基因治療被視為許多治療疾病的方法中最具有潛力的選擇之ㄧ，而最重要的是如何將具有治療性基因有效率的送入特定細胞裡並且釋放出來。以目前來說，傳送基因載體中以病毒性載體效率較好，但是病毒型載體容易引起免疫反應與毒性上的影響，所以許多研究就轉往非病毒載體去探討。因為細胞膜組成與磷脂質相似，所以利用磷脂質將DNA傳送入細胞的方法就像直接將裸露DNA送入細胞一樣。而且DNA為帶負電的分子，所以傳送基因中以陽離子脂質最具有研究潛力。但是微脂粒在血液系統中會被網狀內皮系統當作外來物而被吞噬，加入聚乙二醇可避免這種情形的發生且能有效的穩定微脂粒結構。本論文研究利用金屬親合原理將聚乙二醇連接至陽離子微脂粒上，並利用中國大頰鼠卵巢細胞來探討不同修飾方法對於基因傳遞的影響。
本實驗主要研究重點，一方面是將輔助性脂質DOPE與DSPE分別和組胺酸進行化學接枝，另一方面是經由化學合成將聚乙二醇形成一具有與金屬螯合性質的高分子。實驗中，利用陽離子脂質分別與經由組氨酸修飾前後的輔助性脂質依一定比例製備成正電荷微脂粒，並以螢光基因（pEGFP-C1）為報導基因。實驗利用兩種不同的修飾法藉由金屬螯合將聚乙二醇連接至微脂粒上。此外，以流式細胞儀分析細胞的轉染效率，並用螢光顯微鏡觀察綠色螢光蛋白的表現效率。
若複合物形成前，將聚乙二醇先行螯合在微脂粒上，則有助於轉染效率的提升。但複合物形成後再添加聚乙二醇，其轉染效率相較於前者低。不過，經由聚乙二醇修飾後的微脂粒，其表面電荷會下降且粒徑的相對穩定度會增加。比較經由組氨酸修飾前後的微脂粒，其修飾後發現能提升表面電荷、DNA傳遞與細胞存活率，尤其在DOTAP/DOPE-His形成之複合物相對於DOTAP/DPOE，其轉染效率增加了一倍。
由實驗結果可以觀察到，聚乙二醇能提供微脂粒較佳的穩定度，但是若使用貼附修飾法修飾微脂粒，則可能導致核酸無法完全被釋放出來，進而導致轉染效率變差。而經由組氨酸修飾後的微脂粒相對於未修飾之微脂粒，其能增進基因表現效率。

Gene therapy has been deemed its potential to treat a lot of types of diseases. A major aim of gene therapy is efficient delivery and release of therapeutic gene into specific target cells. At the present, viral vectors have more significantly efficient gene expression than non-viral vectors. However, viral vectors has cruelly limited such as immune response and toxic reaction in vivo gene delivery system. Non-viral vectors are not expected to induce specific immune response and therefore are looked to as the future of gene delivery system. Non-viral vectors include lipid-based encapsulation of DNA as well as the delivery of naked DNA into cells. Furthmore, DNA is an anionic polymer and cationic liposomes are capable of conjugating DNA by condensation. Therefore, cationic liposomes are potentially important non-viral vectors and widely used for gene delivery system. More recently, liposomes carrying PEG on their exterior have ability to avoid uptake by reticuloendothelial system and can effectively stabilize lipid membranes. The purpose of my study is used PEG conjugate liposomes by metal ion affinity and investigated different types of modification affected gene transfer efficiency in Chinese hamster ovary cell.
The aim of this study, on one hand was synthesized novel polymer and its physical and chemical characterization of such metal affinity, therefore, we used NTA-chelator coupled to PEG; and on the other hand was grafting of single histidine functionality in molecular structure of different helper lipid, DSPE and DOPE, respectively. In the experiment, liposomes consisted of cationic lipid, DOTAP, in presence of DSPE, DOPE, DSPE-His, DOPE-His as helper lipid, respectively and the pEGFP-C1 as reported gene. PEG-NTA was linked to vector that employed two approaches to PEGylate liposomes by metal ion affinity. In addition, the different liposomal formulations were further characterized by the flow cytometry analysis and fluorescent microscopy.
