Study On The Effective Reversal Of Drug Resistance Of Tumor Cells Based On "Calcium Ion Burst" Strategy

Multidrug resistance(MDR)of the tumor is the main cause of failure in clinical chemotherapy.The mechanisms in regulating the MDR development of tumor are very complicated.Among those mechanisms,one of the conventional mechanisms is the overexpression of transmembrane ATP-binding cassette(ABC)transporter and function of ABC transports have been extensively studied.Such as P-glycoprotein,the protein is energy dependent drug efflux pump,the energy for which are supplied by ATP,the drug efflux pumps exclude cytotoxic drugs out of the cells,causing low intracellular drug accumulation and final therapeutic failure.Therefore,it is urgent to suppress the expression of P-gp and inhibit the production of ATP to overcome drug resistance.In recent years,strategies to overcome P-gp mediated tumor resistance and limiting the energy supply of MDR tumor cells have been widely explored.Such as using the small-molecule inhibitors of P-gp or small interfering RNA,limiting the energy supply of tumor cells.However,small-molecule inhibitors of P-gp have obvious toxicity,small interfering RNA as a gene-drug still faces the problem of slow onset of action,system needs complicated multi-component carriers,and the preparation is difficult,it is imperative to develop a simple and effective strategy to reverse drug resistance.It was found that intracellular calcium ion abnormal can affect mitochondrial respiratory chain complexes I,thus affecting cell respiration,intracellular oxygen content.The synthesis of P-gp was closely related to the oxygen content of tumor.Hypoxic environment in tumor tissues leads to abnormal expression of HIF-1?,which promotes the synthesis of P-gp.By inhibiting cellular respiration and thereby increasing intracellular oxygen content,down-regulation of HIF-1? is expected to reduce the synthesis of P-gp.In addition,inhibition of cellular respiration is accompanied by decreased energy,blocking P-gp transport and pumping functions.In addition,tumor cells are highly sensitive to calcium ions,which lays a foundation for the selective reversal of tumor cell resistance.Based on the above analysis,we developed a simple and effective calcium nanometer generator to reverse drug resistance by mediating intracellular calcium explosion.1.Preparation of CaP-DOX-RGD nanometer system and its in vitro characterization.In this study,CaP nanoparticles were prepared by double reversed phase emulsion method.The structure of CaP nanoparticles was porous and spherical by TEM observation,with particle size of about 100 nm.ICP-MS detection of calcium ions and TEM observation showed that CaP nanoparticles were weak disintegration and less calcium ion was produced at pH 6.8.At pH 4.5,the CaP nanoparticles were almost completely disintegrated,and the amount of calcium ions produced was about 11 times that under pH 7.4.The anti-tumor drug DOX was loaded into the loose porous CaP by physical stirring.The potential detection showed that the potential increased from-15.5 mv to-8.4 mv after DOX loading.In order to improve the dispersive properties of the nanoparticles and achieve tumor angiogenic targeting function,the DSPE-PEG2000-RGD was modified.The modified structure(CaP-DOX-RGD)was still spherical with a particle size of about 140 nm.The loading rate of DOX was about 25%.Under the conditions of pH 7.4 and pH 6.8,the maximum release of DOX at 24 hours was not more than 25%.At pH 4.5,the release of DOX at 4 h was more than 60%,indicating that it was stable in the blood circulation or tumor microenvironment,and had better release performance in the cell lysosome environment.The hemolysis experiments proved that CaP nanoparticles(0-200 ?g mL-1)have good vascular compatibility,ensuring its safety.2.To investigate the ability of CaP-RGD nanoparticles to reverse drug resistance of MCF-7/ADR cells in vitro.The intracellular calcium ion increase was time-dependent after the CaP-RGD nanoparticles were endocytosis and the distribution characteristics of calcium ions were as follows:2-4 h are more distributed in lysosomes;it was mainly distributed in mitochondria at 6 h.It was found that the activity of mitochondrial complex I(the relative activity decreased from 1.00 to 0.06)was significantly reduced by CaP-RGD,and the intracellular ATP(the relative content decreased from 23.9 to 8.9 nmol/mg)was reduced.We further study the changes in intracellular oxygen content and downstream related molecules.The results