International Journal of Pharmaceutics 474 (2014) 112–122

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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm

Pharmaceutical nanotechnology

Liposome-based co-delivery of siRNA and docetaxel for the synergistic treatment of lung cancer Mei-Hua Qu a,1, Rui-Fang Zeng b,1, Shi Fang c, Qiang-Sheng Dai b , He-Ping Li b , Jian-Ting Long b, * a

Department of Medicinal Oncology, The Third People’s Hospital Of Dalian, Dalian 116033, China Department of Medicinal Oncology, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China c Department of Clinical Nutrition, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou 510080, China b

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 June 2014 Received in revised form 2 August 2014 Accepted 14 August 2014 Available online 17 August 2014

Combination of more than one therapeutic strategy is the standard treatment in clinics. Co-delivery of chemotherapeutic drug and small interfering RNA (siRNA) within a nanoparticulate system will suppress the tumor growth. In the present study, docetaxel (DTX) and BCL-2 siRNA was incorporated in a PEGylated liposome to systemically deliver in a lung cancer model (A549). The resulting nanoparticle (lipo-DTX/siRNA) was stable and exhibited a sustained release profile. The co-delivery of therapeutic moieties inhibited the cell proliferation (A549 and H226) in a time-dependent manner. Moreover, the co-delivery system of DTX and siRNA exhibited a remarkable apoptosis of cancer cells with elevated levels of caspase 3/7 activity (apoptosis markers). Cell cycle analysis further showed remarkable increase in sub-G0/G1 phase, indicating increasing hypodiploids or apoptotic cells. Pharmacokinetic study showed a long circulating profile for DTX from lipo-DTX/siRNA system facilitating the passive tumor targeting. In vivo antitumor study on A549 cell bearing xenograft tumor model exhibited a remarkable tumor regression profile for lipo-DTX/siRNA with 100% survival rate. The favorable tumor inhibition response was attributed to the synergistic effect of DTX potency and MDR reversing ability of BCL-2 siRNA in the tumor mass. Overall, experimental results suggest that co-delivery of DTX and siRNA could be promising approach in the treatment of lung cancers. ã 2014 Published by Elsevier B.V.

Keywords: Docetaxel siRNA Lung cancer Antitumor Liposomes

1. Introduction Lung cancer is the leading cause of cancer-related death in both men and women worldwide with a staggering 28% of total cancer death in United States alone (Jemal et al., 2008). The survival rate of this type of cancer is much less than the other prevalent cancers such as breast and prostate cancers (15%). Although chemotherapy is being the first line of treatment for over 30 years, yet it achieved only limited success with serious dose-limiting side effects (Trussardi et al., 1998; Berger et al., 2005). In addition, intrinsic or acquired multidrug resistance (MDR) is one of the major hurdles of chemotherapy (Meschini et al., 2002). Microtubule depolymerizing agents (docetaxel; DTX) have a great

* Corresponding author at: Department of Medicinal Oncology, The First Affiliated Hospital, Sun Yat-Sen University, No. 58, Zhongshan 2nd Road, Guangzhou, Guangdong 510080, China. Tel.: +86 20 62732276; fax: +86 20 62732276. E-mail address: [email protected] (J.-T. Long). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.ijpharm.2014.08.019 0378-5173/ ã 2014 Published by Elsevier B.V.

potential to inhibit mitotic progression, cell apoptosis, and shown great potency against broad spectrum of cancers including lung cancer (Chowdhury et al., 2007; Fulton and Spencer, 1996). However, MDR decreases the accumulation of drug within the cell and increased the repairing mechanism of DNA damage leading to therapeutic efficiency of single agent (Pecot et al., 2011). In this regard, combination of two or more therapeutic strategies with different mechanism of action is evolving as a promising treatment module in cancer therapy. The duo of small molecule and gene could potentially increase the synergistic action of each therapeutic modality, target selectivity, and effectively counter the drug resistance (Sun et al., 2011; Greco and Vicent, 2009). In this case, BCL-2 siRNA has been reported to synergize the chemotherapeutic potential of anticancer drugs either by suppressing the overexpression of MDR-related mechanism (p-gp, MRP, LRP) or down-regulation of anti-apoptotic genes such as BCL-2 or surviving (Li et al., 2005; Dykxhoorn et al., 2003). The BCL-2 gene is a key apoptosis inhibitor that is overexpressed in many tumors and knockdown of BCL-2 could enhance the chemotherapeutic effect in cancer. siRNA functions by guiding

