| Background and objective:Chemotherapy has been one conventional clinical strategy of curing malignant diseases. However, during chemotherapy process many tumors presented Multidrug resistance (MDR) and cancer cells can experience reduced cytotoxicity in response to a wide range of chemotherapeutics. This phenomenon of multidrug resistance (MDR) in cancer cells is a significant obstacle to successful chemotherapy of human malignancies. Therefore, how to overcome and reverse MDR is a urgent problem in Pharmaceutical and tumor therapeutic category and many researches have been conducted to explore effective strategy to deal with MDR.MDR comprises different mechanisms, and decreased intracellular drug accumulation caused by drug efflux pumps in the cell membrane is one of the most common mechanisms responsible for MDR development in cancer cells. Those membrane efflux pumps belong to the superfamily of ATP-binding cassette transporter proteins and could pump intracellular drug out via adenosinetriphosphate (ATP)-dependent pathways. Many pharmaceutical agents and small molecules as modulators or inhibitors of membrane efflux pumps, have been explored to overcome MDR. These modulators are able to restore the cytotoxicity of anticancer drugs in MDR tumor cells. However, due to unacceptable side effects, their clinical application have been limited.Recently, the nanotechnology-based drug delivery systems have been investigated to overcome MDR phenomena in solid tumors, which has been a hotspot in tumor therapeutic field. These Nano-carriers have been proved able to increase drug cellular uptake, maintain high intracellular drug concentration by modulating drug efflux pumps. Though nanoparticles have shown their potential to enhance the effectiveness of anticancer agents and reverse tumor MDR, influenced by the particle size, it can not be avoided the drug accumulation in undesired tissues except the target organs therefore, novel approaches with low cytotoxity are needed being investigated for overcoming MDR in cancer cells.Ultrasound targeted microbubble destruction (UTMD) technology has proved to be a targeted treatment method with a great potential, which can widen the cell gaps, increase the membrane permeability and form small holes (sonoporation) on the cell surface. Therefore, the medicine ingestion will be enhanced by promoting drugs, proteins and genes reaching to specified part through endothelial barrier and improve the intracellular drug accumulation. Moreover, tumor cells display more sensitivity to ultrasound compared with normal cells, which provides a foundation for targeted therapy mediated by ultrasound. Utilization of sonoporation induced by ultrasound (US) irradiation can increase the permeability of capillaries and cell membrane, promote the permeability of drugs, increase the accumulation of chemotherapy drugs in target tissue, thus acquiring a higher concentration of chemotherapeutic drugs within the tumor cells and a very low concentration in blood, which could increase the cytotoxic effect of chemotherapeutic drugs. Therefore, the ultrasound irradiation combined low-dose cytotoxic drugs can maintain or even improve the curative effect of treatment and decrease the patients’ tolerance to chemotherapy. US irradiation can increase the sensitivity of MDR tumor cells against tumor drugs, and has made some progress in reversal of tumor MDR, showing a good prospect in clinical application.Currently, rarely few research about ultrasound irradiation combined drug-loading nanoparticles reverse tumor MDR has been reported, and related reversal mechanisms and biological effect and interactions between ultrasonic irradiation and nanoparticles need in-depth study. We previously developed heparin-based nanoparticles (NPs) with dual functionalized ligands folate and Tat to deliver paclitaxel, presenting efficient cellular uptake and great anticancer activity in vitro. We envisaged to enhance the cytotoxity of dual-functionized NPs for drug resistant cells by US irradiation effect, and further explore related mechanisms in reversal of tumor MDR.Materials and methods:1. Synthesis and