Method for evaluating cytotoxicity of nanoparticles and application

Abstract

The invention discloses a method for evaluating the toxicity of nanoparticles to cells and application. The method of the present invention comprises the steps of synchronizing the cell cycle of a cell model to a prescribed period of time, and applying the nanoparticles to the synchronized cells. A cell cycle synchronization step is carried out before the amount of nano materials taken by cells and the cytotoxicity are detected, so that the influence of cell cycle heterogeneity on an evaluation result is eliminated.

Description

Technical Field

[0001] The present invention relates to the field of material evaluation, in particular to a method and application for evaluating the cytotoxicity of nanoparticles. Background Art

[0002] With the rapid development of nanotechnology, nanobiomaterials have been widely used in biomedical fields such as imaging, drug delivery, gene delivery, cancer treatment, bone tissue engineering, oral repair and regeneration. The special size range of nanomaterials gives them characteristics such as high specific surface area, active surface activity, and special optical effects. These characteristics enable nanomaterials to react physically and chemically with cells more directly, causing toxicity and harm to cells while performing their functions, limiting their further clinical use. Therefore, actively studying the toxicological effects of nanomaterials and establishing scientific and reasonable safety evaluation methods are crucial to the safe production and application of nanomaterials in the medical field.

[0003] At present, the main methods for evaluating the cytotoxicity of medical devices are GB / T 16886.5 2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test", and YY / T 0993 further describes the in vitro cytotoxicity evaluation methods of nanomaterials and nanomaterial medical devices on this basis.

[0004] According to the above standards and research data, the main methods for evaluating cell compatibility include cell membrane integrity evaluation, proliferation and toxicity detection (including MTT, LDH detection, etc.), oxidative stress response, cell apoptosis, inflammatory response, etc. The cytotoxicity of nanomaterials is affected by physical properties such as material composition, particle size, surface charge, as well as cell type, proliferation rate and other factors.

[0005] As technology advances, more accurate methods for evaluating cytotoxicity are increasingly needed. However, current methods cannot meet this demand.

[0006] The information in the background technology is only for illustrating the general background of the present invention and should not be regarded as admitting or suggesting in any form that such information constitutes the prior art known to a person skilled in the art. Summary of the invention

[0007] At present, the cell compatibility evaluation method of materials generally uses a certain cell line or primary cell colony as a cell model for overall evaluation, and does not take into account the heterogeneity between cells when the cell colony is used as a cell model. These heterogeneities are caused by factors such as gene expression, cell cycle or external environment. The present invention finds that there are significant differences in the uptake and response of nanomaterials in different cell cycles among the causes of heterogeneity, which in turn causes bias in the evaluation results. In order to solve at least some of the technical problems in the prior art, the present invention provides a cytotoxicity evaluation method and application based on a cell cycle synchronization model. Specifically, the present invention includes the following contents.

[0008] In a first aspect, the present invention provides a method for evaluating the cytotoxicity of nanoparticles, comprising the steps of synchronizing the cell cycle of a cell model to a specified cell cycle and applying a suitable concentration of the nanoparticles to the synchronized cells for treatment.

[0009] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles of the present invention, the method comprises the steps of synchronizing the cell cycle of a cell model to the S phase of the cell cycle and applying the nanoparticles to the synchronized cells.

[0010] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles according to the present invention, the nanoparticles are selected from at least one of gold nanoparticles, silver nanoparticles, titanium dioxide nanoparticles, iron oxide nanoparticles and polymer nanoparticles.

[0011] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles described in the present invention, the method further comprises detecting cell proliferation toxicity.

[0012] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles described in the present invention, the cell model is selected from at least one of stem cells, terminal cells and immune cells from different organs.

[0013] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles according to the present invention, cell cycle synchronization comprises treating the cell model with thymidine, then replacing the culture medium with a normal cell culture medium, and then treating the cell model with thymidine again.

[0014] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles of the present invention, the concentration of thymidine during treatment is 1-3 mM, and the treatment time is 10-25 hours.

[0015] A second aspect of the present invention provides a method for evaluating the cytotoxicity of nanoparticles, comprising: (1) determining the proportion of cells in the cell population whose cycles are in the G2 / M phase, S phase, and G0 / G1 phase in the cell population; (2) provide the toxicity values ​​of nanoparticles to cells at different cell cycles; (3) Using the ratio to correct the toxicity values ​​of cells at different cell cycles, an evaluation value of the cytotoxicity of the nanoparticles is obtained.

