二氧化矽奈米空心球之調控及其生物應用

Abstract

二氧化矽奈米空心球近年來在生物醫學及工業應用的潛力漸漸受到重視，主要是因為中空的內部空間可以容納較多及較大的功能性物質，以提高它在應用上的成效。本實驗室過去的研究中，成功地利用油包水的微乳液系統將無機金屬奈米粒子、或是有機的生物酵素在空心球合成的過程中同時包覆在球內部，這是很難得的研究。但是，利用油包水微乳液系統合成的空心球往往因為產率很低並且嚴重的聚集現象，使得應用上常常受到限制，因此這些問題需要被重視並且改善。 在本研究的第一部分，藉由分步添加矽源及一步表面修飾的方法，我們優化了空心球的合成條件，成功地合成出具有高產率、懸浮性佳、且大小均勻的50 nm空心球，由於表面修飾長鏈的親水官能基 (PEG)，空心球在水中、細胞培養液及磷酸鹽緩衝溶液中都能展現良好的懸浮性，大大的提升了空心球在生物醫學的應用潛力。若進一步將空心球的合成概念延伸，我們能夠產生由內而外具有連續性結構差異的實心球，並藉由溫水一步侵蝕的方式，合成出多殼層的空心球，並且可以控制球大小在 100 nm以下，這是文獻上很少見的結果。 本研究的第二部分中，我們希望將50 nm的空心球設計成一個抗癌藥物之奈米載體。我們發展了一個兩相系統能夠高效率地將抗癌藥物阿黴素 (doxorubicin)裝載在空心球內部，此系統主要是將空心球內部先裝填硫酸銨的鹽類水溶液，並利用有機溶劑氯仿維持球內部的鹽類梯度，由於此鹽度梯度會驅使藥物有效地進到空心球內部，而達到高附載量(約8.0 ~ 9.9 wt %) 及高包覆效率(>70 %)。在過去文獻中，很少研究能夠在孔洞二氧化矽奈米粒子上利用這種主動性的裝載藥物方法並且達到如此高的效率。裝載完藥物後，經由後續適當的處理，可以將藥物轉換形式堵住球殼的孔洞，防止藥物在後續的過程中有滲漏的現象。進一步我們也將裝載阿黴素的空心球送到培養的癌細胞及小鼠內，評估其藥物輸送的特性。在細胞的研究中，裝載藥物的空心球可以有效的抑制癌細胞增生，藥物的效果並沒有因為裝載的過程而降低；空心球可以帶著藥物進到細胞內進行釋放，達到一個較具時效性的治療；我們也將空心球(PEG HSNs)送到小鼠體內，分析其循環時間及分布情況，可以發現其在老鼠體內具有良好的循環時間，且不會對老鼠造成毒性；另外在腫瘤鼠中，空心球能夠藉由增強滲透和滯留(enhanced permeability and retention, EPR)效應優先聚集在腫瘤部位，大大提升了空心球作為抗癌藥物運輸載體的可能性。 整體而言，我們從材料的觀點出發建立了一個穩定的微乳液系統，能夠合成出具有良好性質的二氧化矽奈米空心球，這些性質讓我們成功的發展出高效率的藥物裝載方法，並且在細胞及老鼠體內證實其作為抗癌藥物輸送載體的潛力。

Hollow silica nanospheres (HSNs) with large interior space have recently gained increasing interests due to their tremendous potential for biomedical and industrial applications. In previous study, the inorganic metal nanoparticles and organic functional groups, as well as enzymes, have been successfully encapsulated in HSNs by a water-in-oil microemulsion method. However, the applications of HSNs from microemulsion are usually limited due to the problems of low yield and easy aggregation. In the first part of this work, we optimized the synthetic conditions to fabricate a high-yield, size-uniform and well-dispersed PEG HSNs (50 nm) by time-separated addition of silica source and one-step surface modification. The yield of PEG HSNs with uniform size (50 nm) was effectively elevated over 5 times, and PEG HSNs displayed excellent dispersity in water, DMEM (10% FBS), and PBS. By extending the synthetic concept of HSNs, multi-shelled structure can be achieved through layer-by-layer condensation and one-step warm water etching, the morphology of double-shelled HSNs can be controlled below 100 nm which was rare in literatures. In the second part, optimized PEG HSNs (50 nm) displayed increasingly potentials as a nanocarrier for anti-cancer drug. For effective and efficient drug delivery, we developed an active loading method to load doxorubicin (Dox). Inspired by DoxilR, a two-phase system (H2O/CHCl3) was used to establish the (NH4)2SO4 gradient on PEG-HSNs to introduce the amphipathic Dox into the particles with high loading capacity (8.0-9.9 wt %) and entrapment efficiency (>70%). Under proper treatments, Dox could be a capping agent to block the pores on the shell preventing the leakage of drugs. We successfully made good use of the unique structure of PEG HSNs and properties of Dox to accomplish the active loading. Furthermore, in vitro and in vivo studies of Dox loaded HSNs were investigated. Dox loaded HSNs displayed pH-sensitively controlled release character. The cellular uptake, cytotoxicity, and intracellular drug release of Dox@PEG-TA HSNs were evaluated in MDA-MB-231 cells. For in vivo studies, PEG HSNs and PEG-TA HSNs were intravenously injected into animal model. PEG HSNs displayed a better circulation time than PEG-TA HSNs by evaluation of circulation and bio-distribution. Moreover, the passive targeting ability (EPR effect, Enhanced Permeability Retention) were also demonstrated on PEG HSNs in tumor-bearing mice. Overall, we have developed a well-controlled microemulsion system to synthesize the HSNs with desired properties that contribute to the successful development of an active drug loading method and a potential drug delivery system.