Elsevier

Biomaterials

Volume 32, Issue 2, January 2011, Pages 628-638
Biomaterials

Enhanced gene transfection and serum stability of polyplexes by PDMAEMA-polysulfobetaine diblock copolymers

https://doi.org/10.1016/j.biomaterials.2010.09.021Get rights and content

Abstract

Polyethylene glycol or phosphorylcholine is often introduced into polycationic non-viral vectors to inhibit the non-specific protein adsorption. However the ability of vectors to condense DNA and the cellular internalization of complexes are unavoidably compromised. In this work, a polysulfobetaine-cationic methacrylate copolymer: 2-(dimethylamino) ethyl methacrylate-block-(N-(3-(methacryloylamino) propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide) (PDMAEMA-b-PMPDSAH) diblock copolymer was synthesized via atomic transfer radical polymerization method and investigated as a new non-viral vector for gene delivery. Incorporation of polysulfobetaine into cationic methacrylate retained a better DNA condensation capability. MTT assays revealed that the cytotoxicity of PDMAEMA200-PMPDSAHn copolymer was lower than that of PDMAEMA200. PDMAEMA200-PMPDSAH80 which was much superior to its homopolymer in mediating gene transfection demonstrated comparable efficiency to PEI25 kDa at a weight ratio of 8 in the presence of 10% serum. At higher serum contents, the transfection of PDMAEMA200 and PEI25 kDa was deteriorated, whereas PDMAEMA200-PMPDSAH80 still retained better transfection efficiency, 4–5 fold more effective than PEI25 kDa. For the sake of comparative study, we synthesized structurally similar copolymer from DMAEMA and 2-methacryloyloxyethyl phosphorylcholine, PDMAEMA200-PMPC80. PDMAEMA200-PMPDSAH80 exhibited much higher gene transfer levels than PDMAEMA200-PMPC80 under the same conditions. The results of flow cytometry indicated that highly hydrophilic MPC block profoundly impeded the cellular internalization of nanocomplexes; in contrast, incorporation of polysulfobetaine remained the increased cellular uptake. Differential scanning calorimetry assay of thermodynamic phase transition of dipalmitoyl-sn-glycero-3-phosphocholine(DPPC) induced by polymer vectors demonstrated that MPC only marginally contributed to the perturbation of DPPC; polysulfobetaine facilitated more evident perturbation of DPPC bilayer instead, an indication that polysulfobetaine units could aid in the endocytosis of nanocomplexes.

Introduction

Non-viral vectors have become an attractive alternative to viral vectors due to their merits such as safety, reproducibility, large gene-carrying capacity and design flexibility [1]. Although many polycationic delivery systems have been developed and even evaluated for possible application in vivo [2], [3], the positively charged polyelectrolyte/DNA complexes suffer from colloidal aggregation when administered intravenously, which leads to a rapid clearance of DNA-containing particles from bloodstream [4].

Polyethylene glycol(PEG)-based hydrophilic macromolecules are commonly used to form sterically stabilized polycation/DNA complexes. This approach can efficiently reduce non-specific protein adsorption to the polyplexes under in vitro or in vivo conditions [5], [6], [7], [8], [9]. However, the PEGylation has been reported to have a negative effect on transfection efficiency of polyplexes [10], [11], [12], [13], [14]. In vitro studies suggested that the PEGylation showed an inhibitive effect on the cellular uptake of the polyplexes [11], [12], [13], and PEG-modified complex dissociated prematurely under the physiological environment, leading to the degradation of the DNA by serum nucleases [8], [14]. In addition, the susceptibility of PEG to oxidation damage after long-term exposure to biological fluids has limited its applications in vivo [15], [16], [17].

