Potential of pH-Sensitive Polymer-Anchored Cationic Liposomes for Combinatorial Anticancer Therapy with Doxorubicin and siRNA

J. DRUG DEL. SCI. TECH., 24 (1) 27-32 2014

Potential of pH-sensitive polymer-anchored cationic liposomes for combinatorial anticancer therapy with doxorubicin and siRNA U.-H. Jeong1, V.K. Garripelli2, S. Jo2, C.-S. Myung1, S.-J. Hwang3, J.-K. Kim4*, J.-S. Park1* College of Pharmacy and Institute of Drug Research and Development, Chungnam National University, Daejeon, Republic of Korea 2 Department of Pharmaceutics, School of Pharmacy, The University of Mississippi, Oxford, MS, USA 3 College of Pharmacy, Yonsei University, Incheon, Republic of Korea 4 College of Pharmacy and Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan Gyeonggi, Republic of Korea *Correspondence: [email protected], [email protected] 1

To explore a potential of pH-sensitive polymer-liposome complexes for the tumor-specific combinatorial delivery of anticancer agents and siRNA, conventional liposomes (ConL), polymer-liposome complexes (PLC) and polymer-cationic liposome complexes (PCLC) were prepared. Pluronic P104-based multiblock copolymer (MBCP-2) was included as pH-sensitive polymer. Physicochemical properties, release under different pH, cytotoxicity and in vitro cellular uptake of DOX-loaded liposomes were investigated. From the release test, an acidic pH was determined to be an important factor for release from the PLC vehicles. The novel PLC vehicle itself showed low cytotoxicity demonstrating suitable viability. Observing cellular uptake of DOX by confocal microscopy imaging, a greater amount of DOX was delivered to cells with the pH-sensitive polymeranchored vehicles than that with free DOX and ConL. It was verified that the novel vehicles could effectively deliver both DOX and GFP-siRNA. Novel pH-sensitive PCLC have a potential for targeted therapy of anticancer agents and gene therapy under acidic tumor microenvironment. Key words: pH-sensitive polymer – Cationic liposomes – Complexes – siRNA – Doxorubicin.

Doxorubicin (DOX), a DNA-intercalating anthracycline antibiotic, has been used widely as a chemotherapy drug for various cancers including leukemias, breast, liver, and lung. However, chemotherapy has many obstacles such as toxicity to normal cells. To overcome this difficulty, the accurate targeting to cancer cells and the modified release of drugs have been investigated. Accurate cancer cell targeting demands specific requirements, such as acidic conditions [1]. Therefore, acidic-sensitive vehicles are suitable for this approach [2, 3]. Another alternative approach for cancer therapy is gene therapy using small interfering RNA (siRNA), small hairpin RNA (shRNA), or plasmid DNA. In particular, siRNA can initiate RNA interference (RNAi), which produces a gene silencing effect by causing downregulation of the targeted mRNA [4]. Usually associated with multidrug resistance or apoptosis of cancer cells, RNAi, mediated by siRNA, has emerged as one of the most advanced and versatile tools for biological research, as well as one of the most promising therapeutic strategies for various human diseases such as viral infections, genetic diseases, cardiovascular disorders, and cancers. In terms of therapeutics, combinational therapies for cancer have been more effective than single therapies. Co-delivery systems, that could simultaneously deliver drugs and siRNA to the same cells in vitro and in vivo have been proposed to inhibit gene expression or to achieve the synergistic/combined effect of drug and gene therapies [5-9]. Various delivery systems, either viral or nonviral, have been investigated for siRNA delivery in vitro and in vivo. Recently, nonviral vectors such as cationic polymers have received growing interest due to their many advantages over their viral counterparts, including ease of production, improved safety, and low immune responses that enable repeated use. Among various delivery systems, liposomal formulations have been developed for modified release of drugs. In the case of DOX, several products including Myocet, Caelyx (EU), and Doxil (USA) are commercially available. Recent advances in liposome chemistry have resulted in various types of stimulus-responsive polymer-containing liposomes. These liposomes are prepared by simply adding a stimulusresponsive polymer to the liposome dispersion or by mixing lipids and

