Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/16932
標題: 標靶性高分子微胞於光動力治療之應用
Cetuximab Conjugated Polymeric Micelle for Targetable Photodynamic Therapy
作者: 陳穎臻
Chen, Ying-Chen
關鍵字: 標靶
target
光動力治療
微胞
PDT
micelle
出版社: 化學系所
引用: [1] Ackroyd R, Kelty C, Brown N, Reed M. The history of photodetection and photodynamic therapy. Photochemistry and Photobiology. 2001;74:656-69. [2] Daniell M, Hill J. A history of photodynamic therapy. Australian and New Zealand Journal of Surgery. 1991;61:340-8. [3] Hausmann W. Die sensiblisierende Wirkung des Hematoporphyrins. 1911;30:276–316. [4] Meyer-Betz F. Untersuchungen uber die biologische (photodynamische) wirkung des hamatoporphyrins und anderer derivate des blut-und gallenfarbstoffs. Dtsch Arch Klin Med. 1913;112:476-503. [5] LIPSON RL, BALDES EJ. The photodynamic properties of a particular hematoporphyrin derivative. Archives of Dermatology. 1960;82:508. [6] Lipson RL, Baldes, E. J. & Olsen, A. M. The use of an derivative of hematoporphyrin in tumor detection. Journal of the National Cancer Institute. 1961;26:1-11. [7] Dougherty TJ, Gomer CJ, Jori G, Kessel D, Korbelik M, Moan J, et al. Photodynamic therapy. Journal of the National Cancer Institute. 1998;90:889. [8] Diamond I, Mcdonagh AF, Wilson CB, Granelli SG, Nielsen S, Jaenicke R. Photodynamic therapy of malignant tumours. The Lancet. 1972;300:1175-7. [9] Dougherty TJ, Grindey G, Fiel R, Weishaupt K, Boyle D. Photoradiation therapy. II. cure of animal tumors with hematoporphyrin and light. Journal of the National Cancer Institute. 1975;55:115. [10] Kelly J, Snell M, Berenbaum M. Photodynamic destruction of human bladder carcinoma. British Journal of Cancer. 1975;31:237. [11] Dolmans DEJGJ, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer. 2003;3:380-7. [12] Nishiyama N, Morimoto Y, Jang W, Kataoka K. Design and development of dendrimer photosensitizer-incorporated polymeric micelles for enhanced photodynamic therapy. Advanced Drug Delivery Reviews. 2009;61:327-38. [13] Nyman ES, Hynninen PH. Research advances in the use of tetrapyrrolic photosensitizers for photodynamic therapy. Journal of Photochemistry and Photobiology B: Biology. 2004;73:1-28. [14] Truscott SBBaTG. New light on cancer therapy. Chem Br 1933:955-8. [15] H. Yamamoto TO, K. Furukawa, T. Hiyoshi, C. Konaka and H. Kato. Photodynamic therapy for cancers. Current Science. 1999;77:894-903. [16] A. Andreoni RCE. Porphyrins in Tumor Phototherapy. Plenum Press, New York. 1984. [17] Jori G. Far-red-absorbing photosensitizers: their use in the photodynamic therapy of tumours. Journal of Photochemistry and Photobiology A: Chemistry. 1992;62:371-8. [18] Ma L, Moan J, Berg K. Evaluation of a new photosensitizer, meso-tetra-hydroxyphenyl-chlorin, for use in photodynamic therapy: A comparison of its photobiological properties with those of two other photosensitizers. International Journal of Cancer. 1994;57:883-8. [19] Čunderlíková B, Bjørklund EG, Pettersen EO, Moan J. pH-Dependent Spectral Properties of HpIX, TPPS2a, mTHPP and mTHPC¶. Photochemistry and Photobiology. 2001;74:246-52. [20] Kübler AC, Haase T, Staff C, Kahle B, Rheinwald M, Mühling J. Photodynamic therapy of primary nonmelanomatous skin tumours of the head and neck. Lasers in Surgery and Medicine. 1999;25:60-8. [21] G. Bock SHE. Photosensitizing Compounds: Their Chemistry, Biology and Clinical Use Ciba Foundation Symposium, Wiley, Chichester, . 