Visible light-switched cytosol release of siRNA by amphiphilic fullerene derivative to enhance RNAi efficacy in vitro and in vivo

Cationic macromolecules are attractive for use as small interfering RNA (siRNA) carriers due to their performance in non-immunological reactions, customization during synthesis, and low costs compared to viral carriers. However, their low transfection efficiency substantially hinders their application in both clinical practices and academic research, which is mostly attributable to the low capacity of siRNA/cationic macromolecule complexes to escape lysosomes. To address this challenge, we designed an amphiphilic fullerene derivative (C-60-Dex-NH2) for efficient and controllable siRNA delivery. To synthesize C-60-Dex-NH2, terminally aminated dextran was conjugated to C-60. The conjugate was further cationized by covalently introducing ethylenediamine to the dextran. The physicochemical characteristics of C-60-Dex-NH2 was examined with elemental analyses, gel permeation chromatography, solid-state nuclear magnetic resonance (C-13, HPDEC), agarose gel electrophoresis, and dynamic light scattering. The cytotoxicity, cellular uptake, intracellular distribution, and in vitro RNA interference (RNAi) of siRNA/C-60-Dex-NH2 complex was evaluated in the human breast cancer cell line MDA-MB-231. The RNAi efficiencies mediated by C-60-Dex-NH2 in vivo was evaluated in subcutaneous tumor-bearing mice. The results showed that C-60-Dex-NH2 has a specific amphiphilic skeleton and could form micelle-like aggregate structures in water, which could prevent siRNA from destroying by reactive oxygen species (ROS). When exposed to visible light, C-60-Dex-NH2 could trigger controllable ROS generation which could destroy the lysosome membrane, promote the lysosomal escape, and enhance the gene silencing efficiency of siRNA in vitro and in vivo. The gene silencing efficiency could reach a maximum of 53% in the MDA-MB-231-EGFP cells and 69% in the 4T1-GFP-Luc2 tumor-bearing mice. Statement of Significance We designed a novel photosensitive amphiphilic carrier (C-60-Dex-NH2) for efficient and controllable siRNA delivery, which can be used in gene therapy. We showed that C-60-Dex-NH2 could destroy lysosome membrane via controllable generation of ROS when exposed to light, which can help siRNA to escape from lysosome before degradation. This can enhance the gene silencing efficiency significantly and provides a useful way to regulate RNAi efficiency by light. One advantage for C-60-Dex-NH2 system is C-60 has broad absorbance spectrum and can be activated by weak visible light; Furthermore, C-60-Dex-NH2 has a specific amphiphilic structure, which may prevent siRNA from degrading and allows C-60-Dex-NH2 to embed into the lipid membrane of lysosome to improve the ROS induced lysosomal disturbance after internalization. (C) 2017 Published by Elsevier Ltd on behalf of Acta Materialia Inc.