摘要
研究背景和目的:
基因治疗,特别是最具前景的小分子RNA干扰作为基因药的治疗,选择合适的载体及导入方法是一个瓶颈,也是核酸为基础的药物能否在临床成功应用的关键。病毒载体因其具有高效转导基因的特点,是目前基因治疗临床研究的主流载体。但随着时间的推移,病毒载体介导免疫炎症以及激活原癌基因的潜在危险性等自身无法克服的缺陷已日益显现。因此,研发非病毒载体已成为基因治疗研究的热点。
非病毒载体具有低毒、低免疫原性等特点,其中的壳聚糖及其衍生物因具备极高的生物相容性受到科学家们的青睐。我们在前期的研究工作中,已经研制出烷基化壳聚糖纳米核酸载体,并能成功的将靶目标核酸转入肺的上皮细胞,表达相应蛋白。但存在的缺点是转染效率低。精氨酸中的特征基团胍基能够促进细胞的内吞,具有细胞转导肽的作用,因此,对壳聚糖进行胍基化修饰有望改善原壳聚糖纳米载体转染效率低的缺点。同时,β2肾上腺能受体广泛分布于支气管平滑肌和肺组织内,依据配体-受体结合的原理,采纳临床用支气管舒张剂-β2受体兴奋剂作为归巢装置,把沙丁胺醇接枝于胍基化的壳聚糖上,有望通过靶向性修饰增加其核酸转运的效率与特异性。不仅如此,针对特定靶器官的局部给药方式具有吸收迅速、起效快、使用方便和副作用小等特点,是呼吸系统疾病治疗一种常用有效的给药途径。通过探讨超声雾化运送壳聚糖纳米核酸药的给药方式,最终构建一种新型RNA干扰壳聚糖纳米载体靶向归巢气雾治疗装置,将能极大解决RNA干扰等核酸类药物在呼吸系统疾病临床应用的问题。
研究内容和方法:
第一部分胍基化壳聚糖的物化性质和基因转染效率
第一节胍基化壳聚糖物化性质的检测
1.胍基化壳聚糖的合成
2.透射电镜观察Gua-chitosan/DNA的粒径大小和分布;
3.比较中性水溶液中chitosan和Gua-chitosan之间的溶解性,以及完全溶解状态下,两种载体溶液的PH值;
4.凝胶阻滞实验观察两种载体的核酸结合能力;
5.利用WES-8比较各种载体的细胞毒性。
第二节胍基化壳聚糖转染效率的评价与机制探讨
1.应用绿色荧光蛋白报告基因载体(Enhanced green fluorescent protein plasmid, pEGFP)转染293细胞,荧光显微镜观察不同壳聚糖纳米载体的转染效率,并结合流式细胞仪进行转染效率的定量分析;
2.流式细胞仪检测在不同载体的包裹下单个细胞的荧光强度,了解它们对GFP表达影响;
3.观察不同胍基取代度对壳聚糖转染效率的影响;
4.跟踪不同时间点各种载体转染效率的变化。
5.连接Pet质粒和yoyo1荧光染料,观察各种载体向细胞内转运核酸的能力。
6.连接Pet质粒和yoyo1荧光染料,观察各种载体在体转运核酸的能力。
第二部分载体的靶向性改造
1.沙丁胺醇接枝于胍基化的壳聚糖及质谱分析。
2.比较Sal-Gua-chitosan和Gua-chitosan在具有β2受体的293细胞及不表达β2受体的16HBE细胞的转染效率。
第三部分超声雾化对壳聚糖核酸复合物转染效率的影响及其机理
1.凝胶电泳观察超声雾化下,Gua-chitosan对于DNA的保护作用;
2.分别比较载体/DNA复合物经超声雾化以后,pEGFP在293和16HBE细胞当中转染效率的变化;
3.透射电镜观察超声雾化前后,载体和载体/DNA复合物粒径大小和分布的变化。
结果
第一部分胍基化壳聚糖的物化性质和基因转染效率
第一节胍基化壳聚糖粒径在20-70nm之间;壳聚糖经胍基化修饰以后在中性水溶液当中的溶解性大大增加;胍基能降低壳聚糖自身的电荷密度,造成与DNA结合的临界复合比升高;与天然壳聚糖相仿,胍基化改造并没有增加载体的细胞毒性,两者对于细胞生长率均没有明显影响。
第二节胍基化壳聚糖转染pEGFP的效率明显高于天然壳聚糖,这种转染效率的提高在一定范围内并不依赖于胍基化程度的高低。在单个细胞的水平上,胍基化壳聚糖所转染的绿色荧光蛋白的强度高于天然壳聚糖。在转染后24小时,脂质体所介导的基因表达达到峰值,而胍基化壳聚糖所介导的基因转染,蛋白表达的高峰出现在72小时。细胞体外实验和在体实验均显示,胍基化壳聚糖能更有效的地把质粒DNA运送到细胞体内。
第二部分载体的靶向性改造
与不表达β2受体的16HBE细胞比较,293细胞用Sal-胍基化壳聚糖纳米载体转染绿色荧光报告质粒的表达效率显著高于未引入配体的胍基化壳聚糖纳米载体。
第三部分超声雾化对壳聚糖核酸复合物转染效率的影响及其机理
壳聚糖能有效保护被包裹的DNA免受超声振荡的剪切作用。壳聚糖/DNA复合物经超声雾化以后转染效率显著提高。超声处理后复合物粒径分布范围较前变窄,粒径增大,颗粒均匀度较前明显改善。
结论
1.胍基化修饰的壳聚糖纳米载体能有效提高核酸的运送及转染效率,在生理PH值环境下能够溶于水,为临床局部应用的可行性奠定了基础。胍基化修饰的壳聚糖纳米提高转染效率的机制可能是通过提高细胞的内吞作用实现,。该特质性更有利于其在体转送核酸物质。
