新型纳米基因转运体与新型生物荧光纳米颗粒的研制
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摘要
无论是在基因表达、功能与调节等基础研究方面，还是在疾病的治疗、预防，如基因治疗、DNA疫苗等临床应用方面，DNA传递始终是一个强有力的普遍应用工具，而且也是其中的核心技术。DNA传递的效率直接影响到其基础研究与临床应用的成效。传统的DNA传递系统分为病毒载体介导系统和非病毒载体介导系统。病毒载体介导系统是迄今为止最有效的DNA传递工具，其传递效率通常高达90％以上，最近临床基因治疗中75％应用病毒载体介导系统。尽管如此，由于该系统存在的自身局限性，如病毒毒性与免疫原性，有限的DNA装载量，载体组装难度大、花费高等，其应用受到很大限制。非病毒载体介导系统虽无病毒毒性和免疫原性等缺陷，但其传递效率一直不如病毒载体介导系统，如何提高非病毒载体介导系统的传递效率于是成为瓶颈问题。近来，随着纳米生物技术的飞跃发展，以纳米颗粒为基础的非病毒纳米基因传递载体系统的出现有望突破这一瓶颈，实现安全、高效的DNA传递，为基因表达与功能研究及其临床应用创造最佳条件，提供最好工具。
在先前的研究中，Luo D & Saltzman WM使用生物相容性的硅纳米颗粒进行基因转移，尽管它们自身不能传递DNA，但它们可以通过增加DNA—转染试剂复合物到达细胞表面的物理浓度而使最好的商品化转染试剂的基因转染效率增加8.5倍以上。这一研究结果提示，硅纳米颗粒如果用适当的阳离子聚合物，如多聚赖氨酸，改性修饰之
朱诗国博士学位论文 新型纳米基因转运体与新型主物荧光纳米颗粒的矾’制
后有可能获得有效的质粒DNA和反义ODN传递能力。
为此，本研究首先运用OP10／环己烷／氨水微乳液体系，以正硅
酸乙酯为原料，成功研制了一批不同粒径的硅纳米颗粒，并通过正交
试验分析了该体系中各组份对硅纳米颗粒尺寸的影响。结果表明在
OPJ／环己烷／氨水微乳液体系中，水与 OP、 的摩尔比以及水与 TEOS
的摩尔在控制 S i NP尺寸方面起抉定性作用，其次为氨水浓度，且随着
其摩尔比的减小和氨水浓度的下降 SmP的粒径趋于最小。DNA结合滞
留试验表明，在正常生理条件下 S i NP不能结合质粒 DNA。而随后的修
饰研究发现，小粒径的硅纳米颗粒（约20 urn）表面羟基被活化后，它
可与多聚赖氨酸结合形成形态规则、分布均匀的PMSWP，而大粒径的
硅纳米颗粒则不能被修饰或修饰效果较差。这提示多聚赖氨酸对硅纳
米颗粒的修饰与颗粒表面效能和Zeta电位密切相关，这种直接利用颗
粒的表面效能和表面电位进行纳米颗粒改性修饰的方法，避免了复杂
的化学反应和有毒化学物质的涉入，是一种新型的纳米颗粒改性修饰
方法。进一步的细胞毒性研究表明，我们所制备的PMS十P细胞毒性极
低，可能是一种良好的新型基因传递载体。
继而根据正交分析结果，按照最佳的合成方案合成了一批粒径小，
分布更窄，形态更规则的硅纳米颗粒。在此基础上利用PLL进行改性
修饰，制备出良好修饰的PMS—NP。DNA凝胶滞留研究和沉淀试验
表明，随着PMS—NP与质粒DNA结合比例的增加，可逐渐出现的
DNA滞留条带，上清液中 DNA浓度逐渐减少；达到 3 0：1后MA被
完全滞留，上清液中检测不到质粒DNA。而且 PMS—NP与质粒DNA的
12
朱诗国博十学位论义 新型纳爪基旧转运休与新型生物荧光纳米顺粒的汕划
结合在40 Inin后达到平衡。进一步的DNasel消化试验和细胞转染研
究发现，PMS—NP与质粒DNA结合后不仅能有效的保护DNA不被核酶
降解，而且能有效的将质粒DNA转移到细胞内，产生高水平的报道基
因表达。这些研究结果提示，PMS—NP是一新型的非病毒纳米颗粒基
因转移载体，在基因结构与功能研究、基因治疗等领域具有广阔的应
用前景。
反义ILNA是与特异基因互补配对的单链合成DNA序列。它通过
Watson（rick硷基互补配对与靶基因的前mRNA或mRNA杂合，这
种杂合既可直接阻断mMA的翻译，又可以RNA－DNA杂交复合物的
形式激活 RNase H介导的 RNA特异性降解，从而在 mRNA和蛋白水
平抑制基因表达。反义RNA作为肿瘤和感染性疾病的潜在治疗试剂己
经在世界上众多实验室得到了验证，因而在感染性疾病和恶性肿瘤基
因治疗中具有重要的战略意义。但是，为使反义技术广泛应用，有一
个关键性的问题—一极低的的细胞渗透能力，不得不很好的解决。为
增加 ODN的细胞内传递，避兔非特异性反应，颗粒载体，如脂质体或
纳米颗粒的应用可能是解决ODN传递问题的最好方法。在药物载体
中，生物可降解的或不可降解的纳米颗粒以一种有效的行为显示出结
合和传递ODN的巨大潜能。甚至在某些‘倩况下，由于纳米颗粒良好的
稳定性，它们显得比脂质体更为合适。
既然PMS—NP能够传递质粒DNA，自然使人想到它或许也是良
好的反义传递载体。为此，本研究通过反义ODN结合和血浆蛋白切割
实验研究其反义0＊N结合和保护能力：然后利用＊＊C标记的0＊N，
13
朱
DNA delivery has become a powerful popular research tool for elucidating gene structure, regulation, and function, as well as treating and controlling diseases, such as gene therapy and DNA vaccination. The efficiency of DNA delivery directly affects its application in basic research and clinical setting. Traditionally, DNA delivery systems have been classified as viral vector-mediated systems and nonviral vector-mediated systems. Viral systems are by far the most effective means of DNA delivery, achieving high efficiencies (usually > 90%) for both delivery and expression. In fact, around 75% of recent clinical protocols involving gene therapy use recombinant viral-based vectors for DNA delivery. As yet, however no definitive evidence has been presented for the clinical effectiveness of any gene therapy protocol. The impotence of current methodology is attributable to the limitation of viral-mediated delivery, including toxicity, restricted targeting of specific cell line, limited DNA carrying capacity, production and packaging problems, recombination, and high cost. Furthermore, the toxicity and immunogenicity of viral systems also hamper their routine use. For these reasons, nonviral systems have become increasingly desirable in basic research and clinical settings. But nonviral delivery efficiency is always less than viral systems. With the rapid development of nanobiotechnology, nanoparticles-based
nonviral-mediated systems will help, to alleviate the gene delivery bottle-neck, and achieve the ability to safe, efficient, and targeted DNA delivery, and provide the best means for gene structure,, function research, as well as clinical settings.
