Hydrogel crosslinking mechanisms influence the release and functional delivery of lipid nanoparticles

Hydrogels have emerged as attractive vaccine delivery platforms because they enable controlled modulation of antigen availability. However, how different hydrogel environments affect the release and functionality of mRNA-loaded lipid nanoparticles (mRNA-LNPs) remains poorly understood. Here, we investigated the release, stability, cellular uptake, and transfection capability of LNPs released from four hydrogel systems representing distinct crosslinking mechanisms: covalently crosslinked poly(ethylene glycol) (PEG), ionically crosslinked alginate, thermoresponsive Poloxamer 407 (P407), and protein-based Matrigel/collagen hydrogels. All hydrogel types enabled release of LNPs over days, with lower polymer concentrations generally exhibiting faster nanoparticle release. PEG offered greatest tunability of release kinetics; at the same time the recovery of the LNP-incorporated fluorescent dye DiI was reduced to about 80 %, indicating partial dye leakage. Alginate hydrogels exhibited recovery of DiI below 50 % and broader particle size distributions after release, while P407 hydrogels largely preserved LNP characteristics. CyroTEM analysis revealed that mRNA-LNPs kept at 37 , including those released from hydrogels, exhibited a more compact and electron-dense morphology than mRNA LNPs maintained at 4 . Notably, although quantitative recovery from Matrigel/collagen hydrogels was limited, released LNPs achieved approximately 80-90 % of the eGFP expression compared to mRNA-LNPs that have not been embedded. Importantly, cellular uptake and transfection experiments demonstrated that all investigated hydrogels released biologically active mRNA-LNPs capable of mediating protein expression. Moreover, our findings show that hydrogel composition is a critical determinant of mRNA-LNP release, stability, functional delivery and morphology of the released particles. This work provides design principles for the development of hydrogel-based mRNA delivery systems aimed at sustained antigen availability and prolonged vaccine responses.