[0016] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles described in the present invention, step (2) includes: culturing cells using a serum-free culture medium to synchronize the cells in the G0 / G1 phase; after culturing the cells in a serum-free culture medium, treating the cells with a microtubule polymerization inhibitor to synchronize the cells in the G2 / M phase; treating the cells with thymidine, replacing the culture medium with ordinary fresh cell culture medium, and treating the cells with thymidine again to synchronize the cells in the S phase.

[0017] The third aspect of the present invention provides an in vitro cell compatibility evaluation method, which uses the method described in the first aspect of the present invention as a step. In certain embodiments, during the in vitro cell compatibility evaluation, the nanoparticles are derived from particles released or exuded from the surface of a medical device or medical material or from the inside thereof.

[0018] The present invention uses the cell cycle as an influencing factor of the cytotoxicity of nanoparticles, and tries to avoid the influence of different cell cycles in the cell population when evaluating cytotoxicity. Before detecting the amount of nanomaterials taken up by cells and cytotoxicity, a cell cycle synchronization step is first performed to eliminate the influence of cell cycle heterogeneity on the evaluation results. In addition, the cell cycle synchronization scheme is optimized, and a cell cycle synchronization condition that does not affect the cell state and can achieve a higher synchronization rate is explored. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The proportion of cells in each cycle after L929 cells were synchronized.

[0020] Figure 2 The results of MTT cytotoxicity test of L929 cells after being synchronized and stimulated with different concentrations of nanoparticles for 24 hours.

[0021] Figure 3 Results of MTT cytotoxicity test after bone marrow mesenchymal stem cells were synchronized and given different concentrations of nanoparticles.

[0022] Figure 4 Results of MTT cytotoxicity test of L929 cells after being synchronized and stimulated with different concentrations of silver particles. DETAILED DESCRIPTION

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that the upper and lower limits of the scope and each intermediate value therebetween are specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0025] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which the invention belongs. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0026] Herein, the term "nanoparticles" refers to nanometer-sized fine solid particles that can come into contact with or enter the human body, and particularly refers to nanometer-sized fine particles released into a medium by medical devices or medical materials.

[0027] Herein, the term "cell model" is a general term for a class of cell populations, especially model cell populations, which usually have universal biological characteristics and are easy to propagate and culture under experimental conditions, especially immortalized cells. The specific cell type is not limited, and illustratively includes stem cells, terminal cells and immune cells from different organs, such as mesenchymal stem cells, epithelial cells, connective tissue cells, muscle cells, nerve cells, etc. Under normal circumstances, the cells in the cell model of the present invention are heterogeneous, that is, during the cell culture process, the cells are in different proliferation periods at the same time due to different growth rates, starting conditions, etc.

[0028] Method for evaluating the cytotoxicity of nanoparticles (first evaluation method) One aspect of the present invention provides a method for evaluating the cytotoxicity of nanoparticles (hereinafter referred to as the first evaluation method), which includes synchronizing the cell cycle of a cell model to a specified cell cycle and applying the nanoparticles to the synchronized cells. In the present invention, the specified cell cycle can be one period or multiple periods. When one period is selected, the evaluation method of the present invention can be referred to as the first evaluation method. When multiple periods are selected, the evaluation method of the present invention can be referred to as the second evaluation method. The first evaluation method is described in detail below, and the second evaluation method is described in detail later.

[0029] In the present invention, the prescribed cell cycle is not limited. Generally speaking, the prescribed cell cycle is a cycle that is greatly affected by nanoparticles. The art can select the prescribed cell cycle according to different cell types and different nanoparticles. For some cells, for example, L929 cells, the nanoparticles have a greater impact on the S phase, so the prescribed cell cycle can consider selecting S phase cells.

