聚乳酸-甘醇酸 (PLGA) 因具有良好的生物相容性以及生物可降解性，故常應用於控制藥物釋放的載體材料。當PLGA載體被細胞吞噬之後，藥物藉由擴散的方式釋放到細胞內，這過程往往需要數天到數個月的時間，因此，細胞內的藥物濃度無法快速達到藥物的有效濃度。針對以上的問題，在本研究的第一部分，我們提出一個新的策略，製備一智慧型多功能的中空微球 (“smart”, multi-functional hollow microspheres) ，可於酸性胞器中快速將抗癌藥物釋放到細胞內。我們利用雙乳化 (double-emulsion method) 的製備方式，在水相的核心攜帶了抗癌藥物doxorubicin (DOX) 以及小蘇打 (NaHCO3) ，而疏水性的殼層則攜帶了綠色螢光染劑 (DiO)。DiO以及DOX的螢光可用來追蹤此中空微球於細胞內的位置，並同時觀察藥物釋放的情況。當PLGA HMs於酸性胞器的刺激之下，小蘇打會與酸反應產生二氧化碳，提高HMs內部的壓力，撐破PLGA球殼，於癌細胞內快速且大量的釋放抗癌藥物DOX，使藥物濃度能快速高過於藥物有效濃度，進而提高治療的效果。
抗藥性 (multi-drug resistance, MDR) 一直是癌症化療上遇到的一個嚴重的瓶頸。在第二部分的實驗，我們將此PLGA HMs應用於對抗具有過度表現P-glycoprotein (P-gp)的抗藥性人類乳癌細胞 (MCF-7/ADR)，並利用即時影像系統，觀測PLGA HMs於MCF-7/ADR細胞內釋放DOX的情形。PLGA HMs經由macropinocytosis pathway吞噬到MCF-7/ADR細胞內，並隨著時間進入到酸性的胞器early endosomes以及late endosomes/lysosomes。在酸性環境的刺激之下，使小蘇打產生二氧化碳，促使PLGA HMs快速且大量的釋放DOX，造成抗藥性癌細胞的死亡。此PLGA HMs的藥物傳遞系統能有效的投遞抗癌藥物DOX到抗藥性細胞MCF-7/ADR內，克服P-gp造成的抗藥性，提高治療的能力。
此外，我們將PLGA HMs與polyvinylpyrrolidone微針貼片 (PVP MNs) 做結合，提出一個可以經皮兩階段依序投遞兩個模擬藥物，Alexa 488 以及 Cy5的微針貼片系統。於PVP MNs內攜帶第一個模擬藥物Alexa 488以及具有紅色螢光 (DiI) 的PLGA HMs，而PLGA HMs內則攜帶了小蘇打以及第二個模擬藥物Cy5。當此微針系統穿刺到皮膚時，PVP MNs會快速在皮下溶解，釋放出第一個模擬藥物Alexa 488以及PLGA HMs，而釋放出來的PLGA HMs會因為皮膚組織是一偏酸性的環境，酸性環境與小蘇打反應後產生二氧化碳的特點，撐破球殼，進而釋放第二個模擬藥物Cy5。此系統可以有效的於皮膚進行兩階段依序投遞藥物，並可廣泛的適用於多種藥物的釋放治療。

Poly(D,L-lactic-co-glycolic acid) (PLGA) is extensively adopted as a carrier material for drug release, due to its excellent biocompatibility and biodegradability. Once internalized by target cells, the drug release from PLGA carriers is dominated by diffusion, lasting from days to months. Consequently, the concentration of drug released from PLGA-based carriers may fail to reach the therapeutic threshold promptly. To address the above issues, the first part of this study presents a novel approach based on “smart”, multi-functional hollow microspheres (HMs), capable of delivering an anticancer drug into tumor cells and releasing the drug in an acidic organelle promptly. HMs were fabricated from PLGA by using a double-emulsion method, with the shell containing DiO (a fluorescence dye) and the aqueous core containing doxorubicin (DOX) and sodium bicarbonate (NaHCO3). Both DiO (in green) and DOX (in red) could serve as fluorescence probes to localize HMs and monitor the release of DOX intracellularly, while NaHCO3 could react with acid to generate CO2. Once the pressure of CO2 reached a certain level, the PLGA shell ruptured to unload the encapsulated DOX quickly. The efficient uptake of HMs by a tumor cell and the subsequent quick release significantly increased the drug concentration beyond the threshold to kill the cell. Localized delivery of DOX in a prompt manner should help to improve both efficacy and tolerability.
Chemotherapy research highly prioritizes overcoming the multi-drug resistance (MDR) effect in cancer cells. This study also attempts to overcome the drug efflux mediated by P-glycoprotein (P-gp) transporters, owing to the ability of these HMs to deliver DOX into MDR cells (MCF-7/ADR). Real-time confocal images provided visible evidences of the acid-responsive intracellular release of DOX from PLGA HMs in MDR cells. Via the macropinocytosis pathway, PLGA HMs taken up by cells experienced an increasingly acidic environment as they trafficked through the early endosomes and, then, matured into more acidic late endosomes/lysosomes. Progressive acidification of the internalized particles in the late endosomes/lysosomes generated CO2 bubbles, leading to disruption of HMs, prompt release of DOX, its accumulation in the nuclei and, ultimately, the death of MDR cells. Conversely, taken up via a passive diffusion mechanism, free DOX was found mainly at the perimembrane region and barely reached the cell nuclei; therefore, no apparent cytotoxicity was observed. These results suggest that the developed PLGA HMs were less susceptible to the P-gp mediated drug efflux in MDR cells and is a highly promising approach in chemotherapy.
Moreover, this study, also develops a novel approach to co-deliver transdermally two model drugs, Alexa 488 and Cy5, in sequence, based on a system of polyvinylpyrrolidone microneedles (PVP MNs) that contain PLGA HMs. The MN system provides the green fluorescence of Alexa 488 in PVP MNs, the red fluorescence of the DiI-labeled PLGA shell of HMs, and the cyan fluorescence of Cy5 in their aqueous core. Cumulatively, the prepared MN arrays support the localization of HMs and the monitoring of release profiles of model drugs within the skin tissues. The major component of this system is NaHCO3, which can be easily incorporated into HMs. Following treatment of HMs with an acidic solution (i.e. simulating the skin pH environment), protons (H+) can diffuse rapidly through the free volume in the PLGA shells to react with NaHCO3 and form a large number of CO2 bubbles. This effect generates pressure inside the HMs and creates pores inside their PLGA shells, subsequently releasing the encapsulated Cy5. Test MNs were sufficiently strong to be inserted into rat skin without breaking. The PVP MNs were significantly dissolved within minutes, and the first model drug Alexa 488, together with HMs, were deposited into the tissues successfully. Once in the acidic environment of the skin, the released HMs started to release Cy5 and continued to spread throughout the neighboring tissues, in a second step of the release of the drug. This approach can be used clinically to co-deliver sequentially and transcutaneously a broad range of drugs.

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