Recently, zwitterionic polymers such as polyphosphorylcholine [18], polysulfobetaine [19] and polycarboxybetaine [20], arguably a promising alternative to conventional PEG, have been shown to exhibit ultra-high resistance to non-specific protein adsorption [21], [22] and long-term resistance to bacterial biofilm formation [20], [23]. 2-(Dimethylamino)ethyl methacrylate-block-2-(methacryloyloxyethyl phosphorylcholine) (DMAEMA–MPC) diblock copolymer was reportedly synthesized as a new gene delivery vector where the hydrophilic MPC blocks provided steric stabilization to the polymer/DNA complexes and hence ensured a good colloidal stability [24], [25]. However, introducing MPC moieties into the copolymer adversely influenced the transfection efficiency too. Armes et al. suggested that the water molecules around hydrated MPC layer contributed to the weakening of DNA condensation ability [24], [25]. It was also found that the strong hydrophilicity of PEG segments significantly reduced the binding strength of polycation with DNA, thereby rendering DNA more susceptible to serum nucleases [10], [14]. Based on the published literature on designing stealth complexes, it seems that incorporation of hydrophilic macromolecules to enhance the stability of complex unavoidably compromises the cellular internalization as well as the ability of a vector to bind DNA. In our previous work, we demonstrated that polysulfobetaine was able to condense DNA into nanoparticle at an appropriate complexing ratio [26]. This unique behavior led us to reason that we can construct a novel non-viral vector which can maintain a strong ability to condense DNA as well as higher transfection efficiency by incorporating sulfobetaine units into cationic polymers. As far as we know, this is the first example of report on the polyzwitterion-modified vectors without sacrificing the transfection efficiency.

In this study, the cationic 2-(dimethylamino) ethyl methacrylate/zwitterionic (N-(3-(methacryloylamino)propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide) diblock copolymers will be synthesized using atom transfer radical polymerization (ATRP) method, which allows block lengths to be controlled by varying the monomer/initiator molar ratio. The physicochemical properties of copolymer/DNA complexes, cytotoxicity, in vitro transfection as well as the cellular internalization of complexes will be investigated.

Section snippets

Materials

N-(3-(methacryloylamino) propyl)-N,N-dimethyl-N-(3-sulfopropyl) ammonium hydroxide (MPDSAH, 97%), ethyl α-bromoisobutyrate(EbiB,97%),copper(I) chloride (CuCl,99%), N,N,N′,N′,N″-pentamethyldiethylenetriamine (PMDETA, 99%) and dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Sigma–Aldrich Chemical Co. 2-(Dimethylamino)ethyl methacrylate (DMAEMA, 97%) was supplied by Alfa Aesar Co. 2-Methacryloyloxyethyl phosphorylcholine(MPC, 99%) was purchased from Biocompatibles Co. YOYO-1(1 mm

Synthesis and characterization of PDMAEMA200-PMPDSAHn copolymers

PDMAEMA200-PMPDSAHn diblock copolymers were prepared in the same manner as described in our previous work [26]. In general, copolymers with longer cationic blocks results in more stable condensates while complexing DNA, whereas introducing relatively longer PEG or MPC components tends to suppress electrostatic interaction between polycations and DNA [10], [24], [25]. Our experiments revealed that although PMPDSAH was able to condense DNA, it could not be used solely as an effective gene

Conclusions

In summary, PDMAEMA-b-PMPDSAH diblock copolymer synthesized via ATRP method was explored as a non-viral vector for gene delivery. We demonstrated that copolymerization of DMAEMA with sulfobetaine did not compromise gene transfection performance, which offered a promising alternative to polyethylene glycol- and MPC-modified vectors. Introducing polysulfobetaine into cationic methacrylate retained a better DNA condensation capability. PDMAEMA200-PMPDSAHn copolymers showed much higher transfection

Acknowledgements

The authors gratefully acknowledge the support for this work from the National Natural Science Foundation of China (Grants 30770587, 50973082), High Tech Research and Development (863) Programme of China (Grant 2007AA022002) and Tianjin Municipal Natural Science Foundation (Grant 10JCZDJC17400).

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