1989;146. [22] Spikes JD. New trends in photobiology: Chlorins as photosensitizers in biology and medicine. Journal of Photochemistry and Photobiology B: Biology. 1990;6:259-74. [23] Kostenich GA, Zhuravkin IN, Furmanchuk AV, Zhavrid EA. Photodynamic therapy with chlorin e6. A morphologic study of tumor damage efficiency in experiment. Journal of Photochemistry and Photobiology B: Biology. 1991;11:307-18. [24] Kostenich GA, Zhuravkin IN, Zhavrid EA. Experimental grounds for using chlorin [rho]6 in the photodynamic therapy of malignant tumors. Journal of Photochemistry and Photobiology B: Biology. 1994;22:211-7. [25] Choi Y, Weissleder R, Tung C-H. Selective Antitumor Effect of Novel Protease-Mediated Photodynamic Agent. Cancer Res. 2006;66:7225-9. [26] Shieh M-J, Peng C-L, Chiang W-L, Wang C-H, Hsu C-Y, Wang S-JJ, et al. Reduced Skin Photosensitivity with meta-Tetra(hydroxyphenyl)chlorin-Loaded Micelles Based on a Poly(2-ethyl-2-oxazoline)-b-poly(d,l-lactide) Diblock Copolymer in Vivo. Mol Pharm. 2010;7:1244-53. [27] Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer research. 1986;46:6387. [28] Bisht S, Maitra A. Dextran–doxorubicin/chitosan nanoparticles for solid tumor therapy. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 2009;1:415-25. [29] Lin WJ, Juang LW, Lin CC. Stability and release performance of a series of pegylated copolymeric micelles. Pharmaceutical research. 2003;20:668-73. [30] Bechet D, Couleaud P, Frochot C, Viriot M-L, Guillemin F, Barberi-Heyob M. Nanoparticles as vehicles for delivery of photodynamic therapy agents. Trends in Biotechnology. 2008;26:612-21. [31] Konan YN, Gurny R, Allemann E. State of the art in the delivery of photosensitizers for photodynamic therapy. Journal of Photochemistry and Photobiology B: Biology. 2002;66:89-106. [32] Zhang Z, Feng S-S. The drug encapsulation efficiency, in vitro drug release, cellular uptake and cytotoxicity of paclitaxel-loaded poly(lactide)-tocopheryl polyethylene glycol succinate nanoparticles. Biomaterials. 2006;27:4025-33. [33] Carlota, Rangel-Yagui, Pessoa-Jr A, Tavares LC. Micellar solubilization of drugs. J Pharm Pharmaceut Sci. 2005;8:147-63. [34] Chevalier Y, Zemb T. The structure of micelles and microemulsions. Reports on Progress in Physics 1990;53:279-370. [35] Nishiyama N, Kataoka K. Current state, achievements, and future prospects of polymeric micelles as nanocarriers for drug and gene delivery. Pharmacology & Therapeutics. 2006;112:630-48. [36] Sutton D, Nasongkla N, Blanco E, Gao J. Functionalized Micellar Systems for Cancer Targeted Drug Delivery Pharmaceutical Research. 2007;24:1029-46. [37] Riley T, Stolnik S, Heald CR, Xiong CD, Garnett MC, Illum L, et al. Physicochemical Evaluation of Nanoparticles Assembled from Poly(lactic acid)-Poly(ethylene glycol) (PLA-PEG) Block Copolymers as Drug Delivery Vehicles. Langmuir. 2001;17:3168-74. [38] Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov. 2003;2:347-60. [39] Lin WJ, Juang LW, Lin CC. Stability and Release Performance of a Series of Pegylated Copolymeric Micelles. Pharmaceutical Research. 2003;20:668-73. [40] Gaucher G, Dufresne M-H, Sant VP, Kang N, Maysinger D, Leroux J-C. Block copolymer micelles: preparation, characterization and application in drug delivery. Journal of Controlled Release. 2005;109:169-88. [41] Savi, cacute, Radoslav, Eisenberg A, Maysinger D. Block copolymer micelles as delivery vehicles of hydrophobic drugs: Micelle–cell interactions. Journal of Drug Targeting. 2006;14:343 - 55. [42] Maeda H, Wu J, Sawa T, Matsumura Y, Hori K. Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review. Journal of Controlled Release. 2000;65:271-84. [43] Duncan R. Polymer conjugates for tumour targeting and intracytoplasmic delivery. The EPR effect as a common gateway? Pharmaceutical Science & Technology Today. 1999;2:441-9. [44] Ganta S, Devalapally H, Shahiwala A, Amiji M. A review of stimuli-responsive nanocarriers for drug and gene delivery. Journal of Controlled Release. 2008;126:187-204. [45] Park EK, Kim SY, Lee SB, Lee YM. Folate-conjugated methoxy poly(ethylene glycol)/poly([var epsilon]-caprolactone) amphiphilic block copolymeric micelles for tumor-targeted drug delivery. Journal of Controlled Release. 2005;109:158-68. [46] Olivier J-C, Huertas R, Lee HJ, Calon F, Pardridge WM. Synthesis of Pegylated Immunonanoparticles Pharmaceutical Research. 2002;19:1137-43. [47] Paul H. in Control Release Polymeric Formulations. American Chemical Society, Washington, DC. 1976. [48] Wise. D, Fellman T, et al. Drug Carriers in Bioligy and Medicine, Academic Press, London. 1976:237. [49] Marius Murariu, Amalia Da Silva Ferreira, Michael Alexandre, Dubois. P. Polylactide (PLA) designed with desired end-use properties: 1. PLA compositions with low molecular weight ester-like plasticizers and related performances. Polymers for Advanced Technologies. 2008;19:636-46. [50] Burt HM, Zhang X, Toleikis P, Embree L, Hunter WL. Development of copolymers of poly(,-lactide) and methoxypolyethylene glycol as micellar carriers of paclitaxel. Colloids and Surfaces B: Biointerfaces. 1999;16:161-71. [51] Li Y, Lissel T. Synthesis and properties of biodegradable ABA triblock copolymers consisting of poly( -lactic acid) or poly( -lactic-co-glycolic acid) A-blocks attached to central poly(oxyethylene) B-blocks. J Control Rel. 1993;27:247-57. [52] Matsumoto J, Nakada Y, Sakurai K, Nakamura T, Takahashi Y. Preparation of nanoparticles consisted of poly(-lactide)-poly(ethylene glycol)-poly(-lactide) and their evaluation in vitro. International Journal of Pharmaceutics. 1999;185:93-101. [53] Vittaz M, Bazile D, Spenlehauer G, Verrecchia T, Veillard M, Puisieux F, et al. Effect of PEO surface density on long-circulating PLA-PEO nanoparticles which are very low complement activators. Biomaterials. 1996;17:1575-81. [54] Liu H, Farrell S, Uhrich K. Drug release characteristics of unimolecular polymeric micelles. Journal of Controlled Release. 2000;68:167-74. [55] Avgoustakis K, Beletsi A, Panagi Z, Klepetsanis P, Karydas AG, Ithakissios DS. PLGA-mPEG nanoparticles of cisplatin: in vitro nanoparticle degradation, in vitro drug release and in vivo drug residence in blood properties. Journal of Controlled Release. 2002;79:123-35. [56] Riley T, Govender T, Stolnik S, Xiong CD, Garnett MC, Illum L, et al. Colloidal stability and drug incorporation aspects of micellar-like PLA-PEG nanoparticles. Colloids and Surfaces B: Biointerfaces. 1999;16:147-59. [57] Mihir Sheth RAKVDRAGSPM. Biodegradable polymer blends of poly(lactic acid) and poly(ethylene glycol). Journal of Applied Polymer Science. 1997;66:1495-505. [58] Yokoyama M, Miyauchi M, Yamada N, Okano T, Sakurai Y, Kataoka K, et al. Characterization and Anticancer Activity of the Micelle-forming Polymeric Anticancer Drug Adriamycin-conjugated Poly(ethylene glycol)-Poly(aspartic acid) Block Copolymer. Cancer Res. 1990;50:1693-700. [59] Nakanishi T, Fukushima S, Okamoto K, Suzuki M, Matsumura Y, Yokoyama M, et al. Development of the polymer micelle carrier system for doxorubicin. Journal of Controlled Release. 