Simultaneous delivery of Paclitaxel and Bcl-2 siRNA via pH-Sensitive liposomal nanocarrier for the synergistic treatment of melanoma

pH-sensitive drug carriers that are sensitive to the acidic (pH = ~6.5) microenvironments of tumor tissues have been primarily used as effective drug/gene/siRNA/microRNA carriers for releasing their payloads to tumor cells/tissues. Resistance to various drugs has become a big hurdle in systemic chemotherapy in cancer. Therefore delivery of chemotherapeutic agents and siRNA’s targeting anti apoptotic genes possess advantages to overcome the efflux pump mediated and anti apoptosis-related drug resistance. Here, we report the development of nanocarrier system prepared from kojic acid backbone-based cationic amphiphile containing endosomal pH-sensitive imidazole ring. This pH-sensitive liposomal nanocarrier effectively delivers anti-cancer drug (Paclitaxel; PTX) and siRNA (Bcl-2), and significantly inhibits cell proliferation and reduces tumor growth. Tumor inhibition response attributes to the synergistic effect of PTX potency and MDR reversing ability of Bcl-2 siRNA in the tumor supporting that kojic acid based liposomal pH-sensitive nanocarrier as efficient vehicle for systemic co-delivery of drugs and siRNA.

India) and anti β-Actin antibody was purchased from Abcam (Cambridge, MA, USA). HRP conjugated goat anti-rabbit and goat anti mouse secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Cy-3 conjugated anti-rabbit secondary antibody was purchased from Jackson Laboratories (West grove, PA, USA).

Particle size distribution and zeta potential
The hydrodynamic sizes of liposomal formulations were analyzed by dynamic light scattering (DLS) performed on a DynaPro Nano DLS system (Wyatt Technologies, Santa Barbara, CA, USA). A Zetasizer (Malvern, UK) was used to determine the zeta potential of the siRNA encapsulated liposomes. All measurements were performed at a fixed angle of 90 0 at room temperature (25°C). The results were expressed as the size ± standard deviation and zeta potential ± standard deviation. siRNA entrapment and Gel retardation assay 1 µg of scrambled siRNA in was entrapped inside the liposomes by freeze thaw method. On the other hand was liposomes incubated with 1 µg siRNA scrambled siRNA at room temperature for 20 minutes before loading onto the gel. Above mentioned samples and the equivalent amount of naked siRNA was separately loaded on to the gel. The electrophoretic mobility of twas visualized using an ultraviolet illuminator with ethidium bromide staining after electrophoresis on 2% (w/v) agarose gel for 20 minutes at 80 V in TAE buffer (40 mM Tris-HCl, 1% v/v acetic acid, 1 mM EDTA).

RNase protection assay
To prevent digestion of siRNA by endonucleases, we performed a nuclease protection assay, where samples of naked siRNA and the siRNA entrapped liposomal formulations and electrostatic lipoplexes were treated with RNase-A for 30 minutes. The samples were then treated with 8 U of RNase-A inhibitor at 37°C for 15 minutes and run on 2% agarose gel for 20 minutes at 80 V in TAE buffer (40 mM Tris-HCl, 1% v/v acetic acid, 1 mM EDTA). The gel was visualized under an ultraviolet illuminator after staining with ethidium bromide. 1

Morphology
The morphological examination of empty Liposomes, lipo-PTX, lipo-siRNA and lipo-PTX/ siRNA was performed using SEM, Hitachi S-3400N Tokyo, Japan. Liquid sample was placed on a coverslip, dried and coated with gold before observing and analyzing under scanning electron microscopy.

Cellular uptake by flow cytometric analysis
The B16F10 and NIH/3T3 cells were seeded at a density of 2 x 10 5 cells/6-well plate and allowed to adhere for 24 h. The cells were exposed to lipo-PTX/FITC-siRNA and incubated for 4 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).

Confocal microscopy
Confocal Laser Scanning microscopic (CSLM) analysis was carried out to observe the uptake of liposomes within the cells. B16F10 and NIH/3T3 cells were grown on coverslips for 24 h, followed by transfection with FITC-labelled siRNA entrapped liposomal nanparticles for 4 h.
The cells were washed with PBS buffer and fixed with 4% paraformaldehyde (PFA) for 20 min. The cells were then mounted with vectashield mounting media (Vector Laboratories, Inc, Burlingame, CA, USA) and observed under FV1000 confocal laser scanning microscope (FV1000, Olympus, Japan).

Cell cycle analysis by flow cytometry
B16F10 cells were seeded at a density of 2 x 10 5 cells/6 well plate and allowed to adhere for 24 h. Thereafter the cells were treated with liposomes 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 followed by brief centrifugation, and washed with PBS. They were again resuspended in PBS containing 5 mg/mL PI and 50 mg/mL deoxyribonuclease-free ribonuclease A. The suspension was incubated in dark for 25 min and analysed for cell cycle patterns using flow cytometry.

RT-PCR analysis
Total RNA was extracted using RNeasy mini kit (Qiagen, Valencia, CA, USA) and reverse transcribed into cDNA using superscript II reverse transcriptase (Invitrogen, Carlsbad, CA. USA). PCR was carried out with specific primers for GAPDH, Bcl-2 (Table S1) in Eppendorf thermal cycler. The products were electrophoresed on agarose gel (1%) containing ethidium bromide and visualized under U V light. The signal intensity of respective bands was measured by means of the Quantity One version 4.1.1 software using BIORAD image analysis system (Hercules, CA, USA).

Western blot analysis:
To assay changes in apoptotic protein levels, after co-delivery with liposomes in B16F10 cells, both treated and untreated cells were collected and lysed in RIPA lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) . Cell lysate was collected by centrifugation.
To observe the expression of apoptotic proteins both before and after co-delivery of liposomal samples in tumor, 1mg of tumor tissue was collected, washed with PBS and homogenised in RIPA lysis buffer. Protein lysate was collected by centrifugation.
In both in vitro and in vivo experimental condition, total protein concentration was    Synthetic methods and Spectral data of prepared compounds: