| ObjectiveCancer is a leading cause of death worldwide. Chemotherapy is one of the major methods for clinical cancer treatments. However, the resistance to the chemotherapeutic drugs is a major cause of treatment failure in cancer patients.Advances in nanocarrier technology provide more efficacious approaches to overcome drug resistant in cancer chemotherpy. In this study, a nanoparticle system,containing poly(?-amino) ester(PBAE) core and hyaluronic acid(HA) shell, was designed for carrying doxorubicin(DOX), a widely used chemotherapeutic drug in clinical, to overcome drug resistance in breast cancer cells.ContentsThe main contents of this study were divided into two parts. The first part contained the preparation, characterization, drug-carrying capability, and in vitro drug release property of drug-carrying nanosystem, named HA/PBAE/DOX. The second part contained the in vitro study of HA/PBAE/DOX, including cytotoxicity, cell apoptosis and cell cycle arrest, and the preliminary discussion of effective mechanism of the cell apoptosis of HA/PBAE/DOX nanoparticles.Methods1. Preparation, characterization, drug-carrying capability, and drug release preoperty of HA/PBAE/DOX: First, DOX-carried PBAE(PBAE/DOX) nanoparticle cores with different PBAE/DOX weight ratios were prepared the O/W emulsion solvent evaporation method and their morphology, sizes and size distributions, and zeta potentials were characterized using the trancemission electron microscope(TEM)and the analyzer of particle size and zeta potential. The molecular interactions between PBAE and DOX in PBAE/DOX were studied using the infrared(IR) and fluorescence spectroscopic methods. The DOX loading contents and encapsulation efficiencies were determinated by the fluorescence spectrophotometry. Second, HA was coated on the surfaces of PBAE/DOX by the interaction between positive and negative charges using incubation method. Thus obtained DOX-loaded HA/PBAE nanoparticles(HA/PBAE/DOX) were then characterized by TEM and particle size and zeta potential analyzer. Finally, the in vitro drug release properties of PBAE/DOX and HA/PBAE/DOX in the different pH release media were evaluated using the dynamic dialysis method.2. Evaluation of reversal effect of HA/PBAE/DOX on drug resistance in breast cancer MCF-7/ADR cells: The cytotoxicities of free DOX, PBAE/DOX and HA/PBAE/DOX in MCF-7 and MCF-7/ADR cells were measured by CCK-8assay, and their half inhibition rates(IC50) were then calculated. Furthermore, the effects of free DOX, PBAE/DOX and HA/PBAE/DOX on the apoptosis and cell cycle of MCF-7/ADR cells were analyzed by the flow cytometry. Thus, the reversal effects of HA/PBAE/DOX on drug resistance in MCF-7/ADR cells were comprehensively evaluated.3. Mechniasm study of reversal effect of HA/PBAE/DOX on drug resistance: The cellular accumulation and distributiom of HA/PBAE/DOX in MCF-7/ADR cells were analyzed using the flow cytometry and the confocal laser scanning microscope. Moreover, the competition method was used to study the inhibitory effect of free HA on the cellular uptake of HA/PBAE/DOX in MCF-7/ADR cells.Thus, the reversal mechanism of HA/PBAE/DOX against drug resistance in MCF-7/ADR cells was discussed through the investigation of their cell entry route.Results1. Drug-carrying nanosystem, HA/PBAE/DOX, was successfully prepared and exhibited regular spherical shape with a classic “shell-core” structure. When the HA/PBAE/DOX weight ratio was 15/10/3, the size and zeta potential of HA/PBAE/DOX were 185.0 nm and-29.3 m V, respectively. DOX was loaded into PBAE nancores through the hydrophobic interaction and hydrogen bonding.The DOX-loading content in HA/PBAE/DOX nanoparticles was about 7.5%.2. The results of in vitro releases showed that DOX displayed significant p H-sensitve relese from HA/PBAE/DOX, and its release rate gradually increased with the p H of release media decreased from 7.4 to 5.5.3. In breast cancer drug resistant MCF-7/ADR cells, PBAE/DOX and HA/PBAE/DOX both exhibited remarkably higher cytotoxicity than free DOX.Moreover, by comparison to PBAE/DOX, HA/PBAE/DOX displayed selectivity to a certain degree for MCF-7/ADR cells, and significantly induced the cell apoptosis and the cell cycle arrest at S-phase(the phase of DNA synthesis). Thus, we deduced that HA/PBAE/DOX had high reversal effect on drug resistance of MCF-7/ADR cells.4. HA/PBAE/DOX efficiently carried DOX into MCF-7/ADR cells, and this cellular internalization was significantly inhibited by free HA. It indicated that HA/PBAE/DOX could enter MCF-7/ADR cells via CD44-mediated endocytosis,thus avoided the efflux effect of P-glycoprotein(P-gp) and improved the cellular accumulation of DOX, which was very favorable for reversing drug resistance of MCF-7/ADR cells.ConclusionsIn this study, we successfully prepared and characterized a novel drug-carrying nanosystem, named HA/PBAE/DOX, which contained PBAE cores and HA shells.HA/PBAE/DOX displayed obviously in vitro p H-responsive drug release property and remarkably reversed drug resistance of MCF-7/ADR cells. The reveral mechanism of HA/PBAE/DOX against drug resistance was related to their cellular internalization via CD44-mediated endocytosis and subsequent escape from the efflux effect of P-gp. In summary, HA/PBAE/DOX, as a novel therapeutic nanosystem,showed a promising application in clinical treatment of drug resistant breast cancer. |
PDF Full Text Request