Journal of Controlled Release 114 (2006) 153 – 162 www.elsevier.com/locate/jconrel

In vitro and in vivo intracellular liposomal delivery of antisense oligonucleotides and anticancer drug Refika I. Pakunlu a , Yang Wang a , Maha Saad a , Jayant J. Khandare a , Valentin Starovoytov b , Tamara Minko a,c,d,⁎ b

a Department of Pharmaceutics, Rutgers, The State University of New Jersey, Piscataway, NJ, USA Division of Life Sciences, Electron Imaging Facility, Rutgers, The State University of New Jersey, Piscataway, NJ, USA c The Cancer Institute of New Jersey, New Brunswick, NJ, USA d New Jersey Center for Biomaterials, Piscataway, NJ, USA

Accepted 31 May 2006 Available online 15 June 2006

Abstract The specific aims of this investigation were (1) to show that conventional and PEGylated liposomes can penetrate cancer cells in vitro and in vivo; (2) to demonstrate that liposomes can be successfully used both for cytoplasmic and nuclear delivery of therapeutics, including anticancer drugs and antisense oligonucleotides; (3) to examine the specific activity of anticancer drugs and nucleotides delivered inside tumor cells by PEGylated liposomes; and (4) to confirm that simultaneous inhibition of pump and nonpump cellular resistance by liposomal ASO can substantially enhance the antitumor activity of traditional well established anticancer drugs in mice bearing xenografts of human multidrug resistant ovarian carcinoma. Experimental results show that PEGylated liposomes are capable of penetrating directly into tumor cells after systemic administration in vivo and do successfully provide cytoplasmic and nuclear delivery of encapsulated anticancer drug (doxorubicin, DOX) and antisense oligonucleotides (ASO). Encapsulation of DOX and ASO into liposomes substantially increased their specific activity. Simultaneous suppression of pump and nonpump resistance dramatically enhanced the ability of DOX for inducing apoptosis leading to higher in vitro cytotoxicity and in vivo antitumor activity. © 2006 Elsevier B.V. All rights reserved. Keywords: Liposomes; Antisense oligonucleotides; Doxorubicin; Antitumor activity; Intracellular localization

1. Introduction The success of liposomes as drug carriers has been reflected in a number of liposome-based formulations, which are commercially available or are currently undergoing clinical trials. Several formulations of liposomal anthracyclines have been approved [1– 5]. Up to date, virtually all traditional anticancer drugs have been encapsulated in liposomes using different technologies and many of them are included in clinical trials as cancer imaging agents and/or anticancer therapeutics [6]. Several liposomal formulations are currently in different phases of clinical trials. ⁎ Corresponding author. Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, NJ 08854-8020, USA. Tel.: +1 732 445 3831x214; fax: +1 732 445 3134. E-mail address: [email protected] (T. Minko). 0168-3659/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2006.06.010

Some important issues related to liposomal anticancer drugs remain unaddressed. Answers to these questions can result in the development of novel effective anticancer therapeutics. Four areas which require experimental work are: (1) Can conventional and PEGylated liposomes penetrate tumor cells in vivo? (2) Are non-targeted PEGylated liposomes able to provide for cytoplasmic and/or nuclear delivery of anticancer therapeutics? (3) How liposomal delivery influences the activity of encapsulated drugs and other biologically active agents? (4) Whether the simultaneous suppression of pump and nonpump resistance by liposomal antisense oligonucleotides (ASO) will enhance the antitumor activity of a traditional anticancer drug? A positive answer for the first three questions will indicate that PEGylated liposomes can be successfully used for the delivery of anticancer agents and antisense oligonucleotides directly into the cytoplasm and nuclei of tumor cells in vivo preserving or even enhancing their specific activity (e.g. cell

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and BCL2 mRNA as suppressors of pump and nonpump cellular resistance in cancer cells. 2. Methods and materials 2.1. Drug, antisense oligonucleotides and liposomal delivery system

Fig. 1. Complex liposomal drug delivery system. The system consists of PEGylated liposomes containing the anticancer drug (doxorubicin, DOX) in combination with antisense oligonucleotides (ASO) targeted to MDR1 and BCL2 mRNA as suppressors of pump and nonpump resistance respectively.

death induction for anticancer drugs and suppression of targeted proteins for ASO). The positive answer to the last question will open the way for a novel type of drug delivery simultaneously targeted to two intracellular mechanisms: apoptosis induction pathways and cellular resistance against anticancer drugs. The vast majority of anticancer therapeutics induce cancer cell death by apoptosis. However, the efficacy of chemotherapy is limited by intrinsic or rapidly acquired drug resistance. Cancer resistance mechanisms can be subdivided into two distinct classes, pump and nonpump resistance [7–11]. Pump resistance is mediated by membrane transporters that pump out the anticancer agents from cells, decreasing the intracellular drug concentration and thereby the efficacy of the treatment. The main mechanism of nonpump resistance is an activation of cellular antiapoptotic defense. Consequently, to effectively suppress the overall tumor resistance to chemotherapy, it is essential to simultaneously inhibit both mechanisms of cellular resistance by targeting two intracellular molecular targets—proteins which are key players in pump resistance (mainly P-glycoprotein encoded in humans by the MDR1 gene) and antiapoptotic cellular defense (mainly BCL2 protein). The present investigation is aimed at answering the above listed questions and dedicated to experimental testing of a novel liposomal drug delivery system which contains a traditional anticancer drug doxorubicin (DOX) as an apoptosis inducer in combination with antisense oligonucleotides targeted to MDR1

