Study On DOX Delivery By Polymeric Indoximod Prodrug Micelles For Tumor Immunotherapy

Polymeric micelles are a kind of potential drug delivery systems,which have great advantages in increasing the solubility of hydrophobic drugs,improving drug stability,delaying drug release,improving drug efficacy,reducing toxicity and improving targeting.In this paper,PEG-IND was synthesized from polyethylene glycol monomethyl ether(mPEG)and 1-methyl-d-tryptophan(IND),and a micellar drug delivery system(DOX/IND@NPs)was constructed to achieve the simultaneous intracellular delivery of doxorubicin(DOX)and indoleamine 2,3-dioxygenase 1(IDO1)inhibitor IND.It exerts the anti-tumor activity of DOX while inhibiting tumor immune escape,improving the effect of tumor immunotherapy.Using polyethylene glycol monomethyl ether,4-chloromethylstyrene and 1-methyl-D-tryptophan as raw materials,the block copolymer PEG-IND was synthesized.Hydrogen nuclear magnetic resonance(~1H-NMR)and infrared spectroscopy(FT-IR)characterize the copolymer.The results showed that the target product was successfully synthesized.The in vitro fluorescence spectrophotometric analysis method of DOX was established,and the standard curve,precision and recovery rate of the established analysis method met the methodological requirements.The copolymer micelles were prepared by the film dispersion method,and the encapsulation efficiency and drug loading were used as evaluation indicators.The single factor method was used to optimize the formulation and process,and the pharmacological properties of drug-loaded micelles were investigated.The results showed that the particle size of the drug-loaded micelles was about 122nm,and the particle size distribution was narrow,with a spherical shape and uniform size,and the Zeta potential was about-7.58mV.Under the optimal conditions,the encapsulation efficiency and drug loading were 75.36%and6.85%,respectively.The in vitro release results showed that DOX/IND@NPs micelles exhibited a significant sustained-release effect under p H 7.4.Using mouse breast cancer 4T1 cells as a model,flow cytometry and fluorescence microscopy examined the uptake of free DOX and DOX/IND@NPs by tumor cells.It was found that DOX/IND@NPs can greatly increase the uptake of DOX by 4T1 cells.It is about 5times that of free DOX,indicating that DOX/IND@NPs can significantly increase the intracellular DOX content.The MTT method was used to investigate the in vitro cytotoxicity of DOX/IND@NPs micelles.The results showed that,compared with free DOX,DOX/IND@NPs had obvious cytotoxic effects on 4T1 cells,while IND and IND@NPs had almost no cytotoxicity.A 4T1 tumor-bearing BALB/c mouse model was established to investigate the distribution and tumor suppression of the polymer micelle drug delivery system in mice.The results showed that with the extension of time,the DIR-loaded IND@NPs micelles gradually gathered to the tumor site,with good tumor targeting.DOX/IND@NPs can significantly inhibit tumor growth,with the largest area of tumor necrosis and the weakest proliferation.Immunofluorescence staining was used to investigate the changes in the tumor microenvironment.The results showed that DOX/IND@NPs can down-regulate the expression of VEGF,MMP9 and other angiogenic factors and CD31,and reduce the average tumor blood vessel density.In addition,drug-loaded micelles can also reduce the infiltration of tumor-associated macrophages(TAMs),myeloid-derived suppressor cells(MDSCs)and regulatory T cells(Tregs)in the tumor microenvironment and promote the infiltration of tumor killer cells CD8~+T cells,improve anti-tumor effect and reduce immunosuppression.There was no significant change in the weight of the mice during the experimental period,and no significant pathological changes were seen in the main organs,confirming the biological safety of DOX/IND@NPs.