An Altificially Engineered "tumor Bio-magnet" For Collecting Blood-circulating Nanoparticles And Magnetic Hyperthermia

PART?PREPARATION OF THE ALTIFICIALLY ENGINEERED BIO-MAGNET AND PERFORMANCE EVALUATIONSObjective To prepare the bio-magnet and explore the optimal magnetic properties,basic characteristics,and liquid-solid translation abilities by adjusting varied Nd₂Fe₁₄B concentration.Method Three types of bio-magnet without magnetism-charging were prepared via a mechanical vibration process at the mass ratio of Nd₂Fe₁₄B and Fe₃O₄ was 2.5,4.5 and 6.5.The liquid-solid transformation ability and time was observed by ultrasound imaging.The microstructure of solid bio-magnet without magnetism-charging was detected by scanning electron microscope and mapping scanning.The element content was acquired by Mapping examination.The magnetic force of varied mass fraction of Nd₂Fe₁₄B for bio-magnet after charging were measured by Gauss Tesla meter.Result Three types of bio-magnet without magnetism-charging were prepared successfully.Under the ultrasound and morphology observation,the Nd₂Fe₁₄B/Fe₃O₄-PLGA showing the gel-like characters with good injectability and liquid-solid performance,as well as without disintegration and sputtering after transformation.The ultrasound imaging showed the echo increased of solid form bio-magnet.The average transformation time of three varied Nd₂Fe₁₄B/Fe₃O₄-PLGA were 2.71±0.42 min,2.72±0.39 min and 2.69±0.36 min,respectively.The surface of bio-magnet was stable by SEM results,and the element content was equally distributed by mapping outcomes.In addition,Nd,Fe,C,O element percentage are consistent with content percentage.After magnetism-charging,the magnetic force of Nd₂Fe₁₄B/Fe₃O₄-PLGA were increased with mass content of Nd₂Fe₁₄B growing.What's more,the magnetic force of Nd₂Fe₁₄B/Fe₃O₄-PLGA were increased with its volume growing.Due to the 25%-Nd₂Fe₁₄B/Fe₃O₄-PLGA and 45%-Nd₂Fe₁₄B/Fe₃O₄-PLGA endowed with low magnetic force after charging,they were excluded from the subsequent in vivo and in vitro studies.Conclusion The preparation progress of pre-bio-magnet was distinct and within stable output,as well as accompany with equally distributed elementary.In addition,the bio-magnet with good injectability and liquid-solid performance.After charging,the magnetic force of65%Nd₂Fe₁₄B/Fe₃O₄-PLGA was the highest and can be used for the subsequent in vivo and in vitro studies.PART ? EVALUATION OF THERAPEUTIC PERFORMANCE OF THE ARTIFICIAL BIO-MAGNET FOR MAGNETIC HYPERTHERMIA IN VITROObjective To evaluate the therapeutic performance of the artificial bio-magnet for magnetic hyperthermia in vitro,and provide the evidence for further in vivo study.Method According the protocol from the first part,the liquid form of Nd₂Fe₁₄B/Fe₃O₄-PLGA with varied concentration,different mass ratio,and varied volume were prepared.After liquid-solid transformation,shaped them to ball-shaping,put them into 1.5 m L EP tube and place in the center of the electromagnetic induction-heating coil of a homemade magnetic hyperthermia analyzer.After the exposure to external alternating current??a.c.?magnetic field?,the infrared thermal images of the Eppendorf tube and bio-magnets were taken?time interval: 10 s with a total of 180 s?.The Eppendorf tube containing only saline solution and solid PLGA?75 ?L?were tested under the same condition for the blank controls.The time-temperature curves were made for screen out the optimal Nd₂Fe₁₄B content bio-magnet for further study.The fresh ex vivo bovine livers were taken for the tumor mimic assessment.The liquid bio-magnet was injected into the middle of isolated bovine liver tissue.After the liquid-to-solid transformation of Nd₂Fe₁₄B/Fe₃O₄-PLGA followed by magnetism charging,the tissues containing solid Nd₂Fe₁₄B/Fe₃O₄-PLGA bio-magnet were put into the center of the electromagnetic induction-heating coil and was exposed to a.c.magnetic field for 1 min,2 min,3 min,4 min and 5 min,respectively.The temperature variations in tissues were recorded every 10 s and analyzed by using the thermal images.Coagulative necrosis