The complex liposomal drug delivery system included the following components (Fig. 1): (1) a carrier—conventional or PEGylated liposomes; (2) an apoptosis inducer—a traditional anticancer drug doxorubicin; (3) a suppressor of pump resistance— antisense oligonucleotides targeted to MDR1 mRNA and (4) a suppressor of nonpump resistance—ASO targeted to BCL2 mRNA. Doxorubicin was obtained from Sigma (St. Louis, MO). The sequences of the antisense oligonucleotides (ASO) targeted to BCL2 and MDR1 mRNA were: 5′-CAG CGT GCG CCA TCC TTC CC-3′ and 5′-TTC AAG ATC CAT CCC GAC CTC GCG-3′, respectively [12,13]. The DNA backbone of all bases in oligonucleotides was P-ethoxy modified to enhance nuclease resistance and increase incorporation efficacy into liposomes [12]. ASO were synthesized by Oligos Etc. (Wilsonville, OR). ASO and DOX (Sigma, St. Louis, MO) were packaged in liposomes, which were prepared using previously described lipid film rehydration method [10,11]. To prepare conventional liposomes, lipids (Avanti Polar Lipids, Alabaster, AL) were dissolved in chloroform, evaporated to a thin film in a rotary evaporator, and rehydrated with citrate buffer. The lipid ratio for all formulations was 7:3:10 (v/v, egg phosphatidylcholine/1,2dipalmitoyl-sn-glycero-3-phosphatidylcholine/cholesterol). To prepare PEGylated liposomes, egg phosphatidylcholine, cholesterol, and DSPE-PEG (1,2-distearoyl-sn-glycero-3-phosphoetanol amine-N-aminopolyethylene glycol—Mw ∼ 2000 ammonium salt) were dissolved in 4.0 ml of chloroform with 1.85:1:0.15 molar ratio respectively (all compounds were obtained from Avanti Polar Lipids, Alabaster, AL). The clear lipid solution was evaporated at 25 °C under reduced pressure. The thin layer was formed and rehydrated using 2.0 ml of 0.3 M sodium citrate buffer (pH = 4.0). The lipid mixture was sonicated continuously for 3.0 h to obtain stealth liposomes. The liposomes were freeze thawed

Fig. 2. Transmission electron microscopy images of multidrug resistant A2780/AD human ovarian carcinoma cells (A) and tumor tissue obtained from mice bearing xenografts (B) treated with conventional (A) and PEGylated (B) liposomes labeled with osmium tetroxide. Arrows indicate the cytoplasmic and nuclear localization of liposomes.

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under liquid nitrogen and placed to −20 °C overnight. ASO were loaded into the liposomes by dissolving the ASO in the rehydration buffer at a concentration of 0.5 mM when MDR1 and BCL2 ASO were used separately and 0.25 mM of each ASO when they were used in combination. DOX was loaded passively by dissolving in the rehydration buffer with the ASO (0.16 w/ w DOX/lipid ratio). After preparation, free DOX and ASO were separated from conventional liposomes by passing the liposome suspension through a Sephadex G-50 column. PEGylated liposomes with encapsulated DOX were separated from free drug by dialysis, while unbound DOX and ASO were separated from loaded PEGylated liposomes by filtration at 4000×g within 20 min through Amicon Ultra-15 Centrifugal Filter (10,000 WCO, Millipore Corporation, Billerica, MA). The encapsulation efficacy ranged from 51.5% to 58.3% in different series of experiments. The range of liposome diameter was 100–200 nm. A series of in vitro experiments established the stability of the liposomes containing fluorescent ASO and DOX. Liposomes incubated in saline for several weeks did not release any significant amount of labeled ASO. 2.2. Cell line The human multidrug resistant ovarian carcinoma A2780/ AD cell line was obtained from Dr. T. C. Hamilton (Fox Chase Cancer Center). Cells were cultured in RPMI 1640 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Fisher Chemicals, Fairlawn, NJ). Cells were grown at 37 °C in a humidified atmosphere of 5% CO2 (v/v) in air. All experiments were performed on cells in the exponential growth phase. 2.3. Animal tumor model and antitumor activity Animal model of human ovarian carcinoma xenografts was used as previously described [14–16]. Briefly A2780 human ovarian cancer cells (2 × 106) were subcutaneously transplanted

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into the flanks of female athymic nu/nu mice. When the tumors reached a size of about 0.5 cm3 (15–20 days after transplantation), mice were treated intraperitoneally with saline (control), empty PEGylated liposomes, free DOX, PEGylated liposomes containing DOX, PEGylated liposomes containing DOX and ASO targeted to BCL2 mRNA, PEGylated liposomes containing DOX and ASO targeted to MDR1 mRNA and PEGylated liposomes containing DOX and ASO targeted to BCL2 and MDR1 mRNA. The dose of all formulations containing DOX (2.5 mg DOX/kg in 0.1 ml for the single injection) corresponds to the maximum tolerated dose of free DOX. Maximum tolerated dose of DOX was estimated in separate experiments based on animal weight change after the injection of increasing doses of DOX as previously described [14,15]. Animal weight was measured everyday within 1 week after the treatment. Changes in tumor size were used as an overall marker for antitumor activity as previously described [14,15]. 2.4. Cytotoxicity The cellular cytotoxicity of formulations was assessed using a modified MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay as previously described [11,17]. To measure cytotoxicity, cells were separately incubated in microtiter plate with different concentrations of empty PEGylated liposomes, free DOX, PEGylated liposomes containing DOX, PEGylated liposomes containing DOX and ASO targeted to BCL2 mRNA, PEGylated liposomes containing DOX and ASO targeted to MDR1 mRNA and PEGylated liposomes containing DOX and ASO targeted to BCL2 and MDR1 mRNA in the cell growth medium. Control cells received an equivalent volume of fresh medium. The duration of incubation was 24 h. Based on these measurements, IC50 doses of free and liposomal formulations of drug delivery systems (the concentrations of active ingredients necessary to inhibit the cell growth by 50%) were calculated as previously described [11,17].

Fig. 3. Confocal microscopy fluorescent images of human A2780 ovarian carcinoma cells incubated for 24 h with rhodamine-labeled PEGylated liposomes (z-series, from the top of the cell to the bottom).

