聚苯丙氨酸修饰低分子量聚乙烯亚胺制备高效基因载体

分别制备了以支化小分子量聚乙烯亚胺(PEI-1.8k)为引发剂,引发苯丙氨酸-NCA开环聚合得到聚乙烯亚胺-聚苯丙氨酸(PEI1.8k-g-PPhe)以及聚乙烯亚胺接枝苯丙氨酸单体(PEI1.8k-g-Phe)的系列基因载体材料.利用核磁、粒度、zeta电位仪、荧光光度计、流式细胞仪以及激光共聚焦显微镜对PEI1.8k-g-PPhe,PEI1.8k-g-Phe以及PEI1.8k-g-PPhe/DNA和PEI1.8k-g-Phe/DNA复合物颗粒进行了系统的表征.研究结果表明,最佳转染条件下,PEI1.8k-g-PPhe10/DNA复合物颗粒的粒径约为150 nm,表面电位约为16 m V.在人源宫颈癌(He La)和人源乳腺癌(MCF-7)2种细胞系中均具有较高的基因转染效率,且最佳转染效率可达到PEI-25k的12倍.MTT细胞毒性实验分别比较了PEI1.8k-g-PPhe和PEI1.8k-g-Phe对He La细胞毒性的大小.从实验结果可见,苯丙氨酸引入的方式及数量决定着其细胞毒性的大小.PEI1.8k-g-PPhe和PEI1.8k-g-Phe都具有较低的细胞毒性(材料在较高浓度1 mg/m L时的细胞存活率大于70%).内吞实验结果表明,PEI1.8k-g-PPhe由于接入了具有规则聚合链的聚苯丙氨酸,而易于被He La细胞内吞.PEI1.8k-g-PPhe10/DNA复合物颗粒相比于PEI-25k/DNA,PEI-1.8k/DNA和PEI1.8k-g-PPhe/DNA具有更高的细胞内吞效率.     

刺激响应双硒交联聚乙烯亚胺基因微载体

以丙酸二硒醚为交联剂,通过调节交联剂的用量及反应时间,制备了4种双硒交联聚乙烯亚胺(PEISeSe),研究了其对脱氧核糖核酸(DNA)缔合能力、质子缓冲能力和转染效率的影响.结果表明,随着交联程度的增大,质子缓冲能力降低.PEISeSe能有效诱导DNA的缔合,当聚合物与DNA的质量比≥8时,PEISeSe/DNA组装体可形成150 nm的粒子.在模拟细胞内的还原性环境下,双硒键能有效断裂,显示出很好的响应特性.将氯喹与PEISeSe/DNA组装体同时加入到HEK293T细胞中,氯喹的存在有利于PEISeSe/DNA组装体逃离溶酶体.在细胞内高浓度还原型谷胱甘肽的作用下,PEISeSe交联聚合物可被降解为低分子量的PEI片段,有利于释放出DNA并进攻细胞核,提高转染率并降低毒性.     

一种新型聚乙烯亚胺(PEI)载体的制备及表征

设计一种新型的用于传输小干扰核糖核酸(siRNA)聚乙烯亚胺-聚类丙交酯功能聚合物。首先合成一类含功能基团羧基的丙交酯衍生物,对其进行了1 H NMR、GPC、DSC表征,功能基团的密度可以通过与丙交酯共聚的单体的物质的量来调控,利用羧基与聚乙烯亚胺(PEI)分子上的氨基偶合,将小分子量PEI通过共价键连接到可生物降解的高分子主链骨架上,这种方法提供了一条途径来解决分子量与细胞毒性以及转染效率之间相互矛盾的关系。本文进一步研究了此体系在负载siRNA前后粒径及zeta电位的变化,颗粒粒径分布在50~500nm之间,Zeta电位在-9~-35mV之间。     

六氯环三磷腈交联寡聚乙烯亚胺的制备及基因载体应用

将六氯环三磷腈与分子量为600的超支化寡聚乙烯亚胺在干燥氯仿中反应,合成了可降解的交联型聚合物.利用1HNMR表征了聚合物的结构,并用GPC测试了聚合物的分子量.研究了其作为非病毒基因载体的性能,聚合物载体与DNA形成的复合物颗粒粒径为150nm,Zeta电位为30～40mV,凝胶阻滞电泳显示聚合物/DNA在质量比为0.4时能够将DNA完全阻滞.体外转染实验结果表明,载体对HeLa细胞的最佳转染效率为PEI-25K的3倍;聚合物浓度为20μg/mL时,细胞存活率仍然大于80%,材料的细胞毒性低,生物相容性好,具有良好的生物医学应用前景.     