Pre-PEGylation of liposomes by metal ion affinity was able to promote transfection efficiency, whereas Post-PEGylation decreased efficiency. Two types of PEGylation were reduced electric charge of liposomes and raised relative stable of particle size. Compared with unmodified DNA/liposomes complexes（lipoplexes）, DOTAP/DSPE and DOTAP/DOPE, grating of histidine onto helper lipid was found to increase zeta potential, DNA delivery and cellular viability, the DOTAP/DOPE-His lipoplexes increased the transfection activity about 1-fold.
This results showed that PEGylation of liposomes provide better stable of struction than unPEGylation, but Post-PEGylation of liposomes caused to inhibit nucleic acid escape into cytosol. The conjugation of histidine onto helper lipid enhances transgene expression contrast with unmodified liposomes.

1.Anderson, W.F., Prospects for human gene therapy. Science (New York, N.Y.), 1984. 226(4673): p. 401-409.
2.Felgner, P.L. and G. Rhodes, Gene therapeutics. Nature, 1991. 349(6307): p. 351-352.
3.W.T. Godbey , K.K.W., Antonios G. Mikos, Poly(ethylenimine) and its role in gene delivery. Journal of Controlled Release 1999. 60: p. 149-161.
4.Gregoriadis, G.R., BE., Liposomes as carriers of enzymes of drug : a new approach to the treatment of storage diseases. Biochemical Journal, 1971. 124: p. 58.
5.Lasic, D.D. and D. Papahadjopoulos, Liposomes revisited. Science, 1995. 267(5202): p. 1275-1276.
6.Miller, A.D., Cationic lipsomes for gene therapy. Chem.Int.Ed., 1998b. 37: p. 1768-1785
7.Blume, G. and G. Cevc, Liposomes for the sustained drug release in vivo. Biochimica Et Biophysica Acta, 1990. 1029(1): p. 91-97.
8.Allen, T., Long-circulating (sterically stabilized) liposomes for targeted drug delivery. . Trends Pharmacol Sci. , 1994. 15: p. 215-220.
9.Liu, F. and L. Huang, Development of non-viral vectors for systemic gene delivery. Journal Of Controlled Release: Official Journal Of The Controlled Release Society, 2002. 78(1-3): p. 259-266.
10.El-Aneed, A., An overview of current delivery systems in cancer gene therapy. Journal Of Controlled Release: Official Journal Of The Controlled Release Society, 2004. 94(1): p. 1-14.
11.Bangham, A.D., M.M. Standish, and J.C. Watkins, Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal Of Molecular Biology, 1965. 13(1): p. 238-252.
12.http://www.carolguze.com/images/biomolecules/lipid_bilayer.jpg.
13.Pinto-Alphandary, H., A. Andremont, and P. Couvreur, Targeted delivery of antibiotics using liposomes and nanoparticles: research and applications. International Journal Of Antimicrobial Agents, 2000. 13(3): p. 155-168.
14.http://www.endovasc.com/proddev/technology.
15.Felgner, P.L., et al., Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings Of The National Academy Of Sciences Of The United States Of America, 1987. 84(21): p. 7413-7417.
16.Lasic, D., Liposome. Science & Medicine., 1996: p. 34-43. .
17.Stamatatos, L., et al., Interactions of cationic lipid vesicles with negatively charged phospholipid vesicles and biological membranes. Biochemistry, 1988. 27(11): p. 3917-3925.
18.Zabner, J., Cationic lipids used in gene transfer. Advanced Drug Delivery Reviews, 1997. 27: p. 17–28.
19.Mui.B, et al., Membrane perturbation and the mechanism of lipid-mediated transfer of DNA into cells. Biochim Biophys Acta, 2000. 1467(2): p. 281-92.
20.Safinya, C.R., Structures of lipid-DNA complexes: supramolecular assembly and gene delivery. Current Opinion In Structural Biology, 2001. 11(4): p. 440-448.
21.Kostarelos, K. and A.D. Miller, Synthetic, self-assembly ABCD nanoparticles; a structural paradigm for viable synthetic non-viral vectors. Chemical Society Reviews, 2005. 34(11): p. 970-994.
