中文摘要
基因治療的重要關鍵在於是否有一個有效的遞送系統能將具有功能性的基因送至細胞中表現，目前所使用的遞送系統，主要分為兩大類：病毒性載體與非病毒性載體。病毒性載體其遞送效率佳，但是對宿主所造成的毒性以及免疫反應的緣故，有安全上的顧慮，因此致力於非病毒性載體的研究。非病毒性載體具有使用簡單、容易大量製備、不易引起宿主免疫反應的特點，但是其遞送效率差，是目前所面臨到的最大瓶頸。目前已有數種非病毒性載體的遞送系統，其中以微脂體作為載體的遞送效率最好，又以正電荷微脂體最具有潛力，並且已廣泛應用在體外實驗中。但是微脂體的遞送效率仍是有限，而在文獻中有報導指出，利用陽離子聚合物可將DNA纏繞成一緻密結構，再與微脂體所形成之複合體其粒徑比DNA/liposome複合體小，利於細胞的內吞作用，並且會保護DNA避免受到酵素分解，增加DNA到核內表現的機會，提升遞送效率。
本論文主要研究利用不同的陽離子聚合物與正電荷微脂體形成不同劑型的複合體，來提高正電荷微脂體遞送基因至人類肝癌細胞的效率。在體外細胞轉染實驗中，利用陽離子脂質與輔助性脂質以適當比例製成正電荷微脂體，再添加魚精蛋白硫酸胺、聚-L-離胺酸、PEI等陽離子聚合物，以螢光基因（luciferase、EGFP）為報導基因來分析細胞轉染效率。在HepG2細胞中，與DNA/liposome複合體比較，DNA/protamine sulfate/liposome複合體轉染效率提高1.3倍，DNA/poly-L-lysine/liposome複合體提高1.5倍，DNA/PEI/liposome（LED）複合體則提高8.5倍；而在HepG2-HBV細胞中，DNA/protamine sulfate/liposome複合體提高1.4倍，DNA/poly-L-lysine複合體提高1.6倍，LED複合體則提高7倍。再者，以流式細胞儀分析EGFP此報導基因細胞轉染的效率，並用螢光顯微鏡觀察EGFP的表現，可以得知LED複合體遞送基因能力最高。因此利用此最佳劑型的微脂體來遞送反義去氧寡核醣核酸，經由西方墨點法觀察蛋白質改變情形。由實驗結果得知，所遞送的反義去氧寡核醣核酸能夠有效的抑制基因的表現，即使經過藥物作用將基因活化，其基因仍持續被抑制。由此可以知道所發展的遞送系統，不論是遞送質體DNA或是小片段DNA分子，都能有效率的將基因遞送至細胞中表現。
本實驗是利用陽離子聚合物所形成不同劑型的微脂體，增進正電荷微脂體基因遞送的效率，以得到最佳基因遞送系統，能夠有效的遞送完好的基因到細胞中表現或是遞送反義去氧寡核醣核酸抑制基因的表現，達到基因治療的目的。此發展對非病毒性載體有很大的貢獻，期許能夠進一步應用在動物活體的研究。

Abstract
A major aim of gene therapy is the efficient and specific delivery of therapeutic gene into the desired target tissues. At the present time, major gene delivery systems employ either viral or non-viral vectors. Viral vectors can mediate efficient gene transfer. However, there are drawbacks related to the viral elements such as immune and toxic reactions, and the potential for recombination with wild-type virus and insertional mutagenesis. Non-viral vectors, despite their relatively low efficiency, are less immunogenic than viral vectors and ease of manipulation, making them attractive gene delivery systems. Recent reports suggest that cationic liposome are potent non-viral vectors and have been widely used for gene delivery in vitro. In attempt to improve biological activity of the lipoplexes, condensing DNA with polycations prior to its mixture with cationic liposomes was shown to be advantageous. The addition of polycations, such as protamine sulfate、poly-L-lysine and PEI, were showen to condense DNA, to reduce the particle size of the complexes, to render DNA resistant to nuclease activity and to enhance transfection efficiency.