[0030] In the present invention, synchronization to the S phase of the cell cycle can be performed using methods known in the art, and is not particularly limited to this. Preferably, the synchronization method has no substantial effect on cell activity. In a preferred embodiment, the method that has no substantial effect on cell activity includes treating cells with thymine for a period of time, replacing ordinary fresh cell culture medium for a period of time, and then treating cells with thymine again for a period of time to synchronize the cells in the S phase. The concentration of thymine during treatment is 1-3 mM, preferably 1.2-2.8 mM, and preferably 1.4-2.6 mM, and more preferably 1.5-2.5 mM, such as 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mM. The thymine treatment time is 10-25 hours, preferably 12-22 hours, more preferably 14-22 hours, and more preferably 16-20 hours, such as 16, 17, 18, 19, 20 hours. The general cell culture medium treatment time is 2-12 hours, preferably 4-12 hours, further preferably 6-12 hours, and further preferably 8-12 hours, for example 8, 9, 10, 11, 12 hours.

[0031] In the present invention, the nanoparticles are selected from at least one of metal nanoparticles or ceramic nanoparticles and polymer nanoparticles including gold nanoparticles, silver nanoparticles, titanium dioxide nanoparticles, and iron oxide nanoparticles. Examples of ceramic nanoparticles include, but are not limited to, aluminum oxide, zirconium oxide, silicon oxide, zinc oxide, cerium oxide, etc. Examples of polymer nanoparticles include, but are not limited to, polylactic acid, polyglycolic acid, polyvinylidene fluoride, polycaprolactone, polyethylene glycol, chitosan, etc. It will be understood by those skilled in the art that the concentration of nanoparticles varies according to different cell types and nanomaterial compositions. In certain embodiments, when L929 cells are selected for study, when the nanomaterial selects titanium dioxide nanoparticles, the concentration of the nanoparticles has a significant effect on the activity of S phase cells between 200-1000 μg / mL. In certain embodiments, when bone marrow mesenchymal stem cells are selected for study, when the nanomaterial selects titanium dioxide nanoparticles, the concentration of the nanoparticles has a significant effect on the activity of cells in different cell cycles between 5-1000 μg / mL. In certain embodiments, when L929 cells are selected for research, when the nanomaterial is selected as silver nanoparticles, the concentration of the nanoparticles between 100-500 μg / mL has a significant effect on the cell activity of different cell cycles. In certain embodiments, when bone marrow mesenchymal stem cells are selected for research, when the nanomaterial is selected as silver nanoparticles, the concentration of the nanoparticles between 5-500 μg / mL has a significant effect on the cell activity of different cell cycles (when the particle size is less than 100 nm, the concentration can be selected from 5-500 μg / mL, and when the particle size exceeds 100 nm, the concentration is preferably 100-500 μg / mL).

[0032] Those skilled in the art will appreciate that a suitable culture medium can be selected according to the different cells, and this is not particularly limited. The cells used in the present invention include, but are not limited to, at least one of mesenchymal stem cells (e.g., bone marrow-derived mesenchymal stem cells), L929, HaCaT, CaCo-2, NIH-3T3, THP-1, and RAW-294.7.

[0033] In the present invention, the particle size of the nanoparticles is not particularly limited as long as it does not affect the uptake of the nanoparticles by cells. In a specific embodiment, the nanoparticles are at least one selected from the group consisting of silver and titanium dioxide particles.

[0034] The present invention further includes a step of detecting cell proliferation toxicity. Cytotoxicity detection can be performed using, for example, the MTT method, the LDH method, etc. The reagents and detection methods used in the toxicity detection process are known in the art.

[0035] Second evaluation method Considering the influence of cell heterogeneity on cell culture conditions, culture time, age and health status of cell donors, differences in external microenvironment of cells, and different cell cycles, it is necessary to standardize the values ​​measured for cytotoxicity to obtain more accurate and rigorous toxicity evaluation results. Therefore, the present invention also provides a method for evaluating the cytotoxicity of nanoparticles (sometimes referred to herein as the "second evaluation method"), which comprises: (1) determining the proportion of cells in the cell population whose cycles are in the G2 / M phase, S phase, and G0 / G1 phase in the cell population; (2) provide the toxicity values ​​of nanoparticles to cells at different cell cycles; (3) Using the ratio to correct the toxicity values ​​of cells at different cell cycles, an evaluation value of the cytotoxicity of the nanoparticles is obtained.

[0036] Herein, the toxicity value of each cell cycle includes a relative toxicity value or an absolute value. Those skilled in the art will understand that the relative toxicity value refers to the proportion of cells in each cycle in the total cell cycle after the application of nanoparticles, and the absolute value of toxicity refers to the actual number of cells in each cycle (for example, all S phase cells) after the application of nanoparticles.

