The gene transfection efficiency of thermoresponsive N,N,N-trimethyl chitosan chloride-g-poly(N-isopropylacrylamide) copolymer
Introduction
A number of non-viral vectors have been developed for gene therapy because of their higher safety and lower cost. Liposomes and cationic polymers are two major classes of non-viral gene delivery carriers [1]. Although the usage of cationic liposomes can bring the strong expression of an exogenous gene, complexes of cationic liposome/DNA are unstable in vivo [2]. Several cationic polymers such as polyethyleneimine (PEI), chitosan and poly-l-lysine (PLL) have been used to improve the transfection efficiency [3], [4]. They form polyelectrolyte complexes with plasmid DNA, which show stronger ability to protect DNA against enzyme degradation [5].
Chitosan is a biocompatible, biodegradable and low-cost polysaccharide which is nature origin. It shows structure versatility such as covalent binding of targeting moieties, which can mediate the gene expression via the specific recognition [6], [7], [8]. However, an increase in solubility and transfection efficiency is still required for the practical use as a gene vector. Chitosan consists of glucose amine units ((1,4)-linked poly(2-amino-2-deoxy-b-d-glucose or poly(d-glucosamine)) and has an apparent pKa of 5.5, which is soluble in acidic solution (pH 1–6) since most of the amino groups are protonated. It aggregates when the bulk pH is above 6 [9], [10]. Recent studies have also shown that only protonated soluble chitosan, i.e. in its uncoiled configuration, can trigger the opening of the tight junctions, and thereby facilitate the paracellular transport of hydrophilic compounds [10], [11].
To overcome this problem, a cationized chitosan derivative N,N,N-trimethyl chitosan chloride (TMC) has been synthesized and characterized [12], [13], [14]. TMC shows higher solubility in a broader pH range, and is capable of opening tight junctions of cells at physiological pH value, thus increasing the paracellular permeability [13], [14], [15]. The degree of quaternization and the molecular weight of TMC play important roles in its gene transfection efficiency and cytotoxicity [15], [16]. However, TMC still have some drawbacks. Firstly, the strong electrostatic interaction between TMC and DNA deters gene dissociation from its carrier inside cells, impeding the access of RNA polymerase to DNA so that the gene expression level is limited [17]. Secondly, TMC has been shown strong cytotoxicity in an in vitro cell culture experiment using HEK293 cells in our previous study [18] and L929 mouse fibroblasts in Kissel work [15]. Therefore, further improvement of the gene delivery efficiency and biocompatibility of TMC is urgently desirable.
One of the promising approaches to overcome these drawbacks is to graft hydrophilic or hydrophobic side chains to chitosan and TMC backbones. For example, Liu et al. [19] grafted hydrophobic alkyl side chains onto the chitosan molecules. They found that the transfection efficiency was increased when the side chains have larger molecular weight. The explanation is that the hydrophobic chains promote the cell entry and the unpacking of genes from the vectors. Kissel et al. [15] grafted hydrophilic poly(ethylene glycol) (PEG) onto the TMC molecules, resulting in improved biocompatibility.