2.β2受体兴奋剂-沙丁胺醇接枝的胍基化壳聚糖纳米载体通过引入靶向归巢装置,能够利用气道细胞存在大量β2受体的特点,依据配体-受体结合的基本原理,进一步提高细胞对核酸的摄取与转染效率,为在体经气道局部给予核酸类药奠定了重要基础。
3.壳聚糖包裹能有效保护超声振荡下的DNA免受剪切破坏;同时超声雾化通过改变复合物的粒径和分布,可以有效提高核酸的转染效率。
Objective and background
RNA interference (RNAi) is a natural cellular process that regulates gene expression by a highly precise mechanism of sequence-directed gene silencing at the stage of translation by degrading specific messenger RNAs and blocking translation. It has shown more potential than conventional gene-targeting strategies. However, the poor cellular uptake of synthetic small interfering RNA (siRNA) is a major impediment for their clinical use due to its instability, inefficient cell entry, and poor pharmacokinetic profile. Various delivery vectors have thus been developed in order to circumvent these problems. From among the gene vectors that have been studied, non-viral vectors have attracted more and more attention in comparison to viral vectors, although viral vectors have been proven to yield higher transfection efficiency in most cell lines. This is attributed to the advantages of non-viral vectors such as ease of synthesis, low immune response against the vector and unrestricted gene materials size in addition to potential benefits in terms of safety.
In recent years, chitosan-based carriers are one of the non-viral vectors that have gained increasing interest for its beneficial qualities such as low toxicity, low immunogenicity, as well as an excellent biodegradability and biocompatibility. For the treatment of asthma, we previously developed a 12-alkylated chitosan nanoparticle vector that was able to deliver ECE-siRNA into airway epithelial tissues of the OVA-challenged mice, leading to down regulation of the synthesis of ET-1 to some extent. Today’s question remains the delivery of siRNAs. The 12-alkylated chitosan nanoparticle vector cannot be used in human therapy and its delivery efficiency is still the most important obstacle for the siRNA-based treatment.
