A Mechanistic Study of Enhanced Doxorubicin Uptake and ...jpet.aspetjournals.org/.../jpet/early/2006/03/17/jpet.106.101154.full.pdf · 17.03.2006 · JPET #101154 2 Mechanism of

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A Mechanistic Study of Enhanced Doxorubicin Uptake and Retention in Multidrug

Resistant Breast Cancer Cells using a Polymer-Lipid Hybrid Nanoparticle (PLN)

System

Ho Lun Wong, Reina Bendayan, Andrew M. Rauth, Hui Yi Xue, Karlo

Babakhanian, Xiao Yu Wu

Leslie Dan Faculty of Pharmacy, 19 Russell Street, University of Toronto, Ontario,

Canada M5S 2S2 (HLW, RB, HYX, KB, XYW)

Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9

(AMR)

JPET Fast Forward. Published on March 17, 2006 as DOI:10.1124/jpet.106.101154

Copyright 2006 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 17, 2006 as DOI: 10.1124/jpet.106.101154

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Mechanism of Overcoming MDR by Polymer-Lipid Nanoparticles

Corresponding author: Xiao Yu Wu, Tel. 1-416-978-5272; Fax: 1-416-978-8511;

Email: [email protected].

Corresponding address: Graduate Department of Pharmaceutical Sciences, Leslie Dan

Faculty of Pharmacy, 19 Russell Street, University of Toronto, Ontario, Canada M5S 2S2

Number of text pages: 35 (references and table included) Number of table: 1 Number of figures: 9 Number of references: 38 Number of words in Abstract: 243 Number of words in Introduction: 720 Number of words in Discussion: 1494 List of abbreviations: PLN – polymer-lipid hybrid nanoparticles; SLN – solid lipid

nanoparticles; Pgp – P-glycoprotein; MDR – multidrug resistance; Dox – doxorubicin;

Dox-PLN – Dox loaded polymer-lipid hybrid nanoparticles; FSA – fluoresceinamine

labeled stearic acid; EDCH – 1-ethyl-3-3-(3-dimethylaminopropyl)-carbodiimide

hydrochloride; DAPI – 4'-6-diamidino-2-phenylindole

Recommended section assignment: Chemotherapy, Antibiotics and Gene Therapy


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ABSTRACT

The objectives of this study are i) to evaluate the potential of a polymer-lipid

hybrid nanoparticle (PLN) system to enhance cellular accumulation and retention of

doxorubicin (Dox), a widely used anticancer drug and an established P-glycoprotein

(Pgp) substrate, in Pgp-overexpressing cancer cell lines, and ii) to explore the underlying

mechanisms. Nanoparticles containing Dox complexed with a novel anionic polymer

(Dox-PLN) were prepared using ultrasound method. Two Pgp-overexpressing breast

cancer cell lines (a human cell line MDA435/LCC6/MDR1 and a mouse cell line

EMT6/AR1) were used to investigate the effect of nanoparticles on cellular uptake and

retention of Dox. Endocytosis inhibition studies and fluorescence microscopic imaging

were performed to elucidate the mechanisms of cellular drug uptake. Treatment of Pgp-

overexpressing cell lines with Dox-PLN resulted in significantly enhanced Dox uptake

and more substantial increases in drug retention after the end of treatment compared to

free Dox solutions (p < 0.05). Fluorescence microscopic images showed improved

nuclear localization of Dox and uptake of lipid when the drug was delivered in the Dox-

PLN form to MDA435/LCC6/MDR1 cells. Endocytosis inhibition studies revealed that

phagocytosis is an important pathway in the membrane permeability of the nanoparticles.

These findings suggest that some of the Dox physically associated with the nanoparticles

bypass the membrane associated Pgp when delivered as Dox-PLN, and in this form the

drug is better retained within the Pgp-overexpressing cells than the free drug. The present

study suggests a new mechanism for overcoming drug resistance in Pgp-overexpressing

tumor cells using lipid-based nanoparticles formulations.


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INTRODUCTION

Chemotherapy with the use of cytotoxic drugs is commonly implemented in the

management of many cancer types, e.g. breast cancers (Skeel, 2003; Tack et al., 2004).

However, suboptimal therapeutic responses associated with multidrug resistance (MDR)

frequently occur (Longley and Johnston, 2005). Overexpression of P-glycoprotein (Pgp)

is one of the prominent mechanisms that contribute to the MDR phenotype (Endicott and

Ling, 1989; Gottesman, 2002). Pgp is a 170-kDa membrane-associated glycoprotein that

may actively extrude several substrates, including a variety of cytotoxic drugs such as

doxorubicin (Dox), from cell cytoplasm to outside of plasma membrane, thus lowering

the effective drug concentrations within the cells (Endicott and Ling, 1989; Gottesman,

2002). Because cytotoxic drugs typically carry numerous dose-limiting normal tissue

side-effects (Tipton, 2003), it is generally impractical to overcome this form of drug

resistance simply by increasing the drug dose. To improve the therapeutic ratio of cancer

chemotherapy, it is therefore critical to establish alternative approaches that may improve

accumulation and prolong retention of cytotoxic drugs in drug-resistant cancer cells

without causing additional normal tissue side-effects.

Particulate drug delivery systems such as polymeric microspheres (Liu et al.,

2001), nanoparticles (de Verdiere et al., Moghimi and Hunter, 2000), liposomes (Thierry

et al., 1993; Romsicki and Sharom, 1999; Booser et al., 2002) and solid lipid

nanoparticles (SLN) (Wong et al., 2004, 2005) offer great promise to achieve the

aforementioned goal. Particulate systems are well known to be able to deliver drugs with

higher efficiency with fewer adverse side effects (Booser et al., 2002; Lamprecht et al.,

2005). Some encapsulated formulations may be further engineered to deliver substances


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such as Pgp inhibitors or other therapeutic molecules together with cytotoxic drugs to

achieve stronger anticancer activity in drug-resistant cancer cells (Liu et al., 2001; Wong

et al., 2004; Veldman et al., 2005). In addition, some formulations also possess intrinsic

activities to reduce drug resistance (Moghimi and Hunter, 2000; Thierry et al., 1993;

Romsicki and Sharom, 1999; Nori et al., 2003; Kabonov et al., 2002). The mechanisms

that underlie these MDR reversal activities are diversified, and likely vary from one drug

carrier to another. Mechanisms established so far include inhibition of Pgp by the

polymers making up the drug carriers (Moghimi and Hunter, 2000; Romsicki and

Sharom, 1999; Kabonov et al., 2002), local buildup of drug molecules outside the cell

membranes (de Verdiere et al., 1997), and increase of cellular drug uptake by endocytosis

of the drug carriers (Lee et al, 1992; Soma et al, 1999; Nori et al., 2003). At present, there

are few studies devoted to the investigation of the mechanistic issues of MDR reversal by

SLN.

In our previous studies (Wong et al., 2004; 2005, in press), we have developed

polymer-lipid hybrid nanoparticles (PLN), a modified form of SLN that incorporates an

anionic polymer to facilitate loading of water-soluble cationic drugs that contain

lipophilic molecular structures, e.g. Dox and verapamil. Compared to the conventional

free Dox solution treatment, the new nanoparticle system with Dox encapsulated (Dox-

PLN) resulted in approximately an eight-fold increase in cell kill of Pgp-overexpressing

human breast cancer cells in clonogenic assay experiments (Wong et al., in press). This

additional anticancer activity against Pgp-overexpressing cancer cells is not caused by the

non-active ingredients of the nanoparticles, as the lipid, polymer, and surfactants used in

Dox-PLN preparation were shown inoculous in terms of their effects on cancer cell


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proliferation and cell membrane integrity (Wong et al., in press). Furthermore, Dox-

polymer complexes not encapsulated in lipids were not more effective than Dox only.

