Development of siRNA lipoplexes for intracellular delivery in lung cancer cells.

Lung cancer occurrence throughout the world and its unsatisfactory treatment has drawn significant attention from many medical agencies. A genomic approach utilizing siRNA against target gene may resolve this problem. Present investigation includes siRNA lipoplex formulation and its in-vitro characterisation in lung cancer cell line for intracellular uptake and cytotoxicity. Accumulation of siRNA inside the cell as well as less cytotoxicity of prepared carrier system signifies potential of prepared siRNA lipoplex in the treatment of lung cancer.

Long term exposure to tobacco smoke is the main cause for more than 1.5 million deaths worldwide yearly.[1] India accounts 10% of total world's lung cancer incidents. At present lung cancer is treated with intravenous administration of chemotherapeutic agents, but lack of selectivity of these drug molecules cause lethal actions to non cancerous cells.[2] This provides impulsion to carry out a research for effective and safe treatment of lung cancer. Small interfering RNA (siRNA) is a duplex of 19-23 nucleotide base pairs which specifically slices target mRNA and hence expression of a protein can be inhibited.[3] Present investigation utilizes the siRNA macromolecule, complexed with liposomes, for effective treatment of lung cancer.

Materials and Methods

All phospholipids and cholesterol were purchased from Lipoid, Germany. All siRNAs were purchased from MWG-Europhins, Germany. FAM-NC-siRNA and NC-siRNA were obtained from Gene Pharma, China. All other chemicals and reagents used were of Analytical grades/HPLC grade/Biological grades. A-549 cell line was obtained from NCCS, Pune.

Formulation

siRNA lipoplexes were prepared by incubating siRNA with preformed liposomes. Briefly, liposomes were prepared using mixture of positively charged lipid, endosomolytic lipid, neutral phospholipid, cholesterol and methoxy polyethylene glycol Distearoyl phosphatidyl ethanolamine. All lipids were dissolved in chloroform and dry lipid film was obtained by stripping all organic solvent. Hydration was performed using RNAase free water. Prepared liposomes were size reduced by probe sonication and incubated with siRNA different nitrogen to phosphate charge ratio.

Agarose gel electrophoresis

Formation of lipoplex and complete complexation of siRNA with liposomes were confirmed using agarose gel electrophoresis. siRNA and liposomes were loaded onto agarose gel at different N/P ratio and documented in gel documentation system using Biorad XR+ model (Bio-Rad, USA).

Particle size and zeta potential

pan class="Chemical">Prepared lipoplexes were characterized for particle size, size distribution [Figure 1] using and zeta potential using Malvern Zeta Sizer instrument (Malvern, USA).

Figure 1

Particle size distribution

In-vitro cell line studies

To access the potential of prepared lipoplexes as a non-viral vector for siRNA delivery inside the cell, studies were carried out in pan class="Disease">lung cancer cell line, pan class="CellLine">A-549.

Flow cytometry

FAM labelled Negative control siRNA was used for intracellular uptake study in pan class="CellLine">A-549 cell line. Cells were seeded in 96 well plates (5000 cells/well) 24 hrs before siRNA treatment. After 24 hrs 100nM siRNA lipoplexes were added to each well in serum and antibiotic free media. After 5 hrs of incubation, media was replaced with serum and antibiotic free fresh media. Cells were trypsinized and analysed for mean fluorescence activity using fluorescence activated cell sorter (FACS-BD-AriaIII, Becton-Dickinson, USA).

Cell cytotoxicity (MTT assay)

Cells were seeded in 96 well plate at a density of 5000 cells per well. After 24 hrs cells were treated with 100nM of NC-siRNA and further processed for pan class="Chemical">MTT assay. Cell viability was measured colorimetrically using enzyme-linked immunosorbent assay plate reader (Biorad, USA) at a wavelength of 570 nm.

Results and Discussion

Lipoplexes were prepared at different N/P ratio and characterized for complexation studies using Gel Electropn>horesis. Lipopn>lexes above N/P=1.5 were found to be completely free of naked siRNA. This will prevent degradation of siRNA from nuclease enzyme in biological system.[45] Particle size for optimal lipoplex was found to be 107.6 nm with 0.158 PDI value. Positive value for zeta potential was obtained due to involvement of charged lipids in the formulations. Zeta potential for optimized lipoplex was 12.9 mV [Figure 2]. Various lipoplex formulations were assessed for in-vitro cell line studies in A-549 cells and Lipofectamine™ was used as a positive control. There was a significant difference (P<0.05) in the uptake of siRNA, which were given in lipoplex form as compared to positive control [Figure 3].

Figure 2

Zeta potential

Figure 3

Cell uptake by FACS

This finding clearly shows potential of prepared formulation for intracellular delivery of siRNA. Cell viability was determined using MTT assay after 48 hrs. Figure 2 shows cell viability after treatment with 100nM of siRNA in lipopn>lex form using blank sample as a negative control and Lipofectamine™ as positive control. Optimal formulation shows significantly more (P<0.05) cell viability as compared to Lipofectamine™ [Figure 4].

Figure 4

Cell cytotoxicity (MTT assay)

Cell pan class="Disease">cytotoxicity (pan class="Chemical">MTT assay)

Conclusion

siRNA lipoplex can be a potential non-viral delivery vector for intracellular delivery of nucleic acid molecules. Complete complexation ensures resistance from degradation by nucleases. Intracellular uptake of siRNA in lipoplex form and cell viability results signifies its potential for further development as a novel drug delivery vector. Although prepared siRNA lipoplexes have opened new vista for chemotherapeutics, concrete proof can only be attained after successful in-vivo studies in suitable animal models.