It has been found that the arginine-induced perturbation is preferentially ascribed to the presence of the guanidinium group in Arg, indicating that modification of chitosan with guanidinium groups would optimize its delivery efficiency for siRNA. The development of methods for specific delivery of genes into target tissues is also an critical issue for the further progress of gene therapy. Introduction of targeting ligands to nonviral vector. resulted in increased gene expression and allowed to direct transfection complexes more specifically to selected cell types in order to reduce undesired side-effects in non-target cells. The beta2-adrenergic(β2-AR) receptor is found predominantly in bronchial smooth muscle and lung tissue.β2-AR agonists may serve as a suitable targeting ligand to improve receptor-mediated gene delivery. In addition, topical delivery of the drug directly to the site of action offers the advantages of enhanced drug delivery to anatomical target site with maximum therapeutic efficacy but the minimal adverse side effect.. This has led to the widespread use of inhalation therapy in lung diseases. In present study, we developed a targeting gene carrier by covalent linkage of salbutamol to the guanidinylated chitosan(Gua-chitosan).. The effect of ultrasonic aerosol on the vector/nucleic acid complexes was also studied for topical delivery of siRNA in treatment of respiratory diseases.
Methods
Part one
1. Characteristics of Gua-chitosan
Particle size and its distribution of the Gua-chitosan/pDNA complexes were measured by transmission electron microscopy(TEM). The water-solubility of Gua-chitosan was detected under various PH conditions. Gel retardation was evaluated for the pDNA-packaged by Gua-chitosan. The cytotoxicity of Gua-chitosan nanoparticles was tested in HEK 293 cells using CCK-8 assay.
2. Transfection efficiency mediated by Gua-chitosan
HEK29 cells transfected with Gua-chitosan, encapsulating the pEGFP as a reporter were observed under a fluorescence microscope. Transfection efficiency of the complexes and the mean fluorescence intensity of individual cell were further evaluated by flow cytometry. The PET plasmid was labeled with fluorescein Yoyo1 for in vitro and in vivo assessment of the transport of siRNAs across the cell membrane by Gua-chitosan nanoparticle.
Part two Specific modification of Gua-chitosan
Sal-Gua-chitosan was synthesized by covalent linkage of salbutamol to the Gua-chitosan using epichlorohydrin. 1H NMR spectroscopy was performed on investigation of the rate of substitution of salbutamol. The transfection efficiencies of Gua-chitosan and Sal-Gua-chitosan were detected. in both HEK 293 cells and bronchial epithelial cells (16HBE).
Part three Effects of Ultrasonication on the transfection efficiency mediated by the Gua-chitosan
The protection of pDNA by chitosan against ultrasonication was characterized by agarose gel retardation experiment. Transfection efficiency of the Gua-chitosan/pDNA complexes was evaluated by flow cytometry. prior to their treatment of ultrasonic aerosol.. The effects of ultrasonication on the particle diameter and size distribution of Gua-chitosan nanoparticle were determined using TEM.
Results
Part one
1. In TEM images, Gua-chitosan/pDNA complexes assumed spherical particles with diameter ranging from 20nm to 70nm. Chitosan modified by guanidinium group is able to be dissolved in neutral water . In contrast, the natural chitosan is water soluble only at PH below 4.0. We show that the Gua-chitosan can effectively condense DNA in spite of a weaker DNA-binding strength relative to chitosan. Cytotoxicity Assay demonstrate that Gua-chitosan has no any negative impact on the cell proliferation.
2. Expression efficiency for GFP mediated by the Gua-chitosan is greatly enhanced compared with that of chitosan-mediated transfection. The highest transfection efficiency of the both is observed 72 hours after the application of vector/pDNA complexes. The mean fluorescence intensity of GFP is higher in the cells transfected with pEGFP-Gua-chitosan nanoparticle than that in those transfected with pEGFP-chitosan. nanoparticle. The fluorescence microscope shows that Gua-chitosan vector has the capability to deliver much more Yoyo1-labled pDNA into the cells, compared to the natural chitosan. This was not only observed in vitro but also in mice submitted to intracheal instillation of the complex.
