| 摘要: | 角膜是一個透明的構造覆蓋在眼並且沒有血管分佈。角膜血管新生(CNV)是一個常見的疾病，血管會從角膜周圍血管叢向前眼不正常增生。當角膜因為感染、受到刺激或長期配戴隱形眼鏡都會導致角膜缺氧，也就是角膜血管新生的主因。而gp91是一個新興的胜肽，它可以干擾VEGF訊號去抑制血管新生。因此在這項研究中，我們使用gp91作為藥物去抑制角膜血管新生。近年研究發現，由於奈米顆粒體積小、易吸收、具有傳遞及緩慢釋放藥物的效果，可以降低在患部的給藥頻率。故發展含奈米顆粒藥物，以提高治療成效。玻尿酸(HA) 是一個帶負電性的材料，具有高度生物相容性，並在角膜結構中可見。我們將HA與gp91藉由自組裝的機制去形成一個帶有正電電荷奈米顆粒(HA-gp91 NPs)，並作材料測試去觀察其特性。我們結合了材料的優勢去影響角膜的新生血管，並且對人類血管上皮細胞 (HUVECs)和C57BL/6J 小鼠去評估療效。
HA-gp91 NPs 成功地被製備，粒徑為268.433 ± 7.124nm且電位為17.978 ± 0.776 mV，我們也測試了gp91在ＨＡ的載藥率為92.953 ± 0.718 %。TEM 結果顯示 gp91-HA NPs為圓形顆粒且均勻分布也看到gp91均勻分布在顆粒中。NTA顯示濃度為〖1.39 *10〗^10±4.7* 10^8 /1 mL、FTIR和DSC的結果顯示了材料和藥物的結合、藥物釋放測試顯示了藥物的緩慢釋放性。除此之外，從體內細胞攝取量和細胞攝取影像的結果顯示了藥物良好的攝取量再從細胞活性檢測出最適的給藥濃度，並藉由遷移和管柱形成的測試結果證實HA-gp91 NPs對細胞的抑制效果。我們在小鼠在眼球表面給接上FITC螢光的藥物去追蹤藥物滯留情形，並用活體影像系統追蹤發現藥物能在眼表停留。最後將小鼠經化學灼傷後的角膜做一週治療後，所有組別皆有血管從角膜周圍血管從增生的跡象，相較於PBS、HA和gp91組，HA-gp91 NPs具有良好抑制新生血管的生成的成效。組織切片H&E染色的解果也顯示出HA-gp91 NPs組的完整度最接近正常角膜。HA-gp91 NPs在體內外的測試都顯示出對血管良好的抑制成效。這項研究指出了HA-gp91 NPs對於角膜血管新生的治療效果。
Corneal is a transparent structure without blood vessel distribute that covered anterior surface of the eye. Corneal neovascularization (CNV) is one of the most common disease happened on the cornea surface causing by the proliferation of the blood vessels from the corneal edge. Once the cornea got hypoxia by some infections, stimulus or wearing contact lens for long time, which are the main reason causing CNV. Gp91-ds-tat (gp91) is a novel peptide that can distribute VEGF signaling to inhibit angiogenesis. Therefore, in this study, we use gp91 to inhibit angiogenesis of CNV. In the discovery of recent research, due to the small size, easy absorption, delivery and slow release of drugs, nanoparticle can reduce the dose and frequency of injured area. Therefore, the development of nanoparticle drugs is applied for improve the effectiveness of treatment. Hyaluronic acid (HA) is a negatively charge material that normally presence in cornea and highly-biocompatibility. By self-assembling mechanism of HA to form positively charge nanoparticles (HA-gp91 NPs), we combined the advantage of its great capacity and retard the distribution of drug on the eye surface by surface charge attraction to investigate its efficiency on human umbilical vein endothelial cells (HUVECs) and C57BL/6J mice to evaluate the treatment effectiveness.
HA-gp91 NPs was successfully prepared with the size of 268.433 ± 7.124nm, and zeta potential was 17.978 ± 0.776 mV. We also confirmed that the encapsulation rate of gp91 in the NPs was around 92.953 ± 0.718 % and TEM images showed that these particles had spherical morphology and gp91 peptide separate evenly in the nanoparticles. NTA result showed the concentration of nanoparticles was 〖1.39 *10〗^10±4.7* 10^8 /1 mL. The structure of the functional groups and the combination of HA-gp91 NPs were characterized by FTIR and DSC. Result of drug release test showed the slow and stable release ratio of HA-gp91 NPs. Moreover, by the uptake intensity and figure indicated that HA-gp91 NPs had good uptake property, than confirmed the most appropriate drug given concentration by cell viability test. The effect of blood vessel inhibition had been proved by cell migration and tube formation assays. We gave FITC fluorescent drugs on the mice eyeball surface to track the drug retention, and used the bioimaging system to track and find that the drugs can stay on the ocular surface. At last, after one week of treatment on the chemical burned mice cornea, all groups showed signs of proliferation of blood vessels from the peripheral blood vessels. Compared with the PBS, HA and gp91 groups, HA-gp91 NPs had a good inhibitory effect on neovascularization. H&E staining of tissues also showed that the integrity of HA-gp91 NPs group was closest to normal cornea. The tests of HA-gp91 NPs in vivo and in vitro had shown a good inhibition on blood vessels. This study points out the therapeutic effect of HA-gp91 NPs on corneal angiogenesis. |
