| Summary: | 碩士 === 中原大學 === 生物醫學工程研究所 === 103 === 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.
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