The enhancement in anticancer activity was therefore likely attributable to the fact that

Dox was more efficiently delivered to the cells when encapsulated in PLN (i.e., Dox-

PLN).

In the present work, we examined the mechanism by which the PLN system

enhanced the activity of Dox in Pgp-overexpressing cancer cells. Studies were

performed to evaluate quantitatively how Dox, when administered in the form of Dox-

PLN, accumulated in and was retained by Pgp-overexpressing breast cancer cells. Since

endocytosis of lipid formulations by various cell types were previously reported (Lee et

al., 1992; Soma et al., 1999), endocytosis inhibition studies were carried out to determine

if this mechanism was also involved in the uptake of Dox-PLN by cancer cells.

Intracellular distribution of Dox and fluorescence-labeled lipids were examined by

fluorescence microscopy to help further understand the drug uptake processes. Insights

gained from these studies may help to optimize the use of nanoparticle systems for

improved chemotherapy of MDR cancers.

MATERIALS AND METHODS

Materials. Dox hydrochloride, rhodamine-B hydrochloride, stearic acid,

fluoresceinamine isomer I, 1-ethyl-3-3-(3-dimethylaminopropyl)-carbodiimide

hydrochloride (EDCH), 4'-6-diamidino-2-phenylindole (DAPI), and other chemicals

used, unless otherwise specified, were purchased from Sigma-Aldrich Inc. (Mississauga,

ON, Canada). Stearic acid was purified by recrystallizing with 95% ethanol after it was


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received. A new anionic polymer, HPESO (hydrolyzed polymer of epoxidized soybean

oil) was kindly provided by Drs. Z. Liu and S. Erhan. This polymer is an anionic polymer

used to enhance the incorporation of Dox into the lipids by forming relatively lipophilic

drug-polymer complexes (For details please refer to Wong et al, Pharm Res, in press).

Pluronic F68 (i.e. poloxamer 188, a nonionic block copolymer) was supplied by BASF

Corp. (Florham Park, N.J., U.S.A.). For the Western blot analysis, protease inhibitor

cocktail (P8340) and anti-actin antibody AC-40 were purchased from Sigma-Aldrich

(Mississauga, ON, Canada), Pgp antibody C219 and IgG2a:HRP were purchased from ID

Labs Biotechnology (London, ON, Canada) and Serotec (Raleigh, NC, USA),

respectively.

Tumor Cell Lines and Culture. The Pgp-overexpressing human breast

carcinoma cell line MDA435/LCC6/MDR1 and parental cell line MDA435/LCC6/WT

were generous gifts from Dr. Robert Clarke (Georgetown University, Washington, DC,

U.S.A.). The Pgp-overexpressing murine breast carcinoma cell line EMT6/AR1 and

parental cell line EMT6/WT were kindly provided by Dr. Ian Tannock (Ontario Cancer

Institute, Toronto, ON, Canada). Monolayers of all cell types (passages 5-30 in our

hands) were cultured on 75-cm2 polystyrene tissue culture flasks at 37 oC in 5% CO2/95%

air humidified incubator. Cancer cells were maintained in growth medium consisting of

α-minimal essential medium (Ontario Cancer Institute Media Lab, Toronto, ON,

Canada), pH 7.2, supplemented with 10% fetal bovine serum. Cells grown to confluence

(3-5 d for EMT6 cell lines, 5-7 d for MDA435 cell lines after seeding approximately 0.1


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million of cell per dish) were subcultured with 0.05% trypsin-EDTA (Invitrogen Inc.,

Burlington, ON, Canada), diluted (1/10) in fresh growth medium and reseeded.

Preparation of PLN Containing Dox and Fluorescence Probe. PLN

containing Dox (Dox-PLN) were prepared as previously described (Wong et al., 2005, in

press). Typically, a mixture of 100 mg of stearic acid and 0.9 ml of aqueous solution

containing 5 mg of Dox and Pluronic-F68 (2.5 %w/v) was warmed to 72−75 oC.

Following the addition of 2.5 mg of HPESO polymer, the mixture was stirred for 10 min

and then ultrasonicated for 3 min to form sub-micron sized lipid emulsion. The emulsion

was dispersed in water at 4 oC (1 part emulsion to 4 or 9 parts of water) to form PLN.

Blank PLN were similarly prepared except that Dox was omitted. For PLN loaded

with rhodamine-B, rhodamine-B was used in place of Dox. For microscopic imaging that

required co-loading of fluoresceinamine-labeled stearic acid (FSA, see the method of

preparation described below) in Dox-PLN, stearic acid was substituted with FSA in the

PLN preparation.

Dox-PLN and blank PLN used in the present work were characterized for their

physicochemical properties such as particle size, surface charge, drug loading properties

and drug release kinetics in a previous study. Their properties are summarized in Table

1.

Preparation of Fluoresceinamine-Labeled Stearic Acid (FSA) and Evaluation

for Labeling Stability and Fluorescence Properties. FSA was prepared according to

Zhang et al (2004) with a few modifications. Briefly, 0.0439 g of fluoresceinamine

isomer I and 0.0316 g of EDCH were added to 1 mL of dimethyl sulfoxide DMSO, and


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the suspension formed was vortexed until homogeneous. The suspension was added

dropwise to stearic acid solution (1 g of stearic acid in 24 mL of DMSO). The mixture

formed was continuously stirred in darkness for 48 h at room temperature. At the end of

the reaction 100 mL of distilled water was added. The precipitate formed was collected

by filtration, grounded into a fine powder, and washed repeatedly with copious amounts

of ice-cold water until the washout liquid became colorless. The pale yellow product

containing FSA was dried in vacuum and purified by recrystallization in absolute ethanol.

The presence of fluorescence activity in the final product was confirmed by scanning its

ethanolic solution at λex = 430 nm and λem = 498 nm (Delta-Scan fluorometer, PTI Inc.,

NJ., U.S.A.)

The stability of the fluoresceinamine-labeling on PLN was evaluated by

incubating 0.5 mL of PLN suspension (containing 50 mg of FSA) in 10 mL of growth

medium at cell culture conditions for 4 h. The suspension was then centrifuged at

120,000g and the fluorescence signal in the supernatant was measured to examine

possible release of FSA from the FSA-PLN.

Western Blot Analysis. Western blot analysis was performed as previously

described by our laboratory (Ronaldson et al, 2004) to allow comparison of the Pgp

levels of the cell lines used. MDA435/LCC6/MDR1 and EMT6/AR1 cells were harvested

by centrifugation (400g), and the cell pellets formed were lysed for 30 min at 4 oC in a

250 mM sucrose buffer containing 1 mM EDTA and 0.1% (v/v) protease inhibitor

cocktail. Samples were then homogenized on ice for 2 min with a Dounce homogenizer

and aliquots (25 µg protein) from the suspension were resolved on a 10% sodium dodecyl


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sulfate-polyacrylamide gel and electrotransferred onto a polyvinylidene difluoride

membrane. The membrane was blocked overnight (4 oC) in Tris-buffered saline

containing 0.05% Tween 20 and 5% dry skim milk powder. Pgp and β-actin (42 kDa)

were detected by treating the samples with monoclonal Pgp antibody C219 (1:500

dilution) and anti-actin antibody AC-40 (1:500 dilution), following by IgG2a:HRP as the

secondary antibody. Proteins were visualized using enhanced chemiluminescence

according to the manufacturer’s instructions (Pierce Chemical, Rockford, IL, USA).

