磁性纳米颗粒转运体系的构建及其在基因转运中的应用
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摘要
由于非病毒基因载体较低的转染效率，腺病毒受体依赖的宿主趋向性，逆转录病毒载体低的病毒滴度等限制了这些载体在基因转运上的应用。为了克服这些缺陷，本研究以磁性纳米颗粒作为基因载体，磁性纳米颗粒具有超顺磁性，低免疫原性，良好的生物相容性，可以结合大片段的DNA，在外加磁场作用下具有靶向性，可携带核酸发生定向移动，定位富集于细胞表面，从而增加了磁性纳米颗粒/基因复合物与细胞的接触时间和接触量，提高了转染效率，可以实现安全、高效的基因运输，有助于基因表达和基因功能的研究及靶向性基因治疗。本文对PEI包被的磁性纳米颗粒进行了表征，在外加磁场的作用下，使用PEI包被磁性纳米颗粒作为基因载体携带外源基因进行体内外基因的转运，主要结果如下：
1.对PEI包被的磁性纳米颗粒的形貌、粒径和表面电位进行了表征，在电镜和原子力显微镜下观察磁性纳米颗粒为规则球形颗粒，表面光滑，粒径大小均匀一致，分散性良好，颗粒粒径约为100nm。Zeta电位仪测定磁性纳米颗粒表面Zeta电位为29.4mV，呈正电性，能够通过静电吸附与DNA上带负电荷的磷酸基团相结合。能与DNA结合是作为基因载体的必备条件之一，原子力显微镜，凝胶阻滞实验和DNA共沉淀实验表明磁性纳米颗粒具有结合、浓缩DNA的能力。DNA保护实验表明磁性纳米颗粒可以保护DNA免受酶的降解。使用MTT实验来检测磁性纳米颗粒/DNA复合物对不同细胞系的毒性，结果显示磁性纳米颗粒/DNA复合物对细胞的毒性较小。将PEI包被的磁性纳米颗粒与质粒pEGFP-N1以不同比例混合形成磁性纳米颗粒/DNA复合物，在外加磁场的作用下转染293T细胞，倒置荧光显微镜下观察绿色荧光蛋白的表达，使用流式细胞仪检测转染效率，当磁性纳米颗粒与DNA之比为1:1时转染效率最高，高于脂质体介导的转染。
2.将口蹄疫病毒（foot-and-mouth disease virus,FMDV)的2A片段置于串联的RC2(retrocyclin2)和红色荧光蛋白dTomato基因之间，使它们处在一个开放阅读框（ORF）内，将其置于pCDNA4/TO EGFP真核表达载体CMV启动子的下游，构建得到pCDNA4-dTomato-2A-3R2和pCDNA4-dTomato-2A-6R2两个重组质粒，在外加磁场的作用下，磁性纳米颗粒可以携带重组质粒进入293T细胞。结果表明，由2A片段连接的串联的RC2和红色荧光蛋白dTomato可以在293T细胞中进行表达，2A具有自我剪切活性，它在自己的C端切割多聚蛋白，从而得到两个独立而有活性的蛋白。通过dTomato和串联RC2的共表达，使我们很容易的检测到目的蛋白在293T细胞内的表达情况，并且在293T细胞中表达的RC2对禽流感病毒H5N1具有抑制作用，为RC2作为预防和治疗人类流感病毒感染药物的进一步研究奠定了基础。
3.利用9日龄SPF鸡胚建立了鸡胚成纤维细胞系，以pEGFP-N1质粒作为外源DNA，在磁性纳米颗粒的介导下转染CEF细胞，实现了绿色荧光蛋白的表达。经过G418筛选，获得稳定表达绿色荧光蛋白的CEF细胞系，促进了鸡转基因研究的发展。
4.在外加磁场的作用下，磁性纳米颗粒携带质粒pCDNA4-dTomato-2A-3R2/6R2进入CEF细胞和鸡胚，结果表明利用FMDV2A可以实现dTomato和串联RC2在CEF细胞、鸡胚中的共表达，并且表达的RC2可以抑制H5N1在CEF细胞和鸡胚中的增殖，为磁性纳米颗粒作为基因载体进行体内外基因的转移，RC2作为预防和治疗禽类流感病毒感染药物的进一步研究以及生产抗流感病毒的转基因鸡奠定了基础。
The low efficiency of nonviral gene vectors, the receptor-dependent host tropism of adenoviral orlow titers of retroviral vectors limit their utility in gene delivery. To overcome these deficiencies, weused magnetic nanoparticles(MNPS) as gene vectors to transfer genes, which have some characteristicssuch as can combine with big fragment DNA, low immunogenic, good biocompatibility and targetedgene delivery by application of a magnetic field. Nucleic acids carried by magnetic nanoparticles canoccur directional movement and enrich on the cell surface, which increased contact time and exposuredose of the magnetic nanoparticles/gene complexes with cells. It would contribute to study geneexpression, gene function and targeted gene therapy. In this study, PEI-coated magnetic nanoparticleswere characterized, which were used as gene vector to transfer genes in vivo and in vitro by magneticforce.
1.The morphology characteristic, particle size and surface potential of PEI-coated magneticnanoparticles were characterized. The results of electron microscopy and atomic force microscopyshowed that magnetic nanoparticles had100nm diameter with good dispersion, Zeta potential ofmagnetic nanoparticles was29.4mV with positive charge, which can bind with DNA. As the genevector, the DNA binding ability is essential requirement. DNA binding ability of magnetic nanoparticlescan be determined by gel retardation assay, co-sedimentation assay and atomic force microscopy. Theresults proved magnetic nanoparticles had stronger ability to bind DNA. DNA and magneticnanoparticles can form close and tight complex structure. The magnetic nanoparticles can protect DNAagainst the digestion of nuclease. Whether MNPs/DNA complexes influenced cell viability wasinvestigated in several cell lines, including293T, MDCK, Vero and CEF cells by MTT assays.MNPs/DNA complexes showed no obvious negative effect on cell viability. The result of magneticnanoparticles transfection into293T cells showed that the magnetic nanoparticles had high transfectionefficiency, and the highest transfection efficiency was91%when the ratio of magnetic nanoparticles andDNA was1:1, higher than the positive control of lipofectamine transfection efficiency (86%).
2.In the present study, the2A region of foot-and-mouth disease virus (FMDV) was utilized toconstruct a bicistron vector, in which tandem retrocyclin2(RC2) gene and dTomato gene were fusedinto a single open reading frame linked by FMDV2A region. The fused genes were placed under thecontrol of CMV promoter in pCDNA4/TO EGFP vector to construct the recombinant expressionplasmids pCDNA4-dTomato-2A-3R2/6R2. The plasmids pCDNA4-dTomato-2A-3R2/6R2wastransfected into293T cells mediated by MNPs with application of a magnetic field. The results showedthat the fused genes of red fluorescent protein dTomato and muti-copy RC2linked by2A region offoot-and-mouth disease virus were expressed in293T successfully. The2A region mediated aco-translational cleavage event at its own carboxyl-terminus resulting in the release of each individualprotein product. Efficient cleavage was observed and all two proteins were functional, coexpression ofdTomato enabled us to track gene expression in293T cells conveniently. The tandem retrocyclin2can potently inhibit H5N1highly pathogenic avian influenza virus production in293T cells, which providea basis for further development of retrocyclin2for prophylaxis and therapy of H5N1virus infection inhumans.
3.The chicken embryo fibroblast(CEF) cells isolated from the chicken embryo were cultured invitro to construct a cell line. As the exogenous gene the plasmid pEGFP-N1was used to transfect CEFcells mediated by MNPs. G418was employed to screen the drug-resistance cell clones to obtain stablecell lines expressing green fluorescent protein, which laid the foundation for the study of transgenicchickens.
4.The plasmids pCDNA4-dTomato-2A-3R2/6R2were transfected into CEF cells and chickenembryo mediated by MNPs with application of a magnetic field. The results showed that the fusedgenes of red fluorescent protein dTomato and tandem RC2linked by2A region of foot-and-mouthdisease virus were expressed in CEF cells and chicken embryo successfully. The tandem retrocyclin2can potently inhibit H5N1highly pathogenic avian influenza virus production in CEF cells and chickenembryo, which provide a basis for further development of application of MNPs to gene delivery in vitroand in vivo, retrocyclin2for prophylaxis and therapy of H5N1virus infection in poultry, producingtransgenic chickens with suppression of avian influenza transmission.
1.对PEI包被的磁性纳米颗粒的形貌、粒径和表面电位进行了表征，在电镜和原子力显微镜下观察磁性纳米颗粒为规则球形颗粒，表面光滑，粒径大小均匀一致，分散性良好，颗粒粒径约为100nm。Zeta电位仪测定磁性纳米颗粒表面Zeta电位为29.4mV，呈正电性，能够通过静电吸附与DNA上带负电荷的磷酸基团相结合。能与DNA结合是作为基因载体的必备条件之一，原子力显微镜，凝胶阻滞实验和DNA共沉淀实验表明磁性纳米颗粒具有结合、浓缩DNA的能力。DNA保护实验表明磁性纳米颗粒可以保护DNA免受酶的降解。使用MTT实验来检测磁性纳米颗粒/DNA复合物对不同细胞系的毒性，结果显示磁性纳米颗粒/DNA复合物对细胞的毒性较小。将PEI包被的磁性纳米颗粒与质粒pEGFP-N1以不同比例混合形成磁性纳米颗粒/DNA复合物，在外加磁场的作用下转染293T细胞，倒置荧光显微镜下观察绿色荧光蛋白的表达，使用流式细胞仪检测转染效率，当磁性纳米颗粒与DNA之比为1:1时转染效率最高，高于脂质体介导的转染。
2.将口蹄疫病毒（foot-and-mouth disease virus,FMDV)的2A片段置于串联的RC2(retrocyclin2)和红色荧光蛋白dTomato基因之间，使它们处在一个开放阅读框（ORF）内，将其置于pCDNA4/TO EGFP真核表达载体CMV启动子的下游，构建得到pCDNA4-dTomato-2A-3R2和pCDNA4-dTomato-2A-6R2两个重组质粒，在外加磁场的作用下，磁性纳米颗粒可以携带重组质粒进入293T细胞。结果表明，由2A片段连接的串联的RC2和红色荧光蛋白dTomato可以在293T细胞中进行表达，2A具有自我剪切活性，它在自己的C端切割多聚蛋白，从而得到两个独立而有活性的蛋白。通过dTomato和串联RC2的共表达，使我们很容易的检测到目的蛋白在293T细胞内的表达情况，并且在293T细胞中表达的RC2对禽流感病毒H5N1具有抑制作用，为RC2作为预防和治疗人类流感病毒感染药物的进一步研究奠定了基础。
3.利用9日龄SPF鸡胚建立了鸡胚成纤维细胞系，以pEGFP-N1质粒作为外源DNA，在磁性纳米颗粒的介导下转染CEF细胞，实现了绿色荧光蛋白的表达。经过G418筛选，获得稳定表达绿色荧光蛋白的CEF细胞系，促进了鸡转基因研究的发展。
4.在外加磁场的作用下，磁性纳米颗粒携带质粒pCDNA4-dTomato-2A-3R2/6R2进入CEF细胞和鸡胚，结果表明利用FMDV2A可以实现dTomato和串联RC2在CEF细胞、鸡胚中的共表达，并且表达的RC2可以抑制H5N1在CEF细胞和鸡胚中的增殖，为磁性纳米颗粒作为基因载体进行体内外基因的转移，RC2作为预防和治疗禽类流感病毒感染药物的进一步研究以及生产抗流感病毒的转基因鸡奠定了基础。
The low efficiency of nonviral gene vectors, the receptor-dependent host tropism of adenoviral orlow titers of retroviral vectors limit their utility in gene delivery. To overcome these deficiencies, weused magnetic nanoparticles(MNPS) as gene vectors to transfer genes, which have some characteristicssuch as can combine with big fragment DNA, low immunogenic, good biocompatibility and targetedgene delivery by application of a magnetic field. Nucleic acids carried by magnetic nanoparticles canoccur directional movement and enrich on the cell surface, which increased contact time and exposuredose of the magnetic nanoparticles/gene complexes with cells. It would contribute to study geneexpression, gene function and targeted gene therapy. In this study, PEI-coated magnetic nanoparticleswere characterized, which were used as gene vector to transfer genes in vivo and in vitro by magneticforce.