22.New, R.R.C., Liposomes: A Practical Approach. Oxford University Press, 1990.
23.Norman Weiner, F.M., Mohammad Riaz., Liposomes as a drug delivery system. drug development and industrial pharmacy, 1989. 15(10): p. 1523-1554.
24.Janoff, S., Liposomes: Rational Design. Marcel Dekker, 1999: p. 103-107.
25.Gregoriadis, G., Liposome Technology. 1989. 1: p. 139-161.
26.Chiu, D., F. Kuypers, and B. Lubin, Lipid peroxidation in human red cells. Seminars In Hematology, 1989. 26(4): p. 257-276.
27.Fisher, A.A., Immediate and delayed allergic contact reactions with polyethylene glycol. Cont. Derma., 1978. 4: p. 135-138.
28.Yang, Z., et al., PEGylation confers greatly extended half-life and attenuated immunogenicity to recombinant methioninase in primates. Cancer Research, 2004. 64(18): p. 6673-6678.
29.Chaffee, S., et al., IgG antibody response to polyethylene glycol-modified adenosine deaminase in patients with adenosine deaminase deficiency. The Journal Of Clinical Investigation, 1992. 89(5): p. 1643-1651.
30.Conover, C.D., et al., The impact of polyethylene glycol conjugation on bovine hemoglobin's circulatory half-life and renal effects in a rabbit top-loaded transfusion model. Artificial Organs, 1997. 21(8): p. 907-915.
31.Caliceti, P., O. Schiavon, and F.M. Veronese, Biopharmaceutical properties of uricase conjugated to neutral and amphiphilic polymers. Bioconjugate Chemistry, 1999. 10(4): p. 638-646.
32.Deen, W.M., M.P. Bohrer, and B.M. Brenner, Macromolecule transport across glomerular capillaries: application of pore theory. Kidney International, 1979. 16(3): p. 353-365.
33.Brenner, B.M., T.H. Hostetter, and H.D. Humes, Glomerular permselectivity: barrier function based on discrimination of molecular size and charge. The American Journal Of Physiology, 1978. 234(6): p. 455-460.
34.Yamaoka, T., Y. Tabata, and Y. Ikada, Fate of water-soluble polymers administered via different routes. Journal Of Pharmaceutical Sciences, 1995. 84(3): p. 349-354.
35.Yamaoka, T., Y. Tabata, and Y. Ikada, Distribution and tissue uptake of poly(ethylene glycol) with different molecular weights after intravenous administration to mice. Journal Of Pharmaceutical Sciences, 1994. 83(4): p. 601-606.
36.Sherman, M.R.a.W., L. D. , Conjugation of high molecular weight polyethylene glycol to cytokines:granulocyte-macrophage colony stimulating factors as model substrates. polyethylene glycol:chemistry and biological applications, 1997: p. 155-169.
37.Dash, P.R., et al., Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin. The Journal Of Biological Chemistry, 2000. 275(6): p. 3793-3802.
38.Oupicky, D., et al., Importance of Lateral and Steric Stabilization of Polyelectrolyte Gene Delivery Vectors for Extended Systemic Circulation. Molecular Therapy, 2002. 5(4): p. 463-472.
39.Martin-Herranz, A., et al., Surface functionalized cationic lipid-DNA complexes for gene delivery: PEGylated lamellar complexes exhibit distinct DNA-DNA interaction regimes. Biophysical Journal, 2004. 86(2): p. 1160-1168.
40.Hong, K., et al., Stabilization of cationic liposome-plasmid DNA complexes by polyamines and poly(ethylene glycol)-phospholipid conjugates for efficient in vivo gene delivery. FEBS Letters, 1997. 400(2): p. 233-237.
41.Ogris, M., et al., PEGylated DNA/transfrrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Therapy, 1999. 6(4): p. 595-605.
42.Yu, W., et al., A sterically stabilized immunolipoplex for systemic administration of a therapeutic gene. Gene Therapy, 2004. 11(19): p. 1434-1440.
43.Keller, M., et al., Nuclear localisation sequence templated nonviral gene delivery vectors: investigation of intracellular trafficking events of LMD and LD vector systems. Chembiochem: A European Journal Of Chemical Biology, 2003. 4(4): p. 286-298.
