新型可降解PEI衍生物的表征及其在Brl-3A细胞中的毒性和转染研究

目的:对新型可降解高分子进行表征,研究其在Brl-3A细胞中的毒性和转染效率,以及连接剂比例对以上方面的影响。方法:通过化学方法合成不同比例PEI-Tr高分子,考察其包裹质粒DNA形成纳米颗粒的粒径和电位,以CCK-8方法考察Brl-3A细胞中的细胞毒性,以荧光素酶质粒为报告基因考察Brl-3A细胞中的转染效率。结果:PEI-Tr材料能形成200 nm以下带20 mV左右正电荷的纳米颗粒,具有较好的细胞内吞能力和溶液稳定性,细胞毒性实验证明,随着浓度增加PEI-Tr材料显示了远低于PEI-25kDa的细胞毒性,细胞转染实验表明其拥有高效输送质粒的能力。结论:PEI-Tr是一种高效低毒的可降解聚阳离子载体,在基因输送领域有很大的潜力;连接剂的比例在聚阳离子功能中起到重要作用。     

聚乙烯亚胺转基因影响因素的测定及其优化

聚乙烯亚胺 (PEI)为阳离子多聚物 ,可浓缩DNA形成纳米级颗粒 ,作为基因释放载体转染真核细胞 .选用Mr2 5 0 0 0 ,分枝状的聚乙烯亚胺转染质粒 ,比较多种转基因效率的影响因素 .通过MTT法测定PEI对COS 7细胞的细胞毒性 .利用电泳阻滞实验测定PEI与DNA形成复合物时所需的比例 .通过PEI转染增强型绿色荧光蛋白的pEGFP质粒、编码β 半乳糖苷酶的pSVβ质粒 ,探索氯喹、白蛋白、血清、盐离子浓度、质粒剂量、细胞数量等对聚乙烯亚胺转基因效率的影响 .实验发现 ,PEI对细胞的毒性作用与剂量相关 .PEI DNA的N P比在 3 0以上方可完全结合DNA .溶酶体抑制剂氯喹可增加转染效率 .培养液中的白蛋白、血清会降低转染效率 .生理盐溶液作为配制PEI DNA复合物的溶媒 ,转染效率高于 5 %葡萄糖作为溶媒 .随着转染质粒剂量的增加 ,转染效率呈剂量依赖正效应 .聚乙烯亚胺是有效的体外真核细胞转染剂 ,可用于合成更复杂的基因释放载体 .     

聚乙二醇化学修饰的聚乙烯亚胺衍生物的制备及其细胞毒性研究

目的:合成聚乙二醇(PEG)化的聚乙烯亚胺衍生物(PEI-Et)基因输送载体PET 1和PET 2,并考察两个载体材料在He La细胞、MCF-7细胞中的细胞毒性及在He La细胞中的转染效率。方法:将亚乙基二氯甲酸酯与PEI 800 Da交联制备成交联PEI衍生物PEI-Et,进一步将PEI-Et与聚乙二醇(PEG)以不同摩尔比例(1:1,2:1)交联连接,得到PEG化的PET 1和PET 2。采用MTT法检测PEI-Et、PET 1、PET 2对He La细胞、MCF-7细胞的细胞毒性。检测单位质量的荧光强度测定转染效率。结果:PET的细胞毒性随浓度增大而增大,在同一浓度下PET的细胞毒性小于PEI 25 KDa(P0.01);并且与DNA复合后,复合物细胞毒性随质量比的增高而增大,在同一质量比下PET的细胞毒性小于PEI 25 KDa(P0.01),特别是PET 1。并且最佳比例时,PET 1的转染活性最高。结论:作为非病毒基因载体,PET 1具有高的转染效率及低的细胞毒性。     

