Protein microsphere inhibitor as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses a protein microsphere inhibitor and a preparation method and application thereof, the general formula of the protein microsphere inhibitor is XPin beta, XPin represents an XPO1 inhibitor, beta represents a beta-catenin inhibitor, and a gold adjuvant serves as a nano-carrier and carries the XPO1 inhibitor and the beta-catenin inhibitor. The protein microsphere inhibitor provided by the invention can inhibit proliferation of melanoma cells, promote apoptosis of the melanoma cells and inhibit growth of homotransplantation tumors of the melanoma, and can be used for preparing drugs for treating the melanoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a protein microsphere inhibitor and a preparation method and 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 the advancement of 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 usually refers to malignant melanoma, which is a highly malignant tumor derived from melanocytes. It is often found in the skin and can also be seen in mucous membranes and internal organs, accounting for about 3% of all tumors. Cutaneous malignant melanoma ranks third among malignant skin tumors. Malignant melanoma can evolve from congenital or acquired benign melanocytic nevi, or from the malignant transformation of dysplastic nevi, or it can be a new occurrence. In recent years, the incidence and mortality of malignant melanoma have increased year by year, and its age of death is lower than that of other solid tumors. In addition to early surgical resection, malignant melanoma lacks specific treatment and has a poor prognosis. Therefore, early diagnosis and treatment of malignant melanoma are extremely important.

[0004] XPO1 is the most widely studied nuclear export protein. It is abnormally expressed in many tumors and is a new target for tumor treatment. Currently, a variety of XPO1 inhibitors have been developed as tumor treatment drugs, but they have problems such as high toxicity and poor single-drug efficacy. Their effects in melanoma are also not ideal. Therefore, it is urgent to develop a product with good efficacy for the treatment of melanoma. Summary of the invention

[0005] In order to develop a product for treating melanoma, the present invention provides a protein microsphere inhibitor and its preparation method and application. The protein microsphere inhibitor provided by the present invention can inhibit the proliferation of melanoma cells, promote the apoptosis of melanoma cells 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, the general formula of which is 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 value 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 excess reactants are removed by dialysis to form XPinα solution;

[0013] Add carnosic acid solution and VC solution to XPinα solution, react at 24°C-27°C for 50-70 minutes, then add polyallylamine hydrochloride solution, centrifuge and concentrate to remove excess reactants, and 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 was 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 3-4.5:1.

[0021] The present invention also provides an application of the protein microsphere inhibitor in the preparation of 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] The present invention uses a nano-delivery carrier system to self-assemble a polypeptide targeting XPO1 and a β-catenin inhibitor CA into a protein microsphere drug that can dual-target and inhibit XPO1 and β-catenin, and verifies the drug's physicochemical properties, biological functions, and in vitro efficacy, providing new ideas and strategies for the development of more effective and safer tumor therapeutic drugs.

[0025] The protein microsphere inhibitor prepared by the present invention can inhibit the proliferation of melanoma cells, promote the apoptosis of melanoma cells, 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. 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 creative work.

[0027] Figure 1 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) is the HRTEM analysis image 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 element analysis diagram of XPinβ prepared in Example 1;

[0032] (e) is the S element 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 The particle size and potential detection of XPinβ prepared in Example 1 of the present invention;

[0035] In the figure, (a) is the particle size detection 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 was used to detect the uptake of XPinβ 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 is stained);

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

[0042] (c) is a flow cytometric analysis of the uptake rate of XPinβ 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 uptake rate of XPinβ 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 uptake rate of XPinβ 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 For fluorescence experiments 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 graph 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 not treated with any inhibitors;

[0052] (c) is a fluorescence graph of the uptake rate of XPinβ in melanoma cells treated with the inhibitor Chlorpromazine;

[0053] (d) is a fluorescence graph of the uptake rate of XPinβ in melanoma cells treated with the inhibitor Dynasore;

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

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

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

[0057] (h) is a fluorescence graph showing the uptake rate of XPinβ in melanoma cells treated with the inhibitor EIPA.

