抑制致病菌生長的奈米標靶光熱探針之研發

Abstract

抗藥性菌種隨著抗生素的濫用有愈來愈多的趨勢，因此發展新的抑制細菌生長的方法尤其是針對抗藥性菌種的毒殺有實際上的迫切性，而官能化奈米粒子結合光熱療法是針對解決此問題的一種可能方法。因此在本論文中合成並設計二種具有近紅外光吸收能力且對致病菌具有辨識性的奈米標靶探針，使此奈米探針在辨識到細菌後能夠在近紅外光的照射下，快速將光能轉變為熱能，使細菌的生長受到抑制。

本論文中設計的第一種奈米標靶探針是以具有吸收近紅外光能力的多邊形金奈米粒子為基材，此合成方法是本論文開發出的新光化學合成法。實驗中先證實此金奈米粒子具有很好的光熱轉換效率後，接著再進行金奈米粒子的表面官能化，使其具有辨識細菌的能力。萬古黴素對細菌細胞壁上的短五胜肽中的D-Ala-D-Ala具有良好的辨識性，因此利用有機合成的方法將萬古黴素以雙硫基橋接成萬古黴素二聚體，並藉由硫-金鍵結將萬古黴素二聚體修飾至金奈米粒子表面。實驗結果顯示包括革蘭氏陽性菌、革蘭氏陰性菌以及抗藥性細菌都能被此官能化金奈米探針有效的辨識，並且被辨認到的細菌，在808 nm的近紅外光雷射(功率：250 mW/cm2)照射5分鐘内，就能導致99% 以上被此金奈米標靶探針辨識到的細菌生長受到抑制。

而本論文中發展的第二種探針為具有磁性的核殼氧化鐵-金奈米標靶探針，同樣利用硫-金鍵結將萬古黴素二聚體修飾至此奈米粒子表面，所以此奈米粒子除了具備吸收近紅外光的能力、標靶細菌的功能外，更具備有磁性，因此只需要藉由外加磁場的作用就能將被奈米標靶探針辨識到的細菌吸引至一定點，如此可使光熱抑菌的效率因而提昇，並且可降低對細胞的傷害。實驗結果顯示，被此官能化奈米探針辨認到的細菌，在808 nm的近紅外光雷射照射3分鐘下，僅有不到1%的細菌還能存活。此結果證明了磁性核殼氧化鐵-金奈米探針具有很好的光熱抑菌效果，且進一步縮短光熱抑菌過程所需的照光時間。

除此之外，近紅外光因為對於動物細胞組織具有較高的穿透率，因此這種光熱療法應有潛力繼續發展成為可應用在活體受到細菌感染之治療上，但還需要進一步進行毒性及藥物代謝等試驗，才能知道此奈米光熱療法於輔助臨床治療細菌感染是否可行。

The rapid emerging of antibiotic-resistant bacterial strains becomes a serious problem because of the extensive use of antibiotics. Thus, it is urgent to develop a new means for effective inhibition of the cell growth of antibiotic-resistant bacteria. Functional nanoparticles combined with photothermal therapeutics provide a possible solution for solving the problem. In this dissertation, two types of functional nanoparticles, which have the capacity of absorbing light in the near infrared (NIR) region and the ability to recognize target bacteria, were proposed. Upon the attachment of the functional nanoparticles on the cell walls of target bacteria, the cell growth of the bacteria can be effectively inhibited under illumination of NIR light within a short period because the functional nanoparticles attached on the surface of the bacteria can rapidly convert the light they absorbed from NIR laser to heat.

In the first part of this dissertation, polygonal gold nanoparticles with the capacity of absorbing NIR light and elevating the temperature of the solution under irradiation of NIR light were generated via a photochemical reaction. Vancomycin, which can bind with the terminal D-Ala-D-Ala moieties of the peptide units of the cell walls of bacteria, was immobilized on the surface of the gold nanoparticles via S-Au bonding. The results demonstrated that vancomycin-bound gold nanoparticles are capable of selective-binding onto the cell walls of pathogenic bacteria. Therefore, a large portion (>99%) of bacteria targeted by the gold nanoparticles was destroyed under illumination by NIR light (808 nm, ca. 250 mW/cm2) within 5 min. This photothermal approach is effective not only for Gram-positive and Gram-negative bacteria, but also for inhibiting the cell growth of antibiotic-resistant bacteria.

Alternatively, magnetic core/shell gold nanoeggs (Fe3O4@Au) that consisting of gold nanoshells with embedded magnetic Fe3O4 nanoparticles were also developed. Vancomycin bound Fe3O4@Au (Van- Fe3O4@Au) nanoeggs, which posses the absorption capacity in the NIR region, the ability to target bacteria, and magnetic characteristics, play the roles of photothermal agents and targeting probes for bacteria. Therefore, the bacteria targeted by Van-Fe3O4@Au nanoeggs can be readily aggregated under the influence of an external magnetic field. Furthermore, heat transfer from the Van-Fe3O4@Au nanoeggs to the aggregated bacteria under the irradiation of NIR light is more effective. Therefore, the time required for effectively inhibiting the cell growth of the bacteria can be reduced. The possibility of the damage caused during photothermal therapy on animal cells can be diminished. The results show that less than 1% of the bacterial cells targeted by the nanoprobes could survive under illumination of NIR light within 3 min.

NIR light is capable of penetrating deeper tissues, so it is possible that this photothermal approach can be used for treating animals suffering from bacterial infections. However, further studies regarding toxicities and metabolisms of the photothermal agents should be performed to examine the feasibility of using this approach in vivo.