A protein microsphere inhibitor and its preparation method and application

Abstract

The present invention relates to the field of biomedicine and specifically discloses a protein microsphere inhibitor, its preparation method, and application. The protein microsphere inhibitor has the general formula XPinβ, wherein XPin represents an XPO1 inhibitor and β represents a β-catenin inhibitor. A gold adjuvant serves as a nanocarrier to carry the XPO1 inhibitor and the β-catenin inhibitor. The protein microsphere inhibitor provided by the present invention can inhibit melanoma cell proliferation, promote melanoma cell apoptosis, and inhibit the growth of melanoma homografts, and can be used to prepare a drug for treating melanoma.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a protein microsphere inhibitor, a preparation method and an application thereof. Background Art

[0002] Malignant tumors have become a serious threat to human health worldwide. Although emerging therapies such as targeted therapy and immunotherapy have promoted progress in tumor treatment, these methods are generally limited in their effectiveness in prolonging patient survival. The complexity of cancer treatment lies in the fact that there are few effective drugs that can directly target the key factors of the disease. Researchers have found that by combining different drugs, the deficiencies of single therapies can be supplemented, thereby improving the treatment effect of cancer. Therefore, it is particularly urgent to develop new drug combinations that can significantly improve the treatment effect of melanoma.

[0003] Melanoma, commonly referred to as malignant melanoma, is a highly malignant tumor originating from melanocytes. It primarily develops in the skin, but can also occur in mucous membranes and internal organs, accounting for approximately 3% of all tumors. Cutaneous malignant melanoma ranks third among skin malignancies. Malignant melanoma can develop from congenital or acquired benign melanocytic nevi, from malignant transformation of dysplastic nevi, or as a de novo occurrence. In recent years, the incidence and mortality rates of malignant melanoma have increased annually, and the age of death is younger than that of other solid tumors. Malignant melanoma lacks specific treatment options other than early surgical excision, resulting in a poor prognosis. Therefore, early diagnosis and treatment of malignant melanoma are extremely important.

[0004] XPO1 is the most extensively studied nuclear export protein, abnormally expressed in various tumors, making it a novel target for cancer therapy. Currently, several XPO1 inhibitors have been developed as cancer treatments, but they suffer from significant toxicity and poor single-agent efficacy, and their effectiveness in melanoma is also suboptimal. Therefore, there is an urgent need to develop an effective treatment for melanoma. Summary of the Invention

[0005] To develop a product for treating melanoma, the present invention provides a protein microsphere inhibitor, its preparation method, and its application. The protein microsphere inhibitor provided by the present invention can inhibit melanoma cell proliferation, promote melanoma cell apoptosis, and inhibit the growth of melanoma homografts, and can be used to prepare a drug for treating melanoma.

[0006] The present invention provides a protein microsphere inhibitor having the general formula of XPinβ, wherein XPin represents an XPO1 inhibitor, which is a 96-110 functional fragment of the polypeptide Apoptin and is modified with Ac and NH2 at the head and tail; β represents a β-catenin inhibitor, and the XPO1 inhibitor and the β-catenin inhibitor are carried by nanocarriers.

[0007] The amino acid sequence of the XPO1 inhibitor is: Ac-RVSELKESLITTTPSC-NH2.

[0008] The present invention utilizes a nano-delivery carrier system to self-assemble a substance targeting XPO1 and a β-catenin inhibitor into a protein microsphere drug capable of dual-targeted inhibition of XPO1 and β-catenin, thereby achieving safe and effective treatment of melanoma.

[0009] Furthermore, the β-catenin inhibitor is carnosic acid.

[0010] The present invention also provides a method for preparing the protein microsphere inhibitor, comprising the following steps:

[0011] Adding an XPO1 inhibitor to a tetrachloroauric acid solution and adjusting the pH to 6.0-7.0 to obtain a mixture;

[0012] Vitamin C solution is added to the mixture, reacted at 23°C-27°C for 50-70 minutes, and the excess reactant is removed by dialysis to form XPinα solution;

[0013] Add carnosic acid solution and VC solution to XPinα solution, react at 24-27°C for 50-70 minutes, then add polyallylamine hydrochloride solution, centrifuge and concentrate to remove excess reactants to obtain XPinβ.

[0014] Furthermore, the XPO1 inhibitor is an XPin polypeptide;

[0015] The amino acid sequence of the XPin polypeptide is: Ac-RVSELKESLITTTPSC-NH2.

[0016] Furthermore, the molar ratio of the tetrachloroauric acid to the XPO1 inhibitor is 1:1 to 2.5.

[0017] Furthermore, the final concentration of the vitamin C solution is 0.08M-0.12M.

[0018] Furthermore, the concentration of the carnosic acid solution is 3.32 mg / ml.

[0019] Furthermore, the concentration ratio of the XPinα solution to the carnosic acid solution is 1.5 to 2:1.

[0020] Furthermore, the molar ratio of the carnosic acid solution to the polyallylamine hydrochloride solution is 3 to 4.5:1.

[0021] The present invention also provides a use of the protein microsphere inhibitor in preparing a drug for treating melanoma. The protein microsphere inhibitor is used to prepare a drug for inhibiting tumor cell proliferation and / or promoting tumor cell apoptosis.

