Polymeric delivery systems are interesting alternatives to the clinically advanced lipid nanoparticles (LNPs) used in the first-generation messenger RNA (mRNA) vaccines that are characterized by liver tropism and high shear stress sensitivity, which challenges manufacturing, storage, and handling. Here, we uncover design criteria for less studied lipid-polymer hybrid nanoparticle (LPN)-based mRNA vaccines for intramuscular administration, composed of the safe matrix-forming biocompatible polymer poly(D,L-lactic-co-glycolic acid) (PLGA), ionizable lipid, helper lipid, and polyethylene glycol-conjugated lipid. Scalable microfluidics was used for manufacturing, and the influence of critical process and formulation parameters on mRNA-loaded LPNs (mRNA-LPNs) was systematically investigated. Intracellular mRNA delivery and endosomal escape proved to be mainly dependent on the total lipid content and the ionizable lipid:mRNA weight ratio. The mRNA-LPNs induced high protein expression in vivo, which was restricted to the intramuscular injection site, whereas nanoparticles without PLGA comprising only ionizable and helper lipids, also mediated protein expression in the liver. Microscopy analyses suggest that the flow rate ratio (FRR) is of paramount importance for the nanostructure: mRNA-LPNs formulated at lower FRRs adopt a polymer core-shell hybrid structure, whereas higher FRRs result in co-existence of multi-lamellar vesicles and nanospheres, suggesting that lipids and polymer self-assemble at different rates, resulting in a heterogeneous formulation. In mice, LPNs loaded with mRNA encoding the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induced spike-specific CD8T-cell and antibody responses, which were on par with responses induced by mRNA-LNPs. Upon vaccination and SARS-CoV-2 challenge of Syrian golden hamsters, mRNA-LPNs induced high spike-specific IgG responses and were more efficient in reducing the SARS-CoV-2 load in the nasal cavity, compared to mRNA-LNPs. Hence, mRNA-LPN vaccines represent viable alternatives to conventional mRNA-LNPs due to their restricted protein expression at the intramuscular injection site and enhanced ability to reduce the SARS-CoV-2 burden in the nasal cavity of infected hamsters. +
