Enhancement of transfection efficiency for HeLa cells via incorporating arsinine moiety into chitosan

Arginine-rich peptides have attracted considerable attention due to their distinct internalization mechanism. It was reported that arginine and guanidino moieties were able to translocate through cell membranes and played a critical role in the process of membrane permeation. In this work, arginine was conjugated to the backbone of chitosan to form a novel chitosan derivative, arginine modified chitosan （Arg-CS）. Arg-CS/DNA complexes were prepared according to the method of coacervation process. The physicochemical properties of Arg-CS and Arg-CS/DNA complexes were characterized and the transfection activity and efficiency mediated by Arg-CS/DNA complexes were investigated taking HeLa cells as target cells. Arg-CS was characterized by FTIR and ^13C NMR. Arg-CS/DNA polyelectrolyte complexes were investigated by agarose gel retardation, dynamic light scattering （DLS） and atomic force microscopy （AFM）. The results revealed that the Arg-CS/DNA complexes started to form at N/P ratio of 2：1, and the size of particles varied from 100 to 180 nm. The cytotoxicity of Arg-CS and their complexes with plasmid DNA were determined by MTT assay for HeLa cells, and the results suggested that Arg-CS/DNA complexes were slightly less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed significantly lower toxicity than PEI and PEI/DNA complexes, respectively. Taking HeLa cells as target cells and using pGL3-control as reporter gene, the luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan at different pH media. These results suggest that Arg-CS is a promising candidate as a safe and efficient vector for gene delivery and transfection.     

磷酸化壳聚糖-季铵化壳聚糖载体提高体内外基因转染功效

本研究通过磷酸化壳聚糖(PC)包被季铵化壳聚糖(TMC)/质粒(pDNA)纳米复合物,制备PC/TMC/pDNA三元复合物(PTC),用于提高体内外基因转染功效.PTC粒径为100~200nm,Zeta电势为-16~-34mV,可有效缩合pDNA,保护pDNA免遭核酶降解.PC降低PTC中pDNA结合力,增加体外释放量.表面荷负电的PTC抗非特异性蛋白吸附能力强,经小窝蛋白介导的细胞内吞途径入胞后逃避溶酶体降解,细胞核内分布比例高.HEK293细胞体外转染试验结果显示,PTC体外转染效率是TMC/pDNA纳米复合物(TC)的1.5~3.1倍;小鼠胫前肌注射给药试验结果表明,PTC可显著提高基因体内转染效率.因此,合适质量比的PTC有望作为功能性基因药物的递送载体,用于基因治疗.     

基于低分子量PEI的不同疏水链修饰的梳状低聚物基因载体

为了探究基于低分子量聚乙烯亚胺(polyethylenimine, PEI)基因载体的转染效率,通过Michael加成反应将PEI 600 Da接枝于含有疏水链的生物可降解聚酯上形成系列梳状聚合物,并将之应用于基因载体;利用核磁氢谱和凝胶渗透色谱对聚合物的化学结构与分子量进行了测定,此外,还利用凝胶电泳实验和绿色荧光蛋白实验研究了聚合物与DNA的结合能力及其复合物的转染性能。结果表明：本文方法成功合成了系列低聚物,并对DNA表现出较好的包裹能力;油酸修饰后的低聚物与DNA复合物在质量比为6.4时转染效果与PEI 25 kDa相当。可见,油酸修饰后的脂质体低聚物有作为非病毒基因载体的前景。     

纳米谷氨酸壳聚糖制备及其体外质粒转染研究

采用表面修饰方法制备出谷氨酸修饰的壳聚糖纳米基因载体。对样品进行红外分析、粒度分析、zeta电位分析、生物相容性、凝胶阻滞分析、DNA保护性试验、体外细胞转染研究。结果显示所制得的谷氨酸修饰的壳聚糖纳米颗粒平均粒径为170nm,其zeta电位为 4.7mV。红外分析显示谷氨酸已通过酰胺键结合在壳聚糖上。MTT实验结果显示纳米颗粒与细胞有良好的生物相容性。凝胶阻滞分析和DNA保护试验结果表明纳米载体可与DNA通过电性结合作用而结合,并可以有效保护DNA,防止核酸酶对其的降解作用。而体外细胞转染的结果表明,谷氨酸修饰的纳米粒能介导pEGFP-N1质粒转染HepG2细胞并在细胞中表达绿色荧光蛋白。因此,谷氨酸修饰的壳聚糖纳米颗粒可作为一种新型非病毒基因载体介导核酸类生物大分子进入细胞内。     