[0022] Furthermore, the protein microsphere inhibitor is used to prepare a drug for inhibiting the growth of melanoma homografts.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] This study utilizes a nano-delivery system to self-assemble an XPO1-targeting peptide and the β-catenin inhibitor CA into a protein microsphere drug capable of dual-targeted inhibition of XPO1 and β-catenin. The drug's physicochemical properties, biological functions, and in vitro efficacy were then validated. This provides new insights and strategies for developing more effective and safer cancer treatments.

[0025] The protein microsphere inhibitor prepared by the present invention can inhibit melanoma cell proliferation, promote melanoma cell apoptosis, and inhibit the growth of melanoma homografts, and can be used to prepare drugs for treating melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Transmission electron microscopy (TEM) image and HRTEM analysis of XPinβ prepared in Example 1;

[0028] In the figure, (a) is a transmission electron microscope TEM image of XPinβ prepared in Example 1;

[0029] (b) HRTEM analysis of XPinβ prepared in Example 1;

[0030] (c) is the Au elemental analysis diagram of XPinβ prepared in Example 1;

[0031] (d) is the N elemental analysis diagram of XPinβ prepared in Example 1;

[0032] (e) is the S elemental analysis diagram of XPinβ prepared in Example 1;

[0033] (f) is the O element analysis diagram of XPinβ prepared in Example 1.

[0034] Figure 2 Particle size and potential detection of XPinβ prepared in Example 1 of the present invention;

[0035] In the figure, (a) is the particle size test result of XPinβ prepared in Example 1;

[0036] (b) is the potential detection result of XPinβ prepared in Example 1.

[0037] Figure 3 This is the UV-visible spectrum analysis of XPinβ prepared in Example 1.

[0038] Figure 4 This is the Fourier transform infrared spectroscopy analysis of XPinβ prepared in Example 1 of the present invention.

[0039] Figure 5 Flow cytometry analysis of XPinβ uptake in melanoma cells after treatment with different endocytosis inhibitors; ***P<0.001.

[0040] In the figure, (a) is the negative control cell without any treatment (only nuclear DNA was stained);

[0041] (b) is a flow cytometric analysis of the XPinβ uptake rate in melanoma cells not treated with any inhibitors;

[0042] (c) is a flow cytometric analysis of the XPinβ uptake rate in melanoma cells treated with the inhibitor Chlorpromazine;

[0043] (d) is a flow cytometric analysis of the XPinβ uptake rate in melanoma cells treated with the inhibitor Dynasore;

[0044] (e) is a flow cytometric analysis of the XPinβ uptake rate in melanoma cells treated with the inhibitor Genistein;

[0045] (f) is a flow cytometric analysis of the XPinβ uptake rate in melanoma cells treated with the inhibitor Filipin;

[0046] (g) is a flow cytometric analysis of the XPinβ uptake rate in melanoma cells treated with the inhibitor Amiloride;

[0047] (h) Flow cytometric analysis of XPinβ uptake in melanoma cells treated with the inhibitor EIPA.

[0048] (i) Effects of different inhibitors on the uptake rate of XPinβ.

[0049] Figure 6 Fluorescence experiments were performed to detect the uptake of XPinβ in cells after different endocytosis inhibition treatments, 50 μ m ; *** P < 0.001;

[0050] In the figure, (a) is a fluorescence image of the uptake rate of XPinβ by melanoma cells not treated with any inhibitors;

[0051] (b) is a fluorescence graph showing the uptake rate of XPinβ in melanoma cells without any inhibitor treatment;

[0052] (c) Fluorescence image of XPinβ uptake rate in melanoma cells treated with the inhibitor Chlorpromazine;

[0053] (d) Fluorescence image of XPinβ uptake rate in melanoma cells treated with the inhibitor Dynasore;

[0054] (e) is a fluorescence graph showing the uptake rate of XPinβ in melanoma cells treated with the inhibitor Genistein;

[0055] (f) is a fluorescence image of the uptake rate of XPinβ in melanoma cells treated with the inhibitor Filipin;

[0056] (g) is a fluorescence image of the uptake rate of XPinβ in melanoma cells treated with the inhibitor Amiloride;

[0057] (h) Fluorescence image of the uptake rate of XPinβ in melanoma cells treated with the inhibitor EIPA.

[0058] Figure 7 The colocalization of XPinβ and XPO1 is shown, where XPinβ is shown in green, XPO1 is shown in red, and FITC-Drug is FITC-Control and FITC-XPinβ, respectively. Scale bar: 25 μm;

[0059] In the figure, A is a confocal microscopy image of the colocalization of XPinβ and XPO1;

[0060] B is the correlation coefficient of colocalization between control drugs and XPO1;

[0061] C is the correlation coefficient of colocalization between XPinβ and XPO1.

[0062] Figure 8 Western blotting was used to detect the changes in protein levels of np-β-catenin, C-myc and CyclinD1 after different treatments.

[0063] Figure 9 is the effect of XPinβ on melanoma cell viability, CA indicates carnosic acid;

[0064] In the figure, A shows the effect of XPinβ on the viability of melanoma cells A375;

[0065] B shows the effect of XPinβ on the viability of melanoma cells B16F10;

[0066] C shows the effect of XPinβ on the viability of melanoma cells WM35.