Densitometric analysis of protein bands was performed using Image Quant 5.2

(Molecular Dynamics, Piscataway, NJ, USA). Protein concentration of the cell lysate was

determined with Bradford’s protein assay.

Dox Cellular Uptake and Retention Studies. To establish cellular drug uptake

profiles, cells were plated onto 48-well plates at densities of approximately 40,000 to

100,000 cells/well at 37 oC. When cells reached confluence Dox solution or Dox-PLN

suspension was added into each well to initiate cellular drug accumulation. In the

experiments with MDA435 cell lines, treatment with Dox solution/ blank PLN

combination was also included to evaluate the impact of blank PLN and the surfactant.

All treatments were adjusted to 10 µg/ml Dox concentration with Earle’s Balanced Salt

Solution (EBSS). Blank EBSS was used as the negative control. At predetermined time

intervals (0 to 4 h), supernatant was removed, and cells were washed with ice-cold

phosphate-buffered saline (PBS, pH = 7.6) and lysed with PBS containing 1% Triton-X.

Dox concentrations in the cell lysates were measured with SpectraMax Gemini XS

microplate fluorometer (Molecular Devices, Sunnyvale, CA, U.S.A.) at an excitation


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wavelength λex = 478 nm and an emission wavelength λem = 594 nm. To adjust for

background fluorescence from the cellular components, Dox standardization curves were

also prepared using cell lysates. In this case, untreated cells were lysed with PBS/1%

Triton-X containing Dox or Dox-PLN at known concentrations. Separate standardization

for Dox solution and Dox-PLN were prepared in each experiment for more accurate Dox

concentration calibrations.

To evaluate cellular Dox accumulation by cancer cells after drug efflux period,

monolayer cells grown in 48-well plates were loaded with Dox in the form of free Dox

solution or Dox-PLN containing 10 µg/ml Dox for 2 h. Supernatant was removed at the

end of treatment and cells were washed with ice-cold PBS. The wells were refilled with

fresh, drug-free EBSS and cells were incubated at 37 oC to facilitate cellular drug efflux.

At predetermined time intervals (0 to 2 h), supernatant containing the effluxed drug was

removed. Cells were washed and lysed, and the amount of Dox retained by the cells was

measured with a microplate fluorometer as described above.

Cellular Dox uptake or retention is expressed as nanomoles per milligram of

protein. Protein concentrations of the cell lysates were determined by the Bradford

colorimetric assay (Bradford, 1976) using reagents from BioRad (Melville, NY) and

bovine serum albumin (Sigma-Aldrich Inc., Mississauga, ON, Canada) as the standard.

Membrane integrity of the cultured cells in the presence of Dox-PLN and their various

components (e.g. stearic acid, Pluronic surfactant, HPESO polymer) was previously

verified using trypan blue exclusion assay (Wong et al., 2005). There was no significant

loss of cell membrane integrity when the cells were treated with Dox-PLN or their

ingredients.


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Endocytosis Inhibition Studies. The effects of endocytosis inhibitors on cellular

Dox uptakes were evaluated according to Ramge et al (2000) with some modifications.

Cells were pre-treated with 25 µg/ml colchicine or 5 µg/ml cytochalasin B, inhibitors of

pinocytosis and phagocytosis, respectively, in EBSS for 1 h at 37 oC. Following removal

of the pretreatment solution, the cells were washed with PBS, treated with Dox solution

or Dox-PLN containing 10 µg/ml Dox and incubated under the conditions used for cell

culture. Cellular Dox accumulation at 2 h was measured and protein standardization was

performed as described above. Membrane integrity of cells treated with the endocytosis

inhibitors was confirmed by trypan blue exclusion assays. The capacity of the cell lines

tested to perform endocytosis has also been confirmed using lucifer yellow, a well

documented endocytosis or exocytosis substrate (Jayanth and Vinod, 2003). Dox uptakes

of cells pretreated with endocytosis inhibitors are compared to those without

pretreatment, and the reduction in Dox uptake caused by the inhibitors is expressed as %

inhibition of Dox uptake.

Fluorescence Microscopy of Drug and Lipid Uptake into Breast Tumor Cells.

For fluorescence microscopy experiments, MDA435/LCC6 cells at the confluent stage of

growth were re-seeded (approximately 106 cells per dish) and incubated overnight at cell

culture conditions in 10-cm petri dishes, each containing a duplicate of poly-L-lysine

coated coverslips. Cells were then treated with free Dox solution, Dox-PLN, or Dox-

FSA-PLN for 2 h. At the end of treatment cells were washed with ice-cold PBS, and one

of the two coverslips in each dish coated with the treated cells was removed for imaging

within 5 min. The remaining coverslip in each dish was re-immersed in fresh growth


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medium for an additional 2 h to allow cellular drug efflux. The coverslips, after removal

from the Petri dish, were mounted on glass slides, placed on the platform of a differential

interference contrast fluorescence microscope (Axiovert 100TV, Zeiss Inc, Oberkochen,

Germany), and viewed with an oil-immersion objective (100× magnification). λex = 540

nm and λem = 590 nm, and λex = 430 nm and λem = 500 nm filter sets were used for

imaging of Dox and FSA, respectively. “Snap-shot” images were acquired using an

intensified CCD camera under computer control. The same camera settings were used

for the experiments unless otherwise specified.

For comparison, experiments were also conducted using EMT6/AR1 and PLN

loaded with rhodamine-B, as substitutes for MDA435 cells and Dox-PLN, respectively.

λex = 520 nm and λem = 570 nm filter sets were used for rhodamine detection.

Nuclear localization by the Dox delivered using PLN was confirmed by nuclear

staining of Dox-PLN treated cells. MDA435/LCC6/MDR1 cells treated with Dox-PLN

suspension containing 10 µg/mL Dox for 2 h and allowing for 2 h drug free period were

fixed by immersing in 4% paraformaldehyde in PBS for 20 min. The fixed cells were

treated with DAPI solution (2 µM in PBS) for 5 min and rinsed with PBS twice.

Fluorescence microscope images of Dox and DAPI were taken from the same microscope

field. For detection of DAPI, λex = 360 nm and λem = 450 nm filter sets were used. Same

wavelength combination was used for Dox detection.

Data Analysis. Results are presented as mean ± S.D. from a minimum of three

separate experiments in cells pertaining to different passages, unless otherwise specified.

Statistical analysis for unpaired experimental data was performed using Student’s t-test.


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In all analyses, a value of p < 0.05 was considered significant.

RESULTS

Pgp Expression in Breast Carcinoma Cell Lines. Western blot analysis (Fig. 1)

detected a single band of appropriate size for Pgp (≈ 170 kDa product, Evers et al., 1998)

in both cancer cell lines known to overexpress Pgp. Densitometric analysis showed that

the P-gp expression in EMT6/AR1 cells is 1.8 to 2.1-fold higher than

MDA435/LCC6/MDR1 cells. This is consistent with our previous results of cytotoxicity

studies, which demonstrated higher resistance to Dox in the Pgp-overexpressing murine

breast cancer cell line (LD90 > 150 µg/mL) compared to the human cell line (LD90 = 9

µg/mL) (Wong et al., in press; Cheung, 2005). The results of western blot of the wild-

type cell lines (EMT6/WT and MDA435/LCC6/WT) (data not shown) indicated that Pgp

expression in these cells was undetectable (Cheung et al, 2006; Wong et al, unpublished

data).

Enhanced Dox Uptake by PLN in Pgp-overexpressing MDA435/LCC6 Cells

and EMT6 Cells. Figs. 2A, 2B, 2C and 2D present the 4 h time profiles of Dox uptake

by tumor cells treated with Dox formulations, all containing 10 µg/mL (17.2 µM) Dox.

