Anti-tumor Effect Of CBD-loaded Nanomicelles On Breast Cancer

PurposeIn 2020,breast cancer has become the most common cancer in the world,accounting for 11.7%of new cancer cases,which poses a great threat to women’s quality of life.Although the mortality of breast cancer patients has decreased with the progress of examination and treatment,the current chemotherapy or other therapies still have shortcomings,which often have adverse effects on the quality of life of patients.Cannabidiol(CBD)is the main non-psychoactive component of Cannabis sativa L.,which has antiepileptic,immunomodulatory,analgesic,antioxidant,anticonvulsant,anti-anxiety and other functions.In recent years,it has been found that CBD can inhibit the proliferation of tumor cells,induce apoptosis and autophagy of tumor cells,block cell cycle,inhibit invasion and metastasis of tumor cells,regulate tumor microenvironment,have synergistic effect with chemotherapeutic drugs,and can reduce the toxicity of chemotherapeutic drugs.However,CBD has low bioavailability around 6%,measured at 0.1 μg·mL-1,due to its low water solubility.Therefore,this study aims to use the advantages of the new nano drug delivery system to improve the water solubility and bioavailability of CBD,so as to improve the therapeutic efficacy against breast cancer.In this study,different molecular weight diblock copolymers of methoxy poly(ethylene glycol)-polycaprolactone(mPEG2k-PCL2k and mPEG5k-PCL5k)and methoxy poly(ethylene glycol)-poly(lactic acid)(mPEG2k-PLA2k and mPEG5k-PLA5k)were synthesized,and CBD-loaded micelles were prepared by film hydration method.The physicochemical properties of the micelles were characterized,and the cellular uptake of micelles and cytotoxicity of CBD-loaded micelles was evaluated.The pharmacokinetics of the CBD-loaded micelles was studied as well.Methods and Results1.Synthesis and Characterization of Block Copolymers of mPEG-PLA and mPEG-PCLBlock copolymers of mPEG-PLA and mPEG-PCL were synthesized by ring-opening polymerization of D,L-lactide with mPEG(Mw=2000 Da and 5000 Da)as the initiator.By adjusting the molecular weight of mPEG and the mass ratio of mPEG to D,L-LA or ε-CL,block copolymers mPEG2k-PCL2k,mPEG5k-PCL5k,mPEG2k-PLA2k and mPEG5k-PLA5k were obtained.The structure and molecular weight of the products were confirmed by 1H-NMR.The critical micelle concentration of each polymer was determined by pyrene fluorescence probe method.The morphology and particle size of the polymer micelles were characterized by transmission electron microscopy and laser light scattering,respectively.The results showed that the molecular weight of the synthesized polymers was in line with the expectation.The CMC values of mPEG2k-PCL2k,mPEG5k-PCL5k,mPEG2k-PLA2k and mPEG5k-PLA5k were 1.12 μg·mL-1,1.27 μg·mL-1,1.95 μg·mL-1 and 0.87μg·mL-1 respectively.The micelles were spherical and the particle size was between 10-100 nm.The results show that the method of synthesizing polymer is stable and controllable,and the size of micelles formed in aqueous solution conforms to the definition of nano micelle,which has the potential of long circulation and passive targeting in vivo.2.Preparation and Characteriation of CBD-loaded MicellesCBD-loaded Micelles were prepared by film hydration method.The morphology and particle size of the micelles were characterized by TEM and laser light scattering.The stability of the micelles at room temperature and 37℃ was investigated.The drug loading content and encapsulation efficiency of the micelles were determined by UPLC.The in vitro release properties of the micelles were investigated by dialysis method.The results showed that the particle size distribution of each micelle was uniform,and the size of CBD-loaded micelles were larger than blank micelles.The particle size of micelles was very stable,and there was no significant change within 7 days under various conditions.The encapsulation efficiency was more than 90%except for mPEG2k-PLA2k.In vitro release results showed that each micelle released 35.53%and 40.54%respectively within 7 days,indicating that each micelle might have a sustained-release function.3.Antitumor activity of CBD-loaded micelles in vitro and in vivoCoumarin-6 was used as a fluorescent probe to simulate the uptake of insoluble drugs by MCF-7 and MDA-MB-231 breast cancer cells in polymeric micelles.It was found that polymeric micelles could increase the cellure uptake of insoluble drugs,among which mPEG5k-PLA5k-CBD had the greatest effect.The cytotoxicity of blank micelles and drug loaded micelles on MCF-7 and MDA-MB-231 cells was investigated by MTT assay.There was no cytotoxicity on MCF-7 or MDA-MB-231 cells in the concentration range of 0-1000 μg·mL-1,which indicated that the polymers had good biocompatibility.The CBD-loaded micelles could inhibit the proliferation of MCF-7 and MDA-MB-231 cells and the IC50 values was reduced.4.Mechanism of CBD-loaded Micelles against Breast CancerJC-1 was used as fluorescent probe to detect the changes of mitochondrial membrane potential,and the expression levels of autophagy related proteins were detected by western blot.The results showed that mPEG2k-PLA2k-CBD nanomicelles could promote the decrease of mitochondrial membrane potential,inhibit the expression of Bcl-2,inhibit the expression of mTOR,and promote the expression of Beclin 1 and LC-3B-II,thus inducing autophagy.5.Pharmacokinetics of CBD-loaded MicellesAn UHPLC-QqQ-MS method for the determination of CBD in rat plasma was established.The method has good specificity,linearity,precision and recovery.The plasma concentrations of CBD in rats after tail vein injection were investigated,and the pharmacokinetic parameters were calculated.The results showed that the area under the curve(AUC(0-t))increased,the mean residence time(MRT)increased and the clearance rate(CLz)decreased in mPEG2k-PLA2k micelles compared with free cannabidiol,which indicated that nano micelles could prolong time of blood cycle and improve its bioavailability.