新型纳米复合材料作为非病毒基因载体的研究

为了制备理想的非病毒基因载体,合成了甲壳三糖-g-壳聚糖(TCS),并与钙基累托石(REC)插层复合制备纳米复合材料,采用X射线衍射和透射电镜方法对产物进行了表征,考察了其细胞相容性,将REC、TCS和纳米复合材料分别与质粒DNA复合形成复合物,并考察了其体外基因转染效果.结果表明:TCS、REC及纳米复合材料的细胞相容性良好,当N/P比为3时,TCS/DNA复合物粒径大小都在75 nm左右,REC加入后,其粒径不同程度地增大,最大达到191 nm;相对分子质量高的TCS所得DNA复合物较相对分子质量低的TCS所得DNA复合物稳定,而REC的加入降低了TCS/DNA复合物的稳定性;TCS/REC-DNA复合物能介入肝癌细胞中并表达荧光.说明该纳米复合材料可作为潜在的非病毒基因载体.     

壳聚糖/累托石纳米复合材料的结构与性能

采用溶液插层法制备了不同累托石含量的壳聚糖/累托石纳米复合材料,研究了累托石含量、层间距对复合材料性能的影响及不同累托石含量的复合材料的热稳定性和抗菌性能.结果表明:壳聚糖分子链插层进入了累托石层间,累托石均匀地分布在壳聚糖基体中;纳米复合材料的性能与累托石的含量和层间距密切相关,当壳聚糖与累托石的质量比为12∶1时插层效果最好;壳聚糖/累托石纳米复合材料的抗菌能力比单一壳聚糖优异,尤其是对革兰氏阳性菌,其抑菌的最小浓度为单一壳聚糖的1/4～1/2,抑菌时间是单一壳聚糖的3～5倍;复合材料对革兰氏阳性菌的抑制作用比对革兰氏阴性菌的强,且随着累托石含量的增加及层间距的增大,抗菌性能越来越强;纳米复合材料的热稳定性优于壳聚糖.     

有机累托石/环氧树脂纳米复合材料制备及其流变性能研究

采用自制离子液体型累托石,用熔融插层法制备有机累托石/环氧树脂/桐油酸酐纳米复合材料。分别用XRD、DSC、旋转流变仪对复合材料的微观结构、固化动力学和流变性能进行测试。结果表明,有机累托石含量较低时在环氧树脂基体中形成了剥离型纳米复合材料;有机累托石的加入,降低了环氧树脂/桐油酸酐体系固化反应的活化能和频率因子,但环氧树脂/桐油酸酐体系的固化反应不是简单的一元反应,有机累托石的加入并不改变其固化反应机理;随着有机累托石含量的增加,环氧树脂/桐油酸酐体系的黏流活化能逐渐增大。     

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.     

羧甲基壳聚糖磁性纳米复合物的制备与表征

采用共沉淀法制备纳米四氧化三铁磁性粒子，通过一步包埋法将四氧化三铁磁性纳米粒子用羧甲基壳聚糖进行直接包覆，制备羧甲基壳聚糖磁性纳米复合物。透射电镜观察表明：该磁性纳米复合物呈球状，平均粒径为11．6nm；Zeta电位和粒度分析结果表明：其粒度分布比较窄，平均粒径约为12nm，与电镜观察结果一致；红外光谱和热分析结果表明：Fe3O4表面成功地包覆了羧甲基壳聚糖。将该磁性纳米复合物应用于鸡血细胞基因组DNA的分离，结果表明：该磁性纳米复合物非常适合于基因组DNA的分离纯化，避免了传统苯酚一氯仿法中有毒试剂的使用，能够显著简化整个DNA的纯化制备过程。     

用于增强抗肿瘤功效的共载紫杉醇和基因的壳聚糖衍生物载体

本研究对壳聚糖进行亲水修饰、疏水修饰,制得两亲性壳聚糖衍生物(OTC),再用叶酸(FA)修饰OTC得到FOTC.用此FOTC共载难溶性抗肿瘤药物紫杉醇(PTX)和SurvivinshRNA表达质粒(iSur-pDNA)得到FOTC/PTX/pDNA纳米复合物.体外抗肿瘤试验结果表明:与OTC/PTX/pDNA纳米复合物相比,FOTC/PTX/pDNA纳米复合物可提高pDNA释放速率,降低PTX释放速率,显著提高细胞摄取量,提高体外转染效率,增强肿瘤细胞增殖抑制能力.联合给药肿瘤细胞增殖抑制能力强于单独给药.体内抗肿瘤试验结果表明:联合给药可协同增强抗肿瘤作用,FA修饰可增强其主动靶向能力.因此,FOTC有望作为紫杉醇和基因联合给药的递送载体.     

原位插层聚合制备累托石改性酚醛树脂的研究

借鉴原位插层聚合制备聚合物/层状硅酸盐纳米复合材料的方法,进行有机累托石改性热固性酚醛树脂的研究.通过离子交换的方法将天然钙基累托石改性成为有机累托石,使其片状层间由亲水性变为憎水性.以有机累托石为添加物,采用原位插层聚合法完成对酚醛树脂的改性,得到累托石改性酚醛树脂.借助XRD,FTIR及TGA等方法对制备的有机累托石及其改性热固性酚醛树脂作了测定,结果表明：季铵盐阳离子与累托石片层间的可交换阳离子发生了离子交换,故而得到了有机累托石:有机累托石片层在酚醛树脂基体中通过插层和剥离实现了纳米级分散.     

