PO-414 Evaluation and comparison of drug loaded composite nano-emulsions’ cellular uptake in malignant melanoma cell line

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IntroductionMelanoma is multi-drug resistant and is considered as one of the most malignant cancer types. In this context, provision of effective drugs with the right delivery system is highly desirable. A key specification in controlled and targeted delivery is the particle size of the delivery material. Sub-micron sized particles are more likely to provide targeted intracellular delivery, nano-emulsion is a well-recognised preparation.Material and methodsIn this study, chitosan-pullulan, pullulan-alginate and chitosan-alginate composite nano-emulsions were prepared using different ratios (1:2, 2:1, 1:1). Coumarin 6 (a fluorescent dye) was encapsulated in nano-emulsions. The impregnated particles were tracked and cellular uptake was monitored, and compared with doxorubicin uptake in the same delivery system.A range of chitosan, pullulan and alginate nano-emulsions were prepared to optimise the drug release. The polymers were cross-linked using genipin. Genipin stock solution (1 mg/ml) was prepared and 500 microliters of the stock were added to 10 ml of each polymer blend and stirred for 72 hours to complete the crosslinking process. FTIR was used to determine the characteristics of each blend. All the prepared blends were loaded with coumarin 6. Fifteen nano-emulsions were prepared, and UV sterilised before the cellular uptake. A375 malignant melanoma cell line was collected from 4th passage and added to 6 well-plates with the density of 106 cells per well and a 96 well-plate for MTT test. After 80% confluency, 50 micrograms of each nano-emulsion were added to each well. After 72 hours the cells were imaged with confocal microscopy and MTT was done to measure cytotoxicity. Same procedure was applied for encapsulation of doxorubicin to compare, DNA fragmentation ELISA was used to measure the possible apoptosis inducing ability of the blank and drug loaded formulations.Results and discussionsConfocal microscopy observations of the uptake by the cells revealed that release of coumarin impregnated emulsions was slower than the control (coumarin 6 a lone) over a period of 72 hours. Cell viability analysis via MTT indicated that all the cells were viable. ELISA results indicated that doxorubicin loaded formulations are able to induce apoptosis to cells increasingly and starting from 4 hours.Conclusion according to quantification of confocal images the slow release were provided by formulations. Interestingly doxorubicin loaded formulations were observed to be more toxic thatn doxorubicin alone.