Construction Of Linear Accelerator Neutron Genetator And Application In Tumor Therapy

Background:Cancer is a disease which abnormal cells grow out of control and affect different organs.Cancer can spread to any organs.In 2018,there were 9.6 million deaths in the world caused by cancer.The existing cancer treatment methods include surgery,chemotherapy,radiotherapy,targeted therapy and immunotherapy.To minimize the side effects of cancer treatment,researchers have been exploring many way to replace cancer therapy.On March 11,2020,the world's first BNCT treatment system NeucureTM which is AB-BNCT system(Sumitomo Heavy Machinery Industries-cyclotron)and the BNCT Dose calculation program NeucureTM Dose Engine were officially approved for marketing in Japan.On March 25,2020,Japan's Ministry of Health,Labour and Welfare approved boron drugSteboronine(?).In June 2020,BNCT research center of South Tohoku general hospital formally recruited international patients which has locally advanced or recurrent head and neck cancer that could not be surgically resected.The center completed the second phase of clinical treatment for more than 500 patients.Since discovery of neutron in 1932,fast neutron radiation therapy and boron neutron capture therapy(BNCT)for the treatment of tumors have been under continuous exploration.Fast neutron radiation therapy is a relatively mature field,which has certain clinical effects on locally advanced salivary gland tumors,inoperable bone tumors or soft tissue sarcomas.The BNCT remains a constantly updated frontier field.However,the key technologies restricting the development of these two fields are the development of neutron sources suitable for medical and the effective delivery of boron drugs.ObjectiveTo solve the disadvantages of large nuclear reactors and cyclotrons,such as high cost,high energy consumption and low safety,and provide experimental and data support for tumor neutron therapy technology.The linear accelerator type neutron source was prepared by neutron tube technology for radiotherapy of tumor.Fast neutron therapy was combined with chemotherapy using in situ administration strategy and tumor inhibition was evaluated in vivo.Further boric acid was carried by nanofiber for BNCT with this strategy.It provides new experimental and theoretical support for the treatment of tumor.Research methods:1.Build a miniaturized linear neutron source,measure the stability of the neutron source,calculate and simulate neutron dose distribution in tumor cells.2.The optimal irradiation dose and protective device were determined by cytotoxicity test(MTT),apoptosis test and cell clone formation test.The biological safety of neutron radiation was evaluated by blood biochemical analysis.3.The subcutaneous model of mouse cervical cancer U14 was established to evaluate the tumor inhibition effect of fast neutron radiation therapy by tumor inhibition and immunohistochemical test.4.The dichloroacetic acid(DCA)was macromolecularized by ionization and covalency.The drug was loaded by electrospinning technology to realize the controlled release of DCA in vitro and in vivo.5.The sustained release effect of the drug was measured through drug release and tumor penetration experiment in vitro and in vivo.The effect on cells of modified drug was analyzed by ATP content determination.6.The combined effect of drugs and fast neutrons on cell was determined by MTT and apoptosis experiments.The subcutaneous and orthotopic liver tumors models mice were established to measure the combined effects of chemotherapy and radiotherapy by tumor suppression experiment and immunohistochemistry.7.The in situ administration strategy of BNCT was realized by constructing 10BA loaded nanofiber mat and releasing 10BA in situ.The effect of 10BA was evaluated by MTT,cell clone formation test and cell apoptosis test.8.The efficacy of BNCT under in situ administration strategy was systematically evaluated by establishing mouse H22 subcutaneous tumor model and tumor recurrence model.Research results:1.A 14 MeV neutron therapy system was constructed with a neutron output error of less than 5%.A protective device with polyethylene was designed to reduce the toxicity of neutron radiation to normal tissues and improve the survival rate by 33.2%.The colony formation rates of Hela cells were 35.14%,28.78%,0 and 0 for 1.11 Gy,2.23 Gy,3.34 Gy and 4.45 Gy,respectively.The apoptosis rates were 6.81%,7.97%,8.19%and 10.33%,respectively.In the high-dose group with neutron irradiation of 26.73 Gy,the mean platelet count decreased to 50%of that in the control group.It proved that high dose neutron irradiation had certain toxicity.However,in the low-dose group and the medium-dose group(the total dose was 13.35 Gy and 20.04 Gy,respectively),the blood cell count and blood biochemical indexes were maintained within the range of standard values.2.The tumor growth rates were 19.48,13.63,12.31 and 56.07%in the low dose