Part two
On average, about 100 glucosamine units in Gua-chitosan are successfully conjugated with 5 salbutamol molecules. The salbutamol modification of Gua-chitosan increases the expression efficiency of pEGFP in HEK293 cells that expressβ2-AR, but not in 16HBE cells withoutβ2-AR expression.
Part three
The pDNA encapsulated in Gua-chitosan is perfectly protected from a shearing force generated by ultrasonic aerosol. It is found that the Gua-chitosan nanoparticle treated by ultrasonic aerosol results in an elevated transfection efficiency. The particle sizes of Gua-chitosan and Gua-chitosan/pDNA were both increased, accompanied by a narrowing of size distribution after ultrasonication.
Conclusion
1. Guanidinylation modification greatly improve water-solubility of chitosan vector under the physiological PH conditiion, which is important for its clinical use. Guanidinylation modification of the chitosan accelerates the transport of -pDNA across the cell memberance by the nanoparticle vector, leading to an increase of transfection efficiency.
2. Theβ2-AR agonist, salbutamol is coupled to the Gua-chitosan. This made it a homing device that increases nanoparticle’s internalization by the ligand-receptor interactions.
3. The Gua-chitosna protects the DNA from being sheared by ultrasonication. On the other hand, ultrasonic aerosol increases the transfection efficiency of the Gua-chitosan by change of diameter and size distribution. We suggest that ultrasonic aerosol is an available approach for the delivery of gene vector complexes.
引文
[l] FireA, Xu S,Montgomery MK, et al. Potent and specific genetic interference By double-stranded RNA in Caenothabditis elegans [J]. Nature, 1998, 391: 806-811.
[2] Elbashir SM, Martinez J, Lendeckel W, et al. Functional anatomy of siRNAs for Mediating effieient RNAi in Drosophila melanogaster embryo lysate [J]. EMBOJ, 2001, 20(23): 6877-6888.
[3] Elbashir SM, Harborth J, Lendeekel W, et al. Duplexes of 21-nuelcotide RNAs Mediate RNA interference in cultured mammalian cells[J].Nature,2001,411 (6838):494一8,
[4] Hannon GJ. RNA interference [J]. Nature, 2002, 418(6894): 244-251.
[5] Novina CD, Sharp PA. The RNAi revolution [J]. Nature, 2004, 430: 161-168.
[6] Hannon GJ, Rossi JJ. Unlocking the Potential of the human genome with RNA interference [J]. Nature, 2004, 431: 371-378.
[7] Kohn DB, Sadelain M, Glorioso JC, et al. Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer, 2003, 3(7): 477-488.
[8] Marshall E. Second child in French trial is found to have leukemia. Science, 2003, 299(5605): 320.
[9] E Marhsall. Gene Therapy Death Prompts Review of Adenovirus Vector. Science, 1999, 286: 2244-2245.
[10] Malcolm Brenner. The Eyes Have It. Molecular Therapy, 2010, 18(3): 451–452.
[11] Philip R Johnson, Bruce C Schnepp, Jianchao Zhang, et al. Vector-mediated gene transfer engenders long-lived neutralizing activity and protection against SIV infection in monkeys. Nat Med, 2009, 15(8): 901–906.
[12] Sorensen DR, Leirdal M, Sioud M, et al. Gene sileneing by systemic delivery of synthetic siRNA in adult mice [J]. J Mol Biol, 2003, 327(4): 761-766.
[13] Zender L, Hutker S, Liedtke C, et al. Caspase8 small interference RNA prevents Acute liver failure in mice [J]. Proc Natl Acid Sci USA, 2003, 100: 7797-7802.
[14] Morrissey DV, Lockridge JA, Shaw L, et al. Potent and Persistent in vivo anti- HBV activity of chemically modified siRNAs [J]. Nat Biotechnol, 2005, 23(8):1002-1007.
