多聚赖氨酸淀粉纳米颗粒基因载体的研制及应用

以可溶性淀粉为原料, 利用反向微乳液法, 加入交联剂三氯氧磷, 制备了直径为50 nm左右带负电荷的交联淀粉纳米颗粒. 以该纳米颗粒为内核, 经多聚赖氨酸修饰, 得到了多聚赖氨酸淀粉纳米颗粒(PLL-StNP). 对PLL-StNP进行了颗粒粒度、稳定性和电性的表征, 并通过颗粒的体外细胞毒性检测、颗粒与DNA结合能力及细胞转染等方面的分析, 发现多聚赖氨酸淀粉纳米颗粒(PLL-StNP)有可能作为基因载体, 在此基础上发展了多聚赖氨酸淀粉纳米颗粒基因载体的构建与基因转染技术. 作为非病毒基因载体, 赖氨酸淀粉纳米颗粒具有基因装载量大、转染率高、细胞毒性低以及可生物降解等优点.     

非病毒型DNA纳米微球层层组装构建多层膜用于原位基因传递

高效安全的基因传递体系是基因技术发展的关键问题. 基于聚阳离子的基因纳米微球是一种典型的非病毒型基因载体, 能够在体内外有效转染细胞. 本文通过层层组装方法构建装载基因纳米微球的可降解多层膜, 这种固相基因传递体系能实现材料表面的贴壁细胞的原位转染. 与装载裸DNA的多层膜相比, 基因纳米微球多层膜能更有效地原位转染贴壁细胞, 这主要是因为DNA在此多层膜中仍处于与聚阳离子缔合的状态. 构建于聚乳酸三维支架表面的基因纳米微球多层膜亦能实现支架表面贴壁细胞的原位转染. 这种结构可控、易制备的基因纳米微球多层膜为精确控制基因纳米微球传递提供了一种新方法, 也为基因治疗进一步应用于组织工程、介入治疗和医用植入体提供了一种可能的技术手段.     

一种基于二氧化硅微颗粒的基因载体的制备新方法

建立了一种基于二氧化硅微颗粒的基因载体的制备新方法. 首先将正硅酸乙酯在乙醇和氨水环境下水解， 合成得到二氧化硅微颗粒, 然后通过静电作用将多聚赖氨酸修饰到硅微颗粒上, 制备出可有效地结合DNA的基因载体. 所制备的基因载体可将绿色荧光蛋白表达载体pEGFP导入COS-7细胞中, 实现了绿色荧光蛋白的高效表达. 本方法简便、 快速, 在基因转染与基因治疗研究领域具有较好的潜在应用价值.     

含酯键的可降解聚合物基因载体研究进展

基因治疗作为一种极富潜力、用于替代传统化学治疗的方法,为先天性遗传疾病和严重后天获得性疾病的治疗提供了一条富有前景的新途径。有效释放是基因高效表达的关键,将降解基团引入基因载体可有效地提高基因释放效率,且降解后的小片段更易代谢排出体外,从而有效降低细胞毒性。本文依据材料结构及制备方法对新型聚酯材料进行分类,从转染效率、细胞毒性以及降解性能方面综述了可降解聚酯类基因载体的最新研究进展,并对发展前景进行了展望。     

ATRP法制备聚(甲基丙烯酸氨乙酯-co-甲基丙烯酸N,N-二乙基氨乙基酯)基因载体

为得到低毒、高效的聚阳离子基因载体,以甲基丙烯酸氨乙酯(AMA)和甲基丙烯酸N,N-二乙基氨乙基酯(DEAEMA)为单体,以2-溴代异丁酸乙酯(EBIB)为引发剂,通过原子转移自由基聚合(ATRP)制备了两种聚(甲基丙烯酸氨乙酯-co-甲基丙烯酸N,N-二乙基氨乙基酯)阳离子无规共聚物(P(AMA-co-DEAEMA),简称P).琼脂糖凝胶电泳实验结果表明聚合物P作为阳离子载体可以有效地络合DNA,通过粒径仪测定的复合物粒子的尺寸在400 ～ 600 nm之间.扫描电镜观察的P/DNA复合物形貌是分散均匀的球形颗粒.以25kDa PEI为阳性参照,利用MTT比色法考察了聚合物P对HEK293T细胞的毒性.结果表明,聚合物P的细胞毒性低于25 kDa PEI的细胞毒性.以25 kDa PEI和裸质粒DNA作为参照,我们进一步考察了聚合物P与DNA形成的复合物在HEK293T细胞中的转染效率.结果表明P/DNA复合物在HEK293T细胞中的转染效率远远高于裸质粒DNA的转染效率,并且接近于25 kDa PEI/DNA复合物的转染效率.     