Enhancement of the efficiency of PEI/liposome transfection by magnetofectins formed via electrostatic self-assembly

One of the major challenges for successful gene therapy is improving the transfection efficiency of non-viral vectors. Magnetic nanoparticles (MNPs) have been developed as enhancers of non-viral vehicles. We prepared MNPs and modified them with polyethyleneimine (PEI), citric acid (CA) or carboxylmethyl-dextran (CMD). Both positively charged MNPs (MNPs@PEI) and negatively charged MNPs (MNPs@CA, MNPs@CMD) could spontaneously form transfection complexes (magnetofectins) with plasmid DNA and PEI/liposome via electrostatic self-assembly. Our results showed as-prepared magnetofectins apparently enhanced PEI/liposome transfection efficiency and/or gene expression level into COS-7 cells with reduced transfection time from 4 h to 15 min under a magnetic field in vitro. Meanwhile, the effect of magnetofection was cell line-dependant. These results suggest that charged MNPs could improve transfection efficiency for non-viral vectors by simply mixing with them and by exerting a magnetic force. Thus such MNPs provide a convenient platform for further applications of gene delivery.     

Preparation and Adsorption Ability of Polysulfone Microcapsules Containing Modified Chitosan Gel

Chemically modified chitosan beads containing polyethyleneimine （PEI） were prepared to improve the metal ion adsorption capacity of the chitosan beads and their mechanical stability and to limit their biodegradability. The modified beads were encapsulated with the polymer material polysulfone by a novel surface coating method named the emulsion phase inversion method. The adsorption properties of the modified beads and the microstructures of the polysulfone coating layer were then analyzed. The experimental results showed that the PEI was successfully linked onto the chitosan beads. The density of the -NH2 groups in the modified beads was significantly increased, while the water content was reduced. The coating layer thickness was about 200 pm. The modified chitosan gel beads had excellent Cu（ll） adsorption capacity, with a maximum Cu（ll） adsorption capacity 1.34 times higher than that of the unmodified beads. The results show that even with the polysulfone coating the adsorption kinetics of the modified beads is still better than those of the unmodified beads. The modifications improve the mass transfer performance of the chitosan beads as well as the bead stability.     

Non-viral gene carrier mediated short hairpin RNA interference for inhibition of tumor cells growth

A tumor-targeting gene vector G250mAb-PEI-PEG has been prepared by modification of polyethylenimine （PEI） with polyethyleneglycol （PEG） and G250, a monoclonal antibody against the G250 antigen on tumor cell surface. The transfection efficiency was as high as 70% in G250 positive HeLa cells, whereas the transfection efficiency was relatively low （30%） in normal NIH3T3 cells. A plasmid encoding the short hairpin RNA （shRNA） specific for nucleostemin gene （NS） was efficiently transfected into the HeLa cells with this nonviral gene vector. RNA interference down-regulated the expression of NS gene in HeLa cells, inhibited cells proliferation and induced apoptosis. However, the growth and activity of the NIH3T3 cells were not affected under the same treatment. These results indicate that the reported nonviral gene vector, G250mAb-PEI-PEG, can target and efficiently deliver genes into HeLa cells, and has the potential for the cervical cancer treatment.     

甲壳三糖-g-壳聚糖/累托石纳米复合材料作为基因载体的研究

本文合成了甲壳三糖-g-壳聚糖（TCS），并与累托石插层复合制备纳米复合材料，采用XRD和TEM对其进行了表征。粒径分析结果表明，两种分子量的TCS与pDNA复合物的粒径大小都在75nm左右，而累托石加入后，其粒径都不同程度的增加，最大达到191nm。在PBS中的聚集动力学及琼脂糖凝胶电泳实验发现，高分子量TCS /pDNA复合物较低分子量复合物稳定，而累托石的加入降低了其稳定性。体外转染实验初步表明，甲壳三糖-g-壳聚糖/累托石-DNA复合物能介入肝癌细胞中并表达荧光。该纳米复合材料可作为潜在的非病毒基因载体。     

抗人PD-L1单克隆抗体的瞬转表达

利用HEK293 6E细胞瞬转表达抗人PD L1单克隆抗体. 将HEK293 6E细胞无血清悬浮培养, 筛选最佳生长培养基; 考察HEK293 6E细胞的筛选表达培养基、 DNA和PEI质量比及转染时细胞密度等转染条件, 并对收获的抗体进行SDS PAGE和ELISA鉴定. 结果表明: 在A无血清表达培养基中, 以2×106个/mL的细胞密度和m(DNA)∶m(PEI)=1的条件转染, 第4天收获抗体的产量最高, 为170.9 μg/mL; 收获PD-L1抗体的重链分子量约为50 000, 轻链分子量约为25 000.     