2001;74:295-302. [60] Kawano K, Watanabe M, Yamamoto T, Yokoyama M, Opanasopit P, Okano T, et al. Enhanced antitumor effect of camptothecin loaded in long-circulating polymeric micelles. Journal of Controlled Release. 2006;112:329-32. [61] Yokoyama M, Okano T, Sakurai Y, Ekimoto H, Shibazaki C, Kataoka K. Toxicity and Antitumor Activity against Solid Tumors of Micelle-forming Polymeric Anticancer Drug and Its Extremely Long Circulation in Blood. Cancer Res. 1991;51:3229-36. [62] Piskin E, Kaitian X, Denkbas EB, Kucukyavuz Z. Novel PDLLA/PEG copolymer micelles as drug carriers. J Biomater Sci Polym Ed. 1995;7:359-73. [63] Yamamoto Y, Nagasaki Y, Kato M, Kataoka K. Surface charge modulation of poly(ethylene glycol)-poly(,-lactide) block copolymer micelles: conjugation of charged peptides. Colloids and Surfaces B: Biointerfaces. 1999;16:135-46. [64] Danhier F, Feron O, Préat V. To exploit the tumor microenvironment: Passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. Journal of Controlled Release. 2010;148:135-46. [65] Gosk S, Moos T, Gottstein C, Bendas G. VCAM-1 directed immunoliposomes selectively target tumor vasculature in vivo. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2008;1778:854-63. [66] Adams GP, Schier R, McCall AM, Simmons HH, Horak EM, Alpaugh RK, et al. High Affinity Restricts the Localization and Tumor Penetration of Single-Chain Fv Antibody Molecules. Cancer Res. 2001;61:4750-5. [67] Allen TM. Ligand-targeted therapeutics in anticancer therapy. Nat Rev Cancer. 2002;2:750-63. [68] Peer D, Karp JM, Hong S, Farokhzad OC, Margalit R, Langer R. Nanocarriers as an emerging platform for cancer therapy. Nat Nano. 2007;2:751-60. [69] Pang Z, Lu W, Gao H, Hu K, Chen J, Zhang C, et al. Preparation and brain delivery property of biodegradable polymersomes conjugated with OX26. Journal of Controlled Release. 2008;128:120-7. [70] Olivier J-C, Huertas R, Lee HJ, Calon F, Pardridge WM. Synthesis of Pegylated Immunonanoparticles. Pharmaceutical Research. 2002;19:1137-43. [71] Vega J, Ke S, Fan Z, Wallace S, Charsangavej C, Li C. Targeting Doxorubicin to Epidermal Growth Factor Receptors by Site-Specific Conjugation of C225 to Poly(L-Glutamic Acid) Through a Polyethylene Glycol Spacer. Pharmaceutical Research. 2003;20:826-32. [72] Yang J, Lim E-K, Lee HJ, Park J, Lee SC, Lee K, et al. Fluorescent magnetic nanohybrids as multimodal imaging agents for human epithelial cancer detection. Biomaterials. 2008;29:2548-55. [73] Pan XG, Wu G, Yang WL, Barth RF, Tjarks W, Lee RJ. Synthesis of cetuximab-immunoliposomes via a cholesterol-based membrane anchor for targeting of EGFR. Bioconjugate Chemistry. 2007;18:101-8. [74] Sun B, Feng S-S. Trastuzumab-functionalized nanoparticles of biodegradable copolymers for targeted delivery of docetaxel. Nanomedicine. 2009;4:431-45. [75] Cheng C, Wei H, Zhu J-L, Chang C, Cheng H, Li C, et al. Functionalized Thermoresponsive Micelles Self-Assembled from Biotin-PEG-b-P(NIPAAm-co-HMAAm)-b-PMMA for Tumor Cell Target. Bioconjugate Chemistry. 2008;19:1194-201. [76] Chen J, Wu H, Han D, Xie C. Using anti-VEGF McAb and magnetic nanoparticles as double-targeting vector for the radioimmunotherapy of liver cancer. Cancer Lett. 2006;231:169-75. [77] Masui H, Kawamoto T, Sato JD, Wolf B, Sato G, Mendelsohn J. Growth Inhibition of Human Tumor Cells in Athymic Mice by Anti-Epidermal Growth Factor Receptor Monoclonal Antibodies. Cancer Res. 1984;44:1002-7. [78] Wild R, Fager K, Flefleh C, Kan D, Inigo I, Castaneda S, et al. Cetuximab preclinical antitumor activity (monotherapy and combination based) is not predicted by relative total or activated epidermal growth factor receptor tumor expression levels. Molecular Cancer Therapeutics. 