Cellular Dox levels in the four cell lines treated with Dox solution reached a plateau

within 2 h, whereas in cells treated with Dox-PLN, the cellular Dox levels continued to

increase up to 4 h. As expected, MDA435/LCC6/MDR1 and EMT6/AR1, treated with

Dox solution, accumulated 39% and 138% less Dox at 4 h, respectively, than their wild-

type cell lines (Fig. 2A vs 2B; Fig. 2C vs 2D. p < 0.05 in both cases). However, when


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cells were treated with Dox-PLN instead, the differences in the Dox uptake between

wild-type cells and Pgp-overexpressing cells were substantially reduced. In the case of

MDA435 cells, the difference did not even reach statistical significance (p > 0.05).

MDA435/LCC6/MDR1 took up 19% less Dox than its wild-type cells. The use of

different Dox formulations, i.e., Dox solution and Dox-PLN resulted in similar Dox

accumulation levels in both wild-type cell lines (Figs. 2A and 2C, p > 0.05 comparing

Dox to Dox-PLN at all time points). Nevertheless, both Pgp-overexpressing cell lines

treated with Dox-PLN accumulated higher Dox levels than with Dox solution (p < 0.05,

Figs. 2B and 2D). More significant enhancement of Dox uptake is seen in EMT6/AR1.0

cells (Fig. 2D).

To determine if this effect was simply due to the non-drug ingredients of PLN

such as the lipid, polymer and surfactant, we also treated MDA435 cell lines with blank

PLN/ Dox solution combination. In both MDA435 cell lines, the addition of blank-PLN

to Dox solution did not increase cellular Dox uptake. Modest decreases in Dox

accumulation were observed instead. However, the decreases were not statistically

significant.

Enhanced Dox Retention by PLN in Pgp-overexpressing MDA435/LCC6

cells and EMT6 cells. Figs. 3A to D demonstrate the effects of using different Dox

treatments (Dox solution or Dox-PLN) on cellular Dox retention in tumor cells. The

declines in the cellular Dox level as a result of drug efflux into fresh EBSS medium are

presented in form of 2 h time profiles. As expected, after treatment with Dox solution,

cellular efflux was faster in Pgp-overexpressing cell lines than in their corresponding


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wild-type cells. In both wild-type cell lines (Figs. 3A and 3C), the use of Dox-PLN in

place of Dox solution did not improve drug retention. In contrast, both Pgp-

overexpressing cell lines treated with Dox-PLN demonstrated enhanced Dox cellular

retention. Significantly higher cellular Dox levels were observed 1.5 h and 1 h after

initiation of the drug efflux phase in MDA435/LCC6/MDR1 cells (Fig. 3B) and

EMT6/AR1 cells (Fig. 3D), respectively. The enhancement of cellular Dox retention

contributed by Dox-PLN was more prominent in EMT6 cells than in MDA435 cells.

When comparing cells treated with Dox-PLN to those with Dox solution, over 8-fold

enhancement in Dox retention was demonstrated in EMT6/AR1 cells at 2 h, whereas an

approximately 90% enhancement was observed in MDA435/LCC6/MDR1 cells at the

same time point.

Endocytosis Inhibition Study Indicates Bypassing of Pgp by Endocytosis. To

investigate if elevated drug uptake in Pgp overexpressing cells treated with Dox-PLN was

contributed by endocytosis, an endocytosis inhibition study was conducted. Specifically,

the pinocytotic pathway and phagocytotic pathway was respectively inhibited by

colchicine and cytochalasin-B pretreatments, and the impact of the inhibitions of these

pathways on cellular Dox uptake was measured. The results are presented in Figs. 4A and

B. In the wild-type cell lines, cytochalasin-B resulted in significantly stronger inhibition

of Dox accumulation in cells treated with Dox-PLN (30% and 21% inhibition in

MDA435/LCC6/WT and EMT6/WT, respectively) than cells treated with Dox solution

(8% and 4% inhibition in MDA435/LCC6/WT and EMT6/WT, respectively). In

contrast, although colchicine also caused moderate inhibitions in both wild-type cell


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lines, its inhibitory effects were similar regardless of the dosage form of Dox used. These

results suggest that phagocytosis is responsible for the uptake of Dox delivered by Dox-

PLN, but not for Dox in solution, while pinocytosis to some extent contributed to the

uptake of drugs delivered by both dosage forms.

In both Pgp-overexpressing cell lines treated with Dox solution, pretreatment with

colchicine or cytochalasin-B enhanced Dox uptake instead (i.e. negative values obtained).

This is understandable because both agents are Pgp substrates, and probably reduced the

cellular Dox efflux by competition (Zilfou and Smith, 1995). In comparison,

cytochalasin-B demonstrated net inhibitory effect against Dox uptake in both

MDA435/LCC6/MDR1 cells and EMT6/AR1 cells treated with Dox-PLN. In

MDA435/LCC6/MDR1 cells this inhibition was significant compared to cells treated

with Dox solution.

Dox is Better Retained Intracellularly By Pgp-overexpressing Cells and

Tends to Localize in Cell Nuclei when Delivered as Dox-PLN. Figs. 5 to 7present

fluorescence microscopic images of cells treated with Dox (Figs. 5A to 5J), Dox and

DAPI (Figs. 6A and 6B) or rhodamine (Figs. 7A and 7B) in free solutions or PLN form.

The impact of the formulations is compared in terms of the intensity and cellular

distribution of fluorescence. As expected, strong fluorescence intensity was detected in

wild-type cells, and it did not substantially fade after 2 h efflux test (Figs. 5A and B). On

the other hand, though MDA435/LCC6/MDR1 cells treated with Dox solution exhibited

respectable fluorescence intensity after treatment, their intensity became barely detectable

after the 2 h drug efflux period (Figs. 5C and D). In comparison, the fluorescence in


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MDA435/LCC6/MDR1 cells treated with Dox-PLN containing the same Dox

concentration (5 µg/ml) retained moderately high intensity (Fig. 5E vs 5F). The same

trend was observed in MDA435/LCC6/MDR1 cells treated with higher Dox

concentration (10 µg/ml) (Figs. 5G to 5J), where stronger fluorescence was retained in

cells treated with Dox-PLN. When comparing the fluorescence intensities, it should be

noted that the brightness setting was intentionally lowered (to about 50% sensitivity)

during imaging in Figs. 5A, 5B, 5I and 5J to minimize light saturation for improved

image clarity.

Besides fluorescence intensity, the cellular distribution of the fluorescence in

MDA435/LCC6/MDR1 cells was also affected by the dosage form. When Dox solution

was used, even at 10 µg/ml Dox concentration, most of the fluorescence signal was

confined in cytoplasm (Figs. 5G and 5H). However, in MDA435/LCC6/MDR1 cells

treated with Dox-PLN, the nuclear regions apparently show much higher fluorescent

intensity (Figs. 5I and 5J). In addition, the fluorescence appeared more granular, with

some fluorescent particulate forms seen associated with cells treated with Dox-PLN,

while the fluorescent substances in Dox solution treated cells were more evenly and

smoothly distributed.

Figs. 6A and 6B presents the Fluoroescence microscope images of

MDA435/LCC6/MDR1 cells treated with Dox-PLN and DAPI, respectively. The similar

locations of Dox and DAPI seen in the images confirm the nuclear localization of Dox

delivered by the PLN formulation.

To determine if the above trend would also occur in another cell line with a

different P-gp substrate, EMT6/AR1 cells were treated with a solution (Fig. 7A) or PLN


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form (Fig. 7B) of rhodamine for 2 h followed by efflux experiment for another 2 h.

Similarly to the results observed with Dox, a marked improvement of rhodamine

retention is seen when the dye was loaded and administered in the form of PLN.

