| Objective:To explore the physicochemical properties of mPEG-PLGA@ZrO2@(DOX+ILS)multifunctional embolic microspheres and the in vitro experimental study of CT imaging,microwave sensitization,and release of ZrO2@(DOX+ILS).Method:(1)To observe the morphology and size of blank mPEG-PLGA microspheres and mPEG-PLGA@ZrO2@(ILs+DOX)microspheres by an optical microscope(OM UB100i)and a scanning electron microscope(SEM,S-4300,Hitachi).The particle size distribution of 100 microspheres was measured using Nano Measurer(particle size analysis software).SEME-EDS was used to determine the type and surface distribution of the elements.(2)To take different concentrations(0,1.25mg/ml,2.5mg/ml,5mg/ml,10mg/ml,20mg/ml)mPEG-PLGA @ ZrO2 @(DOX + ILS)solution into the PE tube,using Siemens dual source CT scans were performed to observe the CT values at different concentrations.(3)Microspheres of mPEG-PLGA@ZrO2@(ILs+DOX)(30 mg)were dispersed in physiological saline and then radiated by MW(1.8 W)for 5 mins;the temperature change was monitored by an infrared thermal imager,and physiological saline served as a control.The internal environment of the human body was simulated.The materials were uniformly dispersed in hydrogel to give a concentration of 5 mg/m L of materials,and then radiated by MW(4 W)for 2 min.These materials were not used in the control group,and the temperature change was monitored by an infrared thermal imager.After ablation,the ablation area of the hydrogel was calculated and a warming curve,1 cm away from the ablation center,was plotted.Changes in ablation area and warming trend in two groups were observed.(4)In the ZrO2@(ILs+DOX)release test,3 mg/m L microspheres were partially incubated at 37℃ and 65℃ for 20 min,respectively,and then centrifuged at 3000 r/min.Centrifuged substrates and supernatants were observed separately under SEM and TEM by sampling.Result:(1)Under an optical microscope and SEM,blank materials(mPEG-PLGA)were found to be regular sphere,with good dispersibility and a diameter of 35–65 μm.According to statistics for the diameter of 100 microspheres,the mean particle size of blank microspheres was 43.1 μm;after being loaded with ZrO2@(DOX+ILS),the morphology and dispersibility of materials were not influenced,but the mean particle size was slightly increased to 48.66 μm.SEM–EDS testing showed that C,Zr,P,F and N in the materials came from the mPEG-PLGA scaffold,ZrO2,1-butyl-3-methylimidazolium hexafluorophosphate and DOXDOX,respectively.(2)Density and CT values were both elevated,with an increase in concentration showing a linear relationship.(3)The temperature in the material group was higher than that in thesaline group,the differences were statistically significant(30±7.49℃,17±5.63℃,P<0.01).After MW radiation,infrared thermal imaging showed a better heating effect and a larger ablation area in the material group;the heating curve 1 cm away from the ablation center confirmed that the materials significantly improved the marginal heating effect of the hydroge(8.51±7.64℃,6.91±1.91℃).Precise measurements of the hydrogel ablation area showed that the ablation area in the material group was increased by 30.6% compared to the control group.(4)At 37°C,the morphology of the microspheres(Fig.2E)showed no significant change,and only a little of ZrO2@(DOX+ILS)was found in the supernatant after release.At 65°C,the microspheres became deformed and broken.Under a TEM,a large quantity of ZrO2@(DOX+ILS)was observed in the supernatant after release,and substances were present that were suspected to be mPEG-PLGA fragments.Conclusion:(1)It was demonstrated that ZrO2@(DOX+ILS)was successfully encapsulated into mPEG-PLGA blank microspheres.(2)In vitro study,mPEG-PLGA@ZrO2@(DOX+ILS)multi-functional microspheres have CT imaging function;(3)In vitro experimental study,mPEG-PLGA @ ZrO2 @(DOX + ILS)and more functional microspheres have microwave sensitization function;(4)In vitro experimental studies,microwave accelerated mPEG-PLGA @ ZrO2 @(DOX + ILS)multi-functional microspheres ZrO2 @(DOX + ILS)particles and doxorubicin release.Objective: To explore the vascular embolization and doxorubicin-releasing function of mPEG-PLGA@ZrO2@(DOX+ILS)multi-functional microspheres in rabbit VX2 liver tumor model.Method:(1)9 tumor rabbits were selected,with tumor size in 1.2 cm in diameter,and they were randomly divided into 3 groups(1 d,6 d,9 d),each group with three.Hepatic artery injection of mPEG-PLGA@ZrO2@(DOX+ILS)microspheres were executed postoperative 1 d,6 d,9 d.Tumor tissue,heart,lung and kidney and spleen tissues were detected by histology and frozen section.(2)Embolization of arterial blood vessels: DSA was used to observe the hepatic arterial embolization in normal rabbits after hepatic artery was injected into multi-functional microspheres;DSA was used to observe the tumor staining in hepatic arterial superselective tumor feeding arteries;CT scan was used to observe the retention of drugs on tumors within 24 hours after operation.