Virus like particles obtained from the Cowpea Chlorotic Mottle Virus (CCMV) represent an innovative platform for drug delivery applications. Their unique reversible self-assembly properties as well as their suitability for both cargo loading and functionalization make them a versatile scaffold for numerous purposes. One of the main drawbacks of this platform is however its limited stability at physiological conditions. Herein, we report the development of a general reversible cross-linking strategy involving the homobifunctional cross-linker DTSSP (3,3′-dithiobis (sulfosuccinimidylpropionate)) which is suitable for particle stabilization. This methodology is adaptable to different CCMV variants in the presence or absence of a stabilizing cargo without varying neither particle shape nor size thus extending the potential use of these protein cages in nanomedical applications. Cross-linked particles are stable at neutral pH and 37 °C and they are capable of protecting loaded cargo against enzymatic digestion. Furthermore, the reversible nature of the cross-linking ensures particle disassembly when they are taken up by cells. This was demonstrated via the highly effective delivery of active siRNA into cells.

Due to its small size, easy accessibility and immune privileged environment, the eye represents an ideal target for therapeutic nucleic acids in the treatment of posterior segment ocular diseases, such as age‐related macular degeneration (AMD). Among nanocarriers that can be used to achieve nucleic acid delivery, virus‐like particles (VLPs) obtained from the Cowpea chlorotic mottle virus (CCMV) are an appealing platform, because of their loading capacity, ease of manufacture and amenability for functionalization. Herein, antisense oligonucleotide‐loaded CCMV nanoparticles, intended for intravitreal injection, are evaluated for selective silencing of miR‐23, an important target in AMD. CCMV nanoparticles loaded with anti‐miR‐23 locked nucleic acid and stabilized using the 3,3′‐dithiobis(sulfosuccinimidyl propionate) (DTSSP) cross‐linker, are assembled in vitro with a loading efficiency up to 80%. VLPs are found to be stable at 37 °C in the vitreous humor up to 24 hours. Nanoparticle cytotoxicity, cellular uptake and transfection efficacy are evaluated in endothelial cells. Selective miRNA down‐regulation is achieved by the loaded CCMV VLPs both in absence and presence of Lipofectamine, with efficacies of ≈40% and more than 80%, respectively. The authors' findings pave the way for the future development of CCMV nanoparticles as oligonucleotide delivery platform to treat posterior segment ocular diseases.

The N-terminal localization and dynamic intermixing of engineered cowpea chlorotic mottle virus-like particles were studied independently from each other.

Purpose Investigation 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. Methods Anti‐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). Results siRNA‐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. Conclusions CCMV 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.