MMP-2 Enzyme-responsive Liposomes Enhance Chemoimmunotherapy For Cold Tumors

Immunotherapy has become a promising approach for cancer treatment.Despite huge potential in clinic,only a few patients have responded to cancer immunotherapies.Accumulating evidences indicate that the responsiveness is closely related with the immune status of cancer."Cold tumors" show insufficient T cells intratumoral infiltration and failed T cells priming,resulting to limited therapeutic effects.Chemoimmunotherapy can exhibit synergistic mechanisms of chemotherapy and immunotherapy,which is an effective method for cold tumors treatment.Thus,converting cold tumors to hot by chemoimmunotherapy is crucial to achieve successful anti-tumor therapy.Chemotherapy-induced immunogenic cell death(ICD)is an effective way to promote T cells infiltration and is commonly used for converting cold tumors to hot.Chemotherapeutic drugs,such as oxaliplatin(Oxa),mitoxantrone,doxorubicin,etc,can promote tumor cell apoptosis by inducing DNA damage,leading to the sequential release of damage-associated molecular patterns(DAMPs)and activation of antitumor immunity.While,a series of DNA repair pathways in tumor cells could restrict DNA damage and the followed ICD.Therefore,inhibiting DNA repair provides a potentially effective way to strengthen chemotherapy and ICD effect.Among of various DNA repair pathways,bromodomain-containing protein 4(BRD4)is over-expressed in various tumors.BRD4 participates in the repair of damaged DNA and the expression of programmed cell death protein 1 ligand 1(PD-L1).Therefore,the combination of DNA-damaged chemotherapeutic drugs with BRD4 inhibitor is an effective way to increase DNA damage and reinforce ICD,thus promoting T cells infiltration,and ensuring the conversion from cold tumors to hot tumors.Blocking the immune checkpoint is an important means of promoting T cells priming.The immune checkpoint acts as brakes of the T cells,such as programmed cell death protein 1(PD-1),cytotoxic T-lymphocyte-associated protein 4(CTLA-4),PD-1 ligand 1(PD-Ll),etc,inhibiting tumor-infiltrating T cells priming and immune activation.Recently,many immune checkpoint inhibitors have been widely used in clinic for tumor therapy.However,tumor cells could utilize the co-inhibitory pathways to escape the attack of immune system.Therefore,ongoing efforts were studied to combine multiple immune checkpoint inhibitors.T-cell immunoglobulin mucin-3(Tim-3)is a negative regulator of lymphocyte function and survival,which has attracted much attention.It was reported that T cells co-express PD-1 and Tim-3 have been shown to exhibit more severe depletion.It was observed the upregulation of immune checkpoints,notably Tim-3,in PD-1 mAb bound T cells and demonstrated a survival advantage with addition of Tim-3 mAb following failure of PD-1 blockade.Therefore,simultaneously inhibiting PD-1/PD-L1 and Tim-3/galectin-9 pathways is an effective combination strategy to promote T cells priming,which would greatly enhance the effect of immunotherapy.Based on the progress,we combined Oxa,BRD4 inhibitor JQ1,and Tim-3 mAb to convert uninflamed cold tumors to hot tumors,thus strengthening the chemoimmunotherapeutic effect.Oxa is a broad spectrum chemotherapeutic agent causing DNA damage and inducing ICD,and JQ1 is a BRD4 inhibitor inhibiting DNA repair.Combining Oxa with JQ1 could increase DNA damage and augment ICD,ultimately increasing T cells intratumoral infiltration.JQ1 is also widely used as a small molecular PD-L1 inhibitor by reducing the expression of PD-L1.Tim-3 mAb was used to block the immunosuppressive Tim-3/galectin-9 pathway and JQ1 was used to block PD-1/PD-L1 pathway,which would achieve dual immune checkpoint inhibition,thus effectively promoting T cells priming and strengthening immunotherapy.To achieve an ideal therapeutic effect,an ideal co-delivery system for safe and effective delivery of different drugs was essential.Herein,we elaborately designed metalloproteinase-2(MMP-2)enzyme-responsive liposomes(JOT-Lip)that co-loaded Oxa and JQ1(JO-Lip),and coupled Tim-3 mAb on the surface of liposome via using metalloproteinase-2 responsive linkage.Under the high concentration of MMP-2 in tumor microenvironment,JOT-Lip could be disassembled.Tim-3 mAb and JO-Lip could be delivered to T cells and tumor cells respectively to exert their respective effects,which converted cold tumors to hot and enhanced chemoimmunotherapeutic effect.Main researches and results of the study are listed as follows:1.Determination method for Oxa and JQ1HPLC method was successfully established for the determination of Oxa and JQ1.The results showed the determination methods were specific and with good linearity.The within-day and inter-day precisions and method recoveries meet the determination requirements.So the methods can be used for the subsequent determination of Oxa and JQ1.2.Preparation and characterization of MMP-2 enzyme-responsive liposomes(JOT-Lip)co-loaded with Oxa,JQ1 and Tim-3 mAbThe functional materials DSPE-PEG-pep-Tim-3 mAb was firstly synthesized for the subsequent preparation of JOT-Lip.The optimum combination ratio of Oxa and JQ1 was determined by cytotoxicity test.JO-Lip