New venom polypeptide-dendrimer complex, and preparation method therefor and use thereof

Abstract

Provided are a new venom polypeptide-dendrimer complex, and a preparation method therefor and the use thereof. Specifically, provided is a scorpion venom polypeptide, wherein the amino acid sequence of the scorpion venom polypeptide is FLGGLLSSIF. A new scorpion venom polypeptide (designated as C9) is isolated and identified. Moreover, a new polypeptide-polyamidoamine dendrimer complex G5C9 (i.e., nanoparticles) having a glioblastoma (GBM)-targeting effect is constructed. Compared with polypeptide C9, G5C9 exhibits significantly improved cellular uptake efficiency. G5C9 can enter cells via endocytosis and targets lysosomes, and inhibits lysosomal function and mTORC1, thereby promoting nuclear translocation of TFEB and further inducing autophagic cell death. Therefore, G5C9 provides a new targeted anti-GBM approach both in vitro and in vivo.

Description

A novel venom polypeptide composite dendritic macromolecule, its preparation method and application Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a novel venom polypeptide composite dendritic macromolecule, its preparation method, and its application. Background Technology

[0002] Glioblastoma (GBM) is one of the most aggressive malignant central nervous system tumors, accounting for approximately 49% of all intracranial tumors. Currently, chemotherapy and radiotherapy play a crucial role in clinical practice as adjuvant therapy for glioblastoma. However, due to the presence of physiological barriers such as the blood-brain barrier (BBB) ​​and the blood-tumor barrier (BBTB), traditional chemotherapy drugs, including carmustine, nimustine, cisplatin, and cyclophosphamide, have difficulty penetrating GBM tissue, resulting in significantly reduced actual drug concentrations in and around the tumor, thus limiting treatment efficacy. Therefore, there is an urgent need to develop novel antitumor drugs capable of crossing the BBB and BBTB to achieve effective accumulation of therapeutic drugs within the tumor.

[0003] Under this premise, peptides with anti-cancer potential screened and identified from animal venom have shown significant application prospects and enormous medicinal value. Animal venomous organs represent an important evolutionary innovation. To achieve predation and defense capabilities, venomous animals have developed many highly active, highly specific, and structurally diverse venomous peptide molecules during long-term natural evolution. Previous studies have reported the potential anti-glioma activity of animal venom peptides. For example, chlortoxin (CTX), a venomous peptide isolated from the Israeli scorpion, is composed of 36 amino acids. CTX effectively blocks the GCC current (glioma-specific chloride channel) specific to GBM cells, thereby inhibiting GBM cell migration and invasion, achieving a targeted anti-glioma effect. Another CTX-like peptide, AaCTx, purified from the venom of the scorpion Androctonus australis, also inhibits GBM invasion and migration. Furthermore, AaTs-1 from this scorpion venom can regulate formyl peptide receptor 1 (FPRL-1), activating calcium channels in the GBM and thereby inhibiting tumor cell proliferation. However, to date, no animal venom peptide drugs have been approved by the FDA for cancer treatment. Compared to small molecule drugs, peptide drugs lack cell specificity, have short half-lives, and poor solubility. Therefore, developing next-generation peptides with enhanced stability and targeted cell permeability using appropriate modification and delivery methods is an important goal for future peptide research. Summary of the Invention

[0004] The first objective of this invention is to provide a scorpion venom polypeptide.

[0005] The second aspect of the present invention aims to provide a biomaterial of scorpion venom polypeptides similar to those of the first aspect of the present invention.

[0006] The object of a third aspect of the present invention is to provide the application of the scorpion venom polypeptide of the first aspect of the present invention or a pharmaceutically acceptable salt thereof or the biological material of the second aspect of the present invention.

[0007] The fourth aspect of this invention is to provide a nanoparticle.

[0008] The fifth aspect of this invention is to provide a method for preparing nanoparticles according to the fourth aspect of this invention.

[0009] The sixth aspect of this invention aims to provide the application of the nanoparticles of the fourth aspect of this invention.

[0010] The seventh aspect of this invention aims to provide a product.

[0011] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0012] In a first aspect, the present invention provides a scorpion venom polypeptide, wherein the amino acid sequence of the scorpion venom polypeptide is FLGGLLSSIF.

