聚天冬酰胺衍生物药物/基因载体的合成和应用

与碳链聚合物相比,聚氨基酸类高分子由于其生物相容性好、可降解代谢、毒副作用低等优点而被广泛应用于生物医药领域。基于天冬氨酸的聚天冬酰胺衍生物,其合成方法简单多样,通过对其修饰改性可制备出具有各种环境响应性（温度、pH和还原敏感）的智能高分子,得到高效、低毒的药物/基因载体,实现可控释放、增强疗效、降低药物副作用的目的。本文重点介绍了聚天冬酰胺衍生物（特别是刺激响应性聚天冬酰胺衍生物）的合成改性方法、及其在药物和基因载体领域最新的研究进展,并对其发展前景进行了展望。     

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.     

含氟高分子基因载体

阳离子高分子被广泛应用为非病毒类基因载体,但这类高分子材料的转染效率与细胞毒性之间通常存在"恶性"关联,即获得高转染效率时往往会伴随严重的细胞毒性.如何制备兼具高效、低毒特点的高分子载体是成功实施基因治疗的关键.含氟高分子是一类具有独特理化性质的高分子,能够在低电荷密度条件下与核酸形成稳定的复合物,从而实现高效、低毒的基因转染.含氟功能基团可帮助阳离子高分子改善复合物稳定性、细胞内吞、内涵体逃逸、胞内核酸释放等多个环节,从而赋予了含氟高分子在基因递送过程中的氟效应.该专论系统地总结了含氟高分子基因载体的研究,介绍了含氟高分子的基因递送性能、作用机理以及在基因治疗、基因编辑中的应用,并对含氟高分子载体的未来发展进行了展望.     

刺激响应共负载药物/基因微载体的制备

合成了聚乙烯亚胺接枝二茂铁(PEI-Fc)两亲聚合物, 采用水包油法制备包埋疏水性抗癌药阿霉素(DOX)的载药胶束, 并利用胶束表面正电荷的PEI链段有效缔合DNA, 获得尺寸合适、 表面带正电荷的阿霉素与基因共负载微载体. 在磷酸盐(PBS)缓冲溶液中, 共负载微载体能够缓慢释放出DOX. 在硝酸铈铵存在下, 二茂铁从疏水性转变为亲水性, 使载药胶束完全解离, 由于PEI-Fc与DNA之间的静电作用, 使基因超分子组装体稳定存在, 显示出很好的氧化响应特性. 细胞培养结果表明, 表面带正电荷的共负载微载体易被HepG2细胞内吞, 并可转染, 且随着DOX的释放逐渐杀死HepG2肝癌细胞, 为安全稳定、 具有刺激响应的药物与基因共负载微载体的制备提供了可行的途径.     

壳聚糖-g-N-羧甲基-二（2-苯并咪唑）-1,2-乙二醇和纳米金溶胶复合物固定漆酶修饰玻碳电极的直接电化学

制备了壳聚糖-g-N-羧甲基-二（2-苯并咪唑）-1,2-乙二醇（CTS-g-N-CBBIE）,将其与纯化的纳米金溶胶（NGS）共混得到CTS-g-N-CBBIE-NGS复合物。 以此复合物作为固酶载体固定云芝漆酶,固酶量大（31.10 mg/g）,固酶比活力高（1.43 U/mg）;此固酶复合物修饰的玻碳电极在无氧磷酸盐-柠檬酸盐缓冲溶液（pH=5.0）中可以实现无中介酶-电极直接电子迁移（一对准可逆氧化还原峰式电位576 mV(vs.Ag/AgCl)对应于漆酶活性中心T1位的氧化还原）,电子迁移速率常数为228.3 s-1。 当氧气浓度较小时,这种固酶修饰电极对氧气还原具有一定的生物电催化性能（空气饱和缓冲溶液中氧还原峰电位约为320 mV(vs.Ag/AgCl)）。 当氧气浓度增高后,氧还原反应受到抑制;但这种漆酶修饰电极对pH较为敏感,且稳定性和重复使用性欠佳。     

