CN113005171A - Preparation method of titanium dioxide nanoparticle culture medium - Google Patents
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- CN113005171A CN113005171A CN202110296135.7A CN202110296135A CN113005171A CN 113005171 A CN113005171 A CN 113005171A CN 202110296135 A CN202110296135 A CN 202110296135A CN 113005171 A CN113005171 A CN 113005171A
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Abstract
The invention discloses a preparation method of a titanium dioxide nanoparticle culture medium, which comprises the following steps: 1) mixing titanium dioxide nanoparticles with tween-20, and performing ultrasonic treatment on the obtained mixed solution under an ice bath condition; 2) adding the mixed solution of titanium dioxide and tween 20 obtained in the step 1) into a cell culture medium with the mass fraction of serum of 1-10%; 3) carrying out ultrasonic treatment on the mixed solution obtained in the step 2) to obtain the titanium dioxide nano particle culture medium. The invention enables the titanium dioxide nano particles to be evenly and stably dispersed in the culture medium by combining the surfactant and the ultrasonic dispersion method, and the prepared culture medium can be stably maintained for at least 2 days, thereby facilitating the research on the biological effect of the titanium dioxide nano particles in vitro; the preparation method is simple and easy to operate, has strong repeatability, and can provide a good object for the research of the biological effect of the nano particles.
Description
Technical Field
The invention relates to the technical field of culture medium preparation, in particular to a preparation method of a titanium dioxide nano particle culture medium.
Background
Titanium dioxide nanoparticles, also called titanium dioxide, are white loose powder with the diameter of less than 100nm, have the functions of antibiosis and antioxidation, and are widely applied to the fields of cosmetics, functional fibers, plastics, coatings, even foods and the like at present. However, such widespread use has also resulted in titanium dioxide being emitted into the environment in large quantities, being directly or indirectly absorbed by the human body, and becoming a potentially toxic substance. In view of this, research needs to be conducted on the biological effect and the toxic and side effect of titanium dioxide in environmental toxicology.
In vitro cell experiments are one of the main means for evaluating biological effects. However, the titanium dioxide nanoparticles are insoluble in water, and are very easy to agglomerate and precipitate after entering the culture medium, so that the concentration of the nanoparticles in the culture medium is rapidly reduced, and great inconvenience is brought to the biological evaluation test. In the current research, high-salt (8% mass concentration) solution is mostly adopted to be matched with ultrasound, but because the salt concentration in cell culture is limited, the salt concentration is reduced after the original high-salt solution is diluted by a culture medium, the original dispersion effect is further lost, the nano particles still rapidly agglomerate, and the research of biological effect is not facilitated.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a method for preparing a titanium dioxide nanoparticle culture medium, which combines a surfactant and an ultrasonic dispersion method to uniformly disperse titanium dioxide nanoparticles in the culture medium, so as to be used in biological effect research.
In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of a titanium dioxide nanoparticle culture medium comprises the following steps:
1) mixing titanium dioxide nanoparticles with tween-20, and performing ultrasonic treatment on the obtained mixed solution under an ice bath condition;
2) adding the mixed solution of titanium dioxide and tween 20 obtained in the step 1) into a cell culture medium with the mass fraction of serum of 1-10%;
3) carrying out ultrasonic treatment on the mixed solution obtained in the step 2) to obtain the titanium dioxide nano particle culture medium.
Preferably, the ratio of the mass of the titanium dioxide nanoparticles to the volume of the tween in the step 1) is less than 50mg:1 mL.
Preferably, the particle size of the titanium dioxide nanoparticles in step 1) is less than 20 nm.
Preferably, the step 1) is specifically: adding titanium dioxide nanoparticles and tween-20 into a container, mixing, placing the container in an ice bath, and carrying out ultrasonic treatment for 1 minute by using an ultrasonic cell lysis instrument.
Preferably, the specific parameters of the ultrasonic cell lysis instrument for ultrasonic treatment are as follows: sonication was performed at a frequency of 40kHz and a power of 900W for 1s, at 1s intervals, and for 30 cycles.
Preferably, the volume of the titania-tween 20 mixed solution added to the cell culture medium in the step 2) is not more than 0.5% of the volume of the cell culture medium.