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.     

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.     

聚离子亚基化合物结构影响外源基因转染效率

合成了带正电荷密度不同的聚离子亚基化合物,探讨了聚合物结构对外源基因转染真核细胞的影响规律。从实验结果看,聚离子亚基化合物结构影响外源基因转染效率,但其影响程度与靶细胞的种类相关,对ＨｅＬａ细胞,ＮＩＨ３Ｔ３细胞,聚离子亚基化合物的正电荷密度加大,其促基因转移功能加强;而对ＰＡ３１７包装细胞,结果相反。     

具有环状结构的二氧化硅微聚集体的制备、表征及应用

通过溶胶-凝胶法制备二氧化硅溶胶,并采用喷雾干燥法对其进行形态调控.扫描电镜(SEM)结果表明,喷雾干燥使二氧化硅颗粒发生了形态重组,形成均匀的具有环状结构的二氧化硅微聚集体(PC16MS).通过熔融共混制备了二氧化硅/聚丙烯(PP)纳米复合材料,研究了PC16MS的加入对其微观结构、晶体结构、结晶行为、球晶形态、结晶成核密度和球晶生长速率等方面的影响.采用差示扫描量热(DSC)、偏光相差显微镜(POM)和广角X射线衍射(WAXRD)分析表明,在PP结晶初始阶段,PC16MS的加入大幅度提高了基体材料的成核密度,且使晶粒细化,缩短了结晶时间;当添加2%(质量分数)的PC16MS时,复合材料的结晶温度相对于纯PP提高了10.4℃,成核效率达到39.1%,优于大部分无机成核剂的成核效率.在相同条件下,添加2%未经过喷雾干燥处理的纳米二氧化硅(NC16MS),复合材料的结晶温度相对于纯PP提高3.26℃,成核效率达到12.3%.结果表明,喷雾干燥使二氧化硅颗粒发生了形态重组,形成的均匀介稳态微聚集体在熔融挤出过程中重新分散成纳米粒子,从而有效提高了二氧化硅作为成核剂的成核效率.     

用于体外基因转染的氨基硅烷Fe3O4复合纳米粒子的合成及表征

利用2-吡咯烷酮和乙酰丙酮铁为原料制备Fe3O4磁性纳米颗粒, 用XRD和TEM对样品进行了表征. 选择偶联剂γ-氨丙基三乙氧基硅烷[NH2C3H6Si(OC2H5)3]对纳米粒子进行表面修饰, 制得APTTS/Fe3O4复合载体材料. 以此复合粒子作为传递载体, 将CD基因转染U251胶质瘤细胞. 采用RT-PCR, Western blot及免疫荧光等方法检测CD基因的表达及功能. 结果表明, 制备的Fe3O4颗粒粒径为8~10 nm, 结晶度较高; 经表面修饰后, 粒子表面负载—OH, —NH, —NH2, —C—O和—C—OH等多种功能基团. DNA结合分析及DNase-I消化结果表明, APTTS/Fe3O4粒子能够有效地结合和保护DNA. 体外细胞转染实验证实, 该复合纳米颗粒能够高效地传递CD基因进入U251胶质瘤细胞内, 并进行稳定表达.     

Studies of poly-<Emphasis Type="Italic">L</Emphasis>-lysine-starch nanoparticle preparation and its application as gene carrier

Anion starch nanoparticle (StNP) with a diameter of 50 nm was prepared in water-in-oil microemulsion, with soluble starch as raw materials and POCl₃ as crosslinking agent. PLL-StNP was prepared by linking poly-L-lysine (PLL) on the surface of StNP. At the same time, the size of PLL-StNP and its stability in aqueous solution were checked by AFM. The analysis of plasmid DNA binding, DNase I enzymatic degradation, toxicity and transfection were done. We discovered that PLL-StNP may be used as non-virus nanoparticle gene carrier. And we developed the method of preparing PLL-StNP gene carrier and used it in cell transfection. As non-virus gene carrier, PLL-StNP has some advantages, such as large load of DNA, high transfection efficiency, low cell toxicity and biodegradability.     

