藉由發展可轉染幹細胞的基因載體，將可使基因治療方向由抑制不正常細胞增生的被動方向，朝向以幹細胞修復受損組織的主動模式發展，對未來臨床治療提供更有效的策略。然而，目前市面上常見的基因載體能夠有效轉染幹細胞的商品有限，且大部分可轉幹細胞的載體生物相容性仍有疑慮。本研究主要開發一種可有效轉染幹細胞的基因傳輸奈米載體，利用天然高分子─透明質酸 (Hyaluronic Acid) 化學改質接枝上具有氧化還原、酸鹼敏感性的官能基 (硫醇─雙硫鍵結)，再和聚乙烯亞胺 (Polyethylenimine) 利用化學交聯方式形成透明質酸-聚乙烯亞胺 (HA-ss-PEI) 作為材料。材料帶正電的特性可以與帶負電質體 (pDNA) 形成靜電力複合體，顆粒大小約為100奈米左右，可藉由細胞胞吞作用進入細胞中；內吞作用中外來物多傳輸至核內體 (Endosome)，其酸性環境(約pH4-5)及細胞內還原環境使複合體膨漲、崩解，並釋出攜帶的質體。目前對於基因載體的研究十分廣泛，如何提高轉染率及有效降低生物毒性這兩點是目前基因載體發展的兩大方向。研究中使用的透明質酸為構成細胞外基質的成分，能夠促進細胞胞吞作用，雙硫鍵結則由胺基酸組成；在聚乙烯亞胺的部分則選擇小分子的材料，可以有效降低因轉染對細胞造成的傷害，聚乙烯亞胺在核內體可藉由質子海綿效應，搭配環境敏感的雙硫鍵使基因物質有效釋出，同步達成高轉染率及生物相容性的目的。結果顯示本研究開發的透明質酸─聚乙烯亞胺 (HA-ss-PEI) 可有效轉染不易轉染的幹細胞，並且不產生大部分基因載體會使細胞數量減少的問題。除此之外，我們也成功利用此基因載體，將血管生成抑制劑的基因載入間葉幹細胞，使其持續性的製造並釋放抗血管新生因子Endostatin (即血管生成抑制劑)，並在培養環境中模擬軟骨再生環境誘導間葉幹細胞朝向透明軟骨分化確認轉染後的幹細胞仍保有可分化的多功能性。由於此基因載體的主材料成分為透明質酸，與目前治療關節炎注射透明質酸的方法使用相同材料，容易為臨床醫生所接受，具有臨床價值。

Gene therapy strategies can be useful for tissue engineering by modifying stem cells directly using various gene delivery carriers. Gene delivery offers a new and potentially promising treatment modality for the inherited genetic diseases or disorders. The development of delivery systems able to alter the biological profiles of therapeutic agents (viz., pDNA, siRNA, growth factors, drugs, etc.) is considered of utmost importance in biomedical research and the pharmaceutical industry.
In this study, a redox- and pH-sensitive hyaluronic acid (HA) - polyethylenimine (PEI) copolymer with disulfide linkage was synthesized, characterized and examined as a potential non-viral gene vector. The physical and chemical properties of fabricated gene carrier were analyzed via 1H NMR and FT-IR for the demonstration of crosslinking of HA with PEI. TNBS assay and Ellman’s reagent were conducted for the verification of disulfide bond formation and crosslinking degree. The size, morphology and surface charge of nanoparticle were investigated by dynamic light scattering (DLS), transmission electron microscopy (TEM) and zeta potential, respectively. The ability of HA-ss-PEI conjugate to complex with plasmid DNA was observed by gel electrophoresis and the optimal N/P ratio was also determined. Fluorescent microscopy and ELISA spectroscopy were utilized to examine transfection efficiency and protein expression level of therapeutic pDNA. Finally, the ability of transfected hMSCs to differentiate into chondrocyte was investigated by Alcian blue stain and immunocytochemistry (ICC) for type II collagen.