was prepared by thin film dispersion method,and then Tim-3 mAb was modified onto liposome surface by post insertion method to prepare JOT-Lip.The morphology and particle size of JOT-Lip were investigated.The results showed that JOT-Lip was spheres and the size was 155.27 ± 3.30 nm.The X-ray diffraction patterns of Oxa,JQ1,Tim-3 mAb,excipients,physical mixture and JOT-Lip were further studied,and the results showed that JOT-Lip was successfully encapsulated.The enzyme concentration and incubation time of MMP-2 sensitive peptide degradation were investigated by HPLC.The results showed that MMP-2 sensitive peptide was completely degraded when the enzyme concentration was 50 μg/mL and the incubation time was 60 min.The in vitro release behavior of Oxa and JQ1 in JOT-Lip was similar to that in Oxa-Lip or JQ1-Lip,suggesting the modification of Tim-3 mAb did not affect drug release.The cumulative release rate of Tim-3 mAb in JOT-Lip for 24 h in the enzyme-containing medium(93.33 ± 3.95%)was significantly higher(p<0.001)than that in the enzyme-free medium(30.48±2.78%),indicating that the enzyme sensitive release of MMP-2 can be achieved under high concentration of MMP-2 in tumor microenvironment.The particle size and PDI of JOT-Lip did not change significantly,indicating that JOT-Lip had good stability.3.Evaluation of delivery behavior of MMP-2 enzyme-responsive liposomes(JOT-Lip)co-loaded with Oxa,JQ1 and Tim-3 mAbThe in vivo delivery of JOT-Lip was investigated by Real-time imaging in CT26 tumor-bearing mice.The results showed that JOT-Lip had good tumor accumulation ability.The co-localization efficiency was evaluated qualitatively and quantitatively in vitro by fluorescence imaging and flow cytometry.In vivo co-localization of JOT-Lip was further evaluated in CT26 tumor-bearing mice.In vitro and in vivo experiment results showed that the co-localization efficiency of co-liposomes was higher than that of mixed solution group.The expression of Tim-3 on CT26 cells and T cells was determined using flow cytometry.The results showed that the expression of Tim-3 on T cells was significantly higher(p<0.01)than that on CT26 cells.The uptake of drugs and monoclonal antibodies was further investigated by in vitro co-culture experiment and in vivo uptake experiment.The results showed that the drug was mainly taken up by tumor cells and Tim-3 mAb was mainly taken up by T cells,suggesting that JOT-Lip can achieve co-delivery of drugs with different targets.4.In vitro and in vivo efficacy evaluation of MMP-2 enzyme-responsive liposomes(JOT-Lip)co-loaded with Oxa,JQ1 and Tim-3 mAbThe chemical synergistic effect of JOT-Lip was investigated by cytotoxicity test,Pt-DNA adduct assay,DNA damage evaluation and cell apoptosis assay.The results showed that JOT-Lip enhanced cytotoxicity,Pt-DNA binding ability,DNA damage,and promoted tumor cell apoptosis,showing a good chemical synergistic effect.The immune synergistic effect of JOT-Lip was investigated by DAMPs assay,DC maturation assay and PD-L1 expression assay.The results showed that JOT-Lip could further induce CRT exposure,HMGB1 release and ATP secretion,which enhanced ICD effect.JOT-Lip promoted DC maturation,and reduced the expression of PD-L1,showing a good immune synergistic effect.CT26 tumor-bearing mice were used as model to investigate the tumor suppressive effect and the status of immune cells and cytokines in vivo.In vivo efficacy results suggested that JOT-Lip had the strongest inhibitory effect on tumor growth in CT26 tumor-bearing mice.H&E,Ki67 and TUNEL staining showed that JOT-Lip could promote the necrosis and apoptosis of tumor cells and inhibit the proliferation of tumor cells.Flow cytometry was used to measure immune cells in vivo.The results showed that JOT-Lip significantly increased the percentage of CD4+T cells,CD8+T cells and CTL cells,and decreased the percentage of Treg cells.ELISA analysis of cytokines secretion in CT26 tumor tissues showed that JOT-Lip promoted the secretion of IFN-y,IL-12 and TNF-α,and inhibited the secretion of TGF-β.Therefore,JOT-Lip showed synerfistic effects on chemoimmunotherapy in vivo.The results showed that JOT-Lip could significantly inhibit the growth of tumor in 4T1 tumor-bearing mice.Lung bioluminescence images and H&E staining results showed that lung metastasis was the least in the JOT-Lip group.These results suggested that JOT-Lip can inhibit tumor growth and metastasis.Cytotoxicity test,hemolysis test and H&E staining test of the main organs of mice showed that JOT-Lip had good preliminary safety.In summary,the MMP-2 enzyme-responsive liposome JOT-Lip was successfully constructed in this paper.After the accumulation of JOT-Lip at the tumor site,Tim-3 mAb and JO-Lip were separated and performed therapeutic effects,respectively.By intensifying DNA damage,amplifying the ICD effect,and dual immune checkpoint inhibition,the immunosuppressive microenvironment of cold tumors is ultimately reversed.In conclusion,this study provides a reasonably designed combination drug regimen and delivery system,which can convert cold tumors to hot and enhance the efficacy of chemoimmunotherapy,providing a new strategy for clinical tumor treatment.