 2 mM EDTA

that inhibits self-polymerization during the thiolation (Pan

et al., 2007). The unreacted Traut’s reagent was removed by

dialysis in HBS of pH 7.5. The thiolated antibody was then

mixed with liposomes incorporated with maleimide-termi-

nated PEG lipid (DSPE–PEG–MAL) and the mixture was

kept overnight at 4 �C for the formation of C–S covalent link

2 D. Bhavsar et al. Drug Deliv, Early Online: 1–14

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between the antibody and PEGylated hybrid liposome. The

unattached antibodies were removed using Centriprep�

(Darmstadt, Germany) at 4 �C and linking was confirmed

through Fourier transform infrared spectroscopy (FTIR)

(Spectrum 100, Perkin Elmer, Waltham, MA). The reaction

scheme is shown in Figure 1.

The encapsulation efficiency of the si-RNA in liposomes

was quantified using Alexa Fluor–488 (Qiagen, Valencia, CA)

linked fluorescent si-RNA. To quantify the encapsulation

efficiency, the samples containing the liposomes and the

unencapsulated si-RNA was subjected to centrifugation at

15 000 rpm for 30 min at �10 �C. The supernatant was

analyzed using multimode reader at an excitation wavelength

of 490 nm and emission wavelength of 525 nm for determin-

ing the amount of si-RNA present in the liposomes. The

encapsulation efficiency was then calculated using the

formula:

encapsulation efficiency ¼ Total siRNA� Free siRNA

Total siRNA� 100

The particle size and zeta potential of the samples

were measured using dynamic light scattering. The samples

were dispersed in equal volume of deionized water and

loaded in disposable capillary cells. The analysis was

carried using Zetasizer (Nano–ZS, Malvern, UK) at room

temperature.

Thermal behavior of the samples was analyzed in differ-

ential scanning calorimeter (DSC) (Q20, TA instruments,

New Castle, DE). Five milligrams of liposome samples were

weighed and taken in aluminum pan. Heat flow was set to

10 �C per min and samples were analyzed from 10 �C to

90 �C.

The colloidal stability of the samples was recorded using

the laser diffraction method. The extruded samples were

dispersed in 20 mL of PBS (pH 7.4) or PBS containing bovine

serum albumin (BSA) or plasma protein (PP) and incubated

for 24 h at 37 �C and then analyzed using particle size

analyzer (Microtrac Bluewave, Montgomeryville, PA).

For evaluation of the magnitude of protein adsorption,

liposomes were prepared using the thin-film hydration

technique. The pellet having the liposomes was dispersed in

PBS of pH 7.4. The reconstituted liposome samples were

incubated for 2 h with PBS containing BSA or PP (2 mg/mL).

After 2 h incubation, the liposomes were separated by

centrifugation at 15 000 rpm for 30 min at �10 �C and the

Figure 1. Schematic representation of establishment of immunoliposomes.

DOI: 10.3109/10717544.2014.973082 EpCAM-targeted liposomal si-RNA delivery 3

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supernatant was analyzed for determining the non-adsorbed

proteins using Bradford protein estimation assay.

The antibody conjugation to the liposomes was docu-

mented using Fourier transform infrared spectroscopy (FTIR).

To prepare samples for the FTIR analysis, liposomes were

mixed with KBr (IR grade, Merck, NJ, USA) and pelleted

using a pelletizer. The spectra were recorded in a FTIR

spectrometer (Spectrum 100, Perkin Elmer, Waltham, MA)

between 4000 and 400 cm�1.

In vitro studies

For in vitro experiments 2� 105 MCF-7 breast cancer cells

were seeded per well in 24-well plates. Dulbecco’s Modified

Eagle’s Medium (DMEM) and fetal bovine serum (FBS)

(Gibco, NY, USA) were used for maintaining the culture. For

cell uptake studies, MCF-7 cells were seeded 24 h prior to the

experiment. After 24 h, the spent medium was replaced with

fresh DMEM medium. The liposomes containing Alexa

Fluor–488 linked si-RNA dispersed in 0.5 mL PBS of pH

7.4 were added to the respective wells. At pre-determined

time points (0, 1, 2 and 4 h), the cells were counterstained

with Hoechst and imaged using laser scanning confocal

microscope (FV1000, Olympus, Tokyo, Japan). Cell uptake

was also quantified by flow cytometry. Briefly, 2� 105 MCF-

7 cells were treated with Alexa Fluor–488 linked si–RNA

encapsulated liposomes. After 24 h, cells were collected,

centrifuged and washed twice in PBS. The pellet was

dispersed in sheath fluid and cells were analyzed using flow

cytometer (FACS Calibur, BD Biosciences, New Jersey,

USA).