阳离子脂质体介导RNA干扰的效果评价

考察自制的肽型阳离子脂质体CDO14作为RNA转染载体的细胞毒性及其运载si RNA进行RNA干扰的效果。通过MTT法检测脂质体对稳定表达荧光素酶的肺癌A549(Luc-A549)细胞的毒性。以脂质体为载体将荧光素酶si RNA(Luc-si RNA)转染至Luc-A549细胞内,用发光仪检测转染细胞内荧光素酶含量,BCA法检测细胞内总蛋白含量。在裸鼠腋下接种Luc-A549细胞,成瘤后尾静脉注射Luc-si RNA和脂质体的复合物,利用活体成像系统检测模型小鼠体内荧光素酶的表达量。细胞毒性实验表明,自制脂质体的毒性与商品脂质体DOTAP相近,低于商品脂质体Lipo2000;细胞转染实验表明自制脂质体作为基因转染载体的转染效率高于DOTAP;体内转染实验表明CDO14作为载体转染效果优于DOTAP。结果表明,肽型阳离子脂质体CDO14具有毒性小、转染效率高等优点,有望作为转染载体用于基因治疗。     

新型聚乙烯亚胺衍生物在Hep G2 细胞中转染和毒性的研究

目的:研究以对苯二甲醛( Terephthalaldehyde)为连接剂的聚乙烯亚胺(Polyethyleneimine,PEI)衍生物PEI-Tp对肝癌细胞Hep G2的转染活性和细胞毒性的影响.方法:以荧光素酶质粒作为报告基因,研究高分子和DNA的复合物在Hep G2细胞中的转染活性,用MTT的方法研究高分子对Hep G2细胞的毒性.结果:Hep G2细胞转染结果显示构建的聚乙烯亚胺衍生物PEI-Tp具有高效输送质粒的能力;细胞毒性结果显示PEI-Tp随着浓度的增加,其毒性显著低于PEI25 kDa.结论:Hep G2细胞实验数据显示PEI-Tp是一种高效、低毒,在基因治疗领域有相当前景的非病毒载体.     

阳离子脂质体对HepG2.2.15细胞病毒分泌、毒性及凋亡的影响

目的:探讨阳离子脂质体作为基因治疗药物载体对HepG2.2.15细胞的病毒分泌、毒性及凋亡的研究.方法:以2.5、5.0、7.510.0 μL/mL浓度的阳离子脂质体转染HepG2.2.15细胞,共温育10 d,ELISA法检测细胞上清中HBsAg和HBeAg含量的变化.转染48 h后,MTT法检测阳离子脂质体对细胞的毒性、荧光显微镜检测细胞凋亡情况.结果:各个剂量组阳离子脂质体对HepG2.2.15细胞均有抑制作用,其中10 μL/mL组在第10 d对HBsAg、HBeAg抑制率分别达31.5%、29.9%,表现出较高的毒性作用.2.5、5.0、7.5 10.0 μL/mL阳离子脂质体转染HepG2.2.15细胞后,MTT检测结果显示其细胞存活率分别为82.14%、77.62%、77.4%、61.9%.荧光显微镜下可见各浓度组均有凋亡,且随脂质体剂量增加细胞凋亡数增加.结论:阳离子脂质体能抑制HepG2.2.15细胞分泌HBsAg和HBeAg,其毒性及诱导凋亡作用呈剂量和时间依赖性.作为基因治疗药物载体时,剂量宜小于7.5 μL/mL.     

腺病毒载体介导的H9C2心肌细胞外源基因表达系统的构建

本研究旨在探讨不同剂量的脂质体与腺病毒载体对转染H9C2细胞的效率的影响。应用常规剂量脂质体与减半剂量脂质体介导梯度剂量腺病毒载体转染H9C2细胞,并通过观察报告基因GFP表达计算各转染条件下的转染效率。结果发现应用常规剂量减半的脂质体反而能获得更高的转染效率;腺病毒载体与转染效率具有明显的量效关系,但是当载体量过大时转染效率有所下降。因此脂质体与腺病毒载体都具有一定的细胞毒性,细胞处于良好的状态可能更有利于外源基因的转入。     