2006;5:104-13. [79] Hadjipanayis CG, Machaidze R, Kaluzova M, Wang L, Schuette AJ, Chen H, et al. EGFRvIII Antibody–Conjugated Iron Oxide Nanoparticles for Magnetic Resonance Imaging–Guided Convection-Enhanced Delivery and Targeted Therapy of Glioblastoma. Cancer Res. 2010;70:6303-12. [80] Hirsch FR, Scagliotti GV, Langer CJ, Varella-Garcia M, Franklin WA. Epidermal growth factor family of receptors in preneoplasia and lung cancer: perspectives for targeted therapies. Lung cancer (Amsterdam, Netherlands). 2003;41:29-42. [81] Bhattacharyya S, Bhattacharya R, Curley S, McNiven MA, Mukherjee P. Nanoconjugation modulates the trafficking and mechanism of antibody induced receptor endocytosis. Proceedings of the National Academy of Sciences of the United States of America. 2010;107:14541-6. [82] Patra CR, Bhattacharya R, Wang EF, Katarya A, Lau JS, Dutta S, et al. Targeted delivery of gemcitabine to pancreatic adenocarcinoma using cetuximab as a targeting agent. Cancer Res. 2008;68:1970-8. [83] 洪文周. 含NNO三牙Schiff-Base之鋅和鎂錯化合物的合成及結構鑑定:乳酸交酯開環聚合反應的良好催化劑. 中興大學化學所博士論文. 2009. [84] Li Y, Qi XR, Maitani Y, Nagai T. PEG-PLA diblock copolymer micelle-like nanoparticles as all-trans-retinoic acid carrier: in vitro and in vivo characterizations. Nanotechnology. 2009;20. [85] Wilhelm M, Zhao CL, Wang Y, Xu R, Winnik MA, Mura JL, et al. Poly(styrene-ethylene oxide) block copolymer micelle formation in water: a fluorescence probe study. Macromolecules. 1991;24:1033-40. [86] Aliabadi HM, Mahmud A, Sharifabadi AD, Lavasanifar A. Micelles of methoxy poly(ethylene oxide)-b-poly([var epsilon]-caprolactone) as vehicles for the solubilization and controlled delivery of cyclosporine A. Journal of Controlled Release. 2005;104:301-11. [87] Ellman GL. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics. 1959;82:70-7. [88] Debye* P. LIGHT SCATTERING IN SOAP SOLUTIONS. Annals of the New York Academy of Sciences. 1949;51:575-92. [89] Heimbrook DC, Stirdivant SM, Ahern JD, Balishin NL, Patrick DR, Edwards GM, et al. Transforming growth factor alpha-Pseudomonas exotoxin fusion protein prolongs survival of nude mice bearing tumor xenografts. Proceedings of the National Academy of Sciences. 1990;87:4697-701. [90] Kim SC, Kim DW, Shim YH, Bang JS, Oh HS, Kim SW, et al. In vivo evaluation of polymeric micellar paclitaxel formulation: toxicity and efficacy. Journal of Controlled Release. 2001;72:191-202. [91] Stridsberg K, Ryner M, Albertsson A-C. Controlled Ring-Opening Polymerization: Polymers with designed Macromolecular Architecture. Degradable Aliphatic Polyesters: Springer Berlin / Heidelberg; 2002. p. 41-65. [92] Riess G. Micellization of block copolymers. Progress in Polymer Science. 2003;28:1107-70. [93] Pan X, Wu G, Yang W, Barth RF, Tjarks W, Lee RJ. Synthesis of Cetuximab-Immunoliposomes via a Cholesterol-Based Membrane Anchor for Targeting of EGFR. Bioconjugate Chemistry. 2006;18:101-8. [94] Nehilla BJ, Vu TQ, Desai TA. Stoichiometry-dependent formation of quantum dot-antibody bioconjugates: A complementary atomic force microscopy and agarose gel electrophoresis study. J Phys Chem B. 2005;109:20724-30.