area after magnetic-hyperthermia ablation was measured and calculated.The coagulative necrosis area of bovine livers was picked to take H&E staining.The time-ablation volume curves was made to evaluate its preformance synthetically.Result The pure PLGA without magnetic materials showed no significant temperature variations?32.3 ± 0.5 °C and 35 ± 1.3 °C?.The 65% Nd₂Fe₁₄B-bio-magnet achieved the highest temperature by the exposure to a.c.magnetic field only for three minutes.The temperature of 50 ?L,75 ?L,100 ?L of 65%Nd₂Fe₁₄B-bio-magnet after 3 min exposure were determined to be 87.3 ± 1.8 °C,104.4 ± 2.2 °C and 125.9 ± 7.1 °C based on the near infrared thermal measurements,respectively.The high temperatures caused significant tissue ablation.The ablation efficiency strongly depends on the exposure duration and adopted content of the bio-magnet.The ablation area with bovine liver were 1.0 ± 0.3 cm3,1.8 ± 0.2 cm3,2.2 ± 0.3 cm3,2.3 ± 0.1 cm3,2.7 ± 0.3cm3 under explore to a.c.magnetic field for 1-5 minutes,respectively.With the exposure time increasing,the coagulative necrosis area was getting larger.This results would be a indicator for seletcting the treating time.Conclusion The presence of bio-magnet could effectively and quickly increase the temperature of surrounding microenvironment,showing the magnetic thermal property of bio-magnet was controllable.Depanding on these in vitro results,the theraputic time for tumor could be choosen by there volume.PART ? EVALUATION OF TARGETING OF THE CIRCULATING MNPS BY THE ALTIFICIALLY ENGINEERED BIO-MAGNET IN VITRO AND IN VIVOObjective To evaluate the targeted performance of the circulating MNPs by the bio-magnet in vitro and in vivo,as well as explore the advantage of bio-magnet for active targeting in situ.Method The blood vessel model was prepared.According to the first part,65%Nd₂Fe₁₄B/Fe₃O₄-PLGA pre-bio-magnet was prepared and endowed by charging with a magnetizing apparatus.The targeted performance of the circulating magnetic nanoparticles by the bio-magnet was evaluated by the digital photos,fluorescence imaging photos and magnetic absorption range.The SMMC-7721 human liver cells were cultured,and the nude mice bearing the SMMC-7721 xenograft were established.The nude mice were divided into four groups when the tumor volume reached 1±0.1 cm3,including saline as group A,pure PLGA as group B,Nd₂Fe₁₄B/Fe₃O₄-PLGA without magnetizing as group C,Nd₂Fe₁₄B/Fe₃O₄-PLGA bio-injection followed by magnetism charging as group D.Under the ultrasound guidance,the different contents(saline,PLGA,65%Nd₂Fe₁₄B/Fe₃O₄-PLGA and 65%Nd₂Fe₁₄B/Fe₃O₄-PLGA)were injected into the tumor with same volume.Then,the fluorescent superparamagnetic nanoparticles were injected via intravenous administration for different durations?24 h,48 h and 72 h?,and the intensities were recorded for quantitative analysis.The mice were sacrificed after in vivo fluoresce imaging,the bio-magnet was removed from the tumor before cutting the tumor tissues into slices for Prussian-blue staining at different time points?before injecting,after injecting 24 h,48 h,and 72 h?,in order to observe the targeting efficiency of MNPs.Result As the blood vessel model targeting examination showed,the MNPs was collected around the bio-magnet.The magnetic-induction line and magnetic absorption range were clearly observed.As the fluoresce imaging shown,the tumor bio-magnet could effectively collect MNPs from the blood circulating to obtain the active targeting system.The fluorescent intensity increased significantly??11.36 ± 2.46?×108?compared to the group without magnetic targeting??1.94 ± 0.86?