[0037] In certain embodiments, the cells are cultured in a serum-free medium for 30-60 h, preferably 40-50 h, and further preferably 45-50 h, such as 45, 46, 47, 48, 49, 50 h, to synchronize the cells in the G0 / G1 phase.

[0038] In certain embodiments, the cells are placed in a serum-free medium and cultured for 20-30 h, preferably 22-28 h, and preferably 22-26 h, such as 22, 23, 24, 25, 26 h, and then treated with a microtubule polymerization inhibitor for 10-20 h, preferably 12-18 h, and preferably 12-16 h, such as 12, 13, 14, 15, 16 h, to synchronize the cells in the G2 / M phase. The microtubule polymerization inhibitor used in the present invention is known in the art. In a specific embodiment, the microtubule polymerization inhibitor used in the present invention is Nocodazole. The concentration of the microtubule polymerization inhibitor is not particularly limited, for example, its concentration can be 10-100 ng / mL.

[0039] It is well known in the art how to determine cells and their proportions in various cell stages. For example, accurate identification of the cell cycle can be achieved through isotope labeling, flow cytometry, fluorescence detection based on cell imaging (FUCCI method), and immunofluorescence detection.

[0040] Example 1 1. Construction of the cell cycle synchronization model L929 was selected as the cell model. The cells were cultured in serum-free medium for 48 h to synchronize the cells in the G0 / G1 phase; after culturing the cells in serum-free medium for 24 h, the cells were treated with nocodazole (40 ng / mL) for 15 h to synchronize the cells in the G2 / M phase; the cells were treated with thymidine (2 mM) for 18 h, replaced with ordinary fresh cell culture medium for 9 h, and treated with thymidine (2 mM) again for 18 h to synchronize the cells in the S phase.

[0041] Table 1 Treatment conditions of L929 cell cycle synchronization model 2. Detection of Synchronized Cell Model After the cells were treated with synchronized drugs, they were digested with trypsin, washed with PBS, fixed in 70% ethanol and incubated on ice for 15 minutes. The cells were then labeled with PI / RNase staining solution and incubated at room temperature for 15 minutes. The red fluorescence and light scattering were detected using a BD-FACSAriaTM III flow cytometer at an excitation wavelength of 488 nm, and the proportion of cells in the G0 / G1 phase, S phase, and G2 / M phase were analyzed using BD FACSDivaTM software.

[0042] 3. MTT Cytotoxicity Assay The cells were treated with 0.5% MTT for 4 h, the supernatant was discarded, an appropriate amount of dimethyl sulfoxide (DMSO) was added to each well, the shaker was shaken at low speed for 10 min, and the absorbance at 490 nm was detected using an enzyme reader.

[0043] IV. Results 1. Drugs have no real effect on synchronization By continuously exploring the drug concentration, a cell cycle synchronization model was successfully established in L929 mouse fibroblasts. Figure 1 After synchronization, the proportion of each cycle increased significantly, and the cell activity did not change significantly, indicating that the drugs used in this model are not toxic to cells. The cycle synchronization process is reversible.

[0044] Table 2 The proportion of cells in each cycle after L929 cells were synchronized 2. Effects of cell cycle on cytotoxicity of nanomaterials Titanium dioxide nanoparticles were used to stimulate synchronized L929 cells. Figure 2As shown in Table 3, with the increase of material concentration, the cell MTT activity decreased linearly, and the cell activity in each cycle was different. Among them, at the concentrations of 200, 500, and 1000 μg / mL, the cell activity in the S phase was significantly lower than that in other groups.

[0045] Table 3 Results of MTT cytotoxicity test of L929 cells after various synchronization treatments and stimulation with different concentrations of nanoparticles for 24 h Example 2 This example shows a method for synchronizing bone marrow mesenchymal stem cells and a method for evaluating the cytotoxicity of nanoparticles.

[0046] 1. Bone marrow mesenchymal stem cells (mBMSCs) of c57 mice were selected as the cell model. The cells were cultured in serum-free medium for 24 h to synchronize the cells in the G0 / G1 phase. The cells were treated with RO-3306 (10 nM) for 24 hours, and then treated with nocodazole (100 μM) for 2 hours to synchronize the cells in the G2 / M phase. Thymidine double blocking method: cells were treated with thymidine (2 mM) for 18 hours, then treated with ordinary cell culture medium for 9 hours, and then treated with thymidine (2 mM) for 18 hours to synchronize the cells in the S phase.