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ribonucleoprotein complex RNA-induced silencing complex (RISC) to target mRNA and cleaves. Subsequently, mRNA became less stable and degraded by RNase resulting in the reduction in mRNA and total protein level (Davis et al., 2010; Semple et al., 2010). Nevertheless, the biological application of siRNA will be limited by its enzymatic degradation, fast systemic clearance, immunogenicity, and poor internalization/permeability into cell membrane (Castanotto and Rossi, 2009; Kim and Rossi, 2007; Whitehead et al., 2009). Similarly, non-uniform distribution of DTX results in serious systemic side effects in normal healthy cells. Therefore, to stabilize the siRNA in systemic circulation and to ensure its maximum accumulation along with an anticancer drug, an effective delivery system that not only incorporates both the therapeutic agents but can target/deliver deep into the tumor tissue is desired. In this front, many promising approaches such lipidic delivery systems, polymeric system, nanoparticles or polymer conjugates have been developed time and again (Aagaard and Rossi, 2007; Liu et al., 2008; Blum and Saltzman, 2008; Gao and Liu, 2011). Interestingly, liposome represents one of the most versatile nanocarriers with proven systemic performances. Liposome is one of the most studied carriers in clinical trials and reported to be preferentially accumulated in the tumor region by enhanced permeation and retention effects (EPR) (Chen et al., 2010). In this study, we designed a novel liposomal delivery system to co-deliver DTX and BCL-2 siRNA into the tumor cells in the clinical models. The aim of this work was to overcome the multi-drug resistance (by silencing BCL-2 protein) and to increase the therapeutic efficiency of DTX by co-delivering it with BCL-2 siRNA. For this purpose, DTX-loaded cationic liposome was prepared, followed by siRNA conjugation via electrostatic interaction. The drug/gene complex has been extensively characterized by a range of biological studies including apoptosis study, cell cycle analysis and cell proliferation assay. The systemic performance of lipo-DTX/siRNA has been depicted by pharmacokinetic study in experimental animals. Finally, antitumor efficacy of lipo-DTX and lipo-DTX/siRNA has been performed in A549 cell bearing xenograft tumor model. Although, few reports have been published with regard to the combinational approach of anticancer drug and siRNA, yet present study provides a detailed comprehensive approach, and the first attempt to counter multidrug resistance in lung cancer tumor model. 2. Materials and methods 2.1. Materials Docetaxel (DTX) was obtained from Advanced Technology & Industrial Co. Ltd. Hong Kong, China. 1,2-dipalmitoyl-sn-glycero-3phosphocholine (DPPC) was procured form Genzyme Pharmaceuticals, Switzerland. 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethyleneglycol) (DSPEPEG2000) was obtained from NOF corporation (Tokyo, Japan). Cholesterol and dimethyldioctadecyl-ammonium bromide (DDAB) was purchased from Sigma–Aldrich (China). All the chemicals were used without any further purification. Targeting human BCL-2 siRNA (sence: 50 -CCGGGAGAUAGUGAUGAAGdTdT-30 , antisence:50 -CUUCAUCACUAUCUCCCGGdTdT-30 ) was supplied by Shanghai GenePharma Co. Ltd. (Shanghai, China).