Preferably, the cell culture medium is DMEM medium, DMEM/F12 medium, RPMI1640 medium or William's E medium.
Preferably, the step 3) is specifically: putting the mixed solution obtained in the step 2) into a container, and then putting the container into an ultrasonic cleaning instrument for ultrasonic treatment for 1-2 h.
Preferably, in the step 3), the specific parameters of the ultrasonic cleaning apparatus for performing ultrasonic treatment are as follows: frequency 10kHZ, power 100 w.
The invention has the beneficial effects that: the invention enables the titanium dioxide nano particles to be evenly and stably dispersed in the culture medium by combining the surfactant and the ultrasonic dispersion method, and the prepared culture medium can be stably maintained for at least 2 days, thereby facilitating the research on the biological effect of the titanium dioxide nano particles in vitro; the preparation method is simple and easy to operate, has strong repeatability, and can provide a good object for the research of the biological effect of the nano particles.
Drawings
FIG. 1 is a transmission electron microscope result chart of the titanium dioxide nanoparticles in the culture medium prepared in example 2.
FIGS. 2A to 2F are graphs showing the hydration radius at 0h, 24h and 48h of the culture media prepared in example 2 and comparative example 1.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention provides a preparation method of a titanium dioxide nanoparticle culture medium, which comprises the following steps:
1) after weighing commercial titanium dioxide nano particles and tween 20, adding the titanium dioxide nano particles and tween 20 into a container, immediately placing the container into an ice bath, and then carrying out ultrasonic treatment for 1 minute by using an ultrasonic cell lysis instrument.
The ratio of the mass of the titanium dioxide nanoparticles to the volume of the Tween is less than 50mg:1mL, namely the mass of the titanium dioxide nanoparticles added in each 1mL of Tween is not more than 50mg, otherwise, the titanium dioxide nanoparticles cannot be kept suspended in the Tween 20 easily due to overlarge density. Further wherein the titanium dioxide nanoparticles have a particle size of less than 20 nm.
Wherein, need to guarantee the complete submergence of whole container in the supersound in-process in the ice bath, can guarantee the heat dissipation of supersound in-process to make the heating process can not overheat, and lead to titanium dioxide nano particle to be destroyed.
In a preferred embodiment, the specific parameters of the ultrasonic cell lysis instrument sonication are: sonication was performed at a frequency of 40kHz and a power of 900W for 1s, at 1s intervals, and for 30 cycles. Under the parameter, the titanium dioxide nano particles and the Tween can be fully mixed by ultrasonic dispersion treatment for 20, and the Tween 20 covers the surfaces of the titanium dioxide nano particles, so that the titanium dioxide nano particles are not contacted by water molecules to cause agglomeration and precipitation.
2) Adding the mixed solution of titanium dioxide and tween 20 obtained in the step 1) into a cell culture medium with the mass fraction of serum of 1-10%.
Wherein, the quality of the mixed solution of the titanium dioxide and the Tween 20 added into the cell culture medium is not more than 0.5 percent of the quality of the cell culture medium, so as to ensure that the mixed solution of the titanium dioxide and the Tween 20 does not have adverse effect on cells.
Wherein the cell culture medium is DMEM medium, DMEM/F12 medium, RPMI1640 medium or William's E medium, and the source can be Gibco, Thermofeisher and other companies.
Wherein, the serum can be purchased from Hangzhou Sijiqing, Thermofeisher and other companies, the serum concentration is controlled to be 1-10% so as to improve the stability of the titanium dioxide nano particles in the culture medium, otherwise, the titanium dioxide-Tween 20 system can still be gradually destroyed in the serum-free culture medium.
3) Putting the mixed solution obtained in the step 2) into a container, then putting the container into an ultrasonic cleaning instrument, and carrying out ultrasonic treatment for 1-2h to obtain a titanium dioxide nano particle culture medium, wherein the titanium dioxide nano particle culture medium can be stably maintained for 2-3 days.
The specific parameters of ultrasonic treatment of the ultrasonic cleaning instrument are as follows: frequency 10kHZ, power 100 w. The frequency and power setting can ensure that the culture medium cannot lose components due to overheating in the ultrasonic process, and can also ensure that the titanium dioxide-Tween 20 compound in the culture medium is fully dispersed, so that the titanium dioxide nano particle-Tween 20 compound system can be prevented from being broken due to mutual collision when the concentration of the titanium dioxide nano particle-Tween 20 compound in the culture medium is too high.
