A peptide-metal cluster probe and its application in the preparation of kits for sorting drug-resistant cells and / or quantitative detection of cell membrane proteins.

Abstract

This invention provides a peptide-metal cluster probe and its application in the preparation of kits for sorting drug-resistant cells and / or quantitatively detecting cell membrane proteins, belonging to the field of biomedical technology. This invention prepares a peptide-metal cluster probe targeting a multidrug resistance-related protein (P-glycoprotein, P-gp), and utilizes its fluorescence properties to sort mixed tumor cells with different levels of multidrug resistance into different cell subpopulations. Then, laser ablation inductively coupled plasma mass spectrometry (ICP-MS) is used to detect the expression level of P-gp in individual tumor cells within different subpopulations. This rapid and novel optical-mass spectrometry technique can be used for specific tumor cell phenotypic analysis and anti-tumor drug screening. The quantitative analysis of cell membrane protein expression can lay the analytical foundation for drug screening to reverse drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a polypeptide-metal cluster probe and its application in the preparation of kits for sorting drug-resistant cells and / or quantitative detection of cell membrane proteins. Background Technology

[0002] Currently, chemotherapy is the most widely used treatment method in cancer clinical practice, and multidrug resistance is one of the most important reasons for chemotherapy failure in cancer patients. Adriamycin (ADR) is currently a first-line chemotherapy drug for the treatment of breast cancer. Wild-type breast cancer cells (MCF-7) treated with ADR for a long time will obtain breast cancer cells with a multidrug-resistant phenotype (MCF-7 / ADR), with upregulated P-gp protein expression. Dasatinib (DAS), as a multidrug resistance reversal agent with clinical potential, has also been widely reported in its research on reversing multidrug resistance in breast cancer and has entered the clinical trial stage. Using DAS to reduce P-gp expression in drug-resistant breast cancer cells, a reversal model of drug-resistant breast cancer cells (MCF-7 / ADR / DAS) was obtained.

[0003] Cell surface P-glycoprotein (P-gp) acts as an efflux pump and is one of the most important biomarkers of multidrug resistance. Therefore, accurate quantification of P-gp protein is of great significance for studying the multidrug resistance phenotype of tumor cells. Currently, there are two main types of methods for quantifying P-glycoprotein: flow cytometry and antibody-based detection techniques. However, both have certain limitations. Flow cytometry indirectly studies P-gp's role in drug efflux mechanisms through competitive fluorescent substrates, but is limited by the requirement to analyze suspended live cells. Antibody-based detection techniques (enzyme-linked immunosorbent assay and Western blotting) analyze P-gp protein in cell or tissue lysates, inevitably leading to protein loss or loss of detailed information about drug-resistant cells. On the other hand, in the complex tumor microenvironment where drug resistance occurs, various tumor cells with different degrees of resistance exist. Currently, there is no rapid sorting method for tumor cells with different degrees of resistance, nor is there a method for accurately analyzing the expression level of the membrane protein P-gp in cell subsets with specific drug resistance phenotypes. This undoubtedly hinders the progression of tumor drug resistance and the evaluation of the efficacy of drug resistance reversal agents. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a polypeptide-metal cluster probe that specifically targets P-gp for rapid and accurate sorting of cell subpopulations with different drug resistance levels.

[0005] The present invention also aims to provide a polypeptide-metal cluster probe, and to establish a new method for analyzing the expression of cell membrane proteins by using optical and mass spectrometry based on cluster probes to sort tumor cells with different drug resistance levels. This method can also lay the analytical foundation for drug screening to reverse drug resistance.

[0006] This invention provides a polypeptide-metal cluster probe, comprising a metal cluster and a targeting polypeptide connected by covalent bonds;

[0007] The targeted polypeptide includes polypeptides with amino acid sequences as shown in SEQ ID NO:1 or SEQ ID NO:2.

[0008] Preferably, during the synthesis process, the molar ratio of the metal cluster precursor to the targeting peptide is 1:0.7 to 1.33.

[0009] Preferably, the metal element in the metal cluster includes gold, silver, platinum, or palladium.

[0010] This invention provides the application of the peptide-metal cluster probe in the preparation of kits for sorting or evaluating drug-resistant cells.

[0011] This invention provides the application of the polypeptide-metal cluster probe in the preparation of a kit for quantitative detection of cell membrane protein expression.

[0012] This invention provides a method for rapid sorting of drug-resistant cells and quantification of cell membrane protein expression levels for non-diagnostic purposes using optical and mass spectrometry coupled with the aforementioned peptide-metal cluster probe, comprising the following steps:

[0013] 1) Establish a standard curve for single-cell metal content analysis;

[0014] 2) The polypeptide-metal cluster probe is used to label tumor cells, and the labeled tumor cells are sorted to collect tumor cell subpopulations with different phenotypes;

[0015] 3) The metal content of single cells in each tumor cell subpopulation collected in step 2) was quantitatively detected by laser ablation inductively coupled plasma mass spectrometry. The metal content in single cells was converted into the expression level of P-gp protein to obtain the drug resistance of tumor cells.

