疏水修饰聚阳离子高分子SP-Chol 在COS-7 细胞中转染和毒性的研究

目的:研究以精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以胆固醇氯甲酸酯作为疏水基团连接剂合成的疏水修饰聚阳离子高分子SP-Chol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:以荧光素酶质粒为报告基因,研究SP-Chol与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究SP-Chol对COS-7细胞的毒性。结果:COS-7细胞实验显示,SP-Chol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:SP-Chol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。     

新型聚乙烯亚胺衍生物在COS-7 细胞中转染和毒性的研究

目的:研究以乙二醛为连接剂的聚乙烯亚胺(Polyethyleneimine,PEI)衍生物Polyimine-PEI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:以荧光素酶质粒为报告基因,研究高分子与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究高分子对COS-7细胞的毒性。结果:COS-7细胞实验显示,Polyimine-PEI具有很低细胞毒性,其毒性显著低于PEI25kDa,同时也具有高效输送质粒的能力。结论:Polyimine-PEI是一种新型的高效,低毒在基因治疗领域有相当前景的非病毒载体。     

疏水修饰聚阳离子高分子SP-Chol在COS-7细胞中转染和毒性的研究

目的：研究以精胺为单体,以乙二醇二氯甲酸酯作为连接剂,以胆固醇氯甲酸酯作为疏水基团连接剂合成的疏水修饰聚阳离子高分子SP-Chol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：以荧光素酶质粒为报告基因,研究SP-Chol与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究SP-Chol对COS-7细胞的毒性。结果：COS-7细胞实验显示,SP-Chol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：SP-Chol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。     

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。     

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.     

新型聚乙烯亚胺衍生物在COS-7细胞中转染和毒性的研究

目的：研究以乙二醛为连接剂的聚乙烯亚胺（Polyethyleneimine,PEI）衍生物Polyimine-PEI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：以荧光素酶质粒为报告基因,研究高分子与DNA的复合物在COS-7细胞的转染活性,用MTT方法研究高分子对COS-7细胞的毒性。结果：COS-7细胞实验显示,Polyimine-PEI具有很低细胞毒性,其毒性显著低于PEI25kDa,同时也具有高效输送质粒的能力。结论：Polyimine-PEI是一种新型的高效,低毒在基因治疗领域有相当前景的非病毒载体。     

基于间苯二甲醛构建的IPEI在滑膜细胞中的转染与毒性研究

目的:研究以间苯二甲醛(Isophthalaldehyde)作为连接剂构建的新型聚乙烯亚胺(Polyethyleneimine,PEI)衍生物IPEI为非病毒基因载体在治疗类风湿性关节炎(RA)中的应用,主要对阳离子聚合物IPEI在SD大鼠滑膜细胞中进行体外生物学评价。方法:IPEI以不同质量比包裹绿色荧光蛋白(GFP)质粒和白细胞介素-1受体拮抗剂(IL-1Ra)质粒,研究复合物在滑膜细胞中的转染活性,同时用MTT方法研究聚合物对滑膜细胞的细胞毒性。结果:复合物在质量比为2到10范围内,转染效果比较理想,具有高效输送质粒的能力;同时IPEI的细胞毒性也很小,细胞存活率明显高于商业化转染试剂PEI 25 KDa对照组。结论:阳离子聚合物IPEI具有高转染效率和低毒性的特点,从体内水平上证实了IPEI是一种良好的治疗类风湿性关节炎的基因输送载体。     

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 .     

新型PEI衍生物对基因沉默作用的研究

目的:构建出一种新型的高效的非病毒基因输送材料.方法:利用1,4-丁二醇二氯甲酸酯连接小分子PEI,合成新型的PEI衍生物(dPEI),通过体外COS-7和HSC细胞实验检测其对质粒和siRNA的输送情况.结果;COS-7细胞实验显示,dPEI具有高效输送质粒的能力,其转染效率是对照组PEI 25 kDa的10倍以上;同时,HSC细胞实验进一步证实dPEI包裹siRNA转染具有一定的基因沉默作用.结论:本研究合成的新型PEI衍生物既可以用于质粒基因的转染,也可用于siRNA的转染,是一种新型的高效的非毒基因输送材料.     

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.     

Charge density and molecular weight of polyphosphoramidate gene carrier are key parameters influencing its DNA compaction ability and transfection efficiency

A series of polyphosphoramidates (PPAs) with different molecular weights (MWs) and charge densities were synthesized and examined for their DNA compaction ability and transfection efficiency. A strong correlation was observed between the transfection efficiency of PPA/DNA nanoparticles and the MW and net positive charge density of the PPA gene carriers in three different cell lines (HeLa, HEK293, and HepG2 cells). An increase in MW and net positive charge density of PPA carrier yielded higher DNA compaction capacity, smaller nanoparticles with higher surface charges, and higher complex stability against challenges by salt and polyanions. These favorable physicochemical properties of nanoparticles led to enhanced transfection efficiency. PPA/DNA nanoparticles with the highest complex stability showed comparable transfection efficiency as PEI/DNA nanoparticles likely by compensating the low buffering capacity with higher cellular uptake and affording higher level of protection to DNA in endolysosomal compartment. The differences in transfection efficiency were not attributed by any difference in cytotoxicity among the carriers, as all nanoparticles showed a minimal level of cytotoxicity under the transfection conditions. Using PPA as a model system, we demonstrated the structural dependence of transfection efficiency of polymer gene carrier. These results offer more insights into nanoparticle engineering for nonviral gene delivery.     

