A method for evaluating cellular uptake of nanoparticles

Abstract

This invention proposes a method for evaluating cellular uptake of nanoparticles, relating to the field of biomaterials detection. The method includes the following steps: co-incubating standard sample cells and nanoparticles, followed by staining to obtain reference cells containing nanoparticles; performing tomographic imaging on the reference cells to obtain a set of tomographic images; calibrating the background, cells, and nanoparticles in the tomographic image set to obtain corresponding fluorescence values; performing frequency distribution analysis on the fluorescence values ​​to obtain a scale corresponding to cell substructures and nanoparticles; co-incubating the test sample cells and nanoparticles, repeating the above steps, and establishing a three-dimensional visualization; and using the scale to determine the interaction relationship between the test sample cells and nanoparticles. This invention solves the problem in existing technologies of being unable to distinguish between surface-adhered and cell-uptaken nanoparticles, and possesses spatial analysis capabilities, providing a precise determination of whether cells have uptaken nanoparticles under in vitro culture conditions by constructing a scale and a three-dimensional visualization.

Description

Technical Field

[0001] This invention relates to the field of biomaterials detection, and more specifically, to a method for evaluating the uptake of nanoparticles by cells. Background Technology

[0002] Whether biomedical nanoparticles are taken up by cells during practical applications is a key issue that needs to be evaluated in preclinical research on nanoparticles. Evaluating cellular uptake of nanoparticles under in vitro cell culture conditions can reveal, to some extent, the scenarios, mechanisms, and efficiency in which nanoparticles function during cell-cell interaction. Currently, the two most common methods for evaluating cellular uptake of nanoparticles under in vitro cell culture conditions are fluorescence microscopy and electron microscopy. Fluorescence microscopy relies on separately labeling nanoparticles and cells with fluorescence and then imaging using a conventional or laser confocal fluorescence microscope; however, this method has limitations, such as the need for the fluorescent molecules to be modifiable and the tendency for modified fluorescent molecules to quench fluorescence. Furthermore, this method cannot distinguish between surface-adhered nanoparticles and those taken up by cells. Electron microscopy offers high resolution and can determine the distribution of nanoparticles within cellular substructures; however, this method is only suitable for characterizing thin-layer samples and cannot perform spatial analysis.

[0003] Therefore, developing a characterization method with spatial analysis capabilities that does not rely on fluorescent labeling of particles is a key problem that urgently needs to be solved in the study of the process of nanoparticle internalization in cells. Summary of the Invention

[0004] The purpose of this invention is to provide a method for evaluating the uptake of nanoparticles by cells with spatial analysis capabilities. By constructing a scale and a three-dimensional view, it provides an accurate judgment on whether cells have taken up nanoparticles under in vitro culture conditions, solving the problem in the prior art that it is impossible to distinguish between nanoparticles that are adhered to the surface and those that have been taken up by cells.

[0005] The technical problem solved by this invention is achieved by the following technical solution.

[0006] This application provides a method for evaluating the uptake of nanoparticles by cells, comprising the following steps:

[0007] S1: Prepare standard sample cells, co-incubate the standard sample cells and nanoparticles, and obtain reference cells containing nanoparticles after staining;

[0008] S2: Perform tomographic imaging on the reference cells to obtain a set of tomographic images;

[0009] S3: Inspect the background, cells and nanoparticles in the tomographic image set, and record the fluorescence value of the region containing the background, cell substructure outline region and nanoparticle outline region as RLU1, and record the fluorescence value of RLU1 after subtracting the background value as RLU2.

[0010] S4: Frequency distribution analysis of fluorescence value RLU2 was performed to obtain frequency distribution maps of cell substructures and nanoparticles. After analysis, the corresponding scale bars of cell substructures and nanoparticles were obtained.

[0011] S5: Co-incubate the cells and nanoparticles of the test sample, repeat steps S1-S3, and establish a three-dimensional visual image by combining the set of tomographic images obtained in step S2 and the fluorescence values ​​obtained in step S3; use the scale to determine the interaction relationship between the cells and nanoparticles of the test sample.

[0012] Furthermore, in step S1, the nanoparticles are nanoparticles with self-luminous properties or nanoparticles with artificial fluorescent labels.

[0013] Furthermore, the staining process involves staining the substructures of standard sample cells with specific and / or non-specific fluorescent dyes; the substructures include the cell membrane, cytoskeleton, nucleus, and organelles.

[0014] Furthermore, organelles also include mitochondria.

[0015] Furthermore, in step S2, the tomographic imaging includes performing a tomographic scan along the axis of the reference cell using fluorescence imaging software; the tomographic image set includes the stride of the tomographic images and the number of tomographic images.