(3)Frozen section and HE staining were used to observe tumor inside and outside distribution of DOX fluorescence,using Image J software for semiquantitative analysis(4)Post-operation 1-6-9d,frozen section was used observe to DOX fluorescence in tumor tissue,heart,lung,spleen,kidney,and half quantitative analysis with the Image J software.(5)The frozen sections were used to observe the intracardiac,lung,spleen,kidney,and HE staining of cardiac,lung,spleen and kidney tissues on day 1,day 6,and day 9 after operation.Image J software was used to perform semi-quantitative analysis of fluorescence in each tissue.Result:(1)An arteriography showed that the vascular shadows of the left and right hepatic arteries disappeared,indicating a vascular embolization role for mPEG-PLGA @ZrO2@(DOX+ILS)microspheres.Tumor vascular was embolized with mPEGPLGA@ZrO2@(DOX+ILS)microspheres in VX2 liver tumor rabbits,the blood-supply vessels of tumors were partially embolized by mPEG-PLGA@ZrO2@(DOX+ILS)microspheres;the microspheres became aggregated in the area of the tumors,and the embolic agent was residual.CT imaging revealed that the tumor areas with a density lower than that of liver parenchyma showed a significantly high density after the injection of mPEG-PLGA@ZrO2@(DOX+ILS)microspheres via hepatic arteries.(2)After treatment with novel mPEG-PLGA@ZrO2@(DOX+ILS)microspheres,the microspheres became embolized within tumor vessels,so that a large quantity of DOXDOX was released into tumor tissues;only a small amount of DOXDOX was found within adjacent normal liver tissues.HE staining showed that there were a large number of black particles in tumor tissues.(3)Doxorubicin light showed red light under a fluorescence microscope.A small amount of doxorubicin fluorescence was observed in the tumor tissue on day 1,and a large amount of doxorubicin fluorescence was observed on day 6 and day 9.Semi-quantitative analysis using Image showed that within 6 days of oncology was observed the maximum number of doxorubicin.(4)Postoperative 1 day a small amount of DOX fluorescence can be seen in the spleen,heart,lung and kidney not seen obvious DOX in fluorescence.Postoperative 6,DOX fluorescence can be seen in the heart,lung,spleen,kidney.After nine days,no doxorubicin in the myocardial fluorescence,only a small amount of adriamycin can be seen in the lung spleen kidney fluorescence.(5)After 1 day,6 days and 9 days of treatment,HE staining showed no abnormal changes in the myocardium of lung,spleen and kidney.Conclusion:(1)mPEG-PLGA@ZrO2@(DOX+ILS)microspheres can be embolized vascular.(2)The microspheres became embolized within tumor vessels,so that a large quantity of DOX was released into tumor tissues.(3)Sustained release of DOX by mPEG-PLGA@ZrO2@(DOX+ILS)microspheres.Objective:To explore the therapeutic effects of multi-arterial embolization microspheres with hepatic arterial injection of doxorubicin sustained release and microwave sensitization in the VX2 liver tumor model.Method: Twenty-four rabbits were selected and the tumor size was 1.0-1.5 cm.They were randomly divided into 4 groups with 6 in each group.Group A: blank group without any treatment;Group B: microwave ablation alone;Group C: Hepatic artery administration mPEG-PLGA @ ZrO2 @(DOX + ILS)microspheres(embolization group);Group D: mPEG-PLGA @ ZrO2 @(DOX + ILS)microspheres combined with microwave ablation(embolization + ablation group).Microwave ablation(15w,2450 f,2min)was performed on the rabbits in the microwave group and the microwave + embolization group.The temperature of the ablation center area was recorded in real time using an infrared imager,and the temperature change in the 0.5 cm area of the ablation center was calculated using software.The MR DWI sequence was used to compare the size of the residual volume 24 hours after ablation.Fluorescence microscopy was used to observe DOX immunofluorescence within C group and D group in tumor tissue freezing section and biological electron microsocopy(SEM)was used to observe the cell structure and the distribution of ZrO2 in tumor cells in 24 hours after MWA.Blood was collected from the saphenous vein in a standard manner for analysis into EDTA-containing tubes.We analyzed the following markers: RBC,HGB,HCT,MCV,MCH,MCHC,PLT,and WBC.Serum levels of ALT and AST were used to test for liver function,and serum levels of BUN and creatinine(CREA)were used to evaluate kidney function.Biosafety of mPEG-PLGA@ZrO2@(DOX+ILS)microspheres was evaluated by liver function,kidney function,blood routine examination,body weight.After treatment,tumors were measured in the 1,3,6,and 9 d by CT.Residual tumors were measured after the 1,3,6,and 9 d using 3.0T MRI.The width(a)and length(b)of the tumor were obtained from images.Tumor volume(V)was measured using the formula V=a*b2/2.Histology was evaluated by hematoxylin and eosin(H&E stain)and observed by an optical microscope.Result: The time required for the ablation center area temperature of group D and group B to reach 60°C(5.4±0.89°C,10.4±1.14°C)was