Method and application for evaluating cytotoxicity of nanoparticles

Abstract

The present invention discloses a method and application for evaluating the cytotoxicity of nanoparticles. The method of the present invention includes synchronizing the cell cycle of a cell model to a specified period and applying the nanoparticles to the synchronized cells. By performing the cell cycle synchronization step before detecting the amount of nanoparticles taken up by cells and the cytotoxicity, the influence of cell cycle heterogeneity on the evaluation results is eliminated.

Description

Technical Field

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

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

[0003] Currently, the main methods for evaluating the cytotoxicity of medical devices are GB / T 16886.5 2017 "Biological evaluation of medical devices - Part 5: Tests for in vitro cytotoxicity", and YY / T 0993 further describes the in vitro cytotoxicity evaluation methods for nanomaterials and nanomaterial medical devices on this basis.

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

[0005] With technological progress, there is an increasing need for more precise cytotoxicity evaluation methods. However, the current methods cannot meet this requirement.

[0006] The information in the background art is only for explaining the general background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes the prior art known to those of ordinary skill in the art. Summary of the Invention

[0007] The current methods for evaluating the cytocompatibility of materials generally use a certain cell line or primary cell population as a cell model for overall evaluation, without considering the heterogeneity among cells when the cell population is used as a cell model. These heterogeneities are caused by factors such as gene expression, cell cycle, or external environment. The present invention discovers that there are significant differences in the uptake and response of nanomaterials by different cell cycles among the causes of heterogeneity, which in turn causes bias in the evaluation results. To solve at least some of the technical problems in the prior art, the present invention provides a method for evaluating cytotoxicity based on a cell cycle synchronization model and its application. Specifically, the present invention includes the following contents.

[0008] In a first aspect of the present invention, there is provided a method for evaluating the cytotoxicity of nanoparticles to cells, which includes the steps of synchronizing the cell cycle of a cell model to a specified cell cycle and applying an appropriate concentration of the nanoparticles to the synchronized cells for treatment.

[0009] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles to cells of the present invention, wherein the method includes 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 to cells of the present invention, wherein 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 to cells of the present invention, wherein the method further includes detecting cell proliferation toxicity.

[0012] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles to cells of the present invention, wherein the cell model is selected from at least one of stem cells from different organ sources, terminally differentiated cells, and immune cells.

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

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

[0015] In a second aspect of the present invention, there is provided a method for evaluating the cytotoxicity of nanoparticles to cells, which includes:

[0016] (1) determining the proportion of cells in the cell population that are in the G2 / M phase, S phase, and G0 / G1 phase of the cell cycle;

[0017] (2) provide the toxicity values of nanoparticles to cells at different cell cycles;

[0018] (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.

[0019] In certain embodiments, according to the method for evaluating the cytotoxicity of nanoparticles according to the present invention, step (2) comprises: culturing cells in 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 again with thymidine to synchronize the cells in the S phase.

[0020] The third aspect of the present invention provides an in vitro cell compatibility assessment method using the method described in the first aspect of the present invention as a step. In certain embodiments, during the in vitro cell compatibility assessment, the nanoparticles are derived from particles released or exuded from the surface of a medical device or medical material or from within the medical device or medical material.

[0021] The present invention considers the cell cycle as an influencing factor for nanoparticle cytotoxicity and minimizes the influence of varying cell cycle patterns within a cell population when evaluating cytotoxicity. Prior to measuring the nanomaterial uptake and cytotoxicity, a cell cycle synchronization step was performed to eliminate the influence of cell cycle heterogeneity on the evaluation results. Furthermore, the cell cycle synchronization scheme was optimized to identify cell cycle synchronization conditions that achieve a high synchronization rate while maintaining a stable cell state. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The proportion of cells in each cell cycle after L929 cells were synchronized.

[0023] Figure 2 Results of MTT cytotoxicity assays of L929 cells after synchronization and stimulation with nanoparticles at different concentrations for 24 hours.

[0024] Figure 3 Results of MTT cytotoxicity tests on bone marrow mesenchymal stem cells after synchronization and administration of different concentrations of nanoparticles.

