Effects of Amino Side Chain Chemistry on Polyaspartamide-Based Polyplexes for mRNA Delivery

Cationic units in synthetic carriers are a key determinant for the efficient intracellular delivery of mRNA, but their systemic evaluation for mRNA translation efficacy in target cells was not fully understood. A series of amphiphilic polyaspartamide derivatives (PAsp(R/CHE)) with various amino (R) groups and cyclohexylethylamine (CHE) were synthesized to understand the effects of R groups in polyplex formation and their intracellular delivery efficacies of mRNA, including mRNA binding, intracellular uptake, and endosomal escape. All PAsp(R/CHE)s successfully formed polyplexes with firefly luciferase-coded mRNA (FLuc mRNA), having a hydrodynamic diameter of 90–160 nm with a relatively narrow polydispersity index and positive surface charges. PAsp(R/CHE) polyplexes with R = diethylenetriamine (DET) and 2,2′-diamino-N-methyldiethylamine (MDE) exhibited the highest FLuc mRNA expression levels in two cancer cell lines with different endo/lysosomal acidification rates. Interestingly, correlation studies revealed that the FLuc mRNA delivery efficacies were not influenced by intracellular uptake, surface charges, and stabilities of polyplexes, all of which are determined by the cationic units in polymers. Endosomal escape abilities of PAsp(R/CHE) polyplexes mainly determined in vitro mRNA delivery efficacies. Unique buffering capacities of PAsp(DET/CHE) and PAsp(MDE/CHE) induced high endosome escape in the two cancer cells. These findings contribute to the growing understanding of polymer-based delivery platforms and their potential applications for mRNA-based therapeutics.