Intranasal delivery of vaccines is a needle-free route that can induce production of mucosal antibodies and cellular responses to neutralize viruses and pathogens before entering systemic circulation. Moreover, vaccine stimulation of nasal-associated lymphoid tissue can produce persistent immunological memory and provide an easily accessible route to the immune system. However, nasal secretions and mucosa are hydrogel-like biological barriers that inhibit the delivery of vaccine nanoparticles and antigens to nasal-associated lymphoid tissue. Previously, we have determined that protein nanoclusters made from M2E ectodomain protein have elicited high levels of serum antigen-specific IgG via intranasal delivery in mice models. We hypothesized that providing surface modification on protein nanoclusters (PNC) will enable tunable behavior for improved mucosal delivery and mucosal diffusion compared to unmodified nanoclusters. In this study, we engineered several PNC variants using ovalbumin for the model antigen PNC core. Surface charge and muco-adhesiveness were modulated to control the nasal residence time and delivery in mucus. We have modified the surface charge and chemistry of ovalbumin PNC using different surface conjugations: Anionic PNC were fabricated using the native surface charge from ovalbumin PNC after desolvation process. Cationic PNC were fabricated using PNC cores conjugated with ethylenediamine by 1-3-3-dimethlamino-propyl Carbodiimide crosslinking, Neutral PNC were fabricated by pegylation of PNC with O-[(N-Succinimidyl) succinyl-aminoethyl]-O′-Methyl polyethylene glycol, and noncovalent coating was achieved using a layer-by-layer (LBL) PNC approach. Both bulk and multiple particle tracking (MPT) analysis in porcine mucus were used to evaluate the diffusion of the modified PNCs, and periodic acid–schiff (PAS) assay quantified the adsorption of mucus on the engineered PNC. We revealed distinct diffusion profiles for each coating, with a unique bi-modal profile for LBL PNCs, which included a population of fast diffusing nanoclusters. This work provides fundamental understanding of the role of surface chemistry in protein nanoparticle diffusion in mucus and can be used in future research to evaluate immunogenicity and biodistribution of PNCs or other types of protein nanoparticles as intranasal nanoparticle vaccines and coupled with nasal protein corona analysis enable to better understand biological surface changes in nasal solution.

Engineering coatings for protein nanoclusters to understand intranasal biological barriers

ACS Spring 2022

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