The quantitative analysis of silencing of EpCAM gene was

determined using qRT–PCR (AG22331, Eppendorf, Hamburg,

Germany). About 2� 105 MCF-7 cells were incubated with

immunoliposomes and liposomes without antibody containing

100 and 200 nM si-RNA. After 48 h of incubation, the cells

were collected and the total RNA was isolated using RNeasy

mini kit (Qiagen, Valencia, CA) following manufacturer’s

protocol and the RNA was quantified using UV-visible

spectrophotometer (Nano Drop, Thermo Fisher Scientific,

Waltham, MA). Complementary DNA (cDNA) was synthe-

sized using Quantitect Reverse transcription kit (Qiagen, USA)

and random primers. Then, the cDNA samples were subjected

to amplification using specific EpCAM primers with the

following sequence – Forward: CGCAGCTCAGGAAGAA

TGTG and Reverse: TGAAGTACACTGGCATTGACG. The

gene expression was quantified employing the DCt–DCt

method in real time RT-PCR using SYBR Green probe

(Qiagen, USA).

Toxicity of immunoliposomes was evaluated using MTS

assay (Cell Titer 96 AQueous one solution, Promega, Madison,

MI). Ten thousand cells were seeded in a 96-well plate and

incubated at 37 �C in 5% CO2. After the cells attained

confluency, the samples were added. At the end of each time

point, the samples were washed with PBS solution to remove

the non-adherent cells. MTS reagent (20 ml) and 200 ml of

serum-free media were added to each of the samples and

incubated at 37 �C for 2 h. The reaction was stopped by an

addition of 25 ml of 10% sodium dodecyl sulfate (SDS)

solution. The absorbance was measured at 490 nm using a

multimode reader (Infinite 200M, Tecan, Grodig, Austria).

In vivo studies

Female BALB/c SCID mice aged 5–7 weeks were used for

the study. Animal ethics clearance was obtained from the

Institutional Animal Ethics Committee (IAEC approval no.

16/13–(6/4/2013)) to carry out the study. Mice were given

irradiated rodent diet ad libitum (Pet car brand, Bangalore,

India) and were maintained in a sterile setting at 22–25 �Cwith 12 h light/dark cycle. 48 h prior to the tumor induction,

the mice were implanted subcutaneously with 0.18 mg slow

release 17b–estradiol pellets (SE–121, Innovative Research of

America, Novi, MI). About 5� 106 MCF-7 breast cancer cells

in 100 mL matrigel (FACS Calibur, BD Biosciences, New

Jersey, USA) were inoculated subcutaneously in the right

flank of the mice. The animals were observed frequently for

the tumor growth. Palpable tumors were visible from the

second week after cancer induction. The mice were

randomized after they attained a tumor volume of about 70–

90 mm3, and grouped with five mice in each group.

Liposomal formulations were given thorough injection to a

site adjacent to the tumor at 0.15 mg/kg body weight of mice.

The formulations were administered twice a week for four

weeks. The tumor dimensions were measured twice a week

using digital vernier calipers. The tumor volume was

calculated using the relation:

Tumor volume ¼ 0:5� L�W2

where, L is the length and W is the width of the tumor. After

completion of four weeks, animals were sacrificed by CO2

asphyxiation and the tumors were excised, weighed. For

further trials, half of the tumor fragment was snap frozen in

liquid nitrogen and stored at �80 �C. The other half of the

tumor was fixed in 10% neutral-buffered formalin for histo-

pathological analysis. The sections stored in �80 �C were

weighed, crushed in 1 mL of lysis buffer. The lysate were used

for the mRNA isolation following manufacturer’s protocol.

cDNA preparation and qRT-PCR for gene expression was

carried out as mentioned for in vitro studies. Formalin-fixed

tumor samples were used for histopathological analysis.

Thinner sections 2–3 mm thick were cut and processed.

Paraffin-embedded tissues were sectioned to 3–5 micron

thick slices using microtome and stained with haematoxylin

and eosin. The samples were then imaged using light

microscope (Nikon Eclips Ti, Tokyo, Japan) and imaged.