[0067] Figure 10 To observe the effect of XPinβ on the proliferation of different melanoma cells for clone formation experiments;

[0068] In the figure, A shows the effects of CA, XPinα, and XPinβ on the proliferation of melanoma cells A375;

[0069] B shows the effects of CA, XPinα and XPinβ on the proliferation of melanoma cells B16F10;

[0070] C shows the effects of CA, XPinα and XPinβ on the proliferation of melanoma cells WM35.

[0071] Figure 11 The effect of XPinβ prepared in Example 1 of the present invention on apoptosis of melanoma cells A375;

[0072] In the figure, (a) is the flow cytometry graph of the Control group in melanoma A375 cells;

[0073] (b) is a flow cytometry graph of the CA-treated group in melanoma A375 cells;

[0074] (c) is the flow cytometry graph of the XPinα-treated group in melanoma A375 cells;

[0075] (d) is the flow cytometry graph of the XPinβ-treated group in melanoma A375 cells;

[0076] (e) is the flow cytometry statistics of early, late and total apoptosis in Figures (a)-(d).

[0077] Figure 12 The effect of XPinβ prepared in Example 1 of the present invention on apoptosis of melanoma cells B16F10;

[0078] In the figure, (a) is the flow cytometry graph of the Control group in melanoma B16F10 cells;

[0079] (b) is a flow cytometry graph of the CA-treated group in melanoma B16F10 cells;

[0080] (c) is the flow cytometry graph of the XPinα-treated group in melanoma B16F10 cells;

[0081] (d) is the flow cytometry graph of the XPinβ-treated group in melanoma B16F10 cells;

[0082] (e) is the flow cytometry statistics of early, late and total apoptosis in Figures (a)-(d).

[0083] Figure 13 The effect of XPinβ prepared in Example 1 of the present invention on apoptosis of melanoma cells WM35;

[0084] In the figure, (a) is the flow cytometry graph of the Control group in melanoma WM35 cells;

[0085] (b) is a flow cytometry graph of the CA-treated group in melanoma WM35 cells;

[0086] (c) is the flow cytometry graph of the XPinα-treated group in melanoma WM35 cells;

[0087] (d) is the flow cytometry graph of the XPinβ-treated group in melanoma WM35 cells;

[0088] (e) is the flow cytometry statistics of early, late and total apoptosis in Figures (a)-(d).

[0089] Figure 14 The inhibitory effect of XPinβ on melanoma homograft tumor growth;

[0090] In the figure, (a) shows the appearance of tumors in mice treated with different drugs at the end of the experiment, and the area marked by the red dotted circle is the tumor;

[0091] (b) is the curve of mouse tumor volume changes during treatment;

[0092] (c) The weight of the excised tumor tissue was recorded at the end point of the experiment. *P<0.05, **P<0.01, ***P<0.001.

[0093] Figure 15 The curve of body weight changes of mice during treatment.

[0094] Figure 16 H&E staining of tumor tissue, scale bar: 100 μm.

[0095] Figure 17Ki67 immunohistochemical staining and statistical results of tumor tissues;

[0096] In the figure, A shows the results of Ki67 immunohistochemical staining of tumor tissue;

[0097] B shows the effects of CA, XPinα and XPinβ treatment on Ki67 expression.

[0098] Figure 18 TUNEL immunohistochemical staining of tumor tissue and statistical results;

[0099] In the figure, A shows the results of TUNEL immunohistochemical staining of tumor tissue;

[0100] B shows the effects of CA, XPinα and XPinβ treatment on TUNEL expression.

[0101] Figure 19 For tumor proteomic sequencing analysis;

[0102] In the figure, (a) is the proteomic clustering heat map of tumors after XPinβ treatment;

[0103] (b) GSEA enrichment analysis of tumor proteomics after XPinβ treatment, NES, normalized enrichment score.

[0104] Figure 20 Schematic diagram of drug administration for the human melanoma PDX mouse model.

[0105] Figure 21 To observe the efficacy of XPinβ in a human melanoma PDX mouse model;

[0106] In the figure, (a) is a photograph of the mouse tumor in vivo at the end of the experiment, and the area marked by the red dotted circle is the tumor; (b) is a photograph of the ex vivo tumor at the end of the experiment; (c) is the weighing record of the tumor at the end of the experiment; (d) is the curve of the change of mouse tumor volume during treatment, *P<0.05, ***P<0.001.

[0107] Figure 22 Figure 3 is the curve of mouse body weight changes during XPinβ treatment in a human melanoma PDX mouse model.

[0108] Figure 23 H&E staining of tumor tissue during XPinβ treatment in a human melanoma PDX mouse model.

[0109] Figure 24 TUNEL fluorescence staining of tumor tissue after treatment with different drugs.

[0110] Figure 25Ki67 immunohistochemical staining and statistics of tumor tissues after XPinβ treatment, 100 μm; **P<0.01, ***P<0.001;

[0111] In the figure, A is a representative image of Ki67 immunohistochemical staining in tumor tissues of each group;

[0112] B is the Ki67 scoring results in tumor tissues of each group.

[0113] Figure 26 C-myc immunohistochemical staining and statistics of tumor tissues after XPinβ treatment;

[0114] In the figure, A is a representative image of C-myc immunohistochemical staining in tumor tissues of each group;

[0115] B is the scoring result of C-myc in tumor tissues of each group.

