非病毒性基因载体的合成研究

基因治疗是一种有效的治疗先天性遗传性疾病以及后天获得性疾病的手段。它通过激发细胞的生物活性或者抑制细胞非正常的功能来治疗或者预防疾病的发生,例如细胞的基因紊乱,细胞的无序增殖。目前基因治疗所面临的问题是缺乏有效的基因递送载体。基因载体主要分为病毒性基因载体和非病毒性基因载体。与病毒性基因载体相比,非病毒性基因载体具有毒性小、安全性高、易于制备、能够荷载分子量大的DNA等优点。本文综述了非病毒性基因载体的合成研究进展。     

基于纳米材料的基因载体

基因疗法是治疗基因变异引起的先天性遗传疾病和后天获得性疾病以及癌症的新型有效方法。外源基因在细胞中安全、高效、稳定的表达是基因治疗成功的关键,这与基因治疗所使用的载体系统息息相关。基因载体主要分为病毒载体和非病毒载体两大类:病毒载体的转染效率较高,但副作用较大;非病毒载体作为一种新型的基因传递系统,可以弥补病毒载体的缺陷,尽管其转染效率稍逊于病毒载体,但在基因治疗领域具有不可替代的作用。随着纳米技术的出现和蓬勃发展,基于纳米材料的基因载体研究受到越来越多的关注。纳米基因载体具有如下潜在的优势:它制备相对简单,易于对其进行多功能的修饰;具有良好的生物相容性,一般不会引起强烈的机体免疫反应;粒径普遍很小,容易穿过人体的组织间隙而被细胞吸收,基因转运效率较高;可以较有效保护其所携带外源基因,利于基因更高效地表达。本文主要对基于金属、无机非金属、阳离子聚合物和脂质体纳米材料作为基因载体的研究进展进行综述和展望。     

阳离子聚合物基因转染载体的研究进展

安全有效的基因载体是实现基因治疗的必要条件,由于阳离子聚合物易于合成和改性,无免疫原性,可以方便地与DNA形成紧密的超分子复合物,保护DNA免受核酸酶的降解,并促进其进入细胞,从而成为非病毒基因载体中的一个重要类型;但阳离子聚合物基因载体,对细胞具有电荷相关的毒性,转染效率低于病毒载体,这成为限制其进入临床使用的瓶颈.本文从提高阳离子聚合物作为基因载体时的转染效率及降低其毒性方面综述了阳离子聚合物基因载体的研究进展,归纳了改善阳离子聚合物基因载体转染特性的八种方法,预测了阳离子聚合物基因载体的发展前景.     

pH敏感型mPEG-Hz-PLA聚合物纳米载药胶束的制备

以合成的含有腙键的聚乙二醇大分子(mPEG-Hz-OH)为引发剂,以丙交酯为单体引发开环聚合反应,并通过调整投料比,制备出3种不同分子量的含腙键的生物可降解嵌段聚合物(mPEG-Hz-PLA).将腙键引入到聚合物的骨架中,以此构建聚合物胶束并作为pH敏感型纳米药物载体.制备的pH敏感型胶束的CMC值等于或低于5.46×10-4 mg/m L,DLS和TEM显示粒径均小于100 nm,且粒径分布均匀.非pH敏感型胶束在不同pH下的粒径变化不明显,而pH敏感型胶束在酸性环境下(pH=4.0和pH=5.0)胶束粒径出现了明显变化.以阿霉素为模型药物制备了pH敏感型载药胶束,其粒径比空白胶束大(100~200 nm),且粒径分布均匀.药物释放实验表明pH敏感型载药胶束随着释放介质pH降低累积释药量增高.MTT实验表明空白胶束对HeLa细胞和RAW264.7细胞几乎没有抑制作用,而载阿霉素的胶束对2种细胞的抑制作用都随着剂量的增大和时间的延长而增强.     