group,middle dose group,high dose group and control group,respectively.The TUNEL assay indicatied tumors from all the irradiation groups had more extensive regions of apoptosis cells in comparison with those in the control group.The trend of the weight changes of mice clearly exhibited the toxicity to normal tissues and organs caused by neutron irradiation.3.Zeta potential,NMR,XPS and GPC were used to prove the synthesis of two kinds of macromolecular DCA,namely Ion-DCA and Co-DCA.SEM and SEM-EDS proved that the multilayer drug-loading mat was successfully constructed and realized the drug loading.The energy profiles of tumor cell HepG2 have distinct changes after DCA,Ion-DCA,or Co-DCA administration.Combination with FNT significantly increased the apoptosis rate of tumor cells(DCA from 40.49%to 61.5%,Ion-DCA from 54.69%to 62.7%and Co-DCA from 55.4%to 65.66%,respectively).4.At day 19,the tumor volume increased by 6.83,1.78,7.40,1.87,7.65,4.27 and 9.43 times for Co-DCA,Co-DCA+FNT,Ion-DCA,Ion-DCA+FNT,DCA,DCA+FNT and FNT,respectively.On day 19,tumor sizes of the Ion-DCA+FNT and CoDCA+FNT groups were only 11.8%and 10.93%of those of the control group(**P<0.01),respectively.Compared with the Ion-DCA and Co-DCA group without FNT,the tumor volume was also 3.95-fold and 3.84-fold smaller(**P<0.01),respectively.Only little tumor apoptosis was observed in tumor tissues of the control and blank groups.In contrast,the combination of drug-loaded mats and FNT significantly increased the apoptosis of tumor cells.The apoptotic effect of single drug-loaded mats therapy is not as good as that of combined therapy.The treatments with DCA or DCA derivatives and FNT resulted in remarkable decrease in tumor cell proliferation.PCNA and TUNEL confirmed the effect of the combination treatment from the pathological perspective,with increased apoptosis and reduced proliferation of tumor cells.5.The growth of H22 tumors in mice was significantly suppressed by administration of different types of DCA and FNT after 9 days(**P<0.01).After drug-loaded mats implantation and FNT irradiation,the weight of liver and tumor was lighter than that of the control group.The body weight of mice in the treatment groups was decreased in comparison with that in the control group.The survival rate was 20%in the control group,80%in the FNT group,and 60%in the multiple drug-loaded mats in combination with FNT.6.PLA and PEO were used to prepare drug-loaded nanofibers.PLA alone in aqueous solution released less than 70%after 96 h,and about 95%of PEO after 24 h.The PLA80-PEO20 were selected as the drug carrier by adjusting the ratio of PLA to PEO.MTT results showed that the survival rate of HepG2 cells in each group was not significantly reduced after 48 h.The survival rates of HepG2 cells were significantly lower than those in the group without BNCT(**P<0.01).The results of the clone formation experiment showed that the clone rate with the lowest concentration of boric acid(0.0625mg/L)decreased from 86.32%before neutron irradiation to 28.17%after BNCT(**P<0.01).The results showed that the apoptosis of cells in the BNCT group was higher with 97.5%(1 mg/mL),87.8%(0.5 mg/mL),61.7%(0.25 mg/mL),40.0%(0.125 mg/mL)and 17.28%(0.0625 mg/mL),respectively.7.In the H22 subcutaneous tumor,the tumor volume in the BNCT group increased 3.48 times,which showed a significant difference compared with the control group and the 10BA control group(**P<0.01).The body weight of the tumour bearing mice in the 10BA and BNCT groups decreased within 5 days after the treatment.The body weight increase after the irradiation.There was no difference in the body weight of each group in the end.8.The H22 tumor of recurrence model showed a 20-fold increase in the control group and only a 3-fold increase in the BNCT group.The weight of the tumor-bearing mice lost weight after tumor resection.The control group regained and gained weight more quickly and the experimental group did not increase until the seventh day.At the end of the experiment,there was no difference in weight between the two groups.The survival rate results showed that the BNCT treatment group was 100%and the control group was 80%Immunohistochemistry showed that lots of apoptotic cells appeared in the tumor of the BNCT group.The fluorescence intensity was 52.773%.The fluorescence intensity was was only 15.661%in the control group.Conclusion:A neutron therapy system has been constructed.A polyethylene protection de vice as the neutron moderator was designed and connected to the neutron tube to shield normal tissue and organs of the test animals from scatter radiation.It developed a combined therapeutical strategy with multilayered drug-loaded mats and FNT irradiation,for the combination of chemotherapy and radiation therapy of the hepatoma in vivo.Electrospinning technology was used to effectively load 10BA for in-situ administration.The feasibility of in situ administration strategy in BNCT was verified.