| Breast cancer is a malignant tumor with the highest morbidity and mortality among women in the world.It is often accompanied by malignant metastasis,always metastasize to lung,bone,liver and other parts,threatening women’s health and life seriously.Immunotherapy enhances the anti-tumor effect by activating or regulating the immune response of the body,which can produce long-term protection for the body and bring new hope for effective tumor treatment.Immune checkpoint therapy in immunotherapy is to play an anti-tumor effect by blocking the inhibitory pathway of anti-tumor T cell response,including antibody therapy and small molecule therapy.The PD-1 antibody Pembrolizumab and CTLA-4antibody Ipilimumab in antibody therapy have been approved by the US FDA for marketing.The small molecule therapy indoleamine 2,3-dioxygenase 1(IDO-1)inhibitor-NLG919[C18H22N2O,1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol]can inhibit the activity of IDO-1 with high selectivity.It has better stability compared with protein drugs such as antibodies and has become a new hotspot for the research of anti-tumor strategy,which is expected to become a new target drug in immunotherapy.But it has poor water solubility and will cause serious toxic and side effects when distributed in normal tissues.Moreover,due to the low immunogenicity of tumor cells and the existence of immunosuppressive tumor microenvironment,the response rate of anti-tumor drugs is low,which makes the therapeutic effects of most anti-tumor drugs unsatisfactory.Immunogenic cell death(ICD)is a type of cell death that can improve the tumor immunogenicity,by causing calreticulin(CRT)exposure,high-mobility group box 1protein(HMGB1)secretion from the nucleus to the extracellular,and adenosine triphosphate(ATP)release from the cytoplasm to the extracellular.Represented by anthraquinone drugs and oxaliplatin’s chemotherapy and photodynamic therapy can induce ICD in tumor cells,expose tumor cells to related antigens,and stimulate the maturation of antigen-presenting cells,further promote the activation of T cells,produce a specific immune response,and enable the body’s immune system to eliminate tumor cells in the body.Among them,the chemotherapy drug Doxorubicin(DOX),as a typical type I ICD inducer,can improve the immunogenicity of tumor cells and activate the body’s immune response;at the same time,as a broad-spectrum anti-tumor drug,it can be inserted into DNA chain to block the synthesis of tumor DNA,thereby exerting anti-cancer activity.Aiming at the problems of low tumor immunogenicity and poor water solubility of NLG919,a NLG919 micelle composite DOX gel drug delivery system was designed in this research.Firstly,NLG919 is prepared into polymer micelles to improve its water solubility,and then the micelles are added to the temperature-sensitive gel containing DOX·HCl.Taking advantage of its characteristics of keeping the solution state when below the gelling temperature and transforming into a semi-solid gel when at body temperature,it is administered by intratumoral injection to improve tumor cell immunogenicity,then activate the body’s anti-tumor immune response and inhibit tumor metastasis to the lung.The research content of this paper is divided into four parts:the preparation and characterization of NLG919 micelles and micellar/gel drug delivery system,the in vitro pharmacodynamic experiment,and the investigation of the pharmacological effect in mice.The first part is the preparation and characterization of NLG919 micelles.NLG919 is wrapped into the inner core of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](DSPE-m PEG2000)by thin film hydration method to form polymer micelles;the drug-assistance ratio was optimized according to the encapsulation efficiency and loading capacity of NLG919;Particle size,zeta potential,morphology and stability of micelles were determined by Dynamic light scattering and transmission electron microscopy(TEM).Results:After it was prepared into micelles,NLG919 changed from being insoluble in water to being uniformly dispersed in micelles and was in a clear state.When the drug-adjuvant ratio was 1:20,the encapsulation efficiency and drug loading of NLG919 micelles were 79.60±10.64%and 3.88±0.61%respectively;the micelle shape was approximately spherical and the average particle size of NLG919 micelles was 57.55±1.47 nm,meanwhile the Zeta potential is-8.83±2.38 m V;there is no significant changes of the particle size and content of NLG919 micelles after being placed at 4℃for 5 days,which indicating the stability is good.The second part is the preparation and characterization of micellar/gel drug delivery system.Using polylactic acid-glycolic acid copolymer-polyethylene glycol-polylactic acid-glycolic acid copolymer(PLGA-PEG-PLGA)as the temperature-sensitive gel material,the NLG919 micelle composite DOX temperature-sensitive gel(COMPOSITE)is prepared by the cold formulation method;whether gelation can occur under body temperature and the viscosity at room temperature are the inspection indicators to optimize the gel formulation,and the gelation temperature(Tsol-gel),gelation time(tsol-gel)and rheological properties are investigated;the morphology of micelles in the temperature-sensitive gel was observed by TEM;the drug release behavior in micelle at37℃,that of solution state at 4℃and gel state at 37℃of the temperature-sensitive gel were investigated by dialysis method;and its stability was also evaluated.Results:The gelation temperature of 16%PLGA-PEG-PLGA sol was 31.77±0.06℃.It changed from solution state to semi-solid state after 162.3±9.9 s at 37℃,the storage modulus reached the maximum,and the gel strength became largest.And its viscosity measured at 25℃is 617.3±114.1 m Pa·s,reflecting that the sol has a low viscosity and good fluidity at room temperature,which is convenient for injection and administration in clinical applications;the micelles in the TEM image maintain a nearly spherical shape in the gel;in vitro drug release showed that NLG919 micelles released completely after8 hours at 37℃,and then entered the plateau phase;the release rate of NLG919 and DOX·HCl in COMPOSITE at 37℃was slower than