The aim of this study is to develop an effective non-viral gene transfer system by the combination of cationic liposomes and polycations, and increase gene transfer efficiency in human hepatocellular carcinoma cell lines. We used cationic lipid, DOTAP, in the presence of cholesterol as helper lipid. The polycations/cationic liposomes complexes (polyplexes) were used to be vectors and the pCMVLuc, pEGFP as reproter genes. In HepG2 cells, compare with DNA/liposomes complexes (lipoplexes), the protamine sulfate polyplex increased the transfection activity about 1.3-fold, the poly-L-lysine polyplex increased about 1.5-fold, and the LED (DNA/PEI/liposome) polyplex increased up to 8.5-fold. And in the HepG2-HBV cells, the protamine sulfate polyplex increased the transfection activity about 1.4-fold, the poly-L-lysine polyplex increased about 1.6-fold, and the LED polyplex increased up to 7-fold. In addition, the different liposomal formulations were futher characterized by the FAScan analysis and fluorescent microscopy. These data indicated that the LED polyplex is a better formulation in vitro, so we used this liposomal formulation to delivery antisense oligodeoxynucledtides and observed the protein expression by Western bolt analysis. It suggests that the gene expression was inhibited by antisense oligodeoxynucledtides and reduced the protein expression level. The inhibition effect was shown even in treating with anticancer drugs to activate gene expression.
The experimental results demonstrated that the polycations can enhance transfection efficiency, and we will use this delivery system to deliver wild type tumor suppressor gene into a cancer or to deliver antisense molecules to down-regulate the gene expression, achieved the successful gene therapy. The efficiency of this non-viral delivery system in vivo remains to be studied.

目錄
中文摘要……………………………………………………………………1
英文摘要……………………………………………………………………3
縮寫…………………………………………………………………………5
第一章 緒論………………………………………………………………6
1.1 基因治療……………………………………………………….6
1.2 微脂體…………………………………………………………14
1.3 陽離子脂質……………………………………………………24
1.4 魚精蛋白………………………………………………………28
1.5 報導基因………………………………………………………28
1.6 流式細胞儀……………………………………………………29
1.7 唐黴素…………………………………………………………30
1.8 研究動機與目的………………………………………………32
第二章 實驗材料……………………………………………………….33
第三章 實驗方法……………………………………………………….34
3.1 DNA結合能力分析………………………………………………....34
3.2 基因遞送之試驗…………………………………………………...34
3.3 不同劑型正電荷微脂體基因載體之製備………………………...38
3.4 反義去氧寡核醣核酸……………………………………………...41
第四章 實驗結果……………………………………………………….44
4.1 DNA/polycations/cationic liposome複合體對細胞轉染的影響44
4.2 反義去氧寡核醣核酸之遞送對於基因表現的影響……………...48
第五章 討論………………………………………………………………51
第六章 參考文獻…………………………………………………………57
圖表與說明……………………………………………………………….67

參考文獻
1. Anderson WF. Prospects for human gene therapy. Science 226, 401-409, 1984.
2. Felgner PL and Rhodes G. Gene therapeutics. Nature 349, 351-352, 1991.
3. Kukowska-Latallo JF, Raczka E, Quintana A, Chen C, Rymaszewski M, and Baker JR, Jr. Intravascular and endobronchial DNA delivery to murine lung tissue using a novel, nonviral vector. Hum. Gene Ther. 11, 1385-1395, 2000.
4. Li S. and Huang L. Nonviral gene therapy: promises and challenges. Gene Ther. 7, 31-34, 2000.
5. Eglitis MA and Anderson WF. Retroviral vectors for introduction of genes into mammalian cells. Biotechniques 6, 608-614, 1988.
6. Roe T, Reynolds TC, Yu G, Brown PO. Integration of murine leukemia virus DNA depends on mitosis. EMBO J 12, 2099-108, 1993.
7. Danthinne X. and Imperiale MJ. Production of first generation adenovirus vectors: a review. Gene Ther. 7, 1707-1714, 2000.