polymers during the preparation of vesicles [10]. Stimuli-responsive liposomes, especially those sensitive to changes in pH, are attractive for the cytoplasmic delivery of polar drugs because they can be readily internalized by cells. Such liposomes are stably internalized by cells mainly via an endocytic pathway, and they are destabilized at low pH (~5) in the endosome; hence, the drugs can be easily released into the cytoplasm [1, 11]. Stimulus-induced conformational changes are thought to allow the release of drugs at low pH (endosomes) and thus increase the cellular uptake of water-soluble compounds. For this purpose, novel polymer, Pluronic P104-based multiblock copolymer (MBCP-2), was applied as a pH-sensitive polymer that would selectively degrade under locally acidic physiological conditions [2]. A class of thermosensitive biodegradable multiblock copolymers with acid-labile acetal linkages was synthesized from Pluronic triblock copolymers (Pluronic P85 and P104) and di-(ethylene glycol) divinyl ether [2]. In this study, we combined two approaches: the pH-sensitive delivery of DOX and the cationic liposomal delivery of siRNA. We investigated the pH-sensitive properties of polymer-liposome complexes (PLC) attributable to the pH-sensitive polymer and the cationic lipids that allow the delivery of siRNA. The potential for cancer therapy using polymer-cationic liposome complexes (PCLC) as novel therapy-delivery vehicles was investigated.

I. MATERIALS AND METHODS 1. Materials

Egg yolk phosphatidylcholine (EPC), cholesterol (CHOL), and 1,2-dioleoyl-3- trimethylammonium-propane (DOTAP) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Multi-block copolymer (MBCP-2) from triblock copolymer PEO-PPO-PEO (Pluronic P104) was supplied by the School of Pharmacy, the University of Mississippi (Oxford, MS, USA) [3]. DOX-HCl, ammonium phosphate dibasic [(NH4)2HPO4], chloroform (CH3Cl), and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). GFP siRNA (sense, GCA UCA AGG UGA ACU UCA A(dTdT); antisense, UUG AAG UUC 27

Potential of pH-sensitive polymer-anchored cationic liposomes for combinatorial anticancer therapy with doxorubicin and siRNA U.-H. Jeong, V.K. Garripelli, S. Jo, C.-S. Myung, S.-J. Hwang, J.-K. Kim, J.-S. Park

J. DRUG DEL. SCI. TECH., 24 (1) 27-32 2014

ACC UUG AUG C(dTdT)) and Bcl-2 siRNA (sense, CUC UGU GGA UGA CUG AGU A(dTdT); antisense, UAC UCA GUC AUC CAC AGA G(dTdT)) were obtained from Bioneer Co. (Daejeon, Korea). Sephadex G-50 spin-columns were purchased from Geneaid Biotech Ltd. (Taipei, Taiwan). All reagents and chemicals used were reagent grade.

The loading efficiency was determined by UV spectroscopy. The DOX concentration was measured at 495 nm by UV spectrophotometer (Mini 1240, Shimadzu, Kyoto, Japan) after lysis with Triton X-100 (final concentration 1 % v/v).

2. Cell culture

The particle sizes of the empty vehicles and DOX-loaded vehicles were determined by light scattering spectrophotometry (ELS-8000, Photal, Tokyo, Japan). The samples were diluted with deionized water, and then transferred into a quartz cuvette in an ELS-8000 dynamic light scattering instrument. The zeta pontential of the vehicles was measured with an electrophoretic light-scattering spectrophotometer. Data were analyzed using a software package (ELS-8000 software) supplied by the manufacturer.

5. Particle size and zeta potential of vehicles

The rat hepatoma cells (H4II-E), stabilized to express GFP, were a kind gift from Dr. S.K. Kim (Chungnam National University, Daejeon, Korea). Human hepatocellular carcinoma (HepG2), human cervix adenocarcinoma (HeLa), and human breast adenocarcinoma (MDAMB-231, MCF-7) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) in a humidified incubator supplied with 5 % CO2 and maintained at 37 °C for 24 h. All media were supplemented with 10 % heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin (Gibco).

