Injectable Hydrogel Scaffold Loaded With Small Molecule Inhibitors Blocking The CD47-SIRPα Axis For Postoperative Immunotherapy

In recent years,tumor immunotherapy has gradually become a shining star in tumor treatment.Different from radiotherapy and chemotherapy,immunotherapy does not act on tumor cells directly,but exerts tumor inhibition by activating and mobilizing the immune system and regulating the immune microenvironment.To date,most immunotherapies have focused on stimulating the adaptive immune system to attack tumors,including drugs with immune checkpoint inhibitors(ICIs)CTLA-4 and the PD-1/PD-L1 axis,which have made breakthroughs in the treatment of patients with advanced cancer.It’s worth noting that,ICIs still suffers from low objective response rates and drug resistance.Therefore,immunotherapy targeting immune checkpoints in the innate immune system may provide a new solution,and recent therapeutic strategies targeting macrophages and other myeloid immune cells have brought new hope for tumor immunotherapy.CD47-SIRPα axis is a key regulatory factor of myeloid cell activation and plays a broad role as a specific immune checkpoint.Inhibition of this axis can promote the destruction of tumor cells by macrophages and other phagocytes.Current therapies targeting the CD47-SIRPa axis still face several challenges related to effectiveness and safety:(1)CD47 is expressed on the surface of a variety of cells,including red blood cells(RBC),which causes CD47 monoclonal antibody(mab)to be consumed before it reaches tumor tissues,limiting the efficacy of CD47-targeted therapy in solid tumors;(2)Once the CD47-SIRPα signal between RBC and macrophage is blocked by CD47 mab,it will inevitably lead to hematological toxicity such as anemia and thrombocytopenia.The above limitations have led to a focus on developing new approaches to apply the potential of CD47-SIRPα axis immunotherapy to a wider range of patients in a safer and more controlled manner.Based on this,this thesis designed and developed a novel injectable hydrogel targeting CD47-SIRPα treatment axis of small molecule glutaminyl-peptide cyclotransferaselike protein(QPCTL)inhibitor PQ912 for postoperative treatment of solid tumors through local in situ delivery.Firstly,in this paper,postoperative anti-adhesion agents widely used in clinic were used as basic materials,three kinds of two-component hydrogels formed by multi-arm PEGsuccinimide succinate(2 arm-PEG-SS,4 arm-PEG-SS,8 arm-PEG-SS)and albumin(BSA)were designed and prepared,and their internal structure,drug release behavior in vitro and in vivo and swelling rate were investigated.Comprehensive analysis showed that the in vitro release and equilibrium swelling rate of PB-Gel-4 were between PB-Gel-2 and PB-Gel-8,which ensured the effective and continuous release of drugs without causing organizational oppression due to large swelling of hydrogel.Therefore,PB-Gel-4 was selected as the injectable hydrogel carrier for postoperative immunotherapy and was referred to as PB-Gel in the following text.In order to meet the needs of clinical medication,this study continued to investigate the gelation time,morphology,rheological properties,in vivo degradation time and drug release behavior in vivo and in vitro of PB-Gel.The porous network structure of PB-Gel was observed by scanning electron microscope.The rheological study found that when the strain increased from 0.1%to 10%,the storage modulus(G’)of the sample was always one order of magnitude higher than the loss modulus(G"),indicating that the three-dimensional spatial network structure inside PB-Gel was not destroyed,and the prepared sample maintained the gel state.The degradation of PB-Gel in vivo was monitored by observing the size of abdominal gel blocks,and it was found that the gel was completely degraded in vivo after 9 days of in situ injection.Rhodamine B(Rom)was used as a probe to simulate drug release in vivo.The results showed that compared with physical mixture solution,Rom loaded in PB-Gel(Rom/PB-Gel)could significantly prolong the release time of Rom in vivo.PQ912 was released slowly within 12 h in weakly acidic release medium in vitro,and stabilized after 24 h.Studies on the antitumor mechanism at the cellular level showed that the designed PQ/PBGel had certain immunomodulatory effects:(1)Cytotoxicity test found that PQ912 also showed no obvious cytotoxicity when the concentration of PQ912 reached 50μM(only about 50%inhibition rate),indicating that the antitumor effect of PQ912 did not kill tumor cells directly through cytotoxicity.