Western blot analysis

Total protein was isolated from the tumor tissue using cell

lysis buffer (10 mM Tris-HCl of pH 7.2), 2% sodium dodecyl

sulphate (SDS), 10 mM dithiothreitol, 1% protease and

protease inhibitors cocktail (Sigma Aldrich, USA). The

concentration of protein was measured using Lowry’s

method. An aliquot (containing 50 mg protein) of lysate was

used for 12% sodium dodecyl sulfate-polyacrylamide gel. The

blocking of the membrane blots were carried for 1 h with

blocking buffer (5% skimmed milk in Tris-buffered saline

containing 0.1% Tween-20) and then incubated overnight in

primary antibody (EpCAM antibody, dilution 1:150, Cell

Signalling, Beverly, MA) at 4 �C. The blots were washed and

incubated with appropriate anti-mouse horseradish peroxid-

ase-conjugated secondary antibodies (dilution 1:2000, Cell

4 D. Bhavsar et al. Drug Deliv, Early Online: 1–14

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Signaling, USA) for 3 h at room temperature. Tetramethyl

benzidine/hydrogen peroxide (TMB/H2O2, Bangalore Genei,

Bangalore, India) reagent was used for the visualization

according to the manufacturer’s instructions. Membranes

were stripped in 100 mM glycine of pH 2 for 40 min,

reblocked, and re-incubated in primary antibody for the

housekeeping protein b-actin (Sigma Aldrich, USA). The

UVP BioDoc-IT 220 Imaging System (Upland, CA) and

ImageJ software (http://imagej.nih.gov/ij/) were used for the

scanning and densitometry analysis of digitized images. After

the background normalization, b-actin band intensity was

used to normalize the band intensity of the corresponding

EpCAM protein band.

Statistical analysis

Two-way analysis of variance (ANOVA) was employed to

determine the statistical significance. The level of signifi-

cance was determined using Bonferroni’s test (p50.05).

Results

Liposomes were prepared using thin-film hydration method.

The lipid composition was modified to achieve max-

imum encapsulation. The phospholipids used were egg

phosphatidyl choline (egg PC), dioleoyl phosphatidyl etha-

nolamine (DOPE), cholesterol, distearoyl phosphatidyl etha-

nolamine-polyethylene glycol (DSPE-PEG) and distearoyl

phosphatidyl ethanolamine-polyethylene glycol-maleimide

(DSPE-PEG-mal).

Figure 2 shows the encapsulation efficiency liposomes

prepared with different lipid ratios. It is evident from Figure 1

that the lipid composition influences the si-RNA encapsula-

tion significantly. The least encapsulation efficiency of 19%

was exhibited by liposomes prepared using egg PC only

(LA). Introduction of the helper lipid DOPE improved the

encapsulation efficiency. The liposomes with 8:2 ratio of egg

PC and DOPE exhibited 23% efficiency, which increased to

35% when the ratio was changed to 7:3 egg PC:DOPE.

Further changes in the DOPE content did not cause any

significant increase in the encapsulation of si-RNA.

PEGylation of liposomes was found to positively influence

the encapsulation of si-RNA with the liposomes of compos-

ition 7:2:0:1 ratio of egg PC:DOPE:cholesterol:DSPE-PEG

exhibiting the highest encapsulation of 47%. Introduction

of cholesterol reduced the encapsulation efficiency of

si-RNA. However, the 7:1:1:1 ratio of egg PC:DOPE:

cholesterol:DSPE-PEG exhibited an encapsulation efficiency

of 39%. Therefore, for further trials, liposomes with the ratio

of egg PC:DOPE:cholesterol:DSPE-PEG 10:0:0:0, 7:3:0:0,

7:2:0:1 and 7:1:1:1 were chosen. These were designated as

LA, LB, LC and LD, respectively.

Table 1 shows the particle size, zeta potential and phase

transition temperature of the liposomes with four different

lipid compositions (LA, LB, LC and LD).

It is observed from Table 1 that the encapsulation of

si-RNA causes an increase in particle size of LA, LB, LC and

LD liposomes when compared with their blank counterparts.

The zeta potential exhibits a reverse trend where the negative

Figure 2. si-RNA encapsulation efficiency ofliposome formed by different lipid ratios.(*50.05).

Table 1. Particle size, zeta potential and phase transition temperature of liposomes.

Blank liposomes si-RNA liposomes

Liposomes type Particle size (nm) Zeta potential (mV) Tm (�C) Particle size (nm) Zeta potential (mV) Tm (�C)

LA 65 ± 0.126 �29.37 ± 1.7 47.09 95 ± 0.05 �24.37 ± 0.6 48.63LB 73 ± 0.78 �17.53 ± 2.2 33.12 120 ± 0.264 �14.7 ± 1.2 39.69LC 96 ± 0.138 �25.9 ± 1.2 47.25 138 ± 0.286 �21.7 ± 1.1 51.6LD 103 ± 0.006 �32.1 ± 1.1 49.77 142 ± 0.121 �26.03 ± 1.1 51.66

DOI: 10.3109/10717544.2014.973082 EpCAM-targeted liposomal si-RNA delivery 5

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zetapotential was shifted towards less negative values after

the si-RNA encapsulation in all liposome types suggesting

masking and redistribution of the surface charges owing to the

incorporation of si-RNA. The presence of si-RNA in the

liposomes is further confirmed from the shift in the phase

transition temperatures of the blank liposomes with different

lipid compositions after encapsulation of si-RNA. A gen-

eral trend that is discernible from the results is that the

si-RNA-loaded liposomes exhibit a higher phase transition

temperature when compared with their corresponding blank

counterparts.

