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研究生:張哲禕
研究生(外文):Che-Yi Chang
論文名稱:製備RGD胜肽修飾之奈米藥物標的血管內皮細胞作為抑制角膜血管新生之眼藥應用
論文名稱(外文):Preparation of RGD peptide modified nanomedicine as eye-drop for cornea anti-angiogenesis treatment
指導教授:王明誠王明誠引用關係曾靖孋
指導教授(外文):Ming-Chen WangChing-Li Tseng
學位類別:碩士
校院名稱:中原大學
系所名稱:生物醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:中文
論文頁數:93
中文關鍵詞:角膜血管新生
外文關鍵詞:corneaangiogenesis
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中文摘要
角膜是位於眼表上的透明無血管組織,但創傷、發炎、感染或缺氧,都會使角膜形成血管新生而降低其清晰度,嚴重可能導致眼睛失明。而侵入式治療術後會產生副作用;眼藥水中的藥物不能長時間的停留於眼表上,導致治療效果不佳。而奈米藥物的發展,能夠有效的提高藥物停留於眼表上的時間與提高生物利用度,故本研究發展含奈米藥物之眼藥水,以治療角膜血管新生,希望可以提高治療效果。
本研究中,以明膠(Gelatin)作為載體包覆表沒食子兒茶素沒食子酸酯(Epigallocatechin gallate, EGCG)製成奈米顆粒,表面修飾玻尿酸(Hyaluronic acid, HA)與鏈精氨酸-甘氨酸-天門冬氨酸(Arginine-glycine-aspartic acid, RGD),藉此達到與血管內皮細胞(Endothelial cell)上的整合素(αvβ3)特異性標靶的功能,以提高藥物滯留於眼表的時間與專一性抑制血管新生。此標地型奈米顆粒簡稱為GEH-RGD。
而本實驗以核磁共振( Nuclear Magnetic Resonance, NMR)與傅立葉轉換紅外線光譜儀( Fourier transform infrared spectroscopy, FTIR )確認HA上的羧基與RGD上的氨基的化學鍵結反應。利用動態光照射(Dynamic Light Scattering, DLS)、穿透式電子顯微鏡(Transmission Electron Microscopy, TEM)、原子力顯微鏡原理(Atomic Force Microscope, AFM)觀察奈米顆粒的大小、電位與形貌。以螢光取代藥物,進行釋放率測試。並以氧化還原呈色法,測定奈米顆粒中包覆EGCG的含量。表面電漿共振(Surface Plasmon Resonance, SPR)測試RGD與整合素(αvβ3)之間標靶的能力。當確認完奈米顆粒製備後,再與血管內皮細胞做活性測試、螢光影像及顆粒攝取率分析。
而從NMR與FTIR結果表示成功的將RGD與HA做接枝。GEH-RGD奈米顆粒的大小與表面電位分別是168.87 ± 22.50 nm和19.7 ± 2.0 (mv)。透過TEM觀察GEH-RGD奈米顆粒形貌為圓形。而EGCG藥物的包覆率高達97% ; 而藥物釋放率的實驗中也發現能夠緩慢地釋放藥物,SPR實驗中證實GEH-RGD奈米顆粒與整合素(αvβ3)之間具有特異性標靶功能。而細胞活性實驗中,GEH-RGD奈米顆粒以20 μg / ml的EGCG濃度含量能夠有效的抑制血管內皮細胞生長。在細胞攝取率結果中GEH-RGD奈米顆粒,比較於其他組別有較高的攝取率。從以上結果知道GEH-RGD奈米顆粒能夠有效的抑制血管內皮細胞的增生。此研究未來有實際應用於抑制角膜血管新生的潛力。


Abstract
Cornea tissue is a transparent layer on the anterior surface of the eye; it is responsible for light transmittance and it provides most of the refractive ability of the eye. The abnormal vessels may decrease corneal clarity and impede light transmission. Corneal neovascularization (CNV) is a major sight threatening disorder that caused by trauma, infection, inflammation, or corneal hypoxia. Conventional invasive treatments may cause numbers of undesirable side effects. The easiest and most common way for ophthalmic delivery is through eye-drop formulation, but less than 5 % of the administered dose retained in the ocular tissue after five minutes. Nanomedicine can achieve longer retention time and improve the bioavailability of the drug on the ocular surface. Therefore the purpose of this study is to develop an eye drop which containing Nanoparticles (NPs) for treating CNV and hopefully it can be used to increase the treatment efficacy in the future.
In this study, gelatin nanocarriers which encapsulated-EGCG and coated with hyaluronic acid(HA)were used to treat Human Umbilical Vein Endothelial Cells (HUVECs) for evaluate ocular anti-angiogenesis. Arginine-glycine-aspartic acid (RGD) peptide that conjugated nanoparticle could achieve specific binding with integrin (αvβ3) on endothelial cells. This treatment is expected to raise the bioavailability for targeting and long-term ocular drug delivery to inhibit CNV. This RGD modified nanoparticles were called GEH-RGD.
Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared Spectroscopy (FTIR) to confirm the conjugation of carboxyl group on HA and amine group on RGD. Dynamic Light Scattering (DLS), Transmission Electron Microscope (TEM), and Atomic Force Microscope (AFM) were used to observe the size, zeta potentials and morphology. Surface Plasmon Resonance (SPR) can test the targeting ability of RGD peptide with integrin αvβ3. After confirmation of physical and chemical characterization of the nanoparticles, cell viability tests, florescent imaging and cellular uptake tests with HUVECs were followed.
NMR and FTIR indicated conjugation of RGD with HA was successfully prepared. DLS showed the particle size and zeta potential of the GEH-RGD NPs were 168.87 ± 22.50 nm and 19.7 ± 2.0 (mV), respectively. TEM image showed GEHRGD have spherical shape and HA coverage appearance. Encapsulation efficiency of EGCG can achieved up to 97%; drug release experiment confirmed the slow release of the drug. Surface Plasmon Resonance (SPR) experiment confirmed GEH-RGD have a specific targeting ability with integrin αVβ3. In vitro cell viability test showed GEH-RGD could efficiently inhibited HUVECs viability at the EGCG concentration of 20 μg/ml. In cellular uptake experiment, it showed GEH-RGD group had higher cellular uptake. Based on the results we can see that GEH-RGD could effectively inhibit the proliferation of vascular endothelial cells. This study provides evidence for clinical approach by nanoparticles to inhibit corneal neovascularization.