人转铁蛋白偶联交联聚乙烯亚胺体外靶向转染肿瘤细胞

为了提高聚乙烯亚胺(Polythylenimine,PEI)类载体对肿瘤细胞的靶向性同时降低其细胞毒性,先用1800DaPEI制备了交联低分子量PEI,然后将人转铁蛋白与之偶联,得到了新型肿瘤靶向性人转铁蛋白偶联交联聚乙烯亚胺基因载体(TCP)。对所得的TCP的理化特性经行了表征,并检测了其细胞毒性。采用TCP介导pGL-3和pEGFP分别对293T、HepG2和Hela细胞系进行体外转染实验。结果表明:TCP是一种低毒高效的基因载体,在肿瘤细胞中的转染效率显著增强,因为其二硫键可在细胞内还原降解,而且通过偶联的转铁蛋白配体与肿瘤细胞表达的转铁蛋白受体间的相互作用,可增强该载体对肿瘤细胞的转染效率和靶向性。     

PEI-Et输送特异性shRNA对大鼠肝细胞AGT基因表达的影响

目的:研究交联小分子量聚乙烯亚胺衍生物PEI-Et对大鼠肝细胞(BRL-3A)的细胞毒性、转染效率和携带高血压相关基因血管紧张素原(AGT)短发卡RNA(shRNA)沉默AGT表达的能力。方法:MTT法检测PEI-Et/shRNA复合物对BRL-3A细胞的毒性,流式细胞术检测PEI-Et/shRNA复合物对BRL-3A细胞的转染效率,RT-PCR和Western blot检测PEI-Et/shRNA对AGT的基因沉默效果。结果:在相同质量比(w/w)时PEI-Et/shRNA的细胞毒性小于PEI 25kDa/shRNA(P0.01),PEI-Et/shRNA在w/w为30时达到最高转染效率,高于PEI 25 kDa(P0.01),PEI-Et/shRNA能高效沉默BRL-3A细胞中AGT基因的表达。结论:PEI-Et在BRL-3A细胞中是一种低细胞毒性、高转染效率的非病毒基因载体(与商业化的PEI 25kDa比较),能携带AGT shRNA高效沉默BRL-3A细胞中AGT基因的表达,通过用PEI-Et/AGT shRNA来抑制AGT的表达将为高血压的基因治疗提供一种新的思路。     

新型聚乙烯亚胺衍生物在Hep G2细胞中转染和毒性的研究

目的:研究以1,4-丁二醇二氯甲酸酯为连接剂的聚乙烯亚胺(Polyethyleneimine,PEI)衍生物PEI-Bu对肝癌细胞的转染活性和细胞毒性的影响.方法:以荧光素酶质粒作为报告基因,研究高分子和DNA的复合物在HepG2细胞中的转染活性,研究高分子对Hep G2细胞的毒性.结果:Hep G2细胞转染结果显示我们构建的聚乙烯亚胺衍生物PEI-Bu有比阳性对照PEI 25 kDa和Lipofectamine 2000更高的基因表达;细胞毒性结果显示PEI-Bu随着浓度的增加,其毒性显著低于PE125 kDa.结论:HepG2细胞实验教据显示PEI-Bu是一种高效、低毒,在肝脏基因治疗领域有相当前景的非病毒载体.     

PEG修饰的聚阳离子基因载体PEG-Polycarbam-SP在COS-7细胞中转染和毒性的研究

目的：寻找一种转染效率高,细胞毒性低的非病毒基因载体,研究以人体内源性精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以聚乙二醇（PEG）作为亲水基团连接剂合成亲水修饰聚阳离子载体PEG-Polycarbam-SP的基因担载效率,以及对非洲绿猴肾癌细胞COS-7的转染活性和细胞毒性的影响。方法：琼脂糖凝胶电泳方法考察复合物的基因担载效率,检测基因复合物的粒径和电位,以荧光素酶质粒为报告基因,研究PEG-Polycarbam-SP/DNA的复合物在COS-7细胞的转染活性,用MTT方法研究PEG-Polycarbam-SP对COS-7细胞的毒性。结果：聚合物与质粒在质量比5以后形成的复合物粒径稳定在50nm左右,Zate电位在20mV左右。COS-7细胞实验显示PEG-Polycarbam-SP具有低于PEI 25kDa的细胞毒性,同时也具有高效输送DNA的能力。结论：PEG-Polycarbam-SP是一种新型的高效、低毒,在基因治疗领域有潜在应用价值的非病毒基因输送载体。     