Polymers capable of phase transition in response to external stimuli such as temperature represent another type of useful building blocks for gene transfer. Poly(N-isopropylacrylamide) (PNIPAAm) is a typical paradigm of thermosensitive polymers that undergo a coil-to-globule phase transition at 34 °C (lower critical solution temperature, LCST). The affinity of the PNIPAAm copolymers to DNA and the transfection efficiency of delivered DNA can be then controlled by temperature change. When the temperature is below LCST, the gene will be unpacked from its carrier, which is of beneficial to improve the gene transfection level. An easy way to combine the PNIPAAm with cationic vector is copolymerization [20], [21]. For example, NIPAAm, 2-(dimethylamino)ethyl methacrylate and butyl methacrylate were copolymerized, which were used to prepare plasmid DNA complexes. Oupicky et al. [22] synthesized PLL-g-PNIPAAm copolymer, and found that both the structural density and surface charge of the copolymer/DNA complexes could be adjusted by temperature change. Turk et al. [23], [24] reported that the PNIPAAm-PEI copolymer had lower cytotoxicity than pure PEI, which showed also higher transfection efficiency. Liu et al. [25] synthesized PNIPAAm-g-polyarginine vector. With variable temperature the vector showed equivalent transfection efficiency as Lipofectamine 2000. These authors synthesized also a carboxyl-terminated NIPAAm/vinyl laurate copolymer, which was then coupled onto chitosan [26]. The modified chitosan showed an LCST about 26 °C. The dissociation of the modified chitosan/DNA complexes could be tuned by varying the temperature. At 37 °C, the collapse of PNIPAAm is favorable for the formation of compact complexes. While at 20 °C, the hydrophilic and extended PNIPAAm chains facilitate the unpacking of DNA from the complexes.
In our previous study, we synthesized carboxylic end-capped PNIPAAm, which was then grafted onto poly(allylamine) (PAH) [27], [28]. The PAH-g-PNIPAAm copolymers exhibit an LCST in water at 34 °C regardless of their grafting ratios and PNIPAAm lengths, a temperature feasible for practical application. This phenomenon reminds us that incorporation of PNIPAAm in a manner of side chain grafting would produce thermosensitive gene vectors with best performance in terms of gene transfection efficiency and practical application. Driven by this concept, in this study, we shall couple the carboxylic terminated PNIPAAm to TMC. Temperature dependency of the TMC-g-PNIPAAm/DNA complexes shall be elucidated in terms of the physical properties and gene transfection efficiency. We shall show that this TMC-g-PNIPAAm copolymer has great potential to be used as gene vector with low cytotoxicity and high efficiency.
Section snippets
Materials
N-isopropylacrylamide (NIPAAm, Wako Chemical. Co., Japan) was purified by recrystallization in hexane and dried in vacuum at 25 °C. The carboxylic ended initiator, 4,4′-azobis(4-cyanovaleric acid) (75%) was purchased from Sigma-Aldrich. DNA (Fish Sperm, Sodium Salt, used as a model to study the physicochemical property of vector/DNA particles) and methylthiazoletetrazolium (MTT) were purchased from AMRESCO. Chitosan (degree of deacetylation is 90%, Mw∼100 kDa) was purchased from Haidebei Co.
Characterization of TMC-g-PNIPAAm
Following the synthesis routes shown in Scheme 1, TMC, PNIPAAm-COOH and TMC-g-PNIPAAm copolymers were successfully obtained, as characterized by the 1HNMR spectra (Fig. 1). After the quaternization reaction, there appeared a new peak at 3.2 ppm in the TMC spectrum (Fig. 1a), which is assigned to −N(CH3)3+. By comparing the intensity of peak at 2.0 ppm (assigned to –COCH3, deacetylation degree 90%), the quaternization degree was calculated as 12%. Previous study demonstrated that TMC with this
Conclusions
A thermoresponsive copolymer, trimethyl chitosan-g-poly(N-isopropylacrylamide) (TMC-g-PNIPAAm), was successfully synthesized. TMC-g-PNIPAAm copolymers with different grafting ratios show the same LCST (32 °C) as the PNIPAAm-COOH oligomers in PBS. Particles ranging from 200 to 900 nm are formed by mixing the copolymers or TMC with DNA, which are mainly controlled by the N/P ratio while less influenced by the temperature variation. The majority of the particles have spherical morphology. Along with
Acknowledgments
We gratefully acknowledge Dr. Jun Li and Dr. Guoping Sheng, medical school of Zhejiang University, kindly donated pEGFP. This study is financially supported by the Major State Basic Research Program of China (No. 2005CB623902), the Science and Technology Program of Zhejiang Province (2007C23014, 2006C13022), and the National Science Fund for Distinguished Young Scholars of China (No. 50425311).
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