Supramolecular gene carrier base on cyclodextrin and polycation.doc

精品论文Supramolecular gene carrier base on cyclodextrin and polycation5HU Yang, CHAI Mingying, YANG Wantai, XU Fujian(College of Materials Science & Engineering, Beijing University of Chemical Technology, Beijing 100029)Abstract: A series of novel supramolecular pseudo-comb polycations (l-PGEA-Ad/ CD-PGEAs) weresynthesizedbytyingmultiplelow-molecular-weight-cyclodextrin-coredstar10ethanolamine-functionalized poly(glycidyl methacrylate) (PGEA) polymers (CD-PGEAs) with an adamantine-modified linear PGEA (l-PGEA-Ad) backbone via the hostguest interaction. Thepseudo-comb carriers were studied in terms of their DNA binding capability, cytotoxicity, and genetransfection in HepG2 cell line. The pseudo-comb l-PGEA-Ad/CD-PGEAs exhibited better pDNA-condensing abilities than their counterparts, CD-PGEA and l-PGEA. Meanwhile, the15pseudo-comb carriers displayed low cytotoxicity, similar to CD-PGEA and l-PGEA. Moreover, the gene transfection efficiencies of the pseudo-comb carriers were much higher than those of CD-PGEAand l-PGEA at various N/P ratios. Such supramolecular preparation of pseudo-comb gene carriers could provide a flexible approach to adjusting the structure and functionality of supramolecularpolymers via properly using noncovalent interactions.20Key words: Polymer science; Gene delivery; Supramolecular polymer; cyclodextrin0IntroductionThe development of safe and effective polycationic vectors is of crucial importance to the gene therapy.1 In comparison with viral vectors and cationic lipids, polycations as the major type25of nonviral gene delivery vectors show low host immunogenicity and can be produced on a large scale. A large number of polycations, including polyethylenimine (PEI), 2 polyamidoamine, 3 chitosan, 4 and cyclodextrin (CD)-based cationic vectors, 5 have been reported to deliver nucleic acids. We found that ethanolamine (EA)-functionalized poly(glycidyl methacrylate) (PGMA) (or PGEA) with plentiful flanking secondary amine and hydroxyl groups can produce good30transfection efficiency in some cell lines, while exhibiting low toxicity. 6 More recently, thehigh-molecular-weight comb-shaped PGEA gene carriers composed of the low-molecular-weight PGEA backbone and side chains were proposed by a combination of atom transfer radical polymerization (ATRP) and ring-opening reactions.Owing to the dynamic-tunable ability, 7 supramolecular polymers based on noncovalent35interactions8 have been increasingly explored as non-viral gene vectors in preclinical studies9 and even in clinical trials. 10 Among varieties of supramolecular structures, one typical model is the easily assembled and well-demonstrated molecular recognition system based on cyclodextrin and adamantane (Ad). 11 CD applied as host components is particularly appealing because of its excellent biocompatibility, nonimmunogenicity, and low toxicity in animal and human bodies. 1240Furthermore, novel star gene carriers using CDs as cores could be developed when the hydroxyl groups on the outside surfaces of CDs were derivatized to serve as initiation sites for growing cationic branches. The star polymers consisting of -CD cores and cationic poly(2-dimethyl amino)ethyl methacrylate) (or PDMAEMA) arms are capable of efficiently mediating genetransfection. 13 However, its good transfection efficiency was dependent on the high molecular45weight, and the high cytotoxicity of PDMAEMA-based carriers limits their effective applications. Herein, by the synergistical combination of the advantages of the low-molecular-weightFoundations: Specialized Research Fund for the Doctoral Program of Higher Education (No. 20090010120007) Brief author introduction:HU Yang (1991-), Male, Graduate Student, Medical PolymerCorrespondance author: XU Fujian (1976-), Male, Professor, Medical Polymer. E-mail: xufjmail.buct.edu.cn- 8 -CD-cored star PGEA polymers (CD-PGEA) and the dynamic-tunable ability of supramolecular polymers, we prepared a novel class of supramolecular pseudo-comb PGEA gene carriers(l-PGEA-Ad/CD-PGEAs) via the hostguest interaction between CD-PGEA host and three50different adamantine-modified linear PGEA (or l-PGEA-Ad) guests (Scheme 1). Such supramolecular preparation of pseudo-comb l-PGEA-Ad/CD-PGEAs provides a flexible approach to adjusting the structure and functionality of supramolecular polymers by using noncovalent interactions.1Experimental Section551.1 Synthesis of l-PGMA and CD-PGMA via ATRPLinear PGMA (l-PGMA) was rstly prepared via atom transfer radical polymerization (ATRP) of GMA as described in our previous publication. The l-PGMA was synthesized using a molar feed ratio GMA (12 mL):ethyl bromoisobutyrate:CuBr:PMDETA of 140:1:1:1.5 at50oC in 12 mL tetrahydrofuran (THF). The