Polyethylenimine (PEI) is a highly efficient cationic polymer for nucleic acid delivery, and although it is commonly used in preclinical studies, its clinical application is limited because of concerns regarding its cytotoxicity. Poly(β-amino ester)s are a new group of biodegradable and biocompatible cationic polymers that can be used for siRNA delivery. In this study, we synthesized Boc-protected and deprotected poly(β-amino ester)s, P(BSpBAE) and P(SpBAE), respectively, based on spermine and 1,4-butanediol diacrylate to deliver siRNA. The polymers were synthesized by Michael addition in a step-growth polymerization and characterized via 1H NMR spectroscopy and size-exclusion chromatography (SEC). The polymers can encapsulate siRNA as determined by SYBR gold assays. Both polymers and polyplexes were biocompatible in vitro. Furthermore, the cellular uptake of P(BSpBAE) and P(SpBAE) polyplexes was more efficient than for branched PEI (25 kDa) polyplexes at the same N/P ratios. P(BSpBAE) polyplexes achieved 60% eGFP knockdown in vitro, which indicates that the Boc-protection can improve the siRNA delivery and gene silencing efficiency of PBAEs. P(BSpBAE) polyplexes and P(SpBAE) polyplexes showed different cellular uptake mechanisms, and P(BSpBAE) polyplexes demonstrated decreased endosomal entrapment, which could explain why P(BSpBAE) polyplexes more efficiently mediated gene silencing than P(SpBAE) polyplexes. Furthermore, transfection of an siRNA against mutated KRAS in KRAS-mutated lung cancer cells led to around 35% (P(BspBAE)) to 45% (P(SpBAE)) inhibition of KRAS expression and around 33% (P(SpBAE)) to 55% (P(BspBAE)) decreased motility in a migration assay. These results suggest that the newly developed spermine-based poly(β-amino ester)s are promising materials for therapeutic siRNA delivery.
Keywords: KRAS; PBAE; lung cancer; poly(β-amino ester)s; siRNA delivery; spermine.