44.http://www.cnrs.fr/Cnrspresse/n396/html/n396a01.
45.Ritter, S.K., J.; Setlikova, E.; Setlik, I.; Welte, W., Immobilized metal affinity chromatography for the separation of photosystems I and II from the thermophilic cyanobacterium. J. Chromatography 1992. 625: p. 21-31
46.Hansen, P.a.L., G., Immobilized metal ion affinity chromatography of synthetic peptides—binding via the α-amino group. J. Chromatography 1992. 627: p. 125-135.
47.Hubert P, P.J., Metal chelate affinity chromatography. I. Influence of various parameters on the retention of nucleotides and related compounds. . J. Chromatography 1980. 198: p. 247-255.
48.http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Histidine.html.
49.Porath, J., et al., Metal chelate affinity chromatography, a new approach to protein fractionation. Nature, 1975. 258(5536): p. 598-599.
50.Winkler, U., T.M. Pickett, and D. Hudig, Fractionation of perforin and granzymes by immobilized metal affinity chromatography (IMAC). Journal Of Immunological Methods, 1996. 191(1): p. 11-20.
51.Sulkowski, E., The saga of IMAC and MIT. Bioessays: News And Reviews In Molecular, Cellular And Developmental Biology, 1989. 10(5): p. 170-175.
52.Sulkowski, E., Purification of protein by IMAC. Trends Biotech, 1985. 3: p. 1-7.
53.Schmid, E.L. and T.A. Keller, Reversible oriented surface immobilization of functional proteins on oxide surfaces. Analytical Chemistry, 1997. 69(11): p. 1979-1985.
54.Winzerling, J.J., P. Berna, and J. Porath, , How to Use Immobilized Metal Ion Affinity Chromatography. Methods, 1992. 4(1): p. 14-25.
55.Gregoriadis G, R.B., Fate of protein-containing liposomes injected into rats. An approach to the treatment of storage diseases. Eur J Biochem, 1972 Jan 21. 24(3): p. 485–491.
56.Vladimir, T.V.W., Liposomes second edition apractical approach. OXFORD, USA 2003.
57.Perouzel, E., et al., Synthesis and formulation of neoglycolipids for the functionalization of liposomes and lipoplexes. Bioconjugate Chemistry, 2003. 14(5): p. 884-898.
58.VV Kumar, C.P., M Refregiers, B Guerin, P Midoux and A Chaudhuri, Single histidine residue in head-group region is sufficient to impart remarkable gene transfection properties to cationic lipids: evidence for histidinemediated membrane fusion at acidic pH. Gene Therapy, 2003(10): p. 1206–1215.
59.Lutz Schmitt, C.D., and Robert Tampe, Synthesis and Characterization of Chelator-Lipids for Reversible Immobilization of Engineered Proteins at Self-Assembled Lipid Interfaces. J. Am. Chem. Soc., 1994. 116: p. 8485-8491.
60.Zalipsky, S., Synthesis of an end-group functionalized polyethylene glycol-lipid conjugate for preparation of polymer-grafted liposomes. Bioconjugate Chemistry, 1993. 4(4): p. 296-299.
61.ISRAELACHVILI.J.N, Intemolecular and Surface Forces Academic Press, London, 1992.
62.Ilya Koltover, T.S., Joachim O. Rädler, Cyrus R. Safinya An Inverted Hexagonal Phase of Cationic Liposome-DNA Complexes Related to DNA Release and Delivery Science, 1998. 281: p. 78 - 81.
63.Gonçalves C, P.C., Guérin B, Midoux P., Intracellular processing and stability of DNA complexed with histidylated polylysine conjugates. J Gene Med, 2002. 4(3): p. 271-81.
64.Lee, R.J. and Leaf.Huang, Folate-targeted, Anionic Liposome-entrapped Polylysine-condensed DNA for Tumor Cell-specific Gene Transfer The American Society for Biochemistry and Molecular Biology, 1996. 271(14): p. 8481–8487.
65.Zhang YP, R.D., Zhang G, Lee PH, Bally MB., Self-assembling DNA-lipid particles for gene transfer. Pharm Res, 1997. 14(2): p. 190-196.