1.The morphology characteristic, particle size and surface potential of PEI-coated magneticnanoparticles were characterized. The results of electron microscopy and atomic force microscopyshowed that magnetic nanoparticles had100nm diameter with good dispersion, Zeta potential ofmagnetic nanoparticles was29.4mV with positive charge, which can bind with DNA. As the genevector, the DNA binding ability is essential requirement. DNA binding ability of magnetic nanoparticlescan be determined by gel retardation assay, co-sedimentation assay and atomic force microscopy. Theresults proved magnetic nanoparticles had stronger ability to bind DNA. DNA and magneticnanoparticles can form close and tight complex structure. The magnetic nanoparticles can protect DNAagainst the digestion of nuclease. Whether MNPs/DNA complexes influenced cell viability wasinvestigated in several cell lines, including293T, MDCK, Vero and CEF cells by MTT assays.MNPs/DNA complexes showed no obvious negative effect on cell viability. The result of magneticnanoparticles transfection into293T cells showed that the magnetic nanoparticles had high transfectionefficiency, and the highest transfection efficiency was91%when the ratio of magnetic nanoparticles andDNA was1:1, higher than the positive control of lipofectamine transfection efficiency (86%).
2.In the present study, the2A region of foot-and-mouth disease virus (FMDV) was utilized toconstruct a bicistron vector, in which tandem retrocyclin2(RC2) gene and dTomato gene were fusedinto a single open reading frame linked by FMDV2A region. The fused genes were placed under thecontrol of CMV promoter in pCDNA4/TO EGFP vector to construct the recombinant expressionplasmids pCDNA4-dTomato-2A-3R2/6R2. The plasmids pCDNA4-dTomato-2A-3R2/6R2wastransfected into293T cells mediated by MNPs with application of a magnetic field. The results showedthat the fused genes of red fluorescent protein dTomato and muti-copy RC2linked by2A region offoot-and-mouth disease virus were expressed in293T successfully. The2A region mediated aco-translational cleavage event at its own carboxyl-terminus resulting in the release of each individualprotein product. Efficient cleavage was observed and all two proteins were functional, coexpression ofdTomato enabled us to track gene expression in293T cells conveniently. The tandem retrocyclin2can potently inhibit H5N1highly pathogenic avian influenza virus production in293T cells, which providea basis for further development of retrocyclin2for prophylaxis and therapy of H5N1virus infection inhumans.
3.The chicken embryo fibroblast(CEF) cells isolated from the chicken embryo were cultured invitro to construct a cell line. As the exogenous gene the plasmid pEGFP-N1was used to transfect CEFcells mediated by MNPs. G418was employed to screen the drug-resistance cell clones to obtain stablecell lines expressing green fluorescent protein, which laid the foundation for the study of transgenicchickens.
4.The plasmids pCDNA4-dTomato-2A-3R2/6R2were transfected into CEF cells and chickenembryo mediated by MNPs with application of a magnetic field. The results showed that the fusedgenes of red fluorescent protein dTomato and tandem RC2linked by2A region of foot-and-mouthdisease virus were expressed in CEF cells and chicken embryo successfully. The tandem retrocyclin2can potently inhibit H5N1highly pathogenic avian influenza virus production in CEF cells and chickenembryo, which provide a basis for further development of application of MNPs to gene delivery in vitroand in vivo, retrocyclin2for prophylaxis and therapy of H5N1virus infection in poultry, producingtransgenic chickens with suppression of avian influenza transmission.
引文
1.何晓晓.生物亲和性核壳纳米颗粒研究及其在生物/医学中的应用[博士学位论文].长沙：湖南大学，2003.
2.李颖，崔海信，宋瑜，等．PEI介导外源基因进入植物细胞的瞬时表达[J]．中国农业科学，2009,42(6):1918-1923．
3.林霞．纳米基因载体酶切保护机理的研究及新型基因载体的制备与应用[硕士学位论文]．长沙：湖南大学，2005.
4.刘俊,刘选明,肖苏尧等.基于超声波下淀粉纳米颗粒作载体的基因转导[J].高等学校化学学报，2005,26(4):634-637.
5.施卫贤，杨俊，王亭杰等.磁性Fe3O4微粒表面有机改性[J].物理化学学报.2001，17(6):507-510.
6.宋丽贤，卢忠远，刘德春，等.分解沉淀法制备磁性纳米Fe3O4的研究及表征[J].化工进展，2006,25（1）：54-57.
7.谭泽明，基于超顺磁性四氧化三铁磁性纳米颗粒的恶性胶质瘤靶向联合基因治疗[博士学位论文].长沙：中南大学，2009.
8.唐秋莎，张东生，顾宁等.新型纳米基因载体（PEI/Mn0.5Zn0.5Fe2O4）的制备、表征及体外实验[J].功能材料，2007，8(38),1268-1272.
9.肖苏尧,刘选明,童春义等.多聚赖氨酸淀粉纳米颗粒基因载体的研制及应用[J].中国科学B辑，2004,34(6):473-477.
10.徐滨士.纳米表面工程[M].北京:化学工业出版社，2003:
11.张春明，赵梗明，斯庆苏都，谢湘华等.磁性纳米粒子的制备及其细胞分离方面的应用[J].上海师范大学学报（自然科学版）.2008，37（3）291-295.
12.张玲.四氧化三铁纳米颗粒及其复合物的制备和研究[博士学位论文].上海：上海交通大学，
2007.
13.张阳德，张洋，潘一峰.磁性纳米颗粒靶向性肿瘤热疗的研究进展[J].中国医学工程，2006,14（2）148-152.
14.张阳德.纳米生物材料学.北京：化学工业出版社，2005：25.
15.张阳德.纳米生物技术学.北京：科学出版社，2005：62.
16.赵紫来，卞征云，陈朗星等.氧化铁磁性纳米粒子的制备、表面修饰及在分离和分析中的应用[J].化学进展.2006，15(10):1285-1297.
17.朱诗国，李桂源.纳米基因转运体-原理、研制与应用.生物化学与生物物理进展[J].2002,29（6）868-871.
18. Amrani A E, Barakate A, Askari B M,et al. Coordinate expression and independent subcellulartargeting of multiple proteins from a single transgene[J].Plant Physiology,2004,135:16–24.
19. Ang D, Nguyen Q V, Kayal S，et al. Insights into the mechanism of magnetic particle assistedgene delivery[J].Acta Biomaterialia,2011,7:1319-1326.
20. Ao Z, Patel A, Tran K,et al. Characterization of a trypsin-dependent avian influenza H5N1-pseudotyped HIV vector system for high throughput screening of inhibitory molecules[J]. Antiviralresearch,2008,79:12-18.
21. Bartlett D W, Su H, Hildebrandt I J, et al.Impact of tumor-specific targeting on the biodistributionand efficacy of siRNA nanoparticles measured by multimodality in vivo imaging[J].Proc NatlAcad Sci U S A,2007,104:15549–15554.
22. Bertram J. MATra-magnet assisted transfection: combining nanotechnology and magnetic forces toimprove intracellular delivery of nucleic acids[J].Curr Pharm Biotechno,2006,7(4):277-285.
23. Bocalandro Y M, Marin M H, Vallejo R A, et al. Antigenic mixture of synthetic peptides for theimmunodiagnosis of HTLVⅠ/Ⅱin fection[J].Journal of Immunoassay and Immunochemistry,2004,25(3):205-214.
24. Boman H G, Nilsson I, Rasmuson B. Inducible antibacterial defence system in Drasophial[J].Nature,1972,237(5352):232-235.
25. Bowdish D M, Davidson D J, Hancock R E. A re-evaluation of the role of host defence peptides inmammalian immunity[J].Current Protein and Peptide Science,2005,6(1):35-51.
26. Brown M D，Schatzlein A G, Uchegbu I F. Gene delivery with synthetic(nonviral) carriers[J].Int JPharm,2001,229:1-21.
27. Buerstedde J M, Takeda S. Increased ratio of targeted to random integration after transfection ofchicken B cell lines[J].Cell,1991,67(1):179-188.
28. Chan C K，Senden T,Jans D A. Supramolecular structure and nuclear targeting efficieneydetermine the enhancement of transfection by modified polylysines[J]. Gene Therapy,2000,7:1690-1697.
29. Chan P K S. Outbreak of avian influenza A(H5N1) virus infection in Hong Kong in1997[J].Clinical infectious diseases,2002,34(Suppl2):S58-64.
30. Chan W C, Nie S M. Quantum dot bioconjugates for ultrasensitive nonisotopic detection[J].Science,1998,281(25):2016-2018.
31. Chapman S C, Lawson A, MacArthur W C, et al. Ubiquitous GFP expression in transgenicchickens using a lentiviral vector[J].Development,2005,(132):935-940.
32. Chen O R，Zhang L，Stass S A，et al. Co-polymer of histidine and lysine markedly enhancestransfection efficiency of liposomes[J].Gene therapy,2000,7:1698-1705.
33. Chilkoti A, Dreher M R, Meyer D E. Design of thermally responsive,recombinant polypeptidecarriers for targeted drug delivery[J].Adv. Drug Deliv.Rev,2002,54(8):1093-1111.
34. Chinnasamy D, Milsom M D, Shaffer J, et al. Multicistronic lentiviral vectors containing theFMDV2A cleavage factor demonstrate robust expression of encoded genes at limitingMOI[J].Virology Journal,2006,3:14
35. Choi Y H， Liu F，Choi J S，et al. Characterization of a targeted gene carrier lactose-polyethyleneglycol-grafted poly-L-lysine，and its complex with plasmid DNA[J]. Human GeneTheraPy,1999,10:2657-2665.
36. Chul Cho K, Hoon Jeong J, Jung Chung H, et al. Folate receptor-mediated intracellular delivery ofrecombinant caspase-3for inducing apoptosis[J].J. Control. Release,2005,108(1):121–131.
37. Ciftci K, Levy R J.Enhanced plasmid DNA transfection with lysosomotropic agents in culturedfibroblasts[J].Inter J Pharm,2001,218(1-2):81-92.
38. Cole A M, Hong T, Boo L M, et al. Retrocyclin: a primate peptide that protects cells from infectionby T-and M-tropic strains of HIV-1[J].PNAS,2002,99(4):1813-1818.
39. Cole A M, Hong T, Boo L M. Retrocyclin: A primate peptide that protects cells from infection byT-and M-tropic strains of HIV-1[J].PNAS,2002,99(4):1813–1818.
40. Daly N L, Chen Y K, Rosengren K J, et al. Retrocyclin-2: structural analysis of a potent anti-HIVtheta-defensin[J].Biochemistry,2007,46:9920-9928.
41. David A L, David M L, Robert L. Timline: moving smaller in drug discovery and delivery[J].Nat.Rev. Drug Discov,2002,1(1):77-84.
42. de Bruin K, Ruthardt N, von Gersdorff K, et al. Cellular dynamics of EGF receptor-targetedsynthetic viruses[J].Molecular Therapy.2007,157:1297-1305.
43. de Felipe P. Skipping the co-expression problem: the new2A "CHYSEL" technology[J]. Geneticvaccines and therapy,2004,2:13-19.