[0016] Preferably, the method for analyzing P-gp expression levels in tumor cells with different drug resistance levels as described in step 1) includes the following steps:

[0017] A. A series of gold standard points with gold mass gradients were printed using an inkjet printer. By ablating the gold standard points containing different masses, the mass spectrometry intensity signals corresponding to the gold content were statistically analyzed to establish an analytical standard curve.

[0018] B. Using three breast cancer model cell lines with different P-gp protein expression levels as materials, a series of cell line mixtures with different proportions were prepared;

[0019] C. Label the series of cell line mixtures with the peptide-metal cluster probe, perform flow cytometry cell sorting on the labeled series of cell line mixtures, and collect cell subpopulations with different drug resistance levels;

[0020] D. The gold signal intensity of single cells in the cell subpopulations with different drug resistance levels was obtained by laser ablation inductively coupled plasma mass spectrometry.

[0021] E. Based on the single-cell gold signal intensity and analysis standard curve of cell subpopulations with different drug resistance levels, and based on the number of metal atoms contained in the clusters, the expression level of P-gp protein in cell subpopulations with different drug resistance levels was obtained.

[0022] Preferably, the three breast cancer model cell lines with different P-gp protein expression levels include MCF-7, MCF-7 / ADR, and MCF-7 / ADR / DAS.

[0023] Preferably, the ratio of cell lines in step 2) is 1:1.

[0024] Preferably, in step 3), low drug resistance of tumor cells means that the expression level of P-gp protein in a single tumor cell reaches 80-90 amol;

[0025] Moderate drug resistance in tumor cells refers to a P-gp protein expression level of 361–371 amol in a single tumor cell.

[0026] High drug resistance in tumor cells refers to a P-gp protein expression level of 900–904 amol in a single tumor cell.

[0027] This invention provides a polypeptide-metal cluster probe, comprising a metal cluster and a targeting polypeptide covalently linked; the targeting polypeptide includes a polypeptide with an amino acid sequence as shown in SEQ ID NO:1 or SEQ ID NO:2. Since P-gp protein is a biomarker for tumor drug resistance, this invention uses polypeptides that specifically target P-gp protein as templates for biomineralization to synthesize the aforementioned metal cluster probe. This probe exhibits high targeting specificity, fluorescence, and precision in metal content analysis, providing a powerful tool for sorting drug-resistant cells.

[0028] This invention provides a method for rapid sorting of drug-resistant cells and quantification of cell membrane protein expression levels for non-diagnostic purposes using a combination of optical and mass spectrometry based on a peptide-metal cluster probe. Three breast cancer cell models with P-gp protein expression levels ranging from low to high—MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR—are established. Leveraging the targeting, fluorescence, and precise molecular composition of the peptide-metal cluster probe, a novel in-situ method for analyzing tumor protein biomarkers using optical and mass spectrometry is developed. Flow cytometry is used to separate mixed tumor cells with varying degrees of multidrug resistance into different cell subpopulations, and laser ablation inductively coupled plasma mass spectrometry (ICP-MS) is employed to quantify the P-gp expression levels in these subpopulations. This method is expected to provide a powerful analytical tool for the clinical diagnosis of multidrug resistance. Attached Figure Description

[0029] Figure 1 The detection results of the polypeptide-metal cluster 1 involved in this invention are as follows: a) is a high-resolution transmission electron microscope image of the polypeptide-metal cluster 1 aggregated into nanoparticles under electron irradiation; b) is a matrix-assisted laser desorption / ionization time-of-flight mass spectrum of the polypeptide-metal cluster 1.

[0030] Figure 2 The results are for three model cells. a is the immunoblotting analysis of P-gp expressed in the three model cells (MCF-7, MCF-7 / ADR, MCF-7 / ADR / DAS), and b is the corresponding protein grayscale statistical graph.

[0031] Figure 3 This is a laser confocal characterization diagram of the target recognition of P-gp expressed by the polypeptide-metal cluster 1 on three model cells (MCF-7, MCF-7 / ADR, MCF-7 / ADR / DAS);

[0032] Figure 4 These are sorting diagrams of breast cancer cells with different resistance levels labeled with the peptide-metal cluster 1 using a sorting flow cytometer. a) shows the sorting results of MCF-7 & MCF-7 / ADR cells using the metal cluster probe; b) shows the sorting results of MCF-7 / ADR / DAS & MCF-7 / ADR cells using the metal cluster probe.