[0016] Furthermore, it also includes using different fluorescence excitation channels to perform layer scanning on the substructure, including but not limited to DAPI channels, FITC channels, TRITC channels, and TD channels.

[0017] Furthermore, in step S3, the calibration also includes obtaining the calibration distances of the background region, the cell substructure contour region, and the nanoparticle contour region.

[0018] Furthermore, steps S4-S5 include using data processing software to perform frequency distribution analysis and establish a three-dimensional visualization. The data processing software includes, but is not limited to, GraphPad, Origin, and SPSS.

[0019] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects:

[0020] 1. This invention constructs a scale by co-incubating standard sample cells and nanoparticles, and uses the scale to visualize the cell structure and nanoparticles in three dimensions, enabling spatial analysis and effectively evaluating the interaction mode between nanoparticles and cells. This solves the problem in the prior art that it is difficult to evaluate the adhesion of nanoparticles to the cell surface and their phagocytosis by cells.

[0021] 2. This invention uses tomographic imaging to individually label the cell membrane, cytoskeleton, and nucleus, avoiding problems such as fluorescence quenching that occur when using traditional fluorescence observation or electron microscopy. Furthermore, the data obtained through tomographic imaging is more accurate, and combined with a scale, the spatial location of nanoparticles after being taken up by cells can be precisely determined. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 This is a schematic diagram of the overall process of the method of the present invention;

[0024] Figure 2 This is a frequency analysis and scale selection diagram of cell nuclei in Example 1 of the present invention;

[0025] Figure 3 This is a frequency analysis and scale selection diagram of the cytoskeleton in Example 1 of the present invention;

[0026] Figure 4 This is a frequency analysis and scale selection chart of the cell membrane in Example 1 of the present invention;

[0027] Figure 5 This is a frequency analysis and scale selection chart of the nanoparticles in Example 1 of the present invention;

[0028] Figure 6 This is a three-dimensional view of the cytoskeleton in Embodiment 1 of the present invention;

[0029] Figure 7 This is a three-dimensional view of the cell nucleus in Embodiment 1 of the present invention;

[0030] Figure 8 This is a three-dimensional view of the cell membrane in Embodiment 1 of the present invention;

[0031] Figure 9 This is a three-dimensional view of the nanoparticles in Embodiment 1 of the present invention;

[0032] Figure 10 This is a frequency analysis and scale selection diagram of cell nuclei in Example 2 of the present invention;

[0033] Figure 11 This is a frequency analysis and scale selection diagram of the cytoskeleton in Example 2 of the present invention;

[0034] Figure 12 This is a frequency analysis and scale selection chart of the cell membrane in Example 2 of the present invention;

[0035] Figure 13 This is a frequency analysis and scale selection chart of nanoparticles in Example 2 of the present invention;

[0036] Figure 14 This is a three-dimensional view of the cytoskeleton in Embodiment 2 of the present invention;

[0037] Figure 15 This is a three-dimensional view of the cell nucleus in Embodiment 2 of the present invention;

[0038] Figure 16 This is a three-dimensional view of the cell membrane in Embodiment 2 of the present invention;

[0039] Figure 17 This is a three-dimensional view of the nanoparticles in Embodiment 2 of the present invention. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0042] Example 1

[0043] Please refer to Figure 1 , Figure 1 This is a schematic diagram of the overall process of an embodiment of the present invention. This embodiment provides a method for evaluating the uptake of nanoparticles by cells, suitable for determining the short-term interaction between nanoparticles and test cells, including the following steps:

[0044] S1: 600 nm polydopamine nanoparticles were artificially prepared as nanoparticles, sterilized, and dispersed in sterile water to obtain a sterile aqueous dispersion of polydopamine nanoparticles; then diluted with cell culture medium to obtain a polydopamine nanoparticle cell culture medium dispersion with a concentration of 1 μg / mL, which was ready for use.

[0045] RAW264.7 mouse macrophages were cultured in vitro as standard cells and then cultured at a density of 3 × 10⁻⁶ cells / cell. 4 pcs / cm 2 The standard cells were seeded at a density of 1 μg / mL on well plates. After 2 h of seeding, the standard cells were activated with 1 μg / mL LPS (lipopolysaccharide). After 12 h of LPS stimulation, the culture medium of the standard cells was replaced with polydopamine nanoparticle cell culture medium dispersion. After the replacement, the cells were transferred to a cell culture incubator and cultured for another 1 day. After the culture was completed, the cells were fixed.