[0013] This invention isolates a novel hydrophobic decapeptide (FLGGLLSSIF) from the Tibetan hog scorpion Chaerilus tricostatus, with a logP of 3.1, named C9 (i.e., scorpion venom polypeptide). This C9 polypeptide possesses an α-helical structure confirmed by dichroism spectroscopy, exhibits amphiphilicity, and demonstrates a certain degree of cell membrane affinity.

[0014] A second aspect of the invention provides a biomaterial related to the scorpion venom polypeptide of the first aspect of the invention, said biomaterial comprising any one of 1) to 12):

[0015] 1) A nucleic acid molecule encoding a scorpion venom polypeptide of the first aspect of the present invention; 2) An expression cassette comprising the nucleic acid molecule of 1); 3) A vector comprising the nucleic acid molecule of 1); 4) A vector comprising the expression cassette of 2); 5) A transgenic cell line comprising the nucleic acid molecule of 1); 6) A transgenic cell line comprising the expression cassette of 2); 7) A transgenic cell line comprising the vector of 3); 8) A transgenic cell line comprising the vector of 4); 9) A recombinant microorganism comprising the nucleic acid molecule of 1); 10) A recombinant microorganism comprising the expression cassette of 2); 11) A recombinant microorganism comprising the vector of 3); 12) A recombinant microorganism comprising the vector of 4).

[0016] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0017] In some embodiments of the present invention, the vector includes a promoter that is operatively linked to the nucleic acid molecule.

[0018] In some embodiments of the present invention, the vector is independently selected from non-pathogenic viral vectors and viral vectors.

[0019] In some embodiments of the present invention, the viral vector includes at least one of lentiviral vector, adenovirus vector, baculovirus vector, retrovirus vector, poxvirus vector, Sendai virus vector, and herpes simplex virus vector.

[0020] In some embodiments of the present invention, the non-viral vector includes at least one of plasmid vectors, cationic polymer vectors, chitosan, polyethyleneimine, nanoparticle vectors, and liposomes.

[0021] In some embodiments of the present invention, the vector is a plasmid vector, a phage particle, a viral vector, a cell vector, a bacteriophage, a sclerotium, an F sclerotium, or an artificial chromosome.

[0022] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0023] A third aspect of the invention provides the use of the scorpion venom polypeptide of the first aspect of the invention or a pharmaceutically acceptable salt thereof, or the biological material of the second aspect of the invention, in at least one of (a1) to (a4):

[0024] (a1) Inducing tumor cell apoptosis; (a2) Preparing products that induce tumor cell apoptosis; (a3) ​​Inhibiting tumor cell proliferation; (a4) Preparing products that inhibit tumor proliferation.

[0025] In some embodiments of the present invention, the tumor cells include glioblastoma cells.

[0026] In some embodiments of the present invention, the product includes reagents, kits, or drugs.

[0027] A fourth aspect of the present invention provides a nanoparticle prepared from a scorpion venom polypeptide comprising the first aspect of the present invention and a drug delivery carrier.

[0028] Due to the non-specific membrane binding of scorpion venom peptides, these peptides did not exhibit antiproliferative activity against glioblastoma cell lines. Therefore, this invention further developed small nanoparticles (drug delivery systems) to bind peptide C9 to a fifth-generation PAMAM dendritic macromolecule (G5 PAMAM-NHAC), which has terminal modifications of the PBA population. After successful binding to sialic acid on the cell surface, the complex underwent rapid endocytosis and targeted lysosomes, exhibiting significant anti-glioblastoma activity, including cell cycle arrest, inhibition of cell migration, and induction of apoptosis. Furthermore, the mechanism of action of G5C9 against glioblastoma was further elucidated: downregulation of the PI3K / AKT / mTOR signaling pathway, inhibition of mTORC1, and promotion of nuclear translocation of the downstream transcription factor EB (TFEB), thereby inducing lysosomal autophagy.

[0029] In some embodiments of the present invention, the drug delivery carrier comprises PAMAM dendrimers.

[0030] In some embodiments of the present invention, the PAMAM dendrimer includes acetylated fifth-generation PAMAM dendrimer, namely G5 PAMAM-NHAC dendrimer.

[0031] In some embodiments of the present invention, the C-terminal carboxyl group of the scorpion venom polypeptide is linked to the terminal amino group of the PAMAM dendrimer.

[0032] In some embodiments of the present invention, the dendritic macromolecules of the PAMAM are modified with 4-(bromoethyl)phenylboronic acid at their ends.