[0033] Figure 5 After labeling three cell subpopulations with peptide-metal cluster 1, the gold signal and gold content statistics of single cells in the subpopulations were analyzed using laser ablation inductively coupled plasma mass spectrometry. a) shows the gold signal and corresponding gold content statistics of single cells of MCF-7 and MCF-7 / ADR after ablation and sorting; b) shows the gold signal and corresponding gold content statistics of single cells of MCF-7 / ADR / DAS and MCF-7 / ADR after ablation and sorting.

[0034] Figure 6 The detection results of the polypeptide-metal cluster 2 involved in this invention are as follows: a) is a high-resolution transmission electron microscope image of the polypeptide-metal cluster 2 aggregated into nanoparticles under electron irradiation; b) is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometry image of the polypeptide-metal cluster 2.

[0035] Figure 7 This is a laser confocal characterization diagram of the target recognition of P-gp expressed by the polypeptide-metal cluster 2 in three model cells (MCF-7, MCF-7 / ADR, MCF-7 / ADR / DAS);

[0036] Figure 8 These are sorting diagrams of breast cancer cells with different resistance levels to the peptide-metal cluster 2 label using a sorting flow cytometer; a) shows the sorting results of MCF-7 & MCF-7 / ADR cells using the metal cluster probe; b) shows the sorting results of MCF-7 / ADR / DAS & MCF-7 / ADR cells using the metal cluster probe.

[0037] Figure 9 After labeling three cell subpopulations with peptide-metal cluster 2 as a probe, the gold signal and gold content statistics of single cells in the subpopulations were analyzed by laser ablation inductively coupled plasma mass spectrometry. a shows the gold signal and corresponding gold content statistics of single cells of MCF-7 and MCF-7 / ADR after ablation and sorting; b shows the gold signal and corresponding gold content statistics of single cells of MCF-7 / ADR / DAS and MCF-7 / ADR after ablation and sorting. Detailed Implementation

[0038] This invention provides a polypeptide-metal cluster probe, comprising a metal cluster and a targeting polypeptide connected by covalent bonds;

[0039] The targeted polypeptide includes polypeptides with amino acid sequences as shown in SEQ ID NO:1 (3-Methylbutyryl-Val-Val-Sta-Ala-Sta-Lys-Lys-Lys-Lys-Lys-Tyr-Cys-Cys, where 3-Methylbutyryl and Sta are modifying groups) or SEQ ID NO:2 (Cys-Cys-Tyr-Asn-Asp-Gly-Leu-Leu-Phe-Thr-Trp-Gln-Pro-Ser-Pro).

[0040] In this invention, the molar ratio of the metal cluster precursor to the targeting peptide in the synthetic probe is preferably 1:0.7 to 1.33, more preferably 1:0.8 to 1.2, and most preferably 1:1.

[0041] In this invention, the metal element of the metal cluster is preferably a metal element that is not present or has a very low content in the sample to be tested, such as gold, silver, platinum, or palladium. In this invention, the preparation method of the polypeptide-metal cluster probe preferably includes the following steps:

[0042] The solution containing the metal element was mixed with the targeted peptide solution, the pH of the system was adjusted to alkaline, and the mixture was stirred in the dark to obtain peptide-metal clusters.

[0043] In this invention, the solution containing the metal element includes HAuCl4, AgNO3, H2PtCl6, or PdCl2 solution.

[0044] In this invention, the pH value of the system is preferably 12. The temperature during stirring is preferably 37°C.

[0045] This invention provides the application of the peptide-metal cluster probe in the preparation of kits for sorting or evaluating drug-resistant cells.

[0046] In this invention, given that P-gp protein is a biomarker for tumor drug resistance, the polypeptide-metal cluster probe targets P-gp protein on the cell surface, uses the intrinsic fluorescence properties of the metal clusters and sorting flow cytometry to group cells, obtaining cell subpopulations containing different metal clusters, and then uses the quality information of the metal cluster probe to quantitatively detect P-gp protein, thereby obtaining the drug resistance of the cell subpopulations.

[0047] This invention provides the application of the polypeptide-metal cluster probe in the preparation of a kit for quantitative detection of cell membrane protein expression.

[0048] In this invention, a standard curve for single-cell analysis is established by printing a series of gold standard points with varying gold quality gradients using an inkjet printer. By ablating these gold standard points containing different qualities, the gold content signals are statistically analyzed to establish the analytical standard curve. Subsequently, at the single-cell level, the content of P-gp protein expressed in tumor cells within a selected cell subpopulation is analyzed using laser ablation inductively coupled plasma (ICP-PAP) technology.