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2.5. Intracellular localization of liposomes and release of doxorubicin and antisense oligonucleotides To analyze intracellular localization of ASO released from liposomes, a portion of oligonucleotides were labeled by fluorescein isothiocyanate (FITC) prior to the incorporation into the liposomes. The labeling was performed by Oligos Etc. (Wilsonville, OR). These labeled ASO were used only in ASO release and localization experiments. Intracellular localization of ASO and DOX (possesses intrinsic fluorescence) was studied by fluorescent and confocal microscopy. In these experiments cell nuclei were additionally stained by Hoechst 33258 nuclear dye (Sigma, St. Louis, MO). The release of DOX from liposomes was examined by confocal microscopy in living cells at 37 °C within 1 h. Part of the PEGylated liposomes was labeled with rhodamine and osmium tetroxide and visualized by confocal and electron transmission microscopy respectively. Rhodamine red succinimidyl ester (Invitrogen, Molecular Probes, Carlsbad, CA) was covalently conjugated with DSPE-PEG. Rhodamine red succinimidyl ester (RRSE, 2 mg, 0.0026 mM) was dissolved in 1.0 ml of anhydrous dimethylformamide (DMF, Fisher Chemical, Fairlawn, NJ) and 4.0 ml of N,N-diisopropylethylamine (Fisher Chemical, Fairlawn, NJ) was added to adjust alkaline pH and maintain amine group in DSPE-PEG lipid in non-protonated form. DSPE-PEG (14.5 mg, 0.0075 mM) was dissolved separately in 2.0 ml of dimethylformamide and mixed with the RRSE solution. The reaction mixture was stirred for 2 h under subdued light. DSPE-PEG-RRSE conjugate was purified to remove free RRSE using dialysis membrane (Mw cut off ∼ 2000 Da) in DMF as a solvent. The conjugate was further purified by size exclusion G10 sephadex column chromatogra-

phy and solution was dried under the vacuum. DSPE-PEG labeled with rhodamine red was used to prepare liposomes as described earlier. Labeling of liposomes for electron transmission microscopy was done by adding osmium tetroxide (0.5%) to rehydration buffer. Cells and tissue sections were fixed prior to electron microscopy using standard techniques [18,19]. Briefly, cells and tumor tissue were primary fixed for 2 h in Trump's EM Fixative (combination of low concentration of both formaldehyde and glutaraldehyde in 0.1 M Milloning's phosphate buffer, pH 7.3). Postfixation was carried out in 1% osmium tetroxide in buffer for 1 h followed by dehydration in graded Ethanol series and embedded in Spurr's Low Viscosity Resin. Sections were prepared using a diamond knife by LKB-2088 Ultramicrotome (LKB-Produkter, Bromma, Sweden). Observation and micrographs were made with a JEM-100CXII Electron Microscope (JEOL LTD., Tokyo, Japan). Intracellular localization of liposomes was studied in cell culture experiments and in tumor slices. 2.6. Gene expression Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was used for the analysis in tumor tissue homogenates of expression of genes encoding P-glycoprotein (MDR1), BCL2 protein, caspase 3 (CASP3), caspase 9 (CASP9) as previously described [10,11]. RNAwas isolated 24 h after the treatment using an RNeasy kit (Qiagen, Valencia, CA). The following pairs of primers were used: MDR1—5′-CCC ATC ATT GCA ATA GCA GG-3′ (sense) and 5′-GTT CAA ACT TCT GCT CCT GA-3′ (antisense); BCL2—GGATTG TGG CCT TCT TTG AG (sense), CCA AAC TGA GCA GAG TCT TC (antisense); CASP3—TGG

Fig. 4. Fluorescent microscopy images of human ovarian carcinoma A2780 cells before (0 min) and after (2–54 min) incubation with conventional liposomes loaded with doxorubicin.

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AAT TGA TGC GTG ATG TT (sense), GGC AGG CCT GAA TAA TGA AA (antisense); CASP9—TGA CTG CCA AGA AAA TGG TG (sense), CAG CTG GTC CCA TTG AAG AT (antisense); β2-microglobulin (β2-m, internal standard)—ACC CCC ACT GAA AAA GAT GA (sense), ATC TTC AAA CCT CCA TGA TG (antisense). PCR products were separated in 4% NuSieve 3:1 Reliant® agarose gels (BMA, Rockland, ME) in 1× TBE buffer (0.089 M Tris/Borate, 0.002 M EDTA, pH 8.3; Research Organics Inc., Cleveland, OH) by submarine electrophoresis. The gels were stained with ethidium bromide, digitally photographed and scanned using Gel Documentation System 920 (NucleoTech, San Mateo, CA). Gene expression was calculated as the ratio of mean band density of analyzed RT-PCR product to that of the internal standard (β2-m).

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Laboratories, Inc., Burlingame, CA) in combination with DAB substrate kit for peroxidase were used for visualization. After staining the slides were analyzed by light microscopy and photographed. 2.8. Apoptosis The analysis of apoptosis was based on the detection of singleand double-stranded DNA breaks (nicks) by an in situ cell death detection kit (Roche, Nutley, NJ) using terminal deoxynucleotidyl transferase mediated dUTP-fluorescein nick end labeling (TUNEL) method as previously described [14,16]. Briefly, cells were fixed, permeabilized and incubated with the TUNEL reaction mixture. The label incorporated at the damaged sites of the DNA was visualized by a fluorescence microscope.