6. In vitro release test

To investigate pH-dependent release patterns of DOX from vehicles, in vitro release study was performed under two pH conditions. After 100 μL of DOX-loaded vehicles (100 nmole DOX) was added to 96well plates containing 100 μL of two types of buffers: acetate buffer (pH 5.0) and phosphate-buffered saline (PBS, pH 7.4), the plate was stored at 37 °C, with shaking at 100 rpm (n = 3). At the designated time, the samples were withdrawn from the well; the sampled volume was replaced with acetate buffer (pH 5.0) or PBS (pH 7.4). The released DOX was separated from DOX-loaded liposome formulations by ultrafiltration method using Amicon Ultra (3 kDa). To break down the ConL, PLC, and PCLC, a Triton X-100 solution (5 %, v/v) was added to each well. The released DOX was determined with a fluorometer (LS 55, PerkinElmer Inc., San Jose, CA, USA) with excitation at 488 nm and emission at 580 nm [15].

3. Preparation of conventional liposomes and polymer-liposome complexes

Conventional liposomes (ConL), polymer-liposome complexes (PLC), and polymer-cationic liposome complexes (PCLC) were prepared by the lipid film method [12]. All lipids and MBCP were dissolved in chloroform at the ratio of lipid as shown in Table I. The organic mixture was removed using a rotary evaporation under reduced pressure with the temperature of the water bath adjusted to 40 °C. Prior to hydration, the morphology of lipid film was observed. One milliliter of 300 mM (NH4)2HPO4 (pH 7.4) was added to this lipid film and hydrated by vigorous vortexing. The resulting suspension was sonicated for 60 min at 37 °C and was passed 10 times through an extruder (Northern Lipids Inc., Vancouver, BC, Canada) equipped with double-layered 0.2-μm Nucleopore polycarbonate membrane filters (Whatman, Clifton, NJ, USA).

7. Gel retardation assay

To compare the complex formation between vehicles and siRNA, different amounts of ConL and PCLC were added to 10 pmole of GFPsiRNA. The mixture was incubated for 30 min at room temperature with occasional pipetting. After incubation, the complexes were electrophoresed on agarose gel (2 %) and visualized by ethidium bromide staining.

4. Loading of doxorubicin by ion gradient

To evaluate the possibility of using the polymer-coated liposomes to co-deliver a drug and siRNA, DOX was encapsulated as a model drug in the pH-sensitive polymer-coated liposomes by transmembrane ammonium-phosphate ion gradient [13, 14]. A Sephadex G-50 spin-column was used to exchange the outer (NH4)2HPO4 for 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)buffered saline (HBS, pH 7.4). DOX dissolved in HBS (8.5 mg/mL) was added to empty vehicles at a drug-to-lipid ratio of 1:4 (w/w) and incubated at 4 °C for 12 h with occasional mixing. Size-exclusion chromatography was performed using a Sephadex G-50 spin-column again to purify the liposomes and remove the free DOX, according to manufacturer’s purification protocol.

8. Confocal laser scanning microscopy

To visualize the co-delivery of DOX and GFP-siRNA, confocal laser-scanning microscopy was applied. H4ll-E cells were seeded and grown on 22 × 22 mm coverslips placed in 6-well plates at a density of 5 × 105 cells/well. The cells were transfected with siRNA in naked form, complexed with Lipofectamine or DOX-loaded PCLC in basal DMEM. Simultaneously, DOX loaded in ConL or PCLC was delivered. Control cells were untreated and maintained in basal DMEM. At 3 and 24 h post-transfection, the cells were rinsed with PBS and fixed using 4 % paraformaldyde in PBS for 10 min. Following a second rinsing procedure, two drops of mounting solution (Fluoromount aqueous mounting medium, Sigma-Aldrich) were added between the coverslips and the slide glass. Then, the cells were observed under a confocal microscope (Leica TCS NT, Leica Microsystems, Wetzlar, Germany) equipped with a argon laser and associated filters for simultaneous 488 nm excitation.

