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標題: | 以多孔性氫氧基磷灰石作為克崙特羅之藥物控制釋放載體以治療阿茲海默症 Controlled release of Clenbuterol from a hydroxyapatite carrier for the treatment of Alzheimer’s Disease |
作者: | 林意文 Yi-Wen Lin |
指導教授: | 林峯輝 Feng-Huei Lin |
關鍵字: | 阿茲海默症,β澱粉樣蛋白,克崙特羅,氫氧基磷灰石,藥物控制系統, Alzheimer's disease,β-amyloid,clenbuterol,hydroxyapatite,drug delivery system, |
出版年 : | 2023 |
學位: | 博士 |
摘要: | 阿茲海默症(AD)是一種複雜的神經退化性疾病,臨床上以認知功能受損為病理特徵。形態學觀察 AD 的主要病因包括由澱粉樣蛋白(Aβ)形成的細胞外澱粉樣蛋白聚集體(斑塊)和高磷酸化Tau 蛋白的細胞內神經原纖維纏結(NFT)的積累。發炎在AD 的發展中起著重要作用。在本研究中,於澱粉樣蛋白(Aβ)假說,證實阿茲海默症是由異常的Aβ 積累引起,Aβ 會引起氧化壓力,發炎反應及神經元功能障礙之間存在關聯。克崙特羅是一種β2 腎上腺素激動劑,具有顯著抗發炎能力。克崙特羅的半衰期短,患者常因記憶力減退忘記服藥而影響治療效果。為提高
藥物生物利用率,本研究利用共沉澱法將多孔氫氧基磷灰石(meso-HAp)合成出100 nm 大小微粒,並以硬脂酸進行表面改質,修飾其疏水性(SHAP),藉由物理吸附將克倫特羅(CLB)負載到SHAP 奈米顆粒上,形成藥物載體系統(SHAPCLB)。此藥物載體一被巨噬細胞吞噬後,由於滲透壓的改變,溶酶體/核內體複合體破裂,導致CLB 釋放到細胞質。然而,由於高濃度鈣離子(Ca2 +)濃度,CLB被胞吐到細胞外空間,並最終達到血液循環。本研究使用SHAP 製備藥物載體攜載克倫特羅,降低克倫特羅被代謝以提高生物利用率,此載體大小約為100 nm,可有效地被巨噬細胞吞噬,同時利用WST-1 證實SHAP-CLB 具有良好之生物相容性,並設計在體外維持兩週之控制釋放。在體外實驗中,證實搭載20 μM 克倫特羅的SHAP 奈米顆粒可有效調節炎症,包括抑制轉錄因子NF-κB 的作用以及抑 制促炎細胞因子和趨化因子的產生,例如腫瘤壞死因子-α 和介白素及降低Aβ 寡聚體引起的毒性,防止蛋白質聚集。在動物實驗中,利用70 mg/kg 氯化鋁誘導動物模型,將載體注射至細胞活性高的肌肉組織,以細胞吞噬藥物的特性來達到恆定釋放的目的。fMRI 結果顯示可有效降地大鼠的神經退化情形、MWM 結果證實改善記憶能力。組織切片進行H&E 染色及BACE1、6E10 抗體的免疫組織染色,並以光學和共焦顯微鏡觀察切片。分別以西方墨點法和液相層析串聯質譜儀(LCMS/MS)評估克倫特羅的治療效率及藥物動力學。以SHAP-CLB 治療8 週後,測得大鼠大腦中克倫特羅濃度為2.38 ng/mL,上述結果證實載藥載體具有良好防止阿茲海默症之功效。研究結果顯示,SHAP 遞送克崙特羅的藥物控制釋放系統對於AD 治療有臨床上之治療潛力。 Alzheimer's disease (AD) is a complex neurodegenerative disorder characterized clinically by impaired cognitive function. Morphological observations of AD reveal the accumulation of extracellular amyloid-beta (Aβ) fibrillar aggregates (plaques) and intracellular neurofibrillary tangles (NFT) formed by hyperphosphorylated Tau protein as major pathological features. Inflammation plays a crucial role in the development of AD. In this study, based on the Aβ hypothesis, Alzheimer's disease is confirmed to result from abnormal Aβ accumulation, leading to oxidative stress, inflammatory responses, and neuronal dysfunction. Clenbuterol is a β2-adrenergic agonist, exhibits significant anti-inflammatory capabilities. However, its short half-life often leads to treatment inefficacy due to patients forgetting to take the medication. To enhance drug bioavailability, this research utilized a co-precipitation method to synthesize porous hydroxyapatite (mesoHAp) particles of approximately 100 nm. Surface modification using stearic acid improved its hydrophobicity (SHAP). Clenbuterol (CLB) was physically adsorbed onto SHAP nanoparticles, forming a drug delivery system (SHAP-CLB). Once engulfed by macrophages, changes in osmotic pressure cause lysosome/endosome complex rupture, releasing CLB into the cytoplasm. However, due to high concentrations of calcium ions (Ca2+), CLB is expelled into the extracellular space, eventually reaching the bloodstream. This study employed SHAP to prepare a drug carrier for transporting chloroquine, reducing its metabolism to enhance bioavailability. The carrier, with a size of approximately 100 nm, is efficiently phagocytosed by macrophages. WST-1 assays confirmed the good biocompatibility of SHAP-CLB, with controlled release maintained for two weeks in vitro. In vitro experiments demonstrated that SHAP nanoparticles loaded with 20 µM chloroquine effectively modulated inflammation by inhibiting transcription factor NF-κB and suppressing the production of pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and interleukins, while reducing the toxicity induced by Aβ oligomers and preventing protein aggregation. In animal experiments using a 70 mg/kg aluminum chloride-induced model, the carrier was injected into highly active muscle tissue to achieve sustained release through cellular phagocytosis. fMRI results showed effective reduction in neurodegeneration in rats, and Morris Water Maze results confirmed improved memory capacity. Tissue sections were stained with H&E and immunohistochemistry using BACE1 and 6E10 antibodies, observed through optical and confocal microscopy. The therapeutic efficiency and pharmacokinetics of chloroquine were evaluated using Western blotting and liquid chromatography-tandem mass spectrometry (LC-MS/MS). After 8 weeks of SHAP-CLB treatment, the chloroquine concentration in the rat brain was measured at 2.38 ng/mL, confirming the efficacy of the drug-loaded carrier in preventing Alzheimer's disease. The research results suggest that the SHAP-delivered chloroquine drug control release system has clinical potential for AD treatment. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/92663 |
DOI: | 10.6342/NTU202304507 |
全文授權: | 同意授權(限校園內公開) |
顯示於系所單位: | 醫學工程學研究所 |
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