Preparation Of Imperatorin-loaded RH60/mPEG-PLLA Micelles And Its Inhibitory Effect On Tamoxifen Resistance In Breast Cancer

Estrogen receptor（ER）positive breast cancer accounts for 75%of all breast cancer types.Tamoxifen（TAM）is the first-line drug for patients with estrogen-positive breast cancer,and the emergence of secondary TAM resistance has become a major challenge during the an-ticancer treatment of this type of breast cancer.Imperatorin（IMP）,is a linear furan coumarin compound.The previous study found that IMP could enhance the sensitivity of MCF-7/DOX cells to doxorubicin and alleviate the drug-resistance of breast cancer cells,which indicates that IMP has the potential to alleviate the drug-resistance of breast cancer cells.However,IMP is a BCS II compound with poor water solubility,less absorption in gastrointestinal tract after oral administration and low bioavailability,which limit its further application.Therefore,improving the water solubility of IMP is the primary problem.In this paper,we prepared micelles by loading IMP with Cremophor RH60/monomethoxy poly（ethylene glycol）-block-poly（L-Lactide）（RH60/PM-IMP）as the carrier,in order to enhance the solubility of IMP and its reverse drug-resistance activity,and then provide reference and basis for the further development of IMP and the research of other similar drugs.The first part:micelles was prepared by solvent evaporation method.In the process,particle size,PDI,Zeta potential,entrapment efficiency and drug loading were used as indicators to investigate the effects of different factors on the prepared micelles.Box-Behnken response surface method was used to optimize the formulation process of RH60/PM-IMP micelles.The micelles were dried by freeze-drying.The appearance,particle size after resolution,PDI,Zeta potential,encapsulation rate,drug loading,microstructure characterization and physical characterization of freeze-drying samples were used as indexes to optimize the type and dosage of freeze-drying protective agents.The results show that the optimized formula and process are as follows:acetonitrile as organic solvent,28 mg RH60 was added,1.2 mg IMP was added,10 mg m PEG-PLLA was added,6 m L water was added,the ultrasonic treatment was 5 min,the water-bath temperature was 60℃,and the hydration time was 3 min.The verified particle size was 25.48±0.92 nm,the average entrapment efficiency was 85.78±0.84%,and the average drug loading was2.66±0.03%.Determined the technological formula of freeze-drying:the best freeze-drying protective agent was Hydroxypropyl-β-cyclodextrin,and the dosage was 150%（relative to IMP,w/w）,to obtain a loose and porous sheet structure,and the solution was clear and transparent after re-dissolution.Part Ⅱ:Evaluation of biopharmaceutical characteristics of RH60/PM-IMP micelles.The release characteristics of RH60/PM-IMP micelles were investigated by in vitro release method,and the absorption of RH60/PM-IMP micelles in rats was simulated by pharmacokinetic method.Using MCF-7/TAM drug-resistant cells,the drug uptake of RH60/PM-IMP micelles at cell level was studied by LC-MS and fluorescence microscope.The results of in vitro release studies showed that RH60/PM-IMP micelles could significantly increase the in vitro release rate and cumulative release of IMP（P<0.01）.Pharmacokinetic experiments showed that the C_maxand AUC_0-tof RH60/PM-IMP micelles were significantly higher than those of IMP,which were 31.70 times and 23.39 times respectively,indicating that it promoted the absorption of IMP in gastrointestinal tract.Compared with the IMP,the cell uptake of MCF-7/TAM drug-resistant cells was increased by 3.61 times and 4.04 times after RH60/PM-IMP micelles were treated for 24 h and 48 h,respectively,indicating that the cell uptake of micelles was significantly higher than that of IMP（P<0.05）,and RH60/PM-IMP micelles could enhance the cell uptake capacity.Part Ⅲ:Study on the drug-resistance of RH60/PM-IMP micelles to MCF-7/TAM cells.The non-toxic concentration of RH60/PM-IMP micelles on MCF-7/TAM cells was determined by MTT method,and the changes of resistance reversal ratio,mitochondrial membrane potential,migration,invasion,colony formation and the expression of drug-resistant genes MRP1,GST-πand MDR1 on MCF-7/TAM cells after the combination of RH60/PM-IMP micelles with TAM were investigated.The tumor model of BALB/c nude mice was established by using human drug-resistant breast cancer cell（MCF-7/TAM）,and the effects of combined administration on tumor growth,tumor inhibition rate,proliferation and apoptosis of tumor tissue cells were investigated.The results showed that after 24 h and 48 h,the non-toxic concentration of RH60/PM-IMP micelle was 2.09 and 1.04μg/m L,and the reversal ratio of RH60/PM-IMP micelle was 2.14 and 2.12,respectively.After combined administration of RH60/PM-IMP micelles and TAM,the mitochondrial membrane potential of MCF-7/TAM cells decreased,and the ability of migration,invasion and colony formation decreased significantly.q PCR experiments showed that the expression of GST-πand MDR1 drug-resistant genes could be down-regulated.In vivo,the results showed that the growth rate of tumor volume in the model group was faster,and the growth rate of tumor could be slowed down by RH60/PM-IMP micelles and the combination group with TAM.the combination of RH60/PM-IMP micelles with TAM inhibited the proliferation and induced apoptosis of transplanted tumors by inhibiting proliferation antigen Ki-67,and significantly down-regulated the expression of drug-resistant genes GST-πand MDR1,indicating that RH60/PM-IMP micelles had a potential effect of reversing tumor drug-resistance.In this study,the mixed micelles prepared by negative IMP with RH60/PM as carrier were clarified,the preparation process and freeze-drying process were optimized,and its anti-drug-resistant breast cancer activity in vitro and in vivo was evaluated.The results showed that RH60/PM-IMP micelles could improve the bioavailability of IMP,and it could show that RH60/PM-IMP micelles could enhance the sensitivity of MCF-7/TAM cells to TAM by reducing the expression of GST-πand MDR1 drug-resistance genes,which provided a reference for the clinical research and development of anti-tumor drug-resistance preparations.