本研究的目的為開發以透明質酸(hyaluronic acid)接枝 LHRH胜肽作為主動標靶的奈米藥物載體(drug carrier)，此藥物載體為一雙性(amphiphilic)物質，在水溶液中可自我聚合形成奈米顆粒(self-assembling nanoparticle)。抗癌藥物doxorubicin與透明質酸間利用順式烏頭酸鍵(cis-aconityl linkage)及雙硫鍵(disulfide bond)來做鍵結，使之分別具有酸鹼敏感性(pH sensitivity)與氧化還原敏感性(redox sensitivity)，以及緩效釋放(controlled drug release)與抗癌效果(anti-cancer effect)。物化性質分析方面，利用核磁共振頻譜(1H NMR spectra)分析化學結構，以及計算接枝比率(grafting ratio)，結果顯示帶有胺基的長鏈接枝率(grafting ratio)約17.8 %，而藥物的接枝率約6.2%。為了確認奈米顆粒的形成與分布情形(size distribution)，利用動態光散射儀(dynamic light scattering, DLS)來測量所形成的粒徑平均大小(particle size)約為229.0 nm，奈米顆粒的形狀則是藉由穿透式電子顯微鏡(TEM)來觀察，並以分光光度法(spectrophotometric analysis)測量藥物在奈米顆粒中的含量(drug content)。氧化還原與酸鹼敏感性是透過添加二硫蘇糖醇(dithiothreitol, DTT)與改變pH值來量測。螢光影像顯示表面修飾LHRH胜肽有助於提升癌細胞對奈米顆粒的專一性胞吞，此藥物載體可抑制癌細胞OVCAR-3的增生，並且對正常細胞3T3沒有明顯的細胞毒性(cytotoxicity)。本研究證實了透過上述方法能成功製備出以透明質酸為主成分的主動標靶自我聚合奈米藥物載體，該載體具有多功能性(multifunctional)，在酸性環境(pH<7.0)或還原環境(reductive condition)中，可以釋放所包覆的抗癌藥物，達到緩效持續的藥物釋放效果(sustained release)。原位卵巢腫瘤動物模型(orthotopic ovarian tumor model)被建立用以評估活體動物治療與診斷效果，利用IVIS觀測腫瘤生長情形，顯示接枝有LHRH的奈米顆粒可有效抑制腫瘤增生及具有腫瘤顯影功能。

The purpose of this study is to develop a redox and pH-sensitive self-assembling hyaluronic acid nanoparticle with active targeting peptide for anticancer drug delivery. Anti-cancer drug, doxorubicin (DOX), is grafted onto hyaluronic acid (HA) via cis-aconityl linkage and disulfide bond to possess pH sensitivity and redox property, respectively. The conjugate is successfully developed and the grafting ratio of cystamine (cys) is 17.8% with drug loading amount about 6.2% calculated by 1H NMR spectra. This conjugate is amphiphilic and can self-assemble into nanoparticle (NP) in aqueous solution. The particle size is approximately 229.0 nm with narrow size distribution using dynamic light scatting (DLS) measurement and the morphology of nanoparticles is observed as spherical shape by transmission electron microscope (TEM). The pH and redox sensitivity are evaluated by changing either pH value or concentration of dithiothreitol (DTT) in the medium. It is proved that the drug carrier is capable of achieving sustained controlled release of anti-cancer drug to 80% within 120 hours. The intracellular uptake is observed by fluorescent microscope and the images show that conjugating luteinizing hormone-releasing hormone (LHRH) peptide can enhance specific uptake of nanoparticles by OVCAR-3 cancer cells; thus, resulting in inhibitory cell growth less than 20% in 72 hours in vitro. Orthotopic ovarian tumor model is also established to evaluate the therapeutic and diagnostic efficacy using non-invasive in vivo imaging system (IVIS). The representative results demonstrate that LHRH-conjugated NPs possess a preferable tumor imaging capability and an excellent antitumor ability to almost 30% of original size.