目錄
中文摘要……………………………………..…………………….…….....…..I
Abstract………………………………………………..……….….…..………..III
致謝…………………………………………………………..……….……..….V
目錄………………………………………………………………….….....……VII
圖目錄…………………………………………………………………..………IX
表目錄…………………………………………………………………..………XI
附錄………………………………………………………………………..……XII
一、緒論.............................................................................................1
1.1研究背景........................................................................................1
1.2研究目的........................................................................................2
二、理論基礎......................................................................................3
2.1角膜結構與功能.............................................................................3
2.2角膜血管新生狀況 .........................................................................4
2.2.1血管新生過程..............................................................................4
2.2.2角膜血管新生臨床症狀與困擾.....................................................6
2.2.3侵入式手術治療方式....................................................................7
2.2.4非侵入式抑制血管新生用藥.........................................................10
2.2.5 兒茶素於血管新生抑制...............................................................12
2.3奈米藥物的發展..............................................................................14
2.3.1奈米載體的發展...........................................................................14
2.3.2血管標地功能之奈米藥物.............................................................14
2.3.3奈米藥物於眼表應用....................................................................16
三、實驗材料與方法............................................................................19
3.1實驗藥品來源..................................................................................19
3.2實驗儀器.........................................................................................21
3.3實驗流程.........................................................................................23
3.4 RGD修飾之茶多酚奈米顆粒製備...................................................24
3.4.1 HA與RGD共軛接枝反應.............................................................24
3.4.1.1核磁共振(NMR)樣品製備與分析..........................................25
3.4.1.2傅立葉轉換紅外光譜(FTIR)樣品製備與分析........................26
3.4.2奈米顆粒製備過程.......................................................................27
3.4.2.1粒徑分析(DLS)....................................................................29
3.4.2.2穿透式電子顯微鏡(TEM)樣品製備與顆粒形貌分析..............31
3.4.2.3原子力顯微鏡原理(AFM)樣品製備與顆粒形貌分析..............32
3.4.2.4藥物釋放率測試........................................................................34
3.4.2.5兒茶素包覆率測試....................................................................35
3.4.3表面電漿共振(SPR)測試........................................................36
3.4.4血管內皮細胞培養.......................................................................38
3.4.5細胞活性測試..............................................................................39
3.4.6奈米載體對細胞存活之螢光影像..................................................41
3.4.7細胞攝取螢光奈米顆粒影像分析..................................................41
四、結果與討論...................................................................................44
4.1 HA與RGD接枝確認.......................................................................44
4.1.1 NMR數據分析.............................................................................44
4.1.2 FTIR數據分析.............................................................................47
4.2 奈米顆粒特性分析 .........................................................................48
4.2.1動態光照射分析...........................................................................48
4.2.2穿透式電子顯微鏡(TEM)顆粒形貌分析....................................51
4.2.3原子力顯微鏡(AFM)形貌分析..................................................53
4.2.4 兒茶素包覆率結果.......................................................................56
4.3釋放及標地物功能分析....................................................................57
4.3.1藥物釋放率測試結果....................................................................57
4.3.2表面電漿共振分析結果................................................................58
4.4細胞測試.........................................................................................61
4.4.1細胞活性測試結果........................................................................61
4.4.2奈米載體對細胞存活之螢光影像結果...........................................61
4.4.3細胞攝取螢光奈米顆粒影像分析結果...........................................66
五、結論與未來規劃............................................................................69
六、文獻……………………..…………..………..………………………..70