Figure 3 shows the shift in zeta potentials of blank and si-

RNA-loaded liposomes with time. Initially, the blank lipo-

somes exhibit a higher negative zeta potential in all lipid

compositions. These values are found to decrease progres-

sively with time indicating a gradual reduction in the colloidal

stability of the liposomes. A similar trend was observed in the

case of si-RNA-loaded liposomes but the changes were much

more subtle suggesting that encapsulation of si-RNA

improves the colloidal stability of liposomes.

Figure 4(a) and (b) show the percentage adsorption of BSA

and PP on different liposomes after 24 h of incubation in PBS

containing BSA and PP. It was observed that the adsorption of

BSA and PP was less over PEGylated liposomes when

compared to non-PEGylated liposomes. This indicates that

the PEG chains confer protein-repelling property to the

liposomal surface. Adsorption of plasma proteins was greater

on the liposomal surface when compared with bovine serum

albumin.

In all cases, adsorption of BSA on the surface of

si-RNA-loaded liposomes was lesser than that recorded for

their respective blank counterparts. But in the case of PP,

no significant difference was observed between blank and

si-RNA liposomes. Instead, variations in the magnitude of

protein adsorption were found to be solely dependent on the

lipid composition of the liposomes. Figure 4(c) and (d)

show the zeta potential of the liposomes after 24-h incubation

in PBS containing BSA and PP. Zeta potential values

of the blank were decreased considerably than the

si-RNA-encapsulated liposomes. PEGylated liposomes

encapsulating si-RNA were found to exhibit highest zeta

potentials reaffirming their superior stability when compared

with other lipid compositions.

The introduction of a cargo in the liposomal carrier can

either enhance or decrease its stability. This change can be

monitored by measurement of the particle size with time.

Figure 5 depicts the size distribution of different liposomes

after their incubation in PBS, PBS with BSA and PBS with

PP. It was observed that the size of the LA and LB liposomes

progressively increased with time with appearance of a

greater percentage of micron-sized population. However,

PEGylated LC and LD liposomes maintained a narrow size

distribution in the nanodimension even after 24 h. The change

in size was more pronounced in LA, LB and LD liposomes

incubated in the presence of plasma proteins. The si-RNA-

encapsulated liposomes exhibited a slower change in their

dimensions when compared with their blank counterparts in

all the liposome combinations.

Figure 6 shows the release of si-RNA from liposomes with

different lipid compositions. It is seen that the LC liposomes

(7:2:0:1 ratio of egg PC:DOPE:cholesterol:DSPE-PEG)

exhibit the lowest burst release of about 20% and a sustained

release of si-RNA owing, whereas cholesterol incorporated

liposomes and LB liposomes (7:3 ratio of egg PC and DOPE)

show about 50-70% burst release. The cholesterol containing

liposomes display the fastest release when compared to the

Figure 3. Zeta potentials of blank and si-RNA-loaded liposomes after 0, 30, 60 and 120 min of incubation at 37 �C (a) LA, (b) LB, (c) LC and (d) LD(*50.05).

6 D. Bhavsar et al. Drug Deliv, Early Online: 1–14

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other liposomes. The release profile of LA liposomes is not

shown in Figure 6 because of its low-encapsulation efficiency

and poor stability.

As the PEGylated liposomes exhibit better colloidal

stability, the LC liposomes with lipid composition of egg

PC:DOPE:cholesterol:DSPE-PEG in the ratio 7:2:0:1 was

used for in vitro studies. In addition, introduction of the

targeting ligand is possible in PEGylated liposomes and hence

LC liposomes with the maximum encapsulation of si-RNA

and superior colloidal stability were employed for further

trials. Conjugation of the targeting ligand EpCAM antibody

on the surface of LC liposomes was carried out to obtain

EpCAM antibody-tagged liposomes. The presence of EpCAM

antibody in the liposomes was confirmed from the FTIR

spectrum for the tagged liposomes. The significant vibration

bands that appear in the FTIR spectra of LC liposomes

without and with EpCAM antibody modification are sum-

marized in Table 2.

It is evident from Table 2 that conjugation of EpCAM

antibody to the liposome has resulted in the appearance of

new bands at 1634 cm�1 due to the amide carbonyl group and

at 1188 cm�1 due to C-N stretch of the peptide bond.

