Interventional Embolization Of Liver Cancer With Novel Multifunctional Drug-Loading Microspheres Based On Tumor Microenvironment Regulation

Hepatocellular carcinoma（HCC）is a highly prevalent malignancies worldwide,with more than 50%of new cases and deaths occurring in China,which seriously threatens human life and health.At present,surgical excision and interventional ablation are still the preferred treatment for early HCC.However,HCC is characterized by hidden onset,high degree of malignancy and rapid progression.As a result,more than 75%of patients are in the middle and advanced stage when clinically diagnosed,losing the best opportunity for surgical excision.For the patients with advanced HCC,it mainly adopts palliative therapy such as chemoradiotherapy,targeted therapy and interventional therapy in clinic.Among them,the interventional combination therapy based on transcatheter arterial chemoembolization（TACE）has developed rapidly in clinical practice and has become the preferred treatment recommended by different primary liver cancer diagnosis and treatment guidelines,and has gradually developed into a basic treatment strategy for unresectable liver cancer.However,the clinical benefit of HCC patients treated by TACE is still extremely limited in clinical practice,with the objective response rate of tumor only 44%and the short-term recurrence rate as high as 29%,which greatly limits the widespread application of TACE.The basis of TACE treatment is to block the blood supplying artery of the tumor and cut off the nutrient supply of the tumor,and further combine with chemotherapy to play the role of chemoembolization therapy.However,the unique anoxic acid microenvironment created after TACE is closely related to the local progression,recurrence and distant metastasis of the tumor,and is considered to be the key factor affecting the biological process of the tumor.Based on this,how to effectively improve the anoxic microenvironment and acidic microenvironment after TACE is an important strategy to improve its therapeutic efficacy,which will effectively improve the anti-liver cancer ability of TACE,and significantly increase the overall clinical benefits of patients.Drug-loading microspheres are currently commonly used in clinical TACE therapy,compared with other embolic drugs,they have the advantages of high controllability,good reproducibility,precise efficacy and less toxic effects.Currently,the widely used microspheres in clinic are mainly divided into two types:drug-loading microspheres based on Polyvinyl alcohol（PVA）and Superabsorbent polymer（SAP）.Based on this,this study will take drug-loading microspheres as a platform,introduce different drugs into the interventional embolization treatment system,thus endowing drug-loading microspheres with different tumor microenvironment regulation capabilities,and thus play a stronger anti-HCC effect.In this study,we first introduced HIF-2αinhibitor PT-2385 into the microspheres,so that the microspheres could target the inhibition of HIF-2α,thereby improving the tumor hypoxia microenvironment and enhancing the anti-liver cancer efficacy of the microspheres.Secondly,CaCO₃ particles were introduced into the microspheres,so that the microspheres could effectively improve the acidic microenvironment of tumors and have good pH responsiveness.Meanwhile,Erastin,an ferroptosis activator,was further added into the microsphere,which endowed the microspheres with ferroptosis induction ability,thus comprehensively enhancing the anti-liver cancer effect of the microspheres.The main contents of this study are as follows:Part 1:Targeted inhibition of HIF-2αwas used to improve the long-term hypoxic microenvironment of tumors,and PT-2385 was used as HIF-2αinhibitor,and PVA and HA were used as microsphere wall material.Then,the emulsification cross-linking method was applied to achieve efficient co-loading of PT-2385 and DOX,and a novel multifunctional drug-loading microspheres PT/DOX-MS with targeted inhibition of HIF-2αwere prepared.We found that PT-2385 and DOX were efficiently contained in the microspheres with PVA/HA as the wall material,with encapsulation efficiency reaching 83.7±3.07%and 84.3±1.08%,respectively,and their co-loading behavior did not affect their encapsulation efficiency.We further explored the stability and drug release behavior of PT/DOX-MS in PBS（pH 7.4）,PBS（pH 6.5）,and PBS containing 10%FBS.Using PBS（pH 7.4）as the medium,it was found that PT/DOX-MS maintained good stability within 30 days without obvious degradation phenomenon,and both PT-2385 and DOX were able to achieve long-term and effective sustained release,the release amounts were 87.3±6.9%and 61.17±3.22%at 30 days,respectively.It was found that the release behavior of PT-2385 had a certain effect on DOX release.Using PBS（pH 6.5）as the medium to simulate the acidic microenvironment of tumor,it was found that PT/DOX-MS still had good stability under acidic conditions and showed good slow-release performance.After 30 days of incubation,the release amounts of PT-2385 and DOX reached 84.3±4.2%and 68.2±6.1%,respectively.The overall release rate of DOX in PT/DOX-MS was slightly higher than that of DOX-MS.Using PBS containing 10%FBS as the medium to simulate the in vivo blood environment,it was found that PT/DOX-MS still had good stability in the