The present invention is further described in detail by the following examples, which are included to illustrate the present invention.
Example 1
A preparation method of a titanium dioxide nanoparticle culture medium comprises the following steps:
1. weighing 50mg of commercial titanium dioxide nanoparticles (Aladdin reagent (Shanghai) Co., Ltd., 99.8% purity, 5-10nm, anatase, hydrophilic type, Commodity number T104949), adding Tween 20 (Biotechnology engineering (Shanghai) Co., Ltd.), analytically pure, Commodity number A100777)1mL, immediately placing the obtained mixed solution in an ice bath, and treating for 1 minute by using an ultrasonic cell disruptor, wherein the ultrasonic instrument is set as: frequency 40kHz, power 900W, ultrasonic time 1s, interval 1s, 30 times.
2. Adding 20 microliters of the titanium dioxide-tween 20 mixed solution prepared in the step 1 into 10mL of DMEM medium containing 10% fetal calf serum; wherein the fetal bovine serum is from Hangzhou ilex bioengineering materials Co., Ltd, and the DMEM medium is from Gibco Co.
3. Placing the mixed solution obtained in the step 3 in an ultrasonic cleaning instrument for ultrasonic dispersion treatment for 1.5h to prepare the titanium dioxide nano particle culture medium, wherein the ultrasonic cleaning instrument is set as follows: frequency 10kHZ, power 100 w.
The distribution of the titanium dioxide nanoparticles in the obtained titanium dioxide nanoparticle culture medium is detected by a transmission electron microscope, and the titanium dioxide nanoparticles are found to be aggregated to a certain extent, and the aggregation is about 70-80 nm.
Example 2
A preparation method of a titanium dioxide nanoparticle culture medium comprises the following steps:
1. weighing 50mg of commercial titanium dioxide nanoparticles (Aladdin reagent (Shanghai) Co., Ltd., 99.8% purity, 10-20nm, anatase, hydrophilic type, Commodity number T104949), adding 1mL of Tween 20 (Biotechnology engineering (Shanghai) Co., Ltd.), analytically pure, Commodity number A100777), immediately placing the obtained mixed solution in an ice bath, and processing by using an ultrasonic cell disruptor, wherein the ultrasonic instrument is set as: frequency 40kHz, power 900W, ultrasonic time 1s, interval 1s, 30 times.
2. Adding 20 microliters of the titanium dioxide-tween 20 mixed solution prepared in the step 1 into 10mL of DMEM medium containing 10% fetal calf serum; wherein the fetal bovine serum is from Hangzhou ilex bioengineering materials Co., Ltd, and the DMEM medium is from Gibco Co.
3. Placing the mixed solution obtained in the step 3 in an ultrasonic cleaning instrument for ultrasonic dispersion treatment for 1h to prepare the titanium dioxide nano particle culture medium, wherein the ultrasonic cleaning instrument is set as follows: frequency 10kHZ, power 100 w.
The distribution of the titanium dioxide nanoparticles in the obtained titanium dioxide nanoparticle culture medium was examined by transmission electron microscopy and found to be aggregated to a certain extent, approximately 70-80nm, as shown in FIG. 1, which is a graph of the results of examination by transmission electron microscopy (Leica TCS SP5 II, Leica microsystems, Wetzlar, Germany) of the titanium dioxide nanoparticles in the culture medium obtained in example 2.
Comparative example 1
The titania nanoparticles from the same source as in example 2 were directly dissolved in DMEM medium (same source as in example 2) (without serum) according to a conventional method to obtain a conventional titania nanoparticle-containing medium.
The media obtained in example 2 and comparative example 1 were stored in a 37 ℃ incubator and tested for hydration diameter at 0h (as-prepared), 24h, and 48h, respectively, by a Zeta-potentiostat (Malvern Zeta-sizer Nano ZS90, Worcestershire, UK), to test the stability of the nanoparticles in the media, as shown in fig. 2A-2F, fig. 2A, 2C, and 2E are graphs of hydration radius profiles at 0h, 24h, and 48h, respectively, for the media prepared in example 2 (the hydration radius is half the hydration diameter), and fig. 2B, 2D, and 2F are graphs of hydration radius profiles at 0h, 24h, and 48h, respectively, for the media prepared in example 2.