44. Deng R, Yue Y, Jin F, et al. Revisit the complexation of PEI and DNA—How to make lowcytotoxic and highly efficient PEIgene transfection non-viral vectors with a controllable chainlength and structure?[J] Journal of Controlled Release,2009,140(1):40–46.
45. Deng Y H, Wang C C, Hu J H, et al. Investigation of formation of silica-coated magnetitenanoparticles via sol-gel approach[J].Colloids Surf. A,2005,262(1-3):87-93.
46. Divis S S. Biomedical application of nanotechnology-implications for drug targeting and genetheraphy[J].Trends in biotech,1997,15(6):217-224.
47. Dobson J.Gene therapy progress and prospects:magnetic nanoparticle-based gene delivery[J].GeneTherapy.2006,13:283–287.
48. Donnelly M L L, Hughes L E, Luke G,et al. The 'cleavage' activities of foot-and-mouth diseasevirus2A site-directed mutants and naturally occurring '2A-like' sequences[J]. The Journal ofgeneral virology,2001a,82:1027-1041.
49. Donnelly M L L, Luke G, Mehrotra A, et al. Analysis of the aphthovirus2A/2B protein ‘cleavage’mechanism indicates not a proteolytic reaction, but a novel translational effect: a putativeribosomal ‘skip’[J].Journaol of General Virology,2001b,82:1013-1025.
50. Elfinger M, Geiger J, Hasenpusch G, et al.Targeting of the β2-adrenoceptor increases nonviralgene delivery to pulmonary epithelial cells in vitro and lungs in vivo [J].Journal of ControlledRelease,2009,(135):234–241.
51. Elfinger M, Maucksch C, Rudolph C. Characterization of lactoferrin as a targeting ligand fornonviral gene delivery to airway epithelial cells[J].Biomaterial,2007,28(23):3448-3455.
52. Elfinger M, Maucksch C,Rudolph C.Characterization of lactoferrin as a targeting ligand fornonviral gene delivery to airway epithelial cells[J].Biomaterials,2007,28(23):3448–3455.
53. Fan R, Chen X H, Gui Z, et al. A new simple hydrothermal preparation of nanocrystallinemagnetite[J].Mater Res Bull,2011,36(3-4):497-502.
54. Feigner J H, Kumar R, Sridhar C N,et al. Enhanced gene delivery and mechanism studies with anovel series of cationic lipid formulations[J].J Biol Chem,1994,269:2550-2561.
55. Fenske D B, Maclachlan l，Cullis P R，et al. Long-circulating vecots for the systemic delivery ofgenes[J].Curr Opin Mol Ther,2000,3(2):153-158.
56. Fernandez-Pacheco R, Marquina C, Valdivia J G, et al. Magnetic nanoparticles for local drugdelivery using magnetic implants. In:6th international conference on the scientific and clinicalapplications of magnetic carriers[J].Elsevier Science B.V.,2006:318–322.
57. Ferrari M. Vectoring siRNA therapeutics into the clinic[J].Nat Rev Clin Oncol,2010,7:485–486.
58. Fischer Y H，Miletic H， Giroglou T，etal. A retroviral packaging cell line for pseudotype vectorsbased on glioma-infiltrating progenitor cells[J].J Gene Med,2007,9(5):335-344.
59. Fishlock T W, Oral A, Egdell R G, et al. Manipulation of atoms across a surface at roomtemperature[J].Nature,2000,404(6779):743-745.
60. Gao X H, Cui Y Y, Levenson R M, et al. In vivo cancer targeting and imaging with semiconductorquantum dots[J].Nat. Biotech,2004,22(8):969-976.
61. Gich M, Casas Li, Roig A, et al. High-coercivity ultralight transparent magnets[J]. Appl.Phys.Lett,2003,82(24):4307-4310.
62. Gilchrist R K, Medal R, Shorey W D, et al. Selective inductive heating of lymph nodes[J].Annalsof Oncology,1957,146(4):595-606.
63. Guerreiro C I, Fontes C M, Gama M, et al. Escherichia coli expression and purification of fourantimicrobial peptides fused to a family3carbohydrate-binding module (CBM) from Clostridiumthermocellum[J].Protein Expr Purif,2008,59(1):161-168.
64. Halpin C, Cooke S E, Barakate A, et al. Self-processing2A-polyproteins-a system for co-ordinateexpression of multiple protein in transgenic plants[J].The Plant Journal,1999,17(4):453–459.
65. Han M Y, Gao X H, Nie S M, et al. Quantum-dot-tagged microbeads for multiplexed opticalcoding of biomolecules[J].Nat. biotech,2001,19(7):631-635.
66. Harvey A J, Speksnijder G, Baugh L R, et al. Consistent production of transgenic chickens usingreplication-deficient retroviral vectors and high-throughput screening procedures[J].PoultrySci,2002,81(2):202-212.
67. Harvey A J, Speksnijder G, Baugh L R, et al.Expression of exogenous protein in the egg white oftransgenic chickens[J].Nature Biotechnology,2002,20:396-399.
68. Hasebe M, Okano K, Hattori M-A.The incorporation of exgenous DNA into chicken spermatozoa.Proceedings of the8th AAAP Animal science Cogress, Tokyo,1996:424-425.
69. Hasegawa K, Cowan A B, Nakatsuji N, et al. Efficient Multicistronic Expression of a Transgenein Human Embryonic Stem Cells[J].Stem Cells,2007,25:1707–1712.
70. Hashimoto M, Hisano Y. Directional gene-transfer into the brain by an adenoviral vector taggedwith magnetic nanoparticles[J].J. Neurosci. Methods.,2011,194(2):316–320.
71. Hawkins T. M13single-strand purification using a biotinylated probe and streptavidin coatedbeads[J].Mitochondrial DNA,1992,3(2):65-69.
72. He X X，Wang K M，Tan W H et al. Bioconjugated Nanoparticles for DNA Protection fromCleavage [J].J. A m.Chem.Soc,2003,125:7168-716.
73. Hernandez M, Rodriguez I, Pozo L, et al. Chimeric synthetic peptides from the nucleocapsid p24protein of human immunodeficiency virus type-1[J].Biochemical and Biophysical ResearchCommunication,2001,282(1):1-3.
74. Hofmann A, Wenzel D, Becher U M, et al. Combined targeting of lentiviral vectors and positioningof transduced cells by magnetic nanoparticles[J].PNAS,2009,106(1):44-49.
75. Hreczuk-Hirst D, Chicco D, German L, et al. Dextrins as potential carriers for drug targeting:tailored rates of dextrin degradation by introduction of pendant groups[J].Int. J. Pharm,2001,230(1-2):57-66.
76. Hu-Lieskovan S, Heidel J D, Bartlett D W, et al. Sequence-specific knockdown of ews-fli1bytargeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model ofmetastatic ewing's sarcoma[J].Cancer Res,2005,65:8984–8992.
77. Indrajit R, Tymish Y O, Dhruba J B, et al. Optical tracking of organically modified silicananoparticles as DNA carriers: A nonviral, nanomedicine approach for gene delivery[J]. Proc. Natl.Acad. Sci. USA,2005,102(2):279-284.
78. Ivanov V V, Zajats S V, Medvedev A I, et al. Formation of metal matrix composite by magneticpulsed compaction of partially oxidized Al nanopowder[J].J. Mater. Sci,2004,39(16-17):5231-5234.
79. Jackson D A, Juranek S, Lipps H J. Designing nonviral vectors for efficient gene transfer andlong-term gene expression[J].Mol. Ther.,2006,14(5):613–626.
80. James J S, Adam D L, Viswanadham G, et al. Homogeneous detection of unamplified genomicDNA sequences based on colorimetric scatter of gold nanoparticle probes[J]. Nat. biotech,2002,22(7):883-887.
81. Jeong J H, Lee M, Kim W J, et al. Anti-GAD antibody targeted non-viral gene delivery to islet betacells[J].J. Control. Release,2005,107(3):562–570.
82. Jeong J H，Park T G. Poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxicbiodegradable gene delivery carriers[J].J Control Release,2002,82(1):159-166.
83. Joseph F, Poduslo, Thomas M. Wengenack,Geoffry L.Curran, et al.Molecular Targeting ofAlzheimer’s Amyloid Plaques for Contrast-Enhanced Magnetic Resonance Imaging[J]. Neurobi-ology of Disease,2002,11:315-329.
84. Junghans M, Kreuter J, Zimmer A. Phosphodiester and phosphorothioate oligonucleotidecondensation and preparation of antisense nanoparticles[J].Biochim Biophys Acta,2001,1544(1-2):177-188.
85. Junghans M，Kreuter J, Zimmer A. Antisense delivery using protamine-oligonucleotide particles[J].Nucleic Acids Res,2000,28(10):e45.
86. Kaleri H A, Xu S Y, Lin H L. Generation of transgenic chicks using an oviduct-specific expressionsystem[J].Genet. Mol. Res,2011,10(4):3046-3055.
87. Kamei K, Mukai Y, Kojima H, et al. Direct cell entry of gold/iron-oxide magnetic nanoparticles inadenovirus mediated gene delivery[J].Biomaterials,2009,30(9):1809-1814.
88. Kami D, Takeda S, Itakura Y, et al. Application of Magnetic Nanoparticles to Gene Delivery[J].Int.J. Mol. Sci.2011,12:3705-3722.
89. Kaneko Y, Mizuta Y, Nishijima Y, et al. Vickers hardness and deformation of Ni/Cu nano-multilayers electrodeposited on copper substrates[J].J. Mater. Sci,2005,40(12):3231-3236.
90. Kievit F M, Veiseh O, Bhattarai N, et al. PEI-PEG-Chitosan copolymer coated iron oxidenanoparticles for safe gene delivery: synthesis, complexation, and transfection[J]Adv. Funct. Mater,2009,19:2244-2251.
91. Kim TM, Park TS, Shin SSet al.An interclass nuclear transfer between fowl and mammal: in vitrodevelopment of chicken-to-cattle interclass embryos and the detection of chicken geneticComplements[J]. Fertil Steril,2004,82(4):957-959.
92. Kinoshita T, Seino S, Okitsu K, et al. Magnetic evaluation of nanostructure of gold-iron compositeparticles synthesized by a reverse micelle method[J].Journal of Alloys and Compounds,2003,359(1-2):46-50.
93. Kircheis R, Ostermann E, Wolschek M F,et al. Tumor-targeted gene delivery of tumor necrosisfactor-α induces tumor necrosis and tumor regression without systemic toxicity[J].Cancer GeneTher,2002,9:673–680.
94. Kircheis R, Wightman L, Schreiber A, et al. Polyethylenimine/DNA complexes shielded bytransferrin target gene expression to tumors after systemic application[J].Gene Ther,2001,8(1):28–40.
95. Kneuer C, Sameti M, Haltner E G, et al. Silica nanoParticles modified with aminosilanes ascarriers for plasmid DNA[J].International Journal of Pharmaceutics,2000,196(2):257-261
96. Kock N，Kasmieh R，Weissleder R，et al.Tumor therapy mediated by lentiviral expression ofshBcl-2and S-TRAIL[J].Neoplasia,2007,9(5):435-442.
97. Krotz F, de Wit C, Sohn H Y, et al. Magnetofection—a highly efficient tool for antisenseoligonucleotide delivery in vitro and in vivo[J].Mol. Ther,2003a(7):700-710.
98. Krotz F, Sohn H-Y, Glone T,et al. Magnetofection potentiates gene delivery to cultured endothelialcells[J].J. Vasc. Res,2003b,40(5):425-434.
99. Kuhara M, Takeyama H, Tanaka T, et. Al. Magnetic cell separation using antibody binding withprotein A expressed on bacterial magnetic particles [J].Anal Chem,2004,76(21):6207-6213.