[0025] Figure 4Results of MTT cytotoxicity assays of L929 cells after synchronization and stimulation with silver particles at different concentrations. DETAILED DESCRIPTION

[0026] 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.

[0027] It should be understood that the terms described in the present invention are only for describing particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the ranges and each intermediate value therebetween are specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range and any other stated value or intermediate value within the stated range is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0028] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning 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 any conflict with any incorporated document, the content of this specification shall prevail.

[0029] As used herein, the term "nanoparticles" refers to nanoscale fine solid particles that can come into contact with or enter the human body, and particularly refers to nanoscale fine particles released into a medium by medical devices or medical materials.

[0030] As used herein, the term "cell model" is a general term for a class of cell populations, particularly model cell populations, which generally have universal biological characteristics and are easy to propagate and culture under experimental conditions, particularly immortalized cells. Specific cell types are not limited, but illustratively include stem cells, terminal cells, and immune cells from different organ sources, such as mesenchymal stem cells, epithelial cells, connective tissue cells, muscle cells, and neural cells. Typically, the cells within the cell model of the present invention are heterogeneous, meaning that during the cell culture process, cells are in different proliferation phases at the same time due to differences in growth rate, starting conditions, and the like.

[0031] Method for evaluating the cytotoxicity of nanoparticles (first evaluation method)

[0032] In one aspect of the present invention, there is provided a method for evaluating the cytotoxicity of nanoparticles (hereinafter simply 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. In the case of selecting one period, the evaluation method of the present invention can be called the first evaluation method. In the case of selecting multiple periods, the evaluation method of the present invention can be called the second evaluation method. The first evaluation method will be elaborated in detail below, and the second evaluation method will be described in detail subsequently.

[0033] In the present invention, the specified cell cycle is not limited. Generally speaking, the specified cell cycle is the cycle that is more greatly affected by the nanoparticles. Those skilled in the art can select the specified cell cycle according to different cell types and different nanoparticles. For some cells, for example, for L929 cells, the nanoparticles have a greater impact on the S phase. Therefore, the specified cell cycle can be considered to be the S-phase cells.

[0034] In the present invention, synchronizing to the S phase of the cell cycle can be carried out by methods known in the art, and there is no particular limitation thereto. Preferably, a method that does not have a substantial impact on cell viability is used. In a preferred embodiment, the method that does not have a substantial impact on cell viability includes treating the cells with thymidine for a period of time, replacing with normal fresh cell culture medium for a period of time, and then treating the cells with thymidine for a period of time to synchronize the cells to the S phase. The concentration of thymidine during treatment is 1 - 3 mM, preferably 1.2 - 2.8 mM, more preferably 1.4 - 2.6 mM, and further 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 treatment time of thymidine is 10 - 25 hours, preferably 12 - 22 hours, more preferably 14 - 22 hours, and also preferably 16 - 20 hours, such as 16, 17, 18, 19, 20 hours. The treatment time with normal cell culture medium is 2 - 12 hours, preferably 4 - 12 hours, more preferably 6 - 12 hours, and also preferably 8 - 12 hours, such as 8, 9, 10, 11, 12 hours.

[0035] In the present invention, 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. Examples of ceramic nanoparticles include, but are not limited to, aluminum oxide, zirconium oxide, silicon oxide, zinc oxide, and cerium oxide. Examples of polymer nanoparticles include, but are not limited to, polylactic acid, polyglycolic acid, polyvinylidene fluoride, polycaprolactone, polyethylene glycol, and chitosan. Those skilled in the art will appreciate that the concentration of nanoparticles varies depending on the cell type and the composition of the nanomaterial. In certain embodiments, when studying L929 cells and titanium dioxide nanoparticles as the nanomaterial, a nanoparticle concentration of 200-1000 μg / mL has a significant effect on cell viability during the S phase. In certain embodiments, when studying bone marrow mesenchymal stem cells and titanium dioxide nanoparticles as the nanomaterial, a nanoparticle concentration of 5-1000 μg / mL has a significant effect on cell viability during different stages of the cell cycle. In certain embodiments, when L929 cells are selected for research, when silver nanoparticles are selected as the nanomaterial, the concentration of the nanoparticles between 100-500 μg / mL has a significant effect on the cell activity at different cell cycles. In certain embodiments, when bone marrow mesenchymal stem cells are selected for research, when silver nanoparticles are selected as the nanomaterial, the concentration of the nanoparticles between 5-500 μg / mL has a significant effect on the cell activity at 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).

