進階搜尋


   電子論文尚未授權公開,紙本請查館藏目錄
(※如查詢不到或館藏狀況顯示「閉架不公開」,表示該本論文不在書庫,無法取用。)
系統識別號 U0026-2308201317524400
論文名稱(中文) 緩釋疫苗之貼片可溶式幾丁聚醣微針於經皮免疫之應用
論文名稱(英文) Sustained delivery of vaccine using patch-dissolvable chitosan microneedles for transcutaneous immunization
校院名稱 成功大學
系所名稱(中) 化學工程學系碩博士班
系所名稱(英) Department of Chemical Engineering
學年度 101
學期 2
出版年 102
研究生(中文) 賴冠穎
研究生(英文) Kuan-Ying Lai
學號 N36004440
學位類別 碩士
語文別 中文
論文頁數 58頁
口試委員 口試委員-朱純燕
口試委員-陳東煌
口試委員-陳毓宏
口試委員-許梅娟
指導教授-陳美瑾
中文關鍵字 佐劑  幾丁聚醣  鑲嵌式微針  緩慢釋放  經皮免疫 
英文關鍵字 adjuvant  chitosan  embeddable microneedles  sustained delivery  transcutaneous immunization 
學科別分類
中文摘要 現今開發之疫苗微針貼片僅侷限於快速釋放劑型,尚無可緩釋疫苗之微針,本論文為研發一鑲嵌式緩釋疫苗之新型微針系統,由將生物可降解之幾丁聚醣(chitosan, CS)微針與水溶性聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP)和聚乙烯醇(polyvinyl alcohol, PVA)支撐陣列組成,此微針能在穿刺皮膚後,藉由體內組織液將後端支撐陣列溶解,僅留幾丁聚醣針體在皮膚中,緩慢而持續地釋放疫苗;由體外豬皮及體內大鼠穿刺測試證實,此微針貼片具有足夠之機械強度可刺穿角質層,並讓微針鑲嵌於富含抗原呈現細胞之表皮層及真皮層中(深度650700 m),造成之微通道能夠在六小時內癒合,避免傷口感染之風險,穿刺後所產生之些微紅腫,可在三天內完全消失。將微針包覆螢光抗原後刺入老鼠背部皮膚,發現微針能在體內緩慢降解並持續釋放抗原長達28天。大鼠免疫試驗結果證實,微針僅須包覆肌肉注射40 % 之抗原量即可誘發相當或較高之抗體產生長達8週。以小鼠進行流感疫苗之免疫試驗,肌肉注射流感疫苗時,若添加幾丁聚醣溶液作為佐劑可快速增強免疫反應,在第2週即可產生高量抗體,而以微針施打流感疫苗,藉由微針在體內被緩慢降解可持續釋放出疫苗,達到類似多次補強注射(boost)效果,在接種4週時,抗體量明顯高於肌肉注射純病毒組及病毒加幾丁聚醣佐劑組,且在12週時抗體量為肌肉注射純病毒組之2.5倍。本研究所開發之貼片可溶式緩釋型幾丁聚醣微針接種不須長期黏貼貼片,即可達到經皮緩釋疫苗之目的,以較少的疫苗量,即達到類似肌肉注射的免疫反應,可有效節省疫苗量。
英文摘要 Currently developed microneedles for vaccine delivery always rapidly release encapsulated materials. Prolonged antigen release is highly desirable to induce a “depot” effect, which can result in a more potent and persistent immune response. This study introduces an integrated microneedle system, composed of embeddable chitosan microneedles with a dissolvable poly(vinyl pyrrolidone)/poly(vinyl alcohol) (PVP/PVA) supporting array, for complete and sustained delivery of encapsulated antigens to the skin. The strong PVP/PVA supporting array can provide mechanical strength to fully insert the microneedles into the skin. When inserted into rat and porcine skin, the skin interstitial fluid quickly dissolved the supporting array and chitosan microneedles were left within the skin for sustained drug delivery. The microneedle penetration depth was approximately 650-700 m (i.e. the total length of the microneedle), which is beneficial for targeted delivery of antigens to antigen-presenting cells in the epidermis and dermis. When the OVA-loaded microneedles were embedded in rat skin in vivo, histological examination showed that the microneedles gradually degraded and prolonged OVA releasing at the insertion sites for up to 28 days. Compared to traditional intramuscular immunization (500 g OVA), rats immunized by a lower microneedle dose of 200 g OVA showed a significantly higher OVA-specific antibody response on the second week which lasted for at least 8 weeks. Additionally, mice vaccinated with vaccine-loaded microneedles produced 2.5-fold influenza-specific antibody responses compared with those induced by the intramuscular immunization after 12 weeks. These results suggest that embeddable chitosan microneedles are a promising depot for extended delivery of encapsulated antigens, which may provide sustained immune stimulation and improve immunogenicity.