基于原位聚合技术构建细胞内微环境响应型DNA递送系统北大核心CSCD

传统的非病毒载体基于分子间静电自组装作用与核酸结合,组装的复合物在体内复杂的环境中容易发生结构解离,共价结合的交联聚合物载体有望成为解决传统非病毒载体结构稳定性差的有效方案。选择N-(3-氨丙基)甲基丙烯酰胺盐酸盐、1-乙烯基咪唑、2-甲基丙烯酰氧乙基磷酸胆碱与N,N′-双(丙稀酰)胱胺作为多功能性单体,采用原位聚合方法制备包载质粒DNA(pDNA)的交联聚合物-pDNA复合物。其中,共价键为载体提供优异的结构稳定性;1-乙烯基咪唑能够响应胞内溶酶体酸性微环境,触发质子海绵效应便于复合物的溶酶体逃逸;N,N′-双(丙稀酰)胱胺的二硫键可以响应胞内高水平的谷胱甘肽(GSH),实现复合物在细胞内部选择性解聚,释放内含pDNA。研究表明,该复合物平均水合半径约135 nm,ζ电势约−6.5 mV,形貌近似球形。该复合物可在10 mg/mL肝素环境中保持结构稳定性,具有响应细胞内GSH,触发释放包载核酸分子的功能。细胞实验证明该复合物细胞毒性低。细胞摄取、转染能力强。综上所述,基于原位聚合技术制备交联聚合物载体在基因递送领域具有重要应用前景,本研究为新型基因递送载体的开发提供了新思路。     

葡萄糖敏感苯硼酸基高分子纳米药物传输体系

近年来,智能葡萄糖敏感自调式药物传递系统备受关注。这种智能药物释放系统能够模拟胰腺分泌胰岛素的生理模式而精准调控药物释放并控制血糖水平,在糖尿病治疗中具有良好的应用前景。其中,苯硼酸(PBA)功能化的葡萄糖敏感高分子纳米载体成为近年来的研究热点之一。该类材料具有体系稳定、可长期储存、可逆的葡萄糖敏感性能等优势。根据响应因素不同,葡萄糖敏感药物传递系统可分为pH响应、温度响应和光响应等类型。本文重点介绍了基于PBA的葡萄糖敏感高分子纳米药物载体的发展过程、性能和应用,并对该领域的发展前景进行了展望。     

赖氨酸修饰聚酰胺-胺树枝状高分子的制备及性能

通过液相合成法, 用L-赖氨酸(L-Lys)对4代聚酰胺-胺(4.0G PAMAM)进行表面修饰, 制备了新型的PAMAM-Lys树枝状高分子. 采用FTIR、 ¹H NMR、 元素分析和粒径分析等手段进行了结构表征. PAMAM-Lys的C, H, N元素含量分别为53.43%, 9.58%和24.29%, 端氨基测定值为2.18, 接近于理论计算值; 平均粒径约6.35 nm, 多分散系数约0.09. 应用透射电子显微镜和噻唑蓝四氮唑溴化物(MTT)比色法, 探讨了PAMAM和PAMAM-Lys树枝状高分子载体/质粒DNA复合物的形态及体外细胞毒性. 当最佳电荷比R_+/-=4时, PAMAM-Lys与DNA形成复合物, 通过静电作用, 使DNA结构收缩, 质粒粒径介于50~100 nm之间, 分布较均匀, 形态规则; 作用于体外293T细胞时, PAMAM-Lys及其与DNA复合物的细胞毒性明显低于5代聚酰胺-胺(5.0G PAMAM). 研究结果表明, 制备的新型PAMAM-Lys树枝状高分子显著降低了高代数PAMAM树枝状高分子载体的细胞毒性, 具有良好的体外细胞相容性, 有望成为一种DNA疫苗的优良载体.     

