Nano Drug Delivery System Based On The Polymer With UCST For Tumor-targeted Immunotherapy

Cancer is one of the leading causes of death worldwide,and its treatment remains a formidable challenge.Currently,the mainstream clinical treatments for cancer are surgery,chemotherapy,radiotherapy,and interventional treatments.However,these methods commonly lead to limited effects and toxic side reactions,which hindered tumor treatment for a time.In recent years,cancer immunotherapy has developed as an additional treatment regimen to achieve significant efficacy by relieving the tumor immunosuppressive microenvironment and stimulating the antitumor immune responses.Unfortunately,tumor cells usually appear with low antigenicity during the immune escape,which makes it difficult for immune cells to recognize tumor antigens and exert the therapeutic effect.Therefore,improving the immunogenicity of tumor cells while activating immune cells may be an effective strategy to achieve satisfactory tumor immunotherapy.Immunogenicity cell death(ICD)usually occurs simultaneously with tumor cell apoptosis,which is accompanied by the release of damage associated molecular patterns(DAMPs).These DAMPs have been identified to facilitate dendritic cells(DCs)maturation and antigen presentation to na?ve T cells,thereby promoting tumor-specific cellular immunity.So,the ICD of tumors is conducive to immunotherapy efficacy.Bovine lactoferricin(Lfcin B)is a widely studied cationic anticancer peptide,which can induce tumor ICD,stimulate lymphocyte proliferation,and promote cytokine secretion.Nevertheless,when administered intravenously,the cationic Lfcin B is prone to be inactivated and lacks tumor targeting ability,which leads to limited efficacy.Stimuli responsive nano drug delivery system is a promising means to avoid these obstacles because it can maintain the effectiveness of drugs,improve drug distribution,achieve controllable drug release,and reduce side effects.Herein,P-(AAm-co-AN)-PEG with upper critical solution temperature(UCST)of43?was synthesized.The cationic Lfcin B was used as the model drug to interact with anionic hyaluronic acid and cationic P-(AAm-co-AN)-PEG to obtain the ternary composite nanoparticles(LHPN).The encapsulation efficiency and drug loading of Lfcin B were 91.3±0.53%and 13.3±0.38%,respectively.The obtained LHPN appeared as a uniformly spherical shape with a particle size of 52.9±1.31 nm and zeta potential of-10.24±0.98 m V.The in vitro drug release behavior showed that the loaded Lfcin B was liberated immediately under the acidic and thermal conditions,indicating the p H and thermal dual-sensitive characteristics of LHPN.CT26 cells were used as model cells to investigate the cytotoxicity.Blank nanoparticles and the hyperthermia treatment employed in this study exhibited good biosafety.Compared with free Lfcin B,LHPN could be retained in CT26 cells for a longer time without degradation or efflux,which was beneficial for exerting a better efficacy.Also,the p H and thermal dual-sensitive characteristic of LHPN at the cellular level was demonstrated.When LHPN was ingested by CT26 cells,Lfcin B could be rapidly released to exert the function under the acidic condition of lysosomes and the stimulation of external hyperthermia.The released Lfcin B could induce CT26 cell apoptosis by disrupting the mitochondrial membrane potential,and the ICD was also elicited.When the cocultured system of lymphocytes and CT26 cells was exposed to LHPN supplemented with hyperthermia,the strongest immune responses of lymphocytes were stimulated.Compared with free Lfcin B,LHPN exhibited better tumor targeting ability in tumor-bearing mice after intravenous injection.The treatment of LHPN supplemented with microwave hyperthermia could inhibit tumor growth by inducing tumor necrosis and reducing neovascularization,and the survival time of mice was also prolonged.Besides,a higher degree of ICD was also presented in the tumor tissue,the in vivo antitumor immune cells and cytokines were significantly increased while the regulatory T cells were declined,indicating the tumor immune microenvironment was improved and systemic immune responses were effectively activated.Importantly,when the CD8~+T cells were depleted,the antitumor and immune-activating effects were significantly diminished,supporting that CD8~+T cells were indispensable during the antitumor immunity induced by LHPN.Negligible changes in body weight and no