Dual Fluorescence Labeled Dox-FSA-PLN Showed Similar Location and

Retention of Dox and Lipids in Pgp-overexpressing Cells. To delineate whether the

lipid played a role in the drug uptake and retention by the Pgp-overexpressing cancer

cells, the lipid molecules (stearic acid) used for PLN preparation were conjugated with

fluorescence groups and their cellular distribution was examined and compared to that of

the Dox. Examinations using fluorescence microscopy and fluorescence spectroscopy

were performed on PLN labeled with FSA. No noticeable fluorescence intensity in the

incubation medium after 4 h incubation at culture conditions was observed (results not

shown), indicating negligible leakage of FSA from FSA-PLN. Initial studies also

confirmed no interference of the fluorescence signals from Dox and FSA when they were

in a lipid environment. After having been treated with dual labeled PLN, i.e., Dox-FSA-

PLN, for 2 h and re-incubated in a fresh medium for additional 2 h,

MDA435/LCC6/MDR1 cells still exhibited fluorescence signals specific for Dox and

FSA, respectively. The cellular distribution of fluorescence corresponding to Dox (Fig.

8A) and FSA (Fig. 8B) generally overlapped with each other. The fluorescence of Dox,

however, appeared to be slightly more diffusive than FSA.

DISCUSSION


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Many chemotherapeutic agents target intracellular organelles or molecules to

achieve their anticancer activities. For example, Dox may intercalate between the DNA

bases and disrupt the action of topoisomerase II (Dorr 1998). Effective chemotherapy

thus requires a reasonably high level of drug molecules to accumulate within the cancer

cells. In addition, the effectiveness of chemotherapy is also positively correlated to drug

exposure time (Millenbaugh et al., 2000). In Pgp-overexpressing cells, it becomes a

difficult task to maintain a high intracellular drug level for a reasonable length of time.

In the present study, we found that in addition to moderate enhancement of cellular Dox

accumulation (Figs. 2B and 2D), the new nanoparticle formulation resulted in substantial

improvement of Dox retention by two drug-resistant breast cancer cell lines (Figs. 3B and

3D). This may at least in part account for the enhanced in vitro anticancer activity of

Dox-PLN demonstrated in our previous study (Wong et al., in press).

There have been studies showing that some of the lipids and surfactants used in

lipid-based formulations possess intrinsic Pgp inhibitory activities (Romsicki and

Sharom, 1999; Batrakova et al., 1999). However, it does not explain the finding that

blank PLN did not improve Dox accumulation in Dox-treated MDA435/LCC6/MDR1

cells (Fig. 2B). These results indicate that the non-active ingredients of the tested

formulations, at least at the concentrations used, do not suppress Pgp activity by

themselves. In fact, this is consistent with our previous study, which showed that the

cancer-suppressive activity of Dox treatment was also improved only when Dox was an

integral part of the nanoparticles, and not when it was separately added in the presence of

blank PLN or polymer (Wong et al., in press).


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The advantages of nanoparticles over solution appear to be augmented by the

increasing Pgp level of the treated cells. The enhancement in drug accumulation by Dox-

PLN was larger in EMT6/AR1 than in MDA435/LCC6/MDR1. This trend is even more

noticeable in the drug retention data (enhancement by Dox-PLN over Dox solution after

2 h drug efflux: ≈ 90% in Fig. 3B, 900% in Fig. 3D). Because western blot analysis (Fig.

1) shows that EMT6/AR1 expresses a higher Pgp level than MDA435/LCC6/MDR1, and

these drug accumulation and retention enhancing effects by Dox-PLN are practically

non-existent in wild-type cells, it is likely that Dox-PLN are more useful when the cancer

cells express higher levels of Pgp (i.e. more drug-resistant). As discussed before, PLN do

not directly inhibit Pgp. Instead, PLN may improve the treatment by allowing the drug

molecules to bypass the efflux action of Pgp.

In fluorescence microscopy studies, regardless of the dosage form, considerable

amounts of Dox could be detected in MDA435/LCC6/MDR1 immediately after drug

treatment, with Dox-PLN treated cells accumulating moderately higher cellular Dox

levels. The impact of the dosage form was much more noticeable after the 2 h drug

efflux period. Dox was clearly better retained in cells treated with Dox-PLN (Fig. 5F)

than Dox solution (Fig. 5D). A similar trend was observed in cells treated with higher

Dox concentration (Figs. 5G to 5J). These findings generally support the drug uptake and

retention data.

Cell nucleus is the organelle of interest in our study because it is the major target

of many cytotoxic drugs (Dorr, 1998). Considering that DNA may quench the

fluorescence of Dox and low-level Dox-binding in cell nuclei is relatively difficult to be

detected (Lam et al., 2000), it is encouraging to observe strong fluorescence in the nuclei


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of cells treated with Dox-PLN (Fig. 5G). The images show that Dox-PLN is superior to

Dox solution in attaining high nuclear drug concentration in Pgp-overexpressing cells.

Since it has been demonstrated that the nuclear membrane also has Pgp to prevent drug

penetration into the cell nuclei (Baldini et al., 1995), the images suggest that the lipid

nanoparticles may also help overcome this possible source of drug resistance, though the

operative mechanism requires further investigations.

Wielinga et al. (2000) demonstrated that passive permeation also plays an

important and independent role in Dox cellular efflux in drug-resistant cells. Considering

MDA435/LCC6/MDR1 has only moderately high Pgp level, the role of passive diffusion

in drug clearance from these cells should be significant. It may therefore be argued that

the aforementioned findings are attributable to a diminished net outward drug diffusion

rate, for example, as a result of a local buildup of Dox-PLN outside the cell membrane

surfaces. However, had substantial reduction in passive drug diffusion occurred, the

cellular Dox accumulations in wild-type cells treated with Dox-PLN would have been

similarly enhanced like in their Pgp-overexpressing sublines (Figs. 2A and C). The

cellular drug efflux rates of the EMT6/AR1 cell line, which expresses a higher level of

Pgp and its cellular drug efflux rates, should be less reliant on passive diffusion. Instead

EMT6/AR1 cells responded even to a greater extent to Dox-PLN in terms of Dox

accumulation and retention. Furthermore, the microscopic images of EMT6/AR1 cells

treated with rhodamine, an efficient substrate for Pgp, also demonstrated distinctively

higher drug retention in cells when rhodamine was administered in nanoparticle form

(Figs. 7A and 7B). These results all suggest that it is more difficult for Pgp to remove the

drug molecules from the cells when these molecules are associated with the


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nanoparticles. It appears that this is the principal mechanism responsible for the enhanced

cellular drug retention, and to some extent cellular drug uptake. The result with

rhodamine also indicates that this phenomenon is not limited to Dox but to other Pgp

substrates.

We further examined the role of lipids of the nanoparticles. The similarity of the

cellular distributions of FSA and Dox (Figs. 8A and 8B) suggests that it is likely that at

least part of the Dox retained in the cells was physically associated with the lipids of

nanoparticles. This may also explain the granular appearance of the fluorescence in the

images of Dox-PLN treated cells.

As a modified form of SLN, the nanoparticles used in the present study were

prepared with solid lipids. In comparison to liquid lipid-based formulations such as

liposomes, the aforementioned Dox-lipid complexes or aggregates are expected to have

relatively good physical integrity after internalized into the cells. These drug-lipid

complexes are likely too large to be handled by Pgp, and are consequently “trapped”

within the cancer cells. It is possible that other forms of SLN may have similar

properties once they gain entrance into their targeted cells.