characterization of microbubblesThe ultrasound contrast microbubbles (MBs) used in this study were prepared according to the method as follows. Briefly, certain quantities of DSPC、DPPE and PEG4000were well hydrated with ultrapure water in a70℃thermostatic water bath for30min. Subcequently, mixed lipid suspension were added in50mL polypropylene tube, and put the shear cutter below liquid level. Afterward, perfluorobutane gas (C3F8) was dispersed by sonication in the admixture with a certain shear rate for2min. Then lipid microbubbles were stored in schering bottles filled with whole fluorine propane gas. Particle size, size distribution and concentration of MBs were determined using a Coulter counter. Morphologic characteristics of MBs were determined under a light microscope.2. Preparation and characterization of Heparin-Folate-Tat-Paclitaxel nanoparticles (H-F-Tat-P NPs).Succinylated-heparin was dissolved in dry DMSO and maintained by gentle heating. Folate-NH2, Tat peptide, EDC and NHS were successively added to the mixture, and the reaction was allowed to proceed at room temperature. Fluorescence dye Oregon green was also added if needed. After overnight strring, paclitaxel was added in solution at room temperature for30min, DMSO was then removed by a dialysis memberane for48h to get Oregon green488-labled H-F-Tat-P NPs. DiI was regarded as model drug of paclitaxel to prepare for the confocal image study.3. The optimization of ultrasonic irradiation conditions3.1MTT assay to test cell viability after ultrasound (US) with different microbubble concentrationThe human breast carcinoma cell lines MCF-7cells and paclitaxel-resistant MCF-7/Tax cells were divided into four groups:two groups were incubated with NPs or free paclitaxel for48h directly. For the other two groups, the cells were exposured to US with different microbubble concentration (with different volume ratio of MBs to medium:1:40,1:20,1:10) firstly, and the cells were added into NPs or nothing and continued incubated for48h. MTT assay was used to measure the relative cell viability in different groups.3.2Quantitative analysis of cellular Uptake of NPs with and without US by flow cytometerTo optimize the MBs concentration of US, Oregon green488labeled NPs and Oregon green488labeled NPs-loaded MBs were incubated with two cell lines directly or after US exposure with different microbubble concentration (with different volume ratio of MBs to medium:1:40,1:20,1:10) and US intensity was0.6W/cm2with50%duty cycle for80s. After incubation for4h, the fluorescence distribution of two cell lines in different groups were directly introduced to a FACSort flow cytometer equipped with a488nm argonion laser.4. Cell Viability AssayHalf the MCF-7cells and paclitaxel-resistant MCF-7/Tax cells were incubated with paclitaxel and NPs with different drug concentration for48h. For the third groups, the cells were added into NPs with different drug concentration after exposured to US at optimized contidition firstly, and continued incubated for48h. Another group were incubated for48h directly after exposured to US. MTT assay was used to measure the relative cell viability in different groups.5. Qualitative analysis of cellular uptake of nanoparticlesTo investigate the cellular uptake of NPs with and without US, DiI as model drug was entrapped into H-F-Tat to detect drug release into MCF-7/Tax cell lines. Oregon green488labeled H-F-Tat-DiI NPs were incubated with MCF-7/Tax cell lines directly, and half cells were incubated with NPs after exposured to ultrasound. After incubation for4h, cells were incubated with Hochest33342nuclear stain and the fluorescent images were viewed by confocal microscope.6. Intracellular accumulation of Rhodamine123(Rh123)MCF-7/Tax cells were treated with Rh123,US+Rh123, Verapamil+Rh123respectively. After1h incubation, the intracellular Rhl23accumulation in MCF-7/Tax cells was thus measured by flow cytometry.7. The evaluation of lysosome escape ability of NPsIn order to visualize nanoparticles colocalization with lysosomes, MCF-7/Tax cell lines were incubated with Oregon green488labeled NPs for4h with and without US exposure. Then cells