Abstract
AbstractPurposeInvestigation on the applicability of Virus‐like particles (VLPs) based on the Cowpea Chlorotic Mottle Virus (CCMV) for the selective treatment of age‐related macular degeneration (AMD) exploiting RNAi technology.MethodsAnti‐luciferase siRNA was selected as a prototype cargo (Luciferase GL3 Duplex, Dharmacon). CCMV‐siRNA loaded nanoparticles were formulated and stabilization of the protein scaffold was achieved by crosslinking the particle surface with a disulfide containing crosslinking agent DTSSP (3,3′‐dithiobis[sulfosuccinimidylpropionate], Thermo Fisher). Particles were characterized by Transmission Electron Microscopy, Dynamic Light Scattering and Size Exclusion Chromatography. CCMV‐induced siRNA protection against enzymatic digestion with benzonase was assessed exploiting agarose gel electrophoresis. The efficacy of siRNA‐loaded nanoparticles was investigated in vitro exploiting the luciferase assay kit (Promega).ResultssiRNA‐loaded CCMV spherical nanoparticles of 28 nm in diameter were formulated. A 20:1 DTSSP: CP molar ratio was found to be optimal for particle crosslinking and stabilization in physiological conditions. Crosslinked siRNA‐loaded particles showed to be stable at 37°C for at least 12 hr. The drug delivery system showed the capability to protect encapsulated siRNA from degradation. A loading efficiency of 56% corresponding to 22 siRNA molecules per capsid was assessed. Particles incubated with Lipofectamine2000 were able to effectively knock down luciferase expression in HeLa‐Luc cells.ConclusionsCCMV nanoparticles represent an innovative platform for oligonucleotide delivery to the posterior segment of the eye due to their dynamic structure, high loading capacity as well as their cargo protective effect.This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No 722717.
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