Experimental Study On Programmed Targeting Effect Of Surface-modified ZrO₂ Nanocomposite On Mitochondria Of Hepatoma Cells

Objective:Hepatocellular carcinoma?HCC?is one of the most serious medical concerns that threaten human health and life.In particular,the treatment of unresectable liver cancer still remains a difficult and hot topic.Thermal ablation is the first-line solution for unresectable liver cancer.Microwave ablation is more and more widely used in clinical because of its advantages of shorter treatment duration,greater thermal range than radiofrequency ablation,and no heat sink effect.However,Microwave ablation for large tumor is not complete;tumor recurrence after treatment still limits the clinical success of this treatment.Improve the accuracy and completeness of thermal ablation treatment for liver cancer is of great significance.Mitochondria are the powerhouse of cells also a key target for apoptosis of cancer cells,and play an important role in the study of tumor thermal therapy.With the development of nanomedicine,there is a great prospect for the targeted delivery of dr?gs due to the surface modification property of nanomaterials.In this study,hollow mesoporous nanosized zirconia?ZrO₂?was used as a carrier,take advantage of the hollow structure of ZrO₂ to loaded microwave sensitizing dr?g ionic liquid?IL?,tripHenylpHospHine?TPP?and cyclic RGD peptide were attached as targeting Ligands,to synthesize Nanocomposites?MZCNs?with mitochondria-targeting function of tumor cells,and to investigate the mitochondrial targeting ability of tumor cells and the effect of targeted microwave hyperthermia on liver cancer.Methods:Nanosilica spheres were used as templates to synthesize hollow mesoporous nano-sized zirconia by etching.Ionic liquids loaded in the hollow structure of ZrO₂ Nanoparticles.Mitochondrial targeting molecule TPP and tumor targeting peptide iRGD were modified on the surface of ZrO₂ by EDC.NHS reaction.The morphological structure,particle size distribution and surface molecules of the synthesized MZCNs were characterized by Scanning electron microscopy?SEM?,Transmission electron microscopy?TEM?,Fourier transform infrared spectroscopy?FTIR?,dynamic light scattering instrument?DLS?and ZETA potentiometers.The MZCNs were dispersed in PBS and PBS was used as a control.A thermal infrared imager was used to monitor the microwave heating effect of MZCNs.By incubating MZCNs at different concentrations with HepG2 hepatocarcinoma cells,the cell suspension was heated by microwave and the temperature of HepG2 suspension was recorded by thermal infrared imager.After labeled with fluorescein doxorubicin hydrochloride,dual-target?MZCNs?,non-target?IL@ZrO₂?and mono-target nanoggents?IL@ZrO₂-TPP,IL@ZrO₂-iRGD?were incubated with HepG2hepatocarcinoma cells.The cells were stained with DAPI and Mito-Tracker green.The target ability of MZCNs to mitochondrial of HepG2 cells was observed by fluorescence microscopy.The cytotoxicity of MZCNs was detected by CCK-8 assay,and the cytotoxicity of Mitochondrian target hyperthermia on HepG2 hepatoma cells was detected by flow cytometry.BALB/C tumor-bearing mice model was established by injecting H22 mouse hepatoma cells.MZCNs were injected via tail vein of mice.The concentration of Zr⁴⁺in tumor at different time points were analyzed by inductively coupled plasma atomic emission spectrometry?ICP-MS?to calculate the concentration of the nanoagents.Tumor-bearing mice were used to analyze the targeting effects of MZCNs,IL@ZrO₂,IL@ZrO₂-TPP and IL@ZrO₂-iRGD which modified by different target ligands.The BALB/C mice model was established and MZCNs,IL@ZrO₂,IL@ZrO₂-TPP and IL@ZrO₂-iRGD were injected at the same concentrations.8 hours after injection,the mice were treated with microwave?2W,5min?.Temperatures during the treatment were monitored by thermal infrared imager.The growth of tumor and the weight of mice were recorded after treatment to observe the effects of dual-targeted,non-targeted and mono-targeted nanoagents on microwave heating and the mitochondrion target microwave thermal therapy.Results:1.The synthesized MZCNs has a diameter of 158.6±6.9 nm and a hydrated size of about 216 nm,with good uniformity of size distribution