Phosphonium carbosilane dendrimers are efficient non-viral vectors for siRNA cell delivery on model cell lines in vitro

Gene therapy is a rapidly growing field of biomedical research which brought a new hope in the fight against genetic-based diseases. The success depends on the development of suitable vectors for the nucleic acid cell... [ view full abstract ]

Gene therapy is a rapidly growing field of biomedical research which brought a new hope in the fight against genetic-based diseases. The success depends on the development of suitable vectors for the nucleic acid cell targeting. Dendrimers have number of favourable properties, such as good transfection efficiency and relatively low toxicity. This work introduces results from the comparative study of two types of cationic carbosilane dendrimers terminated with ammonium or phosphonium groups for their use as non-viral vectors for transfection of fibroblasts (cell line B14) with siRNA. We present a part of work devoted to characterization of dendriplexes formed from generation 1-3 (G1-3) of carbosilane dendrimers and model siRNA. Dendriplexes were characterized by Gel retardation electrophoresis, DLS, ξ-Potential, Fluorescence Anisotropy, Nuclease Protection Assay and AFM. Transfection efficiency was evaluated by Fluorescence Microscopy.                   We found that both types of G2-G3 dendrimers form stable complexes with siRNA due to positive charge of surface groups of dendrimers and negative charge of siRNA backbone. Formation of dendriplexes was investigated at different charge ratio (1/5 – 10/1 (+/-)) to find an optimal properties of complexes for cell transfection. All types of dendriplexes show quite similar characteristics (stability, surface charge, dimensions etc.), based on the charge ratio and generation of dendrimer used.          In vitro transfection experiments proved an ability of both G3 dendriplex structures to enter the cells, with maximal achieved transfection efficiency at 7/1 (+/-) charge ratio. Ammonium dendrimers achieved max. 30% of transfected cells, contrary more than 70% were transfected at the same conditions with phosphonium terminated dendrimers.

            With the aim to further optimize the properties of phosphonium dendriplexes we incorporated new periphery substituents (P(Et₂)₂(CH₂)₃OH, P(C₆H₄-OMe)₃, P(Ph)3, PBu₃) into the dendrimer structure. Similar cytotoxicity and transfection efficiencies were obtained, with the exception of P(C₆H₄-OMe)₃ and P(Ph)₃ peripheral substituent. These dendrimers exhibits more than 80% transfection efficiency and dendrimer P(C₆H₄-OMe)₃ also very low toxicity at comparable experimental conditions. Therefore, these dendrimers types seems to be “hot” candidates for further improvements of gene delivery by phosphonium carbosilane dendrimer vectors.

Research was supported by project 15-05903S of Czech Science Foundation and partially by Internal Grant Agency UJEP.