From the results, a novel redox- and pH-sensitive gene delivery nanocarrier has been successfully synthesized. The positive HA-ss-PEI conjugate complexed with negative charged plasmid DNA can be achieved via electrostatic attraction to form a stable spherical nanoparticle of 100 nm in diameter. N/P=2 possessed the optimal condition for releasing encapsulated plasmid when triggered by stimuli such as redox reaction and pH change. The nanocarrier could successfully complex with pDNA and transfect stem cells to produce specific protein for therapeutic purposes. hMSCs transfected with HA-ss-PEI/pEndo complexes could produce endostatin, an anti-angiogenic agent, and differentiate into chondrocytes after 7 days chondrogenic induction. In brief, the redox- and pH-sensitive HA-ss-PEI nanoparticles can be used as a promising non-viral gene carrier for stem cell gene therapy.

1. Godbey, W.T., K.K. Wu, and A.G. Mikos, Poly(ethylenimine) and its role in gene delivery. Journal of Controlled Release, 1999. 60(2-3): p. 149-160.
2. Boussif, O., et al., A Versatile Vector for Gene and Oligonucleotide Transfer into Cells in Culture and in-Vivo - Polyethylenimine. Proceedings of the National Academy of Sciences of the United States of America, 1995. 92(16): p. 7297-7301.
3. Behr, J.P., The proton sponge: A trick to enter cells the viruses did not exploit. Chimia, 1997. 51(1-2): p. 34-36.
4. Kim, Y.H., et al., Polyethylenimine with acid-labile linkages as a biodegradable gene carrier. Journal of Controlled Release, 2005. 103(1): p. 209-219.
5. Kunath, K., et al., Low-molecular-weight polyethylenimine as a non-viral vector for DNA delivery: comparison of physicochemical properties, transfection efficiency and in vivo distribution with high-molecular-weight polyethylenimine. Journal of Controlled Release, 2003. 89(1): p. 113-125.
6. Fischer, D., et al., A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and cytotoxicity. Pharmaceutical Research, 1999. 16(8): p. 1273-1279.
7. Aruffo, A., et al., Cd44 Is the Principal Cell-Surface Receptor for Hyaluronate. Cell, 1990. 61(7): p. 1303-1313.
8. Schiffelers, R.M., et al., Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Research, 2004. 32(19).
9. Entwistle, J., C.L. Hall, and E.A. Turley, HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem, 1996. 61(4): p. 569-77.
10. Jiang, G., et al., Target Specific Intracellular Delivery of siRNA/PEI-HA Complex by Receptor Mediated Endocytosis. Molecular Pharmaceutics, 2009. 6(3): p. 727-737.
11. Saito, G., G.L. Amidon, and K.D. Lee, Enhanced cytosolic delivery of plasmid DNA by a sulfhydryl-activatable listeriolysin O/protamine conjugate utilizing cellular reducing potential. Gene Therapy, 2003. 10(1): p. 72-83.
12. Saito, G., J.A. Swanson, and K.D. Lee, Drug delivery strategy utilizing conjugation via reversible disulfide linkages: role and site of cellular reducing activities. Advanced Drug Delivery Reviews, 2003. 55(2): p. 199-215.
13. Meng, F.H., W.E. Hennink, and Z. Zhong, Reduction-sensitive polymers and bioconjugates for biomedical applications. Biomaterials, 2009. 30(12): p. 2180-2198.
14. Fenwick, S.A., P.J. Gregg, and P. Rooney, Osteoarthritic cartilage loses its ability to remain avascular. Osteoarthritis and Cartilage, 1999. 7(5): p. 441-452.
15. Walsh, D.A., Angiogenesis in osteoarthritis and spondylosis: successful repair with undesirable outcomes. Current Opinion in Rheumatology, 2004. 16(5): p. 609-615.
16. Cusack, J.C., Jr. and K.K. Tanabe, Introduction to cancer gene therapy. Surg Oncol Clin N Am, 2002. 11(3): p. 497-519, v.
17. Lv, H., et al., Toxicity of cationic lipids and cationic polymers in gene delivery. J Control Release, 2006. 114(1): p. 100-9.
18. Robbins, P.D. and S.C. Ghivizzani, Viral vectors for gene therapy. Pharmacology & Therapeutics, 1998. 80(1): p. 35-47.
19. Dujardin, N., P. Van der Smissen, and V. Preat, Topical gene transfer into rat skin using electroporation. Pharmaceutical Research, 2001. 18(1): p. 61-66.
20. Li, S., et al., Intramuscular electroporation delivery of IFN-alpha gene therapy for inhibition of tumor growth located at a distant site. Gene Therapy, 2001. 8(5): p. 400-407.