In the previous study, Luo D & Saltzman WM used biocompatible silica nanoparticles, which by themselves do not deliver DNA, to concentrate DNA-transfection reagent complexes at the surface of cell monolayers and enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. Their results suggested if silica nanoparticles were modified with polycations, such as poly-1-lysine, they maybe achieve the ability to efficient deliver DNA and antisense ODN.
In the current research, silica nanoparticles (SiNP) were synthesized firstly in a microemulsion system polyoxyethylene nonylphenyl ether (OP-10)/ cyclohexane/ ammonium hydroxide, at the same time the effects of SiNP size and its distribution were elucidated by orthogonal analysis; then poly-1-lysine (PLL) was linked on the surface of SiNP by nanoparticle surface energy and electrostatically binding; lastly a novel complex nanomaterial-poly-1-lysine-midified silica nanoparticles (PMS-NP) was prepared. The analysis of plasmid DNA binding and DNase I enzymatic degradation discovered that PMS-NP could bind DNA, and protect it against enzymatic degradation. Cell transfection showed PMS-NP could
efficiently transfer pEGFP-C2 or pSV-p-gal plasmid DNA into HNE1 or Hela cell line. These results indicated PMS-NP is a novel nanoparticulate carrier for plasmid DNA delivery, and would probably play an important role in gene structure and function research as well as gene therapy.
Antisense oligonucleotides are single-stranded synthetic DNA sequences designed to be complementary to a specific gene. Hybridization with target pro-mRNA or mRNA through Watson-Crick base-pairing can block sterically the translation of this transcript, or activate RNase H-mediated degradation of the RNA strand in RNA-DNA heteroduplexes and subsequently inhibit gene expression at both mRNA and protein levels. Antisense as a potential therapeutic agent against neoplastic and infectious diseases has been tested in numerous research laboratories around the world. However, one of the crucial problems to broad application of antisense technology, the low intracellular penetration, has to be solved. In order to increase ODN intracellular delivery, to avoid non-specific aptameric effects, the use of particulate carriers such as liposomes or nanoparticles, may be considered as being the more realistic
在先前的研究中，Luo D & Saltzman WM使用生物相容性的硅纳米颗粒进行基因转移，尽管它们自身不能传递DNA，但它们可以通过增加DNA—转染试剂复合物到达细胞表面的物理浓度而使最好的商品化转染试剂的基因转染效率增加8.5倍以上。这一研究结果提示，硅纳米颗粒如果用适当的阳离子聚合物，如多聚赖氨酸，改性修饰之
朱诗国博士学位论文 新型纳米基因转运体与新型主物荧光纳米颗粒的矾’制
后有可能获得有效的质粒DNA和反义ODN传递能力。
为此，本研究首先运用OP10／环己烷／氨水微乳液体系，以正硅
酸乙酯为原料，成功研制了一批不同粒径的硅纳米颗粒，并通过正交
试验分析了该体系中各组份对硅纳米颗粒尺寸的影响。结果表明在
OPJ／环己烷／氨水微乳液体系中，水与 OP、 的摩尔比以及水与 TEOS
的摩尔在控制 S i NP尺寸方面起抉定性作用，其次为氨水浓度，且随着
其摩尔比的减小和氨水浓度的下降 SmP的粒径趋于最小。DNA结合滞
留试验表明，在正常生理条件下 S i NP不能结合质粒 DNA。而随后的修
饰研究发现，小粒径的硅纳米颗粒（约20 urn）表面羟基被活化后，它
可与多聚赖氨酸结合形成形态规则、分布均匀的PMSWP，而大粒径的
硅纳米颗粒则不能被修饰或修饰效果较差。这提示多聚赖氨酸对硅纳
米颗粒的修饰与颗粒表面效能和Zeta电位密切相关，这种直接利用颗
粒的表面效能和表面电位进行纳米颗粒改性修饰的方法，避免了复杂
的化学反应和有毒化学物质的涉入，是一种新型的纳米颗粒改性修饰
方法。进一步的细胞毒性研究表明，我们所制备的PMS十P细胞毒性极
低，可能是一种良好的新型基因传递载体。
继而根据正交分析结果，按照最佳的合成方案合成了一批粒径小，
分布更窄，形态更规则的硅纳米颗粒。在此基础上利用PLL进行改性
修饰，制备出良好修饰的PMS—NP。DNA凝胶滞留研究和沉淀试验
表明，随着PMS—NP与质粒DNA结合比例的增加，可逐渐出现的
DNA滞留条带，上清液中 DNA浓度逐渐减少；达到 3 0：1后MA被
完全滞留，上清液中检测不到质粒DNA。而且 PMS—NP与质粒DNA的
12
朱诗国博十学位论义 新型纳爪基旧转运休与新型生物荧光纳米顺粒的汕划
结合在40 Inin后达到平衡。进一步的DNasel消化试验和细胞转染研
究发现，PMS—NP与质粒DNA结合后不仅能有效的保护DNA不被核酶
降解，而且能有效的将质粒DNA转移到细胞内，产生高水平的报道基
因表达。这些研究结果提示，PMS—NP是一新型的非病毒纳米颗粒基
因转移载体，在基因结构与功能研究、基因治疗等领域具有广阔的应
用前景。
反义ILNA是与特异基因互补配对的单链合成DNA序列。它通过
Watson（rick硷基互补配对与靶基因的前mRNA或mRNA杂合，这
种杂合既可直接阻断mMA的翻译，又可以RNA－DNA杂交复合物的
形式激活 RNase H介导的 RNA特异性降解，从而在 mRNA和蛋白水
平抑制基因表达。反义RNA作为肿瘤和感染性疾病的潜在治疗试剂己
经在世界上众多实验室得到了验证，因而在感染性疾病和恶性肿瘤基
因治疗中具有重要的战略意义。但是，为使反义技术广泛应用，有一
个关键性的问题—一极低的的细胞渗透能力，不得不很好的解决。为
增加 ODN的细胞内传递，避兔非特异性反应，颗粒载体，如脂质体或
纳米颗粒的应用可能是解决ODN传递问题的最好方法。在药物载体
中，生物可降解的或不可降解的纳米颗粒以一种有效的行为显示出结
合和传递ODN的巨大潜能。甚至在某些‘倩况下，由于纳米颗粒良好的
稳定性，它们显得比脂质体更为合适。
既然PMS—NP能够传递质粒DNA，自然使人想到它或许也是良
好的反义传递载体。为此，本研究通过反义ODN结合和血浆蛋白切割
实验研究其反义0＊N结合和保护能力：然后利用＊＊C标记的0＊N，
13
朱
DNA delivery has become a powerful popular research tool for elucidating gene structure, regulation, and function, as well as treating and controlling diseases, such as gene therapy and DNA vaccination. The efficiency of DNA delivery directly affects its application in basic research and clinical setting. Traditionally, DNA delivery systems have been classified as viral vector-mediated systems and nonviral vector-mediated systems. Viral systems are by far the most effective means of DNA delivery, achieving high efficiencies (usually > 90%) for both delivery and expression. In fact, around 75% of recent clinical protocols involving gene therapy use recombinant viral-based vectors for DNA delivery. As yet, however no definitive evidence has been presented for the clinical effectiveness of any gene therapy protocol. The impotence of current methodology is attributable to the limitation of viral-mediated delivery, including toxicity, restricted targeting of specific cell line, limited DNA carrying capacity, production and packaging problems, recombination, and high cost. Furthermore, the toxicity and immunogenicity of viral systems also hamper their routine use. For these reasons, nonviral systems have become increasingly desirable in basic research and clinical settings. But nonviral delivery efficiency is always less than viral systems. With the rapid development of nanobiotechnology, nanoparticles-based
nonviral-mediated systems will help, to alleviate the gene delivery bottle-neck, and achieve the ability to safe, efficient, and targeted DNA delivery, and provide the best means for gene structure,, function research, as well as clinical settings.
