The Evaluation Of ARG1 Inhibitor Based Injectable Temperature-Sensitive Hydrogel For Amplified Tumor Immunotherapy

In recent years,immunotherapy has become an emerging cancer treatment method because of powerful and long-lasting effects and high specificity.This therapy mainly recognizes and kills tumor cells by activating or reactivating the innate immunity system and specific immune system of organisms.However,immunotherapy still has limitations such as low immune response efficacy and immune-related systemic adverse reactions in clinical application.The initiation of the cancer immune cycle(CIC)is the prerequisite for the anti-tumor immune response to effectively recognize and kill tumor cells.The release of tumor-associated antigens(TAAs)is the initial and extremely critical step in the entire cycle,which can be achieved by administrating immunogenic cell death(ICD)inducers.Therefore,ICD inducers have become a research hotspot in recent years.Studies have found that doxorubicin hydrochloride(DOX)not only can directly kill tumor cells,but also has the function of inducing ICD.After DOX treatment,tumor cells in a dying state release damage-related molecular patterns(DAMPs)and TAAs.Then the CIC and T cell-mediated immune response are activated.DOX,as a classic and potent ICD inducer,was widely used in the research of cancer immunotherapy.However,the clinical application of DOX as an ICD inducer was limited due to its adverse reactions.The intratumoral administration is a strategy to reduce the adverse reactions of DOX.Intratumoral administration,as a more efficient local drug delivery method than intravenous injection,can achieve high concentration and long-term retention of the drug in the tumor site,maximize the anti-tumor effect,and minimize systemic side effects.How to improve the immune response rate has become a research hotspot,because immunotherapy was limited by low response rate in practical application.The metabolic pathways of immune cells and tumor cells in the tumor microenvironments(TMEs)have changed significantly,resulting in the formation of immunosuppressive tumor microenvironments(ITMEs).Targeting the metabolic pathways in ITMEs has become an effective strategy to relieve immunosuppression,which could improve the immune response rate.The metabolism of arginine is critical to the process of T cells activation and proliferation and immune regulation.Arginase 1(ARG1),as an immunosuppressive enzyme,can hydrolyze L-arginine to produce L-ornithine and urea.Degradation of L-arginine leads to a significant decrease in the concentration of L-arginine in TMEs,which affects the expression of T cell receptor-related CD3ζchain and the activation and proliferation of T cells.Therefore,supplementing L-arginine in TMEs directly or indirectly will be an effective way to reactivate the immune response mediated by T cells.Inhibiting the activity of ARG1 has become an attractive strategy for indirect supplementation of L-arginine.L-norvaline is an ARG1 inhibitor with low cost,negligible side effects and good water solubility.However,the application of L-norvaline in the blockage of ARG1 pathway is still unsatisfactory because of high half-maximal-inhibitory concentration(IC50).The high IC50 value means that it is necessary to design a drug delivery system with high drug loading in practical application.Therefore,designing an L-norvaline-based system that effectively blocks the ARG1 pathway is still a huge challenge.Smart injectable hydrogels,as promising biomedical materials,are increasingly used in the field of tumor treatment.This kind of hydrogels are usually injected in the solution form,which would then be converted into gels at the injection site upon either endogenous stimuli(such as enzymes,pH)or external stimuli(such as light,temperature).Smart injectable hydrogels have been investigated for the delivery of different small immunotherapeutic drugs to realize sustained and controlled release of drugs after local injection,reduce the number of administrations,and reduce the immune side effects.Unfortunately,most of these hydrogels only as a reservoir of various therapeutic agents,and therapeutic agents are filled in the water channels of the hydrogel microstructure.Inert hydrogels have no anti-tumor function and immune stimulation ability,which result in high risk of potential toxic-side effects and reduction of drug loading content of therapeutic agents.In addition,the water-soluble therapeutic agents are readily to have a burst release from the hydrogel reservoir because there is usually no strong interaction between the therapeutic agents and the inert gelators.In summary,it is significant to design and develop therapeutic smart injectable gels.For example,the hydrogels constructed by therapeutic agents can hold high drug loading content because the gelators themselves are drugs.The system doesn’t involve inert materials,avoiding potential toxic and side effects.Most importantly,the controlled release behavior can be achieved since the therapeutic agents are not located in the water pools of the hydrogel but in the framework of the hydrogel microstructureBased on the above background,we designed an ideal temperature-sensitive hydrogel system with ARG1 inhibitor as the hydrogel framework,which can not only act as a temperature-responsive drug reservoir but also has an immunostimulatory effect.And then DOX,a model drug to induce ICD,is