[0033] In some embodiments of the present invention, 4-(bromoethyl)phenylboronic acid is modified at the ends of PAMAM dendrimers by the following method: a DMSO solution containing acetylated PAMAM dendrimers is mixed with a DMSO solution containing 4-(bromoethyl)phenylboronic acid and reacted to obtain PBA-modified polyamide amine dendrimers.

[0034] In some preferred embodiments of the present invention, the molar ratio of the PAMAM dendrimer and 4-(bromoethyl)phenylboronic acid is 2:(2 to 4); preferably 2:3.

[0035] In some preferred embodiments of the present invention, the reaction conditions are 1400 to 1600 rpm for 20 to 25 hours.

[0036] In some embodiments of the present invention, the nanoparticles are spherical.

[0037] In some embodiments of the present invention, the particle size of the nanoparticles is 10 to 25 nm.

[0038] In some embodiments of the present invention, the zeta potential of the nanoparticles is 7.01 ± 0.77 mV.

[0039] This invention combines the scorpion venom polypeptide (C9) with a PAMAM dendritic macromolecule, which can serve as a targeted delivery system for GBM, enabling rapid internalization into tumor cells. Due to its small size and ability to penetrate tumor cells, G5 PAMAM-NHAC is used as a drug carrier, where C9 acts as a bioactive agent after penetrating tumor cells, enhancing retention within the tumor. To reduce the adverse toxicity of G5 PAMAM-NHAC and enhance its targeting to cancer cells, this invention further links PBA groups to some groups of G5 PAMAM-NHAC. The terminal PBA groups can react with the cis-diol of sialic acid overexpressed on the surface of cancer cells to form a reversible borate ester, thereby achieving specific targeting and binding to tumor cells. Furthermore, the acetyl group of G5-PAMMA-NHAC binds to the C-terminal free carboxyl group of the C9 polypeptide, allowing the hydrophobic peptide chain to embed into the lumen of G5, forming a stable peptide-dendritic macromolecule complex (i.e., the aforementioned nanoparticles, denoted as the G5C9 complex). The G5C9 complex exhibits antitumor activity against two human GBM cell lines, U251MG and U87MG, including inducing cell cycle arrest, inhibiting cell proliferation and migration, and apoptosis.

[0040] The G5C9 complex targets GBM cells and exhibits enhanced cellular uptake. G5C9 targets GBM cells by binding to sialic acid overexpressed on the cell surface, leading to rapid endocytosis and localization to lysosomes, resulting in autophagy-lysosomal apoptosis and cell death of tumor cells. Additionally, G5C9 inhibits GBM cell proliferation by enhancing the nuclear translocation of transcription factor EB (TFEB). TFEB, a key regulator of the autophagy-lysosomal pathway, inhibits GBM cell proliferation by suppressing mTORC1 activity and downregulating the PI3K / AKT / mTOR signaling pathway.

[0041] A fifth aspect of the present invention provides a method for preparing nanoparticles according to the fourth aspect of the present invention, comprising the following steps: mixing scorpion venom polypeptides and a drug delivery carrier in a buffer solution and reacting to obtain nanoparticles.

[0042] In some embodiments of the present invention, the mass ratio of the scorpion venom polypeptide to the drug delivery carrier is 1:(2 to 5).

[0043] In some embodiments of the present invention, the mass ratio of the scorpion venom polypeptide to the drug delivery carrier is 1:(3 to 4).

[0044] In some embodiments of the present invention, the mass ratio of the scorpion venom polypeptide to the drug delivery carrier is 1:4.

[0045] In some embodiments of the present invention, the reaction is carried out at room temperature for 3 to 6 hours.

[0046] In some embodiments of the present invention, the reaction is carried out at room temperature for 4 to 5 hours.

[0047] In some embodiments of the present invention, the buffer solution includes PBS buffer.

[0048] In some embodiments of the present invention, the preparation method further includes purification and drying steps: the solution after reaction is dialyzed, and after dialysis, it is freeze-dried to obtain nanoparticles.