8. Graham FL, and Prevec L. Methods for construction of adenovirus vectors. Mol. Biotechnol. 3, 207-220, 1995.
9. Ali M, Lemoine NR, Ring CJA. The use of DNA virus as vectors for gene therapy. Gene Ther. 1, 367-84, 1994.
10. Gerard RD, Chan L. Adenovirus-mediated gene transfer: strategies and applications in lipoprotein research. Curr. Opin. Lipodol. 7,105-11, 1996.
11. Graham FL, Smiley J, Russell WC, Nairn R. Characteristics of a human cell line transformed by DNA from heman adenovirus type 5. J. Gen. Virol. 36, 59-72, 1977.
12. Hitt M, Bett AJ, Addison CL, Prevec L, Graham FL. Techniques for human adenovirus vector construction and characterization. Methods Mol. Genet. 7, 13-30, 1995.
13. McGrory WJ, Bautista DS, Graham FL. A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5. Virology 163, 614-7, 1988.
14. Ferry N, Heard JM. Liver-directed gene transfer vectors. Hum. Gene Ther. 9, 1975-81, 1998.
15. Starr PA, Lim F, Grant FD, Trask L, Lang L, Yu L, Geller AI. Long-term persistence of defective HSV-1 vectors in the rat brain is demonstrated by reactivation of vector gene expression. Gene Ther. 3, 615-623, 1996.
16. Fink DJ, and Glorioso JC. Herpes simplex virus-based vectors: problems and some solution. Adv. Neurol. 72, 149-156, 1997.
17. Kotin RM. Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum. Gene Ther. 5, 793-801, 1994.
18. Hermonat PL, and Muzyczka N. Use of adeno-associated virus as a mammalian DNA cloning vector: transduction of neomycin resistance into mammalian tissue culture. Proc. Natl. Acad. Sci. USA. 81, 6466-6470, 1984.
19. Kotin RM, Linden RM, Berns KI. Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-homologous recombination. EMBO J. 11, 5071-5078, 1992.
20. Clark KR, Voulgaropopulou F, Johnson PR. A stable cell line carring adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. Gene Ther. 3, 1124-1132, 1996.
21. Xiao X, Li J, Samulski RJ. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J. Virol. 70, 8098-8108, 1996.
22. Acsadi G, Dickson G, Love DR, Jani A, Walsh FS, Gurusinghe A, Wolff JA, Davies KE. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature 352, 815-818, 1991.
23. Katsumi A, Emi N, Abe A, Hasegawa Y, Ito M, Saito H. Humoral and cellular immunity to an encoded protein induced by direct DNA injection. Hum. Gene Ther. 5, 1335-1339, 1994.
24. Yang NS, Burkholder J, Roberts B, Martinell B, McCabe D. In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc. Natl. Acad. Sci. USA. 87, 9568-72, 1990.
25. Rols MP, Delteil C, Golzio M, Dumond P, Cros S, Teissie J. In vivo electrically mediated protein and gene transfer in murine melanoma. Nat. Biotechnol. 16, 168-71, 1998.
26. Yang JP, Huang L. Overcoming the inhibitory effect of serum on lipofection by increasing the charge ratio of cationic liposome to DNA. Gene Ther. 4, 950-60, 1997.
27. Yang JP, Huang L. Time-dependent maturation of cationic liposome-DNA complex for serum resistance. Gene Ther. 5, 380-7, 1998.
28. Crrok K, Stevenson BJ, Dubouchet M, Porteous DJ. Inclusion of cholesterol in DOTAP transfection complexes increases the delivery of DNA to cells in vitro in the presence of serum. Gene Ther. 5, 137-43,1998.
29. Liu F, Qi H, Huang L, Liu D. Factors controlling the efficiency of cationic lipid-mediated transfection in vivo via intravenous administration. Gene Ther. 4, 517-23, 1997.
30. Templeton NS, Lasic DD, Frederik PM, Strey HH, Roberts DD, Pavlakis GN. Improved DNA: liposome complexes for increased systemic delivery and gene expression. Nat. Biotechnol. 15, 647-52, 1997.
