Penetration Of Hafnium Oxide Nanoparticles In Breast Cancer Tumors Based On Synchrotron Radiation Microcomputed Tomography

Objective:Aiming at the problem of insufficient deep penetration of hafnium oxide nanoparticles（HfO₂NPs）as radiation sensitizer,this paper mainly researches four points:First,D-α-tocopheryl polyethylene glycol succinate（TPGS）surface-modified hafnium oxide nanoparticles（s-HfO2 NPs）and unmodified hafnium oxide nanoparticles（l-HfO2NPs）were synthesized and their physical and chemical properties were characterized.Second,the MCF-7 tumor cell sphere model was constructed to simulate the microenvironment of tumor growth in vivo,and the model was used to study the cytotoxicity of NPs.Third,a breast cancer model was established and used for visualization analysis of s-HfO₂ NPs and l-HfO₂ NPs in tumors.Fourth,the penetration depth of NPs in the tumor was quantitatively analyzed and the retention capacity of NPs in the breast cancer tumor was evaluated.Methods:1)Synthesis and characterization of NPs:s-HfO2 NPs and l-HfO2 NPs were synthesized by pyrolysis method and hydrothermal method,respectively,and their physical and chemical properties were characterized.2)In vitro cytotoxicity study:MCF-7 tumor cell sphere model was established in vitro based on the principle of ultra-low adsorption,and monolayer adherent cells and tumor cell sphere were used for cytotoxicity study.3)Establishment of a breast cancer model:18 BALB/c mice were randomly divided into 2groups,group 2 h and group 7 d（three times of 3.7 Gray X-ray irradiation）,with 9 mice in each group.After one week of feeding,20μL（3×10⁷/m L）4T1 cell suspension was inoculated subcutaneously on the fourth pair of breast pads on the left side of the abdominal wall.When the tumor size reached 100 mm³～180 mm³,s-HfO2 NPs and l-HfO2 NPs glucose suspension were injected into the tumor at a dose of 40 mg/m L 50μL.4)Visualization of intratumoral NPs:The penetration of s-HfO2 NPs and l-HfO2 NPs in breast cancer tumor tissues was visualized by synchrotron radiation microcomputed tomography（SR-μCT）.5)Investigation of tumor penetration depth and distribution characteristics:Synchrotron radiation microcomputed tomography（SR-μCT）combined with expectation maximization（EM）algorithm and iterative segmentation method were used to visualize and quantitatively analyze the penetration depth of s-HfO2 NPs and l-HfO2 NPs in breast cancer tumor tissue.Moreover,Inductively coupled plasma-mass spectrometry（ICP-MS）was used to determine the Hf element in the tumor to evaluate the retention effect for one week.Results:1)Physical and chemical properties of NPs:The average particle size of s-HfO2 NPs is2.14±0.06 nm,the average long diameter of l-HfO2 NPs is 53.82±0.32 nm,and the average short diameter of l-HfO2 NPs is 28.66±0.25 nm.2)Cytotoxicity results:The IC₅₀ values of s-HfO2 NPs and l-HfO2 NPs were 447μg/m L and 1356μg/m L,respectively,when using monolayer adherent MCF-7 cells.Toxicity tests using tumor cell spheres showed IC₅₀ values of 1356μg/m L for s-HfO2 NPs and 12618μg/m L for l-HfO2 NPs.3)Establishment of breast cancer model:There was no significant difference in tumor size,and about 88%of tumors could be controlled within the detector after dehydration treatment.4)Visualization of intratumoral NPs:2 h after intratumoral NPs injection,s-HfO2 NPs spread widely throughout the tumor tissue,while the strong absorption signals of l-HfO2NPs in the tumor tissue were mainly concentrated at the tumor injection site.The signals of s-HfO2 NPs and l-HfO2 NPs were significantly enhanced in the deep tumor region after X-ray irradiation.5)Quantitative analysis of permeability and distribution characteristics:The dose ratios of s-HfO₂ NPs in XY,YZ and XZ directions（2200～4400μm）were 26.21%,89.66%and68.12%,respectively,two hours after intratumoral injection of NPs.They were higher than0.76%,63.62%and 42.29%of l-HfO2 NPs.Moreover,at similar imaging volumes,about98.5%of l-HfO₂ NPs were concentrated around 1000μm of the XY tumor volume.These results suggest that s-HfO2 NPs tend to be more concentrated in the central region of the tumor volume than l-HfO2 NPs,whereas l-HfO2 NPs tend to be concentrated in the peripheral region of the tumor.Notably,the pattern of NPs distribution in the tumor changed after three 3.7 Gray X-ray irradiation exposures.The doses of s-HfO2 NPs in the center of the tumor volume 2200～4400μm in XY,YZ and XZ directions were 68.64%,46.96%and79.43%,respectively.The results showed that the distribution of s-HfO2 NPs became more uniform in the tumor volume after X-ray irradiation.The dose ratio of l-HfO2 NPs in the tumor volume center in XY,YZ and XZ directions was 26.56%,80.37%and 46.19%,respectively.The results showed that the distribution of l-HfO2 NPs in the tumor volume was increased after X-ray irradiation.Besides,both NPs have similar accumulation patterns,with both having approximately 65%of the injected dose remaining in the tumor at day 7after intratumoral injection.Conclusion:Compared with l-HfO2 NPs,s-HfO2 NPs is more evenly distributed in breast cancer tumor tissues,with faster diffusion rate and deeper penetration.It was also found that radiotherapy may facilitate the penetration of s-HfO2 NPs and l-HfO2 NPs into tumors.Both s-HfO2 NPs and l-HfO2 NPs had a retention time of at least 7 days in the tumor.