Studies of poly-L-lysine-starch nanoparticle preparation and its application as gene carrier

The gene carrier system is the key factor in genetransfection and gene therapy. Suitable gene carriercan deliver the target gene into the receptor cells safely,highly efficiently, controllably, and then the gene isexpressed, thus accomplishing the gene tr…     

Effects of chirality on gene delivery efficiency of polylysine

Chirality is a key factor in the biological activity of many biomolecules. Poly(L-lysine)(PLL), a polypeptide synthesized from L-lysine, is one of the mostly used cationic polymers for gene delivery. The effect of chirality of polylysine(PL) on its gene delivery remains unknown. Herein, we prepared three polylysines(PLs) with the similar molecular weight but different backbone chiralities including poly(L-lysine)(PLL), poly(D-lysine)(PDL) and poly(DL-lysine)(PDLL). The side chains of each PL were modified with propylene oxide(PO) of different chiralities including(R)PO,(S)PO and(R,S)PO. These PL-POs with distinct chirality in main and side chains could condense p DNA into polyplexes. The polyplexes had approximately the same size, zeta potential and binding ability, but showed distinct gene transfection efficiency. We found that the PLs of L-configuration in the main chain had higher transfection efficiency than that of D or DL configuration due to their faster cellular uptake, while the side chain chirality had no effect on transfection efficiency.     

Monodisperse Poly(lactide‐co‐glycolic acid)‐Based Nanocarriers for Gene Transfection

This contribution describes a simple, aerosol‐based method for fabricating monodisperse particles containing mixtures of poly(lactide‐co‐glycolic acid) [PLGA], protamine sulfate (Prot), and poly(l‐ lysine) [PLL] as nanocarriers for gene transfection. Aqueous solutions of PLGA, Prot, and PLL were collison‐atomized, and the resulting aerosolized droplets were dried “on the fly” to form solid particles, which then were electrostatically size‐classified into 50, 100, and 200 nm mobility diameter samples. Measurements of cell viability and transfection reveal that the fabricated nanocarriers have a lower cytotoxicity (>85% in cell viability) and a higher transfection efficiency [>8.7 × 10⁵ in relative light units (RLU) mg⁻¹] than does 25 kDa polyethyleneimine (≈50% and 6.8 × 10⁵ RLU mg⁻¹).     

Ionic/electronic conductivity regulation of n-type polyoxadiazole lithium sulfonate conductive polymer binders for high-performance silicon microparticle anodes

Low-cost silicon microparticles(SiMP),as a substitute for nanostructured silicon,easily suffer from cracks and fractured during the electrochemical cycle.A novel n-type conductive polymer binder with excellent electronic and ionic conductivities as well as good adhesion,has been successfully designed and applied for high-performance SiMP anodes in lithium-ion batteries to address this problem.Its unique features are attributed to the stro ng electron-withdrawing oxadiazole ring structure with sulfonate polar groups.The combination of rigid and flexible components in the polymer ensures its good mechanical strength and ductility,which is beneficial to suppress the expansion and contraction of SiMP s during the charge/discharge process.By fine-tuning the monomer ratio,the conjugation and sulfonation degrees of the polymer can be precisely controlled to regulate its ionic and electronic conductivities,which has been systematically analyzed with the help of an electrochemical test method,filling in the gap on the conductivity measurement of the polymer in the doping state.The experimental results indicate that the cell with the developed n-type polymer binder and SiMP(~0.5 μm) anodes achieves much better cycling performance than traditional non-conductive binders.It has been considered that the initial capacity of the SiMP anode is controlled by the synergetic effect of ionic and electronic conductivity of the binder,and the capacity retention mainly depends on its electronic conductivity when the ionic conductivity is sufficient.It is worth noting that the fundamental research of this wo rk is also applicable to other battery systems using conductive polymers in order to achieve high energy density,broadening their practical applications.     