Low molecular weight hyaluronan shielding of DNA/PEI polyplexes facilitates CD44 receptor mediated uptake in human corneal epithelial cells

AIM: It was the aim of this study to prepare purified DNA/PEI polyplexes, which are coated with hyaluronan to facilitate CD44 receptor mediated uptake of the DNA/PEI polyplex and to reduce unspecific interactions of the complex with negatively charged extracellular matrix components on the ocular surface. METHODS: Hyaluronans of different molecular weights (<10 kDa, 10-30 kDa and 30-50 kDa) were isolated after enzymatic degradation of high molecular weight hyaluronan via ultrafiltration by centrifugation. The influence of the different hyaluronans used for coating on the stability and transfection efficiency of the complexes was evaluated in vitro. Transfection and uptake studies were performed in human corneal epithelial (HCE) cells. CD44 receptor expression of this cell model was evaluated by immunohistochemistry. RESULTS: Coating of purified DNA/PEI polyplexes with low molecular weight hyaluronan (<10 kDa) facilitated receptor-mediated uptake via the CD44 receptor in HCE cells, increased complex stability in vitro, and effectively shielded the positive surface charges of the polyplex without decreasing its transfection efficiency. Higher molecular weights and larger amounts of hyaluronan in the complexes resulted in lesser improvements in the stability and transfection efficacy of the complexes. CONCLUSIONS: Coating of polyplexes with low molecular weight hyaluronan is a promising strategy for gene delivery to the ocular surface, where CD44 receptor mediated uptake decreased cytotoxicity and reduced non-specific interactions with the negatively charged extracellular matrix components are considered beneficial for increased transfection efficiency of non-viral vectors.     

Biodegradable,endosome disruptive,and cationic network-type polymer as a highly efficient and nontoxic gene delivery carrier

The success of gene therapy is largely dependent on the delivery vector system. Efficient transfection and nontoxicity are two of the most important requirements of an ideal gene delivery vector. To generate both an efficient and nontoxic vector, we rationally constructed polymeric vectors to have simultaneous multiple functions, i.e., controlled degradation, an endosome disruptive function, and positive charges. Remarkably, the transfection efficiency of network poly(amino ester) (n-PAE) synthesized in this manner was comparable to that of polyethylenimine (PEI), one of the most efficient polymeric gene delivery vectors reported to date. However, there was a marked difference in cytotoxicity between the polymers. The majority of PEI-transfected cells were granulated and dead, whereas most of the cells transfected with n-PAE were viable and healthy. Successive events of efficient endosome escape of n-PAE/DNA polyplex and n-PAE biodegradation should result in high transfection efficiency and favorable cell viability response. The n-PAE-mediated transfection was also very efficient in the presence of serum. These data show that the approach we applied is a very appropriate way of making an ideal gene delivery carrier.     

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.     

Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified,long shelf-life polyethylenimine

Introduction of genetic material into cells is an essential prerequisite for current research in molecular cell biology. Although transfection with commercially available reagents results in excellent gene expression, their high costs are obstacles to experimentation with a large number or large scales of transfection. The cationic polymer linear-polyethylenimine (MW 25,000) (PEI), one of the most cost-effective vehicles, facilitates DNA compaction by polyplex formation, which leads to efficient delivery of DNA into cells by endocytosis. However, the use of PEI is still limited because of substantial cytotoxicity and intolerable deterioration in transfection efficiency by its low stability. Here, we show that acidification of PEI is important for its transfection activity. Dissolving PEI powder in 0.2N HCl confers a long shelf-life for PEI storage at 4 and −80 °C, and the polyplex formation of plasmid DNA with PEI is optimized in lactate-buffered saline at pH 4.0. Furthermore, changing the culture medium at 8–12 h posttransfection can minimize the cytotoxicity of PEI without sacrificing the high transfection efficiency comparable to that of commercial reagents. The cost per test using acidified PEI is drastically reduced to approximately 1:10,000, compared with commercial reagents. Thus, we conclude that acidification of PEI satisfactorily accomplishes cost-effective, high-efficiency transfection.     

Nanostructure of polyplexes formed between cationic diblock copolymer and antisense oligodeoxynucleotide and its influence on cell transfection efficiency

Although various cationic polymers have been used to condense anionically charged DNA to improve their transfection efficiency, there is still a lack of fundamental understanding about how to control the nanostructure and charge of the polyplexes formed and how to relate such information to cell transfection efficiency. In this work, we have synthesized a weak cationic and phosphorylcholine-containing diblock copolymer and used it as a model vector to deliver an antisense oligodeoxynucleotide (ODN) into HeLa cells. Small angle neutron scattering (SANS) was used to determine the copolymer/ODN polyplex structure. The SANS data revealed the formation of polyplex nanocylinders at high copolymer (N)/ODN (P) charge ratios, where N symbolizes the amine groups on the copolymer and P symbolizes the phosphate groups. However, the cylindrical lengths remained constant, indicating that the ODN binding over this region did not alter the cylindrical shape of the copolymer in solution. As the N/P ratio decreased and became close to unity the polyplex diameters remained constant, but their lengths increased substantially, suggesting the end-to-end bridging by ODN binding between copolymer cylinders. As the N/P ratios went below unity (with ODN in excess), the polyplex diameters increased substantially, indicating different ODN bridging to bundle the small polyplexes together. Transfection studies from HeLa cells indicated a steady increase in transfection efficiency with increasing cationic charge and decreasing polyplex size. Cell growth inhibition assay showed significant growth inhibition by the polyplexes coupled with weak cytotoxicity, indicating effective ODN delivery. While this study has confirmed the overall charge effect, it has also revealed progressive structural changes of the polyplexes against varying charge ratio, thereby providing useful insight into the mechanistic process behind the ODN delivery.     

Synthesis and characterization of a novel arginine-grafted dendritic block copolymer for gene delivery and study of its cellular uptake pathway leading to transfection

We synthesized a novel arginine-grafted dendritic block copolymer, R-PAMAM-PEG-PAMAM-R G5 (PPP5-R) for gene delivery systems. Its Mw was measured as 2.74 x 104 Da by MALDI-TOF, and approximately 36 arginine residues are found to be grafted to the polymer by 1H NMR. PPP5-R was able to form polyplexes with plasmid DNA, the average size of which was about 200 nm. Positive zeta-potential values (+22 to +28 mV) of PPP5-R polyplex indicate the formation of positively charged stable polyplex particles and suggest that large dendritic blocks with high positive charge may not be fully shielded by PEG chains even after PEG-coated complex formation. PPP5-R polyplex shows enhanced water solubility due to the polymer's PEG core and also shows low cytotoxicity, representing the potential for in vivo application. We identified the greatly enhanced transfection efficiency of PPP5-R in comparison with that of native PPP5 on various cell lines. Moreover, in view of the result of various cellular uptake inhibitor treatments during a transfection step, the cellular uptake of PPP5-R polyplex leading to effective transfection is thought to be not dependent on one exclusive pathway and to have the possibility of multiple pathways (caveolae-, clathrin-, and macropinocytosis-mediated pathways), contrary to the caveolae-dependent uptake of the PPP5 polyplex lacking arginine residues.     

pH-responsive three-layered PEGylated polyplex micelle based on a lactosylated ABC triblock copolymer as a targetable and endosome-disruptive nonviral gene vector

