Dual Transformed Nanocage Co-delivery Abemaciclib And IMD-0354 For Tumor Chemoimmunotherapy

Tumor as a malignant disease has rapid-growing morbidity and mortality.Due to the complexity and heterogeneity of tumor,combination therapy provides a prospective therapeutic strategy,in which the multiple synergistic efficiencies of combined drugs are essential for improving antitumor effects.Based on the theory,drugs with great synergistic effects were selectively in this study.Abemaciclib as a CDK4/6 inhibitors can specifically target CDK4/6 and suppress retinoblastoma protein(RB)phosphorylation to arrest tumor cells in G1 phase and inhibit cell proliferation.CDK4/6-cyclin D pathway is important in the process of malignant tumor cell proliferation.And the expression of cyclin D is regulated by various related signaling factors such as epidermal growth factor receptor(EGFR)and NF-?B.Inhibiting CDK4/6-cyclin D pathway can effectively inhibit cell proliferation and enhance the therapeutic effect.IMD-0354 as a selective IKK? inhibitor can effectively inhibit NF-?B pathway to decrease the expression of cyclin D,thereby inhibiting the formation of CDK4/6-cyclin D complex.Therefore,the synergistic chemotherapy effect can be achieved by combining abmemaciclib and IMD-0354.Recent research has demonstrated that the proliferation of regulator T cell can be inhibited by Abemaciclib to relieve tumor immunosuppressive microenvironment IMD-0354 can effectively inhibit NF-?B pathway,and repolarization tumor-associated macrophage(TAM)from immunosuppressive M2-type to immune-active M1-type to relieve immunosuppression and activate anti-tumor immunity.Taken above,combination of Abemaciclib and IMD-0354 was expected to relieve tumor immunosuppressive microenvironment,stimulate immune microenvironment,exhibiting synergistic immunotherapy effect.In addition,combination of Abemaciclib and IMD-0354 processed synergistic chemoimmunotherapy effect based enhanced chemotherapy and immunotherapy effectsThe biodistributions of Abemaciclib and IMD-0354 are different because of the differences of the physicochemical properties,limiting the efficacy of combination therapy.Co-delivery nano-system has been attracted extensive attention due to the advantages of co-delivery ability and the enhanced tumor accumulation capability.But the traditional nano-delivery systems are generally designed to be negative charged,with particle size in the range of 100-200 nm in order to avoid the clearance effect of reticuloendothelial system and achieve tumor accumulation.The therapeutic effect of traditional nano-delivery system was affected seriously.On the one hand,cellular uptake of negative nano-delivery system is hindered due to the negative cell membrane charge repulsion;On the other hand,it is difficult for nano-delivery system to deep penetrate into tumor interior,because of the dense extracellular matrix and high inter tissue pressure in tumor tissue.Therefore,designing an innovative nano-delivery system which would be realized charge and particle size dual-switch in tumor site is the key to solving the above problemsTherefore,pH responsive charge reversal and size changeable abemaciclib and IMD-0354 co-loaded nanocage was formulated.Firstly,asparagines-glycine-arginine(NGR)modified pH responsive charge reversal materials NGR-PEG-PLL-DMA(ND)was synthesized.Abemaciclib and IMD-0354 loaded PAMAM were prepared(PA and PI),respectively.Then co-loaded nanocage(PA/PI-ND)was self-assembled through electrostatic adsorption among ND,PA and PI.PA/PI-ND exhibited the charge and size dual switchable characteristics.By acting on CDK4/6-cyclin D pathway,the tumor cell cycle could be arrested in G1 phase via both Abemaciclib and IMD-0354,achieving synergistic chemotherapy effect.At the same time,the combination therapy could relieve immunosuppressive microenvironment and active anti-tumor immunity,exhibiting the promotion on immunotherapy.Moreover,the chemoimmunotherapy efficacy could be achieved by the combination therapy based on the synergist mechanism of two drugs.And mediated by NGR as target ligand,PA/PI-ND could active target and co-delivery the two drugs to tumor.Then,PA/PI-ND was switched to positive and triggered disassembly releasing smaller sized PA and PI at tumor acidic microenvironment,to enhance drug accumulation and deep tumor penetration ability.Main researches and results of the study are as follows1.Determination method about Abemaciclib and IMD-0354Methods for the determination of Abemaciclib and IMD-0354 were respectively established through HPLC.The results demonstrated that the determination methods were specific and with good linear relationships within the set concentrations.The precisions and recoveries could meet the methodological requirements2.Preparation and characterization of dual transformed Abemaciclib and IMD-0354 co-loaded nanocageFirst,several charge reversal materials(MAL-PEG-PLL-DMA,MAL-PEG-PLL-TDA,MAL-PEG-PLL-CA)were synthesized in this study.The charge