In the previous study, Luo D & Saltzman WM used biocompatible silica nanoparticles, which by themselves do not deliver DNA, to concentrate DNA-transfection reagent complexes at the surface of cell monolayers and enhanced transfection efficiency by up to 8.5-fold over the best commercially available transfection reagents. Their results suggested if silica nanoparticles were modified with polycations, such as poly-1-lysine, they maybe achieve the ability to efficient deliver DNA and antisense ODN.
In the current research, silica nanoparticles (SiNP) were synthesized firstly in a microemulsion system polyoxyethylene nonylphenyl ether (OP-10)/ cyclohexane/ ammonium hydroxide, at the same time the effects of SiNP size and its distribution were elucidated by orthogonal analysis; then poly-1-lysine (PLL) was linked on the surface of SiNP by nanoparticle surface energy and electrostatically binding; lastly a novel complex nanomaterial-poly-1-lysine-midified silica nanoparticles (PMS-NP) was prepared. The analysis of plasmid DNA binding and DNase I enzymatic degradation discovered that PMS-NP could bind DNA, and protect it against enzymatic degradation. Cell transfection showed PMS-NP could
efficiently transfer pEGFP-C2 or pSV-p-gal plasmid DNA into HNE1 or Hela cell line. These results indicated PMS-NP is a novel nanoparticulate carrier for plasmid DNA delivery, and would probably play an important role in gene structure and function research as well as gene therapy.
Antisense oligonucleotides are single-stranded synthetic DNA sequences designed to be complementary to a specific gene. Hybridization with target pro-mRNA or mRNA through Watson-Crick base-pairing can block sterically the translation of this transcript, or activate RNase H-mediated degradation of the RNA strand in RNA-DNA heteroduplexes and subsequently inhibit gene expression at both mRNA and protein levels. Antisense as a potential therapeutic agent against neoplastic and infectious diseases has been tested in numerous research laboratories around the world. However, one of the crucial problems to broad application of antisense technology, the low intracellular penetration, has to be solved. In order to increase ODN intracellular delivery, to avoid non-specific aptameric effects, the use of particulate carriers such as liposomes or nanoparticles, may be considered as being the more realistic
引文
1．曾益新。肿瘤学。北京：人民卫生出版社，1999。
2. Anderson WF. Human gene therapy. Nature, 1998; 392:25-30.
3. Schatzlein AG. Non-viral vectors in cancer gene therapy: principles and progress. Anti-Cancer Drugs, 2001; 12: 275-304.
4. Luo D & Saltzman WM. Synethetic DNA delivery systems. Nat Biotech, 2000;18:33—36.
5. Fox JL. Gene therapy safety issues come to fore. Nat Biotech, 1999; 17:1153.
6. Hui KM, Chia TF. Eradication of tumor growth via biolistic transformation with allogeneic MHC genes. Gene Ther, 1997; 4: 762-767.
7. Sawarnura D, Ina S, Itai K, et al. In vivo gene introduction into keratinocytes using jet injection. Gene Ther, 1999; 6:1785-1787.
8. Oshima Y, Sakamoto T, Yamanaka I, et al. Targeted gene transfer to comeal endothelium in vivo by electric pulse. Gene Ther, 1998; 5: 1347-1354.
9. Lauer U, Burgelt E, Squire Z, et al. Shock wave permeabilization as a new gene transfer method. Gene Ther, 1997; 4: 710-715.
10. Fenske DB, Maclachlan I, Cullis PR, et al. Long-circulating vecots for the systemic delivery of genes. Curr Opin Mol Ther, 2000; 3(2): 153-158.
11．杜仕国，施冬梅，韩其文。纳米颗粒的液相合成技术。粉末冶金技术，2000；18(1)：46-50。
12. Davis SS. Biomedical application of nanotechnology—implications for drug targeting and gene therapy. Trends in Biotech, 1997;15:217-224.
13. Lambert G, Fattal E, Couvreur P. Nanoparticulate systems for the delivery of antisense oligonucleotides. Adv Drugs Deliv Rev, 2001; 47:99-112.
14. Langer K, Coester C, Weber C, et al. Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid. Eur J Pharm Biopharm, 2000; 49(3):303-307.
15. Moghimi SM, Hunter AC, Murray JC. Long-circulating and target-specific nanoparticles: theory to practices. Pharmacol Rev, 2001; 53:283-318.
16. Moghimi SM, Porter CJH, Muir IS, et al. Non-phagocytic uptake of intravenously injected microspheres in rat spleen: influence of particle size and hydrophilic coating. Biochem Biophys Res Commun, 1991; 177: 861-866.
17. H.Maeda J, Wu T, Sawa Y, et al. Tumor vascular permeability and the EPR effect in macromolecular therapeutic: a review. J Control Release, 2000; 65: 271-284.
18. Vijayanathan V, Thomas T, Thomas TJ. DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry 2002; 41(48): 14085-94.
19. Prabha S, Zhou WZ, Panyam J, et al. Size-dependency ofnanoparticle-mediated gene transfection: studies with fractionated nanoparticles. Int J Pharm 2002; 244(1-2):105-15.
20. Panyam J, Zhou WZ, Prabha S, et al. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB J 2002; 16(10):1217-26.
21. Fattal E, Vauthier C, Aynie I, et al. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J Control Release 1998; 53(1-3):137-43.
22. Maksimenko AV, Mandrouguine V, Gottikh MB, et al. Optimisation of dendrimer-mediated gene transfer by anionic oligomers. J Gene Med 2003; 5(1):61-71.
23. Oh YK, Suh D, Kim JM, et al. Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther 2002 Dec; 9(23): 1627-32.
24. Singla AK, Chawla M. Chitosan: some pharmaceutical and biological aspects-an update. J Pharm Pharmacol 2001; 53(8): 1047-67.
25．向娟娟，朱诗国，吕红斌，等。用氧化铁磁性纳米颗粒作为基因载体的研究。癌症，2001；20(10)：1-6。
26. Scherer F, Anton M, Schillinger U, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther 2002;
9(2): 102-9.
27. Roy I, Mitra S, Maitra A, et al. Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery. Int J Pharm 2003; 250(1):25-33.
28. Luo D, Saltzman NM. Enhancement of transfection by physical concentration of DNA at the surface. Nat Biotech, 2000; 18: 893-895.
29. Lacoste,T.D; Michalet,X; Pinaud,F; C hemla,D.S, Alivisatos,A.P; Weiss,S. Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc Natl Acad Sci USA, 2000; 97(17): 9461.