Endocytosis inhibition studies were conducted to further understand how the drug

or drug-loaded nanoparticles enter the cells. Colchicine mainly inhibits pinocytotic

uptake of liquid and particles smaller than 50 nm in diameter, whereas cytochalasin-B

primarily inhibits phagocytosis of particles greater than 100 nm (Ramge et al., 2000). In

Figs. 4A and 4B, it is evident that Dox-PLN accumulation was inhibited by colchicine in

both wild-type cell lines, and the magnitudes of inhibition were not affected by the type

of Dox formulations. On the other hand, both wild-type cells pretreated with


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cytochalasin-B exhibited stronger inhibition of cellular drug uptake (p < 0.05) when

treated with Dox-PLN, indicating significant phagocytosis of Dox-PLN or their

fragments by the wild-type cancer cells.

The situation is more complicated in Pgp-overexpressing cells, as both colchicine

and cytochalasin-B have Pgp-inhibitory effects (Gottesman, 2002; Zilfou and Smith,

1995), which may lead to various degrees of Dox accumulation enhancement.

Nevertheless, Dox-PLN treated cells were still more responsive to cytochalasin-B,

indicating phagocytosis likely occurred in Pgp-overexpressing cells. Overall, the drug

accumulation was only incompletely inhibited, and there was no further increase in

inhibition when these experiments were repeated at higher concentrations of inhibitors

(data not shown). It is evident that a substantial fraction of drug still enters the cells by

simple diffusion. This may be practically needed, particularly in solid tumor therapy, as

direct contacts between the nanoparticles with all tumor cells are unlikely.

Endocytosis can be receptor-mediated or adsorption-mediated. Endocytosis of

anionic liposomes mediated by scavenger receptors has been demonstrated before (Lee et

al, 1992), although this possibility would be scarce. Adsorption-mediated endocytosis

occurs at a higher efficiency with cationic nanoparticles (Templeton, 2002; Chenevier et

al, 2002), but negatively charged particles can still be captured by cells at a reasonably

high rate as shown in a number of liposomal formulations (Lee et al, 1992; Miller et al,

1998). The high lipophilicity of PLN may also improve their adsorption onto the cell

membranes. Since this adsorption mechanism is non-specific, for improved the efficiency

of endocytosis of Dox-PLN by cancer cells, ligands that specifically target cancer-related

receptors and cationic lipids will be considered in the future PLN formulation designs.


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Concluding Remarks. Drug in PLN probably enters cancer cells by a

combination of simple diffusion and phagocytosis, as schematically illustrated in Fig. 9.

The phagocytotic uptake (pathway (2)) is not necessarily more efficient than simple

diffusion (pathway (1)), as Dox-PLN were shown not more effective than Dox solution in

wild-type cells. However, part of the drug likely remains physically associated with the

solid lipids when internalized by cells. In this form the drug can neither be easily

removed by Pgp efflux nor quickly diffuse out, and may continue to build up in the cells

to serve as intracellular drug sources, which may lead to chronic suppression of the drug-

resistant cancer cell proliferation. All in all, the present study suggests a new mechanism

of action for a novel lipid-based formulation for overcoming the drug resistance of Pgp-

overexpressing tumor cells.

ACKNOWLEDGEMENTS

The authors would like to sincerely thank Dr. Clarke R and Dr. Tannock I for

providing the cancer cell lines, Drs. Liu Z and Erhan S for the HPESO polymer, and Dr.

Pennefather PS for granting the access of fluorescence microscopy instrumentation.


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REFERENCES

Baldini N, Scotlandi K, Serra M, Shikita T, Zini N, Ognibene A, Santi S, Ferracini R and

Maraldi NM (1995) Nuclear immunolocalization of P-glycoprotein in multidrug-resistant

cell lines showing similar mechanisms of doxorubicin distribution. Eur J Cell Biol

68:226-239.

Batrakova EV, Lee S, Li S, Venn A, Alakhov V and Kabanov A (1999) Fundamental

relationships between the composition of pluronic block copolymers and their

hypersensitization effect in MDR cancer cells. Pharm Res 16:1373-1379.

Bradford M (1976) A rapid and sensitive method for the quantitation of microgram

quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248-

254.

Booser DJ, Esteva FJ, Rivera E, Valero V, Esparza-Guerra L, Priebe W and Hortobagyi

GN (2002) Phase II study of liposomal annamycin in the treatment of doxorubicin-

resistant breast cancer. Cancer Chemother Pharmacol 50:6-8.

Chenevier P, Veyret B, Roux D and Henry-Toulme N (2000) Interaction of Cationic

Colloids at the Surface of J774 Cells: A Kinetic Analysis. Biophys J 79:1298-1309.


at ASPE

T Journals on M


Dow

nloaded from


JPET #101154

27

Cheung RY (2005) A study of alternative delivery strategies and systems to enhance the

therapeutic effect and cytotoxic activity of anticancer agents (PhD Thesis) pp. 254-255,

University of Toronto, Toronto.

Cheung RY, Rauth AM, Ronaldson P, Bendayan R and Wu XY (2006) Cytotoxicity of

microsphere-delivered mitomycin C and its combinations with doxorubicin against breast

cancer cells. Eur J Pharm Biopharm 62:321-331.

de Verdiere AC, Dubernet C, Nemati F, Soma E, Appel M, Ferte J, Bernard S, Puisieux F

and Couvreur P (1997) Reversion of multidrug resistance with polyalkylcyanoacrylate

nanoparticles: towards a mechanism of action. Brit J Cancer 76:198-205.

Dorr RT (1998) Antineoplastics, chemoprotectants, and immunosuppressants, in

Handbook of Clinical Drug Data (Anderson PO and Knoben JE eds) pp.157-212,

Appleton & Lange, Stamford.

Endicott JA and Ling V (1989) The biochemistry of P-glycoprotein-mediated multidrug

resistance. Ann Rev Biochem 58:137-171.

Evers R, Kool M, van Deemter L, Janssen H, Calafat J, Oomen LC, Paulusma CC, Oude

Elferink RP, Baas F, Schinkel AH and Borst P(1998) Drug export activity of the human

canalicular multispecific organic anion transporter in polarized kidney MDCK cells

expressing cMOAT (MRP2) cDNA. J Clin Invest 101:1310-1319.


at ASPE

T Journals on M


Dow

nloaded from


JPET #101154

28

Gottesman MM (2002) Mechanisms of cancer drug resistance. Ann Rev Med 53:615-627.

Jayanth P and Vinod L (2003) Dynamics of endocytosis and exocytosis of poly(D,L-

lactide-co-glycolide) nanoparticles in vascular smooth muscle cells. Pharm Res 20:212-

220.

Kabanov AV, Batrakova EV and Alakhov VY (2002) Pluronic block copolymers for

overcoming drug resistance in cancer. Adv Drug Del Rev 54: 759-779.

Lam W, Leung CH, Chan HL and Fong WF (2000) Toxicity and DNA binding of

dextran-doxorubicin conjugates in multidrug-resistant KB-V1 cells: optimization of

dextran size. Anti-Cancer Drugs 11:377-384.

Lamprecht A, Yamamoto H, Takeuchi H and Kawashima Y (2005) Nanoparticles

enhance therapeutic efficiency by selectively increased local drug dose in experimental

colitis in rats. J Pharmacol Exp Ther 315:196-202.

Lee KD, Hong K and Papahadjopoulos D (1992) Recognition of liposomes by cells: in

vitro binding and endocytosis mediated by specific lipid headgroups and surface charge

density. Biochim Biophys Acta 1103:185-197.


at ASPE

T Journals on M


Dow

nloaded from


JPET #101154

29

Liu Z, Bendayan R and Wu XY (2001) Triton-X-100-modified polymer and

microspheres for reversal of multidrug resistance. J Pharm Pharmacol 53:1-12.