交联型聚乙烯亚胺智能基因载体的制备及PEG化影响

使用胱胺双丙烯酰胺(CBA)对低分子量聚乙烯亚胺(PEI)进行交联反应制备智能降解型聚阳离子基因载体.通过与聚乙二醇(PEG)反应得到不同程度PEG化的聚阳离子载体.利用核磁、黏度测试、粒度仪、zeta电位仪和凝胶电泳对聚阳离子载体及其与DNA的复合物进行了表征.研究表明随着PEG含量的增加,聚阳离子载体/DNA复合物颗粒粒径变小、表面正电荷降低,PEG具有明显的屏蔽作用,但过多的PEG也使载体与DNA复合能力下降.通过MTT细胞毒性测试和荧光素酶质粒转染实验得出,含二硫键的交联型阳离子聚合物在测试范围内显示了非常低的细胞毒性,最佳转染效率是PEI25k的4倍,PEG化后其细胞毒性得到进一步改善,转染效率却明显降低.     

京尼平交联低聚乙烯亚胺智能基因载体的制备与表征

使用京尼平与分子量为1800的超支化低聚乙烯亚胺在70％的乙醇溶液中反应,合成了具有荧光的交联型聚合物.利用核磁、凝胶渗透色谱、粒度仪、zeta电位仪和凝胶阻滞电泳对聚合物载体及其与DNA复合物颗粒进行了表征.研究表明,聚合物载体与DNA复合物颗粒粒径为120 ～150 nm,zeta电位为+20～25 mV,聚合物/...     

基于低分子量聚乙烯亚胺和油酸的负电性基因载体

构建负电性的基因载体、发展基于低分子量聚乙烯亚胺(PEI)的基因载体对基因传递研究具有重要意义. 本文基于低分子量聚乙烯亚胺(2 kDa)和油酸构建了负电性的基因载体. 它通过混合聚乙烯亚胺(2 kDa)、dsDNA和油酸胶束而自发形成. 该基因载体在血清中很稳定,细胞毒性非常低,可包封80%以上DNA. 通过1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-N-[甲氧基(聚乙二醇2000)]铵盐(DSPE-PEG)对其表面进行修饰,发现多达90%的基因可被细胞摄取.     

胆固醇修饰的低分子量聚乙烯亚胺接枝化聚[L-天冬酰胺-co-L-赖氨酸]作为基因载体的性能研究

通过将低分子量的聚乙烯亚胺(PEI600)及其胆固醇衍生物与聚(L-天冬酰胺-co-L-赖氨酸)(PSL)进行开环反应, 合成了一类新型的肿瘤靶向基因载体, 研究了这类载体与DNA形成复合物的性质以及介导绿色荧光蛋白质粒pEGFP-C1转染不同细胞的性能. 结果表明, 在复合质量比大于5∶1时, 各载体均能与DNA形成结构稳定的复合物. 同时转染实验结果证明, 通过在侧链引入一定数目的胆固醇, 可以明显提高载体对于癌细胞HepG2和Hela的转染效率. 这类新型的载体具有良好的细胞相容性、较高的转染效率以及易于进行靶向修饰等特点, 在基因治疗研究领域中将具有较好的潜在应用价值.     

聚乙烯亚胺复合铜微粒的制备及表征

以溶液还原法对聚乙烯亚胺（PEI）和Cu2＋形成的配合物进行还原,制备了金属Cu微粒。扫描电镜对微粒的分析表明,低浓度Cu2＋配合物制备的微粒形态规则、分布均匀,粒径约0.98μm。红外光谱、X-衍射对微粒组成的分析表明,PEI键合地包覆Cu微粒,没有Cu的其他氧化物存在,PEI包覆量占微粒质量的14.6%,形成的复合...     