阳离子基因载体的pH敏感遮蔽体系的制备及表征

合成了一种pH敏感的遮蔽体系-谷氨酸苄酯/谷氨酸共聚物(PBLG-co-PGA), 用于对DNA/阳离子基因载体复合物颗粒表面正电荷的遮蔽, 以提高其在体内的稳定性. 研究表明, PBLG-co-PGA (PGA(x), x为PGA占共聚物中摩尔百分数)具有pH敏感性. 并以pH敏感点接近生理pH值的PGA(60)为遮蔽体系进行研究. PGA(60)能够对DNA/PEI(1:1)复合物颗粒表面正电荷进行有效遮蔽. 凝胶阻滞电泳显示, 用PGA(60)对DNA/PEI复合物进行不同比例遮蔽, 没有发生与DNA的链交换作用. MTT细胞毒性测试表明, PGA(60)和三元复合物DNA/PEI/PGA(60) 在测试范围内几乎没有细胞毒性. 荧光素酶转染实验表明, 部分遮蔽后转染效率有所提高; 用PGA(60)对DNA/PEI复合物完全遮蔽为负电后, 由于同细胞表面的电荷排斥作用, 三元复合物不易被细胞内吞, 导致不发生细胞转染. 因其合适的pH响应性, PGA(60)将可能成为一种能随pH值的变化, 实现对聚阳离子基因载体进行电荷遮蔽/智能释放的遮蔽材料.     

基因治疗用病毒载体的化学修饰策略

病毒是目前基因治疗中最常用的载体,但依然不能将治疗基因高效和选择性地输送到特定的靶细胞.因此构建转导效率高、选择性强和免疫原性低的病毒载体是当今基因治疗研究关注的焦点.目前国际上主流的策略是利用不同的技术手段,在病毒载体上偶联各种修饰分子,从而克服病毒载体自身缺陷或赋予其新功能,综合提升病毒载体临床适用性.近十几年来,许多化学手段被报道.本文以基因治疗用病毒载体为切入点,对病毒可利用的化学修饰方法进行了综述,并对基因治疗用病毒载体的未来发展趋势进行了展望.     

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.     

基于树枝状聚合物构建的核酸载体研究进展

安全有效的基因载体对于基因治疗有着重要的应用价值.相对于病毒性基因载体,非病毒基因载体具有低免疫原性、易于大规模生产和成本较低的特性,因而受到越来越多的关注,但是非病毒基因载体在转染效率和选择性方面有一定的限制性.综述了基于树枝状聚合物构建的基因载体的研究进展,包括:(i)树枝状及其衍生材料;(ii)与超分子母体材料,如环糊精、葫芦脲结合的的树枝状聚合物基因载体;(iii)与无机材料结合的树枝状聚合物载体,如金纳米粒子、氧化铁纳米粒子以及石墨烯等结合的树枝状聚合物构建的基因载体;(iv)功能响应性的树枝状聚合物载体,如p H响应型、二硫键响应型、氧化还原型以及氟代化树枝状基因载体.     

以光诱导Fenton反应控制M-PEIs纳米凝胶的粒径

基因治疗,就是将正常基因导入体内以弥补缺失或替换突变基因来医治囊性纤维化和血友病等[1]多种疾病.而制备高效安全的基因转染载体至今仍是基因治疗的一个瓶颈.非病毒类载体的基因转染效率目前虽不高,但由于其具有低毒、低免疫反应、无基因插入片断大小限制、制备方便、易保存     

刺激响应性非病毒转基因载体

常见的阳离子聚合物能够通过静电作用有效缩合DNA,形成聚电解质复合物（PECs）。这些复合物易于细胞内在化,从内涵体中逃逸,并能保护DNA免受DNA酶的降解。但是,强烈的静电作用也限制了基因进入细胞核之后从复合物中的释放,限制了基因的表达。鉴于此,科学家们设计了一类“智能”高分子载体,这些智能高分子载体能够响应外界微环境温度、pH值和氧化还原环境变化的刺激,其自身大分子构象发生改变,进而促进DNA从复合物中逃逸,提高了转染率。本文介绍了近年来有关聚异丙基丙烯酰胺基温度响应性载体以及光、pH和响应胞内谷胱甘肽（GSH）等氧化还原反应的非病毒转基因载体的研究进展。     

Synthesis and characterization of a pH-sensitive shielding system for polycation gene carriers

To increase the in vivo stability of polycation gene carriers,a pH-sensitive shielding system,γ-benzyl L-glutamate-co-glutamate acid polymer(PGA(60)(60 refers to the molar ratio of glutamate acid in the polymer)),was synthesized and characterized.PGA(60) showed pH sensitivity at about pH 6.0.PGA(60) shielded the positive charge of DNA/PEI(1:1) complexes.Gel retardation assay showed that no DNA-strand exchange with PGA(60) occurred after PGA(60) was added to DNA/PEI complexes at different proportions.MTT cyt...     