reaction was performed in a 50 ml flask equipped with60a magnetic stirrer and under the typical conditions of ATRP. GMA, THF, ethyl bromoisobutyrate, and PMDETA were introduced into the 50 mL flask equipped with a magnetic stirrer. Thereaction mixture was degassed by bubbling argon for 20 min. Then, CuBr was added to the mixture, and the ask was then sealed with a rubber stopper under an argon atmosphere. The polymerization was allowed under continuous stirring at 50oC to produce l-PGMA from 2 h of65ATRP (Mn = 1.1 ×104 g/mole, PDI = 1.35). The reaction was stopped by diluting with THF, whilethe catalyst complex was removed by passing the blue dilute polymer solution through a short aluminum oxide column. After removal of THF in a rotary evaporator, PGMA was precipitated in excess n-hexane. The crude polymer was puried by reprecipitation twice in hexane to remove the reactant residues, prior to being dried under reduced pressure.70As shown in Figure 1, in this work, the target arm number of the CD-cored star PGMA (CD-PGMA) is four. The starting bromoisobutyryl-terminated CD (CD-Br) with about four initiation sites was used. The detailed preparation procedures and characterization were described in our previous publication. For the preparation of star CD-PGMA via ATRP, the molar feed ratio GMA (4 mL):CuBr:PMDETA of 100:1:1.5 was used at room temperature in 6 ml of75anhydrous DMF containing 0.2 g of CD-Br. The reaction was conducted in a 25 mL flask equipped with a magnetic stirrer and under the typical conditions for ATRP. CD-Br, PMDETA, and GMA were introduced into the flask containing 6 mL of anhydrous DMF, and the reaction mixture was degassed by bubbling nitrogen for 10 min. Then, CuBr was added into the mixture under a nitrogen atmosphere. The flask was then sealed with a rubber stopper under a nitrogen80atmosphere. The polymerization was allowed to proceed under continuous stirring at room temperature for 10 min (Mn = 8.4 ×103 g/mole, PDI = 1.32). The final reaction mixture was precipitated with excess methanol and washed with deionized water, prior to lyophilization.1.2 Preparation of l-PGEA-Ad and CD-PGEAThe introduction of adamantine (Ad) onto l-PGMA was performed via the direct85ring-opening reactions of the epoxy groups of l-PGMA with Ad-NH2 to produce the Ad-coupledl-PGMA (l-PGMA-Ad). The three l-PGMA-Ads with the different ratios of Ad were prepared by adjusting the l-PGMA/Ad-NH2 feed ratio. With the pre-determined molar feed ratio (2/1, 5/1 or10/1) of the epoxy/Ad units, the Ad-NH2 (5.25, 2.10 or 1.05 mmol) in 3 mL of DMF was addeddropwise at ambient temperature into the flask containing l-PGMA (containing 10.50 mmol GMA90units) in 10 mL of DMF. The reaction was allowed to proceed at 30oC for 24 h to produce95100105110115120125130l-PGMA-Ad, which was directly prepared for the subsequent synthesis of l-PGEA-Ad. DifferentEA-functionalized PGMAs (PGEAs) were prepared by reacting l-PGMA, l-PGMA-Ad orCD-PGMA with EA. 0.3 g of l-PGMA, l-PGMA-Ad or CD-PGMA was dissolved in 7 mL of DMF. 5 mL of EA and 1 mL of triethyleneamine were then added. The reaction mixture was stirred at 37oC for 5 days to produce l-PGEA or CD-PGEA. The final reaction mixture was precipitated with excess diethyl ether. The crude produce was purified by 24 h dialysis against DDW using a dialysis membrane (MWCO 3500) prior to lyophilization.For the preparation of l-PGEA-Ad/CD-PGEA complex, with the 1:1 molar feed ratio of the CD/Ad units, CD-PGEA (containing 0.05 mmol CD units) in 10 mL of H2O was added dropwise at ambient temperature into the flask containing l-PGEA-Ads (containing 0.05 mmol Ad uints) in10 mL of H2O. The mixture was stirred for 12 h, followed by dialysis in water for 24 h and freeze-dried to yield l-PGEA-Ad/CD-PGEA.1.3 Polymer CharacterizationThe molecular weights of polymers were determined by gel permeation chromatography (GPC) and chemical structure by nuclear magnetic resonance (NMR) spectroscopy. GPC measurements of PGMAs were performed on a Waters GPC system equipped with Waters Styragel columns, a Waters-2487 dual wavelength () UV detector, and a Waters-2414 refractiveindex detector. THF was used as the eluent at a low flow rate of 0.5 ml/min at 25oC. 1H NMRspectra were measured by accumulation of 1000 scans at a relaxation time of 2 s on a Bruker ARX300 MHz spectrometer, using CDCl3 (for PGMAs) or D2O (for PGEAs) as the solvent. The chemical shifts were referred to the solvent peaks, = 7.20 ppm for CDCl3 and = 4.70 ppm for D2O, respectively. The 2D-NOESY (two-dimensional nuclear overhauser effect spectroscopy) NMR experiments were performed at 500 MHz in D2O on a Bruker Avance DRX 500 MHz NMR spectrometer.1.4 Characterization of Polycation/pDNA ComplexesThe plasmid (encoding Renilla luciferase) mainly used in this