Skin safety evaluation method on nanometer titanium dioxide based on HaCaT cells

A nano-titanium dioxide, safety evaluation technology, applied in biochemical equipment and methods, color/spectral property measurement, microbial determination/inspection, etc., can solve problems such as complex methods

Inactive Publication Date: 2018-03-02
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
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Problems solved by technology

The method can quickly and accurately find the key metabolites related to the disorder of the metabolic network induced by titanium dioxide nanoparticles, but the method needs to analyze the changes and statistical analysis of multiple metabolic pathways, the method is complicated, and cannot directly reflect the safety assessment results

Method used

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  • Skin safety evaluation method on nanometer titanium dioxide based on HaCaT cells
  • Skin safety evaluation method on nanometer titanium dioxide based on HaCaT cells
  • Skin safety evaluation method on nanometer titanium dioxide based on HaCaT cells

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Experimental program
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Embodiment 1

[0105] Cell culture: HaCaT cells were cultured in DMEM complete medium containing 10% fetal bovine serum, 1% penicillin and streptomycin, and the culture environment was 5% CO 2 and a 37°C constant temperature incubator. The medium was changed every day, and the cells in the logarithmic growth phase were taken for experiments.

[0106] Preparation of nano-titanium dioxide suspension: Accurately weigh 5 mg of nano-titanium dioxide, add 1 mL of LPBS to mix, sonicate for 5 min, and dilute to 10, 50, 100, 500, 1000 μg / mL with DMEM medium for later use.

[0107] Nano-titanium dioxide phototoxicity experiment: take HaCaT cells in the logarithmic growth phase, add 0.25% trypsin to digest, cell suspension 1x10 5 / mL, 100 μL per well was added to a 96-well plate, and cultured in an incubator for 24 hours. Nano-titanium dioxide was diluted with DMEM at different concentrations of 10, 50, 100, 500, and 1000 μg / mL, and 100 μL was added to each well in sequence. At the same time, DMEM cu...

Embodiment 2

[0110] Changes in the expression level of α-2,6 sialic acid in cells: HaCaT cells in the logarithmic growth phase were taken at 3x10 per well 6 The cells were added to 6-well plates containing sterile coverslips and cultured for 24 h. The HaCaT cells were treated with nano-titanium dioxide DMEM medium at a concentration of 0.50 μg / mL for 21 hours. Wash off the supernatant, add PBS, treat with 365nm ultraviolet light for 1 hour, replace PBS with DMEM medium, and incubate for another 2 hours in the incubator. Then discard the supernatant, wash 2-3 times with PBS, fix the cells with 4% paraformaldehyde for 15 minutes, then add 10 μg / mL FITC-labeled SNA and incubate for 1 hour, and wash off excess dye with PBS. DAPI was added for staining for 15 minutes, and excess dye was washed with PBS. Transfer the coverslip to a glass slide, drip anti-fluorescence quencher between the cells and the slide, and then seal the slide with neutral resin. Air-dry with neutral resin, that is, it c...

Embodiment 3

[0113]Changes in the expression level of sialic acid in cells are affected by the ROS produced by nano-titanium dioxide under ultraviolet light: after the cells are treated with 0.50 μg / mL nano-titanium dioxide and ultraviolet light, 10 μmol / L DCFH-DA is added, and the incubation is continued for 30 minutes. After washing 2-3 times, the intracellular ROS level can be detected by fluorescence microscope and flow cytometer, the excitation wavelength is 488nm, and the emission wavelength is 530nm. ROSup is a ROS-positive reagent that produces a large amount of ROS after the cells are treated. Add 50 μg / mL ROSup reagent and incubate the cells for 24 hours, then stain with FITC-labeled SNA, and observe the results. Vitamin C (VC) is an antioxidant that can scavenge ROS. After adding nano-titanium dioxide and ultraviolet treatment cells and adding 1mM vitamin C to incubate together, detect HaCaT by lectin SNA staining

[0114] According to the results of fluorescence microscopy, it ...

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Abstract

The invention provides a skin safety evaluation method on nanometer titanium dioxide based on HaCaT cells, and belongs to the field of external material detection. The method comprises the following steps that the HaCaT cells are sequentially treated by nanometer titanium dioxide solutions with different concentrations and ultraviolet rays, and a cell survival rate is tested; the concentration ofthe nanometer titanium dioxide solution with the HaCaT cell survival rate reaching 50 to 90 percent is selected to be used as potential dangerous concentration; the ultraviolet ray treatment is combined, so that a sialic acid expression level in the cells and an ROS level in the cells generate constant change; finally, the influence of the potential dangerous concentration of nanometer titanium dioxide on the cell injury degree can be judged through detecting the change of the expression level of the sialic acid, so that a set of standard nanometer titanium dioxide skin safety evaluation method is obtained. The invention provides the reliable scientific basis for the safe use of the nanometer titanium dioxide and also provides reference for the safety evaluation of other nanometer materials.

Description

technical field [0001] The invention belongs to the field of external material detection, and in particular relates to a method for evaluating the skin safety of nano titanium dioxide based on HaCaT cells. Background technique [0002] Nano-TiO 2 (nano-TiO 2 ) is a widely used nanomaterial, mainly used in cosmetics, medicine, environmental protection, coatings and other fields. Because of its unique advantages such as large specific surface area and small particle size, nanomaterials are widely used in all walks of life, especially in the cosmetics industry. Because nano-titanium dioxide has the characteristics of absorbing and scattering ultraviolet light, it is widely used in sunscreens. At present, nano-TiO used in cosmetics every year in the world 2 has reached thousands of tons. It is precisely because of these characteristics that it may cause greater harm, and the research on the safety of nano-titanium dioxide on the skin has always been highly valued by research...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/02G01N21/31G01N21/64
CPCG01N21/31G01N21/6458G01N33/5014G01N33/5044
Inventor 刘卫任园园卢群伟饶荣刘旭涵谭熙
Owner HUAZHONG UNIV OF SCI & TECH
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