100. Kuiken T, Rimmelzwaan G, van Riel D, et al. Avian H5N1influenza in cats[J].Science,2004,306:241.
101. Kumar M, Yigit M, Dai G, et al. Image-guided breast tumor therapy using a small interfering RNAnanodrug[J].Cancer Res,2010,70:7553–7561.
102. Kunath K, Merdan T, Hegener O, et al. Integrin targeting using RGD-PEI conjugates for in vitrogene transfer[J].J. Gene. Med,2003,5(7):588–599.
103. Lee R J, Huang L. Folate-targeted，anionic liposome-entrapped polylysine-condensed dna fortumor cell-specific gene transfer[J].The Journal of Biological Chemistry,1996,271:8481-8487.
104. Lehrer R I.Primate defensins[J]. Nature reviews Microbiology,2004,2:727-738.
105. Lehrman S. Virus treatment questioned after gene therapy death[J].Nature,1999,401(6753):517-518.
106. Leikina E, Delanoe-Ayari H, Melikov K, et al. Carbohydrate-binding molecules inhibit viral fusionand entry by crosslinking membrane glycoproteins[J].Nature immunology,2005,6:995-1001.
107. Lewin M, Carlesso N, Tung C H, et al. Tat peptide-derivatized magnetic nanoparticles allow invivo tracking and recovery of progenitor cells[J]. Nat.Biotech.2000,18(4):410-414.
108. Liang Q L, Zhou K, He H X. Retrocyclin2: a new therapy against avian influenza H5N1virus invivo and vitro[J].Biotechnology letters,2010,32:387-392.
109. Liang Z, Susha A, Caruso F.Gold nanoparticle-based core-shell and hollow spheres and orderedassemblies[J].Chem.Mater,2003,15(16):3176-3183.
110. Lin J，Zhou W, Kumbhar A, et al. Gold-coated iron nanoparticles: synthesis, characterization, andmagnetic field-induced self-assembly[J].Journal of Solid State Chemistry,2001,159(1):26-31.
111. Liu S Z, Zhou Z M, Chen T, et al. Blastocysts produced by nuclear transfer between chickenblastodermal cells and rabbit oocytes[J]. Mol Reprod Dev,2004,69(3):296-302.
112. Liu X, Wu J, Yammine M, et al. Structurally Flexible Triethanolamine Core PAMAM DendrimersAre Effective Nanovectors for DNA Transfection in Vitro and in Vivo to the Mouse Thymus[J].Bioconjugate Chem,2011,22(12):2461-2473.
113. Liu Y, Jia S, Wu Q, et al.Studies of Fe3O4-chitosan nanoparticles prepared by co-precipitationunder the magnetic field for lipase immobilization[J].Catalysis Communications,2011,12(8):717–720.
114. Liu Z L, Ding Z H, Yao K L,et al. Preparation and characterization of polymer-coated core-shellstructured magnetic microbeads[J].Magn Mater,2003,265:98-105.
115. Lorens J B, Pearsall D M, Swift S E, et al. Stable, stoichiometric delivery of diverse proteinfunctions[J].Journal of Biochemical Biophysical Methods,2004,58:101-110.
116. Love J, Gribbin C, Mather C,et al.Transgenic birds by DNA microinjection[J].Nature Biotechnology,1994,12(1):60-63.
117. Lungwitz U, Breunig M, Blunk T. Polyethylenimine-based nonviral gene delivery systems[J]. EurJ Pharm and Biopharm,2005,60:247-266.
118. Luo D,Saltzman W M.Synethetic DNA delivery systems[J].Nat Biotech,2000,18:33-37.
119. Luo D，Woodrow-Mumford K，Belcheva N，et al. Controlled DNA delivery systems[J]. PharmRes,1999,16:1300-1308.
120. Lyall J,Irvine R M,Sherman A,et al.Suppression of Avian Influenza Transmission in GeneticallyModified Chickens[J].Science,2011,331(6014):223-226.
121. Ma H, Diamond S L. Nonviral gene therapy and its deliverysystems [J]. Curr PharmBiotechn,2001,2(1):1-17.
122. Maillard S, Ameller T, Gauduchon J, et al. Innovative drug delivery nanosystems improve theanti-tumor activity in vitro and in vivo of anti-estrogens in human breast cancer and multiplemyeloma[J]. J. Steroid Biochem. Mol. Biol,2005,94(1-3):111-121.
123. Mari T, Masanori U, Noritada K, et al. Nanospheres for DNA separation chips[J].Nat. Biotech,2004,22(3):337-340.
124. Markert J M，Parker J N，Buchsbaum D J，etal. Oncolytic HSV-l for the treatment of braintumours[J].Herpes,2006,13(3):66-71.
125. Marumoto T,Tashiro A，Friedmann-Morvinski D，etal.Development of a novel mouse gliomamodel using lentiviral vectors[J].Nat Med,2009,15(1):110-116.
126. Mashall E. Gene therapy death prompts review of adenovirus vectors[J].Science,1999,286(5448):2244-2245.
127. McPhee J B, Hancock R E. Function and therapeutic potential of host defence peptides[J].Journalof Peptide Science,2005,11(11):677-687.
128. Medarova Z, Pham W, Farrar C, et al. In-vivo imaging of siRNA delivery and silencing intumors[J].Nat Med.2007,13:372–377.
129. Medintz I L, Konnert J H, Clapp A R, et al. A fluorescence resonance energy transfer-derivedstructure of a quantum dot-protein bioconjugate nanoassembly[J]. Proc. Natl. Acad. Sci.USA,2004,101(26):9612-9617.
130. Mizuguchi H, Xu Z, Ishii-Watabe A, et al. IRES-dependent second gene expression is significantlylower than cap-dependent first gene expression in a bicistronic vector[J].Molecular Therapy,2000,1(4):376–382.
131. Moghimi S M, Hunter A C, Murray J C. Long-circulating and target-specific nanoparticles: theoryto practices[J].Pharmacol Rev,2001,53(3):283-318.
132. Moore A, Marecos E, Bogdanov A, et al. Tumoral Distribution of Long-circulating Dextran-coatedIron Oxide Nanoparticles in a Rodent Model [J].Radioligy,2000,214:568-574.
133. Moralli D，Simpson K M，Wade-Martins R，etal. A novel human artificial Chromosome geneexpression system using herpes simplex virus typel vectors[J].EMBO Rep,2006,7(9):911-918.
134. Mozdziak P E, Borwornpinyo S, McCoy, D W，et al.Development of transgenic chickensexpressing bacterial β-galactosidase[J]. Dev.Dyn.,2003,226(3):439-445.
135. Müller R H, M der K, Gohla S. Solid Lipid Nanoparticles (SLN) for controlled drug delivery-areview of the state of the art[J].Eur. J. Pharm. Biopharm,2000,50(1):161-177.
136. Munk C, Wei G, Yang O O, et al. The theta-defensin, retrocyclin, inhibits HIV-1entry [J].AIDSresearch and human retroviruses,2003,19:875-881.
137. Naito M, Perry MM.Development in culture of the chick embryo from cleavage to hatch. Br PoultSci,1989,30(2):251-256.
138. Nguyena T X, Cole A M, Lehrer R I. Evolution of primate θ-defensins:a serpentine path to a sweettooth[J].Peptides,2003,(24):1647-1654.
139. Nicholson K G, Wood J M, Zambon M. Influenza[J].Lancet,2003,362:1733-1745.
140. Nicola O. The Antimicrobial Activity of Lactoferrin: Current Status and Perspectives[J].BioMetals,2004,(17):189-196.
141. Ogirs M，Wagner E. Targeting tumors with non-viral gene delivery systems[J].Drug DiscoveryToday,2002,7(8):479-485.
142. Ono T, Wakasugi N. Mineral content of quail embryos cultured in mineral-rich and mineral-freeconditions[J]. Poult Sci,1984,63(1):159-166.
143. Osborn M J, Panoskaltsis-Mortari A, McElmurry R T,et al. A picornaviral2A-like sequence-basedtricistronic vector allowing for high-level therapeutic gene expression coupled to a dual-reportersystem[J].Molecular Therapy,2005,12(3):569-574.
144. Owen S M, Rudolph D L, Wang W, et al. RC-101, a retrocyclin-1analogue with enhanced activityagainst primary HIV type1isolates[J]. AIDS research and human retroviruses,2004,20:1157-1165.
145. Pack D W, Hoffman A S, Pun S,et al. Design and development of polymers for gene delivery[J].Nat. Rev. Drug Discov,2005,4(7):581–593.
146. Panyam J, Zhou W Z, Prabha S, et al. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery[J].FASEB J,2002,16(10):1217-1226.
147. Park J W, Hong K, Kirpotin D B, et al. Anti-HER2immunoliposomes enhanced efficacyattributable to targeted delivery[J].Clin Cancer Res.2002,8:1172–1181.
148. Park T S, Han J Y.Derivation and characterization of pluripotent embryonic germ cells in chicken[J].Mol Reprod Dev,2000,56(4):475-482.
149. Pascal A, Franklin A H, Eric W, et al. Halogen bonds in biological molecules[J]. Proc. Natl. Acad.Sci. USA,2004,101(48):16789-16794.
150. Patil M L, Zhang M, Taratula O, et al.Internally Cationic Polyamidoamine PAMAM-OHDendrimers for siRNA Delivery: Effect of the Degree of Quaternization and Cancer Targeting[J].Biomacromolecules,2009,10(2):258–266.
151. Payungporn S, Chutinimitkul S, Chaisingh A, et al. Single step multiplex real-time RT-PCR forH5N1influenza A virus detection[J].Journal of Virological Methods,2006,131(2):143-147.
152. Peng X H, Qian X M, Mao H, et al. Targeted magnetic iron oxide nanoparticles for tumor imagingand therapy[J].Int J Nanomed,2008,3:311–321.
153. Perry MM.A complete culture system for the chick embryo[J]. Nature,1988,331(6151):70-72.
154. Piao Y， Jiang H，Alemany R， et al. Oncolytic adenovirus retargeted to Delta-EGFR inducesselective antiglioma activity[J].Cancer Gene Ther,2009,16(3):256-265.
155. Rabelo D, Lima E C D, Reis A C, et al. Preparation of magnetite nanoparticles in mesoporouscopolymer template[J].Nano Lett,2001,1(2):105-108.
156. Rhaese S, von Briesen H, Rübsamen-Waigmann H, et al. Human serum albumin-polyethyleniminenanoparticles for gene delivery [J].Control Release,2003,92(1-2):199-208.
157. Robert J C, Sarunya B, Katerina A D, et al. Noncovalent functionalization of carbon nanotubes forhighly specific electronic biosensors[J].Proc. Natl. Acad. Sci. USA,2003,100(9):4984-4989.
158. Roongsawang N, Promdonkoy P, Wongwanichpokhin M, et al. Coexpression of fungal phytaseand xylanase utilizing the cis-acting hydrolase element in Pichia pastoris[J].FEMS YeastResearch,2010,10:909–916.
159. Rowlett K, Simkiss K. Explanted embryo culture: in vitro and in ovo techniques for the domesticFowl[J].British Poultry Science,1987,78:91-101.
160. Roy K, Mao H Q, Huang S K, et al. Oral gene felivery with chitosan-DNA nanoparticles generatesimmunologic protection in a murine model of peanutallergy[J].Nature Med,1999,5(4):387-391.
161. Saccardo P, Villaverde A, González-Montalbán N. Peptide-mediated DNA condensation fornon-viral gene therapy[J]. Biotechnology Advances,2009,27(4)432–438.
162. Safarikova M.“use of Magnetic Techniques for the Isolation of Cells”[J]. J ChromatograhyB,1999,722:33-53.
163. Sauer A M, de Bruin K G，Plank C, et al. Localization and dynamics of magnetic transfectioncomplexes. Journal of Controlled Release.2008,132(3):e15-e16.
