Synthesis and application of spermine-based amphiphilic poly(β-amino ester)s for siRNA delivery

Abstract

Small interfering RNA (siRNA) can trigger RNA interference (RNAi) to therapeutically silence disease-related genes in human cells. The approval of siRNA therapeutics by the FDA in recent years generated a new hope in novel and efficient siRNA therapeutics. However, their therapeutic application is still limited by the lack of safe and efficient transfection vehicles. In this study, we successfully synthesized a novel amphiphilic poly(β-amino ester) based on the polyamine spermine, hydrophobic decylamine and 1,4-butanediol diacrylate, which was characterized by ¹H NMR spectroscopy and size exclusion chromatography (SEC, M_n = 6000 Da). The polymer encapsulated siRNA quantitatively from N/P 5 on as assessed by fluorescence intercalation while maintaining optimal polyplex sizes and zeta potentials. Biocompatibility and cellular delivery efficacy were also higher than those of the commonly used cationic, hyperbranched polymer polyethylenimine (PEI, 25 kDa). Optimized formulations mediated around 90% gene silencing in enhanced green fluorescence protein expressing H1299 cells (H1299-eGFP) as determined by flow cytometry. These results suggest that spermine-based, amphiphilic poly(β-amino ester)s are very promising candidates for efficient siRNA delivery.

Scheme 1. Synthesis route of P(SpDBAE) (8) by reacting tri-boc spermine (4) with decylamine (5) and 1,4-butanediol diacrylate (6) resulting in P(BSpDBAE) (7) followed by deprotection.

Fig. 1. (A) Hydrodynamic diameter (size) and polydispersity index (PDI) of P(SpDBAE) polyplexes and (B) zeta potential of P(SpDBAE) polyplexes at various N/P ratios (mean ± SD, n = 3).

Fig. 2. siRNA encapsulation profiles of P(SpDBAE) polyplexes measured by SYBR gold assays at various N/P ratios. 100% values (N/P = 0) are represented by the determined fluorescence of uncondensed free siRNA (data points indicate mean ± SD, n = 3).

Fig. 3. TEM images of P(SpDBAE)polyplexes at N/P 10.

Fig. 4. Cell viability of P(SpDBAE) and P(SpDBAE) polyplexes determined by dimethylthiazolyl blue diphenyltetrazolium bromide (MTT) assay in H1299 cells (mean ± SD, n = 3, two-way ANOVA with Bonferroni multiple comparison test, ns: p > 0.05, *: p < 0.5).

Fig. 5. Cellular uptake of P(SpDBAE) polyplexes quantified by flow cytometry and presented as median fluorescence intensity corrected for autofluorescence of untreated blank cells (H1299 cells, AF488-siRNA, mean ± SD, n = 3, statistics of the quenched group, one-way ANOVA with Bonferroni multiple comparison test, ***: p < 0.001, ns: p > 0.05).

Fig. 6. Enhanced green fluorescent protein (eGFP) knockdown of P(SpDBAE) polyplexes in H1299 cells expressing eGFP quantified by flow cytometry as median fluorescence intensity, LF: Lipofectamine™ 2000 (mean ± SD, n = 3, one-way ANOVA with Bonferroni multiple comparison test, ***p < 0.001).