21. Liu, F. and L. Huang, Electric gene transfer to the liver following systemic administration of plasmid DNA. Gene Therapy, 2002. 9(16): p. 1116-1119.
22. Muangmoonchai, R., et al., Transfection of liver in vivo by biolistic particle delivery - Its use in the investigation of cytochrome P450 gene regulation. Molecular Biotechnology, 2002. 20(2): p. 145-151.
23. Lin, M.T.S., et al., The gene gun: current applications in cutaneous gene therapy. International Journal of Dermatology, 2000. 39(3): p. 161-170.
24. Amabile, P.G., et al., High-efficiency endovascular gene delivery via therapeutic ultrasound. Journal of the American College of Cardiology, 2001. 37(7): p. 1975-1980.
25. Unger, E.C., et al., Gene delivery using ultrasound contrast agents. Echocardiography-a Journal of Cardiovascular Ultrasound and Allied Techniques, 2001. 18(4): p. 355-361.
26. 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.
27. Gao, X. and L. Huang, Cationic liposome-mediated gene transfer. Gene Therapy, 1995. 2(10): p. 710-722.
28. Safinya, C.R., Structures of lipid-DNA complexes: supramolecular assembly and gene delivery. Current Opinion in Structural Biology, 2001. 11(4): p. 440-448.
29. Karmali, P.P. and A. Chaudhuri, Cationic Liposomes as non-viral carriers of gene medicines: Resolved issues, open questions, and future promises. Medicinal Research Reviews, 2007. 27(5): p. 696-722.
30. De Smedt, S.C., J. Demeester, and W.E. Hennink, Cationic polymer based gene delivery systems. Pharmaceutical Research, 2000. 17(2): p. 113-126.
31. Akinc, A., et al., Parallel synthesis and biophysical characterization of a degradable polymer library for gene delivery. Journal of the American Chemical Society, 2003. 125(18): p. 5316-5323.
32. Leong, K.W., et al., DNA-polycation nanospheres as non-viral gene delivery vehicles. Journal of Controlled Release, 1998. 53(1-3): p. 183-193.
33. Pack, D.W., et al., Design and development of polymers for gene delivery. Nature Reviews Drug Discovery, 2005. 4(7): p. 581-593.
34. Hashimoto, M., et al., Lactosylated chitosan for DNA delivery into hepatocytes: the effect of lactosylation on the physicochemical properties and intracellular trafficking of pDNA/chitosan complexes. Bioconjug Chem, 2006. 17(2): p. 309-16.
35. Sokolova, V. and M. Epple, Inorganic nanoparticles as carriers of nucleic acids into cells. Angewandte Chemie-International Edition, 2008. 47(8): p. 1382-1395.
36. Toole, B.P., Hyaluronan: From extracellular glue to pericellular cue. Nature Reviews Cancer, 2004. 4(7): p. 528-539.
37. Stern, R., A.A. Asari, and K.N. Sugahara, Hyaluronan fragments: An information-rich system. European Journal of Cell Biology, 2006. 85(8): p. 699-715.
38. Plank, C., et al., Activation of the complement system by synthetic DNA complexes: A potential barrier for intravenous gene delivery. Human Gene Therapy, 1996. 7(12): p. 1437-1446.
39. Maruyama, K., et al., Novel receptor-mediated gene delivery system comprising plasmid/protamine/sugar-containing polyanion ternary complex. Biomaterials, 2004. 25(16): p. 3267-3273.
40. Koyama, Y., et al., Novel poly(ethylene glycol) derivatives with carboxylic acid pendant groups: synthesis and their protection and enhancing effect on non-viral gene transfection systems. Journal of Biomaterials Science-Polymer Edition, 2003. 14(6): p. 515-531.
41. Jiang, G., et al., Hyaluronic acid-polyethyleneimine conjugate for target specific intracellular delivery of siRNA. Biopolymers, 2008. 89(7): p. 635-642.
42. Gilbert, H.F., Thiol/Disulfide Exchange Equilibria and Disulfide Bond Stability. Biothiols, Pt A, 1995. 251: p. 8-28.
43. Vert, M., et al., Bioresorbability and Biocompatibility of Aliphatic Polyesters. Journal of Materials Science-Materials in Medicine, 1992. 3(6): p. 432-446.