Similarly, the band at 593 cm�1 may be attributed to the

conjugation of the thiolated antibody to the PEG chain on the

liposome surface. The characteristic bending vibrations due to

aromatic rings from aromatic amino acids present in the

antibody also appear between 750 and 900 cm�1 in the FTIR

spectrum of LC liposomes conjugated with EpCAM antibody.

In vitro studies

Cell uptake of the LC liposomes without and with covalent

linking of EpCAM antibody in MCF-7 breast cancer cell line

was carried out using laser scanning confocal microscopy.

Figure 7 shows the cell uptake of the LC liposomes with and

without EpCAM antibody conjugation over a time period of

4 h. In order to ascertain if the EpCAM antibody modification

directs the uptake of the liposomes in the cells through

receptor-mediated endocytosis, the cells were pre-incubated

with EpCAM antibody. The medium was then replaced with

fresh medium to which the EpCAM antibody conjugated LC

liposomes were added.

It is seen that the cell uptake of the unmodified liposomes

gradually increases with time with more amount of liposomes

(green spots) internalized at 4 h. The immunoliposomes (LC

liposomes conjugated with EpCAM antibody) were found to

exhibit rapid cell uptake and are discernible as intense green

spots in the confocal images of cells even after 30 min of

incubation. The fluorescence intensity was found to increase

with time indicating higher percentage of internalization of

the immunoliposomes. When the cells were pre-incubated

with EpCAM antibody prior to incubation with the immuno-

liposomes, it was observed that the cell internalization of the

immunoliposomes becomes negligible even after 4 h. This

may be attributed to the saturation of the EpCAM receptors

by its antibody during the pre-incubation. This result confirms

that in the case of immunoliposomes, the cell internalization

is mediated through binding to EpCAM receptors on the cell

surface. Quantification of cell uptake using flow cytometry

shows a mean fluorescence intensity 56.47 ± 2.5 and

79.17 ± 0.9 in cells treated with LC liposomes and LC

immunoliposomes encapsulated with fluorescent si-RNA,

respectively.

EpCAM gene silencing efficacy of the LC liposomes in

MCF-7 cells after 48 h is shown in Figure 8. It was observed

that the immunoliposomes containing 100 nM as well as those

Figure 4. Protein adsorption and zeta potentials of blank and si-RNA-loaded liposomes (LA, LB, LC and LD) (a) Bovine serum albumin and (b)plasma protein adsorption after 24 h of incubation. (c and d) Zeta potential of the liposomes after 24 h incubation in PBS containing bovine serumalbumin and plasma proteins (*50.05).

DOI: 10.3109/10717544.2014.973082 EpCAM-targeted liposomal si-RNA delivery 7

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with 200 nM concentrations of si-RNA exhibited better

silencing efficacy when compared with the LC liposomes

containing the same concentrations of si-RNA. The immu-

noliposomes containing 100 nM and 200 nM si-RNA exhibit

1.7- and 3.9-fold down-regulation of EpCAM, respectively. In

contrast, the LC liposomes show only 1.4- and 2-fold decrease

in the EpCAM expression levels at the corresponding

concentrations of si-RNA suggesting that higher cell intern-

alization is a key player in modulating gene silencing efficacy.

Figure 8(b) depicts the cell viability of LC liposomes and LC

immunoliposomes and it reveals no toxic effect of LC

liposomes in MCF-7 cells.

In vivo studies

In order to evaluate the potential of nanocarrier-mediated

EpCAM silencing system on tumor regression, animal studies

were performed using severely compromised immunedefi-

cient (SCID) mice.

Figure 5. Colloidal stability of LA, LB, LC and LD liposomes in PBS, PBS containing BSA and PP after 24 h at 37 �C. BL: Blank and SL: siRNA-loaded liposomes in PES, BLA and SLA: in presence of BSA. BLPP and SLPP: in presence of Plasma protein.

8 D. Bhavsar et al. Drug Deliv, Early Online: 1–14

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Figure 9 shows the results of the in vivo experiments in

xenografted breast cancer SCID mice. Figure 9(a) and (c)

show the change in the body weight of the animals and tumor

volume during the treatment period, respectively. It is seen

that there is no significant change in body weights of the

treated mice during the period and they tend to decrease with

time. The normal mice that served as the negative control,

however, exhibited a slight increase in the body weight during

the same period. Tumor volume was observed to increase

gradually in the control group, whereas it was noticed that the

mice treated with antibody-linked LC liposomes show 32%

reduction in tumor volume when compared with positive

control group after receiving eight doses of 0.15 mg/kg spread

over 28 d.

