| 摘要: | 視網膜病變(黃斑部病變等)，長並隨活性氧增加，進而造成氧化壓力的異常、炎症與細胞凋亡或老化的莊況出現。而免疫調節劑的菸鹼醯胺腺嘌呤二核?酸(Nicotinamide adenine dinucleotide, NAD+)具有調節氧化壓力、協同修復DNA、抑制發炎的能力，但NAD+易受環境影響導致變性，故本實驗採用NAD+的前體，菸醯胺單核?酸(Nicotinamide Mononucleotide, NMN)作為使用藥物。由於傳統眼部藥物遞送屏障效能低，而奈米藥物具有提升眼部藥物生物利用率和長效釋放之優勢，所以本研究將NMN搭載進奈米明膠顆粒，以保護藥物與提升藥物至後眼部之效能。
因此本研究目的是使用奈米明膠做為奈米載體，搭載NMN之奈米藥物，將其應用於視網膜上皮細胞與視網膜神經節細胞，探討其在經過氧化壓力損傷後於不同NMN劑型藥物作用下，其細胞凋亡與衰老狀況。
材料方面，使用粒徑分析儀進行奈米顆粒大小確認與表面電位分析、奈米顆粒追蹤分析檢測奈米材料特性、穿透式電子顯微鏡為顆粒型態觀察、利用HPLC檢測奈米藥物的釋放曲，結果顯示NMN-GNP粒徑大小為303.4 ± 1.2 nm、帶正電賀18.6 ± 0.3 Mv、濃度為3.58×1011 ± 5.05×1010 particles/mL、且通過藥物釋放證實了奈米劑型的緩慢釋放功效。
體外測試，將視網膜色素上皮細胞、視網膜神經節細胞與奈米顆粒進行共培養，觀察經氧化壓力損傷前後的細胞活性與衰老相關基因的表達變化以及凋亡，結果顯示奈米劑型在衰老染色(β-Galactosidase)或是衰老相關基因(Real-time polymerase chain reaction, qPCR)均比未包覆藥物有更顯著的預防細胞在氧化壓力下受損的能力
Retinal diseases (such as macular degeneration) are often accompanied by increased reactive oxygen species (ROS), leading to oxidative stress, inflammation, and resulting in cell apoptosis or aging. Nicotinamide adenine dinucleotide (NAD+), an immunomodulator, has the ability to regulate oxidative stress, aid in DNA repair, and inhibit inflammation. However, NAD+ is prone to environmental degradation. Therefore, this study uses nicotinamide mononucleotide (NMN), a precursor to NAD+, as the therapeutic agent. Traditional ocular drug delivery systems have low efficiency, whereas nano-drugs offer advantages such as increased bioavailability and sustained release in the eye. Consequently, this study aims to use gelatin nanoparticles to deliver NMN, enhancing drug protection and efficacy in targeting the posterior segment of the eye.
The purpose of this study is to apply NMN-loaded gelatin nanoparticles to retinal pigment epithelial cells (RPE) and retinal ganglion cells (RGC), examining their effects on cell apoptosis and aging after oxidative stress injury under different NMN formulations. Materials and methods include using a particle size analyzer to confirm nanoparticle size and surface charge, nanoparticle tracking analysis to examine material properties, and transmission electron microscopy for particle morphology observation. High-performance liquid chromatography (HPLC) was utilized to evaluate drug release profiles. Results showed that NMN-GNP had a particle size of 303.4±1.2 nm, a positive zeta potential of 18.6±0.3 mV, a concentration of 3.58×10?? ± 5.05×10?? particles/mL, and demonstrated sustained release properties.
In vitro tests involved co-culturing RPE and RGC cells with the nanoparticles, observing changes in cell viability, expression of aging-related genes, and apoptosis before and after oxidative stress injury. Results indicated that the nano-formulation significantly improved the prevention of oxidative stress-induced cell damage compared to non-encapsulated drugs, as evidenced by senescence staining (β-Galactosidase) and the expression of aging-related genes (via real-time polymerase chain reaction, qPCR). |