introduced into the hydrogel carrier to improve the efficiency of anti-tumor immune response and minimize systemic adverse effects.L-norvaline based immunomodulating hydrogel could block the ARG1 pathway and thereby alleviate the ITMEs.DOX,as a model drug,was introduced into the immunomodulating hydrogel(PLN-PEG)formed by L-norvaline polymer to trigger ICD,and L-norvaline slowly released.This gel has achieved continuous regulation for the ITMEs.The main research contents,methods and conclusions of this topic were summarized as follows:We designed and successfully constructed an immunomodulating gelator delivery system loaded with ICD inducer DOX(PLN-PEG@DOX)and performed a series of characterizations.Methoxyl polyethylene glycol amine(Mn=2000)was used as macroinitiator to initiate the ring-opening polymerization of L-norvaline n-carboxyanhydride(NCA),resulting in a diblock copolymer PLN-PEG.The synthesized PLN-PEG was determined by 1H-NMR.The polymerization degree of L-norvaline was 16.0.Fourier infrared spectrum(FTIR)and circular dichroism(CD)were used to determine the secondary structure of PLN-PEG in solid and solution states,respectively.Results indicated the predominant β-sheet conformation for PLN-PEG copolymer molecule.At low concentrations,PLN-PEG self-assembled to form micelles,which were measured by DLS and TEM.The results showed that the particle size of PLN-PEG micelles was around 120 nm.At high concentrations,PLN-PEG could form an injectable hydrogel precursor solution.The PLN-PEG solution could transform into hydrogel when the temperature increased.The vial inversion method was used to measure the phase transition temperature of PLN-PEG to determine the polymer concentration to be used in subsequent experiments.The morphological changes of polymer PLN-PEG in gelatinous state and micellar state after being incubated with proteinase K were observed by SEM and TEM,which proved that PLN-PEG with enzyme responsiveness can realize the responsive release of L-norvaline in TMEs.DOX,a classic ICD inducer,was introduced into the PLN-PEG carrier.The vial inversion method was used to measure the sol-gel phase transition temperature to determine the final preparation ratio(6.0 wt%PLN-PEG,0.8 mg·mL-1 DOX).The microscopic morphology of PLN-PEG@DOX hydrogel was observed through SEM.The SEM image showd preparation displayed a continuous network structure.The in vitro release experiment of the preparation proved that the drug release was faster in pH 6.8 with proteinase K than that in pH 7.4 and pH 6.8 without proteinase K.Degradation and intratumoral retention experiments in vivo proved that PLN-PEG@DOX hydrogel had excellent biodegradability and ability to extend drug release.Melanoma cells(B16F10)were selected as the tumor cell models to evaluate the anti-tumor activity and immune effects of PLN-PEG@DOX hydrogel in vitro.MTT results showed that PLN-PEG@DOX hydrogel and free DOX exhibited similar effects.And their cytotoxicity had DOX concentration-dependent,while PLN-PEG hydrogel was almost non-toxic to cells and had high biocompatibility.Cell apoptosis results indicated that the PLN-PEG@DOX hydrogel caused the cell death mainly through apoptosis and necrosis.ARG1 inhibition experiments proved that both PLN-PEG hydrogel and PLN-PEG@DOX hydrogel exhibited obvious ARG1 inhibitory effects.PLN-PEG hydrogel could produce immunomodulatory effects on the TMEs while reduce the systemic toxicity of immunotherapy.It is a feasible strategy for clinical application of immunomodulatory small molecule drugs.CRT and HMGB1 immunofluorescence images showed that PLN-PEG@DOX hydrogel promoted the release of DAMPs and effectively induced the ICD of tumor cells(B16F10).The above results indicated that PLN-PEG@DOX hydrogel displayed significant anti-tumor activity in vitro and could activate CIC.C57BL/6 female mice bearing melanoma(B16F10 cells)were used to study the anti-tumor effect and mechanism of PLN-PEG@DOX hydrogel in vivo.A bilateral tumor-bearing mice model was constructed,and the results of bilateral anti-tumor effects showed that the combination of chemotherapy and immunotherapy was significantly better than chemotherapy or immunotherapy alone.In addition,the preparation exhibited excellent inhibitory effects on both primary tumors and abscopal tumors.The inguinal lymph nodes and tumor tissues of mice were collected,and the flow cytometry(FCM)results showed that the mDCs proportion in PLN-PEG@DOX hydrogel-treated group was significantly increased.The tumor-infiltrating CD8+and CD4+T cells proportions in primary tumors and abscopal tumors were higher than that in the normal saline(NS)group,and the Tregs percentage was observably lower than that in the NS group.Subsequently,the percentage of T lymphocytes in spleen and the concentration of cytokines in serum were measured.The above results thus clearly verified the PLN-PEG@DOX hydrogel could induce an efficient systemic immune response.In summary,the preparation demonstrated excellent anti-tumor effect on tumor-bearing mice and triggered potent systemic immune response.Moreover,the H&E staining results of the main organs and the change curves of mice body weight proved that the toxicity of the preparation was negligible.The pulmonary metastasis model was constructed,and the experimental results further verified that PLN-PEG@DOX hydrogel had a potent inhibitory effect on tumor cells metastasis to lung tissue.