用于基因传递的智能响应性高分子研究进展

智能响应性高分子由于具有优异的环境响应性、多样的功能性、良好的生物可降解性和生物相容性而在生物医用领域备受瞩目.基于特定功能的智能响应性高分子基因载体可以克服基因运载中的障碍,降低对正常组织和细胞的毒副作用,提升靶细胞的基因转染效率.此外,大部分智能响应性高分子能有效结合多种治疗方式以实现更有效的治疗效果.本文综述了近年来智能响应性高分子在基因运载及相关生物医用领域的研究进展,对相关智能响应性高分子的设计及特点进行了介绍,并进一步对其在基因运载及相关生物医用领域的应用前景进行了展望.     

DNA与两性表面活性剂相互作用研究

本文综述了DNA与两性表面活性剂相互作用的研究进展,主要介绍了利用荧光显微镜、动静态光散射、相图及浊度等方法对DNA与两性表面活性剂相互作用的观察与形成复合物的表征。由于两性表面活性剂所具有的独特性质,可以实现在特定pH范围通过静电作用诱导DNA构象发生线圈状向小球状的不连续转变,并可通过调节溶液pH值、离子强度等实现对DNA-两性表面活性剂复合物的稳定性的调控。DNA与两性表面活性剂相互作用形成的复合物在非病毒基因载体研究方面具有潜在的应用价值。     

Comprehensive study on the formation of polyelectrolyte complexes from (quaternized) poly[2‐(dimethylamino)ethyl methacrylate] and poly(2‐acrylamido‐2‐methylpropane sodium sulfonate)

Polyelectrolyte complexes (PECs) have been prepared from well‐defined (quaternized) poly[2‐(dimethylamino)ethyl methacrylate] (PDMAEMA) and high molecular weight poly(2‐acrylamido‐2‐methylpropane sodium sulfonate) (PAMPSNa) after a thorough study of their viscometric properties. The effect of pH and quaternization degree of PDMAEMA on PECs stoichiometry has been examined. PEC‐based materials have been characterized in terms of thermal stability, equilibrium swelling degree, and free/bound water composition. The stoichiometry and swellability of the physically crosslinked hydrogels obtained from fully quaternized PDMAEMA/PAMPSNa complexes do not depend on pH. In contrast, PECs made of non quaternized PDMAEMA and PAMPSNa are highly affected by pH, and could reversibly disintegrate at pH ≥ 9. Partially quaternized PDMAEMA/PAMPSNa PECs exhibit intermediate properties and form stable loose structures in the whole investigated pH range. Finally, stable dispersions of PECs nanoparticles have been successfully produced from dilute solutions of the complementary polyelectrolytes. The nanoparticle average diameter as determined by dynamic light scattering proved to depend on the molar fraction of DMAEMA‐based subunits and on the initial polyelectrolyte concentration. © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 5468–5479, 2006     

New insights into the structure of polyelectrolyte complexes

The formation of polyelectrolyte complexes (PECs) from oppositely charged linear polyelectrolytes (PELs) was studied using static light scattering at various salt concentrations. The PELs used were poly(allylamine hydro chloride) (PAH) and the two polyanions poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA). Physical characteristics such as the radii of gyration, molecular weights, and water contents of the PECs were determined at various molar mixing ratios. Despite relatively small differences in chemical structure between PAA and PMAA, fairly large differences were detected in these physical characteristics. Generally, PECs comprising PMAA were larger and contained more water. Moreover, by using cryogenic transmission electron microscopy, transmission microscopy and atomic force microscopy, shape and structure of the prepared PECs were investigated both in solution and after drying. The PECs were found to be spherical in solution and the shape was retained after freeze-drying. PECs adsorbed on silica surfaces and dried in air at room-temperature still showed a three-dimensional structure. However, the relatively low aspect ratios indicated that the PECs collapsed significantly due to interactions with the silica during adsorption and drying. At intermediate ionic strengths (1-10 mM), stagnation point adsorption reflectometry (SPAR) showed that the adsorption of low charged cationic PAH-PAA PECs on silica surfaces increased if the pH value was increased from pH 5.5 to 7.5.     