[0058] Figure 7 The colocalization of XPinβ and XPO1, 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 of XPinβ and XPO1.

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

[0063] Fig. 9 is the effect of XPinβ on the viability of melanoma cells, 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] Fig.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] Fig.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 the 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 graph of early, late and total apoptosis in Figures (a)-(d).

[0077] Fig.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 graph of early, late and total apoptosis in Figures (a)-(d).

[0083] Fig.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 the 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 graph of early, late and total apoptosis in Figures (a)-(d).

[0089] Fig.14 for 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 point 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] Fig.15 This is the curve of body weight changes of mice during treatment.

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

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

[0096] In the figure, A is the result of Ki67 immunohistochemical staining of tumor tissue;

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

[0098] Fig.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] Fig.19 For tumor proteomics sequencing analysis;

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

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

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

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

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

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

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

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

[0110] Fig.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 scoring result of Ki67 in tumor tissues of each group.

[0113] Fig.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 diagram of C-myc in tumor tissues of each group.

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

[0117] In the figure, A is a representative picture of CyclinD1 immunohistochemical staining in tumor tissues of each group; B is a picture of 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 protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to 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 channels unless otherwise specified.

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

[0120] 1. Preparation of experimental reagents

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

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

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

[0124] 2 mg of XPin polypeptide was weighed and dissolved in 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 for later use.

[0129] (5) PAH solution:

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

[0131] 2. Preparation of protein microsphere inhibitors

[0132] Add 8.5 mL of XPin polypeptide solution to 1 mL of tetrachloroauric acid solution, stir and mix at room temperature of 25° C., adjust the pH value to 6.5, and 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 reacting 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 a preparation method thereof.

[0136] Add 17 mL of XPin polypeptide solution to 1 mL of tetrachloroauric acid solution, stir and mix at room temperature of 25° C., adjust the pH value to 6.5, and 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 a preparation method thereof.

[0140] Add 21.25 mL of XPin polypeptide solution to 1 mL of tetrachloroauric acid solution, stir and mix at room temperature of 25° C., adjust the pH value to 6.5, and 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. Example 1 is used as an example to conduct physical and chemical property detection and application research experiments. 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 1, XPinβ is uniformly distributed near-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 an absorption peak of the characteristic polypeptide XPin at 220nm, and a characteristic absorption peak of carnosic acid at 280nm, indicating that both the 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 shows 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 intracellular endocytosis 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 includes EIPA.

[0155] Melanoma cells were treated with the above inhibitors of different endocytic pathways for 2 hours in advance, and then XPinβ labeled with FITC was added and the cells were incubated for 12 hours. The effects of the above inhibitors on XPinβ uptake were then detected using fluorescence microscopy and flow cytometry.

[0156] The streaming results are as follows Figure 5As shown, 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, 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. Co-localization of XPinβ and XPO1 To study whether XPinβ can bind to XPO1 and inhibit its function, the present invention carried out co-localization analysis.

[0161] FITC-Control and FITC-XPinβ solutions were added to melanoma cells, which were then placed in an incubator (37°C) and incubated for about 24 hours. Subsequently, immunofluorescence staining of XPO1 was performed, and finally, images were observed and collected using a microscope, and the correlation coefficient was further analyzed.

[0162] Preparation of FITC-Control solution: Weigh 5 mg of FITC powder, add 1 mL of DMSO solution to prepare a FITC solution with a concentration of 5 mg / mL, 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, add 1 mL of DMSO solution to prepare a FITC solution with a concentration of 5 mg / mL, take 5 μL of FITC solution and mix with 1 mL of XPinβ drug 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 and XPO1 exhibited good colocalization, with a correlation coefficient R reaching 0.83.

[0165] 2. Study on the inhibition of Wnt / β-catenin signaling pathway by XPinβ In order 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 the intervention of XPinα, XPinβ and carnosic acid.