Table of Content
致 謝 I
Abstract II
摘 要 IV
Chapter 1. Introduction - 1 -
1-1 Hyaluronic acid-based drug carrier - 1 -
1-2 Cis-aconityl linkage & disulfide bond - 3 -
1-3 Self-assembling nanoparticle - 4 -
1-4 Luteinizing hormone-releasing hormone (LHRH) peptide - 8 -
1-5 Optical Imaging with Near-infrared (NIR) fluorescent Cy5.5 - 9 -
1-6 Motivation and purpose of this study - 10 -
Chapter 2. Literature Review - 14 -
2-1. Stimuli-sensitive linkages or covalent bonds - 14 -
2-1.1 Linkages with pH sensitivity - 15 -
2-1.2 Covalent bond with redox sensitivity - 17 -
2-2 Target to cancer tissue - 20 -
2-2.1 Passive targeting - 21 -
2-2.2 Active targeting - 23 -
2-3 Theranostic nanoparticles - 25 -
Chapter 3. Theoretical Basis - 27 -
3-1 Delivery of nanoparticles to pathological site - 27 -
3-2 Intracellular uptake of nanoparticles - 28 -
3-3 Cleavage of covalent bond and release of drug - 31 -
Chapter 4. Materials and Methods - 33 -
4-1 Materials - 33 -
4-2 Synthesis of LHRH-HA-cys-ADOX conjugate - 34 -
4-2.1 Addition of LHRH peptide on hyaluronic acid - 34 -
4-2.2 Modification of hyaluronic acid with cystamine - 35 -
4-2.3 Preparation of HA-cys-ADOX or LHRH-HA-cys-ADOX conjugate - 36 -
4-3 Characterization of HA-cys-ADOX conjugate - 38 -
4-4 Preparation and characterization of NPs - 39 -
4-5 In vitro drug release profile triggered by pH or glutathione - 39 -
4-6 In vitro intracellular uptake study - 40 -
4-7 In vitro cytotoxicity and anticancer effect - 41 -
4-8 Establishment of orthotopic ovarian cancer xenograft model - 42 -
4-9 In vivo therapeutic effect and biodistribution - 43 -
4-10 Statistical analysis - 43 -
Chapter 5. Results - 44 -
5-1 Characterization of LHRH-HA-cys-ADOX conjugate - 44 -
5-2 Characterization of HA-cys-ADOX nanoparticles - 46 -
5-3 In vitro drug release profile triggered by pH or glutathione - 50 -
5-4 In vitro intracellular uptake study - 52 -
5-5 In vitro cytotoxicity and anticancer effect - 54 -
5-6 Establishment of orthotopic ovarian cancer xenograft model - 56 -
5-7 In vivo therapeutic effect and biodistribution - 57 -
5-8 Histological Stain of Tissue Section - 61 -
Chapter 6. Discussion - 63 -
6-1 Characterization of LHRH-HA-cys-ADOX conjugate - 63 -
6-2 Characterization of HA-cys-ADOX nanoparticles - 64 -
6-3 In vitro drug release profile triggered by pH or glutathione - 64 -
6-4 In vitro intracellular uptake study - 65 -
6-5 In vitro cytotoxicity and anticancer effect - 66 -
6-6 Establishment of orthotopic ovarian cancer xenograft model - 67 -
6-7 In vivo therapeutic effect and biodistribution - 67 -
6-8 Histological Stain of Tissue Section - 69 -
Chapter 7. Conclusion - 70 -
Reference - 71 -

[1] Oh EJ, Park K, Kim KS, Kim J, Yang JA, Kong JH, et al. Target specific and long-acting delivery of protein, peptide, and nucleotide therapeutics using hyaluronic acid derivatives. J Control Release. 2010;141:2-12.
[2] Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B. Cd44 Is the Principal Cell-Surface Receptor for Hyaluronate. Cell. 1990;61:1303-13.
[3] Schiffelers RM, Ansari A, Xu J, Zhou Q, Tang Q, Storm G, et al. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucleic Acids Res. 2004;32:e149.
[4] Entwistle J, Hall CL, Turley EA. HA receptors: regulators of signalling to the cytoskeleton. J Cell Biochem. 1996;61:569-77.
