Novel Photoreaction Nucleic Acid Delivery System And Its Application In SiRNA Delivery

Cationic non-viral RNA carriers have attractive advantages and promising application potentials in safety, flexibility and cost in reference to viral carriers. However, the tow transfection efficiency has been a major challenge for the non-viral carriers to be applied in clinic, one of the major reasons is the difficulty of lysosomal escape for the siRNA carrier complex in target cells. ROS at low concentration may disrupt the endosomal membrane and advance the cytoplasmic delivery of macro molecular compounds instead of killing cells. This mechanism is called photochemical internalization (PCI). C60 can be used as a substitute of traditional photosensitizer by its photochemical and modification characteristics, realize lysosome escape under visible light based on PCI mechanism.Therefore, our study synthesised cationic water-soluble fullerene (C60-Dex-NH2) as gene carrier and anionic water-soluble fullerene (C60-Dex-COOH) as anionic coating material, investigated the lysosome escape of water-soluble C60 with LED light in vivo and in vitro experiments.Biocompatibility and interference efficiency taken into consideration, a novel photosensitive carrier (C60-Dex-NH2) for siRNA delivery was designed and prepared in the study. The initial material C60 was water-solublized by terminal aminated Dextran and cationized by covalently introducing ethylenediamine to the polysaccharide skeleton. The amino group content, molecular weight and chemical structure of C60-Dex-NH2 were analyzed by TNBS/elemental analysis, GPC and SSNMR (13C, HPDEC), respectively. Gel electrophoresis and DLS analysis were applied to test the binding capacity of C60-Dex-NH2 with siRNA and the particle size of C60-Dex-NH2/siRNA complex. The reactive oxygen species (ROS) generation was evaluated by conventional cytochrome C method. Lysosome escape ability and RNA interference (RNAi) efficiency with or without light irradiation was evaluated in vitro on the human breast cancer cell line MDA-MB-231 cells. The in vivo gene silence efficiency of the complex intratumor injected to subcutaneous tumor-bearing mice was also measured in situ as well as ex situ.Results showed that C60-Dex-NH2 could be activated effectively by the irradiation of visible light to trigger ROS generation. The produced ROS disrupted the lysosome and promoted the complex escape. These series reactions led to the elevation of RNAi efficiency both in vitro and in vivo, the maximum percentage of gene silence reached 53.02% in the MDA-MB-231-EGFP cells and 69.05% in the 4T1-GFP-Luc2 tumor-bearing mice.In order to confirm the photosensitizer function of water-soluble C60 in different delivery system and prevent cationic polymeric carriers from the adsorption of protein in serum environment, we synthesized carboxylic Dextran (Dex-COOH) and water-soluble carboxylic fullerene (C60-Dex-COOH) anionic polymers, coated on the pullulan-spermine and siRNA complex (PS/siRN A) to reduce the adsorption of serum protein. The particle size and Zeta potential were analyzed by DLS/ELS, respectively. The formation of anion coating and bovine serum albumin (BSA) adsorption of complexes were tested by QCM-D. Cellular uptake, cytotoxicity on the Hela cells and RNAi efficiency on the Hela-EGFP cells was examined by cck-8 reagent and flow cytometry. The results showed that Dex-COOH and C60-Dex-COOH could coat on PS/siRNA to form stable electronegative complexes, prevent the adsorption of seurm effectively. Cytotoxicity of complexes was reduced in the absence of serum environment. Meanwhile, in the serum environment, anionic polymer coated complexes could enter cells insusceptibly and PS/siRNA@C60-Dex-COOH complex with visible light irradiation would escape from the lysosome, increased the interference efficiency by 20% compared with PS/siRNA.