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methoxy (polyethylene glycol) (DSPE-PEG2000-0.5 mmol), cholesterol (4 mmol), and dimethyldioctadecyl-ammonium bromide (DDAB-0.4 mmol) were dissolved in chloroform–methanol (4:1, v/v) in round-bottom flask. The docetaxel was added before the thin-film process along with the lipids. The organic solvent was evaporated using a rotary evaporator under vacuum at 60  C to form a thin lipid dried film. The lipid film was immediately hydrated with phosphate buffered saline solution to obtain drugloaded multilamellar vesicles of the final lipid concentration of 12 mM. The obtained large multilamellar vesicles were extruded using a mini-extruder (using polycarbonate membrane of 0.2 mM pore size) for 11 cycles to obtain a nanosized liposome (unilamellar). siRNA was loaded onto the liposome by a simple mixing method which takes advantage of charge difference between the liposome and siRNA. Optimized concentration of siRNA was mixed with liposome and incubated for 60 min at room temperature (24  C), following which unbound gene was removed by ultrafiltration method. Storage stability of optimized formulation was monitored up to 10 weeks. Moreover, in vitro physical stability of lipo-DTX/siRNA was studied in various specific mediums including phosphate buffered saline (PBS), fetal bovine serum (FBS), heparin, and bronchoalveolar lavage fluid (BALF). Nevertheless, fresh samples were prepared for all the in vitro and in vivo analysis throughout the study. 2.3. Particle size distribution and zeta potential The liposome solutions were suitably diluted to analyze the particle size distribution and zeta potential using dynamic light scattering (DLS) method. Malvern Zetasizer (Malvern, UK) was used to determine the DLS characteristics. The samples were suitably diluted (200 mg/ml) with double distilled water such that mean count rate will be around 300 kcps. All measurements were performed at a fixed angle of 90  C at 25  C room temperature. The results were expressed as the size SD. 2.4. Loading efficiency Loading efficiency was calculated from the total amount of drug added versus amount of drug entrapped in the nanoparticles. Briefly, drug-loaded complex was filtered by Amicon centrifugal filter by centrifuging at a high speed of 5000 rpm for 10 min. The filtrate was analyzed for unentrapped drug by HPLC method. The mobile phase consisting of methanol: water (70:30) was set at 1 ml/min and the effluents were monitored at 227 nm. A standard curve of DTX was plotted. Loading efficiency ¼

Total amount of DTX  Amount of free DTX  100 Total weight of nanoparticles

2.5. Morphology The morphological examination of lipo-DTX and lipo-DTX/ siRNA was carried out through a high resolution transmission electron microscopy (TEM, JEM-2010HR). Briefly, liquid sample was placed in a carbon coated copper grid and counter stained with phosphotungstic acid, followed by air drying for 2 h.

2.2. Methods 2.6. In vitro release study 2.2.1. Preparation of liposomes Cationic liposomes were prepared by thin-film hydration technique as reported/described elsewhere (Davis et al., 2010). Briefly, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC8 mmol), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-

In vitro release of DTX from lipo-DTX and lipo-DTX/siRNA was monitored by means of dialysis method. In this study, 1 ml of NP dispersions was placed in the dialysis bag and both the borders were sealed with a dialysis clip. The dialysis bag (molecular cut-off