30. Uppenbrink,J; Clery, D. Single molecules. Science, 1999; 283: 1667.
31. Moerner, W.E; Orrit,M. Illuminating single molecules in condensed matter. Science, 1999; 283: 1670.
32. Weiss,S. Fluorescence spectroscopy of single biomolecules. Science, 1999; 283: 1676.
33. Taylor JR,Fang MM,Nie S.Probing specific sequence on single DNA molecules with bioconjugated fluorescent nanoparticles.Anal Chem,2000;72(9): 1979-1986.
34. Tiefenauer LX,Tschirky A,Kkne G. et al. Invivo evaluationof magnetic nanoparticles for use as a tumor contrast agent in MRI.Magn Reson Imaging, 1996; 14:391-394.
35. Lewin M, Carlesso N, Tung CH. et al. Tat peptide-derivatizied magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotech, 2000; 18:410-414.
36. Davis SS. Biomedical applications of nanotechnology -implications for drug targeting and gene therapy. Trends Biotechnol, 1997; 15:217-224.
37. Lambert G, Fattal E, Pinto-Alphandary H. et al. Polyisobutylcyanocrylate nanoparticles containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotide. Pharm Res,2000; 17(6):707-714.
38. Kneuer C, Sameti M, Haltner EG. et al. Silica nanoparticles modified with minosilanes ascarrier for plasmid DNA.Int J Pharm,2000;196(2):257-261.
39．瞿其曙，何友昭，淦五二等。超细二氧化硅的制备及研究进展。硅酸盐通报，
2000; 5: 57-63。
40. Arriagada FJ,Osseo-Arare K. Synthesis of nanosize silica in a nonionic water-oil microemulsion: effects of the water /surfactant molar ratio and ammonia concentration. J Colloid Interface Sci, 1999;211:210-220.
41．徐相凌，殷亚东，葛学武等。微乳液聚合研究进展。高等学校化学学报，1999；20(3)：478-481。
42. Kneuer C, Sameti M, Bakowsky V. et al. A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro.Bioconjug Chem, 2000; 11 (6): 926-932.
43. Wolfert MA, Seymour LW. Atomic force microscopic ananlysis of the influence of the molecular weight of poly(L)lysine on the size of polyelectrolyte complexes formed with DNA. Gene Ther, 1996; 3: 269-273.
44. Toncheva V, Wolfert MA, Dash PR, et al. Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers. Biochim Biophys acta, 1998; 1380: 354-368.
45．朱诗国，贺达仁，李桂源。纳米医学的圣殿。医学与哲学，2001：22(7)：41-43。
46. Haensler J.et al.Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug Chem, 1993;4:372-379.
47. Lambert G.et al. Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides.pharm Res.2000;17:707-714.
48. Maruyama A.et al. Nanoparticle DNA carrier with poly(L-lysine) grafted polysaccharide copolymer and poly(D.L-lacticacid). Bioconjug Chem, 1997; 8: 735-742.
49．余鹰，李桂源。CGAP及鼻咽癌相关研究进展。国外医学肿瘤学分册，2000；27(4)：233-235。
50. Fox JL. Gene therapy safety issues come to fore. Nat Biotech, 1999; 17:1153.
51. Schatzlein AG. Non-viral vectors in cancer gene therapy: principles and progress. Anti-Cancer Drugs, 2001; 12: 275-304.
52. Wu G, Wu C. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem, 1987; 262: 4429-432.
53. Cotton M, et al. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfecte DNA or modulate transferrin receptor levels. Proc Natl Acad Sci USA, 1990; 87: 4033-4037. J Biol Chem, 1993; 268: 11265-11271.
54. Chowdhury NR, et al. Fate of DNA targeted to liver by asialoglycoprotein receptor-mediated endocytosis in vivo: prolonged persistence in cytoplasmic vesicles after partial hepatectomy. J Biol Chem,1993; 268:11265-11271.
55. Cristiano RJ, Roth JA. Epidermal growth factor mediated DNA delivery into lung cancer cells via the Epidermal growth factor receptor. Cancer Gene Ther, 1996; 3: 4-10.
56. Zauner W, Ogris M, Wagner E. Polylysine-based transfection sysems utilizing receptor-mediated delivery. Adv Drug Del Rev, 1998;30:97-113.
57. Lechardeur D, Lukacs G. Intracellular barriers to non-viral gene transfer. Current Gene Ther, 2002; 2(2): 195-206.
58. C.A. Stein, Y.C. Cheng. Antisense oligonucleotides as therapeutic ageats-is the bullet really magical?, Science, 261 (1993) 1004-1012.
59. S.T. Crooke. Progress toward oligonucleuotides therapeutics: pharmacodynamic properties, FASEB J, 7 (1993) 533-539.
60. J.L. Mergny, G. Duval-Valentin, G. Nguyen, L. Perouault, B. Faucon, M. Rougee, T. Montenay-Garestier, E. Bisagni, C. Helene. Triple-helix specific ligands, Science 256 (1992) 1681-1684.
61. R.W. Wagner. Gene inhibition using antisense oligodeoxynucleotides, Nature 372 (1994) 333-335.
62. L.A. Yakubov, E.A. Deeva, V.F. Zarytova, E.M. Ivanova, A.S. Ryte, L.V. Yurchenko, V.V. Vlassov. Mechanism of oligonucleotide uptake by cells: Involvement of specific receptors, Proc. Natl. Acad. Sci. USA 86 (1989) 6454-6458.
63. R.E. Clark. Poor cellular uptake of antisense oligodeoxynucleotides: an obstacle
to their use in chronic myeloid leukaemia, Leuk. Lymphoma 19 (1995) 189-195.
64. Q. Hu, C.R. Shew, M.B. Bally, T.D. Madden. Programmable fusogenic vesicles for intracellular delivery of antisense oligodeoxynucleotides: enhanced cellular uptake and biological effects, Biochim. Biophys. Acta 1514 (2001) 1-13.
65. P. Rockwell, W.J. O'Connor, K. King, N.I. Goldstein, L.M. Zhang, C.A. Stein. Cell-surface perturbations of the epidermal growth factor and vascular endothelial grow factor receptors by phosphorothioate oligodeoxynucleotides, Proc. Natl. Acad. Sci. USA 94 (1997) 6523-6528.
66. G. Lambert, E. Fattal, P. Couvreur. Nanoparticulate systems for the delivery of antisense oligonucleotides, Adv. Drug. Deliv. Rev 47 (2001 ) 99-112.
67. E. Fattal, C. Vauthier, I. Aynie, Y. Nakada, G. Lambert, C. Malvy, P. Couvreur. Biodegradable polyalkylcyanoacrylate nanopaticles for the delivery of oligonucleotides, J. Controlled Release 53 (1998) 137-143.
68. E. Fattal, J. Rojas, M. Youssef, P. Couvreur, A. Andremont. Liposome-entrapped ampicillin in the treatment of experimental murine listeriosis and salmonellosis, Antimicrob. Agents. Chemother 35 (1991) 770-772.
69. S.G. Zhu, H.B. Lu, J.J. Xiang, K. Tang, B.C. Zhang, M. Zhou, G.Y. Li. A novel nonviral nanoparticle gene vector: poly-L-lysine silica nanoparticles. Chin Sci Bull. 47(2002) 654-657.