論文目次 摘要 I
Abstract II
致謝 III
目錄 IV
表目錄VI
圖目錄 VI
第一章 緒論 1
1.1經皮藥物傳輸與微針貼片 1
1.1.1 經皮藥物傳輸 1
1.1.2 微針貼片 3
1.2 疫苗緩釋 7
1.2.1 經皮免疫機制 7
1.2.2 流感疫苗 8
1.2.3 免疫佐劑與疫苗之持續釋放 9
1.3 材料 10
1.3.1 幾丁聚醣(chitosan) 10
1.3.2 海藻糖(trehalose) 12
1.3.3 聚乙烯吡咯烷酮(polyvinylpyrrolidone, PVP) 12
1.3.4 聚乙烯醇(Polyvinyl alcohol, PVA) 13
1.4 研究目的 14
第二章 實驗材料與方法 17
2.1 實驗藥品、耗材與動物 17
2.2 儀器設備 19
2.3 鑲嵌式微針貼片 21
2.3.1 幾丁聚醣微針貼片之製備 21
2.3.2 微針穿刺能力測試 23
2.3.3 皮膚穿刺傷口癒合測試 25
2.3.4 皮膚紅腫測試 25
2.4包覆抗原之鑲嵌式微針貼片 26
2.4.1 包覆Alexa Fluor 594-OVA微針貼片之製備 26
2.4.2 體內降解測試 27
2.4.3 體內抗原釋放測試 27
2.4.4 大鼠免疫試驗 28
2.5包覆流感疫苗之鑲嵌式微針貼片 31
2.5.1 包覆流感疫苗之幾丁聚醣微針貼片 31
2.5.2小鼠免疫試驗 31
第三章 結果與討論 33
3.1 鑲嵌式微針貼片 33
3.1.1 鑲嵌式幾丁聚醣微針貼片 33
3.1.2 微針穿刺能力分析 34
3.1.3 皮膚傷口癒合分析 34
3.1.4 皮膚紅腫測試結果 37
3.2包覆抗原之鑲嵌式微針貼片 38
3.2.1 包覆抗原之幾丁聚醣微針貼片 38
3.2.2 微針貼片降解分析 39
3.2.3 體內抗原釋放情形 46
3.2.4 抗原包覆定量與免疫測試 47
3.3包覆流感疫苗之鑲嵌式微針貼片 49
3.3.1微針定量結果 49
3.3.2小鼠免疫試驗結果 49
第四章 結論 51
參考文獻 52
表目錄
表 3-1 幾丁聚醣微針之抗原包覆量 47
表 3-2 幾丁聚醣微針之流感疫苗包覆量 49
圖目錄
圖 1-1 皮膚構造示意圖[11] 2
圖 1-2 應用於經皮給藥之系統與裝置[1] 3
圖 1-3 微針應用於經皮藥物傳輸方法示意圖[13] 3
圖 1-4 固體微針[13] 4
圖 1-5 塗佈型微針[13] 5
圖 1-6 可溶或可降解型高分子微針[13] 6
圖 1-7 中空型微針[13] 6
圖 1-8 經皮免疫機制[28] 8
圖 1-9 幾丁聚醣結構式 11
圖 1-10 海藻糖結構式 12
圖 1-11 聚乙烯吡咯烷酮結構式 13
圖 1-12 聚乙烯醇結構 13
圖 1-13 第二代鑲嵌式幾丁聚醣微針應用示意圖 14
圖 1-14 第一代鑲嵌式幾丁聚醣微針製作流程示意圖 15
圖 1-15 實驗架構 16
圖 2-1 不鏽鋼主結構:(a)金字塔微針;(b)支撐軸 21
圖 2-2 幾丁聚醣微針製備過程示意圖 23
圖 2-3 體外穿刺實驗流程示意圖 24
圖 2-4 體內穿刺實驗流程示意圖 25
圖 2-5 皮膚穿刺傷口癒合實驗流程示意圖 26
圖 2-6 非侵入式活體影像(IVIS)分析圖 27
圖 2-7 免疫試驗流程:IM: intramuscular injection; MN: microneedle; CS: chitosan hydrogel 29