[0046] It is worth noting that in this step, LPS can accurately simulate the "macrophage physiological / pathological activation scenario". It can activate standard sample cells and stimulate endocytosis between standard sample cells and polydopamine nanoparticles.

[0047] In addition, to ensure the accuracy of the experiment, at least three sets of repeated experiments should be set up in this step, and the groups should be classified and labeled according to their group numbers.

[0048] Then, CD86 antibody (excitation wavelength: 495nm, emission wavelength: 519nm), phalloidin (excitation wavelength: 555nm, emission wavelength: 565nm), and DAPI nuclear dye (excitation wavelength: 358nm, emission wavelength: 461nm) were used to stain the cell membrane protein marker CD86, cytoskeleton, and cell nucleus of the reference cells according to the classified group number, so as to obtain reference cells containing nanoparticles.

[0049] S2: Using a Nikon A1R+ confocal microscope, confocal images of stained reference cells containing nanoparticles were taken. A layer scan was performed along the cell axis at a step size of 2µm. The channels used were DAPI (excitation wavelength 405nm, voltage 120V, laser power 60mW, blue), FITC (excitation wavelength 488nm, voltage 50V, laser power 25mW, green), TRITC (excitation wavelength 561nm, voltage 50V, laser power 25mW, red), and TD (voltage 60V, gray). The resulting confocal immunofluorescence images of the reference cells were obtained.

[0050] The confocal immunofluorescence images were then processed using NIS-Elements (Version 4.50.00) software accompanying the Nikon A1R+ confocal microscope. The images were exported as color images for each channel at each step size in Tagged image format, with the scaling option selected to scale from 12 bits to 16 bits. The images were then exported and saved.

[0051] S3: Using the line analysis tool of the fluorescence quantitative analysis software ZEN, specific structures (including image background, cell structure, and nanoparticles) of the processed confocal immunofluorescence images obtained in step S2 were labeled. The labeling included layer-by-layer labeling of bright-field nanoparticles and layer-by-layer labeling of cell structures in the blue, red, and green channels (one image at 2-micrometer intervals). Three or more structures of each image were labeled, and the fluorescence values ​​of the background region, cell substructure contour region, and nanoparticle contour region were recorded as RLU1. The fluorescence values ​​of the background region, the cell substructure contour region after background subtraction, and the nanoparticle contour region were recorded as RLU2. Finally, the fluorescence data were further analyzed using the data analysis software Origin / Graphpad Prism.

[0052] It is worth noting that the background value refers to the fluorescence value obtained by calibrating the same distance between regions in the same image that do not contain particles or cellular structures during the calibration process in each channel of the tomographic image.

[0053] S4: Frequency analysis of the fluorescence value RLU2 was performed using Graphpad Prism software to obtain a frequency distribution map. Then, statistical analysis was performed according to the classification groups to obtain a selection chart for the scale. The analysis results are as follows: Figure 2 - Figure 5 As shown, the DAPI channel has a scale value of 8000 for distinguishing between the cell nucleus and nanoparticles (leading to an increase in fluorescence value exceeding 8000), the TRITC channel has a scale value of 10000 for distinguishing between the cytoskeleton and nanoparticles (leading to an increase in fluorescence value exceeding 10000), the FITC channel has a scale value of 8000 for distinguishing between the cell membrane and nanoparticles (leading to an increase in fluorescence value exceeding 8000), and the TD channel has a scale value of -21000 for distinguishing between nanoparticles and cells (leading to a decrease in fluorescence value exceeding 21000).

[0054] S5: Repeat steps S1-S3, mixing the cells and nanoparticles, staining, performing tomographic imaging, and exporting images for each channel. Each channel's tomographic image is then calibrated layer by layer according to the aforementioned scale calibration method. After calibrating the corresponding structures, the corresponding fluorescence value RμL1 is stored and processed to obtain the fluorescence value RμL2. Then, line analysis is performed on the data obtained from the cells (including step size 2µm, calibration distance, number of tomographic images, and fluorescence value RμL2) to obtain three-dimensional visual images of nanoparticles, the framework, the membrane, and the cell nucleus. Subsequently, the obtained three-dimensional visual images are analyzed using the scale established in the previous steps. The scale allows for a direct visual representation of the outlines of specific structures on the three-dimensional visual image, and the relationship between cells and particles can be analyzed based on the images of specific structures. Experimental results are as follows: Figure 6 - Figure 9 As shown, a three-dimensional visual diagram containing the cytoskeleton, nucleus, cell membrane, and nanoparticles is observed using a scale. Structures with fluorescence intensity below -21000 under bright field are nanoparticles. The red, blue, green, and gray areas in the diagram correspond to the cytoskeleton, nucleus, cell membrane, and nanoparticles, respectively. Furthermore, it can be determined that the nanoparticles are internalized within the cell, thus enabling the assessment of the degree of nanoparticle uptake and their spatial location.

