| OBJECTIVE To increase the active targeted ability to tumor cells, minimize adverse effects of chemotherapy agents, overcome leukemia multidrug resistance (MDR), we developed a novel chemotherapy formulation of polymer nanoparticles which are modified with transferrin (Tf) coloaded with daunorubicin (DNR) and tetrandrin (Tet), DNR/Tet-PLGA-PLL-PEG-Tf, which can be accumulated in the tumor region and prevents drug efflux.METHODS Polylactic-co-glycolic acid (PLGA), poly-L-lysine (PLL) and polyethylene-glycol (PEG) were used as the basic biomaterials to prepare for PLGA-PLL-PEG polymer, in order to deliver a cytotoxic drug, daunorubicin (DNR), a P-gp modulator, tetrandrin (Tet). Then Tf was conjugated to the surface of PLGA-PLL-PEG to construct PLGA-PLL-PEG-Tf which is a novel drug delivery system with active targeted ability to tumor cells. The physical properties of DNR-PLGA-PLL-PEG-Tf and DNR/Tet-PLGA-PLL-PEG-Tf were investigated by fourier transform infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, laser particle size analyzer, high performance liquid chromatography, gel permeation chromatography, scanning electron microscope and in vitro drug release study as well. Their effects on leukemia K562 cells or K562/ADR cells resistant to adriamycin in vitro were evaluated by cytotoxicity assay. Cell apoptosis and intracellular accumulation of DNR were determined by flow cytometry. Furthermore, quantitative real-time polymerase chain reaction and western blotting were performed to study mdrl/P-gp expression, transferrin receptor (TfR) gene transcription and protein expression, apoptosis associated gene transcription and protein expression.RESULTS 1. Our results showed that the prepared polymer nanoparticles were structure identified and highly yield of 61 ± 14%. Further prepared targeted drug-loaded polymer nanoparticles DNR-PLGA-PLL-PEG-Tf and DNR/Tet-PLGA- PLL-PEG-Tf were spherical or nearly spherical under scanning electron microscope, diameter were 229 ± 19 nm and 213 ± 12 nm, the Zeta potential of DNR-PLGA-PLL-PEG-Tf in water was-18.74 ± 0.28 mV and that of DNR/Tet-PLGA-PLL-PEG-Tf was-19.16 ± 0.31 mV. The DNR loading capacity of DNR-PLGA-PLL-PEG-Tf was 5.21 ±0.11%, the DNR and Tet loading capacity of DNR/Tet-PLGA-PLL-PEG-Tf were 3.63 ± 0.15%and 4.27% ± 0.12%respectively. The Tf content of DNR-PLGA-PLL-PEG-Tf and DNR/Tet-PLGA-PLL-PEG-Tf were 2.43 ± 0.26% and 2.18% ±0.11%, respectively. The drug content of nanoparticles before and after coupled with Tf showed that the drug carried by nanoparticles were stable, and there was no leakage during the reaction. A biphasic pattern consisting of an 8h initial burst release followed by a sustained slow release over a prolonged period of time for more than 7 days was detected in vitro drug release of both DNR-PLGA-PLL-PEG-Tf and DNR/Tet-PLGA-PLL-PEG-Tf.2. Proliferation of K562 cells was inhibited in a dose-dependent manner by DNR and DNR-PLGA-PLL-PEG-Tf. The IC50 value of DNR-PLGA-PLL-PEG-Tf was lower than that of DNR (0.91 ±0.10 μg/ml vs 1.31 ± 0.24 μg/ml), (P< 0.05). DNR-PLGA-PLL-PEG-Tf induced more apoptosis in K562 cells than DNR (P< 0.05). Fluorescence intensity of intracellular DNR detection demonstrated that DNR-PLGA-PLL-PEG-Tf can be taken up by K562 cells and DNR was persistently released, DNR-PLGA-PLL-PEG-Tf can significantly increase the intracellular concentration of DNR compared with DNR (P< 0.05). Fluorescent microscope demonstrated fluorescence was observed in the nucleus and cytoplasm localization of K562 cells treated with DNR-PLGA-PLL-PEG-Tf and DNR. Typical features of apoptosis, such as chromatin condensation, nucleolus pyknosis, and nuclear fragmentation could be seen in all groups. TfR gene transcription and protein expression were significantly up-regulated in K562 cells treated with DNR-PLGA-PLL-PEG-Tf.3. The cytotoxicity to K562/ADR cells was also in a dose-dependent manner by DNR/Tet-PLGA-PLL-PEG-Tf, DNR and Tet (DNR/Tet). The IC50 value (total drug) of DNR/Tet-PLGA-PLL-PEG-Tf was lower than that of DNR/Tet (0.87 ± 0.01 μg/ml vs 1.47 ± 0.15μg/ml), there was significant difference between them. Tet can help DNR/Tet-PLGA-PLL-PEG-Tf induce more apoptosis in K562/ADR cells and more intracellular DNR compared with DNR-PLGA-PLL-PEG-Tf. Tf can help DNR/Tet-PLGA-PLL-PEG-Tf induce more apoptosis in K562/ADR cells and more intracellular DNR compared with DNR/Tet-PLGA-PLL-PEG. In cells treated with DNR/Tet, DNR /Tet-PLGA-PLL-PEG and DNR/Tet-PLGA-PLL-PEG-Tf, fluorescent microscope demonstrated fluorescence was observed in the nucleus and cytoplasm localization and typical features of apoptosis,such as chromatin condensation, nucleolus pyknosis, and nuclear fragmentation also could be observed. In K562/ADR cells treated with DNR/Tet-PLGA-PLL-PEG-Tf, TfR gene transcription and protein expression were significantly up-regulated, Mdr1/P-gp expression were down-regulated, the gene transcription and protein expression of Survivin and NF-κB were both decreased, and the ratio of Bax/Bcl-2 and Caspase 3 were all increased. CONCLUSION 1. Our results showed that the new polymer nanoparticles called PLGA-PLL-PEG can be used to load different drugs, after PLGA-PLL-PEG was conjugated by Tf, the constructed PLGA-PLL-PEG-Tf should be a new drug delivery system with active targeted ability to tumor cells.2. Cell cytotoxicity of DNR-PLGA-PLL-PEG-Tf to K562 cells is in a dose-dependent manner, which may consulted to the sustained drug release and induction of apoptosis. DNR-PLGA-PLL-PEG-Tf can promote gene transcription and protein expression of the TfR, which may promote the TfR-mediated drug endocytosis.3. The inhibiton of DNR/Tet-PLGA-PLL-PEG-Tf on leukemia K562/ADR cells is also in a dose-dependent manner by enhancing the accumulation of daunorubicin intracellular via P-gp pathway and inducing apoptosis by increasing gene transcription and protein expression of Bax/Bcl-2 and Caspase 3 and decreasing Survivin and NF-κB. DNR/Tet-PLGA-PLL-PEG-Tf can promote gene transcription and protein expression of the transferrin receptor (TfR), which may promote the TfR-mediated drug endocytosis.4. Our PLGA-PLL-PEG-Tf formulation may be as a promising strategy for overcoming the MDR of leukemia or other drug resistance tumor. |
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