抗坏血酸稳定纳米铜/壳聚糖复合物的原位制备、表征及抗菌性能（英文）

以抗坏血酸为稳定剂,通过原位还原络合于壳聚糖表面的铜离子,制备了平均粒径(2.57±0.5)nm的高稳定的纳米铜/壳聚糖复合物.利用X射线光电子谱(XPS)和傅里叶变换红外光谱(FT-IR)表征了复合物的合成过程及其稳定性,同时采用最低抑菌浓度法(MIC)定量表征了复合物的抗菌性能.结果表明稳定结合于壳聚糖表面的纳米铜具备高效的广谱抗菌性能,特别针对革兰氏阳性菌如金色葡萄球菌以及真菌如白色念珠菌.纳米铜/壳聚糖复合物对金色葡萄球菌的MIC为6.4μg m L-1,该数值优于已报道的其他纳米铜材料.常温常压存放90天后,纳米铜/壳聚糖复合物仍具备稳定的结构和良好的抗菌性能.     

累托石对左氧氟沙星的吸附研究

累托石是一种天然间层黏土矿物,具有强稳定性、高分散性以及良好的吸附性.文章利用累托石良好的吸附特性,以其为吸附材料,通过静态吸附实验研究了累托石对水体中左氧氟沙星的吸附机理;并采用BET、XRF、FT-IR等表征手段对累托石结构和性能进行了分析. 结果表明:累托石主要成分为SiO2和Al2O3,比表面积为7.54 m2/g,孔径为3.62 nm,层间距为2.50 nm,其对左氧氟沙星的吸附过程更加符合Langmuir等温模型和准二级动力学模型,且在pH6时吸附效果最佳,最大吸附量达到63.38 mg/g;主要吸附机理是离子交换.     

有机累托石改性阻燃电缆料的制备

用酸化钠化法对累托石(REC)进行结构修饰,以十六烷基三甲基溴化铵(HDTMA.Br)为插层剂,制备了有机累托石(OREC)。使用X-射线衍射(XRD)、傅里叶红外光谱(FTIR)测试其物相,结果显示HDTMA+已完全插层,累托石的层间距由改性前的2.41 nm增大到4.08 nm。通过熔融插层法制备了无卤阻燃电缆料,对其微观结构、力学性能和阻燃性能进行分析,结果表明:氢氧化镁(MH)及OREC填料深埋于基体树脂乙烯-醋酸-乙烯酯共聚物EVA中,形成硬核软壳的特殊结构;OREC与MH具有协同阻燃作用,当OREC添加量为4%时,复合材料的氧指数达到33.8%,较单独添加MH时高出4.8%;拉伸强度较仅添加MH的复合材料提高17%。经老化测试表明,自制有机累托石改性的EVA无卤阻燃电缆料的性能指标满足行业标准的要求。     

Enhancement of transfection efficiency for HeLa cells via incorporating arginine 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 polye- lectrolyte 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 candi- date as a safe and efficient vector for gene delivery and transfection.     

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.     

靶向多肽功能化阳离子聚合物携载ZNF580质粒对内皮细胞增殖的影响

通过原子转移自由基聚合(ATRP)方法制备了以多面体低聚倍半硅氧烷(POSS)为骨架的POSS-(PDMAEMA)₈(PPD)星型阳离子聚合物,通过巯基-双键化学反应,将特异性靶向EA.hy926内皮细胞的CREDVW多肽键接在PPD阳离子聚合物末端,得到POSS-(PDMAEMA)₈-PPEGMA-CREDVW(PPD-CREDVW)阳离子聚合物.在n(N)/n(P)=5时,PPD-CREDVW阳离子聚合物携载pEGFP-ZNF580(pDNA)质粒自组装形成粒径为125.67±3.31 nm、zeta电位为10.64±1.65 mV的PPD-CREDVW/pDNA基因复合胶束.细胞实验结果显示:与PPD/pDNA基因复合胶束相比,PPD-CREDVW/pDNA基因复合胶束携载基因能力强、细胞毒性低、易被细胞摄取,能够显著提高EA.hy926内皮细胞的转染,促进细胞的增殖.     

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.     

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。     

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。     

聚乙二醇-聚乙烯亚胺负载超顺磁纳米Fe3O4的合成及其基因转染应用

采用单甲基醚聚乙二醇改性聚乙烯亚胺,得到水溶性接枝共聚物聚乙二醇接枝支化聚乙烯亚胺(mPEG-g-PEI),并利用离子交换法制备了PEG-PEI-SPIO. 同时,还合成了mal-PEG-COOH,并用其制备了多功能负载SPIO的抗体-聚乙二醇-聚乙烯亚胺. 负载SPIO的抗体-聚乙二醇-聚乙烯亚胺与pDNA复合后形成的复合物粒径约为105 nm,体外实验结果显示T细胞能特异性的吸收抗体-聚乙二醇-聚乙烯亚胺负载的SPIO,靶向组在基因转染实验中有较好的效果,可用磁共振手段进行实时无创观测.     

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。