[0049] A sixth aspect of the invention provides the use of the nanoparticles of the fourth aspect of the invention in at least one of (b1) to (b13):

[0050] (b1) Preparation of antitumor drugs; (b2) Inhibition of tumor cell proliferation; (b3) Preparation of products that inhibit tumor cell proliferation; (b4) Induction of tumor cell apoptosis; (b5) Preparation of products that induce tumor cell apoptosis; (b6) Inhibition of tumor cell migration; (b7) Preparation of products that inhibit tumor cell migration; (b8) Arrest of tumor cell cycle; (b9) Preparation of products that arrest tumor cell cycle; (b10) Inhibition of p70S6K phosphorylation; (b11) Preparation of products that inhibit p70S6K phosphorylation; (b12) Promotion of TFEB nuclear translocation; (b13) Preparation of products that promote TFEB nuclear translocation.

[0051] In some embodiments of the present invention, the tumors described in (b1) to (b9) include at least one of lung cancer, liver cancer, colon cancer, esophageal cancer, breast cancer, pancreatic cancer, and glioblastoma.

[0052] In some embodiments of the present invention, the tumor cells include glioblastoma cells, such as U251MG and U87MG.

[0053] In some embodiments of the present invention, the product includes reagents, reagent kits, or pharmaceuticals.

[0054] A seventh aspect of the present invention provides a product comprising the nanoparticles of the fourth aspect of the present invention.

[0055] In some embodiments of the present invention, the product includes reagents, kits, or drugs.

[0056] In some embodiments of the present invention, the product further includes at least one of pharmaceutically acceptable excipients, such as fillers, disintegrants, diluents, dispersants, excipients, stabilizers, lubricants, binders, humectants, flavoring agents, solubilizers, suspending agents, solvents, sustained-release agents, emulsifiers, absorption enhancers, surfactants, preservatives, pigments, fragrances, and solvents.

[0057] In some embodiments of the present invention, the drug may also contain other pharmacologically active ingredients, and can be used in combination with other drugs to achieve combination therapy, such as drugs that can be used to treat cancer.

[0058] The beneficial effects of this invention are:

[0059] This invention isolated and identified a novel scorpion venom polypeptide (denoted as C9) and constructed a novel polypeptide-polyamine dendritic complex, G5C9, with GBM-targeting activity. Compared to polypeptide C9, G5C9 exhibits significantly improved cellular uptake efficiency. G5C9 can enter cells via endocytosis and target lysosomes, inhibiting lysosomal function and mTORC1, thereby promoting nuclear translocation of TFEB and inducing autophagic cell death. Furthermore, G5C9 inhibits the PI3K / AKT / mTOR signaling pathway, further inducing cell cycle arrest and inhibiting cell proliferation and apoptosis. Therefore, G5C9 provides a novel targeted anti-GBM mechanism both in vitro and in vivo.

[0060] Specifically, scorpion venom peptide C9 possesses an α-helical structure confirmed by circular dichroism spectroscopy, exhibits some amphiphilicity, and demonstrates a certain affinity for cell membranes, but has not shown significant anticancer activity. The lack of cationicity compared to typical anticancer peptides may be the reason for this phenomenon. In confocal microscopy imaging, C9-FITC is located on the cell membrane, and the peptide's binding to the cell membrane is mainly through hydrophobic non-covalent interactions, which can be easily dissociated; therefore, cellular uptake of C9 could not be detected by flow cytometry. Therefore, this invention proposes that the PAMAM dendrimer incorporating the C9 decapeptide can serve as a targeted delivery system for GBM, enabling rapid internalization into tumor cells. Due to its small size and ability to penetrate tumor cells, G5 PAMAM is used as a drug carrier, where C9, after penetrating deep into tumor cells, acts as a bioactive agent, enhancing retention within the tumor. To reduce the adverse toxicity of G5 PAMAM and enhance its targeting to cancer cells, a PBA group is attached to the end group of G5 PAMAM. The terminal PBA group can react with the cis-diol of sialic acid overexpressed on the surface of cancer cells to form a reversible borate ester, thereby achieving specific targeting and binding to tumor cells. On the other hand, the amino group of G5 dehydrates with the free carboxyl group at the C-terminus of the C9 peptide, and the hydrophobic peptide chain can be inserted into the lumen of G5 to form a stable peptide-dendritic macromolecular complex.

