Elsevier

Journal of Controlled Release

Volume 123, Issue 3, 20 November 2007, Pages 239-246
Journal of Controlled Release

Deposition transfection technology using a DNA complex with a thermoresponsive cationic star polymer

https://doi.org/10.1016/j.jconrel.2007.08.026Get rights and content

Abstract

A novel non-viral gene transfection method in which DNA complexes were kept in contact with a deposition surface (deposition transfection) was developed. We designed a novel aqueous thermoresponsive adsorbent material for DNA deposition, which was a star-shaped copolymer with 4-branched chains. Each chain is comprised of a cationic poly(N,N-dimethylaminopropyl acrylamide) (PDMAPAAm) block (Mn: ca. 3000 g·mol 1), which formed an inner domain for DNA binding and a thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) block (Mn: ca. 6000 g·mol 1), which formed an outer domain for surface adsorption. Complex formation between the copolymer and the luciferase-encoding plasmid DNA occurred immediately upon simple mixing in an aqueous medium; polyplexes approximately 100 nm in size were formed. Because the lower critical solution temperature of the polyplexes was approximately 35 °C, they could deposit on the substrate by precipitation from an aqueous solution upon warming, which was confirmed by quartz crystal microbalance (QCM) method and water contact angle measurement. When COS-1 cells were cultured on the polyplex-deposited substrate in a culture medium, the luciferase activity observed was higher than that observed on a DNA-coated substrate with or without the cationic polymer before and after complete adhesion and by conventional solution transfection using the polyplexes. The activity was enhanced with an increase in the charge ratio (surfactant/pDNA) with permissible cellular cytotoxicity.

Introduction

There are several issues to be resolved regarding clinical applications that use virus vectors for gene therapy, such as the antigenicity and toxicity of the virus or the possibility of disease transfection. On the other hand, cationic polymers such as polyethylenimine (PEI) [1], [2] and dimethylamino group-containing polymers [3], [4], [5], [6], which can generate nanoparticles by the formation of polyion complexes, i.e., “polyplexes,” with a plasmid DNA, have a high potential to be one of the major carriers in non-viral gene delivery systems due to the many advantages they offer over viral systems. However, several research trials using non-viral carriers have not always achieved the expected results in terms of transfection efficiency; this is because the gene expression level is not as high as that obtained with viral carriers [7], [8]. Therefore, an efficient technology and methodology of gene transfection based on the designed carrier materials without the use of virus vectors need to be developed.

The conventional procedure of gene transfection involves complexing the non-viral carrier with a plasmid DNA and then adding it to a cell culture medium. In this case, preincubation of the target cells is necessary for at least 1 day before transfection. In addition, generally, serum cannot be used as the transfection medium because the DNA-carrier complex often interacts with serum components, leading to suppressed gene transfection ability. Therefore, a reverse transfection method was developed as another practical approach for gene transfection [9], [10], [11]. This method is performed by culturing cells on a plasmid DNA-loaded substrate. DNA-loaded substrates are generally prepared by mixing DNA with cationic polymers that have been physically adsorbed or chemically bonded to the substrate. The cells were in direct contact with the DNA-loaded surface during the culture period; this differs from conventional transfection culture in which the contact time is limited. In addition, transfectional microarrays that permit parallel transfer of multiple genes into cultured cells were developed for high-throughput reverse genetics research because the reverse transfection method can be performed with spatial and temporal control [10], [11]. However, it cannot be said that the transfection efficiency of this method is as satisfactory as that of the conventional transfection method. Therefore, an additional physical stimulus, such as an electric pulse, was used to enhance the transfection efficiency [12], [13].

Recently, in order to improve the gene transfection efficiency of methods that use cationic polymers as gene carriers, we designed a series of branched cationic polymers [3]. The series comprised linear and 3-, 4-, or 6-branched PDMAPAAm polymers that functioned as novel high-performance gene carriers termed the star vector. They were prepared by iniferter (initiator-transfer agent-terminator)-based photo-living radical polymerization [14], [15] from their respective multidithiocarbamate-derivatized benzenes (multifunctional iniferters) and by using DMAPAAm as a cationic monomer. These experiments revealed that the transfection efficiency increased with the degree or chain length of branching [3], or by blocking with a nonionic chain [16].

As a continuation of our study on the star vector, in this study, we designed a novel adhesive transfection material to improve the reverse transfection method. The material had 4-cationic PDMAPAAm branching for binding with a plasmid DNA to form polyplexes and was blocked with poly(N-isopropylacrylamide) (PNIPAM) chains for surface deposition on a hydrophobic substrate. PNIPAM is one of the most well-known thermoresponsive polymers that precipitates in water at above 32 °C but is water soluble at room temperature [17]. Due to this unique feature, PNIPAM has been utilized for thermoresponsive tissue culture dishes [18], [19], in drug delivery vehicles [20], [21], for hemostasis [22], [23], and in 3D extracellular matrix materials [24]. The PNIPAM-blocked complex of the star vector and a plasmid DNA was adsorbed onto a culture substrate, and cells were then cultured on the complex-deposited substrate. The transfection efficiency of the newly developed transfection method (deposition transfection), which is an improved reverse transfection method, was evaluated by comparing it with that of the conventional transfection method. Further, the cytotoxicity was evaluated after deposition transfection using COS-1 cells.

Section snippets

Materials

1,2,4,5-Tetrakis(bromomethyl)benzene was obtained from Sigma-Aldrich (Milwaukee, WI). Sodium N,N-diethyldithiocarbamate, N,N-dimethylacrylamide (DMAAm), and N-isopropylacrylamide (NIPAM) were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan). The other chemical reagents were commercially obtained from Wako Pure Chemical Ind., Ltd. The DMAAm was distilled under reduced pressure, and the NIPAM was recrystallized with methanol/hexane prior to use in order to remove the stabilizer. The

Synthesis of PDMAPAAm–PNIPAM 4-branched block copolymers

The PDMAPAAm–PNIPAM 4-branched block copolymer, which was designed as a thermoresponsive adsorbent material for the surface deposition of DNA, was synthesized by the sequential steps of iniferter-based living radical photopolymerization as depicted in the reaction scheme shown in Fig. 1 [3], [25], [26]. As the first step, a PDMAPAAm cationic polymer with 4 branches with an Mn of approximately 3700 g mol 1 was obtained when a methanol solution of DMAPAAm was irradiated with UV for 90 min in the

Discussion

In the present study, we designed a unique gene adsorbent material possessing thermoresponsive properties in order to improve the reverse transfection method [10], [11]. In ordinary reverse transfection, it is necessary to coat pDNA on the culture substrate with cationic matrix materials to ensure that the pDNA is firmly impregnated in the matrix adhered or bonded to the substrate. On the other hand, in our method, the pDNA was precipitated onto a culture substrate using a thermoresponsive

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