圖 2-8 酵素結合免疫吸附法分析血中抗體濃度流程圖 30
圖 3-1 貼片可溶式幾丁聚醣微針:(a)支撐軸陣列光學顯微影像;(b)五倍放大後影像;(c)微針主體與支撐軸黏合後之光學顯微影像;(b)五倍放大後影像 33
圖 3-2 貼片可溶式幾丁聚醣微針對豬皮進行體外穿刺結果:(a)穿刺後之豬皮;(b)豬皮經染色處理結果;(c)微針穿刺前;(d)微針穿刺後 35
圖 3-3 接枝FITC之幾丁聚醣微針對大鼠進行體內穿刺之組織切片結果:(a)可見光影像;(b)螢光影像 35
圖 3-4 幾丁聚醣微針穿刺活體ICR小鼠、SD大鼠及LYD肉豬後,於0、2及6小時之穿刺部位數位影像 36
圖 3-5 大鼠皮膚經微針穿刺部位及未穿刺部位之表皮水分散失值 36
圖 3-6 大鼠皮膚經微針穿刺後不同時間下之紅腫程度:(a)Δa值曲線;(b) 穿刺後0分鐘、15分鐘、2小時、6小時、1天及3天之數位影像 37
圖 3-7 包覆Alexa 594-OVA之貼片可溶式幾丁聚醣微針:(a)光學顯微影像;(b)五倍放大後之光學顯微影像;(c)螢光影像 38
圖 3-8 多光子共軛焦顯微影像分析包覆Alexa 594-OVA幾丁聚醣微針穿刺大鼠背部後第0天,不同z軸深度之x-y平面螢光影像及3D影像圖 40
圖 3-9 多光子共軛焦顯微影像分析包覆Alexa 594-OVA幾丁聚醣微針穿刺大鼠背部後第7天,不同z軸深度之x-y平面螢光影像及3D影像圖 41
圖 3-10 多光子共軛焦顯微影像分析包覆Alexa 594-OVA幾丁聚醣微針穿刺大鼠背部後第14天,不同z軸深度之x-y平面螢光影像及3D影像圖 42
圖 3-11 多光子共軛焦顯微影像分析包覆Alexa 594-OVA幾丁聚醣微針穿刺大鼠背部後第21天,不同z軸深度之x-y平面螢光影像及3D影像圖 43
圖 3-12 多光子共軛焦顯微影像分析包覆Alexa 594-OVA幾丁聚醣微針穿刺大鼠背部後第28天,不同z軸深度之x-y平面螢光影像及3D影像 44
圖 3-13 幾丁聚醣微針鑲嵌於活體SD大鼠皮膚內緩慢降解之組織切片圖 45
圖 3-14 非侵入式活體影像(IVIS)分析體內OVA釋放情形:(a)螢光強度定量結果;(b)不同時間下之螢光影像(每片微針約包覆6.7 µg Alexa 594-OVA) 46
圖 3-15 包覆OVA之幾丁聚醣微針之光學顯微影像(支撐陣列含螢光染劑Rhodamine 6G) 48
圖 3-16 大鼠以抗原OVA免疫試驗產生特異性OVA IgG抗體之結果;IM : intramuscular injection (肌肉注射免疫);saline : 生理食鹽水;CS:幾丁聚醣水膠;MN : microneedle (微針經皮免疫) 48
圖 3-17 包覆流感疫苗H1N1之貼片可溶式幾丁聚醣微針之光學顯微影像 50
圖 3-18 小鼠以WSN流感病毒疫苗免疫試驗產生特異性流感病毒IgG抗體之結果;IM : intramuscular injection (肌肉注射免疫);saline : 生理食鹽水;CS:幾丁聚醣水膠;MN : microneedle (微針經皮免疫) 50

參考文獻 [1] Bal SM, Ding Z, van Riet E, Jiskoot W, Bouwstra JA. Advances in transcutaneous vaccine delivery: do all ways lead to Rome? J Controlled Release 2010;148:266-82.
