Study Of Fabrication Of Methotrexate Loaded Micelles And Their Anti-lymphoma Effects

Background and Objective:The morbidity and mortality of non-Hodgkin’s lymphoma(NHL)in China are increasing year by year,which has caused serious harm to the life and health of our people.At present,methotrexate(MTX)is one of the important drugs for the treatment of NHL.High-dose methotrexate(HD-MTX)is needed in the treatment of highly aggressive lymphoma,such as Burkitt lymphoma,lymphoblastic lymphoma,aggressive mantle cell lymphoma,central nervous system lymphoma,advanced NK/T cell lymphoma and so on.However,MTX has some shortcomings,such as poor water solubility of raw drugs,low concentration of drugs in target tissues,high toxicity and side effects,and tumor drug resistance.Compared with MTX,the indication of the new folic acid antagonist(PDX)is limited,which does not solve the problem of large toxicity and side effects of MTX.At the same time,the cost of PDX is relatively expensive.Therefore,the development of targeted,effective,safe and accessible new methotrexate preparation has important clinical value in the treatment of lymphoma.As a new type of drug delivery system,polymer micelles are loaded with hydrophobic antineoplastic drugs because they self-assemble in water to form the micelle structure of hydrophobic core and hydrophilic shell.The micelle size is in the nanometer level,and it is enriched in the tumor site because of high permeability and retention effect(EPR effect),thus achieving the effect of passive targeting.In recent years,scientists at home and abroad have conducted related studies on the stability,safety,pharmacokinetics and pharmacodynamics of polymer micelles loaded with antineoplastic drugs,which really make the application of polymer nano-micelles in tumor therapy a reality.The physical and chemical properties of MTX are the key factors restricting the construction of its nano-carriers.At present,the basic research on the nano-delivery system of MTX is very limited,and there is no MTX nano-preparation with good safety and definite pharmacodynamics for the treatment of lymphoma.In this study,we intend to use monomethyl polyethylene glycol-polycaprolactone copolymer(MPEG-PCL)to optimize the preparation methods to construct a safe and effective MTX nano-micelle for anti-lymphoma therapy.On this basis,it was combined with(GA),a small molecular drug with chemosensitivity,to construct a stable and efficient nano-micelle loaded with MTX/GA to enhance its anti-lymphoma efficacy.Materials and Methods:MTX was encapsulated by two safe block polymers,monomethylpoly(ethylene glycol)-poly(lactic acid)(MPEG-PDLLA)and monomethyl poly(ethylene glycol)-poly(caprolactone)(MPEG-PCL).Firstly,several processes were optimized,and two kinds of nano-micellar drug delivery systems,MPEG-PDLLA-MTX and MPEG-PCL-MTX,were prepared by thin film hydration method.Secondly,the particle size,particle size distribution(PDI),morphological characteristics(transmission electron microscope photography),crystal properties and drug loading capacity(stability,drug loading,entrapment efficiency)and other pharmaceutical properties of the micelles were characterized.Then the inhibitory effect of MPEG-PCL-MTX nano-micelles on the growth of lymphoma cells was detected by MTT assay,and the apoptosis-inducing effect of nano-micelles on lymphoma cells was analyzed by flow cytometry.Furthermore,the uptake behavior of cells was quantitatively and qualitatively evaluated by flow cytometry and laser confocal microscopy.Finally,the subcutaneous tumor model of Burkitt lymphoma in NOD/SCID mice was established,the tumor growth was closely monitored,and the anti-lymphoma effect of MPEG-PCL-MTX nano-micelles in vivo was evaluated.The proliferation and apoptosis of tumor cells were quantitatively analyzed by Ki-67 staining and TUNEL staining,and the toxicity of nano-micelles and drug-loaded micelles were evaluated by MTT test,body weight monitoring and pathological