were washed with PBS and incubated with Lysotracker-Red for an additional30min. The cells were fixed using4%(w/v) para-formalde-hyde solution followed by Hochest33342staining. The fluorescence distribution in MCF-7cells and MCF-7/Tax cells were observed by confocal microscope.8. Effects of ultrasound on mRNA expression level of ATP-binding cassette transporter genes in MCF-7/Tax tumor cells.Total RNA was extracted from MCF-7cells, MCF-7/Tax cells and US-exposed MCF-7/Tax cells by using the TRIzol reagent according to the manufacturer’s instructions. After reverse transcription-polymerase chain reaction, the mRNA expression of ABCB1, ABCG2and ABCClwere performed using real-time quantitative PCR Detecting System (q-PCR) analysis.9. Western blotting.Total proteins were extracted from MCF-7cells, MCF-7/Tax cells and US-exposed MCF-7/Tax cells. Western blot analy sis was performed to investigate the expression of P-gp, BCRP and MRP1.10. Cell proliferation assessmentTo investigate the US effect on cell proliferation, MCF-7and MCF-7/Tax cells were exposed to US, and cell proliferation was measured1h,12h,24h,48h and72h after US exposure.11. Flow cytometric detection of cell cycleMCF-7/Tax cells were treated with US exposure, nanoparticles and US combined NPs, collected the cells1h,12h,24h,48h after US exposure, and the cells treated with NPs or NPs combined US for4h were also collected. The cell cycle of suspended cells was analyzed by a flow cytometer.12. Statistical Analysis.Statistical analysis was performed using the SPSS13.0software package. All data were performed in triplicate and presented as a mean value with its standard deviation indicated (mean±SD.) Difference of cell viability in different drug delivery system groups were analyzed with one-way ANOVA. Difference of the mRNA expression of ABCB1, ABCG2and ABCC1between MCF-7cells and MCF-7/Tax cells and between MCF-7/Tax cell and US-exposed MCF-7/Tax cells were analyzed with two-sample t-test. Difference of cell proliferation in cells with and without US exposure of two cell lines were analyzed with two-sample t-test. Difference in cell cycle of different time after US exposure and difference in MCF-7/Tax cells after treatment were analyzed with one-way ANOVA. If heterogeneity of variance was observed, the Welch-corrected t test was used to calculate P values evaluating the significance of differences in group means. P<0.05was considered statistically significant.Results:1. characterization of microbubbles and nanoparticlesThe prepared microbubbles had a good shape and uniform distribution without aggregation under the light microscope. The volume-weighted diameter of MBs was2.01±0.93μm by the Coulter counter, and the concentration was (3.93±1.0)×108/mL. The mean diameter of NPs was around120±7nm and zeta potential values were approximately-35±4mV. NPs were showed in round an approximately spherical shape under transmission electron microscopy.2. The optimization of ultrasonic irradiation conditions2.1MTT assay to test cell viability after US with different microbubble concentrationThe results showed that US cytotoxicity was dose-dependent, and an increase of MBs concentration resulted in a decrease in the number of viable cells. Along with the increase of MBs concentration, NPs had a significantly different inhibitory effect on cells than the NPs alone. The cell viability was above80%in the treatment of US alone. At the same MBs concentration the cell viability of MCF-7/Tax cells was lower than that of MCF-7cellls.2.2Quantitative analysis of cellular Uptake of NPs with and without US by flow cytometer:US exposure can enhance the cellular uptake of NPs in MCF-7cells and MCF-7/Tax cells. Along with the increase of MBs concentration, the green fluorescent in MCF-7cells was increased, while the green fluorescent in MCF-7/Tax cells was increased firstly and then decreased. When the volume ratio of medium to MBs was1:10, the green fluorescent in MCF-7cells was highest. When the volume ratio of MBs to medium was1:20, the green fluorescent in MCF-7/Tax cells was highest.3. Cell Viability AssayThe results of cell viability for MCF-7cell lines