[15] Bitko V, Musiyenko A, Shulyayeva O, et al. Inhibition of respiratory viruses by nasally administered siRNA [J]. Nat Med, 2005, 11(l): 50-55.
[16] Schiffelers R, Ansari A, Xu J, et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted stericaly stabilized nanoparticle [J]. Nucleic Acids Res, 2004, 32(19): el49.
[17] Haliza Katasa, H. Oya Alpar. Development and characterisation of chitosan nanoparticles for siRNA delivery. Journal of Controlled Release, 2006, 115(2): 216-225
[18] Morten Ostergaard Andersen, Kenneth Alan Howard, Jorgen Kjems.RNAi Using a Chitosan/siRNA Nanoparticle System: In Vitro and In Vivo Applications. Methods in Molecular Biology, 2009, 555: 77-86.
[19]周雨田,徐军。12-烷基化壳聚糖纳米粒包裹反义内皮素转换酶核酸表达质粒在哮喘基因治疗中的特征。中国组织工程研究与临床康复。2007, 11(5): 829-833.
[20] Green M., Loewenstein P.M.. Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein, Cell, 1988: 55: 1179-1188.
[21] Derossi D., Joliot A.H., Chassaing G., et al. The third helix of the Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem, 1994, 269: 10444-10450.
[22] Fawell S., Seery J., Daikh Y., et al. Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. USA, 1994, 91: 664-668.
[23] Vive`s E., Brodin P., Lebleu B.. A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem., 1997, 272: 16010-16017.
[24] Ezhevsky S.A., Nagahara H., Vocero-Akbani A.M., et al. Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb, Proc. Natl. Acad. Sci. USA, 1997, 94: 10699-10704.
[25] Futaki S., Suzuki T., Ohashi W., et al. Arginine-rich peptides. An abundant sourceof membrane-permeable peptides having potential as carriers for intracellular protein delivery, J. Biol. Chem., 2001, 276: 5836-584.
[26] Luedtke N.W., Carmichael P., Tor Y.. Cellular uptake of aminoglycosides, guanidinoglycosides, and poly-arginine, J. Am. Chem. Soc., 2003, 125: 12374-12375.
[27] Chung H.H., Harms G., Seong C.M., et al. Dendritic oligoguanidines as intracellular translocators, Biopolymers, 2004, 76: 83-96.
[28] Dürrbach A, Angevin E, Poncet P, et al. Antibody-mediated endocytosis of G250 tumor-associated antigen allows targeted gene transfer to human renal cell carcinoma in vitro.Cancer Gene Ther, 1999, 6(6): 564-571.
[29] Peggy C, Motoichi K, Jooe C, et al. Synthesis and characterization of chitosan-g-Poly(ethyleneglycol)-folate as a non-viral carrier for tumor-targeted gene delivery [J]. Biomaterials, 2007, 28: 540-549.
[30] Ralfk Ircheis, Lionel W , Wagner E. Design and gene delivery activity of modified polyethylenimines [J]. Adv Drug Deliver Rev, 2001, 53: 341-358.
[31] IJ Gidebrabdt, Miyer, Wagner E, et al. Optical imaging of transferring targeted PEI/DNA comp lexes in living subjects [J]. Gene Therapy, 2003, 10: 758-764.
[32] Lilh, Kung, Jial, et al. Biologically active core-sell nanoparticles self-assembled from cholesterol-terminated PEG-TAT for drug delivery across the blood-brain barrier [J]. Biomaterials, 2008, 29: 1509-1517.
[33] Lai Li-hua, Jiang Q i-ying, Chen Dan, et al. Peptide TAT modified polyethylenimine-β-cyclodextrin for gene delivery [J]. Journal of Zhejiang University: Medical Sciences, 2009, 38 (1): 15-23.
[34] Liu Jun, Hu Yi-ping, Jiang Q i-ying, et al. Peptide MC10 mediated PEI-β-CyD as a gene delivery vector targeting to Her-2 receptor [J]. Journal of Zhejiang University: Medical Sciences, 2009, 38 (1): 7-14.