showed that intracellular oxygen content increased significantly and HIF-1? decreased;qRT-PCR results showed that multidrug resistance gene(MDRI relative expression decreased from 1.00 to 0.39)decreased;Western blot results show that the protein P-gp(intracellular and on the membrane)encoded by MDR1 was reduced.In addition,under the action of CaP-RGD nanoparticles,the few P-gp remaining on the cell membrane failed to bind enough ATP in the cell and unabled to perform pumping function.It is worth noting that the IC50 value of MCF-7/ADR cells is as high as 168?g mL-1,and they have high resistance to DOX,and the resistance factor is as high as 70 times.It is worth noting that compared to DOX-resistant drug-resistant cells,the IC50 value(5.7 ?g mL-1)of CaP-DOX-RGD group decreased significantly,with a decrease rate of about 96%,and the inhibition rate of cells in vitro was 73%.3.To study the targeting of CaP-RGD and its ability to reverse drug resistance of MCF-7/ADR in vivo.MCF-7/ADR cells were implanted subcutaneously into the back of BALB/c-nu nude mice to construct a drug-resistant tumor model.The near-infrared dye IR 783 was loaded into CaP nanoparticles,and DSPE-PEG-RGD was modified.In vivo imager was used to observe the fluorescence intensity of living and in vitro organs of nude mice,after 36 h of tail vein administration,weaker fluorescence could be observed in the free IR 783 group and CaP-IR 783-PEG group,the tumor site fluorescence intensity of the nude mice in the CaP-IR 783-RGD group was stronger.In addition,the organ and tumor tissue imaging results after 50 h of tail vein administration showed that fluorescence was only observed in the tumor site of the RGD group,confirming the targeting ability of RGD.A single tail vein injection of CaP-RGD into nude mice,and the study of the content of calcium ion(the relative content increased from 1.0 to 3.4),HIF-1?(the relative expression decreased from 1.000 to 0.697),P-gp(the relative expression decreased from 0.91 to 0.74),and ATP(the relative content decreased from 211 to 45 nmol/mg)in the tumor site proved that CaP-RGD has the ability to reverse drug resistance in vivo.4.To study the anti-tumor efficacy and safety evaluation of CaP-DOX-RGD in vivo.The antitumor effect and safety in vivo were evaluated by body weight,tumor volume,H&E and TUNEL staining.Body weight combined with H&E staining results,CaP-DOX-RGD had a better biological safety.The tumor inhibition rate results showed that free DOX tumor inhibition rate was about 5.6%and the tumor inhibition rate of CaP-RGD+DOX group was 27.9%.After the CaP-DOX-RGD group was enriched in tumor tissue,a large number of calcium ions were produced which could down-regulating ATP,reducing P-gp synergistically reverse drug resistance,promote the accumulation of DOX in the cell and exert antitumor effect,and finally the tumor inhibition rate was 92.6%.In summary,we developed a simple-yet-versatile tumor-targeting "calcium ion nanogenerator"(CaP-DOX-RGD)to achieve drug resistance reversal by inducing intracellular Ca2+bursting.In this system,calcium phosphate was prepared by double reversed phase microemulsion method,after loading the antitumor drug DOX,the RGD peptide grafted with lecithin was modified.Due to the tumor vascular targeting function of RGD,the calcium ion nanogenerator system could be enriched to tumor tissues and then absorbed by tumor cells.Nanoparticles rapidly release a large number of calcium ions in the lysosomal acidic environment of tumor cells.Based on the sensitivity of tumor cells to calcium ions,nanoparticles can reverse P-gp mediated multidrug resistance by the following mechanism:intracellular calcium ion selectively accumulates in mitochondria,alleviates cell hypoxia by inhibiting mitochondrial respiration,and down-regulates HIF-1? expression to inhibit P-gp synthesis;Intracellular calcium ion selective gathered in mitochondria,by inhibiting the mitochondrial respiratory chain complexes I,inhibiting cell respiration,alleviating cells lack of oxygen,decreasing HIF-la express inhibit the synthesis of P-gp.At the same time,calcium ion-induced respiratory depression blocks intracellular ATP production and synergistically blocks P-pg mediated cell resistance.As a result,the IC50 value of MCF-7/ADR cells was reduced to 5.7 ?g mL-1,and the reduction range was 96%.The inhibition rate of cells in vitro was 73%,and the tumor-inhibiting rate in vivo was 92.6%.This simple,safe and effective "Ca2+bursting" strategy holds the potential for clinical application in cancer.