[5] Choi KY, Saravanakumar G, Park JH, Park K. Hyaluronic acid-based nanocarriers for intracellular targeting: interfacial interactions with proteins in cancer. Colloid Surf B-Biointerfaces 2012;99:82-94.
[6] Choi KY, Min KH, Na JH, Choi K, Kim K, Park JH, et al. Self-assembled hyaluronic acid nanoparticles as a potential drug carrier for cancer therapy: synthesis, characterization, and in vivo biodistribution. J Mater Chem. 2009;19:4102-7.
[7] Christie RJ, Grainger DW. Design strategies to improve soluble macromolecular delivery constructs. Adv Drug Deliv Rev. 2003;55:421-37.
[8] Ulbrich K, Subr V. Polymeric anticancer drugs with pH-controlled activation. Adv Drug Deliv Rev. 2004;56:1023-50.
[9] D'Souza AJ, Topp EM. Release from polymeric prodrugs: linkages and their degradation. J Pharm Sci. 2004;93:1962-79.
[10] Li J, Huo M, Wang J, Zhou J, Mohammad JM, Zhang Y, et al. Redox-sensitive micelles self-assembled from amphiphilic hyaluronic acid-deoxycholic acid conjugates for targeted intracellular delivery of paclitaxel. Biomaterials. 2012;33:2310-20.
[11] Pan YJ, Chen YY, Wang DR, Wei C, Guo J, Lu DR, et al. Redox/pH dual stimuli-responsive biodegradable nanohydrogels with varying responses to dithiothreitol and glutathione for controlled drug release. Biomaterials. 2012;33:6570-9.
[12] Ganesh S, Iyer AK, Morrissey DV, Amiji MM. Hyaluronic acid based self-assembling nanosystems for CD44 target mediated siRNA delivery to solid tumors. Biomaterials. 2013;34:3489-502.
[13] Liu Y, Sun J, Zhang P, He Z. Amphiphilic polysaccharide-hydrophobicized graft polymeric micelles for drug delivery nanosystems. Curr Med Chem. 2011;18:2638-48.
[14] Oerlemans C, Bult W, Bos M, Storm G, Nijsen JF, Hennink WE. Polymeric micelles in anticancer therapy: targeting, imaging and triggered release. Pharm Res. 2010;27:2569-89
[15] Nicolas J, Mura S, Brambilla D, Mackiewicz N, Couvreur P. Design, functionalization strategies and biomedical applications of targeted biodegradable/biocompatible polymer-based nanocarriers for drug delivery. Chem Soc Rev. 2013;42:1147-235.
[16] Bisht S, Maitra A. Dextran-doxorubicin/chitosan nanoparticles for solid tumor therapy. Wiley Interdiscip Rev-Nanomed Nanobiotechnol. 2009;1:415-25.
[17] Kakar SS, Jin H, Hong B, Eaton JW, Kang KA. LHRH receptor targeted therapy for breast cancer. Adv Exp Med Biol. 2008;614:285-96.
[18] Luo S, Zhang E, Su Y, Cheng T, Shi C. A review of NIR dyes in cancer targeting and imaging. Biomaterials. 2011;32:7127-38.
[19] Ballou B, Ernst LA, Waggoner AS. Fluorescence imaging of tumors in vivo. Curr Med Chem. 2005;12:795-805.
[20] Lavis LD, Raines RT. Bright ideas for chemical biology. ACS Chem Biol. 2008;3:142-55.
[21] Wang W, Ke S, Wu Q, Charnsangavej C, Gurfinkel M, Gelovani JG, et al. Near-infrared optical imaging of integrin alphavbeta3 in human tumor xenografts. Mol Imaging. 2004;3:343-51.
[22] Fleige E, Quadir MA, Haag R. Stimuli-responsive polymeric nanocarriers for the controlled transport of active compounds: concepts and applications. Adv Drug Deliv Rev. 2012;64:866-84.
[23] Torchilin V. Multifunctional and stimuli-sensitive pharmaceutical nanocarriers. Eur J Pharm Biopharm. 2009;71:431-44.