incubation system containing serum,and also showed good slow-release performance.After 30 days of incubation,the release amounts of PT-2385 and DOX reached 84.9±5.4%and 66.63±6.90%,respectively.LM3 and N1S1 cells were used as model cells to study the in vitro pharmacodynamics and mechanism of PT/DOX-MS.It was found that LM3 cells could effectively uptake the DOX released by PT/DOX-MS,and the uptake behavior showed a strong time dependence.The cell viability study found that PVA/HA-MS itself had no obvious killing effect on tumor cells,and had excellent biological safety.In vitro studies on anti-tumor effects found that compared with other microspheres,PT/DOX-MS has the strongest anti-tumor effect,which can effectively induce tumor cell apoptosis and block tumor cells in G2/M phase,preventing the next cell division of tumor cells,thus leading to cell senescence and death.Further studies showed that PT/DOX-MS had strong targeted inhibition of HIF-2α,and could further inhibit VEGF,TGF-α,Cyclin D1 and other cytokines,and synergistic with VEGF inhibition ability and nuclear toxicity of DOX,so as to effectively play the role of anti-liver cancer.We further used RNA-seq to analyze the transcriptome of hepatocellular carcinoma cells treated by PT/DOX-MS,and the results showed that the characteristic regulatory processes involved classical Wnt signaling pathway,histone acetyltransferase complex,transcriptional co-regulatory activity,etc.,whose mechanism is a complex biological process regulated by a variety of signaling pathways.A stable orthotopic HCC model was established in SD rats.Based on this model,interventional embolization was conducted to investigate the in vivo anti-tumor effects,mechanisms and biosafety of different microspheres.In this study,a stable interventional embolization system was established at the animal level,and proved that it could effectively deliver the microspheres to the tumor area and realize the blockage of the tumor blood supply artery.In vivo pharmacodynamic study results showed that PT/DOX-MS showed excellent tumor removal ability compared with other microspheres.Follow-up of six rats treated with interventional embolization by PT/DOX-MS showed that the tumor volume decreased to 35%of the pre-treatment volume 1 week after treatment,and the tumor disappeared in two rats,and also disappeared in three rats after two weeks of follow-up.In the end,only one rat of the tumor was still present after 2 weeks of follow-up,but its volume was also reduced to45%of that before treatment,showing strong tumor clearance ability.Pathological studies also confirmed that PT/DOX-MS group had obvious necrotic cells,and the necrotic area was significantly larger than that of other groups.Meanwhile,obvious lipid deposition at tumor sites indicated that tumor cell metabolism was blocked,and maximum percentage of tumor cell apoptosis was achieved,showing the strongest in vivo tumor inhibition ability.Immunohistochemical results of tumor tissues further indicated that PT/DOX-MS could also effectively target and inhibit the expression of HIF-2α,and then showed a strong inhibitory effect on TGF-α,Cyclin D1 and VEGF.In addition,we also found that PT/DOX-MS interventional embolization therapy did not affect the heart,lung,spleen,kidney and other important organs of rats,and did not cause systemic toxicity of drug-loading microspheres,showing good biological safety.Part 2:In this study,CaCO₃ was introduced into the microsphere to improve the acidic microenvironment of tumors.Gelatin was used as the microsphere wall materials,and Erastin,an ferroptosis activator,was added on the basis of conventional chemoembolization microspheres,thus endowing the microspheres with ferroptosis induction ability.Erastin/DOX@CaCO₃ nanoparticles were first prepared by W1/O/W2 double emulsion method,and Erastin/DOX@CaCO₃ gelatin microspheres（Erastin/DOX@CaCO₃-MS）were further prepared by emulsification-solvent evaporation method.In this study,we thoroughly explored the physicochemical properties,in vitro and in vivo anti-liver cancer efficacy and biosafety of the novel multifunctional microspheres.We first used LM3 cells and Hep1-6 cells as model cells to explore the effects of lactic acid-mediated acid microenvironment on the sensitivity of liver cancer cells to chemotherapy and ferroptosis.Our study confirmed that lactic acid can significantly reduce the sensitivity of liver cancer cells to chemotherapy and ferroptosis.It also indicated that lactic acid can produce protective effects to chemotherapy and ferroptosis on liver cancer cells,and significantly reduce the killing effects of both two drugs on tumor cells.At the same time,we also found that with the addition of CaCO₃,the protective effect of lactic acid was effectively broken,which effectively increased the sensitivity of liver cancer cells to DOX and Erastin,and fully improved the tumor cell killing ability of the two drugs.Our further study confirmed that the expression of ferroptosis markers SLC7A11 and GPX4 in tumor cells was significantly increased after lactic acid treatment,suggesting that lactic acid could inhibit Erastin-induced ferroptosis,and that lactic acid could effectively reverse the resistance of Erastin-induced ferroptosis after rescue by CaCO₃,and the expressions of SLC7A11 and GPX4were significantly