SYNTHESIS AND pH-SENSITIVE SELF-ASSEMBLY OF DENDRITIC POLY(AMIDOAMINE)-b-POLY(L-GLUTAMATE)BIOHYBRIDS

 Dendritic poly(amidoamine)-b-poly(L-glutamate) (PAMAM-b-PLG) biohybrids were synthesized by the ring-opening polymerization of γ-benzyl-L-glutamate N-carboxyanhydride monomer, followed by the deprotection of benzyl groups on poly(benzyl-L-glutamate), and were characterized by ¹H-NMR, FT-IR and gel permeation chromatography. The self-assembly behavior of the PAMAM-b-PLG biohybrid was investigated by means of UV-Vis, dynamic light scattering (DLS), transmission electronic microscopy (TEM) and ¹H-NMR. UV-Vis analysis demonstrated that the critical aggregation concentration of PAMAM-b-PLG was dependent on the pH value of aqueous solutios. DLS results further evidenced that the PAMAM-b-PLG biohybrid exhibited a pH-sensitive self-assembly behavior, and the average size of the self-assembled nanoparticles decreased gradually with the increasing pH value of the PAMAM-b-PLG solution. The self-assembled nanoparticles gave a nearly spherical morphology, and the pH-induced self-assembly had no obvious effect on the morphology of nanoparticles.     

pH响应性聚膦腈水凝胶的制备与表征

以N,N-二甲基乙二胺为阳离子型取代基, 甲基丙烯酸羟乙酯或烯丙胺为共取代基, 制备了同时带有pH响应性侧基和不饱和双键侧基的聚膦腈. 该聚合物的结构和组成经¹H NMR和FTIR确认后, 通过自由基引发交联得到聚膦腈凝胶. 凝胶在不同介质中的吸水率测试结果表明, 该阳离子型聚膦腈水凝胶的溶胀行为受聚合物侧基组成、介质pH、离子强度和阴离子价态影响显著, 有望与葡萄糖氧化酶结合后实现对胰岛素的葡萄糖响应释放.     

A pH-sensitive Modified Polyacrylamide Hydrogel

Intelligent hydrogels which have the capability to respond to small external stimulus changes, such as temperature1, pH2, photo field3, and antigen4, have attracted significant attention from both academia and industry. pH-sensitive polymers are produced …     

Tunable phase transition behaviors of pH-sensitive polyaspartamides having various cationic pendant groups

New pH-sensitive graft copolymers based on poly(2-hydroxyethyl aspartamide) (PHEA) were prepared by attaching various cationic monomers, such as 4-(aminomethyl)pyridine (PY), 1-(3-aminopropyl)imidazole (IM), and N-(3-aminopropyl)dibuthylamine (BU), as pH-sensitive units and octadecylamine (C₁₈) as a hydrophobic segment on poly(succinimide). Phase transition of each copolymer solution occurred at a vicinity of the pK _a value of the cationic groups, and their insoluble pH ranges were broadened as the feed amount of pH-sensitive moieties was increased. Depending on the cationic grafts having different pK _a values, the pH ranges where the copolymer became insoluble could be tuned. Copolymers PHEA-g-C₁₈-PY, PHEA-g-C₁₈-IM, and PHEA-g-C₁₈-BU exhibited phase separations in solutions at pH ranges of 4∼6, 6∼8, and 9∼12, respectively. These polymers have the unique feature of their pH sensitivity profiles being identified to three regimes. Under low pH conditions (below pK _a ), the polymer solution is transparent. At medium pH (around pK _a ), polymer precipitation occurred in solution. At pH > pK _a , the polymer solution is gradually dissolved again.     

Characterization and Evaluation of Acrylic Acid Co-2-acrylamido-2-methylpropane-1-sulfonic Acid Hydrogels for Uranium Recovery

Crosslinked 2-acrylamido-2-methyl-1-propane sulfonic acid -co- acrylic acid (AMPS/AA) hydrogels were synthesized by radical polymerization in the presence of N,N′-methylenebisacrylamide (MBA) as the crosslinking agent using potassium persulfate (KPS) as initiator. Hydrogels with different compositions and crosslinker concentrations were prepared. The structures of hydrogels were characterized by FTIR analysis. Thermal stabilities of the hydrogels were investigated using TGA and DSC analysis. Swelling kinetics and the equilibrium water content (EWC) of the hydrogels were studied. The swelling behavior of these hydrogels was investigated at different pHs at room temperature. Also the swelling behavior of these hydrogels was investigated at different ionic strength. The ability of the prepared hydrogels to bind uranium(VI) was tested under noncompetitive conditions by batch equilibrium procedure. Experimental work using uranyl nitrate hexahydrate bought from the local market was carried out in the safeguards destructive analysis laboratory (KMP-I) in the National Center for Nuclear Safety and Radiation Control.     