Coating of nanoparticles bearing amino groups on the surface with hydrophilic HPMA-based polymers

Two hydrophilic polymer systems, multivalent N-(2-hydroxypropyl)methacrylamide-based copolymers bearing thiazolidine-2-thione (TT) reactive groups randomly distributed along the polymer chain and monovalent semitelechelic pHPMA with the TT end group, were designed for surface modification of gene delivery vectors, namely, DNA polyelectrolyte complexes (PECs) and adenoviruses. In this study, the amino group-modified polystyrene latex nanoparticles were selected as a suitable model of a nanoparticulate delivery system bearing NH₂ groups on the surface. The coating process was monitored by changes in molecular weight and hydrodynamic parameters of the nanoparticles by light scattering methods. It was shown that for study of the coating process the model latex particles are more suitable than the original PEC vectors due to better chemical and physical stability of latexes. The results obtained in the model study (reaction conditions, methods of evaluation) suggest an optimal polymer structure and a method of efficient and complete coating of nanoparticle surface well applicable to the real gene delivery vectors.     

N,N,N-trimethylchitosan iodide complexes with a weak or a strong polyacid and nanoparticles thereof

Polyelectrolyte complexes (PECs) were prepared from N,N,N-trimethylchitosan iodide (TMCh) of different molar mass and a weak polyacid-poly(acrylic acid) (PAA) or a strong polyacid-poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS). The quaternization of the amino groups of chitosan enabled the formation of water-insoluble PECs in a broad pH range—from 3 to 12 and from 1 to 12 for TMCh/PAA and TMCh/PAMPS, respectively. Whereas the stoichiometry of the TMCh/PAA complex was pH dependant, the stoichiometry of the TMCh/PAMPS complex did not depend on pH. The stoichiometry and the yield of the complexes were influenced by the molar mass of TMCh. PEC nanoparticles were produced by mixing dilute solutions of the oppositely charged polyelectrolytes as revealed by dynamic light scattering analyses. The size of the particles was in the range of 135–924 nm and depended on the polyelectrolyte molar mass, the initial polyelectrolyte concentration, and the molar fraction of the TMCh units. Microbiological screening against Staphylococcus aureus and Escherichia coli revealed that PECs between TMCh and PAA or PAMPS have a good antibacterial effect, which is more slowly pronounced than that of the starting TMCh of different molar mass.     

Contribution of flow field flow fractionation with on line static and dynamic light scattering to the study of hydrosoluble polyelectrolyte complexes

Water soluble polyelectrolyte complexes (PECs) formed between polyaspartate (anionic polymer) and poly(trimethylammonium propyl methacrylamide chloride) (cationic polymer) were studied by flow field flow fractionation with on-line coupling multi-angle laser light scattering-quasi elastic light scattering-differential refractive index determination (F4/MALLS/QELS/DRI). The separation technique permits to characterize polydisperse PECs. The molar mass of the polycation (PC) influences the stiffness of the PECs and the proportion between single PECs (i.e. nPA/1PC) and multiple PECs (i.e. nPA/n’PC). High ionic strength with NaCl (>0.1 M) tends to break the multiple PECs while CaCl₂ destroys PECs and leads to the formation of complexes polyaspartate/Ca²⁺. The studied PECs can be used as inhibitors to the calcite formation in the drilling fluids.     

The concept of molecular machinery is useful for design of stimuli-responsive gene delivery systems in the mammalian cell

Since the first generation of molecular machines including photoresponsive crown ethers and its analogues was reported by Shinkai et al., a huge number of molecular machines exhibiting dynamic chemical and physical functions have been designed and developed. On the other hand, non-viral vectors are desired to possess conflicting properties to associate with DNA until reaching the nucleus as their final destination and dissociate from DNA there. In other words, non-viral vectors should work as a sort of molecular machinery. To overcome this dilemma, recently, much attention is focused on the development of the intelligent vectors, also called as ‘stimuli responsive vectors’ working as molecular machines. In this review, stimulus responsive gene delivery systems in which some structural factors and/or physiological properties are regulated in response to extracellular signals such as redox, pH, ultrasound, light, temperature, etc. are introduced as a new generation of non-viral vectors. These extracellular signals such as ultrasound, light, and temperature can be potent stimuli capable of site-, timing-, and duration-specific gene expression. This is a paper selected for “HGCS Japan Award of Excellence 2006”.     