70. R. Heikkila, G. Scheab, E. Wickstrom, S.L. Loke, D.H. Pluznik, R. Watt, L.M. Neckers. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G_0 to G_1, Nature 328 (1987) 445-449.
71. G. Citro, I. D' Agnano, C. Leonetti, R. Perini, B. Bucci, G. Zon, B. Calabretta, G. Zupi. C-myc antisense oligodeoxynucleotide enhance the efficacy of cisplatin in melanoma chemotherapy in vitro and in nude mice, Cancer Res 58 (1998) 283-289
72. C. Leonetti, I. D' Agnano, F. Lozupone, A. Valentini, T. Geiser, G. Zon, B. Calabretta, G. Citro, G. Zupi. Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotide on human melanoma cells in vitro and in vivo, J. Natl. Cancer. Inst 88 (1996) 419-429.
73. E. Wickstrom. Antisense c-myc inhibition of lymphoma growth, Antisense Nucleic Acid Drug Der 7 (1997) 225-228.
74. T. Skorski, D. Perroti, M. Nieborowska-Skorska, S. Gryaznov, B. Calabretta. Antileukemia effect of c-myc N3'-P5' phosphoramidate antisense oligonucleotides in vivo, Proc. Natl. Acad. Sci. USA 94 (1997) 3966-3971.
75. G. Schwab, C. Chavany, I. Duroux, G. Goubin, J. Lebeau, C. Helene. Antisense oligonucleotides absorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice. Proc. Natl. Acad. Sci. USA 91 (1994) 10460-10464.
76. A.E. Gad, B.L. Silver, G.D. Eytan. Polycation-induced fusion of negatively-charged vesicles, Biochim. Biophy. Acta 690 (1982) 124-132.
77. C.Y. Wang, L. Huang. Polyhistidine mediates an acid-dependent fusion of negatively charged liposomes, Biochemistry 23 (1984) 4409-4416.
78. A. Alama, F. Barbieri, M. Cagnoli, G. Schettini. Antisense oligonucleotides as therapeutic agents, Pharm. Res 36 (1997) 171-178.
79. Y. Lavrovsky, S. Chen, A.K. Roy. Therapeutic potential and mechanism of action of oligonucleotides and ribozymes, Biochem. Mol. Med 62 (1997) 11-22.
80. R.Y. Walder, ,J.A. Walder. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 85 (1988) 5011-5015.
81. R.V Giles, D.G. Spiller, DM. Tidd. 1995. Dection of ribonuclease H-generated mRNA fragments in human leukemia cells following reversible membrane permeabilization in the presence of antisense oligonucleotides. Antisense Res Dev. 5:23-31.
82. G.J Veal, S. Agrawal, R.A. Byrn. 1998. Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotides in vitro. Nucleic Acids Res. 26: 5670-5675.
83. M. Manoharan. Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense Nucleic Acid Drug Dev. 12 (2002) 103-128.
84. Moghimi SM, Hunter AC, Murray JC. Long-circulating and target-specific
nanoparticles: theory to practices. Pharmacol Rev, 2001; 53:283-318.
85. Moghimi SM, Porter CJH, Muir IS, et al. Non-phagocytic uptake of intravenously injected microspheres in rat spleen: influence of particle size and hydrophilic coating. Biochem Biophys Res Commun, 1991; 177:861-866.
86. H.Maeda J, Wu T, Sawa Y, et al. Tumor vascular permeability and the EPR effect in macromolecular therapeutic: a review. J Control Release, 2000; 65: 271-284.
87．朱诗国，吕红斌，向娟娟，等。一种新型的非病毒DNA传递载体——多聚赖氨酸-硅纳米颗粒。科学通报，2002：47(3)：193-197。
88. Fenske DB, Maclachlan I, Cullis PR, et al. Long-circulating vecots fbr the systemic delivery of genes. Curr Opin Mol Ther, 2000; 3 (2): 153-158.
89. Kakizawa Y, Kataoka K. Block copolymer micelles for delivery of gene and related compounds. Adv Drugs Deliv Rev, 2002; 54: 203-222.
90. Chonn A, Semple SC, Cullis PR. Association of blood proteins with large unilamellar liposomes in vivo. Relation to circulation lifetime. J Biol Chem, 1992; 267:18759-18765.
91. chughart K, Bischoff R, Rasmussen UB, et al. Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery. Hmn Gene Ther 1999; 10(18): 2891-905.
92. Schughart K, Rasmussen UB. Solvoplex synthetic vector for intrapulmonary gene delivery. Preparation and use. Methods Mol Med 2002; 69:83-94.
93. Pillai R, Petrak K, Blezinger P, et al. Ultrasonic nebulization of cationic lipid-based gene delivery systems for airway administration. Pharm Res 1998; 15(11): 1743-7.
94. Mathiowitz E, Jacob JS, Jong YS, et al. Biologically erodable microspheres as potential oral drug delivery systems. Nature 1997; 386(6623): 410-4.
95. Fooks AR. Development of oral vaccines for human use. Curr Opin Mol Ther 2000; 2(1): 80-6.
96. Page DT, Cudmore S. Innovations in oral gene delivery: challenges and potentials. Drug Discov Today 2001; 6(2): 92-101.
97. Niedzinski EJ, Bennett MJ, Olson DC, et al. Gastroprotection of DNA with a
synthetic cholic acid analog. Lipids 2000; 35(7): 721-7.
98. Jones DH, Clegg JC, Farrar GH. Oral delivery of micro-encapsulated DNA vaccines. Dev Biol Stand 1998; 92:149-55.
99. Paglia P, Terrazzini N, Schulze K, et al. In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene Ther 2000; 7(20): 1725-30.
100. Roy K, Mao HQ, Huang SK, et al. Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nat Med 1999; 5(4): 387-91.
101. Jones DH, Corris S, McDonald S, et al. Poly (DL-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine 1997; 15(8): 814-7.
102. Prokop A, Kozlov E, Newman GW, et al. Water-based nanoparticulate polymeric system for protein delivery: permeability control and vaccine application. Biotechnol Bioeng 2002; 78(4): 459-66.
103. Lemieux P, Vinogradov SV, Gebhart CL, et al. Block and graft copolymers and NanoGel copolymer networks for DNA delivery into cell. J Drug Target 2000; 8(2): 91-105.
104. Gherardi MM, Esteban M. Mucosal and systemic immune responses induced after oral delivery of vaccinia virus recombinants. Vaccine 1999; 17(9-10): 1074-83.
105. Chen SC, Jones DH, Fynan EF, et al. Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles. J Virol 1998; 72(7): 5757-61.
106. Bruchez,M.Jr; Moronne,M; Gin,P; Weiss,S; Alivisatos,A.P. Semiconductor Nanocrystals as Fluorescent Biological Labels. Science, 1998; 281, 2013.
107. Adler, J; Jayan,A; Melia, C.D. A method for quantifying differential expansion within hydrating hydrophilic matrixes by tracking embedded fluorescent microspheres. J Pharm Sci. 1999; 88(3): 371.