2.7. Protein expression 2.9. Statistical analysis To confirm RT-PCR data the expression of P-glycoprotein and BCL2 protein was analyzed. The identification of the above proteins was made by immunohistochemical staining of paraffin embedded tumor tissue sections. After deparaffinization and rehydration, the slides were stained using Vector® M.O.M. Immunodetection Kit (Vector Laboratories, Inc., Burlingame, CA). Mouse monoclonal antibody to P-glycoprotein (ab3366, 1:40 dilution) obtained from Abcam (Cambridge, MA) and mouse monoclonal antibody to BCL2 protein (CP-B201, 1:80 dilution) obtained from Vector Laboratories, Inc. (Burlingame, CA) were used as primary antibody for the detection of P-glycoprotein and BCL2 protein, respectively. Biotinylated anti-mouse IgG Reagent (1:250 dilution, Vector Laboratories, Inc., Burlingame, CA) and HSP-Streptavidine Detection System (1:500 dilution, Vector

Data obtained were analyzed using descriptive statistics, single factor analysis of variance (ANOVA) and presented as mean value ± standard deviation (S.D.) from four to eight independent measurements in separate experiments. 3. Results 3.1. Conventional and PEGylated liposomes penetrate tumor cells To evaluate the penetration of liposomes into tumor interstitium and tumor cells, we labeled both conventional and PEGylated liposomes with osmium tetroxide and treated mice bearing xenografts of multidrug resistant A2780/AD human

Fig. 5. Intracellular localization of antisense oligonucleotides and doxorubicin. Typical images of multidrug resistant A2780/AD human ovarian cancer cells incubated for 24 h with liposomes containing FITC-labeled ASO and DOX. (A) Localization of ASO (green fluorescence); (B) localization of DOX (red fluorescence); (C) fluorescent image of nuclei stained with Hoechst 33258 (blue fluorescence); (D) superposition of fluorescent images of FITC-stained ASO (A) and stained nucleus (C), nuclear localization of ASO gives cyan color; (E) superposition of fluorescent images of DOX (B) and stained nucleus (C), nuclear localization of DOX gives pink color; (F) superposition of fluorescent images of ASO (A), DOX (B) and stained nucleus (C), nuclear co-localization of ASO and DOX gives white color, while colocalization of ASO and DOX in the cytoplasm gives yellow color.

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ovarian carcinoma with these liposomes. In addition we incubated cells with osmium tetroxide-labeled conventional liposomes. Labeled liposomes were visualized in tumor slices by electron transmission microscopy. The mice were treated once by intravenous injection of liposomes. Osmium tetroxidelabeled PEGylated liposomes were found in the cytoplasm, perinuclear regions and nucleus of cancer cells on electron microscopy images as black spheres with average diameter of 100–200 nm (identified by arrows in Fig. 2). Similar results were obtained both for conventional liposomes in vitro (Fig. 2A) and PEGylated liposomes in vivo (Fig. 2B). Theoretically, liposomes could adhere to the surface of cancer cells and erroneously be visualized on microscopic images as internalized within cells. To exclude such type of errors, we analyzed the distribution of labeled liposomes in different cellular layers from the upper to the lower of the fixed cell using confocal fluorescent microscopy (z-sections, Fig. 3). In these experiments PEGylated liposomes were labeled with rhodamine red and incubated with multidrug resistant A2780 human ovarian carcinoma cells. The cells were fixed and subjected to confocal microscopy. The data obtained show that the distribution of labeled liposomes in cellular cytoplasm and nucleus were uniform and very similar in different cell layers. Taken together these data suggest that small (100–200 nm) unilamellar conventional liposomes with fluid membrane as well as PEGylated liposomes can be internalized into cancer cells in both in vitro and in vivo settings and can even penetrate cellular nuclei.

ning of the incubation, a part of liposomes fuses with the plasma membrane releasing DOX from the liposomes near the membrane. However, few minutes later, the fluorescence of released DOX became visible in a perinuclear region and finally in the nucleus itself. Therefore, in the later stages, either liposomes were internalized by endocytosis, DOX was released from liposomes in the perinuclear region and entered the nucleus, or loaded liposomes were internalized into the nucleus and released DOX (Fig. 4). The localization of drugs delivered by liposomes in perinuclear area and inside nuclei was also confirmed in our experiments on human ovarian cancer cells (Fig. 5). In these experiments, antisense oligonucleotides (ASO) labeled with fluorescein isothiocyanate and DOX were incorporated into conventional liposomes with an average diameter of 100–200 nm. Human multidrug resistant A2780/AD cancer cells were incubated for 24 h at 37 °C with those liposomes. Then cells were washed from unbound liposomes, fixed and fluorescence was visualized by fluorescent microscopy. Fluorescent microscopy images and their superposition showed both cytoplasmic and nuclear localization of ASO and DOX after the relatively long-term incubation with liposomes containing these substances (Fig. 5).

3.2. Liposomes can be used for cytoplasmic and nuclear delivery The fact that conventional and PEGylated liposomes penetrate tumor cell cytoplasm and nuclei makes them an attractive vehicle for both cytoplasmic and nuclear delivery. To confirm the fact that liposomes used in the present investigations can release their payload inside cancer cells in the cytoplasm and nucleus, we loaded liposomes with a traditional anticancer drug doxorubicin and antisense oligonucleotides. ASO were labeled with FITC, while DOX possessed an intrinsic fluorescence. Two types of experiments were carried out. In both series, multidrug resistant human A2780/AD ovarian cancer cells were incubated with these liposomes. In the first series, fluorescence of DOX was observed in living cells before and at different time-points after the addition of liposomes into the media (Fig. 4). Doxorubicin was selected as the encapsulated drug because it possesses an easily measurable red fluorescence. Moreover, in contrast to polymeric DOX [15,20,21], the fluorescence of DOX inside liposomes is quenched [11]. Therefore, the presence of red fluorescence is a hallmark of the drug released from the liposomes. Fig. 4 shows the results of in vitro experiments which were carried out on living ovarian cancer cells under 37 °C. No substantial fluorescence has been detected before the application of conventional liposomes with encapsulated DOX to the cells and first few minutes after the incubation of cells with liposomal DOX. Several minutes after the injection of liposomal DOX to the incubation medium, the fluorescence was first seen near the plasma membrane and adjacent cytoplasm. These data show that soon after the begin-

Fig. 6. Effect of treatment with saline (1), empty liposomes (2), DOX (3), PEGylated liposomes loaded with DOX (4) and PEGylated liposomes loaded with DOX and ASO targeted to MDR1 and BCL2 mRNA (5) on the expression of the genes encoding P-glycoprotein (MDR1), BCL2 protein, caspases 9 (CASP9) and 3 (CASP3) in tumor tissue. Mice bearing xenografts of multidrug resistant A2780/AD human ovarian carcinoma were treated with indicated substances and studied 24 h after the treatment. Gene expression was calculated as a ratio of band intensity of studied gene to that in internal standard (β2-m, β2microglobulin). Means ± S.D. from 4–8 independent measurements are shown. *P< 0.05 when compared with control (saline); †P< 0.05 when compared with empty liposomes; ‡P< 0.05 when compared with DOX; ¶P< 0.05 when compared with liposomal DOX.