[0055] Example 2

[0056] The steps in this embodiment are basically the same as those in Embodiment 1. This embodiment provides a method for evaluating the uptake of nanoparticles by cells, which is suitable for determining the long-term interaction between nanoparticles and test cells, and includes the following steps:

[0057] S1: 600 nm polydopamine nanoparticles were artificially prepared as nanoparticles, sterilized, and dispersed in sterile water to obtain a sterile aqueous dispersion of polydopamine nanoparticles; then diluted with cell culture medium to obtain a polydopamine nanoparticle cell culture medium dispersion with a concentration of 1 μg / mL, which was ready for use.

[0058] RAW264.7 mouse macrophages were cultured in vitro as standard cells and then cultured at a density of 3 × 10⁻⁶ cells / cell. 4 pcs / cm 2 The standard sample cells were seeded at a density of 1 μg / mL on well plates. After 2 h of seeding, the standard sample cells were activated with 1 μg / mL LPS (lipopolysaccharide). After 12 h of LPS stimulation, the culture medium of the standard sample cells was replaced with polydopamine nanoparticle cell culture medium dispersion. After the replacement, the cells were transferred to a cell culture incubator and cultured for another 3 days. After the culture was completed, the cells were fixed.

[0059] In this step, similar to Example 1, LPS can accurately simulate the "macrophage physiological / pathological activation scenario". It can activate standard sample cells and stimulate the endocytosis-exocytosis cycle between standard sample cells and polydopamine nanoparticles.

[0060] In addition, to ensure the accuracy of the experiment, at least three sets of repeated experiments are set up in this step, and the groups are classified and labeled according to their group numbers.

[0061] Similar to Example 1, CD86 antibody, phalloidin and DAPI nuclear dye were selected and the cell membrane protein marker CD86, cytoskeleton and cell nucleus of the reference cells were stained according to the classified group number to obtain reference cells containing nanoparticles.

[0062] S2: Using a Nikon A1R+ confocal microscope, confocal images of stained reference cells containing nanoparticles were taken. A layer scan was performed along the cell axis at a step size of 2µm. The channels used were DAPI (excitation wavelength 405nm, voltage 120V, laser power 60mW, blue), FITC (excitation wavelength 488nm, voltage 50V, laser power 25mW, green), TRITC (excitation wavelength 561nm, voltage 50V, laser power 25mW, red), and TD (voltage 60V, gray). The resulting confocal immunofluorescence images of the reference cells were obtained.

[0063] The confocal immunofluorescence images were then processed using NIS-Elements (Version 4.50.00) software accompanying the Nikon A1R+ confocal microscope. The images were exported as color images for each channel at each step size in Tagged image format, with the scaling option selected to scale from 12 bits to 16 bits. The images were then exported and saved.

[0064] S3: Using the line analysis tool of the fluorescence quantitative analysis software ZEN, specific structures (including image background, cell structure, and particles) of the processed confocal immunofluorescence images obtained in step S2 are labeled. The labeling includes layer-by-layer labeling of bright-field nanoparticles and layer-by-layer labeling of cell structures in the blue, red, and green channels (one image at 2-micrometer intervals). Each structure in each image is labeled with three or more labels, and the fluorescence values ​​of the background region, cell substructure contour region, and nanoparticle contour region are recorded as RLU1. The fluorescence values ​​of the background region, the cell substructure contour region after background subtraction, and the nanoparticle contour region are recorded as RLU2. Finally, the fluorescence data are further analyzed using the data analysis software Origin / Graphpadprism.

[0065] S4: Frequency analysis of the fluorescence value RLU2 was performed using Graphpad Prism software to obtain a frequency distribution map. Then, statistical analysis was performed according to the classification groups to obtain a selection chart for the scale. The analysis results are as follows: Figure 10 - Figure 13 As shown, the DAPI channel has a scale value of 8000 for distinguishing between the cell nucleus and nanoparticles (leading to an increase in fluorescence value exceeding 8000), the TRITC channel has a scale value of 10000 for distinguishing between the cytoskeleton and nanoparticles (leading to an increase in fluorescence value exceeding 10000), the FITC channel has a scale value of 8000 for distinguishing between the cell membrane and nanoparticles (leading to an increase in fluorescence value exceeding 8000), and the TD channel has a scale value of -21000 for distinguishing between nanoparticles and cells (leading to a decrease in fluorescence value exceeding 21000).

