1ST INTERNATIONAL CONFERENCE ON NANOBIOTECHNOLOGIES AND BIONANOSCIENCE (NANOBIO2018)”, 24-28 SEPTEMBER, Heraklion, Crete, Greece
Daniela Rebleanu1, Cristina Ana Constantinescu1, Geanina Voicu1, Agneta Simionescu1,2, Ileana Manduteanu1, Manuela Calin1
1Institute of Cellular Biology and Pathology “Nicolae Simionescu” of Romanian Academy, Bucharest, Romania
2Department of Bioengineering, Clemson University, United States of America
RNA-interference (RNAi) holds high potential to silence the expression of a specific target gene. Short hairpin (sh) RNA is an attractive RNAi tool due to its relatively low rate of degradation and has the advantage that it is synthesized within the cell after transfection with a plasmid containing a specific shRNA sequence. The aim of this study is to investigate the transfection efficiency and cytotoxicity of different shRNA plasmid nanocarriers (lipid-based or PEI-based nanoconjugate). PEGylated cationic liposomes comprising different cationic lipids, a helper lipid (e.g. DOPE) and a PEG functionalized lipid, and a nanoconjugate of fullerene-PEI (C60-PEI) were obtained in order to find the best nanocarrier for efficient delivery of a plasmid to human valvular interstitial cells (VIC). Lipoplexes/polyplexes were obtained by complexing cationic liposomes and C60-PEI with a plasmid shRNA at various charge ratios (R) and N/P ratios, respectively and further characterized for size (by dynamic light scattering), zeta potential (by electrophoretic mobility), morphology (by electron microscopy, TEM) and the capacity to protect shRNA (by agarose gel retardation assay). Transfection efficiency was evaluated after exposing VIC, isolated from human aortic valve, for 48 hours to different lipoplexes/polyplexes between cationic liposomes/C60-PEI and a plasmid encoding a fluorescent protein (pEYFP) by fluorescence microscopy and flow cytometry. The cytotoxicity studies were performed by XTT assay after exposing VIC to different types of lipoplexes/polyplexes. The results are: 1) hydrodynamic size of lipoplexes/polyplexes varies as function of R ratio and N/P ratio between 100 and 1600 nm; 2) morphology of lipoplexes analyzed by TEM revealed the appearance of multilamellar vesicles with concentric lamellar structures; 3) packaging and protection of nucleic acids by the complexation with different types of nanocarriers depend on nanocarrier’ s composition and R or N/P ratio 4) transfection efficiency and viability of VIC is dependent on lipoplexes/polyplexes nature and R or N/P ratio. In conclusion, nanocarriers capable to function as efficient transfection vectors for shRNA plasmid delivery in VIC have been developed.
Acknowledgements. This work is supported by the Competitiveness Operational Programme 2014-2020, Priority Axis1/Action 1.1.4/, Financing Contract no.115/13.09.2016/ MySMIS:104362.