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Fig. 7. Typical images of tumor tissue sections stained with antibody against P-glycoprotein (top panel) and BCL2 protein (bottom panel). Dark color indicates high protein concentration. Mice bearing xenografts of multidrug resistant A2780/AD human ovarian carcinoma were treated once with indicated substances and studied 24 h after the treatment.

3.3. The delivery of anticancer drug and ASO by liposomes enhances their specific activity The requirement for a drug delivery system is not only to deliver the payload to the desired location but also deliver therapeutic agents with biological activity. While free DOX possessed anticancer activity, free, non-encapsulated ASO had very limited ability to penetrate inside cancer cells, demonstrated very low toxicity and practically did not change the expression of targeted genes and proteins (data not shown). To investigate the activity of DOX and antisense oligonucleotides delivered by liposomes we carried out the following series of the experiments. In the first series of experiments, we measured the expression of targeted MDR1 and BCL2 genes in mRNA isolated from tumor tissues treated with saline (control), empty liposomes (control 2) and PEGylated liposomes loaded with DOX alone or DOX in combination with ASO targeted to mRNA encoded P-glycoprotein and BCL2 protein. The results

show that treatment with free and liposomal DOX did not significantly influence the expression of the MDR1 gene encoding P-glycoprotein (Fig. 6, bars 3 and 4). Liposomal ASO targeted to MDR1 mRNA significantly suppressed the expression of the targeted gene. In contrast to P-glycoprotein, treatment with free and liposomal DOX led to significant overexpression of the gene encoding BCL2 protein (Fig. 6, bars 3 and 4). Treatment of mice bearing human tumor xenografts with PEGylated liposomes loaded with DOX in combination with ASO targeted to BCL2 mRNA decreased the expression of BCL2 protein back to the normal level. Immunohistochemical analysis of P-glycoprotein and BCL2 protein (Fig. 7) confirms the gene expression data. It shows that ASO targeted to MDR1 mRNA substantially downregulated to the control level the expression of P-glycoprotein in the treated tumor leading to the decrease in the density of color (Fig. 7—upper panel). Similarly, liposomal ASO targeted to BCL2 mRNA suppressed to the normal the expression of targeted protein which was

Fig. 8. Apoptosis induction in multidrug resistant tumor. Typical fluorescent microscopy images of tumor tissue slides labeled by TUNEL 24 h after treatment of mice bearing xenografts of multidrug resistant A2780/AD human ovarian carcinoma with saline, empty PEGylated liposomes, DOX, PEGylated liposomes containing DOX alone and in combination with ASO targeted to MDR1 and BCL2 mRNA.

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overexpressed after the treatment with free and liposomal DOX. Taken together these data show that encapsulation of ASO into liposomes substantially enhanced their specific activity and in turn resulted in the decrease of the expression of targeted genes and proteins. Analysis of cell death induction by DOX showed that encapsulation of the drug into PEGylated liposomes enhanced its ability to induce apoptosis. Such encapsulation led to the significant increase in the expression of caspases responsible for the initiation (caspase 9) and execution (caspase 3) of cell death program (Fig. 6). Activation of caspases led to the apoptosis induction in tumor tissues (Fig. 8). The data show that the encapsulation of DOX into PEGylated liposomes enhanced apoptosis induction in tumor tissues. Taken together these data indicate that liposomal encapsulation substantially enhanced the specific activity of both anticancer drug and antisense oligonucleotides. Finally this led to the increase in DOX toxicity in vitro (Fig. 9) and DOX antitumor activity in vivo (Fig. 10). 3.4. Simultaneous suppression of pump and nonpump resistance enhances toxicity and antitumor efficacy of liposomal doxorubicin Experimental data show that simultaneous suppression of the expression of P-glycoprotein (pump resistance) and BCL2 protein (nonpump resistance, antiapoptotic defense) substantially enhanced the ability of DOX to induce apoptosis. Such simultaneous suppression led to a more pronounced activation of caspases (Fig. 6, bar 5) and apoptosis induction (Fig. 8) when compared with free and liposomal DOX. It also makes the combination of DOX with ASO targeted to MDR1 and BCL2 mRNA in one liposomal delivery system more toxic in vitro for multidrug resistant cancer cells (Fig. 9, curve 5) when compared with free and liposomal DOX (Fig. 9, curves 1 and 2

Fig. 9. Cytotoxicity of DOX and different liposomal formulations in multidrug resistant A2870/AD human ovarian carcinoma cells. Averaged viability curves are presented. 1—DOX, 2—PEG-Lip DOX, 3—PEG-Lip DOX + BCL2 ASO, 4—PEG-Lip DOX + MDR1 ASO, 5—PEG-Lip DOX + MDR1 and BCL2 ASO.