and good stability.target ligands TPP and iRGD was successful attached.In vitro microwave heating experiment the temperature of MZCNs group was 9.6?higher than the control group.After incubated MZCNs with cells,the cell suspension was heating by microwave,the temperature was higher than the control group,and showed a positive correlation with the concentration:Temperature is 42.8°C with MZCNs concentration of 100?g/mL,44.8°C with MZCNs concentration of 200?g/mL and control group is 40.7°C,after 20 minutes microwave irradiation.By analyzing the intracellular fluorescence intensity of the dual-target group,non-target group and mono-target group,it was found that the intracellular fluorescence intensity of MZCNs group was the highest,which was 2.24 times of the non-targeting group.Moreover,there was a strong correlation between dr?g distribution and mitochondria in the MZCNs group?Pearson's r=0.859,p<0.05?,but no correlation in the non-target group?Pearson's r=0.381,p<0.05?.In vivo experiments,the result of zirconium content in the tumor tissue proved that MZCNs began to accumulate in tumor tissue 4 hours after injection,the concentration get to a peak at 8 hours,and began to decline at 12 hours.The concentration of nanomaterials in mice injected with MZCNs was 2.7 times that of the non-target group and 1.9 times that of the TPP mono-target group.The results of CCK-8 showed that MZCNs had no significant inhibitory effect on HepG2 hepatoma cells in the concentration of 10?g/mL to 200?g/mL.The rates of necrotic/apoptotic cells in the different treatment groups were assessed by flow cytometry:control group treated with MZCNs but no microwave irradiation,2.7%;ZrO₂,15.7%;IL@ZrO₂,18.8%;IL@ZrO₂-iRGD,49.4%;IL@ZrO₂-TPP,47.8%;MZCNs,61.9%.5.Analysis of the microwave ablation temperature of the tumor site recorded by the thermal infrared camera:The maximum temperature of the MZCNs group was 56.4°C.,which was 4.3°C.higher than that of the microwave group alone and 3.1°C.higher than that of the non-target group and higher than that of the IL@ZrO₂-TPP group 2.3°C,1.1°C higher than the IL@ZrO₂-iRGD group.The area of the tumor in the ablation picture at temperatures above 52°C was analyzed;in the MZCNs group it was about 2.2 times more than in the non-target group.On the 14th day of observation,the tumors in the MZCNs group remained necrotic due to microwave treatment;in the normal saline+microwave treatment group,tumor recurrence was found on the fourth day after treatment.1989 and 1709 mm³ in the NS+microwave group and the IL@ZrO₂+microwave group,respectively,whereas IL@ZrO₂-TPP+Microwave treatment group and IL@ZrO₂-iRGD+microwave treatment group tumor volume were 1082 and 1035 mm³.The mean progression-free survival time was analyzed in each group:control,2±0 days;NS+microwave treatment,6±1.4days;IL@ZrO₂+microwave treatment,9±1.09 days;IL@ZrO₂-iRGD+microwave treatment,10±2.67 days;IL@ZrO₂-TPP+microwave treatment,10±1.6 days;MZCNs+microwave treatment,14±0 days.Mice were weighed every other day after microwave treatment,and no significant body weight loss was observed in the 5 groups injected with nanoagents.No significant tissue damage or inflammatory response was observed in hematoxylin and eosin?H&E staining?sections of the heart,liver,spleen,lung,kidney and tumor.In the control tumor tissue,mainly deep-stained irregularly shaped cancer cells can be seen.However,necrotic cells accounted for the majority of stained cells in the necrosis tumor of MZCNs plus microwave ablation group.Conclusion:In this study,we successfully synthesized MZCNs,a novel drug delivery system that has the potential of targeting mitochondria in tumor cells.MZCN has good dispersibility and microwave heating performance.MZCNs increase the maximum microwave temperature and enlarge the heating range.MZCNs have the targeting of tumor cells mitochondria;the tumor content in vivo is 2.7 times that of non-targeted nanomaterials.And in vitro and in vivo experiments have confirmed that MZCNs mitochondria-targeted microwave heat therapy enhanced the effect of microwave on tumor,controlled the growth of tumor and prolonged the progression-free survival of the animal model of liver cancer after microwave treatment.