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Abstract
Purpose: To evaluate the cytotoxic effect of pegylated liposomal Recombinant Human Erythropoietinalfa (rHuEPO) nanoparticles synthesized by reverse phase evaporation technique on SH-SY5Y cell line.Methods: To prepare the nanoparticles of the drug, rHuEPO, PEG3000, cholesterol andphosphatidylcholine were dissolved in buffer phosphate. The characteristics of the synthesized nanoparticles were determined by a zetasizer. Encapsulation efficiency, drug loading efficiency and drug release pattern were evaluated spectrophotometrically. The cytotoxicity effect of pegylated nanoliposomal rHuEPO was evaluated on SH-SY5Y cell line by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay.Results: Size and zeta potential of pegylated nanoliposomal drug and blank pegylated nanoliposomes were 571 ± 6.8 nm, -16.5 mV; 280 ± 4.71 nm and -27.1 mV, respectively. Encapsulation and drug loading efficiency were 89 ± 3.9 % and 0.8 ± 0.012 %, respectively. Drug release data indicate that 17 % of rHuEPO was released from pegylated liposomal nanoparticles over a period of 30 h. The difference in cytotoxicity between the free drug (IC50 = 110.1 ± 3.1 μg/ml) and nanodrug (IC50 = 87.2 ± 2.3 μg/ml) was statistically significant (p ˂ 0.05).Conclusion: This study shows that pegylated nanoliposomal rHuEPO has a powerful cytotoxic effect on SH-SY5Y cell line and is therefore a suitable alternative to the standard therapy, rHuEPO, for the chemotherapy of neuroblastoma.Keywords: Recombinant Human Erythropoietin-alfa, Cytotoxicity, Drug delivery, Liposome, Reverse phase evaporation, SH-SY5Y cell line
Highlights
Recombinant human erythropoietin-alfa - a 165 amino acid glycoprotein is manufactured by recombinant DNA technology. rHuEPO is one of the most important drugs used in chemotherapy [1]
The cytotoxicity of pegylated nanoliposomal rHuEPO on neuroblastoma cell line SH-SY5Y was investigated by MTT assay in 96-well plate reader
polyethylene glycol (PEG) was used in the synthesis of nanoparticles, because it is stable in the blood circulatory system and is not removed by the reticulo endothelial system (RES)
Summary
Recombinant human erythropoietin-alfa (rHuEPO) - a 165 amino acid glycoprotein is manufactured by recombinant DNA technology. rHuEPO is one of the most important drugs used in chemotherapy [1]. We synthesized liposomal rHuEPO nanoparticles by reverse phase evaporation technique to increase efficiency and decrease side effect of the drug. Encapsulation and drug loading efficiency were computed as in Eqs 1 and 2 For this purpose, 2 mL of each suspension were centrifuged (15000 rpm, 4 °C, 90 min). RHuEPO, PEG3000, cholesterol and phosphatidylcholine (1:2.8:23:97 weight ratio) were dissolved in 50 mL of 98 % ethanol using magnetic stirrer (150 rpm, 30 min, room temperature) to obtain a transparent yellow suspension. Drug release was determined by membrane diffusion technique For this purpose, 1 mL of each formulation (pegylated nanoliposomal rHuEPO and the standard form) was poured in dialysis bag (cut off 50000 Dalton, Sigma). The amounts of released drug in phosphate buffer were measured using spectrophotometric method at definite time intervals
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