sections of important organs.MTX and GA were encapsulated by MPEG-PCL,and several processes were optimized.The micelles co-loaded with MTX/GA were prepared by thin film hydration method.Then the activity of lymphoma cells treated with co-loaded nano-micelles was detected by MTT experiment,and the combined action index of drugs was calculated.The interaction between MTX and GA in different mass ratios of drug-loaded nano-micelles was analyzed,and the MTX/GA-MPEG-PCL nano-micelles with the best synergistic effect were selected for following research.Finally,the subcutaneous tumor model of Burkitt lymphoma in NOD/SCID mice was established,and the anti-lymphoma effect of co-loaded MTX/GA micelles in vivo was evaluated.Results:1.Polyethylene glycol-polyester copolymer MPEG-PDLLA and MPEG-PCL block copolymers were successfully prepared by ring-opening method.By optimizing several processes,MTX was successfully loaded into MPEG-PDLLA and MPEG-PCL micelles by thin film hydration method,and the entrapment ability of MPEG-PCL to MTX was significantly better than that of MPEG-PDLLA,choosing MPEG-PCL as the carrier of MTX for following research.2.XRD,TEM and particle size distribution analysis showed that MPEG-PCL encapsulated MTX successfully.MPEG-PCL-MTX micelles have the characteristics of small particle size,uniform dispersion,high entrapment efficiency and drug loading,stable pharmaceutical parameters and good repeatability.At 37 °C,MPEG-PCL-MTX micelles have good stability,not tend to aggregate,and have a certain sustained release effect in vitro compared with free MTX.3.MTT assays showed that the tumor inhibitory effect of MPEG-PCL-MTX nano-micelles at high concentration(greater than5000ng/ml)was significantly better than that of free drugs.The results of flow cytometry showed that MPEG-PCL-MTX could induce more apoptosis of human lymphoma Raji cells.Cell uptake assay showed that the uptake of MPEG-PCL-C6 by Raji cells was 1.5 times higher than that of free drugs.4.Pharmacodynamic studies showed that in the Burkitt lymphoma model,the tumor inhibitory effect of MPEG-PCL-MTX was significantly better than that of commercial MTX injection at the same dose.And low dose(3mg/kg)MPEG-PCL-MTX micelles can achieve comparable anti-lymphoma effect as high dose(7mg/kg)MTX injection.Weight monitoring and pathological examination of important organs showed that the nano-micelle preparation had no obvious toxicity in vivo.5.By optimizing several processes,nano-micellar MTX/GA-MPEG-PCL micelles co-loaded with MTX/GA were prepared by thin film hydration method.By adjusting the dosage,a series of MTX/GA-MPEG-PCL nano-micelles with different mass ratios were prepared,and their particle size,PDI,morphology,drug loading,entrapment efficiency,stability and in vitro release behavior were characterized.6.MTT assay was used to detect the cell viability of Raji cells treated with different mass ratios of MTX/GA-MPEG-PCL drug-loaded micelles.The combined action index((CI),)was calculated and compared.It was found that the co-loaded nano-micelles with MTX:GA(W/ W of 1:10 to 10: 1)had better anti-lymphoma synergistic effect,and their CI50 values were0.77,0.69 and 0.76,respectively.7.The results of pharmacodynamic experiments in vivo showed that compared with single micelles(MTX-MPEG-PCL and GA-MPEG-PCL)and mixed free drugs(MTX injection + GA solution),the optimized MTX/GA-MPEG-PCL nano-micelles had stronger anti-lymphoma effect and no obvious toxicity.Conclusions:In this study,a new nano-material MPEG-PCL was used as the carrier,and the chemotherapeutic drug MTX,was optimized to construct MPEG-PCL-MTX nano-micelles,which showed better anti-lymphoma effect in vivo and in vitro.On this basis,combined with small molecular chemosensitizer GA,we optimized the construction of MTX/GA-MPEG-PCL co-loaded nano-micelles,showing a more obvious synergistic effect on anti-lymphoma.No obvious toxicity was found in the two kinds of micelle drug delivery systems.