合成具pH調控性的聚吡咯基奈米粒子應用於治療內皮細胞發炎

Abstract

血栓奈米科技結合光熱療法與pH敏感之靶向性用於治療動脈栓塞近年來備受重視，其具有微創性、可防止非目標區域之損壞並能夠快速復原等優點。我們的研究提供了一具多功能且生物相容性之乙二醇殼聚醣（glycol chitosan）、聚吡咯（polypyrrole）與肝素（heparin）之奈米粒子，由於其好的生物相容性、高穩定性和強近紅外光吸收而成為血栓光熱消融的新型藥物。在本研究中，六水合氯化鐵（FeCl36H2O）在酸性條件下催化吡咯（pyrrole）氧化聚合成聚吡咯（Ppy），接著陽離子乙二醇殼聚醣（GCS）和陰離子肝素（Hep）自組裝成具pH靈敏的抗發炎奈米顆粒。本策略利用了乙二醇殼聚醣的pH敏感性，讓奈米粒子在發炎反應所引起的酸性環境中透過pH值之改變來成功標靶到發炎部位以改善靶向性。而為了更進一步改善靶向性，我們運用了肝素能夠靶向P-選擇素（P-selectin）的能力，P-選擇素是在發炎反應中作為碳水化合物結合細胞粘附的主要分子。此外，近紅外光加熱實驗顯現了奈米粒子優秀的加熱能力，可用於血栓的光熱治療。在體外模擬方面我們是透過脂多醣（lipopolysaccharide）來對牛主動脈內皮細胞引起發炎反應，奈米顆粒在MTT試驗下顯現良好的細胞存活率，抗人類CD62P抗體（PE）的熒光測定則觀察到奈米顆粒靶能夠有效的標靶到發炎的細胞。動物體內實驗結果顯示奈米粒子能夠有效的標靶至血栓並成功地累積，透過光熱後能成功加熱到44.6度，足夠成功的消融血栓。本研究展示了一極具潛力性的奈米顆粒結合了pH敏感並能標靶至P-選擇素且結合光熱效應以消融血栓。

Thrombus nanotechnology is emerging as one of the promising strategies for the treatment of arterial embolism by combining inflammation site targeting and photothermal therapy (PTT). This approach has received considerable attention in the recent years because it is minimally invasive, prevents damage to non-targeted regions and permits fast recovery. The present study demonstrates multifunctional glycol chitosan-polypyrrole-heparin nanoparticles (GCPH NPs) as novel agents for photothermal ablation of thrombus attributed to the biocompatibility, stability, pH-responsiveness, P-selectin targeting and strong near-infrared (NIR) absorbance of the nanoparticles. In this study, iron chloride hexahydrate (FeCl36H2O ) was used to initiate oxidative polymerization of pyrrole to polypyrrole (PPY) in acidic conditions, followed by binding with cationic glycol chitosan (GCS) and anionic heparin (Hep) to self-assemble pH-responsive, anti-inflammatory nanoparticles. The strategy took advantage of glycol chitosan’s pH sensitivity, enhancing the targeting ability of the nanoparticles in acidic microenvironment resulted from inflammation. To further improve targeting, heparin ability to target P-selectin, a carbohydrate-binding cell adhesion molecule that plays a major role in the initiation of inflammatory responses, was utilized. The cytotoxicity of the synthesized nanoparticles was tested using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromidefor (MTT) assay on bovine aortic endothelial cells (BAEC) cell line. The cells were shown to be viable in the presence of the nanoparticles. Cell experiments also revealed the effective targeting ability of the nanoparticles to inflammation sites using anti-human CD62P antibody (PE) fluorescence assay, in tandem with Cyanine5 (Cy5) fluorescence assay, on lipopolysaccharide (LPS)-induced BAEC. The in vivo results further demonstrated successful targeting and accumulation of nanoparticles in thrombosis site compared to corresponding control groups. The accumulation of the nanoparticles was accompanied with NIR absorbance up to 44.6 C, exceeding the temperature required for photothermal ablation of thrombosis. This research highlighted the promising potential use of GCPH NPs for pH-responsive and P-selectin targeting combined with photothermal ablation of thrombosis in preclinical animals.