×108?after 48 h,continuing to 72 h.In addition,Prussian-blue staining of the tumor before and after intravenous administration of MNPs?24 h,48 h and 72 h?shows substantially enhanced Prussian blue-stained tumor tissues could be identified because of the high accumulation of MNPs into tumor.Conclusion The in vitro experiments demonstrated that the bio-magnet with the high magnetic-collecting efficacy of MNPs targeting.The in vivo experiments indicated that the synthesis of tumor bio-magnet was minimally invasive.After magnetism-charging,the active targeting force for bio-magnet was significantly higher than other groups.PART ? EVALUATION OF BIOSAFETY OF THE ALTIFICIALLY ENGINEERED BIOMAGNETObjective To explore the biosafety and bio-degradability of 65%Nd₂Fe₁₄B/Fe₃O₄-PLGA bio-magnet magnetic bone cement in vivo,and provide experimental basis for in vivo therapeutic application.Method According to the previous experiment results,65%Nd₂Fe₁₄B/Fe₃O₄-PLGA was the mass ratio for further therapeutic experiment in vivo,using it for biosafety and bio-degradability study.The as-prepared liquid 75 ?L bio-magnet were subcutaneous injected into Balb/C mice,which were then fed under the guidelines approved by the Chongqing Medical University.The venous blood samples were collected at pre-injection,after injection bio-magnet the fourth week and the eighth week,respectively.The blood and serum samples were used to explore the effects of bio-magnet on hematopoietic system and liver and kidney function,which included white blood cell?WBC?,red blood cell?RBC?and platelet?PLT?counts,alanine aminotransferase?ALT?,aspartate aminotransferase?AST?,creatinine?CR?,blood urea nitrogen?BUN?,creatine kinase?CK?and lactate dehydrogenase levels?LDH-L?.After the biodegradation was completed,the mice were sacrificed,and the main organs?heart,liver,spleen,lung and kidney?were harvested for pathological analysis including hematoxylin and eosin,as well as Prussian blue staining.The biodegradation process was monitored every day until two months feeding.Result There was no significant difference in the results of all hematological and serological examinations before and 1 mouth,2 mouth after injection.The implanted 75 ?L bio-magnet was biodegradable as it was metabolized after the implantation for two months in mice.The Prussian-blue staining results showed that the degraded products were found in the liver and spleen of the mice.The histocompatibility evaluation results based on H&E staining exhibited no obvious pathological changes of the main organs after two months feeding.Conclusion The bio-magnet had no significant influence for blood system,liver function,kidney function and immune system,and also without damage for major organs?heart,liver,spleen,lung,kidney?.These results clearly demonstrate the bio-mangent with good biosafety and biocompatibility.PART ? ULTRASOUND GUIDANCE OF BIO-MAGNET FOR MAGNETIC THERMAL ABLATION OF HEPATOCELLULAR CARCINOMAObjective To prepare the artificial “tumor bio-magnet” to endow magnetic hyperthermia for SMMC-7721 hepatocellular carcinoma by ultrasound guidance.Method According to the results of the above four parts,the 65%Nd₂Fe₁₄B/Fe₃O₄-PLGA is the optimum ration of Nd₂Fe₁₄B containing.The nude mice bearing the SMMC-7721?n=5?xenograft were injected with certain amount of bio-injection?75 ?L?in the center of the tumor under the guidance of ultrasound imaging after anesthesia.The mice were transferred to the center of heating coil for magnetic-induced hyperthermia ablation after anesthesia.The a.c.magnetic field was directly acted at the tumor site for 3 minutes.The tumor volume was monitored before and after magnetic hyperthermia for 5 d,10 d and 15 d,and the tumor image was taken at the selected time intervals.At the end of the experiment,all of mice were euthanized using appropriate approved methods.The mortality a were calculated.Result The SMMC-7721 xenograft models were established.The bio-magnets were injected into tumors under the ultrasound guidance.The tumor could be almost completely treated after the magnetic hyperthermia without obvious reoccurrence.The survival rate of the treatment group was significantly improved compared with that of the control group,and the vital signs of the tumor-bearing nude mice were more stable than other groups.Conclusion The bio-magnet showed the high magnetic-hyperthermia performance of the implanted bio-magnet for tumor treatment.In addition,the whole procedure is convenient,which endow the bio-magnet a strong possibility for future clinical translation.