[2] van der Maaden K, Jiskoot W, Bouwstra J. Microneedle technologies for (trans)dermal drug and vaccine delivery. J Controlled Release 2012;161:645-55.
[3] Pegoraro C, MacNeil S, Battaglia G. Transdermal drug delivery: from micro to nano. Nanoscale 2012;4:1881-94.
[4] Cevc GV, U. Spatial distribution of cutaneous microvasculature and local drug clearance after drug application on the skin. J Controlled Release 2007;118:18-26.
[5] MacNeil S. Biomaterials for tissue engineering of skin. Materials today 2008;11:26-35.
[6] Loan Honeywell-Nguyen P, Wouter Groenink HW, Bouwstra JA. Elastic Vesicles as a tool for dermal and transdermal delivery. J Liposome Res 2006;16:273-80.
[7] Elias PM. Epidermal lipids, barrier function, and desquamation. J Invest Dermatol 1983;80:44-9.
[8] Warner RR, Stone KJ, Boissy YL. Hydration disrupts human stratum corneum ultrastructure. J Invest Dermatol 2003;120:275-84.
[9] Arora A, Prausnitz MR, Mitragotri S. Micro-scale devices for transdermal drug delivery. Int J Phytorem 2008;364:227-36.
[10] Nestle FO, Di Meglio P,Qin JZ, Nickoloff BJ. Skin immune sentinels in health and disease. Nature reviews immunology 2009;9:679-91.
[11] Http://skininfo.org/normal-skin/normal-skin-structure/.
[12] Balmayor ER, Azevedo HS, Reis RL. Controlled delivery systems: from pharmaceuticals to cells and genes. Pharm Res 2011;28:1241-58.
[13] Kim YC, Park JH, Prausnitz MR. Microneedles for drug and vaccine delivery. Adv Drug Delivery Rev 2012;64:1547-68.
[14] Mikszta JA, Alarcon JB, Brittingham JM, Sutter DE, Pettis RJ, Harvey NG. Improved genetic immunization via micromechanical disruption of skin-barrier function and targeted epidermal delivery. Nat Med 2002;8:415-9.
[15] McAllister DV, Wang PM, Davis SP, Park JH, Canatella PJ, Allen MG, et al. Microfabricated needles for transdermal delivery of macromolecules and nanoparticles: fabrication methods and transport studies. Proc. Natl Acad Sci USA 2003;100:13755-60.
[16] Kim YC, Quan FS, Compans RW, Kang SM, Prausnitz MR. Formulation and coating of microneedles with inactivated influenza virus to improve vaccine stability and immunogenicity. J Controlled Release 2010;142:187-95.
[17] DeMuth PC, Su X, Samuel RE, Hammond PT, Irvine DJ. Nano-layered microneedles for transcutaneous delivery of polymer nanoparticles and plasmid DNA. Adv Mater 2010;22:4851-6.
[18] Fernando GJ, Chen X, Primiero CA, Yukiko SR, Fairmaid EJ, Corbett HJ, et al. Nanopatch targeted delivery of both antigen and adjuvant to skin synergistically drives enhanced antibody responses. J Controlled Release 2012;159:215-21.
[19] Park JH, Allen MG, Prausnitz MR. Polymer microneedles for controlled-release drug delivery. Pharm Res 2006;23:1008-19.
