- 學位論文
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
Preparation of RGD peptide modified nanomedicine as eye-drop for cornea anti-angiogenesis treatment
摘要
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。
並列摘要
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.