164. Schatzlein A G. Non-viral vectors in cancer gene therapy: principles and progress[J]. Anti-CancerDrugs,2001,12(4):275–304.
165. Schatzlein A G.Targeting of synthetic gene delivery systems[J].J. Biomed. Biotechnol,2003(2):149–158.
166. Scherer F, Anton M, Schillinger U, et al. Magnetofection: enhancing and targeting gene delivery bymagnetic force in vitro and in vivo[J].Gene Ther,2002,9:102-109.
167. Shi Y, Zhou L, Wang R, et al.In situ preparation of magnetic nonviral gene vectors andmagnetofection in vitro[J].Nanotechnology,2010,21:115-103.
168. Song H P，Yang J Y, Lo S L,et al. Gene transfer using self-assembled ternary complexes ofcationic magnetic nanoparticles, plasmid DNA and cell-penetrating Tat peptide[J]. Biomaterials,2010,31(4)769-778.
169. Sonti S V, Bose A. Cell separation using protein-A-coated magnetic nanclusters[J].J. Colliod.,Interface.Sci,1995,170(1):575-585.
170. Subbarao K, Klimov A, Katz J, et al. Characterization of an avian influenza A (H5N1) virusisolated from a child with a fatal respiratory illness[J].Science,1998,279:393-396.
171. Subramani K, Hosseinkhani H, Khraisat A, et al. Targeting nanoparticles as drug delivery systemsfor cancer treatment[J].Curr Nanosci,2009,5:135-140.
172. Sun M, Shi Q Q,Yin Y H, et al. Generation of antiviral transgenic chicken using spermatogonialstem cell transfected in vivo[J].African Journal of Biotechnology,2011,10(70):15678-15683.
173. Sun P, Zhao C, Li Y. Expression of green fluorescent protein gene in somatic chimeric chickensproduced by transplantation of transfected chicken embryonic fibroblasts[J]. J.Poult.Sci,2009,(46):363-369.
174. Szymczak A L, Workman C J, Wang Y, et al. Correction of multi-gene deficiency in vivo using asingle ‘self-cleaving’2A peptide-based retroviral vector[J].Nature Biotechnology,2004,22(5):589–594.
175. Tran D, Tran P A, Tang Y Q, et al. Selsted ME.Homodimeric theta-defensins from rhesus macaqueleukocytes:isolation, synthesis, antimicrobial activities, and bacterial binding properties of thecyclic peptides[J].J Biol Chem,2002,277:3079–3084.
176. Tresilwised N, Pithayanukul P, Mykhaylyk O,et al. Boosting oncolytic adenovirus potency withmagnetic nanoparticles and magnetic force[J].Mol.Pharmaceutics,2010,7(4):1069-1089.
177. Truong-Le V L，August J T, Leong K W. Controlled gene delivery by DNA-gelatin nanospheres[J].Hum GeneTher,1998,9:1709-1717.
178. Tsapis N, Bennett D, Jackson B, et al. Trojan particles: Large porous carriers of nanoparticles fordrug delivery[J].Proc.Natl.Acad.Sci.USA,2002,99(19):12001-12005.
179. Van Craynest N, Santaella C, Boussif O, et al. Polycationic telomers and cotelomers for genetransfer:synthesis and evaluation of their an vitro transfection efficiency[J]. Bioconjug Chem,2002,13(1):59-75.
180. Varadan V K, Chen L F, Xie J, et al. Nanomedicine: design and applications of magneticnanomaterials, nanosensors and nanosystems.Chichester, UK:Wiley,2008,467.
181. Verkman A S, Sonawane N D, Szoka F C.Chloride accumulation and swelling in endosomesenhances DNA transfer by polyamine–DNA polyplexes[J].J. Biol. Chem,2003,278(45):44826-44831.
182. Vijayanathan V, Thomas T, Thomas T J, et al. DNA nanopartlcles and development of DNAdelivery vehicles for gene therapy[J]. Biochem,2002,41(48):14085-14094.
183. Wadghiri Y Z, Sigurdsson E M, Sadowski M, et al.Detection of Alzheimer’s Amyloid inTransgenic Mice Using Magnetic Resonance Microimaging[J].Magnetic Resonance in Medicine,2003,50(2):293–302.
184. Waite C, Sparks S, Uhrich K, et al.Acetylation of PAMAM dendrimers for cellular delivery ofsiRNA[J].BMC Biotechnology,2009,9:38.
185. Wang J, Zhao G, Li X, et al.Reversible immobilization of glucoamylase onto magnetic chitosannanocarriers[J].Applied Microbiology and Biotechnology,2012,DOI:10.1007/s00253-012-3979-2.
186. Wang S, Yao Q, Tao J, et al. Co-ordinate expression of glycine betaine synthesis genes linked bythe FMDV2A region in a single open reading frame in Pichia pastoris[J]. Applied Genetics andMolecular Biotechnology2007,77:891–899.
187. Webby R J, Webster R G. Are we ready for pandemic influenza?[J] Science,2003,302:1519-1522.
188. Weiss S I, Sieverling N, Niclasen M,et al. Uronic acids functionalized polyethy-leneimine(PEI)-polyethyleneglycol (PEG)-graft-copolymers as novel synthetic gene carriers[J]. Biomaterials,2006,27(10):2302–2312.
189. Wu Q, Moyana T, Xiang J. Cancer gene therapy by adenovirus-mediated gene transfer[J]. CurrGene Ther,2001,(1):101-122.
190. Xiang J J, Tang J Q, Zhu S G, et al. IONP-PLL: a novel non-viral vector for efficient geneDelivery[J].J Gene Med,2003,5:803–817.
191. Yang S Y, Sun J S, Liu C H, et al. Ex vivo magnetofection with magnetic nanoparticles: a novelplatform for nonviral tissue engineering[J]. Artificial Organs,2008,32(3):195-204.
192. Yang Y, Xu Z, Jiang J, et al. Poly(imidazole/DMAEA) phosphazene/DNA self-assemblednanoparticles for gene delivery: Synthesis and in vitro transfection[J].J Control Release,2008,127(3):273-279.
193. Yanson A I, Bollinger G R, Brom H E. Formation and manipulation of a metallic wire of singlegold atoms[J].Nature,1998,395(6700):783-785.
194. Yasin B, Wang W, Pang M, et al. Theta defensins protect cells from infection by herpes simplexvirus by inhibiting viral adhesion and entry[J].Journal of virology,2004,78:5147-5156.
195. Yigit M V, Moore A, Medarova Z.Magnetic nanoparticles for cancer diagnosis and therapy[J].pharmaceutical research,2012,DOI10.1007/s11095-012-0679-7.
196. Yin W K, Feng S S. Effects of particle size and surface coating on cellular uptake of polymericnanoparticles for oral delivery of anticancer drugs[J].Biomaterials,2005,26(15):2713-2722.
197. Zhang H, Lee M Y, Hogg M G,et al.Gene delivery in three-dimensional cell cultures bysuperparamagnetic nanoparticles[J].ACS Nano,2010,4(8):4733–4743.
198. Zhang X, Oulad-Abdelghani M, Zelkin A N, et al. Poly(l-lysine) nanostructured particles for genedelivery and hormone stimulation[J].Biomaterials,2010,31(7):1699–1706.
199. Zhao P，Wang C, Fu Z，et al. Lentiviral vector mediated siRNA knock-down of hTERT results indiminished capacity in invasiveness and in vivo growth of human glioma cells in a telomerelength-independent manner [J].Int J Oncol,2007,31(2):361-368.
200. Zhao X J, Hilliard L R, Mechery S J, et al. From the Cover: A rapid bioassay for single bacterialcell quantitation using bioconjugated nanoparticles[J].Proc. Natl.Acad. Sci. USA,2004,101(42):15027-15032.
201. Ziady A G, Ferkol T， Gerken T, et al. Ligand substitution of receptor targeted DNA complexesaffects gene transfer into hepatoma cells[J].Gene therapy,1998,5:1685-1697.
2.李颖，崔海信，宋瑜，等．PEI介导外源基因进入植物细胞的瞬时表达[J]．中国农业科学，2009,42(6):1918-1923．
3.林霞．纳米基因载体酶切保护机理的研究及新型基因载体的制备与应用[硕士学位论文]．长沙：湖南大学，2005.
4.刘俊,刘选明,肖苏尧等.基于超声波下淀粉纳米颗粒作载体的基因转导[J].高等学校化学学报，2005,26(4):634-637.
5.施卫贤，杨俊，王亭杰等.磁性Fe3O4微粒表面有机改性[J].物理化学学报.2001，17(6):507-510.
6.宋丽贤，卢忠远，刘德春，等.分解沉淀法制备磁性纳米Fe3O4的研究及表征[J].化工进展，2006,25（1）：54-57.
7.谭泽明，基于超顺磁性四氧化三铁磁性纳米颗粒的恶性胶质瘤靶向联合基因治疗[博士学位论文].长沙：中南大学，2009.
8.唐秋莎，张东生，顾宁等.新型纳米基因载体（PEI/Mn0.5Zn0.5Fe2O4）的制备、表征及体外实验[J].功能材料，2007，8(38),1268-1272.
9.肖苏尧,刘选明,童春义等.多聚赖氨酸淀粉纳米颗粒基因载体的研制及应用[J].中国科学B辑，2004,34(6):473-477.
10.徐滨士.纳米表面工程[M].北京:化学工业出版社，2003:
11.张春明，赵梗明，斯庆苏都，谢湘华等.磁性纳米粒子的制备及其细胞分离方面的应用[J].上海师范大学学报（自然科学版）.2008，37（3）291-295.
12.张玲.四氧化三铁纳米颗粒及其复合物的制备和研究[博士学位论文].上海：上海交通大学，
2007.
13.张阳德，张洋，潘一峰.磁性纳米颗粒靶向性肿瘤热疗的研究进展[J].中国医学工程，2006,14（2）148-152.
14.张阳德.纳米生物材料学.北京：化学工业出版社，2005：25.
15.张阳德.纳米生物技术学.北京：科学出版社，2005：62.
16.赵紫来，卞征云，陈朗星等.氧化铁磁性纳米粒子的制备、表面修饰及在分离和分析中的应用[J].化学进展.2006，15(10):1285-1297.
17.朱诗国，李桂源.纳米基因转运体-原理、研制与应用.生物化学与生物物理进展[J].2002,29（6）868-871.
18. Amrani A E, Barakate A, Askari B M,et al. Coordinate expression and independent subcellulartargeting of multiple proteins from a single transgene[J].Plant Physiology,2004,135:16–24.
19. Ang D, Nguyen Q V, Kayal S，et al. Insights into the mechanism of magnetic particle assistedgene delivery[J].Acta Biomaterialia,2011,7:1319-1326.
20. Ao Z, Patel A, Tran K,et al. Characterization of a trypsin-dependent avian influenza H5N1-pseudotyped HIV vector system for high throughput screening of inhibitory molecules[J]. Antiviralresearch,2008,79:12-18.
21. Bartlett D W, Su H, Hildebrandt I J, et al.Impact of tumor-specific targeting on the biodistributionand efficacy of siRNA nanoparticles measured by multimodality in vivo imaging[J].Proc NatlAcad Sci U S A,2007,104:15549–15554.
22. Bertram J. MATra-magnet assisted transfection: combining nanotechnology and magnetic forces toimprove intracellular delivery of nucleic acids[J].Curr Pharm Biotechno,2006,7(4):277-285.
23. Bocalandro Y M, Marin M H, Vallejo R A, et al. Antigenic mixture of synthetic peptides for theimmunodiagnosis of HTLVⅠ/Ⅱin fection[J].Journal of Immunoassay and Immunochemistry,2004,25(3):205-214.