decreased.At the same time,Erastin induced decrease in ROS levels after lactic acid treatment was found,while CaCO₃rescue could effectively reverse the ferroptosis resistance of lactic acid.In addition,we further demonstrated that GSH in tumor cells was significantly increased after lactic acid treatment,and its level was significantly decreased after CaCO₃ rescue,which further indicated that lactic acid can increase GSH level and reduce lipid peroxidation,thereby preventing Erastin-induced ferroptosis in HCC cells.In this study,Erastin/DOX@CaCO₃-NPs was prepared by W1/O/W2 double emulsion method,and Erastin/DOX@CaCO₃-MS was successfully prepared by emulsification and solvent volatilization method.It was found that Erastin/DOX@CaCO₃-MS was spherical and rough due to the adhesion of CaCO₃particles on the surface of the microsphere.The particle size was 30.8±0.12μm,the encapsulation rate and drug loading for DOX were 81.37±3.46%and 0.62±0.45%,respectively,and the encapsulation rate and drug loading for Erastin were 97.24±2.14%and 2.33±0.72%,respectively.Our study found that Erastin/DOX@CaCO₃-MS showed good slow-release performance for both Erastin and DOX,and the presence of CaCO₃ made the drug release behavior of the microspheres have a certain pH response.Under the acid condition,Erastin’s release at 15 days increased from 44.16±0.05%under pH 7.4 condition to 50.56±2.55%,and the DOX increased from 30.62±0.03%at pH 7.4 to 65.54±0.03%,and the cumulative release increased by 114%,which was caused by the continuous neutralization of CaCO₃ on the surface of the microsphere with H⁺in the weak acid condition,thus destroying the microsphere and leading to rapid dissolution of drugs in the inner wall of the microsphere.In vitro pharmacodynamic evaluation of Erastin/DOX@CaCO₃-MS was conducted using LM3 and Hep1-6 cells as model cells.It was found that Erastin/DOX@CaCO₃-MS had strong pH responsiveness and could significantly improve the cytotoxicity of DOX in acidic conditions.It was also found that Erastin/DOX@CaCO₃-MS was significantly stronger than other microspheres in the presence or absence of lactic acid,and its cytotoxicity was significantly enhanced in acidic conditions,and the pH response ability of the microspheres was further confirmed.The results of apoptosis study also showed that Erastin/DOX@CaCO₃-MS treated cells had significantly more apoptosis than other groups of microspheres,indicating it had the strongest anti-tumor ability.In the presence of lactic acid,Erastin/DOX@CaCO₃-MS treated cells had significantly stronger killing ability against tumor cells,indicating that Erastin/DOX@CaCO₃-MS could effectively improve the killing ability of tumor cells in the acidic tumor microenvironment.Meanwhile,Erastin/DOX@CaCO₃-MS treatment resulted in cell membrane breakage,mitochondrial atrophy and ridge reduction,which resulted in cell ferroptosis,confirming that the microspheres could induce ferroptosis.In addition,our study further confirmed that CaCO₃-MS itself does not have the ability to induce ferroptosis.Erastin/DOX@CaCO₃-MS treated tumor cells had the most obvious ability to induce ferroptosis in the presence or absence of lactic acid,and their ability to induce ferroptosis was significantly enhanced under acidic conditions,suggesting that its ability to induce ferroptosis is also pH responsive.In vivo pharmacodynamics and biosafety of Erastin/DOX@CaCO₃-MS were studied using a mouse model of subcutaneous tumor transplantation and a rat model of hepatocellular carcinoma in situ.It was found that Erastin/DOX@CaCO₃-MS had good tumor accumulation ability and could stay at the tumor site for a long time,and no fluorescence signal was observed in other organs,which may be related to the effect of CaCO₃ on the in vivo acidic microenvironment of tumors,and enhanced the uptake of drugs by tumor cells.Using Hep1-6 tumor bearing mice as model animals,Erastin/DOX@CaCO₃-MS was used to evaluate the in vivo anti-liver cancer effects of different microspheres by intratumoral injection.It was found that Erastin/DOX@CaCO₃-MS exhibited significantly smaller tumor volume and better tumor inhibition ability than other microspheres.The in vivo embolization of different microspheres was used to evaluate the anti-HCC ability of different microspheres.It was also found that Erastin/DOX@CaCO₃-MS had the strongest inhibitory effect on tumor volume growth and had the strongest anti-tumor effect compared with other microspheres.Ki-67 immunohistochemical staining and TUNEL staining further confirmed that Erastin/DOX@CaCO₃-MS had the strongest in vivo anti-liver cancer effect.In addition,Erastin/DOX@CaCO₃-MS was also found to have a good biosafety profile with no systemic toxicity.In this study,multi-functional drug-loading microspheres of PT/DOX-MS and Erastin/DOX@CaCO3-MS were designed and prepared to regulate the anoxic microenvironment and improve the acidic microenvironment,respectively.Both of two microspheres can effectively achieve the interventional embolization therapy for liver cancer and the long-term slow release of drugs.Furthermore,the efficacy of interventional embolization therapy with microspheres is effectively improved,and the microspheres have good biological safety,which can achieve safe and efficient interventional embolization therapy for liver cancer.