Table I - Composition of conventional liposomes and polymer-liposome complexes (per mL). Component

ConLa

PLCb-5*

Formulation PLCb-10*

PLCc-5*

PLCc-10*

EPCd CHOLd DOTAPd MBCPe Totald

14 6 20

14 6 5 20

14 6 10 20

14 6 4 5 24

14 6 4 10 24

9. Cytotoxicity

The cytotoxicity of empty vehicles and various DOX-loaded vehicles was determined in HepG2, H4II-E, HeLa, MDA-MB-231, and MCF-7 cells. The cell lines were transferred from a 100-mm cell culture plate into a 96-well plate at a density of 1 × 104 cells per well, except HeLa cells, which were plated at 5 × 103 cells per well. After overnight incubation at 37 °C, the cells were exposed to 200 μL of Bcl2 siRNA, DOX-loaded vehicles, or empty vehicles containing media for 24 h. Quantitatively, 100 pmole or 200 pmole of DOX was treated

Conventional liposome consists of EPC:CHOL = 7:3. Polymerliposome complexes (PLC) were prepared using the molar ratio EPC:CHOL = 7:3 with small amounts of MBCP added. cPolymercationic liposome complexes (PCLC) were prepared using the molar ratio EPC:CHOL:DOTAP = 7:3:2 with small amounts of MBCP added. d µmole. ew/w %. *The numbers 5 and 10 indicate the percentage of MBCP added in formulation. a

b

28

Potential of pH-sensitive polymer-anchored cationic liposomes for combinatorial anticancer therapy with doxorubicin and siRNA U.-H. Jeong, V.K. Garripelli, S. Jo, C.-S. Myung, S.-J. Hwang, J.-K. Kim, J.-S. Park

and 10 pmole or 20 pmole of Bcl-2 siRNA was applied to the cells. The medium was then removed and 100 μL MTT-containing medium (5 mg/mL) was added to the wells. Following 4 h incubation at 37 °C, the MTT-containing medium was carefully aspirated to avoid disturbing any formazan crystals formed, and 100 μL MTT solubilization solution was added to each well. Plates were incubated at room temperature for 30 min and optical densities were determined at 570 nm using a microplate reader (Sunrise, Tecan Trading, Männedorf, Switzerland). Cell viability was expressed as a percentage of the untreated control cells.

J. DRUG DEL. SCI. TECH., 24 (1) 27-32 2014

3. Morphology of lipid films

Most lipid mixture solutions formed a clear and homogeneous film, but PLC and PCLC containing 10 % MBCP showed a different fishnet-patterned heterogeneous film. These phenomena affected the yield of the liposome solutions. After polycarbonate film filtration, the solutions containing 10 % MBCP appeared clearer to the naked eye than other solutions containing lower MBCP or no MBCP, suggesting lipid loss had occured. Cho et al. [16] found that the addition of the polymer does not perturb the bilayer structure or induce a transition to any other type of structure. They also suggested that the polymerliposome complexes have the same bilayer/vesicle structure as polymer-free PC:CHOL liposomes.

10. Statistical analysis

Statistical analysis of the data was performed using Student’s t-test or one-way analysis of variance (ANOVA). A p value of < 0.05 was considered significant. All data are expressed as the mean ± standard deviation of the mean (SD) from three independent experiments.

4. In vitro release

In vitro release study was performed to evalulate the pH-dependent release from at 37 °C under two experimental condition, pH 5.0 and pH 7.4; the release patterns of the formulations under these conditions are illustrated in Figure 1. At the first measured time point, burst release appeared in all formulations, which could be observed when a sustained releasing agent such as chitosan is not used in the lipid formulations [18]. At pH 7.4, the release patterns of all formulations showed no significant difference for 4 h (Figure 1b). A small increase in DOX release was observed at 37 °C compared to at 4 °C (data not shown) and no dramatic release was observed because the amount of MBCP added to formulations was not changed considerably. In other words, the formulations were relatively stable under physiological pH. In contrast, the release at pH 5.0 showed different patterns from those at pH 7.4 (Figure 1a). At 37  °C, most formulations showed fast release of internalized DOX for 4 h. As PLC contained more MBCP, the release was faster under acidic pH. In particular, PLC containing 5 and 10 % MBCP showed the fastest release of DOX, and most of the DOX was released from PLC formulations in 4 h. A slightly lower percentage of DOX was released from PCLC than from PLC. The results indicate that acidic pH is crucial factors in the drug release from novel vehicles.