that at 4℃,and putting the composite gel at 4℃for half an hour can speed up the release of the two drugs;in the stability test,COMPOSITE placed at 4℃for 3 days,the content of DOX·HCl and NLG919 did not change significantly,while the content of the two drugs decreased slightly after being placed for 5 days,on the whole,the COMPOSITE was stable at 4℃for 3-5 days.The third part is the investigation of the in vitro efficacy of the drug delivery system.CCK-8 method was used to investigate the toxicity of different treatments on 4T1 cells;immunofluorescence and flow cytometry were used to investigate the exposure level of calreticulin on the surface of 4T1 cells after different treatments;chemiluminescence method was used to determine the level of extracellular ATP released;immunofluorescence was used to investigate the effects of drugs on the induction of HMGB1 to excrete from the nucleus.Results:In the concentration range of 1-20μM,NLG919 was less toxic to cells,but the toxicity of DOX to cells gradually increased with the increase of DOX concentration;in the concentration range of 1-20μM,NLG919 did not induce CRT exposure,HMGB1 protein efflux and ATP release;in the range of 1-10μM DOX concentration,cell surface CRT expression levels,HMGB1efflux and ATP release level have increased as DOX concentration increased,which indicated the ability of 4T1 cells to induce ICD have been improved.The fourth part is the investigation of the efficacy of micellar/gel drug delivery system in tumor-bearing mice.After establishing a 4T1 mouse subcutaneous breast cancer model,the retention of micellar composite gel and the distribution in main organs were observed by living imaging instrument;on a subcutaneous bilateral tumor model,the cytokine(TNF-α,IFN-γ)in serum of mice after different treatments were determined by Enzyme-Linked Immuno Sorbent Assay;the tumor site CRT exposure was investigated to evaluate the induction of tumor ICD in each group by immunofluorescence method;the expression of IDO-1 enzyme in the tumor was detected by immunofluorescence;the maturation of DC cells was investigated by flow cytometry;mouse tumor volume in situ and distal tumor volume,tumor inhibition rate and mouse body weight on the subcutaneous bilateral tumor model were used as indicators for pharmacodynamic investigation;tumor apoptosis were investigated by TUNEL staining method;pathological examination of heart,liver,spleen,lung,kidney and tumor tissues were performed to investigate the toxicity of COMPOSITE by hematoxylin-eosin staining(H&E);the activation of immune system in mice were evaluated by flow cytometry;the expression of CD4,CD8,and Fox P3 in the tumor were further investigated by immunofluorescence method;the expression of IDO-1enzyme in tumor was detected by immunofluorescence method,and the regulation of tryptophan metabolism was investigated;to investigate the metastasis of tumor tissue,the metastasis indicators Ki67,MMP-2,and MMP-9 were analyzed by immunohistochemical sections.Results:The micelle/gel can be efficiently retained at the tumor site after intratumor injection,and the release of the drug can be accelerated after half an hour of cooling with ice.The mice can absorb and metabolize it by more than 30%after 24 hours.After treatment with COMPOSITE,TNF-αand IFN-γin mouse serum increased from 297.6 and 567.6 pg/m L to 471.2 and 780.4 pg/m L respectively,which initially showed the immune response of the mouse body was activated;the proportion of CRT positive cells in situ tumor in the immunofluorescence section increased from 10.5%to 57.7%,and CRT exposure showed that COMPOSITE induced tumor cells to develop ICD in vivo.At the 10th and 21st days,the proportion of IDO-1 positive cells at the tumor site decreased from 11.3%,13.0%to 5.2%,6.7%respectively,Kyn(n M)/Trp(μM)decreased from 455.8 to 148.0,IDO-1 low expression and reduction of tryptophan in metabolism indicates that COMPOSITE can provide a basis for effectively improving the tumor immunosuppressive microenvironment;the proportion of mature DC cells in lymph nodes and tumor sites has increased from11.4%,10.1%to 63.2%,29.8%respectively,indicating that COMPOSITE can effectively induce DC cell maturation;in the efficacy experiment,COMPOSITE can effectively inhibit the growth of tumors in situ and distal tumors,and the body weight of the mice has no significant change;in the TUNEL section,a large area of apoptotic cells appeared at the tumor site;in the H&E section,the mouse heart,liver,spleen,lung and kidney showed no obvious tissue damage,while the tumor tissue showed extensive necrosis;then in the results of flow cytometry,the ratio of CD4~+T cells and CD8~+T cells in the spleen was changed from 6.0%and 4.8%to 37.1%and 13.8%,respectively;The proportion of CD4~+T cells and CD8~+T cells in the tumor in situ increased from13.8%and 3.9%to 44.1%and 10.6%respectively,the significant increase indicating that COMPOSITE activated the anti-tumor immune response in mice,and increased T cell infiltration at the tumor site.As well as the proportion of regulatory T cells(Treg)in the tumor in situ decreased from 20.6%to 6.2%,indicating that COMPOSITE can effectively improve the immunosuppressive microenvironment of the tumor site;the proportion of CD4 and CD8 positive cells in the in situ tumor immunofluorescence section has increased by 16.4 times and 4.1 times,respectively,and the proportion of Fox P3 positive cells has been reduced from 6.6%to 4.0%,which was consistent with the results of flow cytometry;in the immunohistochemical sections,the brown particles were reduced,and the expression of Ki67,MMP-2,and MMP-9 was reduced,further verifying that COMPOSITE has a good tumor suppression effect.In summary,the micellar/gel drug delivery system prepared in this article has good physical temperature sensitivity and retention,can significantly induce ICD in tumor cells and activate the body’s anti-tumor immune response,and has a good inhibitory effect on both in situ and distal tumors.It provides new ideas and strategies for the combined application of immunotherapy and chemotherapy in clinical breast cancer. |
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