[24] Son YJ, Jang JS, Cho YW, Chung H, Park RW, Kwon IC, et al. Biodistribution and anti-tumor efficacy of doxorubicin loaded glycol-chitosan nanoaggregates by EPR effect. J Control Release. 2003;91:135-45.
[25] Park JH, Kwon S, Lee M, Chung H, Kim JH, Kim YS, et al. Self-assembled nanoparticles based on glycol chitosan bearing hydrophobic moieties as carriers for doxorubicin: in vivo biodistribution and anti-tumor activity. Biomaterials. 2006;27:119-26.
[26] Hu FQ, Liu LN, Du YZ, Yuan H. Synthesis and antitumor activity of doxorubicin conjugated stearic acid-g-chitosan oligosaccharide polymeric micelles. Biomaterials. 2009;30:6955-63.
[27] Zhu S, Qian L, Hong M, Zhang L, Pei Y, Jiang Y. RGD-modified PEG-PAMAM-DOX conjugate: in vitro and in vivo targeting to both tumor neovascular endothelial cells and tumor cells. Adv Mater. 2011;23:H84-9.
[28] Kakinoki A, Kaneo Y, Ikeda Y, Tanaka T, Fujita K. Synthesis of poly(vinyl alcohol)-doxorubicin conjugates containing cis-aconityl acid-cleavable bond and its isomer dependent doxorubicin release. Biol Pharm Bull. 2008;31:103-10.
[29] Lee H, Choi SH, Park TG. Direct visualization of hyaluronic acid polymer chain by self-assembled one-dimensional array of gold nanoparticles. Macromolecules. 2006;39:23-5.
[30] Yeom J, Chang S, Park JK, Je JH, Yang DJ, Choi SK, et al. Synchrotron X-ray bioimaging of bone regeneration by artificial bone substitute of MegaGen Synthetic Bone and hyaluronate hydrogels. Tissue Eng. 2010;16:1059-68.
[31] Lee H, Mok H, Lee S, Oh YK, Park TG. Target-specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels. J Control Release. 2007;119:245-52.
[32] Sharma A, Jain N, Sareen R. Nanocarriers for diagnosis and targeting of breast cancer. BioMed Res Int. 2013;2013:960821.
[33] Li H, Bian S, Huang Y, Liang J, Fan Y, Zhang X. High drug loading pH-sensitive pullulan-DOX conjugate nanoparticles for hepatic targeting. J Biomed Mater Res Part A. 2013;102:150-9.
[34] Engel J, Emons G, Pinski J, Schally AV. AEZS-108 : a targeted cytotoxic analog of LHRH for the treatment of cancers positive for LHRH receptors. Expert Opin Investig Drugs. 2012;21:891-9.
[35] Dharap SS, Wang Y, Chandna P, Khandare JJ, Qiu B, Gunaseelan S, et al. Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide. Proc Natl Acad Sci U S A. 2005;102:12962-7.
[36] Xie J, Lee S, Chen X. Nanoparticle-based theranostic agents. Adv Drug Deliv Rev. 2010;62:1064-79.
[37] Koo H, Huh MS, Sun IC, Yuk SH, Choi K, Kim K, et al. In vivo targeted delivery of nanoparticles for theranosis. Acc Chem Res. 2011;44:1018-28.
[38] Kim K, Kim JH, Park H, Kim YS, Park K, Nam H, et al. Tumor-homing multifunctional nanoparticles for cancer theragnosis: Simultaneous diagnosis, drug delivery, and therapeutic monitoring. J Control Release. 2010;146:219-27.
[39] Chen YC, Lo CL, Hsiue GH. Multifunctional nanomicellar systems for delivering anticancer drugs. J Biomed Mater Res A. 2014;102:2024-38.
[40] Sakhrani NM, Padh H. Organelle targeting: third level of drug targeting. Drug Des Dev Ther. 2013;7:585-99.
[41] Shen WC, Ryser HJ. cis-Aconityl spacer between daunomycin and macromolecular carriers: a model of pH-sensitive linkage releasing drug from a lysosomotropic conjugate. Biochem Biophys Res Commun. 1981;102:1048-54.
