Gag HIV-1 Virus-like Particles and Extracellular Vesicles Functionalization with Spike Epitopes of SARS-CoV-2 Using a...

Area of Science:

• Bioconjugation Chemistry
• Nanomedicine
• Vaccinology

Background:

• Enveloped nanoparticles like extracellular vesicles (EVs) and virus-like particles (VLPs) are promising nanocarriers for drug delivery and vaccination.
• Optimized functionalization is crucial for enhancing nanoparticle performance and increasing their loading capacity for bioactive molecules.

Purpose of the Study:

• To optimize a bioorthogonal copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) reaction for functionalizing human immunodeficiency virus type 1 (HIV-1) Gag-based VLPs and EVs.
• To investigate the functionalization levels and distribution on both VLPs and EVs using Cy5 as a reporter molecule.
• To evaluate the potential of the optimized functionalization approach for developing nanoparticle-based vaccines using SARS-CoV-2 epitopes.

Main Methods:

• Optimization of the SPAAC reaction using reaction kinetics and design of experiments (DoE) with Cy5 as a reporter molecule.
• Functionalization of Gag-VLPs and EVs with Cy5.
• Analysis of nanoparticle functionalization using super-resolution fluorescence microscopy (SRFM) and immunoassay with sera from COVID-19 patients.

Main Results:

• Remarkable differences in functionalization were observed between Gag-VLPs and coproduced EVs.
• EVs functionalized with Cy5 achieved high covalent linkage densities: 3618.63 ± 48.91 molecules/particle for mock transfection and 6498.75 ± 352.71 molecules/particle for cell growth.
• Functionalized nanoparticles carrying SARS-CoV-2 B-cell epitopes were successfully analyzed, validating the epitopes and the functionalization strategy.

Conclusions:

• The optimized SPAAC reaction provides an efficient method for high-level functionalization of EVs and VLPs.
• This approach demonstrates significant potential for the development of advanced nanoparticle-based vaccines and drug delivery systems.
• The study highlights the utility of bioorthogonal chemistry in tailoring nanoparticle properties for biomedical applications.