Synergistic Magnetothermal-chemotherapy For Bone Tumors Based On The Injectable Magnetic Doxorubicin Loaded Polymethyl Methacrylate Bone Cement-Fe₃O₄

PART ? PREPARATION AND DETERMINATION OF MAGNETIC-THERMAL PROPERTIES IN VITRO OF INJECTABLE MAGNETIC DOXORUBICIN-LOADED POLYMETHYL METHACRYLATE BONE CEMENT-Fe₃O₄?DOX/PMMA-Fe₃O₄?Objective : An injectable and rapid phase transitional material DOX/PMMA-Fe₃O₄ was prepared and explored its general physical properties and heating efficiency in vitro.Methods:PMMA powder and Fe₃O₄ magnetic nanoparticles with different concentrations were measured,and then DOX was mixed by mechanical vibration method with a proportion to 1% concentration.Then it was mixed with MMA monomer to prepare DOX-loaded PMMA bone cement containing 2.5%,5%,7.5% and 10% Fe₃O₄?PMMA-2.5c,PMMA-5c,PMMA-7.5c and PMMA-10c?.The fine structure of DOX/PMMA-Fe₃O₄ was observed by scanning electron microscopy,and the magnetic properties of it were investigated by a comprehensive property measurement system at room temperature.Besides the evaluation of the injectivity of DOX/PMMA-Fe₃O₄,the liquid-solid phase transition process was also observed when that was injected into normal saline.The heating characteristics of different proportions of DOX/PMMA-Fe₃O₄ were explored in vitro under an alternating magnetic field.The optimal proportion of DOX/PMMA-Fe₃O₄ was selected and applied in the further experiment.Results:Scanning electron microscopy showed that DOX-loaded Fe₃O₄ nanoparticles were uniformly distributed in PMMA;DOX/PMMA-Fe₃O₄ is a ferromagnetic material with-0.32emug-1 magnetization and 61.5Oe coercivity;liquid DOX/PMMA-Fe₃O₄ can be injected into normal saline and maintain good morphology without disintegration.DOX/PMMA-Fe₃O₄ can produce heat under an alternating magnetic field in saline solution,and the temperature was in direct proportion to the amount of iron and the heating time.The Fe concentration ratio of DOX-loaded PMMA-7.5c was the best and the induction heating efficiency was appropriate and ideal,which could be used in the further experiment.Conclusion:At this part of research,we developed an injectable and rapid phasetransitional material DOX/PMMA-Fe₃O₄,whose Fe₃O₄ nanoparticles uniformly distributing in PMMA,with good solidification and satisfactory magnetism.DOX/PMMA-Fe₃O₄ with a concentration of 7.5% Fe₃O₄ had a appropriate and ideal heating efficiency and could be used in the further in vivo ablation study.PART ? MAGNETIC-INDUCED DOX RELEASE AND EX VIVO BOVINE LIVER ABLATION OF INJECTABLE DOX/PMMA-Fe₃O₄Objective:In vitro and ex vivo bovine liver magnetic-induced DOX release and ablation evaluations of DOX/PMMA-Fe₃O₄.Methods:A total of 100?L DOX-loaded PMMA-7.5c was dropped in a dialysis bag with phosphate-buffered saline?p H 5.0?and then placed in a 45-mlcentrifuge tube with an additional phosphate-buffered saline of the same p H to reach 10 ml.At time points of 0.5 h,2 h,6 h and 21 h,the centrifuge tube was exposed to an alternating magnetic field for 10 s.The efficiency of DOX release was determined using ultraviolet-visible spectrophotometry.Then,the accumulative ratios of the released DOX were calculated as a function of time.The DOX release of phosphate-buffered saline at the same p H without magnetization was assessed with the same method as a control.After an injection of 100 ul DOX-loaded PMMA-7.5c,the bovine liver were placed under an alternating magnetic field,the tissue 1 cm distal from the center of the ablation area were collected for snap-frozen sections.The fluorescence intensity of the inherently fluorescent DOX was then observed via inversed fluorescent microscope to evaluate the effect of magnetic-enhanced the release and distribution of DOX in bovine liver tissues.Different dosages?50?l,100?l,150?l,200?l?of liquid DOX-loaded PMMA-7.5cwas injected into the middle of bovine liver tissue?2 cm×2 cm×2 cm?via syringe.Four pieces of bovine liver were prepared for each dosage.After the liquid DOX-loaded PMMA-7.5c transformed into a solid,the tissue blocks were placed in an alternating magnetic field for 3min,and the temperature variations of the tissue blocks were recorded every 10 s.After magnetization heating for 90 seconds,one piece of bovine liver was selected from the four pieces with the same dosage every 30 seconds,which was cut in half from the center.The average distance of the resected tissue was observed and recorded intuitively with digital photos.Measurements were repeated 3 times,the mean value was taken,and the necrotic volume of bovine liver was calculated.Results:After the magnetization heating,the release percentage of DOX increase from 49% to 88%.With