超支化聚乙烯亚胺-聚天冬氨酸苄酯共聚物的制备及性能

以超支化聚合物聚乙烯亚胺为引发剂, 天冬氨酸苄酯-NCA为单体, 利用热开环聚合法合成了超支化聚乙烯亚胺-聚天冬氨酸苄酯共聚物(PEI-PBLA). 用核磁共振波谱对PEI-PBLA的化学结构进行了表征. 研究了PEI-PBLA在水溶液及有机溶液中的自组装行为. 以芘为荧光探针测定了共聚物在水中形成胶束的临界胶束浓度. 以甲基橙为客体分子, 研究了共聚物胶束的包覆能力, 结果表明, 共聚物对客体分子的吸附与溶液的pH值有关. 研究了共聚物与质粒DNA的复合发现, PEI-PBLA能够很好地与质粒DNA复合, 并且可以避免DNA酶的降解, 对其起到一定的保护作用. 此聚合物在药物传输、可控释放及基因载体等方面都有着潜在的应用价值.     

Design of Serum Compatible Tetrary Complexes for Gene Delivery

A novel gene delivery system, called PoSC, consisting of PEI, PSP, and HA is described. In contrast to the DNA/PEI/HA ternary system whose transfection efficiency decreases significantly with increasing serum concentration, PoSC exhibits a high transfection efficiency of about 51 and 87% for NIH3T3 and HCT116 cells, respectively, at 50% serum concentration. Furthermore, PoSC shows no cytotoxic effect at its working concentration. The overall results suggest that HA adsorption on cationic complexes enhances the transfection efficiency, while PSP is essential for high transfection efficiency at higher serum concentration.

     

pH敏感纳米凝胶的制备及其在siRNA转染中的应用

通过分散聚合的方法,以改性了双键的葡聚糖(Dex-AA)作为交联剂,甲基丙烯酸二甲氨基乙酯(DMAEMA)作为单体,过硫酸铵(APS)和四甲基乙二胺(TEMED)分别作为引发剂和助引发剂,合成了不同交联度的、具有pH敏感内吞增强作用的葡聚糖纳米凝胶(DD-NGs),并测试了其复合siRNA进行转染的能力.实验结果表明,该纳米凝胶表面带有正电荷,具有较好的担载siRNA进入肿瘤细胞并沉默基因的能力,且具有pH响应粒径变化的性质.在pH=7.4的体液环境中,纳米凝胶与基因的复合物粒子较小;在肿瘤酸性(pH=6.8)条件下,纳米凝胶与基因的复合物粒子变大,显著地增强了肿瘤细胞对纳米凝胶与基因复合物的内吞.     

pH响应性超支化星形共聚物的合成及基因转染研究

通过两步ATRP制备了两亲性p H响应性超支化星形共聚物HP(DMAESP-co-BS2MOE)-(POEGMA)n.首先,以2-(2'-溴异丙酰氧基)乙基2″-甲基丙烯酰氧基乙基二硫化物(BS2MOE)为自引发单体,与甲基丙烯酰氧基-3-硫代己酸二甲氨基乙酯(DMAESP)共聚,得到超支化共聚物HP(DMAESP-coBS2MOE).然后以HP(DMAESP-co-BS2MOE)作为大分子引发剂引发甲基丙烯酸寡聚乙二醇酯(OEGMA)的聚合,得到超支化星形共聚物HP(DMAESP-co-BS2MOE)(POEGMA)n.在DMAESP中,叔胺基团和甲基丙烯酸酯基团通过酸敏感的β-硫丙酯键连接,由于该键在酸性条件下能够缓慢水解,形成羧基末端,最终得到的聚合物具有p H响应性.同时,由于BS2MOE中含有二硫键,能够被细胞中的谷胱甘肽还原,该聚合物同时具有氧化还原响应性.HP(DMAESP-co-BS2MOE)(POEGMA)n在水中能够自组装成以HP(DMAESP-co-BS2MOE)为核,以POEGMA为壳的纳米级胶束,并能负载DNA,可作为基因转染的载体.     

基于碳水化合物的阳离子聚合物载体在基因释放中的应用

在基因治疗中,基因释放载体是不可缺少的重要组成部分.近几年来聚合物基因释放载体的研究主要旨在开发低毒或无毒的阳离子聚合物用于安全有效的基因释放.作为一类天然化合物,基于碳水化合物的阳离子聚合物载体由于其良好的生物相容性和低毒性被广泛地研究并应用于基因释放.本文阐述了聚合物基因释放的机理,并对近几年来一些典型的含碳水化合物的阳离子聚合物基因释放载体作一综述.     