24. Boman H G, Nilsson I, Rasmuson B. Inducible antibacterial defence system in Drasophial[J].Nature,1972,237(5352):232-235.
25. Bowdish D M, Davidson D J, Hancock R E. A re-evaluation of the role of host defence peptides inmammalian immunity[J].Current Protein and Peptide Science,2005,6(1):35-51.
26. Brown M D，Schatzlein A G, Uchegbu I F. Gene delivery with synthetic(nonviral) carriers[J].Int JPharm,2001,229:1-21.
27. Buerstedde J M, Takeda S. Increased ratio of targeted to random integration after transfection ofchicken B cell lines[J].Cell,1991,67(1):179-188.
28. Chan C K，Senden T,Jans D A. Supramolecular structure and nuclear targeting efficieneydetermine the enhancement of transfection by modified polylysines[J]. Gene Therapy,2000,7:1690-1697.
29. Chan P K S. Outbreak of avian influenza A(H5N1) virus infection in Hong Kong in1997[J].Clinical infectious diseases,2002,34(Suppl2):S58-64.
30. Chan W C, Nie S M. Quantum dot bioconjugates for ultrasensitive nonisotopic detection[J].Science,1998,281(25):2016-2018.
31. Chapman S C, Lawson A, MacArthur W C, et al. Ubiquitous GFP expression in transgenicchickens using a lentiviral vector[J].Development,2005,(132):935-940.
32. Chen O R，Zhang L，Stass S A，et al. Co-polymer of histidine and lysine markedly enhancestransfection efficiency of liposomes[J].Gene therapy,2000,7:1698-1705.
33. Chilkoti A, Dreher M R, Meyer D E. Design of thermally responsive,recombinant polypeptidecarriers for targeted drug delivery[J].Adv. Drug Deliv.Rev,2002,54(8):1093-1111.
34. Chinnasamy D, Milsom M D, Shaffer J, et al. Multicistronic lentiviral vectors containing theFMDV2A cleavage factor demonstrate robust expression of encoded genes at limitingMOI[J].Virology Journal,2006,3:14
35. Choi Y H， Liu F，Choi J S，et al. Characterization of a targeted gene carrier lactose-polyethyleneglycol-grafted poly-L-lysine，and its complex with plasmid DNA[J]. Human GeneTheraPy,1999,10:2657-2665.
36. Chul Cho K, Hoon Jeong J, Jung Chung H, et al. Folate receptor-mediated intracellular delivery ofrecombinant caspase-3for inducing apoptosis[J].J. Control. Release,2005,108(1):121–131.
37. Ciftci K, Levy R J.Enhanced plasmid DNA transfection with lysosomotropic agents in culturedfibroblasts[J].Inter J Pharm,2001,218(1-2):81-92.
38. Cole A M, Hong T, Boo L M, et al. Retrocyclin: a primate peptide that protects cells from infectionby T-and M-tropic strains of HIV-1[J].PNAS,2002,99(4):1813-1818.
39. Cole A M, Hong T, Boo L M. Retrocyclin: A primate peptide that protects cells from infection byT-and M-tropic strains of HIV-1[J].PNAS,2002,99(4):1813–1818.
40. Daly N L, Chen Y K, Rosengren K J, et al. Retrocyclin-2: structural analysis of a potent anti-HIVtheta-defensin[J].Biochemistry,2007,46:9920-9928.
41. David A L, David M L, Robert L. Timline: moving smaller in drug discovery and delivery[J].Nat.Rev. Drug Discov,2002,1(1):77-84.
42. de Bruin K, Ruthardt N, von Gersdorff K, et al. Cellular dynamics of EGF receptor-targetedsynthetic viruses[J].Molecular Therapy.2007,157:1297-1305.
43. de Felipe P. Skipping the co-expression problem: the new2A "CHYSEL" technology[J]. Geneticvaccines and therapy,2004,2:13-19.
44. Deng R, Yue Y, Jin F, et al. Revisit the complexation of PEI and DNA—How to make lowcytotoxic and highly efficient PEIgene transfection non-viral vectors with a controllable chainlength and structure?[J] Journal of Controlled Release,2009,140(1):40–46.
45. Deng Y H, Wang C C, Hu J H, et al. Investigation of formation of silica-coated magnetitenanoparticles via sol-gel approach[J].Colloids Surf. A,2005,262(1-3):87-93.
46. Divis S S. Biomedical application of nanotechnology-implications for drug targeting and genetheraphy[J].Trends in biotech,1997,15(6):217-224.
47. Dobson J.Gene therapy progress and prospects:magnetic nanoparticle-based gene delivery[J].GeneTherapy.2006,13:283–287.
48. Donnelly M L L, Hughes L E, Luke G,et al. The 'cleavage' activities of foot-and-mouth diseasevirus2A site-directed mutants and naturally occurring '2A-like' sequences[J]. The Journal ofgeneral virology,2001a,82:1027-1041.
49. Donnelly M L L, Luke G, Mehrotra A, et al. Analysis of the aphthovirus2A/2B protein ‘cleavage’mechanism indicates not a proteolytic reaction, but a novel translational effect: a putativeribosomal ‘skip’[J].Journaol of General Virology,2001b,82:1013-1025.
50. Elfinger M, Geiger J, Hasenpusch G, et al.Targeting of the β2-adrenoceptor increases nonviralgene delivery to pulmonary epithelial cells in vitro and lungs in vivo [J].Journal of ControlledRelease,2009,(135):234–241.
51. Elfinger M, Maucksch C, Rudolph C. Characterization of lactoferrin as a targeting ligand fornonviral gene delivery to airway epithelial cells[J].Biomaterial,2007,28(23):3448-3455.
52. Elfinger M, Maucksch C,Rudolph C.Characterization of lactoferrin as a targeting ligand fornonviral gene delivery to airway epithelial cells[J].Biomaterials,2007,28(23):3448–3455.
53. Fan R, Chen X H, Gui Z, et al. A new simple hydrothermal preparation of nanocrystallinemagnetite[J].Mater Res Bull,2011,36(3-4):497-502.
54. Feigner J H, Kumar R, Sridhar C N,et al. Enhanced gene delivery and mechanism studies with anovel series of cationic lipid formulations[J].J Biol Chem,1994,269:2550-2561.
55. Fenske D B, Maclachlan l，Cullis P R，et al. Long-circulating vecots for the systemic delivery ofgenes[J].Curr Opin Mol Ther,2000,3(2):153-158.
56. Fernandez-Pacheco R, Marquina C, Valdivia J G, et al. Magnetic nanoparticles for local drugdelivery using magnetic implants. In:6th international conference on the scientific and clinicalapplications of magnetic carriers[J].Elsevier Science B.V.,2006:318–322.
57. Ferrari M. Vectoring siRNA therapeutics into the clinic[J].Nat Rev Clin Oncol,2010,7:485–486.
58. Fischer Y H，Miletic H， Giroglou T，etal. A retroviral packaging cell line for pseudotype vectorsbased on glioma-infiltrating progenitor cells[J].J Gene Med,2007,9(5):335-344.
59. Fishlock T W, Oral A, Egdell R G, et al. Manipulation of atoms across a surface at roomtemperature[J].Nature,2000,404(6779):743-745.
60. Gao X H, Cui Y Y, Levenson R M, et al. In vivo cancer targeting and imaging with semiconductorquantum dots[J].Nat. Biotech,2004,22(8):969-976.
61. Gich M, Casas Li, Roig A, et al. High-coercivity ultralight transparent magnets[J]. Appl.Phys.Lett,2003,82(24):4307-4310.
62. Gilchrist R K, Medal R, Shorey W D, et al. Selective inductive heating of lymph nodes[J].Annalsof Oncology,1957,146(4):595-606.
63. Guerreiro C I, Fontes C M, Gama M, et al. Escherichia coli expression and purification of fourantimicrobial peptides fused to a family3carbohydrate-binding module (CBM) from Clostridiumthermocellum[J].Protein Expr Purif,2008,59(1):161-168.
64. Halpin C, Cooke S E, Barakate A, et al. Self-processing2A-polyproteins-a system for co-ordinateexpression of multiple protein in transgenic plants[J].The Plant Journal,1999,17(4):453–459.
65. Han M Y, Gao X H, Nie S M, et al. Quantum-dot-tagged microbeads for multiplexed opticalcoding of biomolecules[J].Nat. biotech,2001,19(7):631-635.
66. Harvey A J, Speksnijder G, Baugh L R, et al. Consistent production of transgenic chickens usingreplication-deficient retroviral vectors and high-throughput screening procedures[J].PoultrySci,2002,81(2):202-212.
67. Harvey A J, Speksnijder G, Baugh L R, et al.Expression of exogenous protein in the egg white oftransgenic chickens[J].Nature Biotechnology,2002,20:396-399.
68. Hasebe M, Okano K, Hattori M-A.The incorporation of exgenous DNA into chicken spermatozoa.Proceedings of the8th AAAP Animal science Cogress, Tokyo,1996:424-425.
69. Hasegawa K, Cowan A B, Nakatsuji N, et al. Efficient Multicistronic Expression of a Transgenein Human Embryonic Stem Cells[J].Stem Cells,2007,25:1707–1712.
70. Hashimoto M, Hisano Y. Directional gene-transfer into the brain by an adenoviral vector taggedwith magnetic nanoparticles[J].J. Neurosci. Methods.,2011,194(2):316–320.
71. Hawkins T. M13single-strand purification using a biotinylated probe and streptavidin coatedbeads[J].Mitochondrial DNA,1992,3(2):65-69.
72. He X X，Wang K M，Tan W H et al. Bioconjugated Nanoparticles for DNA Protection fromCleavage [J].J. A m.Chem.Soc,2003,125:7168-716.
73. Hernandez M, Rodriguez I, Pozo L, et al. Chimeric synthetic peptides from the nucleocapsid p24protein of human immunodeficiency virus type-1[J].Biochemical and Biophysical ResearchCommunication,2001,282(1):1-3.
74. Hofmann A, Wenzel D, Becher U M, et al. Combined targeting of lentiviral vectors and positioningof transduced cells by magnetic nanoparticles[J].PNAS,2009,106(1):44-49.
75. Hreczuk-Hirst D, Chicco D, German L, et al. Dextrins as potential carriers for drug targeting:tailored rates of dextrin degradation by introduction of pendant groups[J].Int. J. Pharm,2001,230(1-2):57-66.
76. Hu-Lieskovan S, Heidel J D, Bartlett D W, et al. Sequence-specific knockdown of ews-fli1bytargeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model ofmetastatic ewing's sarcoma[J].Cancer Res,2005,65:8984–8992.
77. Indrajit R, Tymish Y O, Dhruba J B, et al. Optical tracking of organically modified silicananoparticles as DNA carriers: A nonviral, nanomedicine approach for gene delivery[J]. Proc. Natl.Acad. Sci. USA,2005,102(2):279-284.
78. Ivanov V V, Zajats S V, Medvedev A I, et al. Formation of metal matrix composite by magneticpulsed compaction of partially oxidized Al nanopowder[J].J. Mater. Sci,2004,39(16-17):5231-5234.
79. Jackson D A, Juranek S, Lipps H J. Designing nonviral vectors for efficient gene transfer andlong-term gene expression[J].Mol. Ther.,2006,14(5):613–626.
80. James J S, Adam D L, Viswanadham G, et al. Homogeneous detection of unamplified genomicDNA sequences based on colorimetric scatter of gold nanoparticle probes[J]. Nat. biotech,2002,22(7):883-887.
81. Jeong J H, Lee M, Kim W J, et al. Anti-GAD antibody targeted non-viral gene delivery to islet betacells[J].J. Control. Release,2005,107(3):562–570.
82. Jeong J H，Park T G. Poly(L-lysine)-g-poly(D,L-lactic-co-glycolic acid) micelles for low cytotoxicbiodegradable gene delivery carriers[J].J Control Release,2002,82(1):159-166.