[0049] This invention provides a method for rapid sorting of drug-resistant cells and quantification of cell membrane protein expression levels for non-diagnostic purposes using optical and mass spectrometry coupled with the aforementioned peptide-metal cluster probe, comprising the following steps:

[0050] 1) Establish a standard curve for single-cell metal content analysis;

[0051] 2) The polypeptide-metal cluster probe is used to label tumor cells, and the labeled tumor cells are sorted to collect tumor cell subpopulations with different phenotypes;

[0052] 3) The metal content of single cells in each tumor cell subpopulation collected in step 2) was quantitatively detected by laser ablation inductively coupled plasma mass spectrometry. The metal content in single cells was converted into the expression level of P-gp protein to obtain the drug resistance of tumor cells.

[0053] In this invention, the method for analyzing P-gp expression levels in tumor cells with different drug resistance levels preferably includes the following steps:

[0054] A. A series of gold standard points with varying gold mass gradients were printed using an inkjet printer. By ablating these gold standard points containing different masses, the mass spectrometry intensity signals corresponding to the gold content were statistically analyzed to establish an analytical standard curve.

[0055] B. Using three breast cancer model cell lines with different P-gp protein expression levels as materials, a series of cell line mixtures with different proportions were prepared;

[0056] C. Label the series of cell line mixtures with the peptide-metal cluster probe, perform flow cytometry cell sorting on the labeled series of cell line mixtures, and collect cell subpopulations with different drug resistance levels;

[0057] D. The gold signal intensity of single cells in the cell subpopulations with different drug resistance levels was obtained by laser ablation inductively coupled plasma mass spectrometry.

[0058] E. Based on the single-cell gold signal intensity and analysis standard curve of cell subpopulations with different drug resistance levels, and based on the number of metal atoms contained in the clusters, the expression level of P-gp protein in cell subpopulations with different drug resistance levels was obtained.

[0059] In this invention, the preferred three breast cancer cell lines with different P-gp protein expression levels include MCF-7, MCF-7 / ADR, and MCF-7 / ADR / DAS. MCF-7 represents breast cancer cells with low P-gp protein expression. MCF-7 / ADR represents drug-resistant breast cancer cells (MCF-7 / ADR) with upregulated P-gp protein expression. MCF-7 / ADR / DAS represents breast cancer cells treated with DAS to reduce drug resistance; its P-gp protein expression level falls between that of MCF-7 and MCF-7 / ADR.

[0060] In this invention, the mixing ratio of the cell lines is 1:1.

[0061] In this invention, low drug resistance of tumor cells preferably refers to a P-gp protein expression level of 80-90 amol in a single tumor cell; moderate drug resistance of tumor cells preferably refers to a P-gp protein expression level of 361-371 amol in a single tumor cell; and high drug resistance of tumor cells preferably refers to a P-gp protein expression level of 900-904 amol in a single tumor cell.

[0062] Using the method of this invention, cells with different phenotypes can be rapidly sorted from mixed model cells or tissue digestion cells, and the expression levels of membrane proteins related to cell phenotypes can be accurately quantified.

[0063] The following examples illustrate the application of a polypeptide-metal cluster probe provided by the present invention in the preparation of kits for sorting drug-resistant cells and / or quantitative detection of cell membrane proteins. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] A method for preparing a polypeptide-metal cluster probe 1 and for sorting drug-resistant cells and / or quantitatively detecting cell membrane proteins for non-diagnostic purposes.

[0066] A peptide-metal cluster 1 specifically recognizing P-gp was prepared using a peptide sequence targeting the P-gp protein as a template. Based on this metal cluster, breast cancer cell subpopulations with different drug resistance levels were sorted, and P-gp in different cell subpopulations was quantified. The targeting peptide sequence was 3-Methylbutyryl-Val-Val-Sta-Ala-Sta-Lys-Lys-Lys-Lys-Lys-Tyr-Cys-Cys (Peptide 1), and the specific preparation steps are as follows:

[0067] (1) Preparation of a peptide-metal cluster probe that specifically recognizes P-gp protein 1

[0068] 178 μL of chloroauric acid (25 mM aqueous solution) was slowly added dropwise to a peptide solution (1.5 mM) containing magnetic stirring. Subsequently, 1740 μL of sodium hydroxide solution (0.5 M) was added dropwise to the above reaction system. After reacting at 37 °C in the dark for 12 h, the peptide-metal cluster probe 1 was obtained. The probe was further purified by ultrafiltration in an ultrafiltration tube with a molecular weight cutoff of 3 kDa.

[0069] like Figure 1 As shown in Figure a, the peptide-gold clusters form nanoparticles under electron beam bombardment, which proves that the precursor clusters of the nanoparticles have good dispersibility.

[0070] like Figure 1As shown in Figure b, the precise molecular composition of the peptide-gold cluster, characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, is Au. 25 (Peptide)9.

[0071] (2) Validation of the targeting of peptide-metal cluster 1

[0072] First, MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR cell models were established. Cells were lysed, and the relative expression levels of P-gp protein in the three cell types were analyzed using protein immunoblotting. The results are as follows: Figure 2 As shown, the relative expression abundance of P-gp protein indicates the successful establishment of cell models with different levels of drug resistance.