[35] Hu Yi-ping, Jiang Qi-ying, Chen Dan, et al. Peptide CY11 conjugated polyethylenimine-β-cyclodextrin for gene delivery [J]. Journal of Zhejiang University: Medical Sciences, 2009, 38 (1): 24-30.
[36] Wang Y, Gaos J, Yew H, et al. Co-delivery of drug and DNA from cationic core-shell nanoparticles sef-assembled from a biodegradable copolymer [J]. NatureMaterials, 2006, 15: 791-796.
[37]梁剑辉,徐军,钟南山。一种适用于肺脏转基因研究的脂质体运载系统。中国免疫学杂志, 2004, 2: 129-132.
[38]马旭升,何祚光。小儿哮喘防治新进展[J].现代实用医学,2004, 1: 10-12.
[39] Brown AR, Shafiqul I, Chowdhury.Propellant-Driven Aerosols of DNA Plasmids for Gene Expression in the Respiratory Tract. 1997, 10(2): 129-146.
[40] Mohri K, Okuda T, Mori A, Danjo K, et al.Optimized pulmonary gene transfection in mice by spray-freeze dried powder inhalation. J Control Release, 2010 Feb 22.
[41] Okamoto H, Sakakura Y, Shiraki K, et al. Stability of chitosan-pDNA complex powder prepared by supercritical carbon dioxide process.Int J Pharm, 2005, 290(1-2): 73-81.
[42] HIROKAZU OKAMOTO, SEIKO NISHIDA, HIROAKI TODO, et al. Pulmonary Gene Delivery by Chitosan–pDNA Complex Powder Prepared by a Supercritical Carbon Dioxide Process. J Pharm Sci, 2003, 92(2): 371-380.
[43] Nsouri S, Lavigne P, Corsi K, et al. Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to imp rove transfection efficacy [J]. Eur J Pharm Biopharm, 2004, 57 (1): 128.
[44] Bowman K, Leong KW. Chitosan nanoparticles for oral drug and gene delivery [J]. Int J Nanomedicine, 2006, 1 (2): 117-128.
[45] Mitchell D.J., Kim D.T., Steinman L., et al. Polyarginine enters cells more efficiently than other polycationic homopolymers, J. Pept. Res., 2000, 56: 318-325.
[46] Wender P.A., Mitchell D.J., Pattabiraman K., et al. The design, synthesis, and evaluation of molecules that enable or enhance cellular uptake: peptoid molecular transporters, Proc. Natl. Acad. Sci. USA, 2000, 97: 13000-13008.
[47] Calnan B.J., Tidor B., Biancalana S., et al. Arginine-mediated RNA recognition: the arginine fork, Science 1991, 252: 1167-1171.
[48] Desai M.P, Labhasetwar V, Walter E, et al. The mechanism of uptake of biodegradable microparticles in Caco-2 cells is size dependent. Pharm. Res, 1997, 14: 1568–1573.
[49] Desai M.P, Labhasetwar V, Amidon GL, et al. Gastrointestinal uptake of biodegradable microparticles: effect of particle size. Pharm. Res. 1996, 13: 1838–1845.
[50] Wattiaux R, Laurent N., Conninck S.W., et al. Endosomes, lysosomes: their implications in gene transfer. Adv. Drug Del. Rev, 2000, 41: 201–208.
[51] Bottega R, Epand RM. Inhibition of protein kinase C by cationic amphiphiles.Biochemistry, 1992, 31(37): 9025-9030.
[52] Datiles MJ, Johnson EA, McCarty RE. Inhibition of the ATPase activity of the catalytic portion of ATP synthases by cationic amphiphiles.Biochim Biophys Acta, 2008 1777(4): 362-368.
[53] Beavis, AD. On the inhibition of the mitochondrial inner membrane anion uniporter by cationic amphiphiles and other drugs. J Biol Chem, 1989, 264(3): 1508-1515.