Figure 9(b) represents the tumor weight after necropsy of

different groups and the results are in agreement with the

tumor volume data. A significant reduction in the tumor

weight is observed in the mice treated with immunoliposomes

when compared with the positive control. EpCAM gene

expression in excised tumor tissue was analyzed using

qRT-PCR. The results (Figure 9d) reveal that antibody-

linked LC liposomes (immunoliposomes) down-regulate

EpCAM expression by 2-fold in comparison to LC liposomes

where significant silencing of EpCAM was not observed.

Histopathological analysis

The tumor was excised and histopathological analysis was

carried out after 28 days of treatment. Figure 10 shows the

hematoxylin and eosin stained tumor tissue from negative

control, positive control, mice treated with LC liposomes and

antibody tagged LC liposomes. It is observed that the positive

control group show large areas of necrosis and hemorrhage. In

contrast, the tissue sections from the mice treated with LC

liposomes as well as the group treated with immunoliposomes

exhibit densely packed cuboidal to low columnar epithelial

Figure 6. si-RNA release profile of LB, LCand LD lipomses (*50.05).

Table 2. FTIR vibration bands of liposomes and immunoliposomes.

Vibration frequency (cm�1)

Bond Liposomes Immunoliposomes

Asymmetric and symmetric stretchingof –CH2–

2924, 2853 2926, 2852

–CH2– scissoring vibration 1466 1466Rocking of the multiple –CH2– 720 720Asymmetric and symmetric stretching

of the –CH3

2956, 2871 –

–OH stretch Broad band at 3400 Broad band at 3400Ester carbonyl stretch 1734 1736P-O stretch 983 934, 1003Amide carbonyl stretch – 1634C-N stretch – 1188Aromatic ring bend – 750-900C-S bond – 593

DOI: 10.3109/10717544.2014.973082 EpCAM-targeted liposomal si-RNA delivery 9

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cells (arrow) and myoepithelium (arrowhead) cells with

negligible hemorrhage and necrosis. Figure 9(e) shows the

Western blot analysis of excised tumors from the control free

si-RNA, LB liposome and LB immunoliposomes. Band

intensity of immunoliposomes as well as LC liposomes was

lesser than the control and the free si-RNA group.

Discussion

Liposomal carriers have been extensively investigated for the

delivery of oligonucleotides and si-RNA for the treatment of

cancer and many genetic disorders (Ozpolat et al., 2014).

Colloidal stability and protein adsorption on the surface of

liposomes, however, determine the cell uptake and circulation

half-life of the liposomes in serum, which in turn influences

the therapeutic efficacy of the system. The charge on the

liposome determines its colloidal stability, the nature and

magnitude of protein adsorption, which is responsible for the

extent of immune response produced against the carrier and

their clearance from the body (Ishida et al., 2002). Hence,

there exists the need to develop a liposomal carrier that

possesses adequate life-time in the body without compromis-

ing its stability and therapeutic efficacy to realize the true

potential of gene therapy applications.

Figure 7. Confocal images show the uptake of LC liposomes encapsulating fluorescent si-RNA at various time points by MCF-7 cells. Panel aillustrates the uptake of LC liposomes; Panel b illustrates the cell uptake of LC immunoliposomes intervals and Panel c illustrates the uptake of LCimmunoliposomes by MCF-7 cells that have been pre-incubated with anti-EpCAM [Nucleolus: Blue (Hoechst), si-RNA: Green (Alexa flour488)].

Figure 8. (a) EpCAM silencing efficacy of LC liposomes and immunoliposomes with 100 and 200 nM si-RNA concentration after 48 h (*50.05),(b) Cell viability of LC liposomes and LC immunoliposomes.

10 D. Bhavsar et al. Drug Deliv, Early Online: 1–14

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Confinement of a highly charged hydrophilic molecule

like si-RNA into the liposome is chiefly dependent on the

composition of lipids used and their interaction with si-RNA.

DOPE possesses fusogenic property because of its ability to

undergo phase transformation from lamellar to hexagonal

phase in response to pH changes. This property of DOPE can

be invaluable in gene delivery as it enables better cell

internalization and facilitates endosomal escape (Li et al.,

2014). Hence DOPE has been used as a ‘‘helper lipid’’ along

with cationic lipids in many gene delivery systems (Farhood

et al., 1995; Huang et al., 1995). However, use of cationic

lipids tends to increase cytotoxicity and trigger inflammatory

response in biological systems and hence cationic lipid-free

liposomes were explored in this study. Among the nine

different liposomes with different ratios of egg phosphatidyl

choline, DOPE, cholesterol and DSPE-PEG, the liposomes

with ratios 7:3:0:0 (LB), 7:2:0:1 (LC) and 7:1:1:1 (LD)

exhibit significantly high encapsulation of si-RNA when

Figure 9. (a) Body weight of mice from different groups during treatment period, (b) tumor images weight after necrosis, (c) tumor volume duringtreatment period and (d) qRT-PCR data of EpCAM expression in tumor tissue (*50.05).