[20] Kolli CS, Banga AK. Characterization of solid maltose microneedles and their use for transdermal delivery. Pharm Res 2008;25:104-13.
[21] Lee JW, Park JH, Prausnitz MR. Dissolving microneedles for transdermal drug delivery. Biomaterials 2008;29:2113-24.
[22] Matsuo K, Yokota Y, Zhai Y, Quan YS, Kamiyama F, Mukai Y, et al. A low-invasive and effective transcutaneous immunization system using a novel dissolving microneedle array for soluble and particulate antigens. J Controlled Release 2012;161:10-7.
[23] Gardeniers HJGE, Luttge R, Berenschot EJW, de Boer MJ, Yeshurun SY, Hefetz M, van't Oever R,van den Berg A. Silicon micromachined hollow microneedles for transdermal liquid transport. J Microelectromech S 2003;12:855-62.
[24] Wang PM, Cornwell M, Hill J, Prausnitz MR. Precise microinjection into skin using hollow microneedles. J Invest Dermatol 2006;126:1080-7.
[25] Kaushik S, Hord AH, Denson DD, McAllister DV, Smitra S, Allen MG,Prausnitz MR. Lack of pain associated with microfabricated microneedles. Anesthesia and analgesia 2001;92:502-4.
[26] Chen X, Fernando GJ, Raphael AP, Yukiko SR, Fairmaid EJ, Primiero CA, et al. Rapid kinetics to peak serum antibodies is achieved following influenza vaccination by dry-coated densely packed microprojections to skin. J Cntrolled Rlease 2012;158:78-84.
[27] Bachmann MF, Beerli RR, Agnellini P, Wolint P, Schwarz K, Oxenius A. Long-lived memory CD8+ T cells are programmed by prolonged antigen exposure and low levels of cellular activation. Eur J Imunol 2006;36:842-54.
[28] Mills KH. Regulatory T cells: friend or foe in immunity to infection? Nat Rev Immunol 2004;4:841-55.
[29] Moon HJ, Lee JS, Talactac MR, Chowdhury MY, Kim JH, Park ME, et al. Mucosal immunization with recombinant influenza hemagglutinin protein and poly gamma-glutamate/chitosan nanoparticles induces protection against highly pathogenic influenza A virus. Vet Microbiol 2012;160:277-89.
[30] Korteweg C, Gu J. Pandemic influenza A (H1N1) virus infection and avian influenza A (H5N1) virus infection: a comparative analysis. Biochem Cell Biol 2010;88:575-87.
[31] Alving CR, Peachman KK, Rao M, Reed SG. Adjuvants for human vaccines. Curr Opin Immunol 2012;24:310-5.
[32] Andrianov AK, Marin A, DeCollibus DP. Microneedles with intrinsic immunoadjuvant properties: microfabrication, protein stability, and modulated release. Pharm Res 2011;28:58-65.
[33] Rinaudo M. Chitin and chitosan: Properties and applications. Prog Polym Sci 2006;31:603-32.
[34] Dutta PK, Dutta J, Tripathi VS. Chitin and chitosan: Chemistry, properties and applications. J Sediment Petrol 2004;63:20-31.
[35] Jayakumar R, Menon D, Manzoor K, Nair SV, Tamura H. Biomedical applications of chitin and chitosan based nanomaterials—A short review. Carbohydr Polym 2010;82:227-32.
[36] Boucaud A, Machet L, Arbeille B, Machet MC, Sournac M, Mavon A, Patat F, Vaillant L. In vitro study of low-frequency ultrasound-enhanced transdermal transport of fentanyl and caffeine across human and hairless rat skin. Int J Pharm 2001;228:69-77.
[37] Shibata Y, Foster L, Bradfield JF, Myrvik QN. Oral administration of chitin down-regulates serum IgE levels and lung eosinophilia in the allergic mouse. J Immunol 2000;164:1314-21.
[38] Maeda M, Murakami H, Ohta H, Tajima M. Stimulation of IgM production in human-human hybridoma HB4C5 cells by chitosan. Biosci Biotechnol Biochem 1992;56: 427-31.