[0073] 1.0×10 4 MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR cells were uniformly seeded in confocal microscopy dishes with a glass bottom and cultured at 37°C for 24 h. After culturing, the cells were washed three times with PBS. At room temperature, the cells were fixed with 4.0% paraformaldehyde for 15 min and then washed three times with PBS. Non-specific sites on the cells were blocked with 3.0% BSA for 30 min, followed by washing five times with PBS. The cells were then incubated with 15 μM peptide-metal cluster 1 at room temperature for 30 min. Unlabeled cells served as a control group. After completing the above steps, the nuclei of MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR cells were stained with 0.5 μg / mL DAPI for 5 min, followed by washing three times with PBS. 1.0 mL of PBS was added to each culture dish. The labeling effect and fluorescence intensity of the cells under a laser confocal microscope were compared to verify the specificity of the peptide-metal cluster targeting P-gp.

[0074] like Figure 3 As shown, the results indicate that peptide-metal cluster 1 can label the cell membranes of three cell types. Comparison of fluorescence intensity on the cell membranes under the same laser confocal microscopy imaging conditions effectively demonstrates the good targeting ability of the peptide-metal cluster probe 1 for P-gp protein. The probe exhibits weak red fluorescence on the MCF-7 cell membrane, indicating low P-gp expression in this cell. Compared to MCF-7, the fluorescence intensity on the MCF-7 / ADR cell membrane is significantly increased, indicating a significant increase in P-gp protein expression in drug-resistant cells. The red fluorescence intensity on the MCF-7 / ADR / DAS cell membrane is significantly weaker than that on MCF-7 / ADR, indicating that the model reversal agent DAS can reduce the P-gp expression level in drug-resistant tumor cells. Cell fluorescence intensity demonstrates that the peptide-metal cluster probe 1 has the ability to target P-gp protein on the cell membrane.

[0075] (3) Sorting flow cytometry to sort mixed cell subpopulations

[0076] MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR were seeded in 6-well plates. After 24 hours, the culture medium was removed, and the cells were washed three times with PBS. 2 mL of PBS-EDTA solution was added to each well of the 6-well plate to digest the cells. After digestion for 10 min, the cells were centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1 mL of PBS in a flow cytometry tube. The three cell types were counted using flow cytometry, and 2 mL of cell suspension was added to centrifuge tubes at cell ratios of MCF-7:MCF-7 / ADR = 1:1 and MCF-7 / ADR / DAS:MCF-7 / ADR = 1:1. The cells were then collected by centrifugation at 800 rpm for 5 min. Add 1 mL of 4.0% paraformaldehyde to each centrifuge tube to fix cells for 15 min. Centrifuge at 800 rpm to remove the fixative and collect the cells. Wash the cell surface three times with PBS. Next, block with 3.0% BSA at room temperature for 30 min, and wash the cells three times with PBS. Incubate the cells with a 15 μM peptide-metal cluster probe 1 at room temperature for 30 min, and then wash the cells thoroughly with PBS three times to remove excess gold cluster probes after specific binding. After the final centrifugation, resuspend each group of cells in 1 mL PBS in a flow cytometry tube and use sorting flow cytometry to separate the mixed cells.

[0077] like Figure 4 The results show the flow cytometry sorting of equal cell populations MCF-7 & MCF-7 / ADR and MCF-7 / ADR / DAS & MCF-7 / ADR using a sorting flow cytometer. The peptide-metal cluster 1 was used as a probe. Figure 4 Above cell a is the drug-resistant cell subpopulation MCF-7 / ADR with high P-gp protein expression, and below is the wild-type MCF-7 cell subpopulation with low P-gp protein expression. Figure 4 Above b is the drug-resistant cell subpopulation MCF-7 / ADR with high P-gp protein expression. Below is the cell subpopulation MCF-7 / ADR / DAS whose drug resistance was reversed using DAS. These results demonstrate the effectiveness and feasibility of using peptide-metal clusters as probes for drug-resistant cell sorting in this embodiment.

[0078] (4) Content of P-gp protein in single cells of cell subpopulations after laser ablation inductively coupled plasma mass spectrometry quantitative sorting

[0079] First, a standard curve for single-cell analysis was established by printing a series of gold standard points with varying gold quality gradients using an inkjet printer. By ablating these gold standard points containing different qualities, the gold content signals were statistically analyzed to establish an analytical standard curve relating gold quality to gold signal intensity.