Figure 10. Histopathological staining images of (A) normal skin from negative control mice, (B) tumor tissue from positive control, (C) mice treatedwith Liposomes, (C) liposomes and (D) antibody linked liposomes group. Western blot showing EpCAM protein bands and the band intensitycalculated using imagej software.

DOI: 10.3109/10717544.2014.973082 EpCAM-targeted liposomal si-RNA delivery 11

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compared to other lipid ratios. The highly polar si-RNA

favors a polar environment for encapsulation, which is

provided by the hydrophilic dynamic chains of polyethylene

glycol present in LC and LD liposomes. However, introduc-

tion of cholesterol decreased the encapsulation due to its

hydrophobic nature. Interestingly, increasing DOPE concen-

trations beyond 30% was found to decrease the encapsulation

of si-RNA, which may be attributed to the structure of DOPE

that does not aid close packing of the lipids thereby making

the membrane leaky and fluid.

The increase in particle size of the liposomes after

encapsulation confirms the presence of si-RNA in the

liposomes. PEGylated liposomes display more negative zeta

potential values as the PEG chains tend to mask the negative

surface groups from being neutralized by the cations from the

buffer (Ramana et al., 2012). The phase transition tempera-

ture of the liposomes exhibit a positive shift after encapsu-

lation of si-RNA. This may be attributed to the increased

associative forces in the liposomes due to electrostatic

interactions between the phospholipids and si-RNA that

restricts the mobility of the acyl chains. This is reflected in

the increase in the phase transition of the liposomes after

encapsulation with si-RNA. The presence of DOPE was found

to decrease the phase transition temperature owing to its

ability to hinder close packing of the lipid chains, while the

cholesterol tends to increase the phase transition temperature

of liposomes due to its stiff phenanthrene ring structure that

impedes acyl chain mobility.

Colloidal stability of liposomes influences their life-time

in the biological system as agglomeration will lead to immune

recognition (Kapoor & Burgess, 2012). Agglomeration of

liposomes will also be reflected through a change in surface

charges. The zeta potential measurements of the liposomal

samples with time shows a progressive shift to less negative

values indicating neutralization of surface charges due to

aggregation. However, the measurement of zeta potential in

PBS has been reported to mask the surface charges and lower

the value of the zeta potential (Moghaddam et al., 2011). The

shift is less pronounced in the case of si-RNA-loaded

liposomes when compared with their corresponding blank

counterparts implying better colloidal stability. The shift in

zeta potential is also found to be less steep in the case of

PEGylated liposomes containing si-RNA, which may be

attributed to the PEG chains on the liposomal surface that

retard aggregation of liposomal carriers through steric

hindrance.

Opsonins are a class of proteins which promote phagocyt-

osis and subsequent removal of particles from the blood

circulation (Ishida et al., 2002). Therefore, adsorption of these

proteins on liposomes will have an important role in deciding

the fate of the delivery system. Albumin, an abundant

component of plasma, belongs to the opsonin class of proteins

(Aggarwal et al., 2009). In the present study, the adsorption of

albumin and plasma protein with time was found to be least in

PEGylated liposomes when compared to the non-PEGylated

liposomes. This may be attributed to the fast moving chains of

PEG on the surface that tend to repel proteins. Interestingly, si-

RNA encapsulated liposomes also display low levels of

albumin adsorption which suggests that some si-RNA that

is present on the surface of liposomes may electrostatically

repel the anionic albumin thereby retarding its adsorption. In

contrast, the levels of plasma protein adsorbed on the

liposomal carriers over the same period of time were found

to be significantly higher. This may be due to the presence of a

large number of proteins with varying sizes and isoelectric

points in the plasma, which can contribute to an accelerated

adsorption of proteins (Chono et al., 2008; Ramana et al.,

2010).