108. Santra, S; Zhang,P; Wang,K; Tapec,R; Tan, W. Conjugation of biornolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal Chem. 2001; 73(20):4988.
109. Lambert, G; Fattal,E; Pinto-Alphandary, H; Gulik,A; Couvreur, P. Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides. Pharm Res.2000; 17:707.
110. Watt,R.M; Voss,E.W. Jr. Solvent perturbation of the fluorescence of fluorescein bound to specific antibody. Fluorescence quenching of the bound fluorophore by iodide. J Biol Chem. 1979; 254(5):1684.
111. Arriagada,F.J; Osseo-Arare,K. Synthesis of nanosize silica in a nonionic water-oil microemulsion: effects of the water/surfactant molar ratio and ammonia concentration. J Colloid Interface Sci, 1999; 211: 210.
2. Anderson WF. Human gene therapy. Nature, 1998; 392:25-30.
3. Schatzlein AG. Non-viral vectors in cancer gene therapy: principles and progress. Anti-Cancer Drugs, 2001; 12: 275-304.
4. Luo D & Saltzman WM. Synethetic DNA delivery systems. Nat Biotech, 2000;18:33—36.
5. Fox JL. Gene therapy safety issues come to fore. Nat Biotech, 1999; 17:1153.
6. Hui KM, Chia TF. Eradication of tumor growth via biolistic transformation with allogeneic MHC genes. Gene Ther, 1997; 4: 762-767.
7. Sawarnura D, Ina S, Itai K, et al. In vivo gene introduction into keratinocytes using jet injection. Gene Ther, 1999; 6:1785-1787.
8. Oshima Y, Sakamoto T, Yamanaka I, et al. Targeted gene transfer to comeal endothelium in vivo by electric pulse. Gene Ther, 1998; 5: 1347-1354.
9. Lauer U, Burgelt E, Squire Z, et al. Shock wave permeabilization as a new gene transfer method. Gene Ther, 1997; 4: 710-715.
10. Fenske DB, Maclachlan I, Cullis PR, et al. Long-circulating vecots for the systemic delivery of genes. Curr Opin Mol Ther, 2000; 3(2): 153-158.
11．杜仕国，施冬梅，韩其文。纳米颗粒的液相合成技术。粉末冶金技术，2000；18(1)：46-50。
12. Davis SS. Biomedical application of nanotechnology—implications for drug targeting and gene therapy. Trends in Biotech, 1997;15:217-224.
13. Lambert G, Fattal E, Couvreur P. Nanoparticulate systems for the delivery of antisense oligonucleotides. Adv Drugs Deliv Rev, 2001; 47:99-112.
14. Langer K, Coester C, Weber C, et al. Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid. Eur J Pharm Biopharm, 2000; 49(3):303-307.
15. Moghimi SM, Hunter AC, Murray JC. Long-circulating and target-specific nanoparticles: theory to practices. Pharmacol Rev, 2001; 53:283-318.
16. Moghimi SM, Porter CJH, Muir IS, et al. Non-phagocytic uptake of intravenously injected microspheres in rat spleen: influence of particle size and hydrophilic coating. Biochem Biophys Res Commun, 1991; 177: 861-866.
17. H.Maeda J, Wu T, Sawa Y, et al. Tumor vascular permeability and the EPR effect in macromolecular therapeutic: a review. J Control Release, 2000; 65: 271-284.
18. Vijayanathan V, Thomas T, Thomas TJ. DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry 2002; 41(48): 14085-94.
19. Prabha S, Zhou WZ, Panyam J, et al. Size-dependency ofnanoparticle-mediated gene transfection: studies with fractionated nanoparticles. Int J Pharm 2002; 244(1-2):105-15.
20. Panyam J, Zhou WZ, Prabha S, et al. Rapid endo-lysosomal escape of poly(DL-lactide-co-glycolide) nanoparticles: implications for drug and gene delivery. FASEB J 2002; 16(10):1217-26.
21. Fattal E, Vauthier C, Aynie I, et al. Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides. J Control Release 1998; 53(1-3):137-43.
22. Maksimenko AV, Mandrouguine V, Gottikh MB, et al. Optimisation of dendrimer-mediated gene transfer by anionic oligomers. J Gene Med 2003; 5(1):61-71.
23. Oh YK, Suh D, Kim JM, et al. Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA. Gene Ther 2002 Dec; 9(23): 1627-32.
24. Singla AK, Chawla M. Chitosan: some pharmaceutical and biological aspects-an update. J Pharm Pharmacol 2001; 53(8): 1047-67.
25．向娟娟，朱诗国，吕红斌，等。用氧化铁磁性纳米颗粒作为基因载体的研究。癌症，2001；20(10)：1-6。
26. Scherer F, Anton M, Schillinger U, et al. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Ther 2002;
9(2): 102-9.
27. Roy I, Mitra S, Maitra A, et al. Calcium phosphate nanoparticles as novel non-viral vectors for targeted gene delivery. Int J Pharm 2003; 250(1):25-33.
28. Luo D, Saltzman NM. Enhancement of transfection by physical concentration of DNA at the surface. Nat Biotech, 2000; 18: 893-895.
29. Lacoste,T.D; Michalet,X; Pinaud,F; C hemla,D.S, Alivisatos,A.P; Weiss,S. Ultrahigh-resolution multicolor colocalization of single fluorescent probes. Proc Natl Acad Sci USA, 2000; 97(17): 9461.
30. Uppenbrink,J; Clery, D. Single molecules. Science, 1999; 283: 1667.
31. Moerner, W.E; Orrit,M. Illuminating single molecules in condensed matter. Science, 1999; 283: 1670.
32. Weiss,S. Fluorescence spectroscopy of single biomolecules. Science, 1999; 283: 1676.
33. Taylor JR,Fang MM,Nie S.Probing specific sequence on single DNA molecules with bioconjugated fluorescent nanoparticles.Anal Chem,2000;72(9): 1979-1986.
34. Tiefenauer LX,Tschirky A,Kkne G. et al. Invivo evaluationof magnetic nanoparticles for use as a tumor contrast agent in MRI.Magn Reson Imaging, 1996; 14:391-394.
35. Lewin M, Carlesso N, Tung CH. et al. Tat peptide-derivatizied magnetic nanoparticles allow in vivo tracking and recovery of progenitor cells. Nat Biotech, 2000; 18:410-414.
36. Davis SS. Biomedical applications of nanotechnology -implications for drug targeting and gene therapy. Trends Biotechnol, 1997; 15:217-224.
37. Lambert G, Fattal E, Pinto-Alphandary H. et al. Polyisobutylcyanocrylate nanoparticles containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotide. Pharm Res,2000; 17(6):707-714.
38. Kneuer C, Sameti M, Haltner EG. et al. Silica nanoparticles modified with minosilanes ascarrier for plasmid DNA.Int J Pharm,2000;196(2):257-261.
39．瞿其曙，何友昭，淦五二等。超细二氧化硅的制备及研究进展。硅酸盐通报，
2000; 5: 57-63。
40. Arriagada FJ,Osseo-Arare K. Synthesis of nanosize silica in a nonionic water-oil microemulsion: effects of the water /surfactant molar ratio and ammonia concentration. J Colloid Interface Sci, 1999;211:210-220.