Longley DB and Johnston PG (2005) Molecular mechanisms of drug resistance. J

Pathology 205:275-292.

Millenbaugh NJ, Wientjes MG and Au JLS (2000) A pharmacodynamic analysis method

to determine the relative importance of drug concentration and treatment time on effect,

Cancer Chemother Pharmacol 45:265-272.

Miller CR, Bondurant B, McLean SD, McGovern KA and O'Brien DF (1998) Liposome-

cell interactions in vitro: effect of liposome surface charge on the binding and

endocytosis of conventional and sterically stabilized liposomes. Biochemistry 37:12875-

12883

Moghimi SM and Hunter AC (2000) Poloxamers and poloxamines in nanoparticle

engineering and experimental medicine. Trend Biotechnol 18: 412-420.

Nori A, Jensen KD, Tijerina M, Kopeckova P and Kopecek J (2003) Subcellular

trafficking of HPMA copolymer-TAT conjugates in human ovarian carcinoma cells

J Controlled Release 91: 53-59.


at ASPE

T Journals on M


Dow

nloaded from


JPET #101154

30

Ramge P, Unger PE and Oltrogge JB (2000) Polysorbate-80 coating enhances uptake of

polybutylcyanoacrylate (PBCA)-nanoparticles by human, bovine, and murine primary

brain capillary endothelial cells. Eur J Neurosci 12:1935-1940.

Romsicki Y and Sharom FJ (1999) The membrane lipid environment modulates drug

interactions with the P-glycoprotein multidrug transporter. Biochemistry 38:6887-6896

Ronaldson TP, Bendayan M, Gingras D, Piquette-Miller M and Bendayan R (2004)

Cellular localization and functional expression of P-glycoprotein in rat astrocyte cultures.

J Neurochem 89:788-800.

Skeel RT (2003) Selection of treatment for the patient with cancer, in Handbook of

Cancer Chemotherapy (Skeel RT ed) pp 46-52, Lippincott Williams & Wilkins,

Philedelphia.

Soma CE, Dubernet C, Barratt G, Nemati F, Appel M, Benita S and Couvreur P (1999)

Ability of doxorubicin-loaded nanoparticles to overcome multidrug resistance of tumor

cells after their capture by macrophages. Pharm Res 16:1710-1716.

Tack DK, Palmieri FM and Perez EA (2004) Anthracycline vs nonanthracycline adjuvant

therapy for breast cancer. Oncology 18:1367-1376.


at ASPE

T Journals on M


Dow

nloaded from


JPET #101154

31

Templeton NS (2002) Cationic liposome-mediated gene delivery in vivo. Biosci Rep

22:283-295.

Thierry AR, Vige D, Coughlin SS, Belli JA, Dritschilo A and Rahman A (1993)

Modulation of doxorubicin resistance in multidrug-resistant cells by liposomes. FASEB J

7:572-579.

Tipton JM (2003) Side effects of cancer chemotherapy, in Handbook of Cancer

Chemotherapy (Skeel RT ed) pp 561-580, Lippincott Williams & Wilkins, Philedelphia.

Veldman RJ, Koning GA, van Hell A, Zerp S, Vink SR, Storm G, Verheij M and van

Blitterswijk WJ (2005) Coformulated N-Octanoyl-glucosylceramide improves

cellular delivery and cytotoxicity of liposomal doxorubicin. J Pharmacol Exp Ther

315:704-710.

Wielinga PR, Westerhoff HV and Lankelma J (2000) The relative importance of passive

and P-glycoprotein mediated anthracycline efflux from multidrug-resistant cells. Eur J

Biochem 267:649-657.

Wong HL, Bendayan R, Rauth AM and Wu XY (2004) Development of solid lipid

nanoparticles containing ionically-complexed chemotherapeutic drugs and

chemosensitizers. J Pharm Sci 93:1993-2004.


at ASPE

T Journals on M


Dow

nloaded from


JPET #101154

32

Wong HL, Rauth AM, Bendayan R and Wu XY (2005) Novel solid lipid nanoparticles

formulations increase the cytotoxicity and prolong the cellular accumulation of

doxorubicin in human multidrug-resistant breast cancer cells. Proc AACR 45:Abstract

1433.

Wong HL, Rauth AM, Bendayan R and Wu XY. A new solid lipid nanoparticle

formulation increases cytotoxicity of doxorubicin against multidrug-resistant human

breast cancer cells. Pharm Res (in press).

Zhang K, Huang H, Yang G, Shaw J, Yip C and Wu XY (2004) Characterization of

nanostructure of stimuli-response polymeric composite membranes. Biomacromolecules

5:1248-1255.

Zilfou JT and Smith CD (1995) Differential interactions of cytochalasins with P-

glycoprotein. Oncol Res 7:435-443.


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FOOTNOTES This work was financially supported by the Canadian Institutes of Health Research and

the Natural Sciences and Engineering Research Council of Canada.

Please send the reprint request to Xiao Yu Wu; Mail address: Graduate Department of

Pharmaceutical Sciences, Leslie Dan Faculty of Pharmacy, 19 Russell Street, University

of Toronto, Ontario, Canada M5S 2S2; Email: [email protected].


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LEGENDS FOR FIGURES Figure 1

Western blot analysis of Pgp (~170 kDa) in cultured MDA435/LCC6/MDR1 and

EMT/AR1 cells. Whole cell lysate preparations (25 µg of protein) from

MDA435/LCC6/MDR1 (lane 1) and EMT6/AR1 (lane 2) cells were resolved on a 10%

sodium dodecyl sulfate-polyacrylamide gel and transferred to a polyvinylidene difluoride

membrane. Pgp was detected using the monoclonal Pgp antibody C219 (1:500 dilution)

while β-actin (42 kDa) was detected using anti-actin antibody AC-40 (1:500 dilution).

Figure 2

Effect of polymer-lipid nanoparticles containing doxorubicin (Dox) on Dox

uptake by human or murine breast cancer cell lines. MDA435/LCC6/WT (A),

MDA435/LCC6/MDR1 (B), EMT6/WT (C) and EMT6/AR1 (D) cells were treated with

Dox solution or nanoparticles loaded with Dox (Dox-PLN) for up to 4 h. In (A) and (B)

cells were also treated with a combination of blank nanoparticles (blank PLN) and Dox

solution. The total Dox concentration (free + loaded Dox) in all treatments was equally

set at 10 µg/ml. At predetermined time points, cells were washed with PBS buffer and

lysed with 1% Triton-X-100 and the cellular Dox uptake was measured with a microplate

fluorometer. Results were normalized with cellular protein levels and expressed as

means ± S.D. of 3 separate experiments in cells pertaining to different passages. ∗ p <

0.05.


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Figure 3

Effect of polymer-lipid nanoparticles containing doxorubicin (Dox) on the

amount of Dox retained by human or murine breast cancer cell lines.

MDA435/LCC6/WT (A), MDA435/LCC6/MDR1 (B), EMT6/WT (C) and EMT6/AR1

(D) cells were treated with Dox solution or nanoparticles loaded with Dox (Dox-PLN) for

2 h, then re-incubated in fresh EBSS medium for up to 2 h to allow cellular drug efflux.

The total Dox concentration (free + loaded Dox) in all treatments was equally set at 10

µg/ml. Results were normalized with cellular protein levels and expressed as means ±

S.D. of 3 separate experiments in cells pertaining to different passages. ∗ p < 0.05.

