發展幾丁聚醣微粒作為臺灣藜發酵物載體以提升其減緩PM2.5誘導活性氧化物產生

Abstract

懸浮微粒 (PM) 會帶入或誘導細胞產生活性氧化物 (ROS)，過量的 ROS 會活化促發炎轉錄因子 NF- κB 與促發炎相關細胞激素包括 Interleukin family 和TNF-α 的表達。並由於肺泡中的 PM 主要由肺泡巨噬細胞 (AM) 吞噬清除，因此 AM 可被視為抑制 PM 誘導之發炎的主要目標。本實驗室先前的研究發現，發酵臺灣藜萃取出之 G-rich peptide (GP) 能夠有效大幅降低 PM 引發之小鼠肺泡巨噬細胞 (MH-S) 的胞內自由基與提升其細胞存活率。本研究目的為發展幾丁聚醣微粒作為搭載 GP 之生物相容性載體，期望提升 MH-S 對 GP 利用效率，以利未來進一步發展吸入式載體。本實驗測試四種不同 PM 其金屬含量、粒徑大小、界達電位、外觀形態、細胞存活率和胞內外自由基，並發現組成分是否主要為石英及顆粒形狀和大小可能是影響細胞毒性的主要因素。本實驗之幾丁聚醣微粒 (Chitosan particle, CS) 濃度在 1,000 ppm 處理 24 hr 下細胞存活率為 84%，且不會顯著性引發細胞內之自由基，顯示其改善了傳統造粒法之溶劑殘留問題，而具生物可相容性。在經過搭載 G-rich peptide (GP) 後之幾丁聚醣微粒 (CS-GP)，尺寸為 850.8 ± 59.5 nm，界達電位為 +24 ± 0.62 mV，屬於易促進巨噬細胞吞噬作用的表面電荷與大小。在細胞存活率、胞內自由基實驗中，CS-GP group (48.9 ppm GP) 與 GP group (100 ppm GP) 皆能顯著提升細胞存活率從 34% 至 76%，將胞內自由基從 2.44 倍分別降至 1.58 與 1.47 倍。發炎相關基因方面與 PM group 相較下，CS-GP group 與 GP group 能將 TNF-α 基因表現量顯著降低 9.8 與 7.3 倍，TLR2 基因表現量為顯著降低 8.12 與 7.47 倍；IL-1β 基因表現量顯著降低 8.6 與 4.1 倍。此外相較於GP group，CS-GP group 含有之 GP 濃度為其二分之一，顯示搭載幾丁聚醣顆粒可提高 GP 被 MH-S 利用之效率。

Alveolar macrophages (AM) play a crucial role in clearing deposited PM. And particulate matter (PM) elevates the expression of inflammatory cytokines, including the Interleukin family and TNF-α. Therefore, AM is a suitable target for inhibiting PM-induced inflammation. Previous studies have shown that G-rich peptide (GP), extracted from fermented Chenopodium formosanum, significantly reduces PM-induced intracellular ROS and improves cell viability of murine alveolar macrophages (MH-S). In this study, we aimed to develop chitosan particles as a biocompatible carrier for GP, improving the efficiency of MH-S utilization. We measure four types of PMs’ (Standard, Fire plant, Huwei 2.5, Huwei 10) metal content, particle size, zeta potential, morphology and intra-, extracellular ROS. And find that whether component is quartz and it’s shape and size maybe are the dominant factors for cytotoxicity. We select Standard for the subsequent experiments based on its’ lowest viability and highest ROS. Chitosan particles (CS) exhibited a cell survival rate of 84% after 24hr of treatment and not significantly inducing intracellular ROS at 1,000 ppm. This result demonstrates CS improved cytotoxicity problem of conventional granulation methods. Chitosan particles loaded with G-rich peptide (CS-GP), with a size of 850.8 ± 59.5 nm and a zeta potential of +24 ± 0.62 mV, exhibit favorable characters for phagocytosis. In intracellular ROS and cell viability experiment, CS-GP and GP groups both significantly reduce PM-induced intracellular ROS from 2.44 to 1.58 and 1.47-fold of control respectively. And CS-GP (48.9 ppm GP) and GP groups (100 ppm GP) both significantly elevate cell viability from 34 to 76%. In the q-PCR experiment, the CS-GP group significantly reduce TNF-α gene expression from 16.3 to 6.5-fold of ctrl, which GP group is 9-fold of ctrl. And in TLR2 gene expression, the CS-GP group significantly reduce gene expression from 9.9 to 1.78-fold of ctrl, which GP group is 2.43-fold of ctrl. The IL-1β gene expression in PM group is 18.6-fold of ctrl. In CS-GP group is 10-fold of ctrl, which is significantly lower than the GP group’s 14.5-fold of ctrl. It’s worth to notice that CS-GP group only containing half of the GP concentration in the GP group. These results demonstrate that chitosan particle enhance the GP utilization efficiency. And its potential material for further investigating into pulmonary drug delivery.