[54] Zelphati O., Szoka F.C.. Mechanism of oligonucleotide release from cationic liposomes, Proc. Natl. Acad. Sci. U.S.A., 1996, 93: 11493-11498.
[55] Zelphati O.,Szoka F.C.. Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids. Pharm. Res., 1996, 13: 1367-1372.
[56]Wong M, Kong S, Dragowska WH, et al.Oxazole yellow homodimer YOYO-1-labeled DNA: a fluorescent complex that can be used to assess structural changes in DNA following formation and cellular delivery of cationic lipid DNA complexes. Biochim Biophys Acta, 2001, 1527(1-2): 61-72.
[57]Andy Sischka, Katja Toensing, Rainer Eckel, et al. Molecular Mechanisms and Kinetics between DNA and DNA Binding Ligands. Biophysical Journal, 2005, 88(1): 404-411.
[58] Tsuyoshi I, Yoshio O, Toshinori S. Mechanism of cell transfection with plasmid/chitosan complexes. Biochimica et Biophysica Acta, 2001, 1514(1): 51-64.
[59]Akiko Eguchia, Steven F, Dowdy. siRNA delivery using peptide transduction domains. Trends in Pharmacological Sciences, 2009, 30(7): 341-345.
[60] Tamaki Endoha, Takashi Ohtsuki. Cellular siRNA delivery using cell-penetrating peptides modified for endosomal escape. Advanced Drug Delivery Reviews. 2009,61(9): 704-709.
[61] M. Johnson. The beta-adrenoceptor. Am. J. Respir. Crit. Care. Med. 1998, 158 (5 Pt 3): S146–153.
[62] R.B. Clark, B.J. Knoll, R. Barber. Partial agonists and G protein-coupled receptor Desensitization. Trends Pharmacol. Sci. 1999, 20 (7): 279–286.
[63] S.S. Ferguson. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol. Rev. 2001, 53 (1): 1–24.
[64] Markus Elfinger a,b, Johannes Geiger a,b, Günther Hasenpusch, et al. Targeting of theβ2-adrenoceptor increases nonviral gene delivery to pulmonary epithelial cells in vitro and lungs in vivo. Journal of Controlled Release, 2009, 135: 234–241.
[65] Cherng JY, Schuurmans NM, Jiskoot W, et al. Effect of DNA topology on the transfection efficiency of poly [(2-dimethylamino) ethyl methacrylate]-plasmid complexes [J]. J Control Release, 1999, 60: 343-353.
[66] Kawaura C, Noguchi A, Furuno T, et al. Atomic force microscopy for studying gene transfection mediated by cationic liposomes with a cationic cholesterol derivative [J]. FEBS Lett, 1998, 421: 69-72.
[67] OgrisM, Steinlein P, KursaM, et al. The size of DNA/transferring-PEI complexes is an important factor for gene expression in cultured cells [J]. Gene Ther, 1998, 5: 1425-1431.
[68] Ross PC, Hui SW. Lipoplex size is a major determinant of in vitro lipofection efficiency [J]. Gene Ther, 1999, 6: 651-659.
[69] E.S.K. Tang, M. Huang, L.Y. Lim. Ultrasonication of chitosan and chitosan nanoparticles. Inter J Pharm, 2003, 265: 103–114.
[70] Mohammad R. Kasaai, Joseph Arul, Gérard Charlet. Fragmentation of chitosan by ultrasonic irradiation. Ultrasonics Sonochemistry, 2008, 15: 1001–1008.
[71] Giancarlo Cravotto, Silvia Tagliapietra, Bruna Robaldo, et al. Chemical modification of chitosan under high-intensity ultrasound. Ultrasonics Sonochemistry, 2005, 12: 95–98.
[1] Anderson W. Human gene therapy [J]. Science, 1992, 256: 808.
[2] Krepper F, Luther T, Semkova I, et al. Long- term transgene expression in the RPE after gene transfer with a high-capacity adenovirus vector [J]. Invest Ophthalmol Vis Sci, 2002, 43(6): 1965.