Figure 4

Effect of endocytosis inhibitor pretreatment on doxorubicin (Dox) uptake by cells

receiving different forms of Dox treatment. MDA435/LCC6/WT,

MDA435/LCC6/MDR1 (A), EMT6/WT and EMT6/AR1 cells (B) were pre-treated with

colchicine or cytochalasin-B for 1 h, then treated with Dox solution or nanoparticles

loaded with Dox (Dox-PLN) containing 10 µg/ml Dox for 2 h. The amounts of Dox

accumulated in cells were measured with a microplate fluorometer and standardized

against cellular protein levels. Results are normalized against cells receiving the same

form of Dox treatment but no pretreatment. * p < 0.05; Colch = colchicine; Cytoch =

cytochalasin-B.


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Figure 5

Fluoroescence microscope images demonstrating the effect of different

formulations containing doxorubicin (Dox) on drug retention and intracellular drug

distribution in human breast cancer cells. Cells were all incubated with Dox solution or

nanoparticles containing Dox (Dox-PLN) for 2 h. Images were taken either within 5 min

after the end of treatment or 2 h after the end of treatment and re-incubation in EBSS

medium at 37°C. (A) and (B): MDA435/LCC6/WT cells treated with 5 µg/mL Dox

solution, 5 min and 2 h after the end of treatment, respectively. (C) and (D):

MDA435/LCC6/MDR1 cells treated with 5 µg/mL Dox solution, 5 min and 2 h after the

end of treatment, respectively. (E) and (F): MDA435/LCC6/MDR1 cells treated with

Dox-PLN suspension containing 5 µg/mL, 5 min and 2 h after the end of treatment,

respectively. (G) and (H): MDA435/LCC6/MDR1 cells treated with 10 µg/mL Dox

solution, 5 min and 2 h after the end of treatment, respectively. (I) and (J):

MDA435/LCC6/MDR1 cells treated with Dox-PLN containing 10 µg/mL Dox, 5 min

and 2 h after the end of treatment, respectively. λex = 540 nm and λem = 590 nm for Dox.

Magnification of objective = 100×. Bars represent 20 µm.

Figure 6

Fluoroescence microscope images for the confirmation of nuclear localization of

doxorubicin (Dox) delivered by Dox-PLN. MDA435/LCC6/MDR1 cells were treated

with 10 µg/mL Dox as Dox-PLN for 2 h, then allowing 2 h drug-free period for drug

efflux, and incubated in 2 µg/mL DAPI solution for 5 min for nuclear staining.

Fluorescences of (A): Dox and (B): DAPI emitted from the cells were imaged. λex = 540


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nm and λem = 590 nm for Dox, λex = 360 nm and λem = 450 nm for DAPI. Magnification

of objective = 100×. Bars represent 20 µm.

Figure 7

Fluoroescence microscope images demonstrating the effect of different

formulations containing rhodamine on drug retention and intracellular drug distribution in

murine breast cancer cells. EMT6/AR1 cells were treated with (A) rhodamine solution

and (B) rhodamine-PLN suspension containing 1 µg/mL rhodamine-B, respectively, 2 h

after the end of treatment. λex = 520 nm and λem = 570 nm for rhodamine-B.

Magnification of objective = 100×. Bars represent 20 µm.

Figure 8

Fluorescence microscope images of particles containing doxorubicin (Dox) and/or

fluoresceinamine-labeled stearic acid (FSA). (A) and (B) are images of

MDA435/LCC6/MDR1 cells treated with PLN simultaneously loaded with Dox and FSA

for 2 h. Both images were taken in the same microscopy field. Image (A) was taken at λex

= 520 nm and λem = 580 nm for detection of Dox; image (B) was taken at λex = 430 nm

and λem = 500 nm for detection of FSA. Magnification of objective = 100×. Bars

represent 20 µm.


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Figure 9

Schematic representation of the proposed mechanisms underlying the enhanced

anticancer activity of Dox-PLN in a Pgp-overexpressing cancer cell. (1) Diffusion of the

released drug across cell membrane; (2) endocytosis of Dox-PLN. Some of the drug

molecules that diffuse into the cell are removed by the P-glycoprotein (P-gp). The

fraction of Dox molecules that enter cells by endocytosis may be associated with the solid

lipids, and are difficult to be cleared from the cell by the P-gp drug efflux.


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Table 1. Summary of physicochemical properties of Dox-PLN

Type Mean

particle diameter (range in a sample), nm

Zeta potential, mV

% drug loading, %w/w

Drug encapsulation efficiency, %w/w

Average time to release 50% of Dox loaded, h

% of loaded drug released in 72 h, %w/w

Dox-PLN

290 (80 to 350)

−23.1 ± 0.28

3.8 76 3.4 66.2 ± 4.1

Blank PLN

213 (60 to 280)

−19.7 ± 0.65

N.A. N.A. N.A N.A.

Summary of physicochemical properties of Dox-PLN and blank PLN. Dox-PLN were

loaded with 3.4% (w/w). Particle sizes and zeta potentials were measured with photon-

correlation spectroscopy and its surface charge measurement unit. Drug encapsulation

efficiency is defined as the % of drug added into the preparation that is finally

encapsulated by the nanoparticle. For details of the results please refer to Wong et al., in

press.


at ASPE

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Fig. 1

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I: 10.1124/jpet.106.101154 at ASPET Journals on March 28, 2021 jpet.aspetjournals.org Downloaded from


Fig. 2a

0

200

400

600

800

1000

1200

0 1 2 3 4Time of Drug Exposure (h)

Do

x up

take

(nm

ol/m

g pr

ote

in)

Dox-PLN

Dox solution

Dox solution+ Blank PLN




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Fgi.2b

0

200

400

600

800

1000


Dox

upt

ake

(nm

ol/m

g p

rote

in)

Dox-PLN

Dox solution

Dox solution+ Blank PLN




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Fig. 2c

0

200

400

600

800

1000

1200

1400


Dox

upt

ake

(nm

ol/m

g p

rote

in)

Dox-PLN

Dox solution




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0

200

400

600

800


Do

x up

take

(nm

ol/m

g p

rote

in)

Dox-PLN

Dox solution

Fig. 2d




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Fig. 3a

0

200

400

600

800

1000

0 0.5 1 1.5 2Time of drug efflux (h)

Do

x re

tain

ed (

nm

ol/m

g p

rote

in)

.

Dox-PLN

Dox solution




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Fig. 3b

0

100

200

300

400

500

600

700

0 0.5 1 1.5 2

Time of drug efflux (h)

Do

x re

tain

ed (

nm

ol/m

g p

rote

in)

.

Dox-PLN

Dox solution




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Fig. 3c

0

200

400

600

800

1000

0 0.5 1 1.5 2


Do

x re

tain

ed (

nm

ol/m

g p

rote

in)

.

Dox-PLN

Dox solution




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Fig. 3d

0

100

200

300

400

500

600

700

0 0.5 1 1.5 2


Do

x re

tain

ed (

nm

ol/m

g p

rote

in)

.

Dox-PLN

Dox solution




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Fig. 4a

-30

-20

-10

0

10

20

30

40

50

Colch Cytoch-B Colch Cytoch-B

WT MDR1

Pretreatment/Cell line

% in

hib

itio

n of

Dox

acc

um

ulat

ion

. Dox solution

Dox-PLN *

*




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Fig. 4b

-30

-20

-10

0

10

20

30

40

50

Colch Cytoch-B Colch Cytoch-B

WT AR1

Pretreatment/Cell line

% in

hib

ition

of D

ox a

ccum

ulat

ion

Dox solution

Dox-PLN

*

*




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Fig. 5(A) (B) (C) (D)

(E) (G) (H)(F)

(J) (I)




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Fig. 6

(A) (B)




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Fig. 7 (A) (B)




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Fig. 8

(A) (B)




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Fig. 9 This article has not been copyedited and form



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