Fig. 10. Effect of treatment of mice bearing xenografts of human multidrug resistant A2780/AD ovarian carcinoma with saline (1), empty liposomes (2), DOX (3), PEGylated liposomes loaded with DOX (4) and PEGylated liposomes loaded with DOX and ASO targeted to MDR1 and BCL2 mRNA (5) on the size of multidrug resistant tumor.

respectively) and liposomal DOX combined only with one type of ASO (Fig. 9, curves 3 and 4). Simultaneous suppression of pump and nonpump resistance also enhances antitumor activity of DOX. Data in Fig. 10 clearly show that liposomal DOX combined with ASO targeted to MDR1 and BCL2 mRNA suppress tumor growth more effectively (Fig. 10, bars 5) than free (Fig.10, bars 3) and liposomal (Fig. 10, bars 4) DOX. 4. Discussion The specific aims of the present investigation were (1) to show that conventional and PEGylated liposomes can penetrate cancer cells in vitro and tumor cells in vivo; (2) to demonstrate that liposomes can be successfully used both for cytoplasmic and nuclear delivery of therapeutics, including anticancer drugs and antisense oligonucleotides; (3) to examine the specific activity of anticancer drugs and nucleotides delivered inside tumor cells by PEGylated liposomes; and (4) to confirm that simultaneous inhibition of pump and nonpump resistance of multidrug resistant tumors by liposomal ASO can substantially enhance an antitumor activity of traditional well established anticancer drugs. It is generally believed that liposomes, similarly to other macromolecules, are passively accumulated in tumors due to the so-called Enhanced Penetration and Retention (EPR) effect [22,23]. After penetration into the tumor interstitium from the circulation through open fenestrations [24], liposomes are trapped in the tumor tissues because of limited lymphatic drainage. The further destiny of liposomes inside the tumor is still unclear. It is poorly understood as to whether liposomes release their payload without direct penetration into the tumor cells or they are internalized by the cells and deliver encapsulated drugs into the cytoplasm or nuclei. Our present experimental data showed that small (100–200 nm) unilamellar conventional liposomes with fluid membrane as well as PEGylated stealth liposomes can be internalized into cancer

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cells in vitro and into tumor cells in vivo and can even penetrate cellular nuclei. A mechanism which allows liposomes to enter the nucleus still remains unknown. One possible explanation might be that fluorescent and transmission electron microscopy labels used (FITC-labeled antisense oligonucleotides, rhodamine and osmium tetroxide) and an anticancer drug (DOX) might be toxic and could increase permeability of the nuclear membrane which allows liposomes to penetrate into the nucleus. Other possibilities of nuclear translocation of liposomes require either the disassembly of the nuclear envelope or active nuclear transport via the nuclear pore complex or by specific “nuclear import receptors” [25–29]. Most investigators believe that tumor cells internalize liposomes by endocytosis and certain membrane protein and receptors (low and high density lipoprotein receptors, apolipoproteins, etc.) are involved in this process. Membrane fusion mechanisms are also taken into consideration [26,30–34]. According to the theory of membrane fusion interaction [35,36] one expects that after the fusion of the liposomal membrane with cellular plasma membrane, liposomal payload is released into the cytoplasm in close proximity to the plasma membrane. We observed such accumulation of free DOX released from the liposomes near the plasma membrane of cancer cells at the first minutes after the addition of liposomal DOX into the cell incubation media. Therefore, it seems likely that fusion mechanism prevailed at the early stages of the liposome–cancer cell interaction. After endocytosis, external macromolecules, including liposomes, are transported in membrane-limited organelles and released in the perinuclear region [8,11,37,38]. We observed extensive release and accumulation of DOX in the cellular compartments adjacent to the nucleus and in the nuclei themselves at the late stages of liposomal interaction with cancer cells. In addition, we did not find substantial differences in the intracellular distribution of labeled liposomes in different cellular layers from the top to the bottom of a cell. Taken together these experimental results suggest that both mechanisms, membrane fusion and endocytosis, can be involved in the internalization of liposome-encapsulated drugs by tumor cells. Although detailed mechanisms of such interactions require further investigations, the present data show that PEGylated liposomes are able to penetrate into tumor cells after systemic administration and provide for an effective cytoplasmic and nuclear delivery of encapsulated anticancer drugs and DNA fragments into tumor cells. Moreover PEGylated liposomes as non-viral vectors are capable of offering a high degree of nuclear transfection and liposomal ASO might be used for the effective suppression of targeted proteins. Analysis of specific activity of different types of biologically active molecules after their systemic transport and final release inside tumor cells show that liposomal delivery enhances the activity. Liposomal DOX was more effective when compared with its free form and liposomal ASO effectively inhibited the expression of targeted mRNA and proteins, while free ASO in solution did not display such ability. As a result apoptosis induction was more pronounced after the action of liposomal DOX than that of the free drug. While the enhancement of ASO activity can be explained by an increase in bioavailability of ASO