II. RESULTS AND DISCUSSION 1. Particle size and zeta potential

The particle size and zeta potentials of ConL, PLC, and PCLC were determined by dynamic light scattering (Table II). The mean diameter of ConL was 208.4 ± 2.5 nm. Depending on the content of MBCP (5 or 10 %), the addition of MBCP to liposome formulations reduced the mean diameters of PLC to 163.8 ± 3.3 and 165.5 ± 2.2 nm, respectively. Including cationic lipids in the liposome formulation resulted in slight increases in the mean diameters of PCLC as 172.7 ± 2.8 and 186.2 ± 4.2 nm by the content of MBCP (5 or 10 %), respectivley. Compared to ConL, polymer-liposome complexes showed smaller sizes [16]. The mean diameters of PLC containing MBCP were about 20 % smaller than ConL. Although the particle size of PCLC containing 10 % MBCP was larger than PLC, it was still smaller than ConL. As the concentration of MBCP increased, the particle size of PLC and PCLC increased slightly, corresponding to results from a previous study [17]. The addition of the cationic lipid, DOTAP, caused an increase in particle size, but conferred cationic properties to the vehicles, resulting in a value of +35 mV. This value was sufficient for complexing with anionic siRNA [12].

100

100

(a)

80

DOX release (%)

The loading efficiency of DOX in vehicles was determined by UV-spectroscopy at a wavelength of 495 nm. Regardless of the type of vehicle, the encapsulation efficiency was over 90 %. These results correspond to previous research using remote loading by ion gradients [13]. To achieve a high loading efficiency of DOX, having both isotonicity and equal pH values between the outer and inner phases of the liposomes is important. The remote loading of a weak-base drug using ion gradients or pH gradients has been described in previous studies [14]. Using ammonium citrate and ammonium sulfate gradients, similar methods for loading DOX into liposomes have been applied in the formation of various commercial formulations of liposomal DOX, including Myocet, Caelyx, and Doxil.

ConL PLC-5 PLC-10 PCLC-5 PCLC-10

80

DOX release (%)

2. Encapsulation efficiency of doxorubicin

(b)

60

40

20 ConL PLC-5 PLC-10 PCLC-5 PCLC-10

0

0

1

2

3

60

40

20

4

Time (h)

0 0

1

2

3

4

Time (h)

Figure 1 - In vitro pH-dependent release of doxorubicin (DOX) from DOX-loaded formulations at 37 °C in (a) pH 5.0 and (b) pH 7.4 (n = 3).

Table II - Various physicochemical properties of vehicles (n = 3). Type of vehicle

Particle size (nm)

Polydispersity index

Zeta potential (mV)

Encapsulation efficiency (%)

Lipid film morphology

ConLa PLCb-5* PLCb-10* PCLCc-5* PCLCc-10*

208.4 ± 2.5 163.8 ± 3.3 165.5 ± 2.2 172.7 ± 2.8 186.2 ± 4.2

0.161 0.135 0.141 0.136 0.152

-6.77 ± 3.36 5.21 ± 0.46 5.72 ± 0.17 36.33 ± 4.09 35.77 ± 1.25

91.8 93.5 94.8 90.5 91.3

Homogeneous Homogeneous Heterogeneous Homogeneous Heterogeneous

a Conventional liposome. bPolymer-liposome complexes. cPolymer-cationic liposome complexes. *The numbers 5 and 10 indicate the percentage (w/w) of MBCP added in formulation.

29

J. DRUG DEL. SCI. TECH., 24 (1) 27-32 2014

Potential of pH-sensitive polymer-anchored cationic liposomes for combinatorial anticancer therapy with doxorubicin and siRNA U.-H. Jeong, V.K. Garripelli, S. Jo, C.-S. Myung, S.-J. Hwang, J.-K. Kim, J.-S. Park

Lipid membranes are partially destroyed at low pH, causing release of the drugs inside the vesicles and resulting in enhanced cytoplasmic drug delivery [19]. Here, MBCP-2 was included as pH-sensitive polymer, which has thermosensitivity and pH-dependency [3]. However, since our formulations included low content of MBCP-2 such as 5 or 10 % in the composition, it is supposed that DOX release from MBCP-2 based formulation shows pH-dependency rather than thermosensitivity although in vitro release was performed at 37 °C.

