Abstract
Nanomedicine is the medical application of nanotechnology. Combining the basic knowledge of the chemistry, material science and chemical biology, we are able to manipulate the nanoscale materials of interest and extend their uses in cancer treatment and clinical diagnosis. Three nanoparticles tentatively used in bioapplication include (1) liposome, (2) phospholipid－poly lactic acid－co－glycolic acid polymer hybrid nanoparticles (PLGA@Lipid NPs), and (3) quantum dot-conjugated PLGA hybrid nanoparticles (PLGA@QD NPs). Three studies are included in this dissertation: (1) Monitoring the subcellular Localization of doxorubicin in CHO-K1 using MEKC－LIF: liposomal carrier for enhanced drug delivery; (2) Photocontrolled targeted drug delivery: photocaged biologically active folic acid as a light-responsive tumor-targeting molecule; (3) In vivo cell fate tracking of mesenchymal stem cells using PLGA@QD nanoparticles.The aim of the first study is to monitor the subcellular localization of doxorubicin (DOX) delivered in free form and liposomal form, respectively. DOX is an extensively used anthracycline that has proven to be effective against a variety of human malignant tumors, such as ovarian or breast cancer. While DOX was administered into cultured cancer cell targets (such as CHO-K1) in either free drug form or in drug carrier-associated form (i.e., DOX encapsulated in the drug delivery carrier), various action of mechanisms for DOX were initiated, among which, it has been long believed that DOX enters the nucleus, interacts with DNA in numerous ways, and ?nally halts cell proliferation. Aside from its therapeutic effect, regrettably DOX treatment may be accompanied by the occurrence of cardiac and liver toxicity and drug resistance that are attributed from cellular processes involving the parent drug or its metabolites. Liposomal formulation of DOX, known to be capable of attenuating direct uptake of reticuloendothelial system (RES) and prolonging the circulation time in blood, demonstrated reduced toxic side-effects. We herein report the development of a modi?ed MEKC–LIF (Micellar electrokinetic chromatography－Laser induced ?uorescence) method suitable for analyzing DOX in biological samples. The MEKC migration buffer, consisting of 10 mM borate, 100 mM sodium dodecyl sulfate (SDS) (pH 9.3), was found to provide an ef?cient and stable electrophoretic separation and analysis for DOX. Responses were linear in the range of 11.3–725 ng/mL; the limit of quantitation (LOQ) was found to be 43.1 ng/mL (S/N=10) (equivalent to 74.3 nM) and limit of detection (LOD) was calculated as 6.36 ng/mL (S/N=3) (equivalent to 11.0 nM). This approach was subsequently employed to compare the intracellular accumulation in three subcellular fractions of DOX-treated CHO-K1 cells. These fractions form a pellet at <1400g, 1400–14000g, and >14000g and are enriched in nuclei, organelles (mitochondria and lysosomes), and cytosole components, respectively, resulting from treatment of CHO-K1 cells with 25 mM (equivalent to 14.5 mg/mL) of two DOX formats (in free drug form or liposomal form synthesized in current study) for different periods of time. Our results indicated that the most abundant DOX was found in the nuclear-enriched fraction of cells treated for 12 h and 6 h with free and liposomal DOX, respectively, providing direct evidence to con?rm the enhanced ef?ciency of liposomal carriers in delivering DOX into the nucleus. The observations presented herein suggest that subcellular fractionation followed by liquid–liquid extraction and MEKC－LIF could be a powerful diagnostic tool for monitoring intracellular DOX distribution, which is highly relevant to cytotoxicity studies of anthracycline-type anticancer drugs. The second study intends to solve a major problem in current chemotherapy, which is “how to determine the optimum drug dosage given to patients?” A low dosage of drug is ineffective in the treatment of a tumor, whereas a high dosage of chemotherapeutics is intolerable for patients due to toxicity and unwanted side effects. Therefore new designs for anticancer drugs are desirable to increase the local effective therapeutic concentration. Two promising strategies were primed herein to achieve this goal; one is to construct a stimuli-responsive drug delivery system for controlled drug release and the other is to formulate a system for actively targeting the delivery of the therapeutic agent. Both approaches can minimize adverse effect of cytotoxic drugs and improve the therapeutic efficacy of conventional pharmaceuticals. Active targeting that uses specific ligands including monoclonal antibodies, peptides, and aptamers that bind to specific proteins or surface antigens overexpressed on cancer cells is a practical way to enhance the local control of therapeutics. We herein designed the photocaged folate nanoconjugates that selectively target cancer cells upon irradiation with light. The folic acid is masked by a photocleavable o-nitrobenzyl (ONB) group through covalently binding to ��- and ��-carboxylate groups, which interact with folic acid receptors (FRs) on the cell surface. The obtained results revealed that caging and photouncaging can be applied to the FA to potentially improve its targeting specificity. Moreover the application of the caged folate for intracellular drug delivery was examined using a biodegradable PLGA@lipid hybrid nanoparticle. It was confirmed that the cytotoxicity of Taxol encapsulated in PLGA@lipid hybrid nanoparticles increased upon light irradiation. In the final study, a novel PLGA@QD655 NPs were utilized to investigate whether the applied nanoparticles affect the in vivo differentiation of stem cells. Adult stem cells have been intensively studied for their potential use in cell therapies for neurodegenerative diseases, ischemia and traumatic injuries. It was reported previously that the mesenchymal stem cells (MSCs) could be labeled with poly(lactide－co－glycolide) nanoparticles (PLGA NPs) surface-conjugated quantum dots (QDs) (PLGA@QD655 NPs), which were found to exert no toxic effect on the human mesenchymal stem cell (hMSC) after at least 4-wk co-incubation. In addition, it was observed that no significant change of the PLGA@QD655 NPs-labeled hMSCs in their proliferation and differentiation capability toward the production of adipocytes, osteocytes, and chrondrocytes. In the current study, we adapted the synthetic approach previously described by Prof C.S. Yeh’s group at NCKU for the preparation of 100 nm PLGA@QD655 NPs and selected green fluorescence protein (GFP) transgenic mouse MSC (eGFP-MSC) as model stem cell. The morphology and the uptake efficiency of eGFP-MSC labeled with PLGA@QD655 NPs were investigated. The obtained results showed that nearly 66% of PLGA@QD655 NPs were uptaken by eGFP-MSCs that were injected subcutaneously into the flanks of mice, and it was also confirmed that the differentiation capability of eGFP-MSCs to produce adipocytes, osteocytes, and chrondrocytes in vivo remained unaffected.