圖目錄..................................................................................................IX
圖1、角膜構造示意圖..........................................................................3
圖2、血管新生示意圖..........................................................................5
圖3、細針電燒示意圖..........................................................................7
圖4、光動力療法示意圖......................................................................8
圖5、雷射治療示意圖..........................................................................8
圖6、羊膜移植手術示意圖..................................................................10
圖7、玻璃體內注射示意圖..................................................................10
圖8、EGCG結構式.............................................................................12
圖9、不同多酚類抑制血管內皮細胞活性............................................13
圖10、不同多酚類抑制細胞爬行.........................................................13
圖11 (A)、不同藥物濃度治療下的螢光轉染率、圖(B)不同時間點下的螢光轉染率........................................................................................................15
圖12 (A)、不同細胞抑制數據,圖(B)、藥物競爭測試數據,圖(C)、有無血清比較實驗數據.........................................................................................................16
圖13、不同潤滑劑滯留於角膜表面的時間...........................................17
圖14、RGD修飾奈米顆粒應用於標靶血管內皮細胞示意圖.................18
圖15、實驗流程圖................................................................................23
圖16、HA與RGD peptide接枝反應圖………….………..……...............25
圖17、使用之材料其化學結構示意圖...................................................28
圖18、奈米顆粒合成示意圖.................................................................29
圖19、粒徑分析原理與結果圖譜..........................................................31
圖20、電子束與圖像成像路徑..............................................................32
圖21、AFM原理示意圖........................................................................33
圖22、ABTS化學反應示意圖...............................................................36
圖23、SPR訊號及作用原理(1)........................................................37
圖24、SPR訊號及作用原理(2)........................................................37
圖25、Cell Counting Kit-8反應示意圖…………….........…....……….....40
圖26、共軛焦顯微鏡成像示意圖...........................................................43
圖27、HA的NMR分析圖(氫譜).........................................................44
圖28、GRGDSPK的NMR分析圖(氫譜)............................................45
圖29、GRGDSPK混合HA的NMR分析圖(氫譜)................................46
圖30、RGD接合HA 的NMR分析圖(氫譜).........................................46
圖31、傅立葉轉換紅外光譜數據圖........................................................47
圖32、奈米顆粒大小分佈(A) GE、(B) GEH、(C) GEH-RGD................49
圖33、奈米顆粒表面電位分佈(A) GE、(B) GEH、(C) GEH-RGD.........50
圖34、TEM奈米顆粒形貌分析(A) GE、(B) GEH、(C) GEH-RGD.........52
圖35、GE之AFM奈米顆粒形貌分析.......................................................53
圖36、GEH之AFM奈米顆粒形貌分析....................................................54
圖37、GEH-RGD之AFM奈米顆粒形貌分析...........................................55
圖38、EGCG以ABTS+分析之濃度檢量線............................................56
圖39、不同酸鹼度釋放率之影響............................................................57
圖40、奈米顆粒包覆螢光之釋放曲線.....................................................58
圖41、尚未接上抗原訊號分析圖譜.........................................................58
圖42、接上抗原(αVβ3)訊號分析圖譜………………………...........…..59
圖43、比較不同材料之間SPR訊號強度..................................................59
圖44、GEH-RGD、GE之間SPR訊號強度比較.......................................60
圖45、GEH-RGD、GEH之間SPR訊號強度比較.....................................60
圖46、以不同形式及濃度之EGCG與HUVEC共培養一天後之細胞之活性變化.............................................................................................................61
圖47、細胞螢光影像Live &; Dead Day 1 ( EGCG : 200 µg / ml )….....….62
圖48、細胞螢光影像Live &; Dead Day 1(EGCG:20 µg / ml)…......…63
圖49、細胞螢光影像Live &; Dead Day 3(EGCG:200 µg / ml)…..…..64
圖50、細胞螢光影像Live &; Dead Day 3(EGCG:20 µg / ml)……......65
圖51、細胞攝取螢光奈米顆粒之影像(A)DAPI (B)染膜染劑 (C)TAMRA與DAPI之疊圖..............................................................................................................68
圖52、HUVEC細胞攝取螢光奈米顆粒數量化圖........................................68

表目錄.......................................................................................................XI
表1、角膜上不同細胞的整合素分類…………………………………...…….5
表2、角膜新生血管的原因……………………………………..……..….......6
表3、實驗試劑………………………………………………………..…….....19
表4、實驗儀器………………………………………………………..………..21
表5、培養液配置……………………………………………………..…..…...38
表6、磷酸鹽緩衝溶液配置………………………………………..……...…..38
表7、NMR官能機特徵峰值…………………………………….……..….…..45
表8、不同型態之奈米顆粒特徵表(粒徑、電位、分散係數)……….......….48
表9、不同奈米顆粒其EGCG包覆率……………………………………..…..56

附錄...........................................................................................................XII
附錄1、HA的500 MHz-NMR氫譜原始圖譜………………………..…….….74
附錄2、GRGDSPK的500 MHz-NMR氫譜原始圖譜…………………..……75
附錄3、HA混合GRGDSPK的500 MHz-NMR氫譜原始圖譜………..…..…76
附錄4、HA化學接枝GRGDSPK的500 MHz-NMR氫譜原始圖譜…...…….77

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