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of 10 kD) was incubated in 25 ml of release media (PBS, pH 7.4 containing 1% Tween 80 as a solubilizer). The whole set up was placed in an automated shaker maintained at 100 rpm and 37  C. At predetermined time intervals, release media was collected and replaced with equal amount of fresh media. The released drug was quantified using HPLC method as mentioned above. 2.7. Cytotoxicity assay The A549 and H226 cells were cultured in normal RPMI media with 10% FBS and 100 units/ml penicillin, and 100 mg/ml streptomycin in 5% CO2 and 95% humidity atmosphere in humidifier. The cell viability/cytotoxic potential of individual formulation were performed by MTT assay. Briefly, cells were seeded into 96-well plate at a seeding density of 1 104 cells and incubated for 24 h. Following day, medium was removed and cells were incubated with various concentrations of free DTX, lipo-DTX and lipo-DTX/siRNA and incubated for 24, 48 and 72 h, respectively. At designated time intervals, cells were washed with PBS and treated with MTT solution (5 mg/ml in serum free media) and incubated for 3 h. The purple blue formazan crystals were extracted by the addition of DMSO and absorbance was measured at 570 nm using a microplate reader. 2.8. Confocal microscopy observation Confocal microscopic (CSLM) analysis was carried out to know the presence of NP within the cells. The A549 cells were incubated on a confocal dish for 24 h, followed by near infrared (NIR) fluorescent dye (cy5)-loaded lipo-DTX/siRNA was treated to the cell line and allowed for 60 min. The cells were washed with PBS buffer and fixed with 4% paraformaldehyde (PFA) for 20 min. The cells were then stained with DAPI for additional 15 min and subsequently, cells were washed, mounted and sealed with glycerine. The cells were observed under FV1000 confocal laser scanning microscope (Olympus, Japan). 2.9. Cellular uptake by flow cytometer analysis The A549 cells were seeded at a density of 2  105 cells/6-well plate and allowed to attach for 24 h. The cells were exposed to (cy5)-loaded lipo-DTX/siRNA and incubated for 2 h. The cells were washed twice with PBS, trypsinized, collected and re-suspended in PBS. The amount of cellular uptake was confirmed by flow cytometry (Becton Dickinson, Sunnyvale, CA). 2.10. Apoptosis assay The A549 were seeded at a density of 2  105 cells/12-well plate and allowed to attach for 24 h. The cells were treated with free DTX, lipo-DTX and lipo-DTX/siRNA and incubated for 24 h at 37  C in a standard incubator. A control was maintained as untreated cells. After the incubation period, cells were trypsinized with 0.025% trypsin solution, harvested, and resuspended in a 200 ml of binding buffer. Immediately, 5 ml of annexin V-FITC and 8 ml of propidium iodide (PI) was added and gently votexed and kept aside for 15 min. The proportions of apoptotic or stained cells were observed by flow cytometer. 2.11. Caspase 3/7 activity The activity of both caspase 3/7 was determined as reported previously. Briefly, cells were seeded and incubated overnight and treated with lipo-DTX and lipo-DTX/siRNA for 24 h. Cells were washed with cold PBS and lysed in mixed buffer containing hydroxyethylpiperazine-N0 -2-ethanesulphonic acid (HEPES),