44. Arner, E.S.J. and A. Holmgren, Physiological functions of thioredoxin and thioredoxin reductase. European Journal of Biochemistry, 2000. 267(20): p. 6102-6109.
45. Ho, Y.C., et al., Self-organized nanoparticles prepared by guanidine- and disulfide-modified chitosan as a gene delivery carrier. Journal of Materials Chemistry, 2011. 21(42): p. 16918-16927.
46. Godbey, W.T., et al., Improved packing of poly(ethylenimine)/DNA complexes increases transfection efficiency. Gene Therapy, 1999. 6(8): p. 1380-1388.
47. Godbey, W.T., K.K. Wu, and A.G. Mikos, Poly(ethylenimine)-mediated gene delivery affects endothelial cell function and viability. Biomaterials, 2001. 22(5): p. 471-480.
48. Thomas, M., et al., Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proceedings of the National Academy of Sciences of the United States of America, 2005. 102(16): p. 5679-5684.
49. Tang, G.P., et al., Low molecular weight polyethylenimines linked by beta-cyclodextrin for gene transfer into the nervous system. Journal of Gene Medicine, 2006. 8(6): p. 736-744.
50. Yuste, R., Fluorescence microscopy today. Nature Methods, 2005. 2(12): p. 902-904.
51. Komorowski, M., B. Finkenstadt, and D. Rand, Using a Single Fluorescent Reporter Gene to Infer Half-Life of Extrinsic Noise and Other Parameters of Gene Expression. Biophysical Journal, 2010. 98(12): p. 2759-2769.
52. Ren, B., et al., The antiangiogenic and therapeutic implications of endostatin. Methods and Findings in Experimental and Clinical Pharmacology, 2003. 25(3): p. 215-224.
53. OReilly, M.S., et al., Endostatin: An endogenous inhibitor of angiogenesis and tumor growth. Cell, 1997. 88(2): p. 277-285.
54. Yamaguchi, N., et al., Endostatin inhibits VEGF-induced endothelial cell migration and tumor growth independently of zinc binding. Embo Journal, 1999. 18(16): p. 4414-4423.
55. Rehn, M., et al., Interaction of endostatin with integrins implicated in angiogenesis. Proceedings of the National Academy of Sciences of the United States of America, 2001. 98(3): p. 1024-1029.
56. Wickstrom, S.A., et al., Endostatin-induced modulation of plasminogen activation with concomitant loss of focal adhesions and actin stress fibers in cultured human endothelial cells. Cancer Research, 2001. 61(17): p. 6511-6516.
57. Wickstrom, S.A., K. Alitalo, and J. Keski-Oja, Endostatin associates with lipid rafts and induces reorganization of the actin cytoskeleton via down-regulation of RhoA activity. Journal of Biological Chemistry, 2003. 278(39): p. 37895-37901.
58. Hanai, J., et al., Endostatin is a potential inhibitor of Wnt signaling. Journal of Cell Biology, 2002. 158(3): p. 529-539.
59. Lindvall, O. and Z. Kokaia, Stem cells for the treatment of neurological disorders. Nature, 2006. 441(7097): p. 1094-1096.
60. Weissman, I.L., Stem cells: Units of development, units of regeneration, and units in evolution. Cell, 2000. 100(1): p. 157-168.
61. Ragusa, A., I. Garcia, and S. Penades, Nanoparticles as nonviral gene delivery vectors. Ieee Transactions on Nanobioscience, 2007. 6(4): p. 319-330.
62. Khalil, I.A., et al., Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacological Reviews, 2006. 58(1): p. 32-45.
63. Sokolova, V., et al., Tracking the pathway of calcium phosphate/DNA nanoparticles during cell transfection by incorporation of red-fluorescing tetramethylrhodamine isothiocyanate-bovine serum albumin into these nanoparticles. Journal of Biological Inorganic Chemistry, 2007. 12(2): p. 174-179.
64. Friend, D.S., D. Papahadjopoulos, and R.J. Debs, Endocytosis and intracellular processing accompanying transfection mediated by cationic liposomes. Biochimica Et Biophysica Acta-Biomembranes, 1996. 1278(1): p. 41-50.
65. Medina-Kauwe, L.K., J. Xie, and S. Hamm-Alvarez, Intracellular trafficking of nonviral vectors. Gene Therapy, 2005. 12(24): p. 1734-1751.