[42] Srinophakun T, Boonmee J. Preliminary Study of Conformation and Drug Release Mechanism of Doxorubicin-Conjugated Glycol Chitosan, via cis-Aconityl Linkage, by Molecular Modeling. Int J Mol Sci. 2011;12:1672-83.
[43] Su WY, Chen KH, Chen YC, Lee YH, Tseng CL, Lin FH. An injectable oxidated hyaluronic acid/adipic acid dihydrazide hydrogel as a vitreous substitute. J Biomater Sci, Polym Ed. 2011;22:1777-97.
[44] Burdick JA, Prestwich GD. Hyaluronic acid hydrogels for biomedical applications. Adv Mater. 2011;23:H41-56.
[45] Yang JA, Park K, Jung H, Kim H, Hong SW, Yoon SK, et al. Target specific hyaluronic acid-interferon alpha conjugate for the treatment of hepatitis C virus infection. Biomaterials. 2011;32:8722-9.
[46] Park JH, Cho YW, Son YJ, Kim K, Chung H, Jeong SY, et al. Preparation and characterization of self-assembled nanoparticles based on glycol chitosan bearing adriamycin. Colloid Polym Sci. 2006;284:763-70.
[47] Alshamkhani A, Duncan R. Synthesis, Controlled-Release Properties and Antitumor-Activity of Alginate-Cis-Aconityl-Daunomycin Conjugates. Int J Pharm. 1995;122:107-19.
[48] Cordero AB, Kwon Y, Hua X, Godwin AK. In vivo imaging and therapeutic treatments in an orthotopic mouse model of ovarian cancer. J Vis Exp. 2010;17:2125.
[49] Zhang R, Luo K, Yang J, Sima M, Sun Y, Janat-Amsbury MM, et al. Synthesis and evaluation of a backbone biodegradable multiblock HPMA copolymer nanocarrier for the systemic delivery of paclitaxel. J Control Release. 2013;166:66-74.
[50] Choi KY, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, et al. PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo. Biomaterials. 2011;32:1880-9.
[51] Han HS, Lee J, Kim HR, Chae SY, Kim M, Saravanakumar G, et al. Robust PEGylated hyaluronic acid nanoparticles as the carrier of doxorubicin: mineralization and its effect on tumor targetability in vivo. J Control Release. 2013;168:105-14.
[52] Jiang T, Zhang Z, Zhang Y, Lv H, Zhou J, Li C, et al. Dual-functional liposomes based on pH-responsive cell-penetrating peptide and hyaluronic acid for tumor-targeted anticancer drug delivery. Biomaterials. 2012;33:9246-58.
[53] Yao J, Zhang L, Zhou J, Liu H, Zhang Q. Efficient simultaneous tumor targeting delivery of all-trans retinoid acid and Paclitaxel based on hyaluronic acid-based multifunctional nanocarrier. Mol Pharm. 2013;10:1080-91.
[54] Min KH, Park K, Kim YS, Bae SM, Lee S, Jo HG, et al. Hydrophobically modified glycol chitosan nanoparticles-encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy. J Control Release. 2008;127:208-18.
[55] Satpathy M, Wang L, Zielinski R, Qian W, Lipowska M, Capala J, et al. Active Targeting Using HER-2-Affibody-Conjugated Nanoparticles Enabled Sensitive and Specific Imaging of Orthotopic HER-2 Positive Ovarian Tumors. Small. 2014;10:12.
[56] Lv S, Tang Z, Li M, Lin J, Song W, Liu H, et al. Co-delivery of doxorubicin and paclitaxel by PEG-polypeptide nanovehicle for the treatment of non-small cell lung cancer. Biomaterials. 2014;35:6118-29.
[57] Wang Y, Dou L, He H, Zhang Y, Shen Q. Multifunctional Nanoparticles as Nanocarrier for Vincristine Sulfate Delivery To Overcome Tumor Multidrug Resistance. Mol Pharm. 2014; 11:885-894.
[58] Thamake SI, Raut SL, Gryczynski Z, Ranjan AP, Vishwanatha JK. Alendronate coated poly-lactic-co-glycolic acid (PLGA) nanoparticles for active targeting of metastatic breast cancer. Biomaterials. 2012;33:7164-73.