[0066] S5: Repeat steps S1-S3, mixing the cells and nanoparticles, staining, performing tomographic imaging, and exporting images for each channel. Each channel's tomographic image is then calibrated layer by layer according to the aforementioned scale calibration method. After calibrating the corresponding structures, the corresponding fluorescence value RμL1 is stored and processed to obtain the fluorescence value RμL2. Then, line analysis is performed on the data obtained from the cells (including step size 2µm, calibration distance, number of tomographic images, and fluorescence value RμL2) to obtain three-dimensional visual images of nanoparticles, the framework, the membrane, and the cell nucleus. Subsequently, the obtained three-dimensional visual images are analyzed using the scale established in the previous steps. The scale allows for a direct visual representation of the outlines of specific structures on the three-dimensional visual image, and the relationship between cells and particles can be analyzed based on the images of specific structures. Experimental results are as follows: Figure 14 - Figure 17As shown, a three-dimensional visual diagram containing the cytoskeleton, nucleus, cell membrane, and nanoparticles is observed using a scale. Structures with fluorescence intensity below -21000 under bright field are nanoparticles. The red, blue, green, and gray areas in the diagram correspond to the cytoskeleton, nucleus, cell membrane, and nanoparticles, respectively. Furthermore, it can be determined that the nanoparticles are internalized within the cell, thus enabling the assessment of the degree of nanoparticle uptake and their spatial location.

[0067] In summary, the embodiments of the present invention provide a method for evaluating the uptake of nanoparticles by cells. By co-incubating standard sample cells and nanoparticles to construct a scale, and using the scale to visualize the cell structure and nanoparticles in three dimensions, spatial analysis can be performed. This method can effectively evaluate the interaction mode between nanoparticles and cells, solving the problem in the prior art that it is difficult to evaluate whether nanoparticles adhere to the cell surface or are phagocytosed by cells.

[0068] This invention employs tomographic imaging to individually calibrate the cell membrane, cytoskeleton, and nucleus, avoiding problems such as fluorescence quenching that occur when using traditional fluorescence observation or electron microscopy. Furthermore, the data obtained through tomographic imaging is more accurate, and combined with a scale, the spatial location of nanoparticles after being taken up by cells can be precisely determined.

[0069] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for evaluating cellular uptake of nanoparticles, characterized in that, Includes the following steps: S1: Prepare standard sample cells, co-incubate the standard sample cells and nanoparticles, and obtain reference cells containing nanoparticles after staining; The staining is as follows: The substructures of the standard sample cells are stained using specific fluorescent dyes and / or non-specific fluorescent dyes; the substructures include the cell membrane, cytoskeleton, nucleus, and organelles. S2: Perform tomographic imaging on the reference cells to obtain a set of tomographic images; the tomographic imaging includes performing tomographic scanning along the axis of the reference cells using fluorescence imaging software; the set of tomographic images includes the stride of the tomographic images and the number of tomographic images; It also includes performing layer scanning on the substructure using different fluorescence excitation channels, including DAPI channels, FITC channels, TRITC channels, and TD channels; S3: The background, cells and nanoparticles in the set of tomographic images are calibrated, and the fluorescence values ​​of the regions containing the background, cell substructure contours and nanoparticle contours are recorded as RLU1. The background value is subtracted from RLU1 and recorded as RLU2. S4: Frequency distribution analysis of fluorescence value RLU2 was performed to obtain frequency distribution maps of cell substructures and nanoparticles. After analysis, the corresponding scale bars of cell substructures and nanoparticles were obtained. S5: Co-incubate the test sample cells and the nanoparticles, repeat steps S1-S3, and establish a three-dimensional visual image by combining the set of tomographic images obtained in step S2 and the fluorescence values ​​obtained in step S3; determine the interaction relationship between the test sample cells and the nanoparticles by combining the scale.

2. The method for evaluating cellular uptake of nanoparticles according to claim 1, characterized in that, In step S1, the nanoparticles are nanoparticles with self-luminous properties or nanoparticles with artificial fluorescent labels.

3. The method for evaluating cellular uptake of nanoparticles according to claim 2, characterized in that, The organelles also include mitochondria.

4. The method for evaluating cellular uptake of nanoparticles according to claim 1, characterized in that, In step S3, the calibration also includes obtaining the calibration distances of the background region, the cell substructure contour region, and the nanoparticle contour region.

5. The method for evaluating cellular uptake of nanoparticles according to claim 1, characterized in that, Steps S4-S5 include using data processing software to perform frequency distribution analysis and establish a three-dimensional visualization. The data processing software includes GraphPad, Origin, and SPSS.

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