theincrease of time exposed to magnetic field,the fluorescence signal in the ablation center of bovine liver tissue,associated with the DOX molecule,enhanced distinctly.After injection of different doses?50 ?l,100 ?l,150 ?l,200 ?l?of DOX-loaded PMMA-7.5c,with the increase of time exposed to magnetic field,the temperature of bovine liver also rose.At the time point of 180 s after heating,the temperature were28.1 ± 2.8,46.4 ± 5.3,63.4 ± 5.6 and 79.7 ± 3.1,respectively.Meanwhile,the ablation volumes of bovine liver were 0.41±0.08,1.21±0.11,1.89±0.19,4.34±0.38cm³,respectively.Conclusion:The alternating magnetic field can enhance the releasing and dispersing of DOX from DOX-loaded PMMA-7.5c.The increase of bovine liver temperature and ablation volume was positively correlated with the increasing dosage of injected iron and the extension of magnetization heating time.The temperature and necrotic volume of 100?l DOX-loaded PMMA-7.5c were appropriate,and can be used for the further in vivo experiments.PART ? IN VIVO SYNERGISTIC THERMAL-CHEMOTHERAPY OF DOX/PMMA-Fe₃O₄ FOR BONE TUMORSObjective:To observe the effect of DOX/PMMA-Fe₃O₄ synergistic thermal-chemotherapy on tibial plateau graft tumor in nude rabbits.Methods:The preparation method of injectable DOX/PMMA-Fe₃O₄ was followed the aforementioned methods.40 New Zealand white rabbits were selected for establishing the tumor model of VX2 on the tibial plateau.Then40 tumor rabbits with tumor were randomly divided into four groups?10rabbits in each group?as the control group,the chemotherapy group,the magnetic hyperthermia group and the thermal-chemotherapy group according to the different treatment methods.In addition to the magnetic hyperthermia group,100?l DOX-loaded PMMA-7.5c was injected into the tumor model under the guidance of CT?magnetic hyperthermia group was injected only with DOX,while the control group was only injected with the same amount of saline?,and the distribution of bone cement in the tibial plateau of tumor rabbits was observed by CT after injection.The groups were placed in an alternating magnetic field for 90 s expect chemotherapy group and the control group,and the tumor surface temperature was observed by an infrared imager.Immediately,at 7 d,14 d,21 d and 28 d after the magnetic heating,the bone destruction volume of the tibial plateau of all surviving rabbits was calculated by CT images,and the maximum perigenicular diameter of the rabbits was measured by soft tape to evaluate the tumor growth trend.During the observation period,all the dead rabbits were dissected,and at the end of the experiment,all surviving rabbits were executed to evaluate the visceral metastasis.At the same time,the weight of rabbits in each group was recorded.Results:Under the alternating magnetic field,the temperature of tumor surface increased with the extension of heating time in the magnetic hyperthermia group and the thermal-chemotherapy group.Additionally,the mean maximum perigenicular diameter of the rabbits was decreased in the thermal-chemotherapy group,which was increased in the other three groups.Compared with the control group before and after injection,the bone defect volume was significantly and continuously increased in the chemotherapy group and the control group?165.8±6.58mm³and164.8±5.3mm³,401.1±21.62mm³ and 403.0±24.2mm³,respectively,P<0.05?.The weight of the thermal-chemotherapy group was significantly higher than that of the magnetic hyperthermia group,the chemotherapy group and the control group?2.6±0.2 kg,2.4±0.2kg,1.8± 0.3kg,1.7±0.3kg,respectively,P<0.05?.In terms of tumor metastasis,there were 8 rabbits with lung metastasis and 2 rabbits showed kidney and lung metastasis in the control group,these all died in 28 days;there were 6 rabbits with lung metastasis and 1 rabbits showed kidney metastasis in the chemotherapy group,8 rabbits died in 28 days;there were 2 rabbits with lung metastasis in the magnetic hyperthermia group,2 rabbits died,one died of diarrhea,the other from metastasis;and no distal tumor metastasis was found in the thermal-chemotherapy group,2rabbits died of diarrhea in 28 days.Conclusion:As a new magnetic material,DOX/PMMA-Fe₃O₄ can be used not only to deal with orthotopic bone tumors,but also to provide structural support after bone tumor ablation.Compared with single magnetic hyperthermia or chemotherapy,the synergistic thermal-chemotherapy can play a synergistic role.The magnetization heating effect can promote the release of DOX and enhance its cytotoxic,which helps to remove residual tumor after ablation,significantly improving the theraputic effect and reducing the distant metastasis of tumor in rabbits.