PB2207: IN VITRO VALIDATION OF DENDRIMER-BASED APPROACH FOR MICRO-RNA DELIVERY INTO LEUKEMIA CELLS

Abstract

Background: microRNAs are known to be important players in the epigenetic regulation of many cell processes. Some of them demonstrate anti-tumor properties (e.g. miR-34), others are associated with the enhancing of the tumor growth (e.g. miR-21), therefore being prospective effectors and targets for the gene therapy. For this aim, synthetic microRNAs or microRNA inhibitors can be used. We suggest to use dendrimers - highly symmetric hyperbranched polymers - as a novel type of nanocarriers for therapeutic RNAs delivery and gene therapy. Aims: In this work we assessed the effects of microRNA-34a (miR-34a) and microRNA-21 inhibitor (anti-miR-21) on Jurkat and 1301 cells (human T-cell leukemia) upon the dendrimer-mediated delivery. Methods: As carriers for microRNAs, amphiphilic second- and third generation triazine-carbosilane dendrons (DG2 and DG3 respectively) and fully symmetrical 3rd generation carbosilane (BDEF33) and phosphorus (AE2G3) dendrimers have been taken. Oligonucleotides were complexed onto the surface of dendrimer vesicles by means of electrostatic interactions, forming dendriplexes. To prove the dendriplexes can be sufficiently internalized, we performed FACS analysis after 3 h of incubating cells with FITC-labelled microRNA. Cells were cultivated with free dendrimers or complexes (25, 50, 100, 150 nM RNA, 10-fold excess of cations) in the complete cell medium for 72 h. We investigated cell viability (MTT assay, Annexin V/PI apoptosis assay) in comparison with non-tumor cells (PBMCs), IL-10 secretion and expression of several markers, characterizing interaction of tumor with immune microenvironment - PD-L1, TIM-3, CD47. Statistical analysis was done by using Mann-Whitney and Wilcoxon tests, differences between groups were considered significant if p<0.05. This work was supported by RFBR grant No.18-33-20109, by the grant of the President of the Russian Federation No.2278.2019.4, by the HHU STIBET Scholarship grant support and based upon work from COST Action CA 17140 “Cancer Nanomedicine from the Bench to the Bedside” supported by COST (European Cooperation in Science and Technology). Results: Free dendrimers have their own dose-dependent toxic effects, which is slightly higher for tumor cells, than for PBMCs. Treatment by free dendritic molecules led to increasing of expression of PD-L1, TIM-3 and CD47 on Jurkat cells, but decreased the IL-10 secretion. Dendriplexes can be efficiently internalized into tumor cells, transport activity of dendrimers was higher than for Lipofectamine 3000 or was comparable. Dendron-based dendriplexes (DG2, DG3) had pro-apoptotic effect on leukemia cells. PD-L1 expression did not change after treatment by dendriplexes. Summary/Conclusion: Dendrimers not onle have their own toxic effect per se, but also they can be used as effective nanocarriers for therapeutic microRNAs. Using of complexes, containing microRNAs with anti-tumor activity, leads to induction of apoptosis of leukemia cells. Treatment by free dendrimers, but not by dendriplexes, increases expression of PD-L1, TIM-3, CD47 on Jurkat cells, but IL-10 secretion was found to be decreased. We suppose, that dendrimer-based approach can be useful and perspective tool for delivery of therapeuthic microRNAs into tumor cells. At the same moment, characterization of their effects on interactions between tumor and immune microenvironment demands the further studies.