83. Joseph F, Poduslo, Thomas M. Wengenack,Geoffry L.Curran, et al.Molecular Targeting ofAlzheimer’s Amyloid Plaques for Contrast-Enhanced Magnetic Resonance Imaging[J]. Neurobi-ology of Disease,2002,11:315-329.
84. Junghans M, Kreuter J, Zimmer A. Phosphodiester and phosphorothioate oligonucleotidecondensation and preparation of antisense nanoparticles[J].Biochim Biophys Acta,2001,1544(1-2):177-188.
85. Junghans M，Kreuter J, Zimmer A. Antisense delivery using protamine-oligonucleotide particles[J].Nucleic Acids Res,2000,28(10):e45.
86. Kaleri H A, Xu S Y, Lin H L. Generation of transgenic chicks using an oviduct-specific expressionsystem[J].Genet. Mol. Res,2011,10(4):3046-3055.
87. Kamei K, Mukai Y, Kojima H, et al. Direct cell entry of gold/iron-oxide magnetic nanoparticles inadenovirus mediated gene delivery[J].Biomaterials,2009,30(9):1809-1814.
88. Kami D, Takeda S, Itakura Y, et al. Application of Magnetic Nanoparticles to Gene Delivery[J].Int.J. Mol. Sci.2011,12:3705-3722.
89. Kaneko Y, Mizuta Y, Nishijima Y, et al. Vickers hardness and deformation of Ni/Cu nano-multilayers electrodeposited on copper substrates[J].J. Mater. Sci,2005,40(12):3231-3236.
90. Kievit F M, Veiseh O, Bhattarai N, et al. PEI-PEG-Chitosan copolymer coated iron oxidenanoparticles for safe gene delivery: synthesis, complexation, and transfection[J]Adv. Funct. Mater,2009,19:2244-2251.
91. Kim TM, Park TS, Shin SSet al.An interclass nuclear transfer between fowl and mammal: in vitrodevelopment of chicken-to-cattle interclass embryos and the detection of chicken geneticComplements[J]. Fertil Steril,2004,82(4):957-959.
92. Kinoshita T, Seino S, Okitsu K, et al. Magnetic evaluation of nanostructure of gold-iron compositeparticles synthesized by a reverse micelle method[J].Journal of Alloys and Compounds,2003,359(1-2):46-50.
93. Kircheis R, Ostermann E, Wolschek M F,et al. Tumor-targeted gene delivery of tumor necrosisfactor-α induces tumor necrosis and tumor regression without systemic toxicity[J].Cancer GeneTher,2002,9:673–680.
94. Kircheis R, Wightman L, Schreiber A, et al. Polyethylenimine/DNA complexes shielded bytransferrin target gene expression to tumors after systemic application[J].Gene Ther,2001,8(1):28–40.
95. Kneuer C, Sameti M, Haltner E G, et al. Silica nanoParticles modified with aminosilanes ascarriers for plasmid DNA[J].International Journal of Pharmaceutics,2000,196(2):257-261
96. Kock N，Kasmieh R，Weissleder R，et al.Tumor therapy mediated by lentiviral expression ofshBcl-2and S-TRAIL[J].Neoplasia,2007,9(5):435-442.
97. Krotz F, de Wit C, Sohn H Y, et al. Magnetofection—a highly efficient tool for antisenseoligonucleotide delivery in vitro and in vivo[J].Mol. Ther,2003a(7):700-710.
98. Krotz F, Sohn H-Y, Glone T,et al. Magnetofection potentiates gene delivery to cultured endothelialcells[J].J. Vasc. Res,2003b,40(5):425-434.
99. Kuhara M, Takeyama H, Tanaka T, et. Al. Magnetic cell separation using antibody binding withprotein A expressed on bacterial magnetic particles [J].Anal Chem,2004,76(21):6207-6213.
100. Kuiken T, Rimmelzwaan G, van Riel D, et al. Avian H5N1influenza in cats[J].Science,2004,306:241.
101. Kumar M, Yigit M, Dai G, et al. Image-guided breast tumor therapy using a small interfering RNAnanodrug[J].Cancer Res,2010,70:7553–7561.
102. Kunath K, Merdan T, Hegener O, et al. Integrin targeting using RGD-PEI conjugates for in vitrogene transfer[J].J. Gene. Med,2003,5(7):588–599.
103. Lee R J, Huang L. Folate-targeted，anionic liposome-entrapped polylysine-condensed dna fortumor cell-specific gene transfer[J].The Journal of Biological Chemistry,1996,271:8481-8487.
104. Lehrer R I.Primate defensins[J]. Nature reviews Microbiology,2004,2:727-738.
105. Lehrman S. Virus treatment questioned after gene therapy death[J].Nature,1999,401(6753):517-518.
106. Leikina E, Delanoe-Ayari H, Melikov K, et al. Carbohydrate-binding molecules inhibit viral fusionand entry by crosslinking membrane glycoproteins[J].Nature immunology,2005,6:995-1001.
107. Lewin M, Carlesso N, Tung C H, et al. Tat peptide-derivatized magnetic nanoparticles allow invivo tracking and recovery of progenitor cells[J]. Nat.Biotech.2000,18(4):410-414.
108. Liang Q L, Zhou K, He H X. Retrocyclin2: a new therapy against avian influenza H5N1virus invivo and vitro[J].Biotechnology letters,2010,32:387-392.
109. Liang Z, Susha A, Caruso F.Gold nanoparticle-based core-shell and hollow spheres and orderedassemblies[J].Chem.Mater,2003,15(16):3176-3183.
110. Lin J，Zhou W, Kumbhar A, et al. Gold-coated iron nanoparticles: synthesis, characterization, andmagnetic field-induced self-assembly[J].Journal of Solid State Chemistry,2001,159(1):26-31.
111. Liu S Z, Zhou Z M, Chen T, et al. Blastocysts produced by nuclear transfer between chickenblastodermal cells and rabbit oocytes[J]. Mol Reprod Dev,2004,69(3):296-302.
112. Liu X, Wu J, Yammine M, et al. Structurally Flexible Triethanolamine Core PAMAM DendrimersAre Effective Nanovectors for DNA Transfection in Vitro and in Vivo to the Mouse Thymus[J].Bioconjugate Chem,2011,22(12):2461-2473.
113. Liu Y, Jia S, Wu Q, et al.Studies of Fe3O4-chitosan nanoparticles prepared by co-precipitationunder the magnetic field for lipase immobilization[J].Catalysis Communications,2011,12(8):717–720.
114. Liu Z L, Ding Z H, Yao K L,et al. Preparation and characterization of polymer-coated core-shellstructured magnetic microbeads[J].Magn Mater,2003,265:98-105.
115. Lorens J B, Pearsall D M, Swift S E, et al. Stable, stoichiometric delivery of diverse proteinfunctions[J].Journal of Biochemical Biophysical Methods,2004,58:101-110.
116. Love J, Gribbin C, Mather C,et al.Transgenic birds by DNA microinjection[J].Nature Biotechnology,1994,12(1):60-63.
117. Lungwitz U, Breunig M, Blunk T. Polyethylenimine-based nonviral gene delivery systems[J]. EurJ Pharm and Biopharm,2005,60:247-266.
118. Luo D,Saltzman W M.Synethetic DNA delivery systems[J].Nat Biotech,2000,18:33-37.
119. Luo D，Woodrow-Mumford K，Belcheva N，et al. Controlled DNA delivery systems[J]. PharmRes,1999,16:1300-1308.
120. Lyall J,Irvine R M,Sherman A,et al.Suppression of Avian Influenza Transmission in GeneticallyModified Chickens[J].Science,2011,331(6014):223-226.
121. Ma H, Diamond S L. Nonviral gene therapy and its deliverysystems [J]. Curr PharmBiotechn,2001,2(1):1-17.
122. Maillard S, Ameller T, Gauduchon J, et al. Innovative drug delivery nanosystems improve theanti-tumor activity in vitro and in vivo of anti-estrogens in human breast cancer and multiplemyeloma[J]. J. Steroid Biochem. Mol. Biol,2005,94(1-3):111-121.
123. Mari T, Masanori U, Noritada K, et al. Nanospheres for DNA separation chips[J].Nat. Biotech,2004,22(3):337-340.
124. Markert J M，Parker J N，Buchsbaum D J，etal. Oncolytic HSV-l for the treatment of braintumours[J].Herpes,2006,13(3):66-71.
125. Marumoto T,Tashiro A，Friedmann-Morvinski D，etal.Development of a novel mouse gliomamodel using lentiviral vectors[J].Nat Med,2009,15(1):110-116.
126. Mashall E. Gene therapy death prompts review of adenovirus vectors[J].Science,1999,286(5448):2244-2245.
127. McPhee J B, Hancock R E. Function and therapeutic potential of host defence peptides[J].Journalof Peptide Science,2005,11(11):677-687.
128. Medarova Z, Pham W, Farrar C, et al. In-vivo imaging of siRNA delivery and silencing intumors[J].Nat Med.2007,13:372–377.
129. Medintz I L, Konnert J H, Clapp A R, et al. A fluorescence resonance energy transfer-derivedstructure of a quantum dot-protein bioconjugate nanoassembly[J]. Proc. Natl. Acad. Sci.USA,2004,101(26):9612-9617.
130. Mizuguchi H, Xu Z, Ishii-Watabe A, et al. IRES-dependent second gene expression is significantlylower than cap-dependent first gene expression in a bicistronic vector[J].Molecular Therapy,2000,1(4):376–382.
131. Moghimi S M, Hunter A C, Murray J C. Long-circulating and target-specific nanoparticles: theoryto practices[J].Pharmacol Rev,2001,53(3):283-318.
132. Moore A, Marecos E, Bogdanov A, et al. Tumoral Distribution of Long-circulating Dextran-coatedIron Oxide Nanoparticles in a Rodent Model [J].Radioligy,2000,214:568-574.
133. Moralli D，Simpson K M，Wade-Martins R，etal. A novel human artificial Chromosome geneexpression system using herpes simplex virus typel vectors[J].EMBO Rep,2006,7(9):911-918.
134. Mozdziak P E, Borwornpinyo S, McCoy, D W，et al.Development of transgenic chickensexpressing bacterial β-galactosidase[J]. Dev.Dyn.,2003,226(3):439-445.
135. Müller R H, M der K, Gohla S. Solid Lipid Nanoparticles (SLN) for controlled drug delivery-areview of the state of the art[J].Eur. J. Pharm. Biopharm,2000,50(1):161-177.
136. Munk C, Wei G, Yang O O, et al. The theta-defensin, retrocyclin, inhibits HIV-1entry [J].AIDSresearch and human retroviruses,2003,19:875-881.
137. Naito M, Perry MM.Development in culture of the chick embryo from cleavage to hatch. Br PoultSci,1989,30(2):251-256.
138. Nguyena T X, Cole A M, Lehrer R I. Evolution of primate θ-defensins:a serpentine path to a sweettooth[J].Peptides,2003,(24):1647-1654.
139. Nicholson K G, Wood J M, Zambon M. Influenza[J].Lancet,2003,362:1733-1745.
140. Nicola O. The Antimicrobial Activity of Lactoferrin: Current Status and Perspectives[J].BioMetals,2004,(17):189-196.
141. Ogirs M，Wagner E. Targeting tumors with non-viral gene delivery systems[J].Drug DiscoveryToday,2002,7(8):479-485.
142. Ono T, Wakasugi N. Mineral content of quail embryos cultured in mineral-rich and mineral-freeconditions[J]. Poult Sci,1984,63(1):159-166.
143. Osborn M J, Panoskaltsis-Mortari A, McElmurry R T,et al. A picornaviral2A-like sequence-basedtricistronic vector allowing for high-level therapeutic gene expression coupled to a dual-reportersystem[J].Molecular Therapy,2005,12(3):569-574.