[39] Koide SS MD. Chitin-chitosan:properties, benefits and risks. Nutr Res 1998;18:1091-101.
[40] Esteban MA, Mulero V, Cuesta A, Ortuno J, Meseguer J. Effects of injecting chitin particles on the innate immune response of gilthead seabream. Fish Shellfish Immun 2000;10: 543-54.
[41] Rauw F, Gardin Y, Palya V, Anbari S, Gonze M, Lemaire S, van den Berg T, Lambrecht B. The positive adjuvant effect of chitosan on antigen-specific cell-mediated immunity after chickens vaccination with live Newcastle disease vaccine. Vet Immunol Immunop 2010;134:249-58.
[42] Higashiyama T. Novel functions and applications of trehalose. Pure Appl Chem 2002;74:1263-9.
[43] Leslie SB, Israeli E, Lighthart B, Crowe JH, Crowe LM. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol 1995;62:3592-7.
[44] Leslie SB, Teter SA, Crowe LM, Crowe JH. Trehalose lowers membrane phase transitions in dry yeast cells. Biochim Biophys Acta 1994;1192:7-13.
[45] Bieganski RM, Fowler, A, Morgan JR, Toner M. Stabilization of active recombinant retroviruses in an amorphous dry state with trehalose. Biotechnol Prog 1998;14: 615-20.
[46] Amorij JP, Meulenaar J, Hinrichs WL, Stegmann T, Huckriede A, Coenen F, et al. Rational design of an influenza subunit vaccine powder with sugar glass technology: preventing conformational changes of haemagglutinin during freezing and freeze-drying. Vaccine 2007;25:6447-57.
[47] Carpenter JF, Crowe JH. An infrared spectroscopic study of the interactions of carbohydrates with dried proteins. Biochemistry 1989;28:3916-22.
[48] Kim YC, Quan FS, Compans RW, Kang SM, Prausnitz MR. Stability kinetics of influenza vaccine coated onto microneedles during drying and storage. Pharm Res 2011;28:135-44.
[49] Song JM, Kim YC, Lipatov AS, Pearton M, Davis CT, Yoo DG, Park KM, Chen LM, Quan FS, Birchall JC, Donis RO, Prausnitz MR, Compans RW, Kang SM. Microneedle delivery of H5N1 influenza virus-like particles to the skin induces long-lasting B- and T-cell responses in mice. Clinical and vaccine immunology 2010;17: 1381-9.
[50] Haaf F, Sanner A, Straub F. Polymers of N-Vinylpyrrolidone-synthesis, characterization and uses. Eur Polym J 1985;17:143-52.
[51] Wang H, Yu T, Zhao C, Du Q. Improvement of hydrophilicity and blood compatibility on polyethersulfone membrane by adding polyvinylpyrrolidone. Fiber Polym 2009;10:1-5.
[52] Donnelly RF, Singh TR, Garland MJ, Migalska K, Majithiya R, McCrudden CM, et al. Hydrogel-forming microneedle arrays for enhanced transdermal drug delivery. Adv Funct Mater 2012;22:4879-90.
[53] Bal SM, Caussin J, Pavel S, Bouwstra JA. In vivo assessment of safety of microneedle arrays in human skin. Eur J Pharm Sci 2008;35:193-202.
[54] Bortolatto J, Borducchi E, Rodriguez D, Keller AC, Faquim-Mauro E, Bortoluci KR, et al. Toll-like receptor 4 agonists adsorbed to aluminium hydroxide adjuvant attenuate ovalbumin-specific allergic airway disease: role of MyD88 adaptor molecule and interleukin-12/interferon-gamma axis. Clin Exp Allergy 2008;38:1668-79.
論文全文使用權限
  • 同意授權校內瀏覽/列印電子全文服務,於2018-08-30起公開。


  • 如您有疑問,請聯絡圖書館
    聯絡電話:(06)2757575#65773
    聯絡E-mail:etds@email.ncku.edu.tw