(2)After macrophages were treated with physical mixture of hydrogel substrates under weakly alkaline conditions(BSA&4 arm-PEG-SS)and PQ912 respectively,the proportion of M1 to M2 macrophages was 1.1 and 1.5 times as that of Untreated group,respectively.After BSA&4 arm-PEG-SS and PQ912 combined treatment,the ratio of M1 to M2 macrophages was 1.7 times as that of the Untreated group,suggesting that the combination of weak alkaline gel matrix and PQ912 could promote the macrophages polarization to M1type.The postoperative recurrence and proximal melanoma models of C57BL/6 mice were established.The postoperative recurrence rate of primary tumor and the size of proximal tumor were used as indicators to determine the optimal time of administration for postoperative treatment.A postoperative melanoma recurrence model of C57BL/6 mice was established.The Untreated group,PB-Gel group and PQ/PB-Sol group were used as control groups to evaluate the efficacy of PQ/PB-Gel against postoperative melanoma recurrence.Compared with the other three groups,mice treated with PQ/PB-Gel showed almost no tumor growth in tumor resection lumen 5 days after surgery,and tumor growth was still effectively controlled 10 days after surgery.After 40 days of tumor inoculation,the survival rate of mice treated with PQ/PBGel could still reach 62.5%,while all mice in other groups had died.On this basis,the regulation of melanoma immune microenvironment and remodeling of melanoma immunosuppressive microenvironment by PQ/PB-Gel were continued to study,and the blood safety was evaluated.The results showed that PQ/PB-Gel treatment could down-regulate the expression of CD47 on tumor surface,reversed the postoperative immunosuppressive tumor microenvironment(TME),and enhanced the function of antigen presenting cells(APCs),thus stimulated T lymphocytemediated antitumor immunity.PQ912 had little effect on the expression of CD47 on RBC and could avoid phagocytosis of RBC by macrophages without hematological toxicity.No necrosis and damage were observed in the main tissues of mice treated with PQ/PB-Gel,which further proved the safety of PQ/PB-Gel.The distal melanoma model of C57BL/6 mice was established and the time when the immune memory function reached its peak after PQ/PB-Gel administration was evaluated using the proportion of immune memory cells in the spleen as an indicator.After local injection of PQ/PB-Gel,the immune memory function of spleen was enhanced in a time-dependent manner,and reached the highest level after hydrogel degradation(day 9).The Untreated group was used as a control group to investigate whether PQ/PB-Gel could inhibit distal melanoma,and whether it was related to the generation of memory cells.It was found that the continuous release of PQ912 indirectly promoted the phagocytosis of tumor cells by phagocytes,indicating that the tumor cells phagocytized at this point could be used as antigens for the in situ vaccine to further activate T lymphocytes and induce memory cells to fight against new tumors.The postoperative recurrence model of breast cancer ectopic tumor in Balb/c mice was established.The Untreated group,PQ/PB-Sol group and anti-PD-1 antibody(aPD-1)group were used as control groups to evaluate the efficacy of PQ/PB-Gel against postoperative recurrence of breast cancer.The results showed that the local injection of PQ/PB-Gel could effectively inhibit the recurrence of "cold" tumor and prolong the survival time of tumor bearing mice,and the survival rate of 60 days after treatment was 75%.Immune mechanism research showed that PQ/PB-Gel significantly down-regulated the expression of CD47 on the surface of 4T1 tumor cells,about 1.6 times lower than the Untreated group.In addition,both PB-Gel and PQ/PB-Gel treatment could effectively improve the infiltration of CD4+and CD8+T cells in"cold" tumors.The antitumor efficacy of PQ/PB-Gel,anti-mouse CD47 antibody(aCD47)and PB-Gel loaded with aCD47(aCD47/PB-Gel)was compared in mouse models of breast cancer and melanoma recurrence.The results showed that there was almost no tumor recurrence in the PQ/PB-Gel group at 21 days after tumor cell inoculation,and the 50-day survival rate was 85.7%.These results suggested that local injection of PQ/PB-Gel had better anti-recurrence effect than intravenous injection of aCD47 and local injection of aCD47/PB-Gel.The pharmacodynamic results in the melanoma recurrence model were similar to those in the 4T1.model.The effect of improving the low lymphocyte infiltration of "cold" tumor of PQ/PB-Gel was significantly stronger than the other two groups.However,PQ/PB-Gel had no significant difference with aCD47 in reducing CD47 expression on tumor cell surface.In addition,the innate and adaptive immune responses induced by PQ/PB-Gel were significantly stronger than those induced by aCD47 and aCD47/PB-Gel.In conclusion,in this study,a reasonable combination of tumor surgery and immunotherapy was achieved by in situ injection of PQ912-loaded two-component TME remodeling hydrogel on the wound surface after tumor resection in tumor-bearing mice.Compared with many CD47-SIRPα axis inhibitors aCD47 reported at present,the use of small molecule inhibitor PQ912 significantly reduced the production cost,relieved the economic burden of cancer patients and was beneficial to realize clinical transformation.The twocomponent TME remodeling hydrogel loaded with PQ912 not only showed outstanding performance in effectiveness and safety,but also had simple preparation and administration methods,which had good practicability.It is expected to become the "successor" of aCD47 in the future and provide a new method for postoperative treatment of cancer patients.