[0116] Figure 27 CyclinD1 immunohistochemical staining and statistics of tumor tissues after XPinβ treatment;

[0117] In the figure, A is a representative image of CyclinD1 immunohistochemical staining in tumor tissues of each group; B is a graph showing the scoring results of CyclinD1 in tumor tissues of each group. DETAILED DESCRIPTION

[0118] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0119] Example 1: A protein microsphere inhibitor and its preparation method.

[0120] 1. Preparation of experimental reagents

[0121] (1) Preparation of tetrachloroauric acid solution:

[0122] 2.25 mL of HEPES (100 mM) was added to 2.25 mL of deionized water and 500 μL of HAuCl4 (10 mM), mixed, and stirred at 50° C.-55° C. until the color of the reaction solution turned light purple to form a tetrachloroauric acid solution.

[0123] (2) Preparation of XPin polypeptide solution:

[0124] 2 mg of XPin polypeptide was weighed and dissolved in 500 μL of NH2-PEG-SH (500 μL, MW: 2000, concentration 4 mg / mL in deionized water) and 500 μL of anhydrous ethanol, and completely dissolved by ultrasound.

[0125] (3) Preparation of 0.1M Vitamin C solution:

[0126] Weigh 10 mg of pure vitamin C and dissolve it in 1% oxalic acid solution to make up to 100 mL.

[0127] (4) Carnosic acid (CA) solution:

[0128] Weigh 1 mg of carnosic acid and dissolve it in 0.3008 mL of dimethyl sulfoxide (DMSO) solution, and store it at -20°C until use.

[0129] (5) PAH solution:

[0130] Weigh 4 mg of PAH solution and dissolve it in 1 mL of deionized water, then sonicate to dissolve.

[0131] 2. Preparation of protein microsphere inhibitors

[0132] 8.5 mL of XPin polypeptide solution was added to 1 mL of tetrachloroauric acid solution, stirred and mixed at room temperature of 25° C., and the pH value was adjusted to 6.5 to obtain a mixture.

[0133] A freshly prepared 0.1 M vitamin C solution was added to the obtained mixture, and the mixture was reacted at 25° C. for 1 h. Excess reactants were removed using a 10 kDa dialysis tube (Millipore, UFC901008) to obtain an XPinα solution.

[0134] 9.46 mL of XPinα solution was added with 0.02 mL of freshly prepared carnosic acid (CA) and 0.02 mL of VC solution. After the reaction at 25 °C for 1 h, 0.5 mL of PAH solution (MW: 20 kDa, 2 × 10 -3 M, pH 7.4), and concentrated by filtration through 50 kDa (Millipore, UFC905008) to remove excess reactants and obtain XPinβ.

[0135] Example 2: A protein microsphere inhibitor and its preparation method.

[0136] 17 mL of XPin polypeptide solution was added to 1 mL of tetrachloroauric acid solution, stirred and mixed at room temperature of 25° C., and the pH value was adjusted to 6.5 to obtain a mixture.

[0137] A freshly prepared 0.08 M vitamin C solution was added to the obtained mixture, and the mixture was reacted at 27° C. for 50 min. Excess reactants were removed using a 10 kDa dialysis tube (Millipore, UFC901008) to obtain an XPinα solution.

[0138] 9.42 mL of XPinα solution was added with 0.04 mL of freshly prepared carnosic acid (CA) and 0.04 mL of VC solution. After reacting at 27°C for 50 min, 0.5 mL of PAH solution (MW: 20 kDa, 2×10 -3 M, pH 7.4), and concentrated by filtration through 50 kDa (Millipore, UFC905008) to remove excess reactants and obtain XPinβ.

[0139] Example 3: A protein microsphere inhibitor and its preparation method.

[0140] To 1 mL of tetrachloroauric acid solution, 21.25 mL of XPin polypeptide solution was added, the mixture was stirred at room temperature of 25°C, and the pH value was adjusted to 6.5 to obtain a mixture.

[0141] A freshly prepared 0.12 M vitamin C solution was added to the obtained mixture, and the mixture was reacted at 23° C. for 70 min. Excess reactants were removed using a 10 kDa dialysis tube (Millipore, UFC901008) to obtain an XPinα solution.

[0142] 9.4 mL of XPinα solution was added with 0.05 mL of freshly prepared carnosic acid (CA) and 0.05 mL of VC solution. After reacting at 24°C for 70 min, 0.5 mL of PAH solution (MW: 20 kDa, 2 × 10 -3 M, pH 7.4), and concentrated by filtration through 50 kDa (Millipore, UFC905008) to remove excess reactants and obtain XPinβ.

[0143] Examples 1-3 can all prepare protein microsphere inhibitors with similar structural properties. Taking Example 1 as an example, physical and chemical property testing and application research experiments were carried out. The details are as follows:

[0144] 1. Physical and chemical property testing of XPinβ prepared by the present invention

[0145] 1. XPinβ appearance characteristics and elemental analysis

[0146] The morphology and elements of XPinβ prepared in Example 1 were detected using a high-resolution transmission electron microscope. The results are as follows: Figure 1As shown in Figure 2, XPinβ is uniformly distributed and nearly spherical particles under transmission electron microscopy. High-resolution transmission electron microscopy shows that XPinβ is mainly composed of gold, nitrogen, sulfur, and oxygen.