41．徐相凌，殷亚东，葛学武等。微乳液聚合研究进展。高等学校化学学报，1999；20(3)：478-481。
42. Kneuer C, Sameti M, Bakowsky V. et al. A nonviral DNA delivery system based on surface modified silica-nanoparticles can efficiently transfect cells in vitro.Bioconjug Chem, 2000; 11 (6): 926-932.
43. Wolfert MA, Seymour LW. Atomic force microscopic ananlysis of the influence of the molecular weight of poly(L)lysine on the size of polyelectrolyte complexes formed with DNA. Gene Ther, 1996; 3: 269-273.
44. Toncheva V, Wolfert MA, Dash PR, et al. Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers. Biochim Biophys acta, 1998; 1380: 354-368.
45．朱诗国，贺达仁，李桂源。纳米医学的圣殿。医学与哲学，2001：22(7)：41-43。
46. Haensler J.et al.Polyamidoamine cascade polymers mediate efficient transfection of cells in culture. Bioconjug Chem, 1993;4:372-379.
47. Lambert G.et al. Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides.pharm Res.2000;17:707-714.
48. Maruyama A.et al. Nanoparticle DNA carrier with poly(L-lysine) grafted polysaccharide copolymer and poly(D.L-lacticacid). Bioconjug Chem, 1997; 8: 735-742.
49．余鹰，李桂源。CGAP及鼻咽癌相关研究进展。国外医学肿瘤学分册，2000；27(4)：233-235。
50. Fox JL. Gene therapy safety issues come to fore. Nat Biotech, 1999; 17:1153.
51. Schatzlein AG. Non-viral vectors in cancer gene therapy: principles and progress. Anti-Cancer Drugs, 2001; 12: 275-304.
52. Wu G, Wu C. Receptor-mediated in vitro gene transformation by a soluble DNA carrier system. J Biol Chem, 1987; 262: 4429-432.
53. Cotton M, et al. Transferrin-polycation-mediated introduction of DNA into human leukemic cells: stimulation by agents that affect the survival of transfecte DNA or modulate transferrin receptor levels. Proc Natl Acad Sci USA, 1990; 87: 4033-4037. J Biol Chem, 1993; 268: 11265-11271.
54. Chowdhury NR, et al. Fate of DNA targeted to liver by asialoglycoprotein receptor-mediated endocytosis in vivo: prolonged persistence in cytoplasmic vesicles after partial hepatectomy. J Biol Chem,1993; 268:11265-11271.
55. Cristiano RJ, Roth JA. Epidermal growth factor mediated DNA delivery into lung cancer cells via the Epidermal growth factor receptor. Cancer Gene Ther, 1996; 3: 4-10.
56. Zauner W, Ogris M, Wagner E. Polylysine-based transfection sysems utilizing receptor-mediated delivery. Adv Drug Del Rev, 1998;30:97-113.
57. Lechardeur D, Lukacs G. Intracellular barriers to non-viral gene transfer. Current Gene Ther, 2002; 2(2): 195-206.
58. C.A. Stein, Y.C. Cheng. Antisense oligonucleotides as therapeutic ageats-is the bullet really magical?, Science, 261 (1993) 1004-1012.
59. S.T. Crooke. Progress toward oligonucleuotides therapeutics: pharmacodynamic properties, FASEB J, 7 (1993) 533-539.
60. J.L. Mergny, G. Duval-Valentin, G. Nguyen, L. Perouault, B. Faucon, M. Rougee, T. Montenay-Garestier, E. Bisagni, C. Helene. Triple-helix specific ligands, Science 256 (1992) 1681-1684.
61. R.W. Wagner. Gene inhibition using antisense oligodeoxynucleotides, Nature 372 (1994) 333-335.
62. L.A. Yakubov, E.A. Deeva, V.F. Zarytova, E.M. Ivanova, A.S. Ryte, L.V. Yurchenko, V.V. Vlassov. Mechanism of oligonucleotide uptake by cells: Involvement of specific receptors, Proc. Natl. Acad. Sci. USA 86 (1989) 6454-6458.
63. R.E. Clark. Poor cellular uptake of antisense oligodeoxynucleotides: an obstacle
to their use in chronic myeloid leukaemia, Leuk. Lymphoma 19 (1995) 189-195.
64. Q. Hu, C.R. Shew, M.B. Bally, T.D. Madden. Programmable fusogenic vesicles for intracellular delivery of antisense oligodeoxynucleotides: enhanced cellular uptake and biological effects, Biochim. Biophys. Acta 1514 (2001) 1-13.
65. P. Rockwell, W.J. O'Connor, K. King, N.I. Goldstein, L.M. Zhang, C.A. Stein. Cell-surface perturbations of the epidermal growth factor and vascular endothelial grow factor receptors by phosphorothioate oligodeoxynucleotides, Proc. Natl. Acad. Sci. USA 94 (1997) 6523-6528.
66. G. Lambert, E. Fattal, P. Couvreur. Nanoparticulate systems for the delivery of antisense oligonucleotides, Adv. Drug. Deliv. Rev 47 (2001 ) 99-112.
67. E. Fattal, C. Vauthier, I. Aynie, Y. Nakada, G. Lambert, C. Malvy, P. Couvreur. Biodegradable polyalkylcyanoacrylate nanopaticles for the delivery of oligonucleotides, J. Controlled Release 53 (1998) 137-143.
68. E. Fattal, J. Rojas, M. Youssef, P. Couvreur, A. Andremont. Liposome-entrapped ampicillin in the treatment of experimental murine listeriosis and salmonellosis, Antimicrob. Agents. Chemother 35 (1991) 770-772.
69. S.G. Zhu, H.B. Lu, J.J. Xiang, K. Tang, B.C. Zhang, M. Zhou, G.Y. Li. A novel nonviral nanoparticle gene vector: poly-L-lysine silica nanoparticles. Chin Sci Bull. 47(2002) 654-657.
70. R. Heikkila, G. Scheab, E. Wickstrom, S.L. Loke, D.H. Pluznik, R. Watt, L.M. Neckers. A c-myc antisense oligodeoxynucleotide inhibits entry into S phase but not progress from G_0 to G_1, Nature 328 (1987) 445-449.
71. G. Citro, I. D' Agnano, C. Leonetti, R. Perini, B. Bucci, G. Zon, B. Calabretta, G. Zupi. C-myc antisense oligodeoxynucleotide enhance the efficacy of cisplatin in melanoma chemotherapy in vitro and in nude mice, Cancer Res 58 (1998) 283-289
72. C. Leonetti, I. D' Agnano, F. Lozupone, A. Valentini, T. Geiser, G. Zon, B. Calabretta, G. Citro, G. Zupi. Antitumor effect of c-myc antisense phosphorothioate oligodeoxynucleotide on human melanoma cells in vitro and in vivo, J. Natl. Cancer. Inst 88 (1996) 419-429.
73. E. Wickstrom. Antisense c-myc inhibition of lymphoma growth, Antisense Nucleic Acid Drug Der 7 (1997) 225-228.
74. T. Skorski, D. Perroti, M. Nieborowska-Skorska, S. Gryaznov, B. Calabretta. Antileukemia effect of c-myc N3'-P5' phosphoramidate antisense oligonucleotides in vivo, Proc. Natl. Acad. Sci. USA 94 (1997) 3966-3971.