31. Stein CA, Cohen JS. Oligodeoxynucleotides as inhibitors of gene expression: A review. Cancer Res. 48, 2659-68, 1998.
32. Durko M, Brodt P. Suppression of type I collagenase expression by antisense RNA in melanoma cells results in reduced synthesis of the urokinase-type plasminogen activator receptor. Biochem. Biophys. Res. Commun. 247, 342-8, 1998.
33. James HA, Gibson I. The therapeutic potential of ribozymes. Blood 91, 371-82, 1998.
34. Boussif O, Lezoualc’h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc. Natl. Acad. Sci. USA. 92, 7297-301, 1995.
35. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent ions across the lamellae of swollen phospholipids. J. Mol. Biol. 13, 238-252, 1965.
36. Sessa G, and Weissmann G. Phospholipid spherules (liposomes) as a model for biological membranes. J. Lipid Res. 9, 310-318, 1968.
37. Albrecht O, Johnston DS, Villaverde C, Chapman D. Stable biomembrane surfaces formed by phospholipid polymers. Biochim. Biophys. Acta 687, 165-169, 1982.
38. Pinto-Alphandary H, Andremont A, Couvreur P. Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications. Int. J. Antimicrob. Agents 13, 155-168, 2000.
39. Maurer N, Mori A, Palmer L, Monck MA, Mok KW, Mui B, Akhong QF, Cullis PR. Lipid-based systems for the intracellular delivery of genetic drugs. Mol. Membr. Biol. 16, 129-140, 1999.
40. Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc. Natl. Acad. Sci. USA. 84, 7413-7414, 1987.
41. Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561, 1944a.
42. Li S, and Huang L. In vivo gene transfer via intravenous administration of cationic lipid-protamine-DNA (LPD) complexes. Gene Ther. 4, 891-900, 1997.
43. Gabev EE, Svilenov DK, Poljakova-Krusteva OT, Vassilev I. Brain, liver and spleen detection of liposomes after rectal administration. J. Microencapsul. 2, 85-89, 1985.
44. Lasic DD. “Liposomes” Science/Medicine May/June, 34-43, 1996.
45. Mary K. Campbell. “Biochemistry”, Saunder College Publishing.
46. R.R.C. New. “liposome: a practial approach”. Oxford University press, New York, 1990.
47. Norman Weiner, Frank Martin, Mohammad Riaz. “Liposomes as a drug delivery system” drug development and industrial pharmacy, 15 (10), 1523-1554, 1989.
48. Gregory Gregoriadis edited, “Liposome Technology”, vol. 1, Chapter 2 lipid peroxidation in liposome, 139-161, 1989.
49. Ecsedi GG, Amanuma K, Imanaka T, Aoyagi T, Ohkuma S, Takano T. Effect of esterastin, an acid lipase inhibitor, on the free and esterified cholesterol contenets of cultured aortic smooth muscle cells treated with LDL and cholesterol ester liquid crystals. Biochem. International, 10 (3), 337-42, 1985.
50. Chiu D, Kuypers F, Lubin B. Lipid peroxidation in human red cells. Seminars in Hematology, 26, 257-276, 1989.
51. Wrobel I, and Collins D. Fusion of cationic liposomes with mammalian cells occurs after endocytosis. Biochim. Biophys. Acta. 1235, 296-304, 1995.
52. Felgner PL. The evolving role of the liposome in gene delivery. J. liposome research, 5 (4), 725-734, 1995.
53. Goyal K, and Huang L. Gene therapy using DC-Chol liposome. J. liposome research, 5 (1), 49-60, 1995.
54. Felgner PL, and Ringold GM. Cationic liposome-mediated transfection. Nature 337, 387-8, 1989.
55. Allen TM. Long-circulating (sterically stabilized) liposomes for targeted drug delivery. TiPS, 15, 215-219, 1994.
56. Lasic DD. “Liposome: from physics to applications”, Chapter 1 General introduction to liposome, 1-40, 1993.
57. Woodle MC, and Lasic DD. Sterically stabilized liposomes. Biochim. Biophys. Acta 1113, 171-199, 1992.
58. Chesnoy S, and Huang L. Structure and function of lipid-DNA complexes for gene delivery. Annu. Rev. Biophys. Biomol. Struct. 29, 27-47, 2000.