Amphiphilic Block Copolymer Micelles for Gene Delivery

Gene therapy is a promising method to treat acquired and inherited diseases by introducing exogenous genes into specific recipient cells. Polymeric micelles with different nanoscopic morphologies and properties hold great promise for gene delivery system. Conventional cationic polymers, poly(ethyleneimine)(PEI), poly(L-lysine)(PLL), poly(2-dimethyla-minoethyl methacrylate)(PDMAEMA) and novel cationic polymers poly(2-oxazoline)s(POxs), have been incorporated into block copolymers and decorated with targeting moieties to enhance transfection efficiency. In order to minimize cytotoxicity, nonionic block copolymer micelles are utilized to load gene through hydrophilic and hydrophobic interactions or covalent conjugations, recently. From our perspective, properties(shape, size, and mechanical stiffness, etc.) of block copolymer micelles may significantly affect cytotoxicity, transfection efficiency, circulation time, and load capacity of gene vectors in vivo and in vitro. This review briefly sums up recent efforts in cationic and nonionic amphiphilic polymeric micelles for gene delivery.     

低分子量壳聚糖制备与应用研究进展

低分子量壳聚糖因具有特殊的生物活性而日益受到人们的关注。本文概要介绍了近5年来国内外低分子量壳聚糖的研究进展,包括低分子量壳聚糖的制备方法,如酶降解法、酸水解法、氧化降解法、物理法等,以及低分子量壳聚糖在农业、医药、抗菌、化妆品、食品等方面的应用。     

DOPC‐Detergent Conjugates: Fusogenic Carriers for Improved In Vitro and In Vivo Gene Delivery

Phospholipid‐detergent conjugates are proposed as fusogenic carriers for gene delivery. Eleven compounds are prepared and their properties are investigated. The ability of the conjugates to promote fusion with a negatively charged model membrane is determined. Their DNA delivery efficiency and cytotoxicity are assessed in vitro. Lipoplexes are administered in the mouse lung, and transgene expression Indeterminate inflammatory activity are measured. The results show that conjugation of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPC) with C₁₂E₄ produces a carrier that can efficiently deliver DNA to cells, with negligible ­associated toxicity. Fusogenicity of the conjugates shows good correlation with in vitro transfection efficiency and crucially depends on the length of the polyether moiety of the detergent. Finally, DOPC‐C₁₂E₄ reveals highly potent for in vivo DNA delivery and favorably compares to GL67A, the current golden standard for gene delivery to the airway, opening the way for further promising developments.

     

Synthesis and Application of Low Molecular Weight PEI-Based Copolymers for siRNA Delivery with Smart Polymer Blends

Polyethylenimine (PEI) is a commonly used cationic polymer for small-interfering RNA (siRNA) delivery due to its high transfection efficiency at low commercial cost. However, high molecular weight PEI is cytotoxic and thus, its practical application is limited. In this study, different formulations of low molecular weight PEI (LMW-PEI) based copolymers polyethylenimine-g-polycaprolactone (PEI–PCL) (800 Da–40 kDa) and PEI–PCL–PEI (5–5–5 kDa) blended with or without polyethylene glycol-b-polycaprolactone (PEG–PCL) (5 kDa-4 kDa) are investigated to prepare nanoparticles via nanoprecipitation using a solvent displacement method with sizes ≈100 nm. PEG–PCL can stabilize the nanoparticles, improve their biocompatibility, and extend their circulation time in vivo. The nanoparticles composed of PEI–PCL–PEI and PEG–PCL show higher siRNA encapsulation efficiency than PEI–PCL/PEG–PCL based nanoparticles at low N/P ratios, higher cellular uptake, and a gene silencing efficiency of ≈40% as a result of the higher molecular weight PEI blocks. These results suggest that the PEI–PCL–PEI/PEG–PCL nanoparticle system could be a promising vehicle for siRNA delivery at minimal synthetic effort.     

Synthesis of Multivalent Galactosides as Targeting Ligand for Gene Delivery

A series of novel bifunctional glycolipid ligands designed to bind with high affinity and specificity to the asialoglycoprotein receptor (ASGP-R) has been synthesized and assayed in vitro on human hepatoma cells, HepG2, derived from parenchymal liver cells. The compounds bear five β-linked Gal moieties linked to the core scaffold, hexa-antennary alcohol, for interaction with the binding site of the ASGP-R. The liposome/DNA complexes containing the glycolipid ligands are efficiently recognized by ASGP-R and exhibited high affinity and transfection activity.