[0080] Following the flow cytometry cell sorting steps described above, the cell subpopulations obtained by sorting the mixed cell populations MCF-7 & MCF-7 / ADR were MCF-7 and MCF-7 / ADR, and the cell subpopulations obtained by sorting the mixed cell populations MCF-7 / ADR / DAS & MCF-7 / ADR were MCF-7 / ADR / DAS and MCF-7 / ADR. 10 μL of cell suspension from each sorted cell subpopulation was pipetted onto a clean glass slide. The slide was then allowed to air dry at room temperature. Due to adsorption, the air-dried cells adhered tightly to the glass slide. Laser ablation inductively coupled plasma mass spectrometry (ICP-MS) was used to quantitatively analyze the gold content on individual cells within the sorted cell populations, thereby quantifying the expression level of P-gp protein on individual cells.

[0081] like Figure 5 Figure a shows the gold mass spectrometry signal on the MCF-7 and MCF-7 / ADR subpopulations after sorting from the mixed cell populations MCF-7 and MCF-7 / ADR. The average Au mass bound to the surface of each MCF-7 cell is 405 ± 105 fg, and the average Au mass bound to the surface of each MCF-7 / ADR cell is 4399 ± 565 fg. Based on the composition of peptide-metal cluster 1, which is Au... 25 (Peptide)9, the average amount of P-gp protein expressed on the surface of each MCF-7 cell was 82±21 amol, and the average amount of P-gp protein expressed on the surface of each MCF-7 / ADR cell was 893±114 amol.

[0082] Figure 5 Image b shows the gold mass spectrometry signal on single cells in the sorted subpopulations MCF-7 / ADR / DAS and MCF-7 / ADR of the mixed cell population MCF-7 / ADR / DAS & MCF-7 / ADR. The average Au mass on the surface of each MCF-7 / ADR / DAS cell is 1826±168 fg, and the average Au mass on the surface of each MCF-7 / ADR cell is 4376±531 fg. The composition of the bound clusters is Au. 25 (Peptide)9, the average amount of P-gp protein expressed on the surface of each MCF-7 / ADR / DAS cell was 370±34 amol, and the average amount of P-gp protein expressed on the surface of each MCF-7 / ADR cell was 888±107 amol.

[0083] Example 2

[0084] A method for preparing a polypeptide-metal cluster probe 2 and for sorting drug-resistant cells and / or quantitatively detecting cell membrane proteins for non-diagnostic purposes.

[0085] A peptide-metal cluster 2 specifically targeting the P-gp protein was prepared using the target peptide sequence as a template. Based on this cluster, different drug-resistant breast cancer cell subpopulations were sorted, and P-gp in each subpopulation was quantified. The targeting peptide sequence was Cys-Cys-Tyr-Asn-Asp-Gly-Leu-Leu-Phe-Thr-Trp-Gln-Pro-Ser-Pro.

[0086] (1) Preparation of peptide-metal cluster probes that specifically recognize P-glycoprotein 2

[0087] 200 μL of chloroauric acid (25 mM aqueous solution) was slowly added dropwise to a 1.3 mM peptide 2 solution containing magnetic stirring. Subsequently, 1860 μL of sodium hydroxide solution (0.5 M) was added dropwise to the reaction system. The reaction was carried out at 37°C in the dark for 12 h to obtain the peptide-metal cluster probe 2. The probe was further purified by ultrafiltration in an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Figure 6 As shown in Figure a, the polypeptide-gold clusters form nanoparticles under electron beam bombardment, demonstrating the good dispersibility of the precursor clusters used as nanoparticles. Figure 6 As shown in Figure b, the precise molecular composition of the peptide-gold cluster, characterized by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry, is Au. 26 (Peptide) 10 .

[0088] (2) Validation of the targeting ability of peptide-metal clusters

[0089] 1.0×10 4 MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR cells were uniformly seeded in confocal microscopy dishes with a glass bottom and cultured in a cell culture incubator for 24 h. After culturing, the cells were washed three times with PBS. At room temperature, the cells were fixed with 4.0% paraformaldehyde for 15 min and then washed three times with PBS. Non-specific sites were blocked with 3.0% BSA for 30 min, followed by washing five times with PBS. Cells were incubated with 15 μM peptide-metal cluster 2 at room temperature for 30 min. Unlabeled cells were used as a control group. After completing the above steps, the nuclei of MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR cells were stained with 0.5 μg mL⁻¹ of the nuclear dye DAPI for 5 min. 1.0 mL of PBS was added to the culture dish. The labeling effect of each group of cells and the fluorescence intensity of the cells under a laser confocal microscope were compared to verify the targeting specificity of peptide-metal clusters to P-gp.