A facile route for fabricating novel polyelectrolyte complex membrane with high pervaporation performance in isopropanol dehydration

Polyelectrolyte complexes (PECs) of sodium carboxymethyl cellulose (CMCNa) and poly(diallyldimethylammonium chloride) (PDDA) were prepared in dilute hydrochloric acid (HCl) aqueous solution and obtained in its solid form. Element analysis and FT-IR showed that the composition of PECs could effectively be tuned by the concentration of HCl in parent polyelectrolyte solution. The PECs were then dissolved in 0.1 mol/L aqueous NaOH and subsequently cast onto polysulfone ultra-filtration membrane. This composite membrane, which has a unique homogeneous PECs separation layer, was subjected to pervaporation test for the first time and gave a performance of J = 3.0 kg/m² h, α = 960 for 10 wt% water–isopropanol feed at 75 °C. Meanwhile, performance of the PECs membrane displays good stability and unique dependence on feed temperature. These findings, together with its ultra-high performance, are primarily explained by the structure characteristic of PECs.     

Formation and properties of positively charged colloids based on polyelectrolyte complexes of biopolymers

Formation of colloids based on polyelectrolyte complexes (PECs) was mainly studied with synthetic polyelectrolytes. In this study, we describe the elaboration of positively charged PEC particles at a submicrometer level obtained by the complexation between two charged polysaccharides, chitosan as polycation and dextran sulfate (DS) as polyanion. The complexes were elaborated by dropwise addition of default amounts of DS to excess chitosan. Quasi-elastic light scattering was used to investigate in detail the influence of the characteristics of components (chain length, degree of acetylation) and parameters linked to the reaction of complexation (molar mixing ratio, ionic strength, concentration in polymer) on the sizes and polydispersity of colloids. Chain length of chitosan is the major parameter affecting the dimensions of the complexes, high molar mass chitosans leading to the largest particles. Variations of hydrodynamic diameters of PECs with the molar mass of chitosan are consistent with a mechanism of particle formation through the segregation of the neutral and then hydrophobic blocks of the polyelectrolyte complexed segments. Resulting particles display probably a structure constituted by a neutral core surrounded by a chitosan shell ensuring the colloidal stabilization. Such a structure was evidenced by measurements of electrophoretic mobilities revealing that the positive charge of particles was decreasing with pH, in relation with the neutralization of excess glucosamine hydrochloride moieties.     

Metal ion-enriched polyelectrolyte complexes and their utilization in multilayer assembly and catalytic nanocomposite films

The mixing of Ag ion-doped poly(ethyleneimine) (PEI) and poly(acrylic acid) (PAA) produced Ag ion-doped polyelectrolyte complex particles (PECs) in solution. Positively charged Ag ion-doped PECs (Ag ion PECs) with a spherical shape were deposited alternatively with PAA to form a multilayer assembly. The multilayered film containing Ag ion PECs was reduced to generate a composite nanostructure. Metal nanoparticle (NP)-enriched nanocomposite films were formed by an additional process of the postadsorption of precursors on PECs within the nanocomposite films, which resulted in the enhancement of the catalytic and electrical properties of the composite films. Because the films contain PECs that are responsive to changes in pH and most of the NPs are embedded in the PECs, interesting catalytic properties, which are unexpected in a particle-type catalyst, were observed upon pH changes. As a result of the reversible structural changes of the films and the immobilization of the NPs within the films, the film-type catalysts showed enhanced performance and stability during catalytic reactions under various pH conditions, compared to particle-type catalysts.