[0166] like Figure 8 As shown, 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 are selected. The specific experimental steps are as follows: 1) After digestion and centrifugation of cells in the logarithmic growth phase, they are 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 to the wall, 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 prepare CCK8 solution with serum-free basic culture medium DMEM to make the concentration of CCK8 solution 10%. 5) Add 100 μL of the prepared solution containing CCK8 to the cells, mix well, place in an incubator, and detect the absorbance every 1 h, setting the wavelength of the microplate reader to 450 nm.

[0169] like Fig. 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 clone formation experiment detects the long-term effects of CA, XPinα and XPinβ on cells, and the untreated melanoma cells are used as the control. The specific experimental steps are as follows: 1) After digestion and centrifugation of the cells in the logarithmic growth phase, they are inoculated into a 6-well plate, with 500 cells in each well, and incubated in an incubator overnight. 2) On the second day, after observing the cells completely attached to the wall under a microscope, they are divided into 4 groups: Control group, CA group, XPinα group and XPinβ group, and then the corresponding drugs are added respectively, and the cells are placed in the incubator for further incubation. 3) Observe the cell growth under a microscope every day, pay attention to the clone formation; and replace the culture medium every 2-3 days. 4) After about 10 days of culture, the clone formation can be clearly seen with the naked eye, and the culture can be terminated. 5) Prepare to collect cells, discard the old culture medium, and wash the cells with PBS 2-3 times. 6) Wash the residual PBS solution, then add 1mL of 4% paraformaldehyde solution to each well, and fix at room temperature for 15-20min. 7) Then, discard the fixative and wash the cells twice with PBS solution. 8) Prepare crystal violet staining solution, dilute 1% crystal violet staining solution to 0.2% concentration 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 take the 6-well plate under tap water, rinse with tap water at a low flow rate to remove surrounding colors. 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: Fig.10 As shown, XPinβ has a significant inhibitory effect on the clone formation ability of all melanoma cells, and its inhibitory effect is 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 of different melanoma cells was detected by flow cytometry. The specific experimental steps are as follows: 1) After digestion and centrifugation of cells in the logarithmic growth phase, they were inoculated into a 6-well plate with a cell number of about 300,000, and then incubated in an incubator. 2) When the cell density reached about 50%-60%, they were divided into 4 groups: Control group, CA group, XPinα group and XPinβ group, and then new DMEM culture medium was replaced, and then the corresponding drugs were added respectively, and the cells were placed in an incubator for further incubation for 24 hours. 3) The cells were collected, the original culture medium was recovered into a centrifuge tube, and then 0.25% trypsin without EDTA was added, and digestion was performed on ice for about 3-4 minutes. The cell morphology was observed to shrink and become round under a microscope, and then the digestion was immediately terminated with complete DMEM culture medium, and the cell suspension was gently blown and recovered into a centrifuge tube containing the original old culture medium. 4) Centrifuge in a pre-cooled (4°C) centrifuge at 1000rpm for 5 minutes. 5) Pour off the supernatant and keep the cell pellet. Then, add 1 mL of PBS solution, gently pipette to mix, and centrifuge at 1000 rpm for 5 min at 4°C. 6) Prepare 1×Binding buffer. Take 1.8 mL of PBS solution and add 0.2 mL of 10×Binding buffer solution. Mix well and place on ice. 7) Discard the supernatant of the centrifuged cells, use a pipette to dry the remaining PBS solution, then add 100 μL of 1×Binding buffer solution to each sample and gently pipette to resuspend the single cells in the solution. 8) Then, add 5 μL of PE Annexin V and 5 μL of 7AAD solution to each sample in sequence. 9) Vortex at low speed on a vortex machine with a sponge pad to mix well, and then incubate at room temperature in the dark for 15 min. 10) After the incubation, add 300 μL of 1×Binding buffer solution to each tube and mix appropriately to terminate the staining. 11) Finally, the apoptotic cell ratio was detected on a flow cytometer within 1 hour. Fig.11 As shown in the figure, in A375 cells, CA, XPinα and XPinβ caused cell apoptosis rates of 9.2%, 11.0% and 21.9%, respectively. In B16F10 cells, compared with the Control group, CA, XPinα and XPinβ could promote tumor cell apoptosis, P<0.001, but XPinβ caused the highest apoptosis rate of 31.6%; in WM35 cells, CA and XPinα alone caused cell apoptosis rates of 6.5% and 10.6%, respectively, while XPinβ caused cell apoptosis rate of 21.3%. It can be seen that XPinβ caused the most obvious phenomenon of cell 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 melanoma homograft tumor growth