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after incorporation into liposomes, such simple explanation does not work in case of high water-soluble DOX. Moreover, it was found that the incorporation of DOX inside PEGylated liposomes decreased its availability for tumor cells [39]. One can speculate that similar to HPMA copolymer-bound DOX [15,21,40,41], incorporated into liposomes DOX is transferred inside cells in membrane-limited organelles. This protects DOX and substantially limits drug degradation by intracellular enzymes and environment and therefore preserves its anticancer activity. Our present experimental data clearly show that DOX released from liposomes into nuclei is more active as a cell death inducer when compared with free drug. This in turn leads to the enhancement in the toxicity of DOX after the incorporation into liposomes and provides for higher tumor growth suppressive ability of liposomal DOX when compared with free DOX. The last, but not least, aim of the present investigation included experimental support of the proposed idea about the simultaneous suppression of both pump and nonpump resistance in order to enhance the efficacy of traditional anticancer drugs [7–11,14,42–44]. Liposomal carriers provide for a relatively easy way of combining in one drug delivery system an anticancer drug with suppressors of pump and nonpump resistance. In the present study we used DOX as a model of a traditional effective anticancer drug and ASO targeted to MDR1 and BCL2 mRNA as suppressors of pump and nonpump resistance respectively. The results of the application of such complex liposomal delivery system to cancer cells in vitro and as a systemic treatment in vivo clearly support the concept. We found that concurrent suppression of P-glycoprotein drug efflux pump (the main cause of pump resistance in multidrug resistant A2780/AD human ovarian carcinoma cells) and BCL2 protein (the key player in cellular antiapoptotic defense, i.e., nonpump resistance in most cancer cells) led to an additional activation of caspase dependent apoptotic pathway and enhanced apoptosis induction by the anticancer drug—DOX. This enhancement led to the dramatic increase in the toxicity in vitro and tumor suppression activity in vivo of liposomal DOX. Taken together all results of the present investigation clearly suggest that liposomal drug delivery system containing an anticancer drug as an apoptosis inducer in combination with antisense oligonucleotides targeted to MDR1 and BCL2 mRNA as suppressors of pump and nonpump resistance respectively has a high potential as a novel antitumor therapeutic agent. Acknowledgement The research was supported by NIH Grant CA111766 from the National Cancer Institute. References [1] P. Goyal, K. Goyal, S.G. Vijaya Kumar, A. Singh, O.P. Katare, D.N. Mishra, Liposomal drug delivery systems—clinical applications, Acta Pharm. 55 (1) (2005) 1–25. [2] D.E. Hernandez-Morales, A.E. Hernandez-Zaccaro, Gastrointestinal and cutaneous AIDS-related Kaposi's sarcoma: different activity of liposomal doxorubicin according to location of lesions, Eur. J. Cancer Care (Engl.) 14 (3) (2005) 264–266.

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[3] R.D. Hofheinz, S.U. Gnad-Vogt, U. Beyer, A. Hochhaus, Liposomal encapsulated anti-cancer drugs, Anticancer Drugs 16 (7) (2005) 691–707. [4] M. Lichterfeld, N. Qurishi, C. Hoffmann, B. Hochdorfer, N.H. Brockmeyer, K. Arasteh, S. Mauss, J.K. Rockstroh, Treatment of HIV-1associated Kaposi's sarcoma with PEGylated liposomal doxorubicin and HAART simultaneously induces effective tumor remission and CD4+ T cell recovery, Infection 33 (3) (2005) 140–147. [5] J.M. Siehl, E. Thiel, A. Schmittel, G. Hutter, P.M. Deckert, H. Szelenyi, U. Keilholz, Ifosfamide/liposomal daunorubicin is a well tolerated and active first-line chemotherapy regimen in advanced soft tissue sarcoma: results of a phase II study, Cancer 104 (3) (2005) 611–617. [6] V.P. Torchilin, Recent advances with liposomes as pharmaceutical carriers, Nat. Rev. Drug Discov. 4 (2) (2005) 145–160. [7] T. Minko, S.S. Dharap, A.T. Fabbricatore, Enhancing the efficacy of chemotherapeutic drugs by the suppression of antiapoptotic cellular defense, Cancer Detect Prev. 27 (3) (2003) 193–202. [8] T. Minko, S.S. Dharap, R.I. Pakunlu, Y. Wang, Molecular targeting of drug delivery systems to cancer, Curr. Drug Targets 5 (4) (2004) 389–406. [9] S.S. Dharap, B. Qiu, G.C. Williams, P. Sinko, S. Stein, T. Minko, Molecular targeting of drug delivery systems to ovarian cancer by BH3 and LHRH peptides, J. Control. Release 91 (1–2) (2003) 61–73. [10] R.I. Pakunlu, T.J. Cook, T. Minko, Simultaneous modulation of multidrug resistance and antiapoptotic cellular defense by MDR1 and BCL-2 targeted antisense oligonucleotides enhances the anticancer efficacy of doxorubicin, Pharm. Res. 20 (3) (2003) 351–359. [11] R.I. Pakunlu, Y. Wang, W. Tsao, V. Pozharov, T.J. Cook, T. Minko, Enhancement of the efficacy of chemotherapy for lung cancer by simultaneous suppression of multidrug resistance and antiapoptotic cellular defense: novel multicomponent delivery system, Cancer Res. 64 (17) (2004) 6214–6224. [12] M. Konopleva, A.M. Tari, Z. Estrov, D. Harris, Z. Xie, S. Zhao, G. LopezBerestein, M. Andreeff, Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosinearabinoside, and induce apoptosis independent of other antiapoptotic proteins, Blood 95 (12) (2000) 3929–3938. [13] S. Motomura, T. Motoji, M. Takanashi, Y.H. Wang, H. Shiozaki, I. Sugawara, E. Aikawa, A. Tomida, T. Tsuruo, N. Kanda, H. Mizoguchi, Inhibition of P-glycoprotein and recovery of drug sensitivity of human acute leukemic blast cells by multidrug resistance gene (mdr1) antisense oligonucleotides, Blood 91 (9) (1998) 3163–3171. [14] S.S. Dharap, Y. Wang, P. Chandna, J.J. Khandare, B. Qiu, S. Gunaseelan, S. Stein, A. Farmanfarmaian, T. Minko, Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide, Proc. Natl. Acad. Sci. U. S. A. 102 (36) (2005) 12962–12967. [15] T. Minko, P. Kopeckova, J. Kopecek, Efficacy of the chemotherapeutic action of HPMA copolymer-bound doxorubicin in a solid tumor model of ovarian carcinoma, Int. J. Cancer 86 (1) (2000) 108–117. [16] S.S. Dharap, P. Chandna, Y. Wang, J.J. Khandare, B. Qiu, S. Stein, T. Minko, Molecular targeting of BCL2 and BCLXL proteins by synthetic BH3 peptide enhances the efficacy of chemotherapy, J. Pharmacol. Exp. Ther. 316 (3) (2006) 992–998. [17] T. Minko, P. Kopeckova, J. Kopecek, Chronic exposure to HPMA copolymerbound adriamycin does not induce multidrug resistance in a human ovarian carcinoma cell line, J. Control. Release 59 (2) (1999) 133–148. [18] J.J. Bazzola, L.D. Russel, Principal and Techniques for Biologists, Electron Microscopy, Jones and Bartlett Publishers, Boston, 1991. [19] M. Hayt, Fixation for Electron Microscopy, Academic Press, New York, 1981. [20] J. Kopecek, P. Kopeckova, T. Minko, Z. Lu, HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action, Eur. J. Pharm. Biopharm. 50 (1) (2000) 61–81. [21] T. Minko, P. Kopeckova, V. Pozharov, K.D. Jensen, J. Kopecek, The influence of cytotoxicity of macromolecules and of VEGF gene modulated vascular permeability on the enhanced permeability and retention effect in resistant solid tumors, Pharm. Res. 17 (5) (2000) 505–514.

