合成以表面修飾之金奈米粒子的生物特性及在癌症治療上的研究評估

Abstract

金奈米粒子因具備特異的理化性質和良好的生物相容性，而曾被應用於音波呈像的增強、基因的運送及光誘導的加熱治療等。然而其生物特性所知仍有限。本實驗中我們則以不同的製程生產出球形 (AuNPs) 和桿狀(AuNRs)兩種大小和形狀相異之金奈米，且作不同之表面修飾，並分別於體外及活體評估其生物性質及探索其醫學上之應用性。在細胞毒性的實驗中，發現表面修飾聚乙二醇 (PEG) 後的金奈米較之原始奈米粒子具有較低的細胞毒性。此外，將金奈米由BALB/c品系之小鼠尾靜脈注射後，藉由原子吸光光譜檢測其在器官分佈情形得知，金奈米粒子主要堆積在脾臟其次是肝臟，而在心臟、肺臟及腎臟的量都相對得少，並且其生物分布與奈米形狀、大小、表面修飾皆有顯著相關。在對尿液和糞便的檢測中也發現，注射後24小時後，這些奈米金粒子能持續從腎臟和消化道排出體外。這些生物相容性和生物降解性的觀察結果，將有助於我們對金奈米的藥物動力及毒性的了解，並藉以設計更佳之奈米載體。 此外，我們進一步設計並評估以金奈米為載體之癌症化學治療的新平台。由上述實驗發現球形金奈米之生物相容性最佳，因此以球形金奈米為平台，藉由端修飾硫醇 (SH) 的寡核苷酸與金奈米的共價鍵結，並以鹽離子梯度環境控制，成功的將寡核苷酸與奈米金作高密度自組裝結合。我們並以DNA雙股間的氫鍵特異結合方式，來鍵結類核苷酸的抗癌藥物- 5-FU。實驗結果顯示表面經核甘酸修飾過的金奈米比沒修飾過者能攜帶更多5-FU，並且隨著金奈米直徑從 25 nm 增加到35nm時，表面電荷也由-9.58降低到-21.66mv，顯示更多的寡核苷酸與之結合。在磷酸緩衝液的酸鹼值為環境改變之藥物釋放試驗中，發現藥物由金奈米釋出程度與環境酸鹼指數有正相關性。此種特性顯示，這類新穎的奈米載體藥物聯結方式，在發展對後半段消化道的病變之投藥系統上具有相當的潛力，因其微環境傾向由酸性轉鹼性之故也。我們更進一步在大腸癌細胞株藉由四唑鹽比色法 (MTT) 檢視其癌細胞之毐殺特性，結果顯示金奈米-抗癌藥物的結合體對癌細胞的毒殺能力隨著劑量增加而加強，且確實比單獨投藥效果來得好。此種金奈米藥物的結合，可藉著環境條件的調控來釋放藥物，極適合以口服的方式來對結腸癌進行化學治療。整體結果顯示金奈米具備相當好的生物相容性和生物降解性，且可攜帶分子到特定區，未來結合金奈米之超音波顯影特性，將能達到癌症的診斷和治療之新穎應用，也能發展成為相當優異且有極具潛力的奈米運送者。

Gold nanoparticles (AuNPs) with its distinctive physical and chemical properties and well as biocompatibility have been utilized in the fields of photoacoustic imaging, delivery of genes and laser induced photothermal therapy in recent years. In this study, we fabricated two types of gold-nanoparticles with various surface modifications. Their biological properties and potential biomedical applications were explored. In the in vitro biocompatibility evaluation, those PEG modified nanoparticles exhibit better compatibility in the MTT assays. The in vivo biodistribution analysis was performed using BALB/c mice through I.V. injection via tail vein followed by Atomic Absorption Spectroscopy analysis. We discovered that most nanoparticles accumulated in the spleen and liver, and few of them were found in heart, lung and kidney. The nanoparticles were then excreted via kidney and gastrointestinal tract and can be detected in urine and feces 24 hr after injection. The conformation, size and surface modifications all contribute to their in vivo biodistribution. Together with the results from biocompatibility, biodegradation and biodistribution will contribute to our understanding of the pharmacokinetic and toxicity of nanoparticles and to develop better nano carrier. To evaluate the chemotherapeutic efficiency of our AuNPs, oligonucleotide conjugated AuNPs was applied as the carrier for simultaneous DNA and anti-cancer nucleoside delivery. The polynucleotide was modified with thiol group in the 5’ end. Under a salt gradient, these oligonucleotides form high-density self-assembled monolayer on the particle surface. The nanoparticle-oligonucleotide complex presented higher capacity in carrying 5-FU anti-cancer compounds than the original gold particles. The hydrodynamic size of the AuNPs increased from 25 to 35nm with an increase in the negative surface charge from -9.58 to -21.66mV after polynucleotide conjugation and drug loading. A positive association between environmental pH and the drug release was observed in PBS, which implied their potential use in the controlled localized drug release in the lower GI tract. The MTT assay revealed a dose dependent cytotoxicity of the nanoparticle- oligonucleotide-anti-cancer drug complex to colon cancer cell line than the free compounds of the same dosage. This new polynucleotide-AuNPs complex presented great potential to be used as the environmental controlled anti-cancer nanocapsule, especially for per oral colon cancer chemotherapy. As gold nanoparticles have been shown to be a good photoacoustic imaging contrast agent, their potential as an excellent nano vehicles for combined molecular imaging and cancer drug are highly expected.