posted on 2024-01-18, 20:00authored byAhmed Ali, Suwannee Ganguillet, Yagmur Turgay, Timothy G. Keys, Erika Causa, Ricardo Fradique, Viviane Lutz-Bueno, Serge Chesnov, Chia-Wei Tan-Lin, Verena Lentsch, Jurij Kotar, Pietro Cicuta, Raffaele Mezzenga, Emma Slack, Milad Radiom
Virus-like particles (VLPs) are emerging as nanoscaffolds
in a
variety of biomedical applications including delivery of vaccine antigens
and cargo such as mRNA to mucosal surfaces. These soft, colloidal,
and proteinaceous structures (capsids) are nevertheless susceptible
to mucosal environmental stress factors. We cross-linked multiple
capsid surface amino acid residues using homobifunctional polyethylene
glycol tethers to improve the persistence and survival of the capsid
to model mucosal stressors. Surface cross-linking enhanced the stability
of VLPs assembled from Acinetobacter phage AP205
coat proteins in low pH (down to pH 4.0) and high protease concentration
conditions (namely, in pig and mouse gastric fluids). Additionally,
it increased the stiffness of VLPs under local mechanical indentation
applied using an atomic force microscopy cantilever tip. Small angle
X-ray scattering revealed an increase in capsid diameter after cross-linking
and an increase in capsid shell thickness with the length of the PEG
cross-linkers. Moreover, surface cross-linking had no effect on the
VLPs’ mucus translocation and accumulation on the epithelium
of in vitro 3D human nasal epithelial tissues with
mucociliary clearance. Finally, it did not compromise VLPs’
function as vaccines in mouse subcutaneous vaccination models. Compared
to PEGylation without cross-linking, the stiffness of surface cross-linked
VLPs were higher for the same length of the PEG molecule, and also
the lifetimes of surface cross-linked VLPs were longer in the gastric
fluids. Surface cross-linking using macromolecular tethers, but not
simple conjugation of these molecules, thus offers a viable means
to enhance the resilience and survival of VLPs for mucosal applications.