Fabrication of Gold Nanoparticles-Based Platforms for Assaying Peptidase Activity and Loading Drugs
|關鍵字:||奈米金球;光學檢測平台;螢光;基質金屬蛋白酶;表面修飾;Gold nanoparticles;Surface modification;Fluorescence;Matrix metalloproteinase;Optical biosensing platform|
|摘要:||奈米金球 (gold nanoparticles; AuNPs) 具有獨特的光學特性和良好的穩定性，因此在生物醫學領域中有前瞻性的發展與應用。在本研究中，我們利用AuNPs的表面電漿共振 (sufaceplasmon resonance; SPR) 的特性，建構光學與螢光生物檢測系統，應用於檢測胜肽酶 (peptidase) 之活性。當AuNPs的大小改變或聚集時，其SPR光譜會改變，基於這個特性，AuNPs可被發展成特定生物分子的光學檢測平台。另外，藉由AuNPs的表面修飾技術，AuNPs可以應用於藥物的裝載與傳輸。
在本研究中，我們在AuNPs修飾上特定胜肽 (peptide) 後，peptide除了是基質金屬蛋白酶-2 (matrix metalloproteinase-2; MMP-2) 的受質之外，也會在AuNPs表面提供足夠的排斥力，避免AuNPs在緩衝液中聚集。當加入MMP-2於AuNPs/peptide後，MMP-2會將AuNPs表面上peptide降解，使AuNPs失去保護進而產生聚集現象。據此，AuNPs/peptide聚集後和聚集前的A625 nm與A530 nm吸收峰比值 (A625 nm/A530 nm) 可用於測定MMP-2活性。實驗結果顯示，所建立的AuNPs可見光學檢測平台用於MMP-2活性的檢測低限為100 ng/mL，而當MMP-2活性位於100 - 1,500 ng/mL之間時，量測結果呈一線性相關 (R2 = 0.9703)。
為了提升本AuNPs/peptide之檢測靈敏度，我們將AuNPs/peptide上的peptide置換成peptide-FITC，以建構一AuNPs為基礎的螢光檢測平台。當peptide-FITC修飾在AuNPs時，FITC的螢光會被AuNPs遮蔽；然而，加入MMP-2於AuNPs/peptie-FITC後，peptide會被MMP-2降解並釋放出FITC以顯現螢光，透過螢光強度偵測便可量化MMP-2的活性。此AuNPs螢光檢測平台用於MMP-2活性檢測的低限為0.01 ng/mL，而當MMP-2活性位於 0.01 – 2 ng/mL之間時，量測結果呈一線性相關 (R2 = 0.9759)。由於我們所建立的AuNPs螢光檢測平台靈敏度是AuNPs可見光學檢測平台的10,000倍，因此我們已將之運用於單一細胞中peptidase活性的測定。
另一方面，由於AuNPs擁有生物相容性與表面修飾的特性，因此可以利用AuNPs建立多功能藥物裝載與傳輸平台。據此，我們將人類生長荷爾蒙 ( human growth hormone; hGH) 與有拉曼 (Raman) 報導功能的孔雀綠 (malachite green isothiocyanate; MGITC) 修飾在AuNPs上，使AuNPs同時具有標靶和報導的功能。因此，我們以AuNPs/hGH-MGITC處理人類肝癌細胞HepG2後，可以用表面增強拉曼散射來偵測AuNPs被傳輸至細胞內的位置。此外，AuNPs也透過修飾上抗癌藥物阿黴素 (doxorubicin) 與hGH後，具有成為癌症標靶藥物的可行性，因為利用AuNPs/hGH-doxorubicin來處理HepG2，其對HepG2的毒殺性顯著高於單獨使用doxorubicin的處理結果。|
Gold nanoparticles (AuNPs) have received interests due to their characteristics, especially optical and physical properties. In this research, the optical biosensing and fluorescent platforms were developed. Both AuNPs-based biosensing platforms were set up based on the surface plasmon resonance (SPR) property of AuNPs to detect certain peptidase activity. Additionally, the surface-modified techniques of AuNPs were also utilized on the drug load and delivery. The AuNPs-based optical biosensing system was established by means of the varied SPR spectra of AuNPs, while AuNPs changed their sizes, included aggregation or modified with functional molecules. According to the mechanism, AuNPs modified with peptide (AuNP/peptide) that was used as a peptidase (matrix metalloprotease-2; MMP-2) substrate and also a shelter to protect AuNPs from aggregation. After MMP-2 digested, AuNPs became aggregation because of decreasing the steric repulsion among AuNPs. The aggregation of AuNPs was measured via the red-shift of SPR absorption. Therefore, the MMP-2 activity could be quantitatively estimated by the absorption ratio, A625 nm/A530 nm. The results show that the detection limit of the established platform was 100 ng/mL, a linear correlation between MMP-2 was ranging from 100 to 1,500 ng/mL, and the changes of A625 nm/A530 nm was observed (R2 = 0.9703). For improving the sensitivity of AuNPs-based platform, the peptide was exchanged with peptide-FITC as substrate modified on AuNPs to establish AuNPs-based fluorescence platform. The FITC would be quenched by AuNPs when the peptide-FITC modified on AuNPs surface. The fluorescence intensity of FITC was detected after MMP-2 digested the peptide leading peptide-FITC released from AuNPs surface. According to the concept, the MMP-2 activity could be analyzed by measuring the change of fluorescence intensity (at emission of FITC, 515 nm). The AuNPs-based fluorescence platform performed a detection limit as 0.01 ng/mL, with a linear correlation ranging from 0.01 to 2 ng/mL of MMP-2 (R2 = 0.9759). Additionally, both AuNPs-based optical and fluorescence platforms showed the ability to assay the efficiency of MMPs inhibitors with high specificity. Especially, the AuNPs-based fluorescence platform could apply in cellular peptidase activity analysis through bio-image (confocal) that revealed a promising potential to utilize in in vivo peptidase detection. On the other hand, the AuNPs-based delivery platform was fabricated due to the biocomplementary and various surface modifications of AuNPs through special molecules with their functional groups. Based on the concept, the AuNPs were conjugated with human growth hormone protein (hGH) used to target the hGH receptor of HepG2 cells, and Raman reporter (malachite green isothiocyanate; MGITC) as tags. After incubating the AuNPs-complexes with HepG2 cells, the AuNPs specifically targeted to the cells and could be traced in cells by surface-enhanced Raman scattering through Raman confocal. In addition, AuNPs were also used as drug delivery carrier by modifying AuNPs with hGH and anticancer drug, doxorubicin. Using AuNPs/hGH-doxorubicin could bind HepG2 cells precisely and inhibit the growth of cancer cells at the same time. This result indicates that the multifunctional AuNPs improved the effective of the medicine in vitro according to selective targeting and treating drug to the objective in once.
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