144. Owen S M, Rudolph D L, Wang W, et al. RC-101, a retrocyclin-1analogue with enhanced activityagainst primary HIV type1isolates[J]. AIDS research and human retroviruses,2004,20:1157-1165.
145. Pack D W, Hoffman A S, Pun S,et al. Design and development of polymers for gene delivery[J].Nat. Rev. Drug Discov,2005,4(7):581–593.
146. Panyam J, Zhou W Z, Prabha S, et al. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery[J].FASEB J,2002,16(10):1217-1226.
147. Park J W, Hong K, Kirpotin D B, et al. Anti-HER2immunoliposomes enhanced efficacyattributable to targeted delivery[J].Clin Cancer Res.2002,8:1172–1181.
148. Park T S, Han J Y.Derivation and characterization of pluripotent embryonic germ cells in chicken[J].Mol Reprod Dev,2000,56(4):475-482.
149. Pascal A, Franklin A H, Eric W, et al. Halogen bonds in biological molecules[J]. Proc. Natl. Acad.Sci. USA,2004,101(48):16789-16794.
150. Patil M L, Zhang M, Taratula O, et al.Internally Cationic Polyamidoamine PAMAM-OHDendrimers for siRNA Delivery: Effect of the Degree of Quaternization and Cancer Targeting[J].Biomacromolecules,2009,10(2):258–266.
151. Payungporn S, Chutinimitkul S, Chaisingh A, et al. Single step multiplex real-time RT-PCR forH5N1influenza A virus detection[J].Journal of Virological Methods,2006,131(2):143-147.
152. Peng X H, Qian X M, Mao H, et al. Targeted magnetic iron oxide nanoparticles for tumor imagingand therapy[J].Int J Nanomed,2008,3:311–321.
153. Perry MM.A complete culture system for the chick embryo[J]. Nature,1988,331(6151):70-72.
154. Piao Y， Jiang H，Alemany R， et al. Oncolytic adenovirus retargeted to Delta-EGFR inducesselective antiglioma activity[J].Cancer Gene Ther,2009,16(3):256-265.
155. Rabelo D, Lima E C D, Reis A C, et al. Preparation of magnetite nanoparticles in mesoporouscopolymer template[J].Nano Lett,2001,1(2):105-108.
156. Rhaese S, von Briesen H, Rübsamen-Waigmann H, et al. Human serum albumin-polyethyleniminenanoparticles for gene delivery [J].Control Release,2003,92(1-2):199-208.
157. Robert J C, Sarunya B, Katerina A D, et al. Noncovalent functionalization of carbon nanotubes forhighly specific electronic biosensors[J].Proc. Natl. Acad. Sci. USA,2003,100(9):4984-4989.
158. Roongsawang N, Promdonkoy P, Wongwanichpokhin M, et al. Coexpression of fungal phytaseand xylanase utilizing the cis-acting hydrolase element in Pichia pastoris[J].FEMS YeastResearch,2010,10:909–916.
159. Rowlett K, Simkiss K. Explanted embryo culture: in vitro and in ovo techniques for the domesticFowl[J].British Poultry Science,1987,78:91-101.
160. Roy K, Mao H Q, Huang S K, et al. Oral gene felivery with chitosan-DNA nanoparticles generatesimmunologic protection in a murine model of peanutallergy[J].Nature Med,1999,5(4):387-391.
161. Saccardo P, Villaverde A, González-Montalbán N. Peptide-mediated DNA condensation fornon-viral gene therapy[J]. Biotechnology Advances,2009,27(4)432–438.
162. Safarikova M.“use of Magnetic Techniques for the Isolation of Cells”[J]. J ChromatograhyB,1999,722:33-53.
163. Sauer A M, de Bruin K G，Plank C, et al. Localization and dynamics of magnetic transfectioncomplexes. Journal of Controlled Release.2008,132(3):e15-e16.
164. Schatzlein A G. Non-viral vectors in cancer gene therapy: principles and progress[J]. Anti-CancerDrugs,2001,12(4):275–304.
165. Schatzlein A G.Targeting of synthetic gene delivery systems[J].J. Biomed. Biotechnol,2003(2):149–158.
166. Scherer F, Anton M, Schillinger U, et al. Magnetofection: enhancing and targeting gene delivery bymagnetic force in vitro and in vivo[J].Gene Ther,2002,9:102-109.
167. Shi Y, Zhou L, Wang R, et al.In situ preparation of magnetic nonviral gene vectors andmagnetofection in vitro[J].Nanotechnology,2010,21:115-103.
168. Song H P，Yang J Y, Lo S L,et al. Gene transfer using self-assembled ternary complexes ofcationic magnetic nanoparticles, plasmid DNA and cell-penetrating Tat peptide[J]. Biomaterials,2010,31(4)769-778.
169. Sonti S V, Bose A. Cell separation using protein-A-coated magnetic nanclusters[J].J. Colliod.,Interface.Sci,1995,170(1):575-585.
170. Subbarao K, Klimov A, Katz J, et al. Characterization of an avian influenza A (H5N1) virusisolated from a child with a fatal respiratory illness[J].Science,1998,279:393-396.
171. Subramani K, Hosseinkhani H, Khraisat A, et al. Targeting nanoparticles as drug delivery systemsfor cancer treatment[J].Curr Nanosci,2009,5:135-140.
172. Sun M, Shi Q Q,Yin Y H, et al. Generation of antiviral transgenic chicken using spermatogonialstem cell transfected in vivo[J].African Journal of Biotechnology,2011,10(70):15678-15683.
173. Sun P, Zhao C, Li Y. Expression of green fluorescent protein gene in somatic chimeric chickensproduced by transplantation of transfected chicken embryonic fibroblasts[J]. J.Poult.Sci,2009,(46):363-369.
174. Szymczak A L, Workman C J, Wang Y, et al. Correction of multi-gene deficiency in vivo using asingle ‘self-cleaving’2A peptide-based retroviral vector[J].Nature Biotechnology,2004,22(5):589–594.
175. Tran D, Tran P A, Tang Y Q, et al. Selsted ME.Homodimeric theta-defensins from rhesus macaqueleukocytes:isolation, synthesis, antimicrobial activities, and bacterial binding properties of thecyclic peptides[J].J Biol Chem,2002,277:3079–3084.
176. Tresilwised N, Pithayanukul P, Mykhaylyk O,et al. Boosting oncolytic adenovirus potency withmagnetic nanoparticles and magnetic force[J].Mol.Pharmaceutics,2010,7(4):1069-1089.
177. Truong-Le V L，August J T, Leong K W. Controlled gene delivery by DNA-gelatin nanospheres[J].Hum GeneTher,1998,9:1709-1717.
178. Tsapis N, Bennett D, Jackson B, et al. Trojan particles: Large porous carriers of nanoparticles fordrug delivery[J].Proc.Natl.Acad.Sci.USA,2002,99(19):12001-12005.
179. Van Craynest N, Santaella C, Boussif O, et al. Polycationic telomers and cotelomers for genetransfer:synthesis and evaluation of their an vitro transfection efficiency[J]. Bioconjug Chem,2002,13(1):59-75.
180. Varadan V K, Chen L F, Xie J, et al. Nanomedicine: design and applications of magneticnanomaterials, nanosensors and nanosystems.Chichester, UK:Wiley,2008,467.
181. Verkman A S, Sonawane N D, Szoka F C.Chloride accumulation and swelling in endosomesenhances DNA transfer by polyamine–DNA polyplexes[J].J. Biol. Chem,2003,278(45):44826-44831.
182. Vijayanathan V, Thomas T, Thomas T J, et al. DNA nanopartlcles and development of DNAdelivery vehicles for gene therapy[J]. Biochem,2002,41(48):14085-14094.
183. Wadghiri Y Z, Sigurdsson E M, Sadowski M, et al.Detection of Alzheimer’s Amyloid inTransgenic Mice Using Magnetic Resonance Microimaging[J].Magnetic Resonance in Medicine,2003,50(2):293–302.
184. Waite C, Sparks S, Uhrich K, et al.Acetylation of PAMAM dendrimers for cellular delivery ofsiRNA[J].BMC Biotechnology,2009,9:38.
185. Wang J, Zhao G, Li X, et al.Reversible immobilization of glucoamylase onto magnetic chitosannanocarriers[J].Applied Microbiology and Biotechnology,2012,DOI:10.1007/s00253-012-3979-2.
186. Wang S, Yao Q, Tao J, et al. Co-ordinate expression of glycine betaine synthesis genes linked bythe FMDV2A region in a single open reading frame in Pichia pastoris[J]. Applied Genetics andMolecular Biotechnology2007,77:891–899.
187. Webby R J, Webster R G. Are we ready for pandemic influenza?[J] Science,2003,302:1519-1522.
188. Weiss S I, Sieverling N, Niclasen M,et al. Uronic acids functionalized polyethy-leneimine(PEI)-polyethyleneglycol (PEG)-graft-copolymers as novel synthetic gene carriers[J]. Biomaterials,2006,27(10):2302–2312.
189. Wu Q, Moyana T, Xiang J. Cancer gene therapy by adenovirus-mediated gene transfer[J]. CurrGene Ther,2001,(1):101-122.
190. Xiang J J, Tang J Q, Zhu S G, et al. IONP-PLL: a novel non-viral vector for efficient geneDelivery[J].J Gene Med,2003,5:803–817.
191. Yang S Y, Sun J S, Liu C H, et al. Ex vivo magnetofection with magnetic nanoparticles: a novelplatform for nonviral tissue engineering[J]. Artificial Organs,2008,32(3):195-204.
192. Yang Y, Xu Z, Jiang J, et al. Poly(imidazole/DMAEA) phosphazene/DNA self-assemblednanoparticles for gene delivery: Synthesis and in vitro transfection[J].J Control Release,2008,127(3):273-279.
193. Yanson A I, Bollinger G R, Brom H E. Formation and manipulation of a metallic wire of singlegold atoms[J].Nature,1998,395(6700):783-785.
194. Yasin B, Wang W, Pang M, et al. Theta defensins protect cells from infection by herpes simplexvirus by inhibiting viral adhesion and entry[J].Journal of virology,2004,78:5147-5156.
195. Yigit M V, Moore A, Medarova Z.Magnetic nanoparticles for cancer diagnosis and therapy[J].pharmaceutical research,2012,DOI10.1007/s11095-012-0679-7.
196. Yin W K, Feng S S. Effects of particle size and surface coating on cellular uptake of polymericnanoparticles for oral delivery of anticancer drugs[J].Biomaterials,2005,26(15):2713-2722.
197. Zhang H, Lee M Y, Hogg M G,et al.Gene delivery in three-dimensional cell cultures bysuperparamagnetic nanoparticles[J].ACS Nano,2010,4(8):4733–4743.
198. Zhang X, Oulad-Abdelghani M, Zelkin A N, et al. Poly(l-lysine) nanostructured particles for genedelivery and hormone stimulation[J].Biomaterials,2010,31(7):1699–1706.
199. Zhao P，Wang C, Fu Z，et al. Lentiviral vector mediated siRNA knock-down of hTERT results indiminished capacity in invasiveness and in vivo growth of human glioma cells in a telomerelength-independent manner [J].Int J Oncol,2007,31(2):361-368.
200. Zhao X J, Hilliard L R, Mechery S J, et al. From the Cover: A rapid bioassay for single bacterialcell quantitation using bioconjugated nanoparticles[J].Proc. Natl.Acad. Sci. USA,2004,101(42):15027-15032.
201. Ziady A G, Ferkol T， Gerken T, et al. Ligand substitution of receptor targeted DNA complexesaffects gene transfer into hepatoma cells[J].Gene therapy,1998,5:1685-1697.