The statistical results of the hydrated diameters are shown in table 1 below.
TABLE 1
From the above results, it can be seen that the hydrated diameter of the nanoparticles in the culture medium obtained in example 2 is smaller, and the nanoparticles can be well stabilized within at least 48h, and the performance thereof is significantly better than that of comparative example 1.
While embodiments of the invention have been disclosed above, it is not limited to the applications listed in the description and the embodiments, which are fully applicable in all kinds of fields of application of the invention, and further modifications may readily be effected by those skilled in the art, so that the invention is not limited to the specific details without departing from the general concept defined by the claims and the scope of equivalents.
Claims (9)
1. A preparation method of a titanium dioxide nanoparticle culture medium is characterized by comprising the following steps:
1) mixing titanium dioxide nanoparticles with tween-20, and performing ultrasonic treatment on the obtained mixed solution under an ice bath condition;
2) adding the mixed solution of titanium dioxide and tween 20 obtained in the step 1) into a cell culture medium with the mass fraction of serum of 1-10%;
3) carrying out ultrasonic treatment on the mixed solution obtained in the step 2) to obtain the titanium dioxide nano particle culture medium.
2. The method for preparing the titanium dioxide nanoparticle culture medium according to claim 1, wherein the ratio of the mass of the titanium dioxide nanoparticles to the volume of tween in the step 1) is less than 50mg:1 mL.
3. The method for preparing the titanium dioxide nanoparticle culture medium according to claim 2, wherein the particle size of the titanium dioxide nanoparticles in the step 1) is less than 20 nm.
4. The method for preparing the titanium dioxide nanoparticle culture medium according to claim 3, wherein the step 1) is specifically as follows: adding titanium dioxide nanoparticles and tween-20 into a container, mixing, placing the container in an ice bath, and carrying out ultrasonic treatment for 1 minute by using an ultrasonic cell lysis instrument.
5. The preparation method of the titanium dioxide nanoparticle culture medium according to claim 4, wherein the specific parameters of the ultrasonic cell lysis instrument for ultrasonic treatment are as follows: sonication was performed at a frequency of 40kHz and a power of 900W for 1s, at 1s intervals, and for 30 cycles.
6. The method for preparing a titania nanoparticle culture medium according to claim 1, wherein the volume of the titania-tween 20 mixed solution added to the cell culture medium in the step 2) is not more than 0.5% of the volume of the cell culture medium.
7. The method for preparing a titania nanoparticle culture medium according to claim 6, wherein the cell culture medium is a DMEM medium, a DMEM/F12 medium, an RPMI1640 medium, or a William's E medium.
8. The method for preparing the titanium dioxide nanoparticle culture medium according to claim 1, wherein the step 3) is specifically: putting the mixed solution obtained in the step 2) into a container, and then putting the container into an ultrasonic cleaning instrument for ultrasonic treatment for 1-2 h.
9. The method for preparing the titanium dioxide nanoparticle culture medium according to claim 8, wherein in the step 3), the specific parameters of the ultrasonic cleaning instrument for ultrasonic treatment are as follows: frequency 10kHZ, power 100 w.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671718A (en) * | 2009-10-27 | 2010-03-17 | 北京师范大学 | Detection method of toxicity of nanomaterials |
| CN107746873A (en) * | 2017-11-16 | 2018-03-02 | 华中科技大学 | A kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671718A (en) * | 2009-10-27 | 2010-03-17 | 北京师范大学 | Detection method of toxicity of nanomaterials |
| CN107746873A (en) * | 2017-11-16 | 2018-03-02 | 华中科技大学 | A kind of cutaneous safety evaluation method based on HaCaT cells to nano titanium oxide |
Non-Patent Citations (1)
| Title |
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| NOUF M.AI-RASHEED等: "Potential impact Of quercetin and idebenone against immune- inflammatory and oxidative renal damage induced in rats by titanium dioxide nanoparticles toxicity", 《JOURNAL OF OLEO SCIENCE》 * |
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