| [Background]Lung cancer is one of the most frequent cancers among males and females in China and in Western countries. Non-small cell lung cancer (NSCLC) accounts for85%of lung cancer cases. Chemotherapy is a method most widely applied in cancer treatment, however, prognosis is relatively poor with a median time for progression ranging from three to five months. Antitumor drugs, such as doxorubicin, are widely used in cancer therapy. However, multidrug resistance (MDR) and side effects, such as heart toxicity, bone marrow suppression, and mucositis, limit the use of doxorubicin.Nanocarriers such as polymeric nanoparticles, nanoshells, liposomes, microemulsion and dendrimers have been studied to reverse the MDR of the tumor cells. Especially micelles, which are developed hydrophobic/hydrophilic self-assembly, have gained significant attention from medical fields for their various advantages in drug delivery applications. Poorly water-soluble drugs can be incorporated into the micelle by means of chemical, physical or electrostatic interactions. To reduce the toxicity of these drugs and improve their therapeutic efficacy, various polymeric micelle systems have been designed as delivery vehicles. Except for a simple preparation, micelles have several advantages, micelles have high stability and biocompatibility, via the enhanced permeability and retention effect of drugs towards tumor cells, with prolonged circulation times, reduced toxicity, and are capable of controlled release of the drug for optimal targeting.[Objective]The purpose of this experiment was focused on the preparation of a doxorubicin-loaded composite polymer micelle. The doxorubicin-encapsulated micelles not only have the potentiality in reversing MDR, but also radiosensitize non-small cell lung cancer line to achieve reduced toxicity and a higher therapeutic effect. The pathway of cellular internalization as well as the subcellular localization of the composite micelles were also evaluated.[Method]1. We used three kinds of polymer polyethylene glycol (PEG), polycaprolactone (PCL) and P105as drug carriers to synthesis DOX-loaded composite micelle. Particle size of the micelles was measured by dynamic light scattering, and the morphology was observed under transmission electron microscopy (TEM). An analytical high performance liquid chromatograph (HPLC) was developed to quantify DOX.2. Human lung adenocarcinoma A549was selected to model non-small cell lung cancer. The cytotoxicity of the composite PEG-PCL/P105micelle in A549cells have been evaluated by a standard MTT assay.3. Doxorubicin is a fluorescent compound, so the intensity of cellular doxorubicin fluorescence can be measured by confocal laser scanning microscopy (CLSM). The CLSM with an emission wavelength of488nm was applied to determine the accumulation and release of the drug.4. Flow cytometry was used to quantitate the doxorubicin fluorescence signal intensity. Data were analyzed using Flowjo7.6software.5. Cells were incubated with MitoTracker Deep Red FM, LysoTracker Blue DND-22, and Hoechst33342to visualize mitochondria, lysosomes, and nuclei, respectively. Confocal microscoppy was used to determine the subcellular localization of doxorubicin fluorescence.6. A549cells were incubated with drug-loaded micelles, free doxorubicin, or blank composite micelles and then irradiated. Colony-forming assays were used to evaluate the radiosensitivity of composite doxorubicin-loaded micelles following ionizing radiation. The cell survival curves were generated using GraphPad Prism5.0based on the multitarget/single-hit model.[Results]1. A novel doxorubicin-loaded composite micelle consisting of PEG-PCL/Pluronic P105has been developed. The particle size of the composite copolymers micelles was around100-200nm as defined by the mean diameter.2. Doxorubicin-loaded PEG-PCL/P105composite micelles show notable improvement of cytotoxicity compared with free DOX or blank composite micelle. Drug-loaded micelle was proved to inhibit the drug resistance of A549cell line. Almost no cytotoxicity was observed for the blank composite micelle, indicating that cytotoxicity was caused by DOX rather than the carrier material.3. Confocal laser scanning microscopy and flow cytometry data showed that the envelopment of doxorubicin in the composite micelles significantly increased cellular accumulation of the drug. Following a recovery time, the use of micelle carriers also produced significantly sustained cellular release of the drug.4. Confocal images indicating that the intracellular distribution of DOX-loaded composite micelles and free doxorubicin was similar. Colocalization of blue and red fluorescence was not observed when cells were labeled with Hoechst33342, suggesting that doxorubicin was not specific for nuclei. After treatment with DOX-loaded composite micelles and free doxorubicin, most of the doxorubicin was localized to mitochondria and lysosomes, as evidenced by colocalization of red fluorescence with that from MitoTracker Deep Red FM and LysoTracker Blue DND-22, respectively.5. Colony-forming assays demonstrated that both composite doxorubicin-loaded micelles and free doxorubicin were radiosensitive. The surviving fraction confirmed the increased radiosensitivity of cells treated with composite doxorubicin-loaded micelles compared with those treated with free doxorubicin. There was no difference between the group receiving irradiation only and the group receiving irradiation together with blank composite micelles.[Conclusion]In this study, composite doxorubicin-loaded micelles consisting of PEG-PCL/Pluronic P105were developed. We observed that doxorubicin-loaded micelles were more cytotoxic towards A549cells than free DOX or blank composite micelle. Confocal laser scanning microscopy and flow cytometry data demonstrated that drug-loaded micelles stayed in intracellular much longer than DOX solution. Confocal images indicating that the intracellular distribution of DOX-loaded composite micelles and free doxorubicin was similar. Increased radiosensitivity of cells treated with composite doxorubicin-loaded micelles was observed, comparing with those treated with free doxorubicin or blank composite micelle. |
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