Construction Of Nano-micelles Co-loaded With Immune Checkpoint Blockades And Evaluation Of Anti-breast Cancer In Vivo And In Vitro

Immunological checkpoint blockade therapy,represented by programmed cell death 1/programmed cell death ligand 1(PD-1/PD-L1),is the new type of cancer treatments in recent years.The treatment method,which has been extensively and deeply studied,is part of the main ways of tumor immunotherapy.Although it applies in respect of some solid tumor patients,the patients who benefit from PD-1/PD-L1 therapy are still a minority.Because a variety of other immunosuppressive molecules,such as indoleamine2,3-dioxygenase(IDO),are also triggered during tumor cell immune escape.Studies have shown that dual-blockade immunological checkpoints can enhance anti-tumor immunotherapy,but the simultaneous delivery of two immunological checkpoint blockades remains a challenge.This study we generated a peptide assembling tumor-targeted nano delivery system based on a breast cancer homing and penetrating peptide for the co-delivery of a programmed cell death ligand 1(PD-L1)small interfering RNA(si RNA)(siPD-L1)and an idoleamine 2,3-dioxygenase inhibitor(1MT)as a dual blockade of an immune checkpoint,exert anti-tumor effect.This topic is mainly composed of three parts.The first part is the construction of polypeptide nano-micelles co-loaded with immune checkpoint blockades.That is,the synthesis of cholesterol-modified peptide monomers,the construction of targeted nano-micelles co-loaded with immune checkpoint blockade,and the measurement of the structure and characteristics of nano-micelles.First,we synthesized the cholesterol-modified 16 amino acid peptide monomer structure Chol-HHHHHHH-AKRGARSTA(CHL)by solid phase synthesis,and then purified it by high performance liquid chromatography.The molecular weight was identified by mass spectrometry.The results showed that we successfully synthesized CHL monomer.Then we used dialysis to prepare 1MT-CHL by self-assembly of CHL in deionized water.Co-CHL was then prepared by co-incubation of siPD-L1 and 1MT-CHL.Finally,the particle size,zetaThe second part is the in vitro study of anti-breast cancer cells co-loaded with immune checkpoint blockades nano-micelles.That is,to study the in vitro characteristics of nano-micelle Co-CHL co-loaded with immune checkpoint blockades at the cellular level.In this section,the cytotoxicity,cell uptake,intracellular distribution,lysosomal escape and gene transfection experiments and kynurenine production inhibition experiments were carried out in mouse breast cancer 4T1 cells,respectively.The results indicate that si RNA can be prevented from being degraded and delivered into the cell to achieve lysosomal escape through the mediation of CHL,which effectively achieve cell transfection andreduce the expression of PD-L1 in tumor cells.Moreover,cells can uptake more 1MT to inhibit cell IDO activity,and reduce kynurenine production.On the basis of these,we constructed a lymphocyte-tumor cell co-culture system at the cellular level.Through different drug stimulation,we found that Co-CHL can relieve the tumor cells from immunosuppressing situation and reactive lymphocytes.The third part is the in vivo evaluation of the anti-breast cancer effect of nano-micelles co-loaded with immune checkpoint blockers.That is,to verify the in vivo distribution,safety and anti-tumor effect of Co-CHL on the in situ breast cancer model of4T1 mice.Di R-CHL micelles were prepared using fluorescent Di R,to demonstrate the distribution of CHL micelles in vivo optical imaging.The distribution of CHL micelles in vivo showed that the nanomicelles were mainly concentrated in the tumor site.The fluorescence in the main organs was also very limited indicating that the nanomicelles have a better targeting ability in vivo.The tumor growth inhibition experiment also showed that Co-CHL could significantly inhibit tumor growth under the experimental conditions,while the results of H&E staining showed that the toxicity of nano-micelles was low and the safety was high.On this basis,we further studied the anti-tumor immune mechanism of Co-CHL.The study found that the proportion of CD8+/CD4+ in tumor infiltrating lymphocytes was significantly increased,while the levels of IL-2 and IFN-γ in tumor tissues were also significantly increased.In this study,we constructed a polypeptide micelle CHL,containing two kinds of immune checkpoint blockade drugs,which can specifically accumulate in the tumor site of breast cancer,siPD-L1 can escape from the endosome and transfect after being endocytosis by tumor cells mediated by CHL.The drug within these cells then acts to block tryptophan metabolism.The results showed that locally released siPD-L1 and 1MT favor the survival and activation of cytotoxic T lymphocytes(CTLs),resulting in apoptosis of breast cancer cells.The results of in vitro and in vivo experiments in mice showed that the polypeptide micelles with two immune checkpoints could effectively inhibit the growth of breast cancer cells.Therefore,this study can provide a reference for the immunotherapy of breast cancer.