Chitosan-Graft-Polyethylenimine/DNA Nanoparticles as Novel Non-Viral Gene Delivery Vectors Targeting Osteoarthritis

The development of safe and efficient gene carriers is the key to the clinical success of gene therapy. The present study was designed to develop and evaluate the chitosan-graft-polyethylenimine (CP)/DNA nanoparticles as novel non-viral gene vectors for gene therapy of osteoarthritis. The CP/DNA nanoparticles were produced through a complex coacervation of the cationic polymers with pEGFP after grafting chitosan (CS) with a low molecular weight (Mw) PEI (Mw = 1.8 kDa). Particle size and zeta potential were related to the weight ratio of CP:DNA, where decreases in nanoparticle size and increases in surface charge were observed as CP content increased. The buffering capacity of CP was significantly greater than that of CS. The transfection efficiency of CP/DNA nanoparticles was similar with that of the Lipofectamine™ 2000, and significantly higher than that of CS/DNA and PEI (25 kDa)/DNA nanoparticles. The transfection efficiency of the CP/DNA nanoparticles was dependent on the weight ratio of CP:DNA (w/w). The average cell viability after the treatment with CP/DNA nanoparticles was over 90% in both chondrocytes and synoviocytes, which was much higher than that of PEI (25 kDa)/DNA nanoparticles. The CP copolymers efficiently carried the pDNA inside chondrocytes and synoviocytes, and the pDNA was detected entering into nucleus. These results suggest that CP/DNA nanoparticles with improved transfection efficiency and low cytotoxicity might be a safe and efficient non-viral vector for gene delivery to both chondrocytes and synoviocytes.     

Fabrication and Optimization of Linear PEI-Modified Crystal Nanocellulose as an Efficient Non-Viral Vector for In-Vitro Gene Delivery

Background:One of the major challenges in gene therapy is producing gene carriers that possess high transfection efficiency and low cytotoxicity (1). To achieve this purpose, crystal nanocellulose (CNC) -based nanoparticles grafted with polyethylenimine (PEI) have been developed as an alternative to traditional viral vectors to eliminate potential toxicity and immunogenicity.Methods:In this study, CNC-PEI10kDa (CNCP) nanoparticles were synthetized and their transfection efficiency was evaluated and compared with linear cationic PEI10kDa (PEI) polymer in HEK293T (HEK) cells. Synthetized nanoparticles were characterized with AFM, FTIR, DLS, and gel retardation assays. In-vitro gene delivery efficiency by nano-complexes and their effects on cell viability were determined with fluorescent microscopy and flow cytometry.Results:Prepared CNC was oxidized with sodium periodate and its surface cationized with linear PEI. The new CNCP nano-complex showed different transfection efficiencies at different nanoparticle/plasmid ratios, which were greater than those of PEI polymer. CNPC and Lipofectamine were similar in their transfection efficiencies and effect on cell viability after transfection.Conclusion:CNCP nanoparticles are appropriate candidates for gene delivery. This result highlights CNC as an attractive biomaterial and demonstrates how its different cationized forms may be applied in designing gene delivery systems.Key Words: Crystal Nanocellulose, Gene transfection, Nanoparticle, Nano-complex     

Dextran-g-PEI nanoparticles as a carrier for co-delivery of adriamycin and plasmid into osteosarcoma cells

Combination of chemotherapy and gene therapy of cancer has synergistic effects on overcoming drug resistance. Macromolecular materials such as dextran and PEI have been a potential module for chemotherapeutics and gene delivery. Herein, we hypothesize the combinational strategy of chemotherapy and gene therapy in a single dextran-PEI nanoplatform. The physicochemical properties, cytotoxicity, transfection efficiency were investigated in vitro. Ultra-violet spectrum and ¹H NMR revealed adriamycin and PEI were grafted to dextran chain. Agarose gel electrophoresis demonstrated that the migration of plasmid was completely retarded when the N/P ratio of complex was 4. The sizes of DEX-ADM-PEI/DNA nanoparticles decreased and the zeta potentials enhanced with the increasing N/P ratio. Transmission electron microscope indicated a round morphology of the nanoparticles. DEX-ADM-PEI conjugation has higher cytotoxicity, compared to free adriamycin, in MG-63 and Saos-2 osteosarcoma cells but DEX-PEI maintained over 65% cell viability at the concentration of 8 mg/mL. The transfection efficiency of DEX-ADM-PEI/pEGFP-N1 at N/P ratio of 4:1 both in MG-63 and Saos-2 cell were slightly low than that of PEI 25k. But our nanoplatform efficiently delivered both plasmid pEGFP-N1 and adriamycin into osteosarcoma cells. This study demonstrated that DEX-ADM-PEI efficiently and selectively delivered both plasmid pEGFP-N1 and adriamycin to osteosarcoma cells with low cytotoxicity.     