Size marker Naked (100 bp) siRNA 1:1

Ratio of vehicle to siRNA (w:w) 5:1

10:1

15:1

20:1

25:1

30:1 40:1

50:1

70:1

(a)

(b)

5. Gel retardation

To confirm that siRNA had adhered to the vehicles, gel retardation assays were performed in 2 % agarose gels (Figure 2). Because ConL did not have cationic charges, and rather showed a small anionic surface charge, they could not complex with siRNA despite the high weight ratio. Although PLC-5 shows slight positively charged zeta potential, it is thought that low positive zeta potential of PLC-5 would not be enough to form complex with siRNA easily. In contrast, PCLC formed complexes with siRNA perfectly over the weight ratio of 30:1, but these complexes faded upon the weight ratio of vehicle to siRNA decreased. Gene materials like siRNA have an anionic charge due to phosphates, and thus it can electrostatically interact and form complexes with cationic entities provided by the addition of cationic DOTAP [12].

Figure 2 - Gel retardation of siRNA complexes. The siRNA complexes were prepared using (a) conventional liposomes or (b) PCLC-5. 3h GFP

DOX

24 h Merge

GFP

DOX

Merge

(a)

(b)

6. Confocal microscopy

Confocal laser scanning microscopy was performed to observe the cellular uptake of DOX-loaded vehicles complexed with GFP-siRNA (Figure 3), and the images of H4ll-E cell were obtained at each 3 h and 24 h. In images taken at 3 h, the reduced GFP expression of H4ll-E was not observed due to the lack of sufficient time for RNA interference. In contrast, a significant decrease in green fluorescence, signifying RNA interference, was observed at 24 h. From the results, the naked GFP siRNA seems to be as efficient as complexed siRNA to silence GFP (Figure 3b). However, it is not treated with siRNA alone, but with naked siRNA and DOX. At physiological pH, DOX is protonated and positively charged which can complex with siRNA and help the cytosolic localization of siRNA. In all images, the nuclei were stained with the strong red fluorescence of DOX and condensation of the nucleus before cell death was also observed at 24 h. For a quantitative analysis of DOX in the cells, it would be more appropriate to use fluorescence microplate reader. However, our confocal microscopic studies showed that the polymer-liposome complex generally delivered more drugs into the cell than ConL or the drug solubilized in water.

(c)

(d)

(e)

Figure 3 - Cellular uptake of doxorubicin and inhibition of green fluorescence protein (GFP) expression in H4ll-E cells. Green fluorescence represents the cytoplasm and the cell shape, and red fluorescence indicates doxorubicin (DOX). The H4II-E cells were treated with (a) negative control, (b) free DOX and naked siRNA, (c) free DOX and siRNA/Lipofectamine complex, (d) DOX-loaded in conventional liposomes and siRNA/Lipofectamine complex, and (e) DOX-loaded in PCLC-5 complexed with siRNA. Scale bar, 20 μm.

7. Cytotoxicity

The carrier-induced toxicity has been another challenge for cationic lipids and polymers. In various cancer cell lines, most empty vehicles showed higher cell viability than the widely used cationic agent Lipofectamine (more than 72.3 % in all tested cell lines, Table III). Because ConL, PLC and PCLC consist of natural lipids and biocompatible polymers, they do not significantly affect cell growth. In accordance with DOX-release data, empty PLC containing 10 % MBCP showed higher cytotoxicity (over 87 % cell viability) than empty PLC containing 5 % MBCP (over 92 % cell viability). More DOTAP was added, higher cytotoxicity was observed than PLC (over 80 and 83 % cell viability when PCLC with 5 or 10 % MBCP were added, respectively). However, cell viability profiles of PCLC were still higher than observed with Lipofectamine. Moreover, since siRNA is sometimes rather toxic, it is required to perform control experiment with a siRNA that targets no gene. In our previous report [12], the cytotoxicity of intact GFP siRNA was observed. Although the DOX-susceptibility for each cell line was tested, all DOX-loaded PLC showed higher cytotoxicity than that observed with free DOX or DOX-loaded ConL in all cell lines (Table IV). All liposomal DOX shows better cytotoxicity than free DOX, which