59. Pinnaduwange P, Schmitt L, Huang L. Use of quaternary ammonium detergent in liposome mediated DNA transfection of mouse L-cells. Biochim. Biophys. Acta 985, 33-7, 1989.
60. Rose JK, Buonocore L, Whitt MA. A new cationic liposome reagent mediating nearly quantitative transfection of animal cells. Biotechniques 10, 520-525, 1991.
61. Farhood H, Bottega R, Epand RM, Huang L. Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity. Biochim. Biophys. Acta 1111, 239-246, 1992.
62. Stamatatos L, Leventis R, Zuckermann MJ, Silvius JR. Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochemistry 27, 3917-3925, 1988.
63. Behr JP, Demeneix B, Loeffler JP, Perez-Mutul J. Efficient gene transfer into mammalian primary endocrine cells with lipopolyamine-coated DNA. Proc. Natl. Acad. Sci. USA. 86, 6982-6986, 1989.
64. Gao X, Huang L. A novel cationic liposome reagent for efficient transfection of mammalian calls. Biochem. Biophys. Res. Commun. 179, 280-285, 1991.
65. Litzinger DC, and Huang L. Phosphatidyethanolamine liposomes: drug delivery, gene transfer and immunodiagnostic applications. Biochim. Biophys. Acta 1113, 201-227, 1992.
66. Sternberg B, Hong K, Zheng W, Papahadjopoulos D. Ultrastructural characterization of cationic liposome-DNA complexes showing enhanced stability in serum and high transfection activity in vivo. Biochim. Biophys. Acta 1375, 23-35, 1998.
67. Sternberg B, Sorgi FL, Huang L. New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy. FEBS Lett. 356, 361-366, 1994.
68. Kawakami S, Fumoto S, Nishikawa M, Yamashita F, Hashida M. In vivo gene delivery to the liver using novel galactosylated cationic liposomes. Pharm. Res. 17, 306-313, 2000.
69. Mahato RI, Rolland A, Tomlinson E. Cationic lipid-based gene delivey systems: pharmaceutical perspectives. Pharm. Res. 14, 853-859, 1997.
70. Xu L, Pirollo KF, Tang WH, Rait A, Chang EH. Transferrin-liposome-mediated systemic p53 gene therapy in combination with radiation results in regression of human head and neck cancer xenografts. Hum. Gene Ther. 10, 2941-2952, 1999.
71. Sorgi FL, Bhattacharya S, Huang L. Protamine sulfate enhances lipid-mediated gene transfer. Gene Ther. 4, 961-968, 1997.
72. de Wet JR, Wood KV, DeLuca M, Helinski DR, Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol. Cell Biol. 7, 725-737, 1987.
73. Kain SR, Adams M, Kondepudi A, Yang TT, Ward WW, Kitts P. Green fluorescent protein as a reporter of gene expression and peotein localization. Biotechniques 19, 650-655, 1995.
74. Cashion LM, Bare LA, Harvey S, Trinh Q, Zhu Y, Devlin JJ. Use of enhanced green fluorescent protein to optimize and quantitate infection of target cells with recombinant retroviruses. Biotechniques 26, 924-930, 1999.
75. Bierhuizen MF, Westerman Y, Visser TP, Dimjati W, Wognum AW, Wagemaker G. Enhanced green fluorescent protein as selectable marker of retroviral-mediated gene transfer in immature hematopoietic bone marrow cells. Blood 90, 3304-3315, 1997.
76. Orrmerod MG. Flow cytometry: A practial approach. IRL press N. Y. 1-28, 1990.
77. Areamone F. Anthracycline antibiotic isolated from Streptomyces peucetius var caesius. Biotechnol. Bioeng. 11, 1101, 1969.