基于HPMCP包覆介孔SBA-15的pH敏感药物缓释系统

以肠溶性包衣材料羟丙甲纤维素邻苯二甲酸酯(HPMCP)为原料,在负载法莫替丁(Famo)的SBA-15药片表面包覆聚合物膜,成功制备了一种新型的pH敏感药物缓释系统, 并考察了此缓释系统在不同pH释放环境中的释放行为. 结果表明: 在模拟胃液中(SGF, pH=1.2),HPMCP能致密包覆在药片表面,从而明显延缓Famo的释放速度;而在模拟肠液中(SIF, pH=7.5),HPMCP能够迅速溶于缓释溶液中,因而对Famo释放速度的影响甚微. 因此,可以将这种新型智能药物缓释系统应用于肠道靶向给药.     

pH敏感型聚合物PEG-b-PHEMA(His)的合成及胶束性能研究

通过点击化学(Click chemistry)与原子转移自由基聚合(ATRP)一锅法合成AB型嵌段聚合物聚乙二醇嵌段聚甲基丙烯酸羟乙酯(PEG-b-PHEMA), 之后对所得聚合物进行组氨酸(His)修饰得到pH敏感型聚合物PEG-b-PHEMA(His), 运用核磁共振(¹H NMR)及凝胶渗透色谱(GPC)表征了该嵌段聚合物及组氨酸修饰后的产物的结构和分子量分布(M_n= 8.2×10³～13.0×10³ g/mol, PDI=1.27～1.51), 说明聚合物被成功地合成且具有较小的多分散系数. 之后对嵌段聚合物PEG-b-PHEMA(His)进行了动态光散射(DLS)、透射电镜(TEM)和临界胶束浓度(CMC)的测试, 结果表明, 聚合物胶束呈球形且粒径分布均匀, 并且粒径随组氨酸接枝率增大而减小. 最后我们做不同接枝比的PEG-b-PHEMA(His)的酸碱滴定曲线, 结果表明, 组氨酸的接入使聚合物具备明显的pH响应能力, 进一步的释药试验也证明了这一结果.     

Pharmacokinetics and Tissue Distribution of Vinorelbine Bitartrate after Intraveous Administration of Liposomal and Injectable Formulations

A hydrophilic and temperature-induced degradation drug, vinorelbine bitartrate (VB)-loaded phosphatidylethanolamin liposomes (PSLs), was prepared by the thin-film hydration method. Liposomes were made of phosphatidylethanolamine: cholesteryl: oleic acid (PE: CHOL: OA, 3:3:1 mass/mass). The mean particle size of the PSLs ranged from 293.06 nm. The transmission electron microscope (TEM) images displayed that the shape of the PSLs was multilamellar vesicles with smooth surface. The highest entrapment efficiency (EE) and drug loading capacity (DL) could reach up to 68.5% and 6.23%, respectively. The PSLs was evaluated by comparing the rate of release of encapsulated VB in different phosphate buffer solution (PBS), and the result showed that the rate of drug release in acid medium was faster than in pH 7.4. Pharmacokinetic characteristics in vivo and the tissue distribution in mice were investigated, which provided experimental and theoretical basis for utilizing liposomes in malignant tumor chemotherapy.     

Design, synthesis and characterization of a novel pH-sensitive hydrogel

A novel degradable pH-sensitive hydrogel with pendent carboxyl groups was designed and synthesized from ethylenediaminetetraacetic dianhydride （EDTAh） and butanediamine （BDA） with dicyclohexylcarbodiimide （DCC） as a condensating agent and BDA as a crosslinking agent. The obtained polymers were characterized by ^13C NMR, ^1H NMR and FTIR. The swelling experiments of the hydrogel in pH 3, 7, and 12 media indicated much higher swelling ratio in pH 12 media than in pH 3 and pH 7 media, exhibiting sound pH sensitivity. The pH sensitivity of this type of hydrogel may be regulated through controlling the type and the dose of the crosslinking agent.