摘要: 光動力治療在臨床上較傳統外科手術具有非侵入式治療的優勢,可以在特定位置持續做治療並且在治療後不易留有疤痕,並且保有原組織或器官的功能性;相反地,傳統的外科手術則必須切除病灶及周圍組織,除了容易留下大範圍的傷口及疤痕外,術後傷口的復元需要較長與較多的照護與時間,另外,組織或器官的功能性或外觀有可能因此受到影響。光動力治療為一光化學反應,係由三個基本的要素所構成,主要是由光、光感藥物、和氧氣交互作用而產生的反應,其原理主要是由光來激發光感藥物,使光感藥物和組織間的氧氣發生能量交換或是電子轉移,進而產生單態氧(Singlet oxygen, 1O2)物質或是活性氧物質(Reactive oxygen species, ROS)來達到治療的效果。目前已有多種光感藥物在臨床上被使用,像是Foscan®、Photofrin®、ALA 等,用於肺癌、皮膚癌、口腔癌、頭頸癌等治療。 單株抗體,像是cetuximab、herceptin、Bevacizumab等等,在臨床標靶癌症治療已經行之有年,單株抗體會主動標靶在特定的接受器上並使腫瘤生長受到抑制。癌細胞上常過度表現某些蛋白接受器,如大量表皮生長因子接受器(Epidermal growth factor receptor, EGFR)的癌細胞,則可使用具有選擇性的單株抗體,如cetuximab,進行標靶治療。常見的單株抗體,如cetuximab多用在轉移性大腸癌、非小細胞肺癌、頭頸癌,而herceptin多用於乳癌、乳腺癌之標靶治療。 在目前光動力治療和標靶治療,是未來癌症醫療發展的趨勢,若能結合兩者治療的優勢,期望能為癌症醫療或研究有所助益。在本篇論文中,備製cetuximab (C225)結合高分子微胞包覆光感藥物並運用在主動標靶性光動力治療上為本篇論文之目的。首先,包覆光感藥物的高分子微胞是由聚乳酸聚乙二醇之共聚物和光感藥物經由溶劑蒸發方法所製備,接著,利用改質後C225上的硫醇基和高分子微胞表面上的順丁烯二醯亞胺形成共價鍵結將C225修飾於包覆光感藥物的高分子微胞表面。此篇論文中的結果顯示,修飾了C225的高分子微胞具有良好的選擇性,並能標靶至表皮生長因子接受器過度表現的癌細胞 (A431)上,若再經由光的照射後能有效的殺死癌細胞,此系統在主動標靶性光動力治療用於癌症治療上可能具有卓越的療效。
Monoclonal antibodies, such as cetuximab(C225), herceptin, etc., have been utilized for target cancer therapy in clinic. In this study, C225 conjugated polymeric micelles with photosensitizer loading were prepared for active targeting photodynamic therapy (PDT). First, mixed micelle composed of poly(ethylene glycol)-b- poly(lactide) and maleimide-poly(ethylene glycol)-b-poly(lactide) with photosensitizer loading were prepared by solvent evaporation method. Next, the C225 antibody was conjugated to the micelle surface via thiol group and maleimide group coupling. Our results show that the photosensitizer-loaded C225-conjugated micelle can target selectively to the epidermal growth factor receptor (EGFR) overexpressed cancer cells with efficient cell killing ability after irradiation whereas most EGFR-negative cells survived under the same treatment. Thus, this active-targetable photosensitizer delivery system may have great potential for cancer therapy.
URI: http://hdl.handle.net/11455/16932
其他識別: U0005-3008201122204000
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