reversal material MAL-PEG-PLL-DMA was selected by investigating charge switchable behaviors and M AL-PEG-PLL-DMA could switch to positive under pH 6.5(simulating tumor acidic microenvironment).Then NGR modified pH sensitive charge reversal material NGR-PEG-PLL-DMA(ND)was synthesized,Abemaciclib and IMD-0354 loaded PAMAM(PA and PI)were prepared by thin film hydration method,respectively PA/PI-ND was prepared by electrostatic adsorption method.Then the physicochemical properties such as particle size,potential,morphology and charge switch ability were evaluated by dynamic light scattering,TEM and so on.The results showed PA/PI-ND was elliptic with particle size of 177.7±5.8 nm and zeta potential of-11.17±0.45 mV.PA/PI-ND could change from-11 mV to about 7 mV and particle size changed from 180 nm to about 5 nm under pH 6.5,realizing the dual charge and size switch.In vitro release results showed the accumulative release rates of abmaciclib and IMD-0354 released from PA/PI-ND were significantly increased to 50%and 45%approximately under pH 6.5 while these were about 35%and 32%under pH 7.4 after incubation for 48 h,indicating the disassembly of PA/PI-ND could speed up the release of Abemaciclib and IMD-0354.3.Evaluation of delivery behaviors of dual transformed Abemaciclib and IMD-0354 co-loaded nanocageThe ability of active targeting,co-delivery efficacy and deep tumor penetration of PA/PI-ND were investigated in vitro and in vivo,respectively.The results of cellular uptake and real-time imaging assays demonstrated thatPA/PI-ND could actively target to tumor tissues and enhance drugs accumulation in tumor mediating by NGR.The co-localization efficacy was investigated in CT-26 cells and CT-26 bearing Balb/c mice,respectively.The results demonstrated that PA/PI-ND exhibited great co-localization efficiency in tumor tissues compared with the mixture of PA loaded nanocage and PI loaded nanocage.Subsequently,the results of cellular uptake in vitro and deep tumor penetration showed that PA/PI-ND could be uptake by tumor cells and M2 macrophages respectively,and the cellular uptake was significantly increased under pH 6.5 compared with that under pH 7.4.Meanwhile,PA/PI-ND could penetrate into tumor interior which was caused by the switch of charge and size of PA/PI-ND in tumor acid microenvironment.Taken above,it was demonstrated that the enhanced cellular uptake and deep tumor penetration were mainly caused by the pH triggered disassembly of PA/PI-ND.4.Study of antitumor efficacy and safety of dual transformed Abemaciclib and IMD-0354 co-loaded nanocageThe enhanced chemotherapy effect was investigated through cytotoxicity assay and cell cycle arrest experiments on CT-26 cells and MCF-7 cells,respectively.The synergy indexes on CT-26 and MCF-7 are 0.412 and 0.539,respectively when the mass ratio of Abemaciclib and IMD-0354 was 5:1,indicating the good synergestic effects.The results showed that the half maximal inhibitory concentration(IC50)of PA/PI-ND was about 0.235 ?g/mL,exhibiting great cytotoxicity compared with PA-ND and PI-ND(p<0.01,p<0.001).And G1 phase proportion of cell cycle was increased to 62.46%and 73.26%in CT-26 cells and MCF-7 cells,respectively.The expressions of p-RB and cyclin D were decreased in PA/PI-ND group.The above results ensured that PA/PI-ND exhibited expected promoting on chemotherapy.In immunological evaluation experiments,PA/PI-ND could stimulate tumor associated macrophage(TAM)repolarization from M2-type to M1-type,inhibit regulatory T cells(Tregs)proliferation.increase the amounts of CD4+T cells and CD8+ T cells,enhance immune-active cytokines secretion such as INF-??IL-12 and TNF-? meanwhile decrease the immunosuppressed cytokines secretion such as IL-10 and TGF-?,inferring that the immunosuppressive tumor microenvironment could be relieved and to stimulate anti-tumour immune responses by PA/PI-ND.In vivo anti-tumor results showed that PA/PI-ND could significantly improve therapeutic efficacy compared with PA-ND and PI-ND(p<0.001),demonstrating PA/PI-ND could ensure the synergetic chemoimmunotherapeutic effect.And the results of H&E stain,Ki67 stain and TUNEL stain demonstrated that PA/PI-ND could significantly promote tumor cell necrosis and apoptosis,inhibit tumor cell proliferation,and enhance the tumor chemoimmunotherapeutic effect.No obvious lesions were observed in H&E staining of major organs of mice treated by PA/PI-ND,and no obevious change was observed in hematological index compared with normal saline groupIn summary,this study constructed pH responsive charge reversal and size changeable Abemaciclib and IMD-0354 co-loaded nanocage based on the concept of chemoimmunotherapy.The nanocage exhibited multi-functions including active targeting,charge and particle size dual-switch,co-delivery,deep tumor penetration and so on.The nancage could realize synergistic chemoimmunotherapy based on chemotherapy and immunotherapy,providing a promising strategy for further delivery system design and combined therapy pattern.