[0090] like Figure 7As shown, the results indicate that peptide-metal cluster 2 can label the cell membranes of three cell types. Comparison of fluorescence intensity on the cell membranes under the same laser confocal microscopy imaging conditions effectively demonstrates the good targeting ability of the peptide-metal cluster probe 2 for P-gp protein. The probe exhibits weak red fluorescence on the MCF-7 cell membrane, indicating low P-gp expression in this cell. Compared to MCF-7, the fluorescence intensity on the MCF-7 / ADR cell membrane is significantly increased, indicating a significant increase in P-gp protein expression in drug-resistant cells. The red fluorescence intensity of the MCF-7 / ADR / DAS cell membrane is significantly weaker than that of MCF-7 / ADR, indicating that the model reversal agent DAS can reduce the P-gp expression level in drug-resistant tumor cells. Cell fluorescence intensity demonstrates that the peptide-metal cluster probe 2 has the ability to target P-gp protein on the cell membrane.

[0091] (3) Sorting flow cytometry to sort mixed cell subpopulations

[0092] MCF-7, MCF-7 / ADR / DAS, and MCF-7 / ADR were seeded in 6-well plates. After 24 hours, the culture medium was removed, and the cells were washed three times with PBS. 2 mL of PBS-EDTA solution was added to each well of the 6-well plate to digest the cells. After digestion for 10 min, the cells were centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cells were resuspended in 1 mL of PBS in a flow cytometry tube. The three cell types were counted using flow cytometry, and 2 mL of cell suspension was added to centrifuge tubes according to cell ratios of MCF-7:MCF-7 / ADR = 1:1 and MCF-7 / ADR / DAS:MCF-7 / ADR = 1:1. The cells were then collected by centrifugation at 800 rpm for 5 min. Add 1 mL of 4.0% paraformaldehyde to each centrifuge tube to fix cells for 15 min. Centrifuge at 800 rpm to remove the fixative and collect the cells. Wash the cell surface three times with PBS. Next, block with 3.0% BSA at room temperature for 30 min, and wash the cells three times with PBS. Incubate the cells with a 15 μM peptide-metal cluster probe 2 solution at room temperature for 30 min, and then wash the cells thoroughly three times with PBS to remove excess gold cluster probes after specific binding. After the final centrifugation, resuspend each group of cells in 1 mL PBS in a flow cytometry tube and use sorting flow cytometry to separate the mixed cells into clusters.

[0093] like Figure 8 The results show the flow cytometry sorting of equal cell populations MCF-7 & MCF-7 / ADR and MCF-7 / ADR / DAS & MCF-7 / ADR using a sorting flow cytometer. The peptide-metal cluster 2 was used as a probe. Figure 8Above cell a is the drug-resistant cell subpopulation MCF-7 / ADR with high P-gp protein expression, and below is the wild-type MCF-7 cell subpopulation with low P-gp protein expression. Figure 8 Above b is the drug-resistant cell subpopulation MCF-7 / ADR with high P-gp protein expression. Below is the cell subpopulation MCF-7 / ADR / DAS whose drug resistance was reversed using DAS. These results demonstrate the effectiveness and feasibility of using peptide-metal clusters as probes for drug-resistant cell sorting in this embodiment.

[0094] (4) Content of P-gp protein in single cells of cell subpopulations after laser ablation inductively coupled plasma mass spectrometry quantitative sorting

[0095] First, a standard curve for single-cell analysis was established by printing a series of gold standard points with varying gold quality gradients using an inkjet printer. By ablating these gold standard points containing different qualities, the gold content signals were statistically analyzed to establish an analytical standard curve relating gold quality to gold signal intensity.

[0096] Following the flow cytometry cell sorting steps described above, the cell subpopulations obtained by sorting the mixed cell populations MCF-7 & MCF-7 / ADR were MCF-7 and MCF-7 / ADR, and the cell subpopulations obtained by sorting the mixed cell populations MCF-7 / ADR / DAS & MCF-7 / ADR were MCF-7 / ADR / DAS and MCF-7 / ADR. 10 μL of cell suspension from each sorted cell subpopulation was pipetted onto a clean glass slide. The slides were then allowed to air dry at room temperature. Due to adsorption, the air-dried cells adhered tightly to the glass slide. Laser ablation inductively coupled plasma mass spectrometry (ICP-MS) was used to quantitatively analyze the gold content on individual cells within the sorted cell populations, thereby quantifying the expression level of P-gp protein on individual cells. Figure 9 Figure a shows the gold mass spectrometry signal on the MCF-7 and MCF-7 / ADR subpopulations after sorting from the mixed cell populations MCF-7 and MCF-7 / ADR. The average Au mass bound to the surface of each MCF-7 cell is 407±195 fg, and the average Au mass bound to the surface of each MCF-7 / ADR cell is 3983±736 fg. Based on the composition of peptide-metal cluster 1, which is Au... 26 (Peptide) 10 The average amount of P-gp protein expressed on the surface of each MCF-7 cell was 80±38 amol, and the average amount of P-gp protein expressed on the surface of each MCF-7 / ADR cell was 783±143 amol. Figure 9Image b shows the gold mass spectrometry signal on single cells in the sorted subpopulations MCF-7 / ADR / DAS and MCF-7 / ADR of the mixed cell population MCF-7 / ADR / DAS & MCF-7 / ADR. The average Au mass on the surface of each MCF-7 / ADR / DAS cell is 1857±271 fg, and the average Au mass on the surface of each MCF-7 / ADR cell is 3966±831 fg. The composition of the bound clusters is Au. 26 (Peptide) 10 The average amount of P-gp protein expressed on the surface of each MCF-7 / ADR / DAS cell was 362±53 amol, and the average amount of P-gp protein expressed on the surface of each MCF-7 / ADR cell was 778±144 amol.

