Artificial Macrophage-imitating Nano-delivery System Co-loading Black Phosphorus Quantum Dot And CDK4/6 Inhibitors For Colorectal Cancer Therapy

Exploring the effective tumor therapy to improve the cure rate and mortality in patients is the primary problems and challenges for the treatment of tumor.With the continuous deepening of research,the role of tumor microenvironment in promoting tumor development has attracted attention.Tumor-associated macrophages(TAMs)are one of the most abundant infiltrating immune cells in the tumor microenvironment,and they are the crucial cells that affect the tumor microenvironment and participate in tumor immunity.TAMs in the tumor tissue are main M2 TAMs(M2 TAMs),which exhibits tumor-promoting effects.However,TAMs have strong plasticity,its phenotype can be repolarized into M1 TAMs(M1 TAMs)under external intervention,which has anti-tumor effects.Therefore,regulating the phenotype and function of TAMs is an important method in tumor therapy.More importantly,macrophages have the function of directly recognizing and engulfing tumor cells,so they are widely concernd by researchers as therapeutic cellsHowever,due to the complexity of tumor microenvironment,the effectiveness of TAMs based therapy strategy is still unable to meet the needs of the requirements in clinical treatment.It is imperative to develop a new immunotherapy strategy integrating the superior function of macrophages.Based on this,an "artificial macrophage-mimetic" strategy was proposed in this paper to construct an artificial macrophage-imitating nano delivery system to simulate the three capabilities of macrophages:1)Tumor targeting;2)The ability of macrophages to activate the immune response and reverse the tumor immunosuppressive microenvironment;3)The ability of antitumor.In this paper,the photosensitizers Black phosphorus(BP)quantum dots(BPQDs)and Abemaciclib(Ab)were selected as therapeutic drugs.BPQDs can not only directly kill tumors through the PDT/PTT effect,but also cause immunogenic cell death(ICD)effect to activates the tumor immune response.Ab can inhibit the tumor cell proliferation through arresting cell cycle at G1 phase through specifically inhibit retinoblastoma protein(RB)phosphorylation.Meanwhile,Ab can also restrain the proliferation of immunosuppressive regulatory T cells(Treg)to modulate immune-suppressive microenvironment caused by Tregs.First,thin film dispersion method was used to prepare Ab-loaded liposomes(Ab-Lip),and then fused with the macrophage membrane to obtain the artificial macrophage-imitating co-loading BPQDs and Ab(AB@LM).1)AB@LM could achieve the active targeting to tumor tissues mediated by macrophage membrane;2)AB@LM could achieve immune activation and the regulation of immunosuppressive microenvironment by inducing ICD effect and inhibiting Tregs,including promoting DCs maturation,antigen presentation,T cells proliferation,inhibiting the proliferation of Tregs and the enhanced immune killing effect,mimicking the immunotherapy function of macrophages;3)phototherapy/chemotherapy/immunotherapy of AB@LM could induce tumor cell arrest and cell apoptosis which further augment the antitumor efficiency.In summary,the new strategy of "artificial macrophage-imitating" can imitate the functions of macrophages,combine tumor targeting,regulate the immune microenvironment,and efficiently kill tumors,significantly improve tumor treatment effects.The main research contents and results of this topic are as follows:1.Determination method for for AbThe determination method for Ab was established by HPLC.The results show that the established determination method was specific,and had good linear relationship within the set concentration range.The precision and recovery of the method meet the requirements of methodologica,and can accurately determine Ab content.2.Preparation and characterization of the artificial macrophage-imitating nano delivery system co-loading BPQDs and AbIn this study,BPQDs with uniform particle size were prepared,which showed good photothermal heating and ROS properties in vitro.Then,macrophage membrane(MMs)was extracted and prepared from RAW264.7 macrophages.Ab-Lip was prepared by thin film dispersion method.Finally,AB@LM were prepared using the above materials by extrusion method.The results showed that the morphology of AB@LM was elliptic with the particle size of 152.2±0.989 nm and the zeta potential of-17.4±0.424 mV.The in vitro heating experiment showed that the temperature of AB@LM could be increased by 25℃ after being irradiated by 808 nm laser for 10 min,which met the need of photothermal therapy.In vitro photodynamic experiments showed that the light absorption of AB@LM decreased by about 40%after 5 min irradiation with 660 nm laser in vitro,which indicated that the particles had photodynamic activity.The results of in vitro release experiments showed that the cumulative release rate of AB@LM was 81.07%under NIR laser irradiation,which was significantly higher than that under non-laser irradiation(p<0.01),indicating that laser irradiation could accelerate drug release.3.Evaluation of in vitro uptake and in vivo targeted delivery behavior of the artificial macrophage-imitating nano delivery system co-loading BPQDs and AbUsing colorectal cancer(CT26)as a model,the ability of AB@LM in targeting delivery was investigated at the cellular and animal levels.The results of in vivo imaging suggested the active targeting and enhanced tumor accumulation capacity of AB@LM mediated by macrophage membrane.The results of cellular uptake experiments showed that AB@LM can promote the uptake of tumor cells.The above results show that AB@LM had good tumor targeting ability.4.In vivo and in vitro evaluation antitumor efficacy of the artificial macrophage-imitating nano delivery system co-loading BPQDs and AbTaking CT26 as the cell model,the anti-tumor effect of AB@LM was investigated by in vitro cytotoxicity assay,cell cycle arrest assay and apoptosis assay.AB@LM could effectively block CT26 cells cycle in G1 phase(62.33%),and increase the apoptosis rate of tumor cells(41.0%),indicating that AB@LM can kill CT26 cells effectively.The result of in vivo immunological evaluation experiment shows that AB@LM could significantly inhibit the proliferation of Tregs,promote the infiltration of T cells.These results showed that AB@LM can reverse the tumor immunosuppressive microenvironment,activate the immune response and improve the anti-tumor immune effect.The tumor weight of AB@LM could group mice was lower than that in A@LM and B@LM groups(p<0.005,p<0.005),demonstrating the great antitumor effect of AB@LM.The results of H&E staining of main organs in vivo showed that there was no obvious lesion in main organs of AB@LM groupIn conclusion,an artificial macrophage-imitating nano delivery system co-loaded BPQDs and Ab to mimic the three abilities of macrophages and achieve anti-tumor therapy.The artificial macrophage-imitating can actively target the tumor tissue and reverse the immunosuppressive tumor microenvironment in vivo and activate the immune response after laser irradiation.At the same time,the nanoparticle integrated photo/chemo/immunotherapy,to enhance the effect of tumor treatment.This paper provides an experimental and theoretical basis for the further design and development of a new model of tumor therapy based on macrophages.The strategy of artificial macrophage-mimicing is an effective new strategy of tumor immunotherapy.