normally happened when there is significant uptake of liposomes whether due to cationic or specific surface targeting property since the release of drug from liposomes required time. However, release of DOX from PLC or PCLC reached over 70 % within 4 h. Moreover, it is supposed that MBCP contributed to the specific surface property except pH-sensitivity. In comparing the cytotoxicity of DOX-loaded PLC and PCLC, PLC resulted in higher cytotoxicity than PCLC, but the difference was not significant. From the results, the optimum PCLC-5 formulation was selected and compared with a mixture of ConL for DOX delivery as well as Lipofectamine delivery of siRNA. To investigate the capability of co-delivering DOX and siRNA, we chose a siRNA against B-cell lymphoma 2 (Bcl-2) gene, a key gene that is overexpressed in many malignant tumors. Note that siRNAmediated silencing of Bcl-2, an anti-apoptotic gene, synergized with even small amounts of DOX was successful in inducing cancer cell death [20,21]. Thus, the combination of RNAi (RNA interference)mediated downregulation of Bcl-2 and DOX may be a reliable therapeutic model for efficient combination cancer therapy. First, DOX was loaded in the pH-sensitive cationic vehicle PCLC-5, which showed higher cytotoxicity than that loaded in ConL, regardless of 30

Potential of pH-sensitive polymer-anchored cationic liposomes for combinatorial anticancer therapy with doxorubicin and siRNA U.-H. Jeong, V.K. Garripelli, S. Jo, C.-S. Myung, S.-J. Hwang, J.-K. Kim, J.-S. Park

J. DRUG DEL. SCI. TECH., 24 (1) 27-32 2014

Table III - Cell viability (%) of blank vehicles in various cell lines (n = 3). Vehicles Lipofectamine ConLa PLCb-5* PLCb-10* PCLCc-5* PCLCc-10*

Cell viability (%) H4IIE

HeLa

HepG2

MCF-7

MDA-MB-231

75.7 ± 1.03 91.4 ± 6.10 92.2 ± 15.0 88.7 ± 3.91 92.9 ± 2.76 89.1 ± 3.37

73.6 ± 3.97 92.4 ± 8.34 95.9 ± 5.78 91.9 ± 7.80 90.8 ± 1.02 89.1 ± 1.48

74.3 ±1.18 97.3 ± 1.25 94.1 ± 6.99 102.4 ± 2.87 83.6 ± 2.84 80.3 ± 2.94

72.3 ± 3.68 99.9 ± 3.73 92.3 ± 1.26 103.1 ± 3.39 88.7 ± 3.25 94.3 ± 7.68

83.1 ±7.51 98.0 ± 10.7 96.1 ± 9.52 87.3 ± 3.70 89.8 ± 3.44 81.4 ± 3.90

a Conventional liposome. bPolymer-liposome complexes. cPolymer-cationic liposome complexes. *The numbers 5 and 10 indicate the percentage (w/w) of MBCP added in formulation.

Table IV - Cell viability (%) of DOX-loaded vehicles in various cell lines (n = 3). Vehicles Free DOX ConLa PLCb-5* PLCb-10* PCLCc-5* PCLCc-10*

Cell viability (%) H4IIE

HeLa

HepG2

MCF-7

MDA-MB-231

78.5 ± 1.33 67.6 ± 9.04 58.3 ± 5.11 50.4 ± 7.12 65.5 ± 1.10 64.0 ± 2.32

71.4 ± 2.24 57.5 ± 0.99 36.6 ± 0.58 36.8 ± 4.80 54.5 ± 1.84 50.8 ± 1.09

86.2 ± 2.97 66.8 ± 6.02 45.3 ± 3.63 46.3 ± 1.75 49.0 ± 1.72 48.1 ± 4.13

62.9 ± 3.39 48.9 ± 4.21 56.2 ± 1.88 48.2 ± 4.10 62.9 ± 7.27 44.8 ± 7.01

43.8 ± 0.28 30.5 ± 0.56 22.0 ± 15.3 15.3 ± 0.79 24.2 ± 3.22 21.4 ± 1.84

a Conventional liposome. bPolymer-liposome complexes. cPolymer-cationic liposome complexes. *The numbers 5 and 10 indicate the percentage (w/w) of MBCP added in formulation.