[0176] About 10 days after 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, CA, XPinα, and XPinβ. The drugs corresponding to the different groups were injected into the tail vein for 5 times. 3 At 4 hr, the mice were euthanized and data were collected for analysis.

[0177] At the end of the experiment, take photos to record the morphology and size of the tumor nodules in vivo. Fig.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 inhibition effect of the XPinβ group is the most significant; at the end of the experiment, the tumor tissue was peeled off and weighed. Consistent with the above results, the tumor tissue weight of the XPinβ group was the smallest, P<0.001. In addition, during the drug treatment, the changes in the weight of the mice were monitored and recorded and a curve was drawn. The results are shown in Fig.15 As shown in Figure 2, there was no abnormal change in the body weight of mice in each group. Fig.16 As shown, it was found that the tumor cells all showed the characteristics of large and dark nuclei. The above results suggest that XPinβ can significantly inhibit the growth of melanoma B16F10 homologous transplanted tumors without causing weight loss in mice.

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

[0179] Immunohistochemical staining was performed on tumor tissues to detect the effects of XPinβ on cell proliferation and apoptosis. Ki67 is a cell cycle-related protein that is mainly expressed during the cell proliferation phase. By detecting the expression of Ki67, active proliferating cells in tissues can be evaluated.

[0180] The results are as follows Fig.17 As shown in the figure, compared with the Control group, the Ki67 expression in the other three groups was significantly reduced, among which the Ki67 expression 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 breaks. It shows nuclear staining of apoptotic cells by marking the 3'-OH end of the DNA break end. It is a commonly used method for apoptosis detection. Fig.18As shown in the figure, compared with the Control group, CA group, and XPinα group, the number of apoptotic cells in the XPinβ group increased significantly, indicating that XPinβ promoted tumor cell apoptosis. The above 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 effect of XPinβ on the Wnt / β-catenin signaling pathway, proteomic sequencing was performed on tumors in the XPinβ group and the Control group. Proteomic sequencing can provide comprehensive protein expression data, and through subsequent biological analysis, a more comprehensive understanding of the regulation of the Wnt / β signaling pathway can be achieved, while identifying potential therapeutic targets.

[0183] The results are as follows Fig.19 As shown in the figure, there were a large number of differentially expressed proteins in the XPinβ group compared with the Control group. The results of GSEA enrichment analysis showed that the Wnt signaling pathway in the XPinβ group was significantly downregulated compared with the Control group, P < 0.001. This suggests 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] In order to further evaluate the efficacy of XPinβ and predict the clinical response of patients to XPinβ treatment, the present invention constructed a human melanoma PDX model. The tumor in this model was derived from the human melanoma PDX tissue that had been constructed by our research group in the early stage, and was published in: Drug Resist Updat.2024;73:101037. At the same time, in order to compare the efficacy of XPinβ with that of clinical XPO1 inhibitors, the KPT-330 group was added to observe the efficacy simultaneously.

[0186] Melanoma tissue from patients was inoculated into NOD / SCID female mice until the tumor volume reached 800 mm 3 At P0, the tumor was excised and inoculated into nude mice. When the tumor volume reached 800 mm again, 3 , i.e. P1 generation, continued to be subcultured 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 Fig. 20 Mice were photographed at the end point of the experiment for the corresponding drug interventions as indicated.