78. Cole MP, Todd ID, Wilkinson PM. A preliminary trial od DOXORUBICIN in advanced breast cancer and other malignant disease. British J. Cancer 29, 114, 1974.
79. Vincent T, DeVita JR, Hellman S, Steven AR. Cancer Principlesand Practice of Oncology. 376-381, 1993.
80. Einhorn LH, Fee WH, Farber MO, Livingston RB, Gottlieb JA. Improved chemotherapy for small-cell undifferentiated lung cancer. JAMA 234, 1223, 1976.
81. Alley MC, Scudiero DA, Monks A, Hursey ML, Czerwinski MJ, Fine DL, Abbott BJ, Mayo JG, Shoemaker RH, Boyd MR. Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Research 48, 589, 1988.
82. Plumb JA, Milroy R, Kaye SB. Effects of the pH dependence of 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay. Cancer Research 49, 4435, 1989.
83. 曾錦鴻 利用輸鐵蛋白促進基因遞送之研究 國立台灣大學醫學院生化學研究所八十八學年度碩士論文, 2001.
84. Pedroso de Lima MC et al. Cationic lipid-DNA complexes in gene delivery: from biophysics to biological applications. Adv. Drug. Deliv. Rev. 47, 277-294, 2001.
85. Pires P, Simões S, Nir S, Gaspar R, Düzgüneş N, Pedroso de Lima MC. Interaction of cationic liposomes and their DNA complexes with monocytic leukemia cells. Biochim. Biophys. Acta 1418, 71-84, 1999.
86. Zabner J, Fasbender AJ, Moninger T, Poellinger KA, Welsh MJ. Cellular and molecular barries to gene transfer by a cationic lipid. J. Biol. Chem. 270, 18997-19007, 1995.
87. Noguchi A, Furuno T, Kawaura C, Nakanish M. Membrane fusion plays an important role in gene transfection mediated by cationic liposomes. FEBS Lett. 433, 169-173, 1998.
88. Wagner E, Plank C, Zatloukal K, Cotton M, Birnstiel ML. Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-polylysine-DNA complexes: Toward a synthetic virus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. USA 89, 7934-7938, 1992.
89. Kichler A, Mechtler K, Behr JP, Wagner E. Influence of membrane-active peptides on lipospermine/DNA complex mediated gene transfer. Bioconjug. Chem. 8, 213-221, 1997.
90. Plank C, Oberhauser B, Mechtler K, Koch C, Wagner E. The influence of endosome-disruptive peptides on gene transfer using synthetic virus-like gene transfer systems. J. Biol. Chem. 269, 12918-12924, 1994.
91. Simões S, Slepushkin V, Pretzer E, Gaspar R, Pedroso de Lima MC, Düzgüneş. Transfection of human macrophages by lipoplexes via the combined use of targeting ligands and fusogenic peptides. J. Leukoc. Biol. 65, 270-279, 1999.
92. Subbarao NK, Parente RA, Szoka FC, Nadasdi L, Pongracz K. pH-dependent bilayer destabilization by an amphipathic peptide. Biochemistry 26, 2964-2972, 1987.
93. Bally MB, Harvie P, Wong FMP, Kong S, Wasan EK, Reimer DL. Biological barriers to cellular delivery of lipid-based DNA carriers. Adv. Drug. Deliv. Rev. 38, 291-315, 1999.
94. Harashima H, Shinohara Y, Kiwada H. Intracellular control of gene trafficking using liposomes as drug carriers. Euro. J. Pharma. Sci. 13, 85-89, 2001.
95. Yoneda, Y. Nuclear pore-targeting complex and its role on nuclear protein transport. Arch. Histol. Cytol. 59, 97-107, 1996.
96. Jans DA, Hubner S. Regulation of protein transport to the nucleus: central role of phosphorylation. Physiol. Rev. 76, 651-685, 1996.
97. Gao X, Huang L. Potentiation of cationic liposome-mediated gene delivery by polycations. Biochemistry 35, 1027-1036, 1996.
