Fabrication Of Innovative Bioreactors For Rational Cancer Combination Therapy

As negatively impaired by hostile tumor microenvironment(TME)and other factors,the treatment effectiveness of most currently used cancer therapeutics(e.g.immunotherapy,radiofrequence ablation therapy)is far from statisfactory.In recent years,tremendous biomaterials enabling direct cancer cell killing via innovative action mechanisms or TME modulation has been elaborately fabricated to achieve more efficient cancer treatment.Enzyme as a type of natural catalyst in liveinig things palys an irreplaceable role in maintaining the normal metabolism,and its abnormal expression alway results in severe diseases.Apart from being explored for dignosising diseases by utilizing their high effective and specific catalytic capacity,diverse enzymes have shown great promses to enable effective treatment different diseases including cancer.However,as a type of biomacromolecules,enzyme is prone to be denatured and thus lost its catalytic capacity during the isolation process and/or the inappropriate working environment.Therefore,it is urgent to develop innovative strategies to retain the catalytic activity of enzymes outside their producing sites for promoting the advance of enzyme-based cancer treatments.In this doctoral thesis,we have designed several enzyme-based bioreactors by according to the previous experience on biomaterials and enzyme immobilization for innovative cancer treatments.Additionally,the effects of these bioreactors on the TME modulation and cancer treatment enhancement has also clearly elucidated.The main research contents and results of the full text are summarized as follows:1.In situ fixation of PLA1 and COD to enable catalytic cancer treatment via enzymatically destructing cancer cell membrane and amplifying tumor oxidative stress.In this work,a thermal-responsive hydrogel composed of chitosan and β-glycerol phosphate is prepared and utilized for in situ immobilization of phospholipase A1(PLA1)and cholesterol oxidase(COD).It is shown that PLA1 can destruct the integreity of the cancer cell membrane by hydrolyzing phospholipids,the main building block of the cell membrane,and thus promote the release of cholesterol,which would be converted to cytotoxic 4-choleston-3-one and H2O2 with COD.As the result,in situ fixed PLA1 and COD would work together to inhibite the growth of CT26 tumors in mice via the combined cell memberane destruction and oxidative stress amplification.2.Tumor-killing nanoreactors fueled by tumor debris can enhance radiofrequency ablation therapy and boost antitumor immune responses.In this work,we prepared a type of tumor debris fueled tumor-killing nanoreactors(HLCaP NRs)by encapsulating both LOXs and hemin with PLGA via the CaCO3 assisted double emulsion process.We show the HLCaP NRs with pH-dependent catalytic capacity can continuously produce cytotoxic lipid radicals via the lipid peroxidation chain reaction using cancer cell debris or linoleic acid as the fuel to induce ferroptosis.Upon being fixed inside the residual tumors post RFA,HLCaP NRs exhibit a suppression effect on residual tumors in mice and rabbits by triggering ferroptosis.Moreover,treatment with HLCaP NRs post RFA can prime antitumor immunity to effectively suppress the growth of both residual and metastatic tumors,particularly in combination with immune checkpoint blockade.3.Engineering bioluminescent bacteria to boost photodynamic therapy and systemic anti-tumor immunity for synergistic cancer treatment.In this work,we utilize the bioluminescence emitted from engineered luciferase-expressing Luc-S.T.△ppGpp in the presence of D-luciferin as tumor-implantable light source to sustainably activate photosensitizer Ce6 to produce cytotoxic singlet oxygen.It is uncovered that such LucS.T.△ppGpp boosted PDT could not only induce tumor cell death and thus promote the release of tumor associated antigens(TAA),calreticulin(CRT),high mobility group box 1(HMGB1).Additionally,such treatment can also kill some bacteria to release pathogen associated molecular patterns(PAMPs).As the result,such treatment can activate potent innate and adaptive antitumor immunity to synergistically inhibit the growth of different treated tumors at greatly improved therapeutic efficacies over traditional PDT and bacteria besed cancer treatment.With its high efficacy in priming antitumor immune response,our Luc-S.T.△ppGpp boosted PDT can also effectively suppress tumor metastasis and prevent tumor recurrence.In summary,in this doctoral thesis,we have developed three multifunctional bioreactors with well protected and retained catalytic performance and clairufy their pontencies in TME modulation and cancer treatment enhancement.Based on these innovative bioreactors,we have developed three dinstinct rational cancer combination treatments at significantly improved therapeutic effectiveness and safety.Furthermore,these studies collectively highlight that these enzymes based cancer treatments hold great promise for future clinical translation.