Synthesis and characterization of dexamethasone‐conjugated linear polyethylenimine as a gene carrier

Linear polyethylenimine (25 kDa, LPEI25k) has been shown to be an effective non‐viral gene carrier with higher transfection and lower toxicity than branched polyethylenimine (BPEI) of comparable molecular weight. In this study, dexamethasone was conjugated to LPEI25k to improve the efficiency of gene delivery. Dexamethasone is a synthetic glucocorticoid receptor ligand. Dexamethasone‐conjugated LPEI25k (LPEI–Dexa) was evaluated as a gene carrier in various cells. Gel retardation assays showed that LPEI–Dexa completely retarded plasmid DNA (pDNA) at a 0.75:1 weight ratio (LPEI/pDNA). LPEI–Dexa had the highest transfection efficiency at a 2:1 weight ratio (LPEI–Dexa/DNA). At this ratio, the size of the LPEI–Dexa/pDNA complex was approximately 125 nm and the zeta potential was 35 mV. LPEI–Dexa had higher transfection efficiency than LPEI and Lipofectamine 2000. In addition, the cytotoxicity of LPEI–Dexa was much lower than that of BPEI (25 kDa, BPEI25k). In conclusion, LPEI–Dexa has a high transfection efficiency and low toxicity and can therefore be used for non‐viral gene delivery. J. Cell. Biochem. 110: 743–751, 2010. © 2010 Wiley‐Liss, Inc.     

新型PEI 衍生物在COS-7 细胞中的转染与毒性研究

目的:寻找一种新型的转染效率高,毒性低的非病毒基因载体.方法:通过化学方法合成Polyimine-MPEI,然后以不同质量比包裹绿色荧光蛋白质粒,检测在COS-7细胞中的转染效率和毒性.结果:在比例从5到100之间,转染效率均比较理想,能达到1.00E+07以上,Polyimine-MPEI的毒性也很小,细胞的生长率均在80％以上,明显高于PEI25KDa对照组.结论:Polyimine-MPEI是一个很有研究前景的聚合物载体,具有高转染效率低毒性的特点,可以通过延长反应时间,增加分子量,增大转染能力.     

新型阳离子基因传递载体PEI-PBLG的细胞转染研究

对新型阳离子聚合物PEI(10kD)-PBLG进行研究,重点考察其基因转染效率与细胞毒性,探讨其作为基因载体的可能性。通过粒径分析及扫描电镜(SEM)观察PEI(10kD)-PBLG与质粒pEGFP自组装形成的颗粒形态及粒径,预测其进入细胞的可能性。使用MTT比色法分析PEI(10kD)-PBLG、PEI(25kD)-PBLG、PEI(10kD)和PEI(25kD)的细胞毒性差异。选用表达增强型绿色荧光蛋白的质粒pEGFP作为报告基因模型,将其与PEI(10kD)-PBLG自组装后,分别转染真核细胞株Hela、COS-7、Vero-E6和ECV304,应用流式细胞术检测细胞转染效率,并比较了血清、缓冲液、细胞谱等多种因素对基因转染效率的影响。PEI(10kD)-PBLG可包裹质粒形成粒径100~120nm的纳米复合物,适合介导质粒进入细胞。该纳米粒复合物对转染缓冲液的敏感度较低,并能够在10%血清存在的条件下,转染全部实验用细胞株,尤其对Hela的转染效率最高,其次是COS-7、Vero-E6和ECV304;其中PEI-PBLG(10kD)/pEGFP复合物转染Hela细胞的比率为45.02%,高于PEI(10kD)/pEGFP的29.16%;PEI(10kD)-PBLG的细胞毒性作用显著低于PEI(25kD)、PEI(10kD)和PEI(25kD)-PBLG。新型阳离子多聚物PEI(10kD)-PBLG在提高PEI介导的基因转染效率的同时降低了其细胞毒性,提高了生物相容性,有望成为基因转移的有效载体。     

Characterization of (aminoethyl)chitin/DNA nanoparticle for gene delivery

Nonviral gene delivery systems have been increasingly proposed as a safer alternative to viral vehicles. In the present study, we synthesized water-soluble chitin by aminoalkylating onto chitin at the C-6 position, and its transfection efficiency was investigated. Aminoethyl-chitin (AEC) was complexed with DNA, and AEC/DNA nanoparticles were characterized. AEC/DNA nanoparticles showed good DNA binding ability, high protection of DNA from nuclease and serum, and low cytotoxicity. Mean particle size decreased from 367 to 290 nm and zeta potential increased from -4.58 to 22.87 mV when the AEC/DNA charge ratio (N/P) increased from 1.15 to 18.5. The transfection efficiency of AEC/DNA nanoparticles was investigated in a human embryonic kidney cell line (HEK293), and the results showed that AEC/DNA nanoparticles were much enhanced compare with naked DNA.