[0147] 2. XPinβ particle size analysis

[0148] The particle size and surface potential of XPinβ were measured by dynamic light scattering instrument. Figure 2 As shown, at room temperature, in PBS solution with pH = 7.4, XPinβ presents a unimodal distribution with an average hydrodynamic diameter of 55.61 nm and a surface potential of 42.1 mV.

[0149] 3. UV-visible spectroscopy

[0150] like Figure 3 As shown, the UV-visible spectrum shows that XPinβ has a characteristic absorption peak of polypeptide XPin at 220 nm and a characteristic absorption peak of carnosic acid at 280 nm, indicating that both polypeptide XPin and carnosic acid have been loaded into XPinβ.

[0151] 4. Fourier transform infrared spectroscopy detection

[0152] like Figure 4 As shown, Fourier transform infrared spectroscopy showed that SR-Au + The characteristic peaks of -CO-NH- and Ph-OH further confirmed the successful loading of XPin and CA.

[0153] 5. Determination of the endocytic type of XPinβ

[0154] Inhibitors of different endocytic pathways include the clathrin-mediated endocytosis inhibitor Chlorpromazine, denoted as CPZ; the dynamin-mediated endocytosis inhibitor Dynasore, denoted as DYN; two cytoplasm-mediated endocytosis inhibitors Filipin and Genistein, denoted as GEN; two macropinocytosis inhibitors Amiloride, denoted as AMI; and also EIPA.

[0155] Melanoma cells were treated with the above-mentioned inhibitors of different endocytic pathways for 2 hours before being incubated with FITC-labeled XPinβ for another 12 hours. The effects of the above-mentioned inhibitors on XPinβ uptake were then examined using fluorescence microscopy and flow cytometry.

[0156] The streaming results are as follows Figure 5As shown in the data, in the absence of any inhibitors, the uptake rate of XPinβ by melanoma cells was as high as 88.48%. After using inhibitors Chlorpromazine, Dynasore, Genistein, Filipin, Amiloride and EIPA, the uptake rates of XPinβ were 87.42%, 83.11%, 85.70%, 77.23%, 37.39% and 20.80%, respectively.

[0157] The fluorescence experiment results are as follows Figure 6 As shown in Figure 3, the macropinocytosis inhibitors Amiloride and EIPA both significantly inhibited the endocytosis of XPinβ, while other endocytic pathway inhibitors had no obvious effect on the uptake of XPinβ.

[0158] The above results indicate that the uptake of XPinβ mainly depends on the macropinocytosis pathway.

[0159] 2. Effects of XPinβ Prepared by the Present Invention on Melanoma Cells

[0160] 1. Colocalization of XPinβ and XPO1 To investigate whether XPinβ can bind to XPO1 and inhibit its function, the present invention conducted colocalization analysis.

[0161] FITC-Control and FITC-XPinβ solutions were added to melanoma cells, which were then incubated in an incubator (37°C) for approximately 24 hours. Immunofluorescence staining of XPO1 was then performed, and images were collected and analyzed using a microscope.

[0162] Preparation of FITC-Control solution: Weigh 5 mg of FITC powder and add 1 mL of DMSO solution to prepare a 5 mg / mL FITC solution. Take 5 μL of FITC solution and 1 mL of Control drug and mix them in an ultrasonic machine for about 30 minutes to obtain FITC-Control solution.

[0163] FITC-XPinβ solution: Weigh 5 mg of FITC powder and add 1 mL of DMSO solution to prepare a 5 mg / mL FITC solution. Take 5 μL of FITC solution and mix with 1 mL of XPinβ in an ultrasonic machine for about 30 minutes to obtain a FITC-XPinβ solution.

[0164] The results are as follows Figure 7 As shown, FITC-XPinβ exhibited good colocalization with XPO1, with a correlation coefficient R reaching 0.83.

[0165] 2. Study on the inhibition of Wnt / β-catenin signaling pathway by XPinβ To clarify the effect of XPinβ on the Wnt / β-catenin signaling pathway, the present invention used Western blotting to detect the changes in the Wnt / β-catenin pathway after intervention with XPinα, XPinβ and carnosic acid.

[0166] like Figure 8 As shown in the results, after administration of the inhibitory peptide XPinα that only targets XPO1, the expression of np-β-catenin and its downstream molecules C-myc and CyclinD1 were upregulated, indicating the activation of the Wnt / β-catenin signaling pathway; while after the use of CA and XPinβ, the expression of the above proteins was downregulated, suggesting that the Wnt / β-catenin pathway was inhibited.

[0167] 3. Effect of XPinβ on melanoma cell viability

[0168] The present invention evaluates the anti-tumor effect of XPinβ in melanoma cells. CCK8 is used to detect the effects of XPinα, XPinβ and carnosic acid on cell viability. Melanoma cells A375, B16F10 and WM35 were selected. The specific experimental steps are as follows: 1) After digestion and centrifugation of cells in the logarithmic growth phase, they were inoculated into 96 wells, with about 1,500 cells in each well, and then cultured in an incubator overnight. 2) After the cells are completely attached, the cells are divided into 4 groups: Control group, CA group, XPinα group and XPinβ group, and then the corresponding drugs are prepared according to different concentration gradients and added to the cells, and then placed in an incubator for continued incubation for 24 hours. 3) After the culture time is over, the old culture medium is discarded with a spray gun. 4) Prepare CCK8 solution, and use serum-free basic culture medium DMEM to prepare CCK8 solution so that the concentration of CCK8 solution is 10%. 5) Add 100 μL of the prepared CCK8 solution to the cells, mix well, and place in an incubator. Measure the absorbance every 1 h using a microplate reader set to a wavelength of 450 nm.