Nonviral vectors for gene therapy have recently received an increased impetus because of the inherent safety problems of the viral vectors, while their transfection efficiency is generally low compared to the viral vectors. The lack of the ability to escape from the endosomal compartments is believed to be one of the critical barriers to the intracellular delivery of noviral gene vectors. This study was devoted to the design and preparation of a novel ABC triblock copolymer for constructing a pH-responsive and targetable nonviral gene vector. The copolymer, lactosylated poly(ethylene glycol)-block-poly(silamine)-block-poly[2-(N,N-dimethylamino)ethyl methacrylate] (Lac-PEG-PSAO-PAMA), consists of lactosylated poly(ethylene glycol) (A-segment), a pH-responsive polyamine segment (B-segment), and a DNA-condensing polyamine segment (C-segment). The Lac-PEG-PSAO-PAMA spontaneously associated with plasmid DNA (pDNA) to form three-layered polyplex micelles with a PAMA/pDNA polyion complex (PIC) core, an uncomplexed PSAO inner shell, and a lactosylated PEG outer shell, as confirmed by 1H NMR spectroscopy. Under physiological conditions, the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles prepared at an N/P (number of amino groups in the copolymer/number of phosphate groups in pDNA) ratio above 3 were found to be able to condense pDNA, thus adopting a relatively small size (< 150 nm) and an almost neutral surface charge (zeta approximately +5 mV). The micelle underwent a pH-induced size variation (pH = 7.4, 132.6 nm --> pH = 4.0, 181.8 nm) presumably due to the conformational changes (globule-rod transition) of the uncomplexed PSAO chain in response to pH, leading to swelling of the free PSAO inner shell at lowered pH while retaining the condensed pDNA in the PAMA/pDNA PIC core. Furthermore, the micelles exhibited a specific cellular uptake into HuH-7 cells (hepatocytes) through asialoglycoprotein (ASGP) receptor-mediated endocytosis and achieved a far more efficient transfection ability of a reporter gene compared to the Lac-PEG-PSAO/pDNA and Lac-PEG-PAMA/pDNA polyplex micelles composed of the diblock copolymers and pDNA. The effect of hydroxychloroquine as an endosomolytic agent on the transfection efficiency was not observed for the Lac-PEG-PSAO-PAMA/pDNA polyplex micelles, whereas the nigericin treatment of the cell as an inhibitor for the endosomal acidification induced a substantial decrease in the transfection efficiency, suggesting that the protonation of the free PSAO inner shell in response to a pH decrease in the endosome might lead to the disruption of the endosome through buffering of the endosomal cavity. Therefore, the polyplex micelle composed of ABC (ligand-PEG/pH-responsive segment/DNA-condensing segment) triblock copolymer would be a promising approach to a targetable and endosome disruptive nonviral gene vector.     

Exploring N-imidazolyl-O-carboxymethyl chitosan for high performance gene delivery

Chitosan shows good biocompatibility and biodegradability, but the poor water solubility and low transfection efficiency hinder its applications as a gene delivery vector. We here report the detailed synthesis and characterization of a novel ampholytical chitosan derivative, N-imidazolyl-O-carboxymethyl chitosan (IOCMCS), used for high performance gene delivery. After chemical modification, the solubility of the resulting polymer is enhanced, and the polymer is soluble in a wide pH range (4-10). Gel electrophoresis study reveals the strong binding ability between plasmid DNA and the IOCMCS. Moreover, the IOCMCS does not induce remarkable cytotoxicity against human embryonic kidney (HEK293T) cells. The cell transfection results with HEK293T cells using the IOCMCS as gene delivery vector demonstrate the high transfection efficiency, which is dependent on the degree of imidazolyl substitution. Therefore, the IOCMCS is a promising candidate as the DNA delivery vector in gene therapy due to its high solubility, high gene binding capability, low cytotoxicity, and high gene transfection efficiency.