work was pRL-CMV (Promega Co., Cergy Pontoise, France), which was cloned originally from the marine organism Renilla reniformis. The plasmid DNA (pDNA) was amplified in Escherichia coli and purified according to the suppliers protocol (Qiagen GmbH, Hilden, Germany). The purity and concentration of the purified DNA were determined by absorption at 260 and 280 nm and by agrose gel electrophoresis. The purified pDNA was resuspended in tris-EDTA (TE) buffer and kept in aliquots of 0.5 mg/mL in concentration. All polycation stock solutions were prepared at a nitrogen concentration of 10 mM in distilled water. Solutions were filtered via sterile membranes(0.2 m) of average pore size and stored at 4oC. Polycations to DNA ratios are expressed as molarratios of nitrogen (N) in PGEAs to phosphate (P) in DNA (or as N/P ratios). The average mass weight of 325 per phosphate group of DNA was assumed. All polycation/pDNA complexes were formed by mixing equal volumes of polycation and pDNA solutions to achieve the desired N/P ratio. Each mixture was vortexed and incubated for 30 min at room temperature. Each polycation was examined for its ability to bind pDNA through agarose gel electrophoresis using the similarprocedures as those described earlier.5 The particle sizes and zeta potentials of thepolycation/pDNA complexes were measured using a Zetasizer Nano ZS (Malvern Instruments, Southborough, MA) using the procedures as described earlier.1.5 Cell ViabilityThe cytotoxicity of the polycations was evaluated using the MTT assay in HepG2 and135140145150155160165170175HEK293 cell line. The cells were cultured in Dulbecco's modified eagle medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/mL of penicillin and100 g/mL of streptomycin at 37oC, under 5% CO2, and 95% relative humidity atmosphere. Thecells were seeded in a 96-well microtiter plate at a density of 104 cells/well and incubated in 100L of DMEM/well for 24 h. The culture media were replaced with fresh culture media containing10 µL polyplex solutions at various N/P ratios, and the cells were incubated for 24 h. Then, 10 L of sterile-filtered MTT stock solution in PBS (5 mg/mL) was added to each well, reaching a final MTT concentration of 0.5 mg/mL. After 5 h, the unreacted dye was removed by aspiration. The produced formazan crystals were dissolved in DMSO (100 L/well). The absorbance was measured using a Bio-Rad Model 680 Microplate Reader (UK) at a wavelength of 570 nm. The cell viability (%) relative to control cells cultured in media without polycations was calculated from Atest/Acontrol ×100%, where Atest and Acontrol are the absorbance values of the wells (with the polycations) and control wells (without the polycations), respectively. For each sample, the final absorbance was the average of those measured from six wells in parallel.1.6 Transfection AssayTransfection assays were performed first using plasmid pRL-CMV as the reporter gene in HepG2 and HEK293 cell lines in the presence of serum. In brief, the cells were seeded in 24-well plates at a density of 5 ×104 cells in the 500 L of medium/well and incubated for 24 h. The polycation/pDNA complexes (20 L/well containing 1.0 g of pDNA) at various N/P ratios wereprepared by adding the polycation into the DNA solutions, followed by vortexing and incubation for 30 min at room temperature. At the time of transfection, the medium in each well was replaced with 300 L of fresh normal medium (supplemented with 10% FBS). The complexes were added into the transfection medium and incubated with the cells for 4 h under standard incubator conditions. Then, the medium was replaced with 500 L of the fresh normal medium (supplemented with 10% FBS). The cells were further incubated for an additional 20 h under the same conditions, resulting in a total transfection time of 24 h. The cultured cells were washed with PBS twice, and lysed in 100 L of the cell culture lysis reagent (Promega Co., Cergy Pontoise, France). Luciferase gene expression was quantified using a commercial kit (Promega Co., Cergy Pontoise, France) and a luminometer (Berthold Lumat LB 9507, Berthold Technologies GmbH. KG, Bad Wildbad, Germany). Protein concentration in the cell samples was analyzed using a bicinchoninic acid assay (Biorad Lab, Hercules, CA). Gene expression results were expressed as relative light units (RLUs) per milligram of cell protein lysate (RLU/mg protein). PGEA-mediated gene transfection was also assessed at their optimal N/P ratios using Plasmid pEGFP-N1 encoding green fluorescent protein (GFP) (BD Biosciences, San Jose, CA) as the reporter gene in HepG2 cell line using the same procedures as those described above. The transfected cells were imagedby using a Leica DMIL Fluorescence Microscope. The percentage of the EGFP positive cells wa