[0036] Those skilled in the art will appreciate that appropriate 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.

[0037] 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 selected from at least one of the group consisting of silver and titanium dioxide particles.

[0038] 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.

[0039] Second evaluation method

[0040] Considering the effects of differences in cell culture conditions, culture time, age and health status of cell donors, differences in the external microenvironment of cells, and different cell cycle stages on cell heterogeneity, it is necessary to standardize the measured values of 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 simply referred to as the "second evaluation method" in this article), which includes:

[0041] (1) Determining the proportions of cells in the G2 / M phase, S phase, and G0 / G1 phase in the cell population among the cell population;

[0042] (2) Providing the cytotoxicity values of the nanoparticles for cells in different cycles;

[0043] (3) Using the proportions to correct the cytotoxicity values of the cells in different cycles to obtain the evaluation value of the cytotoxicity of the nanoparticles to the cells.

[0044] In this article, the cytotoxicity values of each cell cycle, including relative cytotoxicity values or absolute values, those skilled in the art will understand that the relative cytotoxicity 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 cytotoxicity refers to the actual number of cells in each cycle (for example, all S-phase cells) after the application of nanoparticles.

[0045] In some embodiments, cells are cultured in a serum-free medium for 30 - 60 h, preferably 40 - 50 h, more preferably 45 - 50 h, such as 45, 46, 47, 48, 49, 50 h, so as to synchronize the cells in the G0 / G1 phase.

[0046] In some embodiments, cells are placed in a serum-free medium and cultured for 20 - 30 h, preferably 22 - 28 h, more preferably 22 - 26 h, such as 22, 23, 24, 25, 26 h, and then the cells are treated with a microtubule polymerization inhibitor for 10 - 20 h, preferably 12 - 18 h, more 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.

[0047] Those skilled in the art are familiar with how to measure the cells and their proportions in each cell stage. For example, precise identification of the cell cycle can be achieved by isotope labeling, flow cytometry, fluorescence detection based on cell imaging (FUCCI method), and immunofluorescence detection.

[0048] Example 1

[0049] 1. Construction of a cell cycle synchronization model

[0050] L929 cells were selected as a cell model. Cells were cultured in serum-free medium for 48 hours to synchronize them in the G0 / G1 phase. After 24 hours of culture in serum-free medium, cells were treated with nocodazole (40 ng / mL) for 15 hours to synchronize them in the G2 / M phase. Cells were treated with thymidine (2 mM) for 18 hours, replaced with fresh normal cell culture medium for 9 hours, and then treated with thymidine (2 mM) again for 18 hours to synchronize them in the S phase.

[0051] Table 1 Treatment conditions of L929 cell cycle synchronization model

[0052]

[0053] 2. Detection of Synchronized Cell Model

[0054] After synchronization with drug treatment, cells were trypsinized, washed with PBS, fixed in 70% ethanol, and incubated on ice for 15 minutes. Cells were then labeled with PI / RNase staining solution and incubated at room temperature for 15 minutes. Red fluorescence and light scatter were measured using a BD-FACSAria™ III flow cytometer at an excitation wavelength of 488 nm. The proportions of cells in the G0 / G1, S, and G2 / M phases were analyzed using BD FACSDiva™ software.

[0055] 3. MTT Cytotoxicity Assay

[0056] The cells were treated with 0.5% MTT for 4 h, the supernatant was discarded, and an appropriate amount of dimethyl sulfoxide (DMSO) was added to each well. The cells were shaken at low speed for 10 min, and the absorbance at 490 nm was measured using a microplate reader.