DOX dose. The pH-sensitive PLC mediated more cellular uptake of drugs than ConL. Meanwhile, no difference between PCLC-5 and Lipofectamine was observed in delivering siRNA. However, when cells were treated with the combinations of DOX and Bcl-2 siRNA, a synergistic effect of co-delivery of DOX and Bcl-2 by PCLC-5 occurred (Figure 4). The co-delivery of DOX further enhanced the anticancer effect of Bcl-2 siRNA from approximately 16 to 45 % for the control and about 60 % for PCLC. However, the cytotoxicity of 200 pmole DOX and 20 pmole Bcl-2 was similar between the treated groups by ConL+Lipofectamine or PCLC-5. It is suggested that an excess amount of DOX is cytotoxic enough to exclude the effect of Bcl-2 siRNA. We found that the pH-sensitive polymer-coated liposome increased the bioactive efficiency of DOX and siRNA, which was higher than when delivered separately without PCLC. Cationic liposomes are widely used for delivery of siRNA [22]. Not only can it complex with siRNA by electrostatic interaction, it also encapsulates the entity into its core and phospholipid bilayer. Therefore, many attempts have been made to deliver drugs and DNA simultaneously using liposomes [23, 24]. Moreover, the suppression of the anti-apoptotic activity of Bcl-2 by the siRNA may have made the cells more sensitive to DOX. The Bcl-2 gene is known to prevent DOX-induced apoptosis in human cancer cell-lines [25]. Therefore, downregulation of the Bcl-2 gene is an effective cancer therapy tool. The co-delivery of drugs and DNA has been proposed to enhance gene expression and to achieve the synergistic/combined drug effects and gene therapies. Paclitaxel and Bcl-2-targeted siRNA were also used as a pair to demonstrate the synergistic effect of drug and gene delivery in the same vehicle [26].

Cell viability (%)

100

ConL + Lipofectamine PLCL-5

80 60

*

*

*

40

*

20 0

DOX (pmole) 100 siRNA (pmole) −

200 − − 10

− 20

100 100 200 10 20 20

Figure 4 - Cell viability of H4II-E cells treated with doxorubicin (DOX) and Bcl-2 siRNA. DOX was loaded in ConL or PCLC-5 formulation, and Bcl-2 siRNA was complexed with Lipofectamine or DOX-loaded PCLC-5 (n = 5). *Significant difference compared to the treatments of DOX-loaded ConL or siRNA/Lipofectamine complex.

ConL. Therefore, novel PCLC could be promising vehicles for the combinational delivery of anticancer siRNA and drugs.

REFERENCES

*

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In conclusion, the PCLC had better physicochemical properties than ConL and PLC, owing to their size reduction and cationic properties. However, the addition of too much MBCP hindered the formation of the homogeneous lipid film, resulting in low yield and wasted lipid mixtures. From the results, PCLC containing 5 % MBCP provided the best results in terms of physicochemical properties and high yield. Moreover, the release pattern from PLC and PCLC showed acidsensitive characteristics that are suitable for targeting cancer cells. As demonstrated by the in vitro MTT assay, DOX-loaded PLC and PCLC resulted in higher cytotoxicity than free DOX and DOX-loaded

2.

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ACKNOWLEDGEMENTS This work was supported by Basic Science Research Program (No. 2010-0003083) and the Priority Research Centers Program (No. 20090093815) through the National Research Foundation of Korea (NRF) funded by the Korea government (Ministry of Education, Science and Technology).

MANUSCRIPT Received 6 August 2013, accepted for publication 18 September 2013.

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