were:MBs+US+NPs<NPs<free paclitaxel (P<0.05). The IC50of NPs alone and US combimed NPs on MCF-7cells were6.30ng/mL and1.43ng/mL, respectively. For MCF-7/Tax cells, the cell toxity of NPs alone (IC501966.3ng/mL) was not better than free palitaxel (IC50826.3ng/mL), while US exposure combined NPs had a higher cytotoxity than NPs alone (P>0.05).4. Qualitative analysis of cellular Uptake of NPs by confocal laser scanning microscopyIt was obviously detected only few model drug DiI was detected in the cytoplasm of MCF-7/Tax cells, while the red fluorescent signal was increased after US exposure and DiI was observed in not only cytoplasm but also nucleus of cells.5. Intracellular accumulation of Rhodamine123(Rh123)After1h of incubation, intracellular Rh123accumulation in MCF-7/Tax cells after US exposure was increased compared to that in MCF-7/Tax cells without US exposure.6. The evaluation of lysosome escape ability of NPs The position of nucleus in each cell was labeled with Hochest33342staining (blue). It could be observed the colocalization of NPs with lysosome produced a yellow fluorescence in merged images. Almost all Oregon labeled NPs was distributed in the cytoplasm and represented yellow fluorescence merged with lysosome in MCF-7/Tax cells. For both two cell lines, US exposure could significantly enhance the lysosome escape of NPs and a part of green NPs distributed in nucleus.7. Effects of ultrasound on mRNA expression level of ATP-binding cassette transporter genes in MCF-7/Tax tumor cells.To gain further insight into the mechanisms related to the effects of US exposure on MDR cancer cells, we investigated the possibility that US might regulate, directly or indirectly, the mRNA expression of ABCB1, ABCG2and ABCC1genes using Q-PCR. Compared to MCF-7cells, the mRNA expression ratio of ABCB1, ABCG2and ABCC1genes in MCF-7/Tax cells were284.3,5.3,2, respectively. US could significantly suppress the mRNA expression of ABCB1and ABCG2genes in MCF-7/Tax cells (P<0.01). However, no significant change was observed in the mRNA expression of ABCC1gene between MCF-7/Tax cells and US-exposed MCF-7/Tax cells.8. Western blotting.In accordance with the Q-PCR results, the expression of P-gp, BCRP and MRP1in MCF-7/Tax by western blot analysis also revealed that P-gp and BCRP were downregulated, but MRP1was not differently expressed under the optimum irradiation.9. Cell proliferation assessmentCompared to the control groups, the growth rate of MCF-7and MCF-7/Tax cells were slowing down in12h,24h,48h and72h after US exposure.10. Flow cytometric detection of cell cycleCompared with MCF-7/Tax cells, the ratio of cells after US exposure in S phase and G2M phase decreased, and cells in G1phasee increased obviously with significant difference (G1phase:F=19.474, P=0.002; S phase:F=35.330,P< 0.001; G2phase:F=35.330, P<0.001). The results of cell cycle showed that after the4h treatment of NPs alone and US exposure combined NPs the ratio of cells in G1phase decreased and G2M phase increased significantly compared to MCF-7/Tax cells without US exposure.Conclusion:1. The optimal ultrasonic irradiation conditions for MCF-7cells, irradiation intensity0.6W/cm2,50%duty cycle, irradiation time80s, and the MBs concentration with volume ratio of MBs to medium was1:10; and for MCF-7/Tax cells, irradiation intensity0.6W/cm2,50%duty cycle, irradiation time80s, and the MBs concentration with volume ratio of MBs to medium was1:20.2. US exposure could enhance cytotoxity of the dual-functionized NPs, indicating that US irradiation could reverse or overcome tumor MDR3. The acoustic effects in a biological milieu offer several scenarios for the reversal of MDR. The related mechanisms of MDR reversal induced by US combined with dual-functionized NPs were:sonoporation enhance the cell membrane permeability, improvement of the lysosome escape ability of NPs thus increasing the drug accumulation in MCF-7/Tax cells, downregulation the expression of drug-resistant related protein to enhance the sensitivity of MCF-7/Tax cells to NPs, affect the cell cycle and slow down the cell growth rate. |
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