| Summary: | 博士 === 國立清華大學 === 化學工程學系 === 100 === 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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