[0187] The results are as follows Fig.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; Fig.21 This result can be more clearly seen in the exfoliated tumor tissue in (b). Fig.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 rates in the KPT-330 group and the XPinα group are slower than those in the Control group and the CA group. Fig.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 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. Fig. 22 As shown in Figure 3, there was no significant weight loss in each group of mice during drug treatment and monitoring. Fig.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 clinical practical application and promotion.

[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. Fig.24 As shown, the number of apoptotic cells in XPinβ, KPT-330 and XPinα groups was significantly increased compared with that in Control and CA groups, and the increase of apoptotic cells in 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. Fig.25 All treatment groups could inhibit the proliferation of melanoma cells, but the inhibitory effect of XPinβ group was the most obvious, P<0.001. The above results suggest that XPinβ effectively promotes cell apoptosis and inhibits cell proliferation in vivo.

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

[0193] After fixing, embedding and sectioning the excised tumor tissue, we used immunohistochemistry to detect downstream target molecules of the Wnt / β-catenin signaling pathway. Fig.26 and Fig. 27 As shown in the figure, compared with the Control group, after treatment with XPO1 inhibitor KPT-330 and XPinα, respectively, the expression of C-myc and CyclinD1 in tumor tissues was significantly upregulated (P<0.001), indicating that the Wnt / β-catenin signaling pathway was activated. In the CA group, the expression levels of C-myc and CyclinD1 were significantly downregulated, indicating that the Wnt / β-catenin signaling pathway was inhibited by CA. Similarly, after treatment with XPinβ, a phenomenon similar to that in the CA group was observed, that is, the Wnt / β-catenin signaling pathway was inhibited. The above results show 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 may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0195] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these 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 the XPO1 inhibitor, which is the 96-110 functional fragment of the polypeptide Apoptin, and is modified with Ac and NH2 at the head and tail; β represents the β-catenin inhibitor, and the gold adjuvant is used as a nanocarrier to carry the XPO1 inhibitor and the β-catenin inhibitor.

2. The protein microsphere inhibitor according to claim 1, characterized in that The β-catenin inhibitor is carnosic acid.

3. A method for preparing the protein microsphere inhibitor according to any one of claims 1 to 2, characterized in that: The steps include: Adding an XPO1 inhibitor to a tetrachloroauric acid solution and adjusting the pH value to 6.0-7.0 to obtain a mixture; Vitamin C solution is added to the mixture, reacted at 23°C-27°C for 50-70 minutes, and excess reactants are 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 minutes, then add polyallylamine hydrochloride solution, centrifuge and concentrate to remove excess reactants, and obtain XPinβ.

4. The method for preparing the protein microsphere inhibitor according to claim 3, characterized in that: The XPO1 inhibitor is XPin polypeptide; The amino acid sequence of the XPin polypeptide is: Ac-RVSELKESLITTTPSC-NH2.

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

5.

6. The method for preparing the protein microsphere inhibitor according to claim 3, characterized in that: The final concentration of the vitamin C solution is 0.08M to 0.12M.

7. The method for preparing the protein microsphere inhibitor according to claim 3, characterized in that: The concentration ratio of the XPinα solution to the carnosic acid solution is 1.5 to 2:

1.

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

1.

9. Use of the protein microsphere inhibitor according to any one of claims 1 to 2 in the preparation of a drug for treating melanoma, characterized in that: The protein microsphere inhibitor is used for preparing a drug for inhibiting tumor cell proliferation and / or promoting tumor cell apoptosis.

10. Use of the protein microsphere inhibitor according to claim 9 in the preparation of a drug for treating melanoma, characterized in that: The protein microsphere inhibitor is used for preparing a drug for inhibiting the growth of melanoma homograft tumors.

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