5 mmol/l dithio-threitol (DTT), 5 mmol/l ethylene glycol-bis(baminoethyl ether)-N,N,N0 ,N0 ,-tetra-acetic acid (EGTA), 0.04% Nonidet P-40 and 20% glycerol; pH 7.4. The lysed mixture was centrifuged at 4000 rpm for 10 min at 4  C. The protein content of lysate was measured by Bradford method (Pierce, Rockford, IL) and bovine serum albumin was used as a standard. The cell lysate was mixed with 100 ml of Apo-ONE caspase-3/7 reagent containing bis-(N-CBZ-L-aspartyl-L-glutamyl-L-valyl-aspartic acid amide (Z-DEVD-R110). Z-DEVD-R110 is a caspase substrate known to cleave caspase-3 and caspase-7. The resultant fluorescence signal was measured using a spectrofluorometer at the excitation/ emission wavelength pair of 499Ex/521Em. 2.12. Cell cycle analysis by flow cytometry The A549 and H226 cells were seeded at a density of 2  105 cells/12-well plate and allowed to attach for 24 h. The cells were treated with free DTX, lipo-DTX and lipo-DTX/siRNA and incubated for 24 h at 37  C in a standard incubator. The cells were trypsinized, harvested, and centrifuged using a microcentrifuge at 1500 rpm for 4 min. The cell pellets were washed twice with PBS buffer and fixed in 75% ethanol solution at 4  C. Cells were centrifuged, washed twice, and resuspended in PBS containing 5 mg/ml PI and 50 mg/ml deoxyribonuclease-free ribonuclease A. This suspension was incubated in dark atmosphere for 25 min and then cell cycle patterns were analyzed using flow cytometry (Becton Dickinson, Sunnyvale, CA). 2.13. Pharmacokinetic study The animal study protocol was approved by ‘Animal Ethics Committee’, Sun Yat-Sen University, China. The animals were given good human care with access to 12 h day/light pattern. Sprague-Dawley (SD) rats were divided into 3 experimental groups (6 rats each). group I received free DTX, group II received lipo-DTX while group III received lipo-DTX/siRNA formulation at a DTX equivalent dose of 10 mg/kg via tail vein. DTX suspension was prepared by mixing it with PEG400 as a suspending agent. At predetermined time (0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 h), 0.4 ml of blood was collected (via femoral artery) into a small Eppendorf tube containing heparin. The heparinized blood samples were centrifuged at 10,000 rpm for 10 min; plasma supernatant was collected and stored at 80  C until further analysis. HPLC machine was used to analyze the plasma samples. The plasma was mixed with methanol to precipitate the unwanted proteins, centrifuged, and supernatant was injected into the HPLC column. 2.14. In vivo antitumor study The antitumor efficacy of free DTX, lipo-DTX and lipo-DTX/ siRNA was investigated on 6-weeks BALB/c nude mice model. For this, 4  106 A-549 human lung cancer cells were subcutaneously injected into the right flank of mice. When tumor volume approximately reached 150 mm3, respective formulations were injected at a dose of 5 mg/kg for 4 times during 2 weeks period. Subsequently, tumor volume was measured at specified time using vernier caliper in two dimensions. Tumor volume (V) was measured by the formula: V = (L  W2)/2, wherein length (L) is the longest diameter and width (W) is the shortest diameter perpendicular to length. Simultaneously body weight of individual mice was noted to interpret the toxicity or safety profile of each formulation. The tumor mass was surgically removed at the end of the study period and compared. Additional survival rate of each group was monitored and a graph was plotted using graphpad prism software.

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2.15. Statistical analysis Data were expressed as mean  standard deviations. The statistical significance was determined using T test and analysis of variance (ANOVA). p < 0.05 was considered statistically significant. 3. Results and discussion DTX is a potential cytotoxic drug with a strong antimitotic action against broad spectrum of cancers including lung cancer. Its cytotoxic effect is related with its enormous potential to inhibit cell proliferation and cell apoptosis (Chowdhury et al., 2007; Fulton and Spencer, 1996). However, DTX-based single agent therapy often associated with unwanted severe systemic toxicity including bone marrow suppression, hypersensitivity reactions, peripheral neuropathy, and musculoskeletal disorders. Besides, MDR effect greatly reduces the therapeutic efficacy and limits the therapeutic dose. Small RNA interference (siRNA) based therapy considered as

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powerful technique with remarkable ability in silencing target genes in cancer (Li et al., 2005; Dykxhoorn et al., 2003). siRNA involves in the knockdown of proteins involved in cancer cell proliferation or survival thereby inhibiting its drug compromising property. Earlier, reports have been published that siRNA treatment follows the reduction in the Bcl-2 mRNA expression. A reduction in Bcl-2 mRNA will be followed by a decrease in the protein expression of BCL-2. However, siRNA cannot permeate into cell membrane on its own due to the presence of negative charge compared to that of some small molecules which can easily diffuse (Whitehead et al., 2009). In addition, relatively short half-life and systemic instability hinders the clinical success of siRNA based therapy. Therefore, co-delivery of drug and gene is becoming a standard strategy in cancer chemotherapy which results in substantial reduction in the dose of DTX and increase the over therapeutic benefits by promoting the synergistic action, target selectivity, and overcoming drug resistance. We hypothesized that suppression of BCL-2 protein in combination with cell death induced by DTX can influence the apoptotic response of A-549.

