Abstract 2063: Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells
Authors: Lalani Rohan A. , Bhatt Priyanka , Rathi Mohan , Misra Ambikanandan 
Published: July 2016

Aim of the study was to prepare and characterize RGD grafted PEGylated liposomes of gemcitabine (PLGs) and to evaluate its cellular uptake, in vitro anti-proliferative activity and apoptotic effect in siRNA pretreated lung cancer cells.

PLGs were prepared by thin film hydration method and optimized for particle size, zeta potential and entrapment efficiency. Functionalization of liposomes was done by coupling reaction between DSPE mPEG and cRGD peptide by maleimide based reaction. Similarly, RGD grafted RRM1 siRNA liposomes were also formulated and evaluated. MTT assay was done to determine IC50 values of RGD grafted PLGs in A549 and H1299 cancer cells which were pre-treated with RGD grafted RRM1 siRNA loaded liposomes at a concentration of 50 pM of siRNA for gene silencing. DNA content analysis was done by flow cytometry using rhodamine in A549. The mode of cell death at different time and concentration was determined by FITC-AnnexineV assay in A549 cells and confocal microscopy was performed to assess the potential of RGD grafting on cellular uptake.

RGD conjugated and unconjugated PLGs were found Nano sized and had negative zeta potential with entrapment of 65%. H1299 cell line showed more amount of viable cells after 48 hr as compared to A549 cells in RGD conjugated PLGs. siRNA pre-treated PLGs exposed cells showed significantly less IC50 values as compared to cells without siRNA pretreatment and non-grafted liposomes. The results showed that the RGD conjugated liposomes at the concentration of 7nM showed 46% G1 phase arrest in siRNA pretreated cells as compared to 22% G1 phase arrest without prior siRNA treatment at 16hr. Two types of mode of cell death were found during the FITC-Annexine V assay. At 24 hr, the treatment with RGD grafted PLGs resulted in 17% & 4.4% necrotic & apoptotic cell death respectively. While at equivalent drug concentration, the PLGs and drug solution showed 5.3% & 32.2% and 4.2% & 29.6% necrotic & apoptotic cell death respectively. Furthermore, the apoptosis was found to be time and concentration dependent. Results substantiate the sensitization effect by pre-exposure of siRNA in liposomal forms at Pico molar concentration along-with phagocytosis as mechanism of uptake of RGD-grafted liposomes.

To conclude, prior silencing of the resistance imparting gene can manifest the effect of therapy by conferring improved sensitivity in cancer cell lines. The effect can further be augmented by employing receptor targeting peptides such as RGD. Hence, Nano-constructs of chemotherapeutic drugs conjugated with RGD can effectively target lung cancer cells and pretreatment of RRM1 siRNA can probably reduce the limitation of drug resistance associated with lung cancer chemotherapy.

Citation Format: Rohan A. Lalani, Priyanka Bhatt, Mohan Rathi, Ambikanandan Misra. Improved sensitivity and in vitro efficacy of RGD grafted PEGylated gemcitabine liposomes in RRM1 siRNA pretreated cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2063.