[0169] like Figure 9 As shown, CA, XPinα and XPinβ all have inhibitory effects on the growth of melanoma cells, among which XPinβ has the most obvious inhibitory effect on the vitality of melanoma cells.

[0170] 4. Effect of XPinβ on melanoma cell proliferation

[0171] The long-term effects of CA, XPinα, and XPinβ on cells were examined in a colony formation assay. Untreated melanoma cells served as a control. The specific experimental steps are as follows: 1) Cells in the logarithmic growth phase were digested and centrifuged, then seeded into 6-well plates at 500 cells per well and incubated overnight in an incubator. 2) The next day, after complete cell attachment under a microscope, the cells were divided into four groups: control, CA, XPinα, and XPinβ. The corresponding drugs were then added and the cells were placed in the incubator for further incubation. 3) Cell growth was observed daily under a microscope, noting the presence of colonies. The culture medium was changed every 2-3 days. 4) After approximately 10 days of culture, colonies were clearly visible and the cells were terminated. 5) Prepare the cells for harvest, discard the old culture medium, and wash the cells 2-3 times with PBS. 6) Rinse thoroughly to remove any remaining PBS solution, then add 1 mL of 4% paraformaldehyde solution to each well and fix at room temperature for 15-20 minutes. 7) Discard the fixative and wash the cells twice with PBS. 8) Prepare crystal violet staining solution and dilute 1% crystal violet staining solution to a concentration of 0.2% with PBS. 9) Add 1 mL of 0.2% crystal violet staining solution to each well, stain at room temperature for about 15 minutes, and observe the staining. 10) After staining, recover the crystal violet staining solution, then place the 6-well plate under tap water and rinse with low-flow tap water to remove surrounding color. 11) Place the 6-well plate upside down on filter paper, let it dry naturally, and then take a picture. The results are as follows: Figure 10 As shown in the results, XPinβ significantly inhibited the clone formation ability of all melanoma cells, and its inhibitory effect was significantly better than that of CA and XPinα.

[0172] 5. Effect of XPinβ on apoptosis of melanoma cells

[0173] The effect of XPinβ on apoptosis in different melanoma cells was examined by flow cytometry. The specific experimental steps were as follows: 1) Cells in the logarithmic growth phase were digested and centrifuged, then seeded into 6-well plates at a cell density of approximately 300,000 and incubated in an incubator. 2) When the cell density reached approximately 50%-60%, the cells were divided into four groups: control, CA, XPinα, and XPinβ. Fresh DMEM medium was then replaced, and the corresponding drugs were added. The cells were then incubated in an incubator for another 24 hours. 3) The cells were harvested, the original medium was returned to a centrifuge tube, and 0.25% EDTA-free trypsin was added. Digestion was continued on ice for approximately 3-4 minutes. Cell morphology was observed under a microscope to confirm shrinkage and rounding. The digestion was then immediately terminated with complete DMEM medium. Gently pipetting the cell suspension was then collected and transferred to a centrifuge tube containing the original medium. 4) The cells were centrifuged in a pre-cooled (4°C) centrifuge at 1000 rpm for 5 minutes. 5) Discard the supernatant and retain the cell pellet. Next, add 1 mL of PBS solution and gently pipette to mix thoroughly. Centrifuge at 1000 rpm for 5 minutes at 4°C. 6) Prepare 1× Binding buffer by adding 0.2 mL of 10× Binding buffer to 1.8 mL of PBS solution, mix thoroughly, and place on ice. 7) Discard the supernatant from the centrifuged cells and aspirate any remaining PBS solution. Then, add 100 μL of 1× Binding buffer to each sample and gently pipette to resuspend the cells in the solution. 8) Next, add 5 μL of PE Annexin V and 5 μL of 7AAD solution to each sample in that order. 9) Vortex at low speed on a sponge pad to mix thoroughly. Incubate at room temperature in the dark for 15 minutes. 10) After the incubation period, add 300 μL of 1× Binding buffer to each tube and mix thoroughly to terminate the staining. 11) Finally, the apoptotic cell ratio was detected by flow cytometry within 1 hour. Figure 11 As shown, in A375 cells, CA, XPinα, and XPinβ caused apoptosis rates of 9.2%, 11.0%, and 21.9%, respectively. In B16F10 cells, CA, XPinα, and XPinβ all promoted apoptosis compared to the control group (P < 0.001), with XPinβ leading to the highest apoptosis rate of 31.6%. In WM35 cells, CA and XPinα alone caused apoptosis rates of 6.5% and 10.6%, respectively, while XPinβ led to a 21.3% apoptosis rate. This suggests that XPinβ is the most potent agent in promoting apoptosis.

[0174] The above in vitro cell experiment results show that XPinβ can inhibit cell proliferation, promote apoptosis, and has a good anti-tumor effect in vitro.

[0175] 6. XPinβ inhibits the growth of melanoma homografts

[0176] About 10 days after the mouse melanoma cells B16F10 were inoculated into the buttocks of C57BL / 6 mice, the volume of the transplanted tumor reached about 80 mm. 3 The animals were randomly divided into 4 groups: Control group, CA group, XPinα group and XPinβ group. The drugs corresponding to the different groups were injected into the tail vein for 5 times. 3 At 3 hr, the mice were euthanized and the data were collected for analysis.