66. Wu, G.Y., et al., Glutathione metabolism and its implications for health. Journal of Nutrition, 2004. 134(3): p. 489-492.
67. Go, Y.M. and D.P. Jones, Redox compartmentalization in eukaryotic cells. Biochimica Et Biophysica Acta-General Subjects, 2008. 1780(11): p. 1271-1290.
68. Kafedjiiski, K., et al., Synthesis and in vitro evaluation of thiolated hyaluronic acid for mucoadhesive drug delivery. International Journal of Pharmaceutics, 2007. 343(1-2): p. 48-58.
69. Ellman, G.L., Tissue Sulfhydryl Groups. Archives of Biochemistry and Biophysics, 1959. 82(1): p. 70-77.
70. Snyder, S.L. and P.Z. Sobocinski, Improved 2,4,6-Trinitrobenzenesulfonic Acid Method for Determination of Amines. Analytical Biochemistry, 1975. 64(1): p. 284-288.
71. Jeng, L., B.R. Olsen, and M. Spector, Engineering Endostatin-Producing Cartilaginous Constructs for Cartilage Repair Using Nonviral Transfection of Chondrocyte-Seeded and Mesenchymal-Stem-Cell-Seeded Collagen Scaffolds. Tissue Engineering Part A, 2010. 16(10): p. 3011-3021.
72. Li, Z.T., et al., Chitosan-graft-polyethylenimine with improved properties as a potential gene vector. Carbohydrate Polymers, 2010. 80(1): p. 254-259.
73. Liu, Y.M. and T.M. Reineke, Hydroxyl stereochemistry and amine number within poly(glycoamidoamine)s affect intracellular DNA delivery. Journal of the American Chemical Society, 2005. 127(9): p. 3004-3015.
74. Duceppe, N. and M. Tabrizian, Factors influencing the transfection efficiency of ultra low molecular weight chitosan/hyaluronic acid nanoparticles. Biomaterials, 2009. 30(13): p. 2625-2631.
75. Schafer, F.Q. and G.R. Buettner, Redox environment of the cell as viewed through the redox state of the glutathione disulfide/glutathione couple. Free Radical Biology and Medicine, 2001. 30(11): p. 1191-1212.
76. Park, K., et al., Target specific systemic delivery of TGF-beta siRNA/(PEI-SS)-g-HA complex for the treatment of liver cirrhosis. Biomaterials, 2011. 32(21): p. 4951-4958.
77. Miyashita, M., et al., Serum levels of vascular endothelial growth factor, basic fibroblast growth factor and endostatin in human metastatic liver tumors. Hepato-Gastroenterology, 2003. 50(50): p. 308-309.
78. Bono, P., L. Teerenhovi, and H. Joensuu, Elevated serum endostatin is associated with poor outcome in patients with non-Hodgkin lymphoma. Cancer, 2003. 97(11): p. 2767-2775.
79. Johnstone, B., et al., In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Experimental Cell Research, 1998. 238(1): p. 265-272.
80. Kramer, J., et al., Embryonic stem cell-derived chondrogenic differentiation in vitro: activation by BMP-2 and BMP-4. Mechanisms of Development, 2000. 92(2): p. 193-205.
81. Monagle, J.J., Carbodiimides .3. Conversion of Isocyanates to Carbodiimides - Catalyst Studies. Journal of Organic Chemistry, 1962. 27(11): p. 3851-&.
82. Kolf, C.M., E. Cho, and R.S. Tuan, Mesenchymal stromal cells - Biology of adult mesenchymal stem cells: regulation of niche, self-renewal and differentiation. Arthritis Research & Therapy, 2007. 9(1).
83. Meirelles, L.D., A.I. Caplan, and N.B. Nardi, In search of the in vivo identity of mesenchymal stem cells. Stem Cells, 2008. 26(9): p. 2287-2299.
84. Tierney, E.G., et al., The development of non-viral gene-activated matrices for bone regeneration using polyethyleneimine (PEI) and collagen-based scaffolds. Journal of Controlled Release, 2012. 158(2): p. 304-311.
85. Dagdas, S., et al., Assessment of Plasma Endostatin Levels in Patients with Acute Myeloblastic and Lymphoblastic Leukemia. Uhod-Uluslararasi Hematoloji-Onkoloji Dergisi, 2011. 21(4): p. 210-216.