[0047] 2. After the cells are treated with synchronized drugs, trypsin is used to digest them, the cells are washed with PBS, and the cells are counted using a cell counter or a blood cell counting plate. The cells are then fixed in 70% ethanol and incubated on ice for 15 minutes. The cells are then labeled with PI / RNase staining solution and incubated at room temperature for 15 minutes. The red fluorescence and light scattering are detected using a BD-FACSAriaTM III flow cytometer at an excitation wavelength of 488 nm, and the proportion of cells in the G0 / G1 phase, S phase, and G2 / M phase are analyzed using BD FACSDivaTM software.

[0048] 3. After the cells were treated with synchronized drugs, they were treated with 0.5% MTT for 4 hours, the supernatant was discarded, an appropriate amount of dimethyl sulfoxide (DMSO) was added to each well, the shaker was shaken at low speed for 10 min, and the OD 490 value was detected using a microplate reader.

[0049] The results are as follows Figure 3 As shown, there are differences in cytotoxicity after BMSC cells take up nanoparticles at different cycles.

[0050] Example 3 This example shows a method for synchronizing L929 cells and a method for evaluating the cytotoxicity of nanoparticles. Different from Example 1, the detected nanoparticles are silver nanoparticles. The results are shown in FIG. Figure 4As shown, there are differences in cytotoxicity after L929 cells take up nanoparticles at different stages.

[0051] Although the present invention has been described with reference to exemplary embodiments, it should be understood that the present invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments of the present specification without departing from the scope or spirit of the present invention. The scope of the claims should be based on the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims

1. A method for evaluating the cytotoxicity of nanoparticles, characterized in that: The method comprises the steps of synchronizing the cell cycle of the cell model to a specified cell cycle, and applying the nanoparticles of appropriate concentration to the synchronized cells for treatment.

2. The method for evaluating the cytotoxicity of nanoparticles according to claim 1, characterized in that: The nanoparticles are selected from at least one of metal nanoparticles including gold nanoparticles, silver nanoparticles, titanium dioxide nanoparticles, and iron oxide nanoparticles, or ceramic nanoparticles and polymer nanoparticles.

3. The method for evaluating the cytotoxicity of nanoparticles according to claim 1, characterized in that: Further comprising the step of detecting the toxicity of the nanoparticles to cell proliferation.

4. The method for evaluating the cytotoxicity of nanoparticles according to claim 1, characterized in that: The cell model is selected from at least one of stem cells, terminal cells and immune cells derived from different organs.

5. The method for evaluating the cytotoxicity of nanoparticles according to claim 1, characterized in that: The cell cycle synchronization includes treating the cell model with thymidine, then replacing the cell culture medium with a normal cell culture medium, and then treating the cell model with thymidine again.

6. The method for evaluating the cytotoxicity of nanoparticles according to claim 5, characterized in that: The concentration of thymidine during treatment is 1-3 mM.

7. The method for evaluating the cytotoxicity of nanoparticles according to claim 6, characterized in that: The thymidine treatment time is 10-25 hours.

8. A method for evaluating the cytotoxicity of nanoparticles, characterized in that: include: (1) determining the proportion of cells in the cell population whose cycles are in the G2 / M phase, S phase, and G0 / G1 phase in the cell population; (2) provide the toxicity values ​​of nanoparticles to cells at different cell cycles; (3) Using the ratio to correct the toxicity values ​​of cells at different cell cycles, an evaluation value of the cytotoxicity of the nanoparticles is obtained.

9. The method for evaluating the cytotoxicity of nanoparticles according to claim 8, characterized in that: Step (1) comprises: culturing the cells in a serum-free medium to synchronize the cells in the G0 / G1 phase; or After culturing the cells in serum-free medium, treat the cells with a microtubule polymerization inhibitor to synchronize the cells in the G2 / M phase; or Treat the cells with thymidine, change to normal fresh cell culture medium, and treat the cells with thymidine again to synchronize the cells in S phase.

10. An in vitro cell compatibility evaluation method, characterized in that: Use the method according to any one of claims 1 to 9 as a step.

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