[0177] At the end of the experiment, take photos to record the morphology and size of the tumor nodules. Figure 14 As shown in the figure, it can be directly observed that the tumor volume of the XPinβ group is smaller than that of the Control group, CA group, and XPinα group; the tumor growth curve results show that compared with the Control group, CA, XPinα, and XPinβ can inhibit the growth of mouse tumors, but the inhibitory effect of the XPinβ group is the most significant; at the end of the experiment, the tumor tissue was removed and weighed, and consistent with the above results, the tumor tissue weight of the XPinβ group was the smallest, P < 0.001. In addition, during the treatment period, the changes in the weight of the mice were monitored and recorded and a curve was drawn. The results are shown in the figure. Figure 15 As shown in Figure 2, there was no abnormal change in the body weight of mice in each group. Figure 16 As shown in the figure, it was found that all tumor cells showed large and darkly stained nuclei. These results suggest that XPinβ can significantly inhibit the growth of melanoma B16F10 homograft tumors without causing weight loss in mice.

[0178] 7. XPinβ inhibits proliferation and promotes apoptosis of melanoma homograft cells

[0179] Immunohistochemical staining of tumor tissues was performed to examine the effects of XPinβ on cell proliferation and apoptosis. Ki67 is a cell cycle-associated protein primarily expressed during the cell proliferation phase. Detecting Ki67 expression can assess actively proliferating cells in tissues.

[0180] The results are as follows Figure 17 As shown in the figure, compared with the control group, the expression of Ki67 in the other three groups was significantly reduced, among which the expression of Ki67 in the XPinβ group was the lowest, P < 0.001, indicating that the proliferation ability of tumor cells in this group was significantly inhibited. TUNEL staining is used to detect DNA fragmentation. It shows nuclear staining of apoptotic cells by labeling the 3'-OH end of DNA fragmentation. It is a commonly used method for apoptosis detection. Figure 18As shown in the figure, compared with the Control, CA, and XPinα groups, the number of apoptotic cells in the XPinβ group increased significantly, suggesting that XPinβ promotes tumor cell apoptosis. These results suggest that XPinβ inhibits melanoma growth by inhibiting cell proliferation and promoting cell apoptosis.

[0181] 8. XPinβ inhibits the Wnt / β-catenin signaling pathway in melanoma

[0182] To further clarify the regulatory role of XPinβ on the Wnt / β-catenin signaling pathway, proteomic sequencing was performed on tumors in the XPinβ and control groups. Proteomic sequencing provides comprehensive protein expression data, which, through subsequent biological analysis, can provide a more comprehensive understanding of the regulation of the Wnt / β signaling pathway and identify potential therapeutic targets.

[0183] The results are as follows Figure 19 As shown in the figure, there are a large number of differentially expressed proteins in the XPinβ group compared with the Control group. GSEA enrichment analysis results show that the Wnt signaling pathway in the XPinβ group is significantly downregulated compared with the Control group (P < 0.001), suggesting that XPinβ intervention can significantly inhibit the activity of the Wnt / β-catenin signaling pathway.

[0184] 9. XPinβ effectively inhibits melanoma growth in the Hu-PDX mouse model

[0185] To further evaluate the efficacy of XPinβ and predict patient responses to treatment with XPinβ, we constructed a human melanoma PDX model. The tumors in this model were derived from previously established human melanoma PDX tissues, which were published in Drug Resist Updat. 2024;73:101037. Furthermore, to compare the efficacy of XPinβ with that of a clinical XPO1 inhibitor, we also added a KPT-330 group for simultaneous observation.

[0186] Melanoma tissue from patients was inoculated into NOD / SCID female mice until the tumor volume reached 800 mm 3 , i.e. P0 generation, the tumor was removed and inoculated into nude mice for passage, and the tumor volume reached 800 mm again. 3 , i.e. P1 generation, continued to be passaged in nude mice, and the tumor tissue with strong vitality was inoculated into nude mice. After about 3 weeks, the tumor volume reached 80-100mm 3 After that, i.e., the P2 generation, they were divided into 5 groups: Control group, CA group, KPT-330 group, XPinα group and XPinβ group, and the following Figure 20 The mice were photographed at the end of the experiment according to the corresponding drug interventions as indicated.

[0187] The results are as follows Figure 21 As shown, the tumor volume in the XPinβ group was significantly smaller than that in the Control group, CA group, KPT-330 group, and XPinα group; Figure 21 This result can be more clearly seen in the peeled tumor tissue in (b). Figure 21 The mouse tumor growth curve in (c) also shows that the tumor growth rate in the XPinβ group is the slowest, while the tumor growth rate in the KPT-330 group and the XPinα group is slower than that in the Control group and the CA group. Figure 21 As shown in (c), it is consistent with the results of naked eye observation, that is, the tumor tissue weight of the XPinβ group is the smallest, and the tumor weight has significant statistical differences compared with the other groups. It is worth noting that although the tumors of the KPT-330 group and the XPinα group are smaller than those of the Control group and the CA group, there is no significant difference between the KPT-330 group and the XPinα group. Figure 22 As shown in Figure 3, there was no significant weight loss in the mice during drug treatment and monitoring. Figure 23 As shown, the tumor cells were found to have large and darkly stained nuclei.