statistically different between the two groups(P<0.001).The statistical significance was different.A comparison of mean temperature at the ablation center between two groups showed a statistical difference(111.75± 17.16°C,84.58±17.41°C,P<0.01,).A comparison of mean temperature 0.5 cm away from the ablation center between D groups and B groups showed a statistically significant difference(77.17±16.97°C,47.2±4.57°C,P<0.01,).A comparison of residual tumor size 24 h after MW ablation between D groups and B groups showed a statistically significant difference(0.272±0.052cm3,0.487±0.064cm3,P<0.01,).Twenty-four hours after the operation,a large quantity of DOX fluorescence was found within tumors in the D group and some DOX fluorescence was found within tumors in the C group.BEM of samples from the D group,cells had obvious pyknotic nuclei,thickened nuclear membrane and condensed chromatins.Black electron-dense particles were found in the nuclei,which were ZrO2 nanoparticles 200 nm in diameter.In the C group,cells had an intact structure and irregular nuclei with mitosis(10,000×).One day before the operation,a comparison of tumor size among the four groups showed no statistical difference(F=0.249,P=0.861);at the 3,6 and 9 d after the operation,there were statistical differences in tumor size between the D group and the other three groups(P<0.01);however,no statistical difference was observed between the C and B groups(P=0.349;P=0.235;P=0.806),and there was a statistical difference between the C or B groups,and the control group(P<0.01).Before the operation,there were no statistical difference in tumor size among the four groups(F=0.249,P=0.861).After the operation,in vivoresidual tumor tissues in samples from the D group markedly decreased;at 1,3,6 and 9 d,there were statistical differences in in vivo tumor sizes between the D group and the other three groups(P<0.01),with a statistical difference found between the C or B group,and the control group(P<0.01).In A group,HE staining showed a large quantity of tumor cells without obvious necrosis.In the B group,stained tumor cells were found,in which there were massive necrotic areas.In the C group,stained tumor cells were found,in which necrotic areas were evident.In the D group,no obvious,stained tumor cells were found,but necrotic tumor cells were observed,comparing the liver and renal functions among the four groups.3 days after the operation,aspartate aminotransferase(AST)levels in the C and D groups markedly increased compared with the control and MW groups;the differences were statistically significant(169±12.62;177±12.626,P<0.01).A statistically significant difference between C and D groups(18.62±12.62,P=0.164)was not found.At 6 d,the AST level was significantly decreased;at 9 d,there were no statistical differences among the four groups(F=1.457,P=0.264).3 d after the operation,alanine aminotransferase(ALT)levels in the C and D groups were markedly increased compared with the control and MW groups,with the differences statistically significant(302±73.32;281±73.32,P<0.01).There was no statistically significant difference between the C and D groups(19.80±73.32,P=0.791).After 6 d,ALT levels were significantly decreased;after 9 d,there were no statistical differences among the four groups(F=3.229,P=0.051).After 3,6 and 9 d,there were no statistical differences in BUN and Cr levels among the four groups(P> 0.05).Blood routine examinations of the four groups at 3,6 and 9 d after the operation.3 d after the operation,the white blood cell count(WBC)of the B and C and D groups was markedly increased compared with the control group;differences were statistically significant(F=8.152,P=0.002).After 6 d,WBC was significantly decreased.At 9 d,there were no statistical differences between these three groups and the control group(F=0.217,P=0.883).After the operation,there were no statistical differences in red blood cells(RBC),hematocrit(HCT),mean corpuscular volume(MCV),mean corpuscular hemoglobin(MCH),mean corpuscular hemoglobin concentration(MCHC),and platelet(PLT)and hemoglobin(HGB)levels among the four groups(P> 0.05).Body weight changes of the four groups after the operation.Before the operation and 3,6 and 9 d after the operation,there were no statistical differences(P> 0.05).The body weights of the C and D groups were decreased 3 d after the operation,and nearly recovered to a preoperative level by 6–9 dConclusion:(1)mPEG-PLGA @ ZrO2 @(DOX + ILS)multi-functional microspheres can play a role in microwave sensitization,increase the volume of ablation.(2)Microwaves can accelerate the release of doxorubicin in mPEG-PLGA@ZrO2@(DOX+ILS)multi-functional microspheres.(3)mPEG-PLGA @ ZrO2 @(DOX + ILS)multi-functional microspheres in vivo is safe,non-toxic side effects.(4)mPEG-PLGA @ ZrO2 @(DOX + ILS)assisted microwave ablation,can play a role in embolization,chemotherapy,microwave trinity,greatly improving the efficacy of cancer treatment. |
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