[0057] IV. Results

[0058] 1. Medications have no substantial effect on synchronization

[0059] 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 cells in each cycle increased significantly, and cell activity did not change significantly, indicating that the drugs used in this model are not toxic to cells. This cycle synchronization process is reversible.

[0060] Table 2 The proportion of cells in each cell cycle after L929 cells were synchronized

[0061]

[0062] 2. Influence of cell cycle on the cytotoxicity of nanomaterials

[0063] Synchronized L929 cells were stimulated with titanium dioxide nanoparticles. As Figure 2 shown in and Table 3, with the increase of material concentration, the MTT activity of cells decreased linearly, and there were differences in the activity of cells in each cycle. Among them, at the concentrations of 200, 500, and 1000 μg / mL, the activity of S-phase cells was significantly lower than that of other groups.

[0064] Table 3 Results of MTT cytotoxicity test of L929 cells after different synchronization treatments and stimulation with different concentrations of nanoparticles for 24 h

[0065]

[0066] Example 2

[0067] This example shows a method for synchronizing bone marrow mesenchymal stem cells and a method for evaluating the cytotoxicity of nanoparticles to cells.

[0068] 1. Select the bone marrow mesenchymal stem cells (mBMSCs) of c57 mice as the cell model, culture the cells in a serum-free medium for 24 h to synchronize the cells in the G0 / G1 phase; treat the cells with RO-3306 (10 nM) for 24 hours, and then treat the cells with nocodazole (100 μM) for 2 hours to synchronize the cells in the G2 / M phase; thymidine double-blocking method: treat the cells with thymidine (2 mM) for 18 hours, change to a normal cell culture medium and treat for 9 hours, and then treat the cells with thymidine (2 mM) for 18 hours to synchronize the cells in the S phase.

[0069] 2. After treating the cells with synchronization drugs, digest the cells with trypsin, wash the cells with PBS, and count the cells using a cell counter or a hemocytometer. Then fix the cells in 70% ethanol and incubate on ice for 15 minutes. Then label the cells with PI / RNase staining solution and incubate at room temperature for 15 minutes. Use a BD-FACSAriaTM III flow cytometer to detect the red fluorescence and light scattering at an excitation wavelength of 488 nm, and use BD FACSDivaTM software to analyze the proportions of cells in the G0 / G1 phase, S phase, and G2 / M phase.

[0070] 3. After treating the cells with synchronization drugs, treat the cells with 0.5% MTT for 4 hours, discard the supernatant, add an appropriate amount of dimethyl sulfoxide (DMSO) to each well, shake at low speed on a shaker for 10 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to detect the OD 490 value.

[0071] The results are as Figure 3 shown, and there are differences in cytotoxicity after different cycle BMSC cells take up nanoparticles.

[0072] Example 3

[0073] This example shows a method for synchronizing L929 cells and a method for evaluating the cytotoxicity of nanoparticles. Different from Example 1, the tested nanoparticles are silver nanoparticles. The results are as Figure 4 shown, and there are differences in cytotoxicity after different cycle L929 cells take up nanoparticles.

[0074] 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 can be made to the exemplary embodiments of the present invention 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, It includes the steps of synchronizing the cell cycle of the cell model to the specified S phase of the cell cycle, and treating the synchronized cells with the appropriate concentration of the said nanoparticles. The cell cycle synchronization includes treating the cell model with thymidine, then replacing it with normal cell culture medium for treatment, and then treating the cell model with thymidine again.

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

3. The method for evaluating the cytotoxicity of nanoparticles according to claim 1, wherein It further includes 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, wherein The said cell model is selected from at least one of stem cells from different organ sources, terminally differentiated cells, and immune cells.

5. The method for evaluating the cytotoxicity of nanoparticles according to claim 1, wherein The concentration during thymidine treatment is 1 - 3 mM.

6. The method for evaluating the cytotoxicity of nanoparticles according to claim 1, characterized in that, The time of the said thymidine treatment is 10 - 25 hours.

7. An in vitro cell compatibility evaluation method, characterized in that, Use the method according to any one of claims 1 - 6 as a step.

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