Bis-Thioether-Containing Lipid Chains in Cationic Amphiphiles: Physicochemical Properties and Applications in Gene Delivery

Cationic amphiphiles featuring two thioether functions in each lipid chain of bicatenar cationic amphiphiles are reported here for the first time. The physicochemical properties and transfection abilities of these new amphiphiles were compared with those of already reported analogues featuring either (i) saturated, (ii) unsaturated or (iii) mono-thioether containing lipid chains. The homogeneity of the series of new compounds allowed to clearly underscore the effect of bis-thioether containing lipid chains. This study shows that besides previous strategies based on unsaturation or ramification, the incorporation of two thioether functions per lipid chain constitutes an original complementary alternative to tune the supramolecular properties of amphiphilic compounds. The potential of this strategy was evaluated in the context of gene delivery and report that two cationic amphiphiles (i. e. 4 a and 4 b) can be proposed as new efficient transfection reagents.     

聚乙二醇嵌段树枝状聚赖氨酸共聚物的合成及其基因载体研究

采用液相多肽合成法制备得到窄分子量分布、结构可控的生物相容性聚乙二醇嵌段共聚树枝状聚赖氨酸阳离子功能大分子(PEG-b-Dendritic PLL). 运用¹H NMR核磁共振、凝胶电泳以及荧光淬灭滴定手段对所得阳离子两嵌段大分子的化学结构及其与质粒DNA (pDNA)结合作用与复合行为进行了研究. 结果表明聚乙二醇嵌段树枝状聚赖氨酸与pDNA分子可以在缓冲溶液中形成稳定的胶束, pDNA与阳离子树枝赖氨酸嵌段通过静电相互作用形成胶束核, 其水溶性聚乙二醇嵌段形成水溶性胶束壳, 提高了阳离子大分子/pDNA复合胶束的稳定性. 同时发现随着阳离子嵌段树枝状赖氨酸代数的增加, 阳离子两嵌段大分子与pDNA的结合作用增强, 有利于其作为基因转染生物功能载体的应用.     

γ-Ray-Radiation-Scissioned Chitosan as a Gene Carrier and Its Improved in uitro Gene Transfection Performance

Chitosan (CS) is expected to be an ideal gene carrier for its high biosafety. In this work, CS with low molecular weight were prepared through the γ-ray radiation on the acetic acid solution of CS. The CS chains were scissioned under the γ-ray radiation, and the molecular weight (MW) of CS decreased with the absorbed dose. When the absorbed dose was above 30 kGy, the molecular weight of CS decreased about an order of magnitude. The γ-ray-radiation-scissioned CS can e ectively bind with plasmid (pEGFP) through complex coacervation method, forming pEGFP/ γ-ray-radiation-scissioned CS complex particles with a size of 200-300 nm. The complex particles have good stability and little cytotoxicity. The in uitro gene transfection efficiencies of the pEGFP/ γ-ray-radiation-scissioned CS complex particles were investigated by fluorescence microscope and flow cytometry. The results showed that the gene vectors using γ-ray-radiation-scissioned CS as the carrier will possess better gene transfection efficiency than those using natural high-MW CS as the carrier. The higher the absorbed dose, the smaller the MW of CS and the better transfection efficiency of the corresponding gene vector. This work provides a green and simple method on the preparation of CS-based gene vectors with high efficiency and biosafety.     

Effects of Various Cell Surface Engineering Reactions on the Biological Behavior of Mammalian Cells

Cell surface engineering technologies can regulate cell function and behavior by modifying the cell surface. Previous studies have mainly focused on investigating the effects of cell surface engineering reactions and materials on cell activity. However, they do not comprehensively analyze other cellular processes. This study exploits covalent bonding, hydrophobic interactions, and electrostatic interactions to modify the macromolecules succinimide ester-methoxy polyethylene glycol (NHS-mPEG), distearoyl phosphoethanolamine-methoxy polyethylene glycol (DSPE-mPEG), and poly-L -lysine (PLL), respectively, on the cell surface. This work systematically investigates the effects of the three surface engineering reactions on the behavior of human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts, including viability, growth, proliferation, cell cycle, adhesion, and migration. The results reveals that the PLL modification method notably affects cell viability and G2/M arrest and has a short modification duration. However, the DSPE-mPEG and NHS-mPEG modification methods have little effect on cell viability and proliferation but have a prolonged modification duration. Moreover, the DSPE-mPEG modification method highly affects cell adherence. Further, the NHS-mPEG modification method can significantly improve the migration ability of HUVECs by reducing the area of focal adhesions. The findings of this study will contribute to the application of cell surface engineering technology in the biomedical field.