Injectable And Thermally Contractible Hydroxypropyl Methyl Cellulose/Fe₃O₄ For Magnatic Hyperthermia Ablation Of Tumors

PART ? PREPARATION OF HYDROXYPROPYL METHYL CELLULOSE/FE3O4 AND EVALUATIONS OF THE IN VITRO INDUCTION HEATING EFFICIENCYObjective To develop a injectable and thermal response in situ implant using Hydroxypropyl methyl cellulose containing Fe₃O₄?HPMC/Fe₃O₄?,and investigate the optimum mass fraction of Fe₃O₄ in HPMC and evaluate of its heating efficiency in an altering magnetic field in vitro.Methods Five groups of HPMC/Fe₃O₄?50 ?l?containing different amounts of Fe₃O₄ nanoparticles?50%,60%,70%,80% and 90%?were developed in the 10 ml EP tubes with PBS and then exposed them in an altering magnetic field?AMF?for 3min.Their peak surface temperatures were continuously recorded every 20 s by a far-infrared thermometer to make time temperature curve.Different Fe₃O₄ mass fraction of HPMC/ Fe₃O₄ were injected with the same force to evaluate their injectability.When the mass fraction of Fe₃O₄ was 60%?Fe₃O₄,HPMC and PVA mass ratio was 6:3:1?,the heating performance and injection performance were the best.The method that incubated with PBS at 37°C for 25 h to evaluate the dispersion of HPMC/60%Fe₃O₄.Results The in vitro heating efficiency of HPMC/Fe₃O₄ increased with the heating time and the injection dosage.Comprehensive consideration of the heating rate,injection and safety,the HPMC/Fe₃O₄ has higher heating efficiency and better injection performance when the mass fraction of Fe₃O₄ is 60%.HPMC/Fe₃O₄,with a loose structure,its basic elements are carbon,hydrogen,oxygen and iron which are all safe to our body.Then,HPMC/Fe₃O₄ has good anti-washout property for 25 h.Conclusion In this experiment,we have developed a simple,safe,and easy prepared material?HPMC/Fe₃O₄?,which has a higher heat production efficiency in an AMF.The HPMC hydrogel have a better cohesive property which can prevent the leakage of HPMC/Fe₃O₄ particles to the surrounding tissues.PART ? THE THERMALLY RESPONSIVE CONTRACTION AND BIOSAFETY OF HPMC/FE3O4 EVALUATIONObjective To investigate the thermally responsive contraction and the biological safety of HPMC/60%Fe₃O₄ in vitro and in vivo.Methods The prepared HPMC/60%Fe₃O₄ was injected into bovine liver and subcutaneous xenograft?breast cancer?and exposed to the AMF for 2 min,the volume changes of the material before and after heating ware observed by the ultrasound and micro-CT.Macroscopically observation of the contraction changes was administrated in a gel module to make volume-time curve.Four methods to evaluate the biosafety of HPMC/Fe₃O₄: 1)we incubated the materials with normal human SMCs;2)Cytotoxicity based on the MTT assay;3)Flow cytometry evaluation of cell apoptosis;4)Serological indexes detection and inverted microscope observation.Results Ultrasound imagings and CT imagings showed that the volume of HPMC/Fe₃O₄ in fresh bovine liver and tumor was significantly smaller after heating than that before heating.The “tail” of HPMC/Fe₃O₄ implant?caused by various reasons,such as the injection technique,the characteristics of the material and the pressure in the tumor?was disappeared in the CT imagings.After incubation of the HPMC/Fe₃O₄ with normal human SMCs,some of the HPMC/Fe₃O₄ particles?most may be the Fe₃O₄ particles?were engulfed by the cells as a result of the cellular endocytosis and some gathered around the cell membranes,but no cell death was observed.The results of MTT showed that the survival rate of cells decreased with the increase of HPMC/Fe₃O₄ concentration,and the cell survival rate of 60%Fe₃O₄ group was 91.6 ± 2.7%.We can see from the results of FMC and the column chart of the serological indicators,there is no significant statistic difference between the experimental group and the control group,which means that the material was safe.One week after magnetic hyperthermia ablation of tumor with HPMC/Fe₃O₄,no nanoparticles were found in the vital organs such as liver,spleen,lung and kidney?H&E staining?.Conclusion HPMC/Fe₃O₄,is a high biosafety material,can shrink in an AMF,this thermally responsive contraction can avoid potential damage which caused by the "tail" of HPMC/Fe₃O₄ in the ablation process,this performance will greatly enhance the safety of magnetic material.This novel magnetic material HPMC/Fe₃O₄ will further improve the safety to promote the rapid bench-to-bed translation of magnetic-hyperthermia technology,and the developed “thermally contractible biomaterial” is also expected to bring a new concept for the biomaterial research field.PART ? EVLUATION OF THE ULTRASOUND VISUAL AND INJECTABLE HPMC/FE3O4 MAGNETIC HYPERTHERMAL ABLATION OF TUMORSObjective Preparation of different dosage of injectable HPMC/Fe₃O₄ and study the ablation efficiency in bovine liver and xenograft tumor.Methods Five different doses of HPMC/Fe₃O₄?0.02 ml,0.04 ml,0.06 ml,0.08 ml,0.1 ml?were injected along the long axis of the prepared excised bovine liver and then the bovine liver was exposed to an AMF for different time periods?1min,2 min,3 min,4 min,5min?.The ablation range?the distance from the outer edge of the black implant to the outer edge of the necrotic area?of the bovine liver was macroscopically measured using a ruler.The peak surface temperature of the liver was continuously recorded using a far-infrared thermometer.The tumor-bearing mice were randomly divided into three groups.Mice in the treated group were subjected to intratumoral injection of HPMC/Fe₃O₄ and exposure to an AMF with magnetic heating.For comparison,we prepared the following control groups: mice without any treatment,and mice subjected to intratumoral administration of HPMC/Fe₃O₄.A far-infrared thermometer was applied in situ to monitor the temperature changes and acquire thermal images every 10 s.The volume changes of the implant and tumor were monitored by the ultrasound.The in vivo therapeutic efficiency of the HPMC/Fe₃O₄ implant after magnetic field irradiation was evaluated by measuring the tumor volumes.The microscopic therapeutic outcome for the tumors was evaluated using typical H&E staining.After 15 d,the mice in the control groups were euthanized,and the mice in the experiment group were kept feeding to observe the treatment and recurrence of tumor.Results The temperature and the ablation distance of the bovine liver increased with the heating time.The tumor appeared hypoechoic in the ultrasound images,the material was hyperechoic,and the HPMC/Fe₃O₄ implant had obviously contracted after heating for 3 min.The echo of the tumor tissue around the material was higher than that of before heating,which exhibited the ablation process of the tumor.After ablation 14 d,the high echo of the implant and tumor were all disappeared and left a local skin depression.The tumor tissues in the treated group that had received HPMC/Fe₃O₄ followed by magnetic ablation suffered from apoptosis and coagulation necrosis,and their cytoplasm was uniformly stained red.Conclusion Ultrasound visualization of injectable HPMC/Fe₃O₄ can responded to an external AMF and exhibited high efficiency for tumor therapy,which is a minimally invasive,safe and effective technique for magnetic hyperthermia ablation of the tumor.