聚乙烯亚胺在锰锌铁氧体纳米粒子表面定量吸附

以可自动恒温控温的锰锌铁氧体磁性纳米粒子(MZF-NPs)为核心,在其表面修饰聚乙烯亚胺(PEI)以制备一种新型纳米基因载体.利用扫描电镜、红外光谱仪、Zeta电位仪对其形貌、表面包覆功能团、电位等进行表征;UV/vis光谱仪及DNA体外结合、释放、转染实验研究了PEI在MZF-NPs上的定量吸附与修饰效果.电镜下修饰后粒子之间的聚集明显减轻、等电点由pH 7.0移至pH 11.5.PEI质量、介质的pH值以及离子强度均可影响PEI在锰锌铁氧体上的吸附.不同的吸附量也影响了纳米粒子的DNA结合、释放与转染能力.     

Restoration of rat calvarial defects by poly（lactide-co-glycolide）/ hydroxyapatite scaffolds loaded with bone mesenchymal stem cells and DNA complexes

A composite construct comprising of a bone mesenchymal stem cell (BMSC) sheet, plasmid DNA, encoding human bone morphogenic protein-2 (hBMP-2), and poly(lactide-co-glycolide)/hydroxyapatite (PLGA/HA) sponge was designed and employed in the restoration of rat calvarial defects. To improve gene transfection efficiency, a cationic chitosan derivative, N,N,N,-trimethyl chitosan chloride (TMC), was employed as the vector. The TMC/DNA complexes had a transfection efficiency of 13% in rat BMSCs, resulting in heterogeneous hBMP-2 expression in a 10-d culture period in vitro. In vivo culture of the composite constructs was performed by implantation into rat full-thickness calvarial defects, using constructs lacking pDNA-hBMP-2 or BMSC sheets as controls. Significantly higher heterogeneous expression of hBMP-2 was detected in vivo at 2 weeks for the cell sheet/DNA complex/scaffold constructs, compared with the constructs lacking pDNA-hBMP-2 or BMSC sheets. New bone formation was evident as early as 4 weeks in the experimental constructs. At 8 weeks, partial bridging of calvarial defects was observed in the experimental constructs, which was significantly better than the constructs lacking pDNA-hBMP-2 or BMSC sheets. Therefore, the combination of the PLGA/HA scaffold with BMSC sheets and gene therapy vectors is effective at enhancing bone formation.     

Compatibility of Chitosan-Gelatin Films with Adipose Tissue Derived Stromal Cells

Introduction Chitosan, the partially N-deacetylated form of chitin, is the second most abundant biomaterial after cellulose. Due to its biocompatibility, biodegradability, and bio- activity, chitosan and its derivant complexes have been studied in various…     

O-硬脂酰化壳聚糖作为DNA的递送载体材料

以邻苯二甲酰基预先保护壳聚糖分子中-NH_2,以硬脂酰氯进行可控酯化,制备O-硬脂酰化壳聚糖.通过超声振荡与溶剂结合制备O-硬脂酰化壳聚糖/λDNA复合物,采用动态激光散射及原子力显微镜分析复合物的粒径分布及形态.凝胶电泳考察该载体与λDNA的结合能力.L929细胞培养对O-硬脂酰化壳聚糖进行细胞毒性评估.FT-IR和~1H NMR结果表明,可以成功制备不同酯化度的O-硬脂酰壳聚糖;O-硬脂酰化壳聚糖与λDNA结合可形成不规则球形复合物,平均粒径可达到86 nm,并且对DNase显示高稳定性.同时O-硬脂酰化壳聚糖具有促L929细胞增殖能力,安全性较高,可作为一种新型的非病毒型基因载体.     

