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 (C₆₀-Dex-NH₂) for efficient and controllable siRNA delivery. To synthesize C₆₀-Dex-NH₂, terminally aminated dextran was conjugated to C₆₀. The conjugate was further cationized by covalently introducing ethylenediamine to the dextran. The physicochemical characteristics of C₆₀-Dex-NH₂ was examined with elemental analyses, gel permeation chromatography, solid-state nuclear magnetic resonance (¹³C, HPDEC), agarose gel electrophoresis, and dynamic light scattering. The cytotoxicity, cellular uptake, intracellular distribution, and in vitro RNA interference (RNAi) of siRNA/C₆₀-Dex-NH₂ complex was evaluated in the human breast cancer cell line MDA-MB-231. The RNAi efficiencies mediated by C₆₀-Dex-NH₂in vivo was evaluated in subcutaneous tumor-bearing mice. The results showed that C₆₀-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, C₆₀-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 (C₆₀-Dex-NH₂) for efficient and controllable siRNA delivery, which can be used in gene therapy. We showed that C₆₀-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 C₆₀-Dex-NH₂ system is C₆₀ has broad absorbance spectrum and can be activated by weak visible light; Furthermore, C₆₀-Dex-NH₂ has a specific amphiphilic structure, which may prevent siRNA from degrading and allows C₆₀-Dex-NH₂ to embed into the lipid membrane of lysosome to improve the ROS induced lysosomal disturbance after internalization.