One of the contributing factors for liposome aggregation

with time is a phenomenon called ‘‘Ostwald ripening’’ (Keck

et al., 2012). Colloidal stability of liposomes systems can

be explained by the Derjaguin-Landau-Verwey-Overbeek

(DLVO) theory, which describes that the stability of the

colloidal systems is determined by the net effect of repulsive

electrical double layer and the attractive van der Waals forces

that the particles experience as they contact one another

(Sabın et al., 2006). Factors like adsorption of proteins and

neutralization of surface charges promote van der Waal’s

associative forces and reduce the repulsive forces. Therefore,

the energy barrier must not be overcome by van der Waal’s

forces and the repulsive forces should stay dominant to

prevent the formation of aggregates. In the present study,

effect of albumin and plasma protein adsorption on the

particle size distribution of blank and si-RNA-loaded lipo-

somes of all the four lipid combinations (LA, LB, LC and LD)

shows interesting outcomes. PEGylated liposomes (LC and

LD) with si-RNA incubated with albumin or plasma protein

do not show significant changes in the particle size distribu-

tion with time, which may be attributed to the steric repulsion

offered by the fast moving PEG chains on the surface. But,

non-PEGylated liposomes formed from only egg PC (LA) and

egg PC and DOPE (LB) show a discernible change in the

particle size distribution with an appearance of distinct

micron sized populations suggesting progressive adsorption

of the proteins on the liposomal surface promoting their

aggregation.

The release of si-RNA from the LC liposomes show a

sustained release profile, which may be attributed to the

presence of fast moving PEG chains on the surface that

impede the diffusion of the si-RNA from the liposomes. The

low burst release of si-RNA from LC liposomes also imply

that most of the si-RNA is localized in the interior due to the

presence of PEG chains on the surface. In contrast, the DOPE

containing non-PEGylated liposomes (LB) show a pro-

nounced burst release and a rapid release. This may be due

to the presence of the unsaturated acyl chains of DOPE that

hinder the close packing of lipids leading to creation of

defects that facilitate the diffusion of si-RNA from the

liposomes. Cholesterol containing liposomes (LD) also dis-

play a high-burst release and rapid release of si-RNA

suggesting that the si-RNA is localized near the periphery

of the liposomes owing to the presence of the rigid

phenanthrene rings of cholesterol.

MCF-7 breast cancer cell line is a type of epithelial cancer

that over-expresses the EpCAM receptor. Hence, LC lipo-

somes linked with the EpCAM antibody exhibited better cell

internalization when compared to the LC liposomes without

antibody. When the cells were pre-incubated with EpCAM

antibody, the internalization of the immunoliposomes was

significantly reduced. This is because the surface EpCAM

12 D. Bhavsar et al. Drug Deliv, Early Online: 1–14

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receptors are bound to the EpCAM antibody preventing the

binding of the immunoliposomes. This result also proves that

the internalization of the immunoliposomes is mediated

through receptor-mediated endocytosis. The enhanced

down-regulation of EpCAM gene expression levels after

treatment with immunoliposomes is also a direct consequence

of the better internalization of the immunoliposomes when

compared with the unmodified LC liposomes. This is also

reflected in the in vivo studies where the immunoliposomes-

treated mice show better reduction in tumor volume with no

significant change in body weight. This can be ascribed to the

ability of the immunoliposomes to deliver its cargo more

efficiently into the cancer cells when compared with the non-

targeted liposomes as well as free si-RNA. The results also

suggest that targeting cancer cells using EpCAM antibody can

enhance the efficiency of the tumor treatment. The histo-

pathological staining of tumor tissue depicts less necrotic

regions in mice treated with immunoliposomes with presence

of densely packed cuboidal to low columnar epithelial cells.

Qualitative and quantitative analysis of EpCAM silencing in

tumor samples are in agreement with the earlier in vitro

inference that the immunoliposomes possess better cell

internalization and higher EpCAM silencing efficiency

when compared to its non-targeted counterpart and free si-

RNA. It was also observed in PCR and Western blot analysis

of tumor tissues from various treatment groups that the

immunoliposomes significantly silence the EpCAM gene and

reduces the EpCAM protein levels in cells. As the down-

regulation of EpCAM has been associated with up-regulation

of apoptotic genes and down-regulation of proliferation genes

(Mitra et al., 2010), silencing of EpCAM through immuno-

liposome-mediated delivery resulted in a better tumor

regression when compared to other treatment groups. The

si-RNA concentrations used in this study (0.15 mg/kg) are

among the lowest reported for gene silencing in vivo (Casals

et al., 2003). Increase in the si-RNA concentrations or

increase in the duration of treatment can result in complete

regression of the tumor.

Conclusion

The development of cationic lipid-free liposomes targeting

EpCAM expressed on the surface of epithelial cancers for

silencing the EpCAM gene has been proved to be a feasible

strategy to bring about tumor regression even at very low

concentrations. This proof-of-concept could be taken forward

to evaluate its efficacy against other types of epithelial cancers.

The use of PEGylated nanoliposomes has enabled realization

of the promise of gene silencing strategies for cancer therapy.

Declaration of interest

The authors have no conflict of interest to declare.

The authors wish to acknowledge funding from

Department of Biotechnology Government of India (BT/

PR11210/NNT/28/2008) and SASTRA University for infra-

structural support.

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