98. Nishikawa M, Huang L. Nonviral vectors in the new millennium: Delivery barriers in gene transfer. Hum. Gene Ther. 12, 861-870, 2001.
99. Wu GY, Wu CH. Receptor-mediated gene delivery and expression in vivo. J. Biol. Chem. 263, 14621-14624, 1988.
100. Wagner E, Zenke M, Cotton M, Beug H, Birnstiel ML. Transferrin-polycation conjugates as carriers for DNA uptake into cells. Proc. Natl. Acad. Sci. USA 87, 3410-3414, 1990.
101. Diebold SS, Kursa M, Wagner E, Cotton M, Zenke M. Mannose polyethyleneimine conjugates for targeted DNA delivery into dendritic cells. J. Biol. Chem. 274, 19087-19094, 1999.
102. Ferkol T, Kaetzel CS, Davis PB. Gene transfer into respiratory epithelial cells by targeting the polymeric immunoglobulin receptor. J. Clin. Invest. 92, 2394-2400, 1993.
103. Li S, Tan Y, Viroonchatapan E, Pitt BR, Huang L. Targeted gene delivery to pulmonary endothelium by anti-PECAM antibody. Am. J. Physiol. Lung Cell Mol. Physiol. 278, L504-L511, 2000.
104. Ross GF, Morris RE, Ciraolo G, Huelsman K, Bruno M, Whitsett JA, Baatz JE, Korfhagen TR. Surfactant protein A-polylysine conjugates for delivery of DNA to airway cells in culture. Hum. Gene Ther. 6, 31-40, 1995.
105. Ando T, Yamasaki M, Suzuki K. Protamines: Isolation, Characterization, Structure and Function. Springer-Verlag: New York, pp 3-87, 1973.
106. Warrant RW, Kim SH. Alpha-helix-double helix interaction shown in the structure of a protamine-trnansfer RNA complex and a nucleoprotamine model. Nature 271, 130-135, 1978.
107. Liu F, Huang L. Development of non-viral vectors for systemic gene delivery. J. controlled Release 78, 259-266, 2002.
108. Peterson BR, Sun LJ, Verdine GL. A critical arginine residue mediates coopoerativity in the contact interface between transcription factors NFAT and AP-1. Proc. Natl. Acad. Sci. USA 93, 13671-13676, 1996.
109. Hui SW, Langner M, Zhao YL, Ross P, Hurley E, Chan K. The role of helper lipids in cationic liposome-mediated gene transfer. Biophys. J. 71, 590-599, 1996.
110. Lewis JG, Lin KY, Kothavale A, Flanagan WM, Matteucci MD, DePrince RB, Mook RA, and Wagn RW. A serm-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA. Proc. Acad. Sci. USA 93, 3176-3181, 1996.
111. Oku N, Yamazaki Y, Matsuura M, Sugiyama M, Hasegawa M, Nango M. A novel non-viral gene transfer system, polycations liposomes. Adv. Drug. Deliv. Rev. 52, 209-218, 2001.
112. Akhtar S, Hughes MD, Khan A, Bibby M, Hussain M, Nawaz Q, Double J, Sayyed P. The delivery of antisense therapeutics. Adv. Drug. Deliv. Rev. 44, 3-21, 2000.
113. Lee RJ, Huang L. Folate-targeted, anionic liposome-entrapped polylysine-condensed DNA for tumor cell-specific gene transfer. J. Biol. Chem. 271, 8481-8487, 1996.
114. Staggs DR, Burton DW, Deftos LJ. Importance of liposome complexing volume in transfection optimization. Biotechniques 21, 792, 784, 796, 798.
115. Zhang YP, Reimer DL, Zhang G, Lee PH, Bally MB. Self-assembling DNA-lipid particles for gene transfer. Pharm. Res. 14, 190-196, 1997.
116. Felgner JH, Kumar R, Sridhar CN, Wheeler CJ, Tsai YJ, Border R, Ramsey P, Martin M, Felgner PL. Enhanced gene delivery and mechanism studies with a novel series of cationic lipid formulations. J. Biol. Chem. 269, 2550-2561, 1994b.