茶多酚奈米藥物抑制眼球表面發炎反應於乾眼症治療評估

Evaluation of Anti-inflammatory Effect of Tea Polyphenol-contained Nanoparticles for Dry Eye Syndrome Treatment

黃馨儀(Hsin-Yi Huang)

指導教授 ： 王明誠 ； 曾靖孋

中原大學/工學院/生物醫學工程研究所/碩士(2015年)

https://doi.org/10.6840/cycu201500890

摘要

乾眼症是一種常見之眼科疾病，主要是因為淚腺分泌功能減退，產生不穩定的淚膜，導致眼睛表面的發炎現象。現行之治療主要採局部給藥的方式，但是眼部的特殊結構在藥物釋放上產生了很大的阻礙，導致藥物於眼表停留時間短，生物利用度低，故發展一有效之眼科用藥為近年研究話題。本研究的目的在於開發一款新型的乾眼症奈米藥物劑型，透過包覆具有抗發炎效果之表沒食子兒茶酸鹽(Epigalocatechin gallate, EGCG)達到治療乾眼症的效果。將明膠、EGCG和透明質酸均勻混合後，製備出茶多酚奈米載體。以動態光散射儀及穿透式電子顯微鏡檢測其粒徑大小和顆粒型態，並且以人類角膜上皮細胞為測試對象，利用WST-8檢測其細胞活性，再以rt-PCR檢測細胞發炎因子（IL1-β、IL-6、IL-8和TNF-α）的表現量；而體內實驗的部分，透過活體影像分析系統檢測藥物於活體眼表的停留，並且以紐西蘭大白兔建立乾眼症動物模型，利用茶多酚奈米藥水治療3週，最後透過角膜螢光染色、淚液分泌量量測以及組織H&E染色來檢測其治療效果。本研究所製備之茶多酚奈米載體(Gelatin-EGCG-HA,GEH)，當HA濃度為62.5 μg/ml 時，粒徑大小為253.4 ± 7.3 nm ，表面電位為9.2 ± 1.8 mV，透過細胞活性測試，此藥物濃於20 μg/ml以下無細胞毒性 ; 利用藥物濃度0.2 μg/ml治療發炎細胞後，其細胞發炎因子表現量皆有下降。製成茶多酚奈米眼藥水後，其滲透壓和酸鹼值皆與淚液相似，對於眼睛不會造成刺激感 ; 而動物實驗證實，奈米茶多酚載體可在活體眼表停留較長時間，再以乾眼症動物證實，GEH可有效修復受損的角膜細胞，並使淚液分泌量提升，達到治療乾眼症之效果。本研究已成功證明奈米茶多酚劑型，可有效減少細胞發炎因子之表現量。透過眼藥水方式給予，可有效治療紐西蘭白兔乾眼症，未來可應用於人體試驗，成為治療乾眼症之另一選擇。

關鍵字

乾眼症 ； 表沒食子兒茶素 ； 透明質酸 ； 奈米載體 ； 發炎

並列摘要

Dry eye syndrome (DES) is a common disease that causing unstable tear film and inducing ocular surface inflammation. In ophthalmological treatment, eye drops is the common topical formulation for ocular drug delivery. However, ocular barriers impede the utility of pharmaceutical drugs, hence it results in low local bioavailability of the drug. Nanocarriers offer a promising alternative for the treatment of ocular diseases. The objective of this study is to develop a new nanomedicine for dry eyes syndrome treatment. One of the major tea polyphenol, epigallocatechin gallate (EGCG), was encapsulated in the nanocarriers as the anti-inflammation agent to treat DES.
Gelatin, EGCG and hyaluronic acid solution were mixed homogenously to form self-assembling nanoparticles by electroattraction. These nanoparticles were named as GEH(Gelatin-EGCG-HA). The particle size and morphology of GEH were examined by dynamic light scattering spectrometry (DLS) and transmission electron microscope (TEM). The tolerable concentration of EGCG for culture with human corneal epithelium cells (HCET) was evaluated by WST-8 assay. Gene expression of inflammation cytokine (IL1-β, IL-6, IL- 8 and TNF-α) were evaluated by real time-Polymerase chain reaction (rt-PCR). In vivo study, the ocular retention of nanoparticles were examined by an in vivo imaging system (IVIS). DES animal model was established on New Zealand White Rabbit, 0.1‰ benzalkonium chloride (BAC) was applied on the rabbit eyes for 4 weeks to induce DES. GEH eye drops were administered into both healthy and experimental DES rabbits eyes twice a day for 3 weeks. Treatment results were evaluated by corneal fluorescein staining, Schirmer strips for tear production. Corneal tissues were harvested for H&E staining..
The particle size and zeta potential of GEH, when HA conc. , at 62.5 μg/ml, were 253.4 ± 7.3 nm and 9.2 ± 1.8 mV, respectively. HCET treated by GEH at EGCG con. at 20 μg/ml showed no cytotoxicity. When HCET treated by GEH at EGCG 0.2 μg/ml, all the inflammation cytokine (IL1-β, IL-6, IL- 8 and TNF-α) were reduced.When GEH particles were prepared into eye-drops formulation, its pH value, osmolality and refractive index were similar to human tear hence it is less likely to cause irritation. In the in vivo experiment, higher fluorescent particles accumulated in the anterior part of eyes in the GEH group were observed by IVIS image. Anterior eye segment of treated rabbits showed normal integrity. The tear production in DES group showed the average wetted length was 3.67 ± 1.46 mm, but the GEH treated one was almost the same as normal group. In addition, there was no obvious fluorescein staining on the cornea observed in GEH treated group.
In conclusion, we successfully prepared EGCG-contained gelatin nanoparticles with surface coating of HA. This study confirmed the reduction of the inflammation cytokine expression. From DES animal test, we can see ocular damage decreased, corneal thickness recovered and tear secretion recover to normalby the treatment of GEH nanomedicine twice a day. These results identify a potential alternative treatment option by EGCG nanomedicine as a new therapeutic modality for patients of dry eye disease in the future.

並列關鍵字

Dry eye syndrome ； Epigallocatechin gallate ； Hyaluronic acid ； Nanocarrier ； Inflammation

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