Evaluation of RNA transfection efficiency using novel lipoplexes in three different kinds of cells

CARLUCCI A; PEREZ S; BONAZZOLA P; LOMBARDI MG; MIRANDA S

CABA

Congreso; IV International Congress in Translational Medicine.; 2018

Facultad de Farmacia y Bioquímica UBA

Nowadays, the term ?lipoplexes? defined lipid-based delivery systems able to load/release oligonucleotides. Previously, soya phosphatidylcholine?sodium cholate (SPC:SC) nanoparticles were prepared in molar ratios 1:2 and 1:4 (2 % w/v) using isotonic phosphate buffer pH 7.4 or 50 mM isotonic acetate buffer pH 5.0 as dispersion media and were characterized upon their physicochemical properties. In this work, we evaluated the Transfection Efficiency (TE) of dsRNA delivered from the respective lipoplexes to three different kinds of cells using Lipofectamine RNAiMAXTM (Invitrogen) as a control. Tested cell types were: 1- 112D5 Hybridoma Cells (hybrids from mouse B lymphocytes and non-secretory tumor NSO cells), 2- MCF-10A (non-tumoral human breast epithelial cell line) and 3- primary culture of rat myocardiocytes/fibroblasts. As transfection control, dsRNA conjugated with Alexa 546 (BLOCK-iTTM, Invitrogen) was used. Other variables were culture conditions and the dsRNA/nanovehicle rates (1.0, 1.5 or 2.0μl of dsRNA 20 μM/ 1.0 or 1.5μl of nanovehicle). Transfection complexes were prepared by adding dsRNA to vehicles in serum and antibiotic free media and incubating 20 minutes at room temperature. Then, lipoplexes were added to cells and incubated for 20 hours at 37ºC, 5% CO2. Cell viability was assessed by exclusion of Trypan Blue dye.TE was calculated by flow cytometry (Partec Past III cytometer) determining the percentage of cells that acquired the fluorescent label and the mean fluorescent signal (MFS) employing the Cyflogic v 1.2.1 software. For 112D5 cells, the highest TE and MFS obtained were: nearly 40% and 46.0, respectively employing 2μl dsRNA + 1.5μl SPC-CS 1:4, even after freezing and thawing (SPC-CS 1:2 have not been tested yet). For myocardiocytes and ventricle fibroblasts, best results were obtained using 1.5 µL of vehicle: TE: 39.2%/MFS: 20.0 (for SPC-CS 1:2 pH 7.0) and TE: 32.6%/MFS: 21.4 for SPC-CS 1:2 pH 7.0. In both cases, TE and MFS data were similar to those observed with Lipofectamine. Moreover, no modifications in cell viability were observed. Instead, MCF-10A cells could not be transfected either with Lipofectamine or employing these lipoplexes. Previous results demonstrated that only electroporation turned out to be an efficient transfection method for these cells. These experiments, in addition to previous transfection ones carried out using MCF-7 and MDCK cells, showed that 1:2, 1:4 SPC-CS pH 5.0/ 7.4 lipoplexes could be used as efficient nanodelivery systems for dsRNA transfection, even though results depend on the target cell and on the pharmaceutical composition of the vehicle. In addition, this work showed that flow cytometry can provide an accurate and quantitative analysis for the screening of new transfection agents, a major issue for improving gene therapy products´ translation.