| Background:Hepatocellular carcinoma(HCC)is one of the most common diagnosed malignancy worldwide and the fourth leading cause of death in cancer.Characterized by concealed pathogenesis,over 70%of patients(>70%)are already in advanced stages when diagnosed,which causes the unavailability for surgical resection or liver transplantation.Based on the main blood supply of HCC tissue by hepatic artery,transarterial radioembolization(TARE)is a conventional treatment for intermediate HCC(BCLC B).The anti-tumor effect of TARE is functioned by embolization and brachytherapy of internal irradiation of radionuclide labeled microspheres.Currently,90Y labeled glass microspheres(TheraSphere)and 90Y labeled resin microspheres(SIR-Spheres)have been available for treatment of HCC commercially.Although 90Y has the advantages of high-energy β-rays,ideal radiation radius and half-life,90Y cannot be detected in vivo because of lacking of γ-rays.Glass and resin microspheres are nonbiodegradable and dense,which leads to uneven intrahepatic distribution,infeasibility for multiple treatments and increase in the difficulty of administration.As one of the most commonly used nuclides in clinical practice,iodine-131 has half-life of 8.04 days and radiation radius of 0.9 mm.It emits both high-energyβ-rays for treatment and γ-rays for in vivo imaging.Radiolutetium-177 is another promising nuclide for theranostics and has been approved to be used in the treatment of gastrointestinal,intestinal and pancreatic neuroendocrine tumors.Both 131I and 177Lu are ideal nuclides for preparing radionuclide labeled microspheres.Collagen contains a large number of tyrosine residues for labeling with 131I.Chitosan has been available for developing microspheres and has good biostability.Polydopamine(PDA)is widely used in the biomedical field for strong adhesion properties.These three biomaterials are ideal carriers for the preparation of radionuclide microspheres.Objective:The purpose of this study was to synthesize novel biodegradable collagen-chitosan composite microspheres(CCMs).We also evaluate the feasibility in the treatment of liver cancer by TARE after labeling with 131I.Subsequently,we explored other biodegradable novel micro spheres based on the experience of 131I-CCMs and developed PDA coated chitosan composite microspheres(PDA-CMs).PDA-CMs was then labeled with 177Lu,which laid a foundation for the future treatment of HCC.Part 1:This part was to prepare and characterize of CCMs,as well as investigation of its biodegradability and compatibility in vitro.Methods:(1)CCMs was prepared by emulsification cross-linking method using type I collagen and chitosan.Several parameters were investigated for the optimum protocol of CCMs synthesis.(2)CCMs was characterized by light microscopy,electron microscopy,density analysis,fourier infrared analysis and zeta potential.(3)CCMs was incubated in serum for 12 weeks and cultured with human umbilical vein endothelial cells and liver cancer cells for 3 weeks.The morphology changes of microspheres were observed under electron microscopy at different time points.Results:(1)Composite CCMs had desirable settlement and diameter of 7.5-15 μm(95.4%).Every milligram contains approximate 3.6×105 particles.(2)CCMs showed gradually trend of degradation in serum and almost completely degraded at 12 weeks since incubation.No significant change in morphology of CCMs after culturing in human umbilical vein endothelial cells and HCC cells for 3 weeks.Part 2:This part was to label CCMs with iodine-131 and assess the stability in vitro.In addition,in vivo distribution of 131I-CCMs and the biosecurity were also evaluated.Methods:(1)131I labeling of CCMs was achieved by chloramine T method.The in vitro stability of 131I-CCMs was investigated by comparing the radiochemical purity after incubation in serum and PBS solution for 768 hours.(2)Biodistribution of 131I-CCMs was analyzed by detecting the radioactivity of different organs at 1,3 and 7 days after interventional administration of 131I-CCMs.(3)The heart rate,blood pressure and electrocardiogram(ECG)of rats were monitored for biosecurity under 10 times of the therapeutic dose of CCMs.Pathological analysis of selected organs was also performed for further evaluation of biosecurity.Results:(1)131I-CCMs was successfully developed with high 131I labeling rate and the radiochemical purity of 131I for each micro sphere was 196 Bq.Moreover,131I-CCMs manifested ideal in vitro stability over 768 hours of incubation in serum.(2)After TARE,131I-CCMs was mainly concentrated in the liver,whereas the radioactivity in other organs were quite low.(3)No significant changes in heart rate,blood pressure and ECG were observed after administration of 131I-CCMs.Pathological results showed plenty of CCMs in the hepatic artery without significant pathological changes in adjacent tissues and other organs.Part 3:The purpose of this part was to explore therapeutic efficacy of 131I-CCMs against HCC by means of TARE.Methods:(1)Rats HCC model in situ was established by chemical induction using N-nitroso diethylamine.131I-CCMs were administered by means of TARE for HCC treatment.SPECT/CT imaging was performed to determine the biodistribution of 131I-CCMs in vivo.(2)Therapeutic efficiency evaluation was performed after surgery through the following experiments:MRI imaging,liver function,body weight monitor,monitor of serum γ count,gross observation,pathological analysis,and survival analysis.Results:(1)48 rats models of HCC in situ were successfully established via chemical inducing process.(2)131I-CCMs were diffusely distributed throughout the liver,while no significant radioactivity was observed in other organs.131I-CCMs demonstrated favorable anti-tumor effect and significantly prolonged the overall survival of rats.Part 4:On the basis of 131I-CCMs,this part was to synthesize novel biodegradable microspheres-PDA-CMs with chitosan and polydopamine,and explored the feasibility of labeling with 177Lu as well as stability in vitro.Methods(1)Chitosan microspheres were prepared by emulsification crosslinking method,and then PDA coated chitosan microspheres to form PDA-CMs.(2)177Lu labeling was labeled under the strong adsorption ability of PDA.Afterwards,177Lu-PDA-CMs was placed in PBS solution throughout 14 days for investigation of the stability in vitro.Results(1)PDA-CMs were well spherical and the size of 85%microspheres were between 7.5-20 μm,and microspheres smaller than 2.5 μm in size were not found.(2)177Lu labeling rate of PDA-CMs was 40.58%.After incubation in PBS solution for 14 days,the radiochemical purity of 177Lu decreased from 96.1%to 79.7%.ConclusionTwo novel radionuclide labeled microspheres,131I-CCMs and 177Lu-PDA-CMs,were prepared.131I-CCMs demonstrated high 131I loading efficiency,good biocompatibility and biodegradability,ideal in vitro and in vivo stability.131I-CCMs also showed favorable therapeutic effect against HCC and significantly prolonged OS,suggesting its potential in the treatment of HCC.177Lu-PDA-CMs was also successfully prepared which has good in vitro stability in PBS solution.The preparation and labeling protocol of PDA-CMs will be further investigated for optimized microspheres for TARE of HCC. |
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