[0188] The above results show that in the human melanoma PDX model, XPinβ still exhibits a significant inhibitory effect on tumor cell growth, and the efficacy of this drug is significantly better than the clinical XPO1 inhibitor KPT-330, highlighting its potential value in practical application and promotion in clinical practice.

[0189] 10. XPinβ inhibits tumor cell proliferation and promotes apoptosis in the Hu-PDX mouse model

[0190] After the tumor tissue was peeled off, TUNEL fluorescence staining was performed to observe the apoptosis of tumor cells. Figure 24 As shown in the figure, the number of apoptotic cells in the XPinβ, KPT-330 and XPinα groups was significantly increased compared with the Control and CA groups, among which the increase of apoptotic cells in the XPinβ group was the most obvious.

[0191] After the tumor tissue was peeled off, Ki67 immunohistochemical staining was performed to evaluate the proliferation of melanoma cells. Figure 25 All treatment groups could inhibit the proliferation of melanoma cells, but the inhibitory effect of XPinβ group was the most significant (P<0.001). These results suggest that XPinβ effectively promotes cell apoptosis and inhibits cell proliferation in vivo.

[0192] 11. XPinβ inhibits the Wnt / β-catenin signaling pathway in the Hu-PDX mouse model

[0193] After fixing, embedding and sectioning the excised tumor tissues, we used immunohistochemistry to detect downstream target molecules of the Wnt / β-catenin signaling pathway. Figure 26 and Figure 27 As shown in the results, compared with the control group, treatment with the XPO1 inhibitor KPT-330 and XPinα, respectively, significantly upregulated the expression of C-myc and CyclinD1 in tumor tissues (P<0.001), suggesting activation of the Wnt / β-catenin signaling pathway. In contrast, the expression levels of both C-myc and CyclinD1 were significantly downregulated in the CA group, indicating that the Wnt / β-catenin signaling pathway was inhibited by CA. Similarly, treatment with XPinβ also observed inhibition of the Wnt / β-catenin signaling pathway, similar to the CA group. These results demonstrate that XPinβ significantly inhibits the Wnt / β-catenin signaling pathway in the Hu-PDX model.

[0194] Although preferred embodiments of the present invention have been described, additional changes and modifications to these embodiments may occur to those skilled in the art once the basic inventive concepts become known.

[0195] Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, if such modifications and variations fall within the scope of the claims and their equivalents, the present invention is intended to include such modifications and variations.

Claims

1. A protein microsphere inhibitor, characterized in that The general formula of the protein microsphere inhibitor is XPinβ, wherein XPin represents an XPO1 inhibitor, which is an XPin polypeptide; the amino acid sequence of the XPin polypeptide is: Ac-RVSELKESLITTTPSC-NH2; β represents a β-catenin inhibitor, which is carnosic acid; and the gold adjuvant serves as a nanocarrier to carry the XPO1 inhibitor and the β-catenin inhibitor. The preparation method of the protein microsphere inhibitor comprises the following steps: adding an XPO1 inhibitor to a tetrachloroauric acid solution, adjusting the pH value to 6.0-7.0, and obtaining a mixture; Vitamin C solution is added to the mixture, reacted at 23°C-27°C for 50-70 min, and the excess reactant is removed by dialysis to form XPinα solution; Add carnosic acid solution and VC solution to XPinα solution, react at 24°C-27°C for 50-70 min, then add polyallylamine hydrochloride solution, centrifuge and concentrate to remove excess reactants to obtain XPinβ.

2. A method for preparing the protein microsphere inhibitor according to claim 1, characterized in that: The steps include: Adding an XPO1 inhibitor to a tetrachloroauric acid solution and adjusting the pH to 6.0-7.0 to obtain a mixture; the XPO1 inhibitor is an XPin polypeptide; the amino acid sequence of the XPin polypeptide is: Ac-RVSELKESLITTTPSC-NH2; Vitamin C solution was added to the mixture and reacted at 23-27°C for 50-70 min. Excess reactants were removed by dialysis to form XPinα solution. Add carnosic acid solution and VC solution to XPinα solution, react at 24°C-27°C for 50-70 min, then add polyallylamine hydrochloride solution, centrifuge and concentrate to remove excess reactants to obtain XPinβ.

3. The method for preparing the protein microsphere inhibitor according to claim 2, characterized in that: The molar ratio of the tetrachloroauric acid to the XPO1 inhibitor is 1:1 to 2.

5.

4. The method for preparing the protein microsphere inhibitor according to claim 2, characterized in that: The final concentration of the vitamin C solution is 0.08 M to 0.12 M.

5. The method for preparing the protein microsphere inhibitor according to claim 2, characterized in that: The concentration ratio of the XPinα solution to the carnosic acid solution is 1.5-2:

1.

6. The method for preparing the protein microsphere inhibitor according to claim 2, characterized in that: The molar ratio of the carnosic acid solution to the polyallylamine hydrochloride solution is 3-4.5:

1.

7. Use of the protein microsphere inhibitor according to claim 1 in the preparation of a drug for treating melanoma, characterized in that: The protein microsphere inhibitor is used to prepare a drug for inhibiting tumor cell proliferation and / or promoting tumor cell apoptosis.

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