Fig. 1. (A) Schematic illustration of preparation of lipo-DTX/siRNA nanoparticles. siRNA was physically entrapped to the cationic liposome via electrostatic interactions wherein negatively charged gene will bind with the positively charged liposome. Particle size distribution of (B) lipo-DTX/siRNA (C) lipo-DTX/siRNA via dynamic light scattering technique. Morphological imaging of (B) lipo-DTX/siRNA (C) lipo-DTX/siRNA using transmission electron microscopy (TEM).

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Fig. 2. (A) Gel electrophoresis analysis of siRNA incorporation on the cationic liposomes. The siRNA encapsulation was studied in the weight ratio of NP to siRNA up to 1:25. (B) Release kinetics of DTX from lipo-DTX and lipo-DTX/siRNA incubated in phosphate buffered saline (pH 7.4) at 37  C. The samples are collected at specified time points for up to 24 h and analyzed using sophisticated HPLC method. (C) Stability analysis of lipo-DTX and lipo-DTX/siRNA for 10 weeks. (D) In vitro stability of lipo-DTX/siRNA in phosphate buffered saline (PBS), fetal bovine serum (FBS), heparin, and bronchoalveolar lavage fluid (BALF). Date represents mean  SD (N = 3).

Generally treatment with anti-cancer drug induces BCL-2 up-regulation in cancer cells, and therefore suppressed BCL-2 knockdown ability of siRNA along with DTX could result in synergistic outcome. In the present study, therefore DTX and siRNA was co-delivered by incorporating in a cationically charged PEGylated liposomal carrier. Our objective was to enhance the blood circulation of therapeutic carrier, target to the tumor region, and to improve the synergistic potential of delivery system. 3.1. Preparation and characterization of DTX and siRNA-loaded liposomes The DTX loaded liposome was prepared by thin-film hydration technique. The DTX was added in the thin-film followed by the hydration and extrusion resulting in the formation of DTX-loaded liposomes. siRNA was adsorbed on the surface of positively charged liposome via electrostatic interactions resulting in the formation of DTX and siRNA-loaded liposome (lipo-DTX/siRNA) (Fig. 1a). The PEG moiety present on the surface of liposome will mask its recognition from opsonins and other blood proteins (reticuloendothelial system) via its anti-fouling property and thereby prolong its circulation subsequent clearance (Hobbs et al., 1998). Dynamic light scattering technique revealed that the average particle size of DTX-loaded liposome (lipo-DTX) was 132.5  2.5 nm with a Narrow Polydispersity Index of 0.095. The

lipo-DTX exhibited a zeta potential was around 24 mV. The positive charge was attributed to the presence of dimethyldioctadecyl-ammonium bromide (DDAB) as a surfactant in addition to DSPE-PEG which is present on the surface of liposomes. Moreover, loading of siRNA slightly increased the size of nanoparticles (165.4  1.7 nm) with a uniform distribution of particles (PDI-0.115) (Fig. 1b,c). As expected the surface charge of lipoDTX/siRNA nanoparticles decreased from around 24 mV to 13 mV. This could be due to the surface neutralization of siRNA on the liposome surface. Transmission electron microscopy (TEM) was used to characterize the morphology of both the nanoparticle systems (Fig. 1d,e). As can be seen, lipo-DTX exhibited a dense black core containing spherical shaped particle with clear edges. The particles are present as aggregates although clear round shaped boundary. This aggregation might occur during drying process wherein rapid removal water resulted in particle aggregation. Lipo-DTX/siRNA, however well-separated and presented a clear circular shaped morphology. This further suggest that adsorption of siRNA did not disturb the already existing morphology and still maintained the intact shape. The TEM particle sizes were consistent with the DLS observation. The particle size plays a vital role in in vivo systemic circulation, tissue distribution and its clearance from the central compartment. It has been reported that tumor possess impaired lymphatic drainage and interstitial spaces (Hobbs et al., 1998). Therefore, nanoparticles with appropriate size (

Liposome-based co-delivery of siRNA and docetaxel for the synergistic treatment of lung cancer.

Combination of more than one therapeutic strategy is the standard treatment in clinics. Co-delivery of chemotherapeutic drug and small interfering RNA...
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