75. G. Schwab, C. Chavany, I. Duroux, G. Goubin, J. Lebeau, C. Helene. Antisense oligonucleotides absorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice. Proc. Natl. Acad. Sci. USA 91 (1994) 10460-10464.
76. A.E. Gad, B.L. Silver, G.D. Eytan. Polycation-induced fusion of negatively-charged vesicles, Biochim. Biophy. Acta 690 (1982) 124-132.
77. C.Y. Wang, L. Huang. Polyhistidine mediates an acid-dependent fusion of negatively charged liposomes, Biochemistry 23 (1984) 4409-4416.
78. A. Alama, F. Barbieri, M. Cagnoli, G. Schettini. Antisense oligonucleotides as therapeutic agents, Pharm. Res 36 (1997) 171-178.
79. Y. Lavrovsky, S. Chen, A.K. Roy. Therapeutic potential and mechanism of action of oligonucleotides and ribozymes, Biochem. Mol. Med 62 (1997) 11-22.
80. R.Y. Walder, ,J.A. Walder. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 85 (1988) 5011-5015.
81. R.V Giles, D.G. Spiller, DM. Tidd. 1995. Dection of ribonuclease H-generated mRNA fragments in human leukemia cells following reversible membrane permeabilization in the presence of antisense oligonucleotides. Antisense Res Dev. 5:23-31.
82. G.J Veal, S. Agrawal, R.A. Byrn. 1998. Sequence-specific RNase H cleavage of gag mRNA from HIV-1 infected cells by an antisense oligonucleotides in vitro. Nucleic Acids Res. 26: 5670-5675.
83. M. Manoharan. Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action. Antisense Nucleic Acid Drug Dev. 12 (2002) 103-128.
84. Moghimi SM, Hunter AC, Murray JC. Long-circulating and target-specific
nanoparticles: theory to practices. Pharmacol Rev, 2001; 53:283-318.
85. Moghimi SM, Porter CJH, Muir IS, et al. Non-phagocytic uptake of intravenously injected microspheres in rat spleen: influence of particle size and hydrophilic coating. Biochem Biophys Res Commun, 1991; 177:861-866.
86. H.Maeda J, Wu T, Sawa Y, et al. Tumor vascular permeability and the EPR effect in macromolecular therapeutic: a review. J Control Release, 2000; 65: 271-284.
87．朱诗国，吕红斌，向娟娟，等。一种新型的非病毒DNA传递载体——多聚赖氨酸-硅纳米颗粒。科学通报，2002：47(3)：193-197。
88. Fenske DB, Maclachlan I, Cullis PR, et al. Long-circulating vecots fbr the systemic delivery of genes. Curr Opin Mol Ther, 2000; 3 (2): 153-158.
89. Kakizawa Y, Kataoka K. Block copolymer micelles for delivery of gene and related compounds. Adv Drugs Deliv Rev, 2002; 54: 203-222.
90. Chonn A, Semple SC, Cullis PR. Association of blood proteins with large unilamellar liposomes in vivo. Relation to circulation lifetime. J Biol Chem, 1992; 267:18759-18765.
91. chughart K, Bischoff R, Rasmussen UB, et al. Solvoplex: a new type of synthetic vector for intrapulmonary gene delivery. Hmn Gene Ther 1999; 10(18): 2891-905.
92. Schughart K, Rasmussen UB. Solvoplex synthetic vector for intrapulmonary gene delivery. Preparation and use. Methods Mol Med 2002; 69:83-94.
93. Pillai R, Petrak K, Blezinger P, et al. Ultrasonic nebulization of cationic lipid-based gene delivery systems for airway administration. Pharm Res 1998; 15(11): 1743-7.
94. Mathiowitz E, Jacob JS, Jong YS, et al. Biologically erodable microspheres as potential oral drug delivery systems. Nature 1997; 386(6623): 410-4.
95. Fooks AR. Development of oral vaccines for human use. Curr Opin Mol Ther 2000; 2(1): 80-6.
96. Page DT, Cudmore S. Innovations in oral gene delivery: challenges and potentials. Drug Discov Today 2001; 6(2): 92-101.
97. Niedzinski EJ, Bennett MJ, Olson DC, et al. Gastroprotection of DNA with a
synthetic cholic acid analog. Lipids 2000; 35(7): 721-7.
98. Jones DH, Clegg JC, Farrar GH. Oral delivery of micro-encapsulated DNA vaccines. Dev Biol Stand 1998; 92:149-55.
99. Paglia P, Terrazzini N, Schulze K, et al. In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene Ther 2000; 7(20): 1725-30.
100. Roy K, Mao HQ, Huang SK, et al. Oral gene delivery with chitosan-DNA nanoparticles generates immunologic protection in a murine model of peanut allergy. Nat Med 1999; 5(4): 387-91.
101. Jones DH, Corris S, McDonald S, et al. Poly (DL-lactide-co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine 1997; 15(8): 814-7.
102. Prokop A, Kozlov E, Newman GW, et al. Water-based nanoparticulate polymeric system for protein delivery: permeability control and vaccine application. Biotechnol Bioeng 2002; 78(4): 459-66.
103. Lemieux P, Vinogradov SV, Gebhart CL, et al. Block and graft copolymers and NanoGel copolymer networks for DNA delivery into cell. J Drug Target 2000; 8(2): 91-105.
104. Gherardi MM, Esteban M. Mucosal and systemic immune responses induced after oral delivery of vaccinia virus recombinants. Vaccine 1999; 17(9-10): 1074-83.
105. Chen SC, Jones DH, Fynan EF, et al. Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles. J Virol 1998; 72(7): 5757-61.
106. Bruchez,M.Jr; Moronne,M; Gin,P; Weiss,S; Alivisatos,A.P. Semiconductor Nanocrystals as Fluorescent Biological Labels. Science, 1998; 281, 2013.
107. Adler, J; Jayan,A; Melia, C.D. A method for quantifying differential expansion within hydrating hydrophilic matrixes by tracking embedded fluorescent microspheres. J Pharm Sci. 1999; 88(3): 371.
108. Santra, S; Zhang,P; Wang,K; Tapec,R; Tan, W. Conjugation of biornolecules with luminophore-doped silica nanoparticles for photostable biomarkers. Anal Chem. 2001; 73(20):4988.
109. Lambert, G; Fattal,E; Pinto-Alphandary, H; Gulik,A; Couvreur, P. Polyisobutylcyanoacrylate nanocapsules containing an aqueous core as a novel colloidal carrier for the delivery of oligonucleotides. Pharm Res.2000; 17:707.
110. Watt,R.M; Voss,E.W. Jr. Solvent perturbation of the fluorescence of fluorescein bound to specific antibody. Fluorescence quenching of the bound fluorophore by iodide. J Biol Chem. 1979; 254(5):1684.
111. Arriagada,F.J; Osseo-Arare,K. Synthesis of nanosize silica in a nonionic water-oil microemulsion: effects of the water/surfactant molar ratio and ammonia concentration. J Colloid Interface Sci, 1999; 211: 210.