[0097] As described in the above embodiments, this invention relates to a method for rapidly sorting cells with specific phenotypes and quantifying the expression levels of membrane proteins associated with specific cell phenotypes using an optical-mass spectrometry technique based on metal cluster probes. Specifically, it involves utilizing the fluorescence properties of peptide-metal cluster probes targeting multidrug resistance-related proteins (P-glycoprotein, P-gp). First, flow cytometry is used to separate mixed tumor cells with different levels of multidrug resistance into different cell subpopulations. Then, using the precise mass information of the peptide-metal cluster probes, laser ablation inductively coupled plasma mass spectrometry is employed to quantify the expression levels of P-gp in individual tumor cells from different subpopulations. This invention is of great significance for assessing tumor drug resistance progression, screening multidrug resistance reversal agents, and optimizing cancer treatment regimens.

[0098] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide-metal cluster probe, characterized in that, This includes metal clusters and targeting peptides linked by covalent bonds; The targeted polypeptide includes polypeptides with amino acid sequences as shown in SEQ ID NO:1 or SEQ ID NO:

2.

2. The polypeptide-metal cluster probe according to claim 1, characterized in that, During the synthesis process, the molar ratio of the precursor of the metal cluster to the targeting peptide is 1:0.7 to 1.

33.

3. The polypeptide-metal cluster probe according to claim 1, characterized in that, The metal elements in the metal clusters include gold, silver, platinum, or palladium.

4. The use of the polypeptide-metal cluster probe according to any one of claims 1 to 3 in the preparation of a kit for sorting or evaluating drug-resistant cells.

5. The use of the polypeptide-metal cluster probe according to any one of claims 1 to 3 in the preparation of a kit for quantitative detection of cell membrane protein expression.

6. A method for rapid sorting of drug-resistant cells and quantification of cell membrane protein expression levels for non-diagnostic purposes using optical and mass spectrometry coupled with the polypeptide-metal cluster probe according to any one of claims 1 to 3, characterized in that, Includes the following steps: 1) Establish a standard curve for single-cell metal content analysis; 2) The polypeptide-metal cluster probe is used to label tumor cells, and the labeled tumor cells are sorted to collect tumor cell subpopulations with different phenotypes; 3) The metal content of single cells in each tumor cell subpopulation collected in step 2) was quantitatively detected by laser ablation inductively coupled plasma mass spectrometry. The metal content in single cells was converted into the expression level of P-gp protein to obtain the drug resistance of tumor cells.

7. The method according to claim 6, characterized in that, The method for analyzing drug resistance based on P-gp expression levels in tumor cells includes the following steps: A. A series of gold standard points with gold mass gradients were printed using an inkjet printer. By ablating the gold standard points containing different masses, the mass spectrometry intensity signals corresponding to the gold content were statistically analyzed to establish an analytical standard curve. B. Using three breast cancer model cell lines with different P-gp protein expression levels as materials, a series of cell line mixtures with different proportions were prepared; C. Label the series of cell line mixtures with the peptide-metal cluster probe, perform flow cytometry sorting on the labeled series of cell line mixtures, and collect cell subpopulations with different drug resistance levels; D. The gold signal intensity of single cells in the cell subpopulations with different drug resistance levels was obtained by laser ablation inductively coupled plasma mass spectrometry. E. Based on the single-cell gold signal intensity and analysis standard curve of cell subpopulations with different drug resistance levels, and based on the number of metal atoms contained in the clusters, the expression level of P-gp protein in cell subpopulations with different drug resistance levels was obtained.

8. The method according to claim 7, characterized in that, Three breast cancer cell lines with different levels of P-gp protein expression include MCF-7, MCF-7 / ADR, and MCF-7 / ADR / DAS.

9. The method according to any one of claims 6 to 8, characterized in that, In step 3), low drug resistance in tumor cells refers to a P-gp protein expression level of 80–90 amol in a single tumor cell. Moderate drug resistance in tumor cells refers to a P-gp protein expression level of 361–371 amol in a single tumor cell. High drug resistance in tumor cells refers to a P-gp protein expression level of 900–904 amol in a single tumor cell.

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