合成可注射交聯透明質酸添加兒茶素作為人工玻璃體之研究

Abstract

許多與玻璃體視網膜病變相關的眼睛疾病，像是視網膜剝離、黃斑部病變等，都可能需要玻璃體切除術來改善視力，藉由灌注替代物填充切除後的玻璃體腔室，然而手術後容易引起發炎反應，造成手術失敗產生其他眼睛疾病副作用，或是替代物的降解。因此本研究在替代物中添加表沒食子兒茶素沒食子酸酯（EGCG），其為天然的茶葉萃取物並有許多研究指出具有抗發炎特性，並且以1,4-丁二醇二縮水甘油醚（BDDE）交聯透明質酸（HA）製備玻璃體替代物，此材料已經通過測試具有良好生物安全性，期望能夠模擬原生玻璃體性質且減少發炎反應的產生。 本研究以 FTIR 和 1H-NMR 檢驗交聯官能基的改變與交聯程度，檢測交聯劑 BDDE 的殘餘量，再鑑定折射率、黏彈性質以及滲透壓，以酵素模擬體外降解，測定降解速率；細胞實驗部分，使用 WST-1 測定材料細胞毒性，Live/Dead 染色觀察細胞活性，而後同時給予細胞脂多醣（LPS）以及本實驗材料觀察誘導發炎情況以及材料抗發炎效果，利用即時定量反轉錄聚合酶連鎖反應（RT-qPCR）測定細胞發炎基因表現變化；動物實驗部分，進行玻璃體切除術並注入本實驗替代物，測量眼壓與角膜厚度，以及視網膜電流圖進行視網膜功能性檢驗，未來將以眼球組織切片染色觀察眼睛結構與發炎現象。 本實驗成功製備出透明易注射之膠體溶液，BDDE 殘餘量符合安全標準，材料的物化性質也與原生玻璃體極為相似，具有較長的降解時間能在術後維持填充效果；體外實驗則證實材料具有良好生物相容性，添加 50 μM EGCG 具有抑制發炎作用；動物實驗選用 50 μM EGCG 為基準而調動低、中、高三種的濃度劑量，藉此讓注射後被未切除完全玻璃體液稀釋的 EGCG 能夠發揮抗發炎作用，結果顯示注射含有 100 μM EGCG 之替代物具有較佳的替代效果，術後眼壓和角膜厚度沒有顯著變化，視網膜電訊號振幅強度也與術前無顯著差異。

A range of vitreoretinopathy complications such as retinal detachment and macular degeneration can be treated with vitrectomy. After surgery, vitreous substitutes should be injected to maintain properties and structure. However, high incidence rate of postoperative inflammation, which cause the failure of surgery and the early degradation of substitute, has been reported following surgery. To alleviate this condition, EGCG, which is present in green tea extract with anti-oxidant and anti-inflammatory properties, was added into our substitute materials. We investigated BDDE-crosslinked HA for a vitreous substitute and expected to mimic the properties of the vitreous humor and reduce the postoperative inflammatory response. We successfully developed a transparent and injectable gel solution. The results of FTIR and 1H-NMR confirmed the cross-linking reaction between BDDE and HA. BDDE residue in the materials was lower than 2 ppm. The refractive index, dynamic viscosity, and osmolality were similar to the vitreous humor. The materials underwent gradual degradation and the complete degradation occurred after one month. For the in vitro study, the cytotoxicity was evaluated by WST-1 assay and Live/Dead stain. High biocompatibility were presented in both test. In the anti-inflammation study, we used LPS to induce inflammation co-treated with our materials and assessed cytokines mRNA expression by RT-qPCR. Substitutes with 50 μM EGCG inhibited the expression of inflammatory cytokines significantly. To avoid dilution of EGCG of injected substitutes by remaining vitreous humor after surgery and the aqueous humor circulation in vivo, we proposed to use three dosages of EGCG based on the in vitro study. The results showed that no significant change in intraocular pressure and cornea thickness of operated eyes of the substitutes with 100 μM EGCG. The amplitudes of electrical signal on electroretinograms also remained normal compared with preoperative signal.