obvious lesions in all organs suggested the good biocompatibility of LHPN.Tumor cells can release large amounts of adenosine triphosphate(ATP)during the ICD process,which is subsequently metabolized to adenosine(ADO)by ectonucleotidases.The engagement of ADO and ADO 2A receptors(A2AR)on various immune cell surfaces hampers the immune reaction toward tumor cells,further exacerbating tumor immunosuppression.Antagonists of A2AR can block the interaction between ADO and A2AR,thereby alleviating tumor immunosuppression.So,the combined application of ICD inducers and A2AR antagonists provides a method to eliminate the paradox between ICD-induced antitumor immunity and ADO-mediated immunosuppression.The artificial nano drug delivery system exhibits unsatisfied tumor targeting efficiency due to the short systemic circulation time,whereas leukocytes hold great promise as the delivery vehicle for nanoparticles due to their natural tropism to tumors.Based on previous work,thermal sensitive NTA-PEG-p-(AAm-co-AN)with UCST of43?was synthesized,which could self-assemble into micelles in the aqueous medium with a critical micelle concentration of 33.2?g/m L.The hydrophobic ICD-inducer doxorubicin(DOX)and A2AR antagonist SCH 58261 were applied as model drugs to prepare drug loaded micelles(DSM),the particle size and zeta potential were 164.0±7.0 nm and 3.93±0.05 m V,respectively.After introducing E-selectin onto the surface of DSM,the E-selectin modified drug loaded micelles(ES-DSM)were obtained with a particle size of 247.7±15.6 nm and zeta potential of-1.2±0.09 m V.The encapsulation efficiency and drug loading of DOX were 92.9±0.61%and 2.7±0.01%,respectively,while those of SCH 58261 were 41.8±0.97%and 0.41±0.005%,respectively.Moreover,the thermal sensitive in vitro drug release behavior of ES-DSM was also demonstrated,the release rates of drugs were accelerated significantly under the hyperthermia condition.4T1 cells were used as model cells to investigate the cytotoxicity.Blank micelles and the hyperthermia treatment employed in this study exhibited good biosafety.ES-DSM exhibited thermal sensitive drug release behavior at the cellular level.When tumor cells were exposed to ES-DSM supplemented with hyperthermia,the micelles were destructed and the drugs were released immediately.The DOX could effectively kill tumor cells,accompanied by the occurrence of ICD.In the cocultured system with immune cells,tumor cells with ICD could effectively stimulate the maturation and antigen presentation ability of DCs,further promote the proliferation and differentiation of T cells.Besides,in the cocultured systems containing the adenosine mimetic NECA,the existence of SCH 58261 could antagonize the binding of NECA to A2AR on the surface of DCs or T cells,thereby relieving their immunosuppressive state.After intravenous injection in 4T1 tumor-bearing mice,ES-DSM could be delivered to the tumor site by leukocytes,therefore exhibited better tumor accumulation than DSM.Under the stimulation of local microwave hyperthermia,the drugs were immediately released from micelles to induce tumor cell apoptosis and necrosis,thereby inhibiting the tumor growth and spontaneous lung metastases.However,the depletion of CD8~+T cells would diminish the antitumor effect.Moreover,ES-DSM supplemented with microwave hyperthermia could induce a high degree of ICD in the tumor site,which favored the infiltration of DCs and promoted their maturation and antigen presentation.Ultimately,the T cell immune response was activated,the contents of cytotoxic T cells and antitumor cytokines in vivo were significantly upregulated,as were the numbers of memory T cells,whereas the proportion of regulatory T cells was decreased,indicating that the tumor immune microenvironment had been improved and the systemic immune system was effectively activated,thereby preventing lung metastases,inhibiting recurrent and rechallenged tumor growth.Negligible changes in body weight and no lesions in major organs were observed during the ES-DSM treatment,supporting the biocompatibility of ES-DSM.Both of the two stimuli responsive nano drug delivery systems based on the polymer with UCST can achieve tumor targeted drug delivery and controlled drug release,enhance tumor immunogenicity by inducing ICD of tumor cells,alleviate the tumor immunosuppressive microenvironment,stimulate the activation of immune cells,and achieve highly effective and safe tumor immunotherapy.