[3] Lundstrom K. Latest development in viral vectors for gene therapy [J]. Trends Biotechnol, 2003, 21 (3): 117-122.
[4] Kohn D B, Sadelain M, Glorioso J C, et al. Occurrence of leukaemia following gene therapy of X-linked SCID. Nat Rev Cancer, 2003, 3(7): 477-488.
[5] Marshall E. Second child in French trial is found to have leukemia. Science, 2003, 299(5605): 320.
[6] Majhen D, Ambriovic-Ristov A. Adenoviral vectors-How to use them in cancer gene therapy? [J]. Virus Res, 2006, 119(2): 121-133.
[7] E Marhsall. Gene Therapy Death Prompts Review of Adenovirus Vector. Science, 1999, 286: 2244-2245.
[8] Vorburger SA, Hunt KK. Adenoviral gene therapy [J]. Oncologist, 2002, 7 (1): 46-59.
[9] Carter BJ. Adeno-associated virus vectors in clinical trials [J]. Hum Gene Ther, 2005, 16(5): 541-550.
[10]李振宇,徐开林,潘秀英,等. HIV-1慢病毒载体的构建及结构改造。[J].中华血液学杂志,2004, 25(9): 571-572.
[11] Indraccolo S, Habeler W, Tisato V, et al. Cene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors [J]. Cancer Res, 2002, 62(21): 6099-6107.
[12] AL Dosarims, Knappje, Liud. Hydrodynamic delivery [J]. Adv Genet, 2005, 54: 65-82.
[13] North S, Butts C. Vaccination with BLP25 liposome vaccine to treat non-small cell lung and prostate cancers [J]. Expert Rev Vaccines, 2005, 4(3): 249-257.
[14] Richards RL, Raom, Vancotttc, et al. Liposome-stabilized oil-in-water emulsions as adjuvants : increased emulsion stability promotes induction of cyto-toxic Tlymphocytes against an H IV envelope ant igen [J]. Immunol Cell Biol, 2004, 82 (5): 531-538.
[15] Kouraklis G. Progress in cancer gene therapy. Acta Oncol, 1999, 38(6): 675-683.
[16] Cristiano RJ. Viral and non-viral vectors for cancer gene therapy. Anticancer Res, 1998, 18(5A): 3241-3245.
[17] Midoux P, Monsigny M, Efficient gene transfer by histidylated polylysine/pDNA complexes. Bioconjug Chem, 1999, 10(3): 406-411.
[18] McKenzie DL, Kwok KY, Rice KG. A potent new class of reductively activated peptide gene delivery agents. J Biol Chem, 2000, 275(14): 9970-9977.
[19] Holger Perersen, Petram Fechn ER, et al. Synthesis, characterization, and biocompatibility of Polyethylenimin-graft-Poly(ethylene glycol)block copolymers [J ]. Macromolecules, 2002, 35: 6867-6874.
[20] Kazuyo Shis, Kims W. A new synthsis galactose-poly ( ethylene glyco l)-polyethylenimine for gene delivery to hepatocytes [J]. J Controlled Release, 2002, 79: 271-281.
[21] Sato T, Shirakawa N, Nishi H, et al. Formation of a DNA/polygala cocaine complex cocaine complex and its interaction with cells. Chen lett, 1996, 9: 725-726.
[22] Maclaughlin FC, Mumper RJ, Wang J, et al. Chitosan and depdymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. Control Rel, 1998, 56: 259-272.
[23] Mao HQ, Roy K, Troung-Le VL. Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency. Control Release, 2001, 70(3): 399-421.
[24] Roy K, Mao HQ, KW Leng, et al. DNA-chitosan nanospheres: transfection efficiency and cellular uptake. Proc Int Symp Control Rel Bioact Mater, 1997, 24: 673-674.
[25]杜芝燕,黄伟,李峰生,等.新型纳米转染试剂转染PNP自杀基因体外杀伤实验,研究报告,2003: 10004.