构建HMGB1/PEI非病毒载体介导高效基因传递的研究

 研究了以聚乙烯亚胺（PEI）为骨架复合高迁移率族蛋白 B1 (HMGB1 )的复合型载体HMGB1/PEI的性能,以期提高非病毒基因载体的转染效率。透射电镜观察pDNA/HMGB1/PEI复合物粒子形态呈球形;动态光散射法测定粒径与表面电位,结果显示复合HMGB1后,复合物粒径降低,且随HMGB1加入量的增大表面电位有增大的趋势;凝胶电泳阻滞试验表明HMGB1可协助PEI与pDNA结合;MTT试验结果显示HMGB1/PEI复合载体的细胞毒性低于PEI;HMGB1/PEI复合载体的转染率较PEI的转染率增大2.9~4.0倍,且HMGB1可以弱化血清对转染的阻碍作用。所以HMGB1被证实能有效提高PEI的体外转染效率。     

乙二胺羟丙基壳聚糖固定化天门冬酰胺酶的研究

研制了新型壳聚糖胺基衍生物-乙二胺羟丙基壳聚糖（EDA-HPCS）,并将其用于固定天门冬酰胺酶,分别考察了甲醛活化载体和戊二醛活化载体对固定化酶活力的影响,结果表明,在适当的固定化条件下,甲醛活化EDA-HPCS的固定化酶活力约为30U/g载体,而戊二醛活化EDA-HPCS固定化酶活力约为16U/g载体。     

壳聚糖与DNA相互作用的单分子方法研究

利用原子力显微镜与磁镊技术研究不同浓度的壳聚糖与多种DNA分子的作用,对其凝聚形态及力谱曲线进行观察。在恒力情况下,壳聚糖与DNA的电荷比k分别为3和4时,从DNA的力谱曲线上可以看见0.2~0.5μm左右的跳跃阶梯及线性增长区域,原因可能是壳聚糖-DNA聚合物中出现了环状、棒状及球状结构;利用原子力显微镜观察壳聚糖与λ-DNA及bpr322-DNA作用后的凝聚形态,发现随着壳聚糖与DNA电荷比k的增加,球状与棒状聚合物的比例明显减小,而且凝聚物的尺寸也整体减小。最后根据实验结果,建立了壳聚糖导致DNA构象变化的直观模型。     

一种新型两亲性壳聚糖衍生物的合成及性质的研究

首次合成了一种新型非离子型两亲性壳聚糖衍生物(2-羟基-3-丁氧基)丙基-丁二酰化壳聚糖(HBP-SCCHS)。用红外光谱和元素分析等手段对产物结构进行了表征，证明在壳聚糖分子上已成功地引入了疏水和亲水基团。研究表明，该新型壳聚糖衍生物具有良好的表面活性、泡沫性和乳化性，可以作为高分子表面活性剂使用。     

Preparation and characterization of chitosan/nano-hydroxyapatite composite used as bone substitute materials

Chitosan/nano-hydroxyapatite composites with different weight ratios were prepared through a co-precipitation method using Ca（OH）2, H3PO4 and chitosan as starting materials. The properties of these composites were characterized by means of TEM, IR, XRD, TGA, burn-out tests and universal matertial testing machine. The results showed that the HA synthesized here was poorly crystalline carbonated nanometer crystals and dispersed uniformly in chitosan phase and there was no phase-separation between the two phases. The addition of n-HA resulted in a decrease of decomposing temperature of chitosan. Because of the interactions between chitosan and n-HA, the mechanical properties of these composites were improved, and the maximum value of the compressive strength was measured to be about 120MPa corresponding to the chitosan/n-HA composite with a weight ratio of 30/70.     

N—苄基壳聚糖的合成和液晶性表征

合成了新的液晶性壳聚糖衍生物－N－苄基壳聚糖,N－苄基壳聚糖／二氯乙酸浓溶液呈现液晶相。用偏光显微镜测得形成液晶有序的临界浓度为9％（重量百分数）。在溶液的红外光谱中,二氯乙酸的羰基吸收谱带1738cm^-1在相变时位移到1731cm^-1,利用这一突变测得N－苄基壳聚糖的临界浓度为10％,与光学显微镜法基本相符。     

树状分子接枝壳聚糖的合成与热致液晶性

甲壳素/壳聚糖是拥有多个反应性官能团的天然多糖,而树状分子则是一类可以在分子水平上控制和设计大小、形状、结构和功能基团的化合物,将其作为侧链通过接枝反应引入到壳聚糖主链上有望获得新型的天然高分子液晶材料.我们利用经典的收敛法合成了DOBOB酸(3,4,5-三[对-(十二烷氧基)苄氧基]苯甲酸)树枝状分子,并且以对甲苯磺酰氯为催化剂,在吡啶/无水氯化锂的溶剂体系中将其接枝到壳聚糖分子链上,产物的化学结构经红外1、H-NMR1、3C-NMR以及元素分析表征验证.通过DSC和热台偏光显微镜研究表明,这种树枝状分子接枝壳聚糖化合物同时具有热致和溶致液晶性,是一种全新的壳聚糖衍生物液晶.