[22] J. Fang, T. Sawa, H. Maeda, Factors and mechanism of “EPR” effect and the enhanced antitumor effects of macromolecular drugs including SMANCS, Adv. Exp. Med. Biol. 519 (2003) 29–49. [23] H. Maeda, The enhanced permeability and retention (EPR) effect in tumor vasculature: the key role of tumor-selective macromolecular drug targeting, Adv. Enzyme Regul. 41 (2001) 189–207. [24] J.A. Kamps, G.L. Scherphof, Receptor versus non-receptor mediated clearance of liposomes, Adv. Drug Deliv. Rev. 32 (1–2) (1998) 81–97. [25] B. Alberts, D. Bray, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, Essential Cell Biology, Garland Publishing, Inc., New York, 1998. [26] D. Lechardeur, G.L. Lukacs, Intracellular barriers to non-viral gene transfer, Curr. Gene Ther. 2 (2) (2002) 183–194. [27] S. Jakel, W. Albig, U. Kutay, F.R. Bischoff, K. Schwamborn, D. Doenecke, D. Gorlich, The importin beta/importin 7 heterodimer is a functional nuclear import receptor for histone H1, EMBO J. 18 (9) (1999) 2411–2423. [28] G. Jenster, J. Trapman, A.O. Brinkmann, Nuclear import of the human androgen receptor, Biochem. J. 293 (Pt 3) (1993) 761–768. [29] K.S. Ullman, M.A. Powers, D.J. Forbes, Nuclear export receptors: from importin to exportin, Cell 90 (6) (1997) 967–970. [30] M.R. Niesman, G.A. Peyman, M.V. Miceli, Liposome uptake by human retinal pigment epithelial cells in culture, Curr. Eye Res. 16 (11) (1997) 1073–1080. [31] I.S. Zuhorn, D. Hoekstra, On the mechanism of cationic amphiphilemediated transfection. To fuse or not to fuse: is that the question? J. Membr. Biol. 189 (3) (2002) 167–179. [32] H. Harashima, Y. Shinohara, H. Kiwada, Intracellular control of gene trafficking using liposomes as drug carriers, Eur. J. Pharm. Sci. 13 (1) (2001) 85–89. [33] M.R. Almofti, H. Harashima, Y. Shinohara, A. Almofti, Y. Baba, H. Kiwada, Cationic liposome-mediated gene delivery: biophysical study and mechanism of internalization, Arch. Biochem. Biophys. 410 (2) (2003) 246–253. [34] A. Lakkaraju, Y.E. Rahman, J.M. Dubinsky, Low-density lipoprotein receptor-related protein mediates the endocytosis of anionic liposomes in neurons, J. Biol. Chem. 277 (17) (2002) 15085–15092. [35] G. Knoll, K.N. Burger, R. Bron, G. van Meer, A.J. Verkleij, Fusion of liposomes with the plasma membrane of epithelial cells: fate of incorporated lipids as followed by freeze fracture and autoradiography of plastic sections, J. Cell Biol. 107 (6 Pt 2) (1988) 2511–2521. [36] J.H. Prestegard, M.P. O'Brien, Membrane and vesicle fusion, Annu. Rev. Phys. Chem. 38 (1) (1987) 383–411. [37] Y. Wang, R.I. Pakunlu, W. Tsao, V. Pozharov, T. Minko, Bimodal effect of hypoxia in cancer: the role of hypoxia inducible factor in apoptosis, Mol. Pharmacol. 1 (2004) 156–165. [38] Y. Wang, T. Minko, A novel cancer therapy: combined liposomal hypoxia inducible factor 1 alpha antisense oligonucleotides and an anticancer drug, Biochem. Pharmacol. 68 (10) (2004) 2031–2042. [39] K.M. Laginha, S. Verwoert, G.J. Charrois, T.M. Allen, Determination of doxorubicin levels in whole tumor and tumor nuclei in murine breast cancer tumors, Clin. Cancer Res. 11 (19 Pt 1) (2005) 6944–6949. [40] T. Minko, P. Kopeckova, J. Kopecek, Comparison of the anticancer effect of free and HPMA copolymer-bound adriamycin in human ovarian carcinoma cells, Pharm. Res. 16 (7) (1999) 986–996. [41] T. Minko, P. Kopeckova, J. Kopecek, Preliminary evaluation of caspasesdependent apoptosis signaling pathways of free and HPMA copolymerbound doxorubicin in human ovarian carcinoma cells, J. Control. Release 71 (3) (2001) 227–237. [42] T. Minko, Drug targeting to the colon with lectins and neoglycoconjugates, Adv. Drug Deliv. Rev. 56 (4) (2004) 491–509. [43] T. Minko, S.S. Dharap, S. Gunaseelan, S. Stein, P.J. Sinko, R.I. Pakunlu, Y. Wang, B. Qiu, Targeted proapoptotic anticancer drug delivery system, 31 International Symposium on Controlled Release of Bioactive Materials, Honolulu, Hawaii, 2004, p. 108. [44] T. Minko, Soluble polymer conjugates for drug delivery, Drug Discov. Today 2 (2005) 15–20.