| 【Background】At present,a large number of studies have shown that nanomaterials,as a chemical pollutant in the environment,have become an important carcinogenic factor[1].Zinc oxide nanoparticles（Zn O NPs）,a fine inorganic product with particle size between 1-100nm,exhibit many special properties such as non-migration,fluorescence,piezoelectricity,ability to absorb and scatter UV light It has been widely used in biopharmaceuticals,cosmetics,bioimaging,coatings and other fields.Due to these special properties,Zn O NPs currently used in many fields have entered industrial production,so the toxicity research of nano-zinc oxide has attracted extensive attention[2].Studies have shown that when Zn O NPs are applied topically on the skin surface,the content of zinc ions in the stratum corneum will increase,which can then enter the systemic circulation and urine[3].Airway inhalation exposure is the primary mode of exposure for workers in the chemical,cosmetic or paint industries,with nanoparticles able to reach peripheral airway sites such as bronchioles and alveolar areas.If not carried away by the mucociliary transport mechanism,NPs may affect alveolar cells and cause toxic,genotoxic or inflammatory effects[4],Vermylen et al.[5]argued that inhalation or ingestion of ultrafine structures has profound negative local and Systemic side effects such as increased risk of cardiovascular disease,these very small particles are able to penetrate the tracheobronchial tree.As a typical representative of non-persistent NPs,Zn O NPs have high reactivity,and their special physicochemical properties are easily transformed when they are released into the environment or ingested by animals,thus significantly affecting their toxicological effects.Previous studies by our group found that Zn O NPs underwent physicochemical transitions after aging for 40–120 d in ultrapure water.This shows that in the natural environment,nano-zinc oxide can undergo an aging process.Compared with fresh Zn O NPs,the crystal structure of aged Zn O NPs undergoes a drastic change,transforming from a clear crystal structure to amorphous or sheet-like and showing the formation of new compounds.In our group’s previous in vitro experiments,it was found that aged nano-zinc oxide has weaker cytotoxicity and stronger genotoxicity[6];in nude mouse tumorigenesis experiments,we found that chronic induction of nano-zinc oxide makes MEF cells have the tumorigenic ability of nude mice,and the tumor volume formed in the aging group was significantly larger than that in the fresh group,indicating that nano-zinc oxide is carcinogenic[7].More and more studies have shown that the toxicological exploration of nano-zinc oxide needs to be further explored,and further exploration between aging nano-zinc oxide and tumors is very necessary.According to the latest global cancer statistics,the global cancer incidence and mortality in 2020,in the global cancer incidence,female breast cancer accounted for 11.7%more than lung cancer to become the most common cancer in the world,and the incidence rate among female patients and mortality rates are ranked first[8].Therefore,there are many related studies using different types of nanomaterials to evaluate and study the metastasis of breast cancer.For example,studies have shown that long-term induction of the lung microenvironment in mice using multi-wall carbon nanotubes can promote the metastasis of breast cancer[9];silver nanoparticles can promote the EMT-like transformation of breast cancer cells[10].However,there is no research to explore the effect of nano-zinc oxide on breast cancer metastasis,so we thought,whether the aged nano-zinc oxide discovered by our research group can promote the malignant metastasis of breast cancer?Therefore,we made the following experimental design.First,in order to better observe the metastasis of breast cancer,we selected female Balb/C mice and used non-invasive transtracheal instillation to instill PBS and PBS-diluted fresh or aged Zn O NPs between nano-zinc oxide will release zinc ions.As Zn O NPs release zinc ions in the fluid,we added a control group of zinc chloride to reduce possible experimental errors.After the lung instillation,the mouse breast tumor cell line 4T1-Luci cells with high and stable expression of luciferase were injected into the tail vein,and the tumor metastasis after the influence of different materials was observed by in vivo imaging.【Methods】1.First of all,it is necessary to prepare fresh nano-zinc oxide materials and aged nano-zinc oxide to verify the reliability of the materials,and at the same time,cultivate and verify whether the 4T1-Luci cells have the ability to efficiently and stably express.2.The diluted material was instilled into the lungs of mice by non-invasive transtracheal instillation,once a week for a total of three times.During this period,the body weight of the mice was recorded and the state of the mice was observed.3.The mice were euthanized one week after three pulmonary instillations,and the venous blood of the mice was collected for routine blood examination.4.The m RNA expression of IL-1β,IL-4,IL-10 and other related factors in the lung tissue of mice after three instillations was detected by Q-PCR.5.Detect related gene expression in mouse lung tissue after three instillations using flow cytometry.6.HE staining was used to detect the degree of damage to the lungs of mice by different types of materials,and immunofluorescence was used to detect the expression of Gr1 in the lung tissue of mice.7.After the four groups of mice were instilled with different materials in the lungs,4T1-Luci cells were injected into the tail vein of the mice,and the metastasis of breast cancer cells in the mice was detected by in vivo imaging.【Results】1.The aged nano-zinc oxide was successfully prepared,and its crystal structure was transformed into an amorphous structure and new compounds were formed.2.During the mouse modeling process,the weight loss of the mice in the aging group was the most obvious.3.The results of Q-PCR showed that the expression of inflammatory factors in the lungs of mice in the aging group was stronger.4.The results of HE showed that the lung damage of the mice in the aging group was more obvious,and the results of immunofluorescence showed that the expression of Gr1in the lungs of the mice of the aging group was more obvious.5.5.In vivo imaging results show that instillation of aged nano-zinc oxide in the lungs of mice can accelerate the death of mice with metastatic breast cancer.【Conclusions】1.Zn O NPs can be aged in pure water.2.Compared with fresh nano-zinc oxide,aged nano-zinc oxide has more serious damage to alveolar space,terminal bronchioles and capillaries of mouse lungs.3.Metastatic colonization and growth of 4T1 in an experimental mouse model of metastasis can be accelerated after instillation of aged nano-zinc oxide in mouse lungs. |
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