Structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles in siRNA delivery

Abstract

The multiformity in polymer structure and conformation design provides a great potential in improving the gene silencing efficiency of siRNA by polymer vectors. In order to provide information on the polymer design for siRNA delivery, the structural contributions of blocked or grafted poly(2-dimethylaminoethyl methacrylate) on PEGylated polycaprolactone nanoparticles (NPs) in siRNA delivery were studied. Herein, two kinds of self-assembly nanoparticles (NPs) formed by amphiphilic cationic polymers, methoxy poly(ethylene glycol)-block-polycaprolactone-block-poly(2-dimethylaminoethyl methacrylate) (mPEG-PCL-b-PDMAEMA, PECbD) and methoxy poly(ethylene glycol)-block-(polycaprolactone-graft-poly(2-dimethylaminoethyl methacrylate)) (mPEG-PCL-g-PDMAEMA, PECgD), were used to deliver siRNA for in vitro and in vivo studies. The physiochemical properties including size and zeta potential of PECbD NPs/siRNA and PECgD NPs/siRNA complexes were characterized. In vitro cytotoxicity, cellular uptake and siRNA knockdown efficiency were evaluated in HeLa-Luc cells. The endosome escape and intracellular distribution of PECbD NPs/siRNA and PECgD NPs/siRNA in HeLa-Luc cells were also observed. In vivo polymer mediated siRNA delivery and the complexes distribution in isolated organs were studied using mice and tumor-bearing mice. At the same total degree of polymerization (DP) of DMAEMA, PECgD NPs/siRNA complexes possessed higher zeta potentials than PECbD NPs/siRNA complexes (at the same N/P ratio), which may be the reason that PECgD NPs/siRNA complexes can deliver more siRNA into the cytoplasm and lead to higher in vitro luciferase and lamin A/C silencing efficiency than PECbD NPs/siRNA complexes. The in vivo imaging measurement and histochemical analysis also confirmed that siRNA could be delivered to lungs, livers, pancreas and HeLa-Luc tumors more efficiently by PECgD NPs than PECbD NPs. Meanwhile, the PDMAEMA chains of PECgD could be shortened which provides benefits for clearing. Therefore, PECgD NPs have great potential to be used as efficient non-viral carriers for in vivo siRNA delivery.

Introduction

RNA interference, a natural mechanism of gene silencing in both plant and mammalian cells, has been proven an effective way to inhibit mammalian gene function [1], [2], [3], [4]. Small interfering RNA (siRNA), which is generally composed of 21–23 nucleotide (nt) double-stranded RNA (dsRNA) segments and is able to suppress gene expression for therapeutic purposes [5], has already been demonstrated as a potential therapeutic agent in vivo. Whereas, there are delivery limitations of naked siRNA in vivo, such as: (a) it tends to be degraded by Rnases; (b) naked siRNA can’t be transported through the cell membrane freely; and (c) targeting siRNA at a specific organ is problematic [6]. Therefore, there remains a significant challenge for the delivery of siRNA to realize its full therapeutic potential.

To help the active siRNA access the cytoplasm of the target cell, various delivery systems have been developed, which can typically be classified into two groups: viral and non-viral delivery systems. Viral vectors are highly efficient delivery systems for nucleic acids; however, the potential of host immune response and the high cost of production limit their applications [7]. Non-viral delivery systems include liposome systems [8], [9] and polymer systems. Synthetic cationic polymers have drawn much attention in the past decades because of low immunoreaction in vivo, controllable structure and easy production, such as branched or linear polyethylenimine (PEI) and its derivatives [10], [11], polyamidoamine (PAMAM) [12], [13], poly(l-lysine) (PLL) [14], poly(β-amino ester) (PAE) [15], [16] and poly(2-dimethylaminoethyl methacrylate) (PDMAEMA) [17] etc.

The gene silencing efficiency of polymer vectors/siRNA complexes is lower than that of the viral vectors, but the flexibility in conformation design provides great promising potential in improving the siRNA delivery efficiency of polymer vectors to a level comparable to that of viral vectors. In this work, block and graft structural contributions of cationic segments on PEGylated PCL NPs for siRNA delivery were discussed, in order to provide some suggestions in the rational formulation design of polymer vectors.

Our previous research has showed that PEGylated PDMAEMA for DNA vaccine increased the immunogenicity of the intranasal administered DNA vaccine compared to the homopolymer PDMAEMA [18], due to the reduction in the cytotoxicity and prolongation in circulation time [19]. However, on the other hand, PEGylation also reduced gene transfection efficiency in vitro. In order to improve the gene transfection efficiency, we tried to introduce hydrophobic segments between PEG and PDMAEMA and prepared methoxy poly(ethylene glycol)-block-polycaprolactone-block-poly(2-dimethylaminoethyl methacrylate) (mPEG-PCL-b-PDMAEMA, PECbD) [20] and methoxy poly(ethylene glycol)-block-(polycaprolactone-graft-poly(2-dimethylaminoethyl methacrylate)) (mPEG-PCL-g-PDMAEMA, PECgD) [21]. Fortunately, our research results indicated that the introduction of the hydrophobic chains can improve cellular uptake and the DNA transfection efficiency in vitro was largely increased [22]. However, the delivery efficiency of PECbD and PECgD in vivo had not been studied. Herein, we aimed to resolve (1) if PECbD and PECgD are also suitable for siRNA delivery, since the molecular topography, conformational flexibility and action mechanism of siRNA are completely different from DNA [23], (2) how PECbD and PECgD perform in vivo as siRNA vectors, and (3) the distinction between the structural contributions of the blocked and grafted PDMAEMA of PECbD and PECgD. It could be expected that different structures of cationic segments may have different performance in siRNA delivery and present different knockdown efficiencies.