[0061] Experiments revealed that the G5C9 complex exhibits antitumor activity against two human GBM cell lines, U251MG and U87MG, including inducing cell cycle arrest, inhibiting cell proliferation and migration, and apoptosis. These findings also confirm the hypothesis that an intracellular antitumor target for peptide C9 does indeed exist. From the perspective of mechanism of action, this invention reveals two modes of action of G5C9 on GBM cells. First, after entering tumor cells via endocytosis, G5C9 targets the lysosomal membrane within a short period (~2 hours) due to its hydrophobic and weakly basic nature. The polycyclic aromatic structure of the PBA group and the phenylalanine (Phe) residues in the C9 sequence may further promote the capture of G5C9 within the hydrocarbon core of the lipid bilayer and the protonation of free amino groups in PAMAM dendritic molecules. Second, mTORC1, located on the lysosomal membrane, is a central serine / threonine protein kinase that responds to cellular stress signals, thereby promoting cell growth by regulating anabolism and catabolism. As a nutrient sensor, mTORC1 closely regulates the inactivation of TFEB through phosphorylation. Depending on the cell's nutritional status, TFEB follows two distinct cycles: when amino acids are abundant, it shuttles continuously between lysosomes and the cytoplasm; conversely, in the absence of amino acids, TFEB is dephosphorylated by serine / threonine phosphatase (calcineurin), thereby inducing its transport from the cytoplasm to the nucleus, where it activates the expression of genes essential for lysosomal and autophagic biogenesis. In the experiments of this invention, G5C9 reduced the phosphorylation levels of mTOR and the mTORC1 substrate p70S6k, subsequently promoting the nuclear translocation of TFEB. Therefore, G5C9 induces lysosomal membrane dysfunction, leading to mTORC1 inactivation and reduced TFEB phosphorylation levels.

[0062] More than 400 downstream target genes of TFEB have been identified, including 10 lysosomal biogenesis regulatory genes and 17 autophagy regulatory genes, namely the autophagosome-lysosomal pathway. G5C9 induces the degradation of p62 and the accumulation of LC3B II through this process, leading to autophagic cell death.

[0063] Furthermore, lysosomal drugs such as cilassine and sunitinib, once entering the acidic lysosomal lumen, undergo protonation and become trapped within the lysosomal membrane, leading to drug resistance. The proton sponge effect of PAMAM may induce lysosomal alkalization, thereby inhibiting lysosomal function and mTORC1 activity. Therefore, upon entering the lysosome, G5C9 cells with the proton sponge effect can absorb protons accumulated in the lysosomal lumen via V-ATPase, preventing lysosomal acidification. This may lead to lysosomal membrane rupture, promoting lysosomal escape of G5C9 cells, which then distribute into the cytoplasm.

[0064] In tumor cells, overactivation of the PI3K / AKT signaling pathway is closely associated with enhanced tumor cell growth and survival, with phosphorylated AKT levels being strongly correlated with poor prognosis. Many therapies targeting PI3K / AKT signaling inhibition involve indirectly inhibiting PI3K / AKT signaling by targeting mTOR, a key downstream target of PI3K / AKT and a core component of mTORC1. In this study, we demonstrated that G5C9 inhibits the PI3K / AKT / mTOR signaling pathway, an activity that also contributes to its anti-cancer effects. Furthermore, the inhibition of mTORC1 can also be achieved through this inhibited axis. Specifically, the mechanism of action of G5C9 against glioblastoma involves downregulating the PI3K / AKT signaling pathway, inhibiting the nuclear translocation of mTORC1 and its downstream transcription factor EB (TFEB) to induce lysosomal autophagy.

[0065] In summary, G5C9 exhibits potent activity against GBM both in vivo and in vitro. The drug delivery strategy of this invention not only enhances GBM uptake of the peptide but also provides a novel therapeutic approach for targeted GBM clearance. Attached Figure Description

[0066] Figure 1 shows the identification of the novel polypeptide Ctri9495 (denoted as C9 polypeptide); in Figure 1, A is the nucleotide sequence of the cloned polypeptide Ctri9495 encoding cDNA and the open reading frame of translation. The signal peptide is shown in green, and the mature peptide is marked with a single underline. The start and stop codons are marked in blue; Figure 1, B is the helical wheel diagram of polypeptide Ctri9495 predicted by the Heliquest online server; Figure 1, C is the three-dimensional structural model of polypeptide Ctri9495; Figure 1, D is the chemical structure of polypeptide Ctri9495; Figure 1, E is the circular dichroism spectrum of polypeptide Ctri9495 (100 μM) in 50% TFE / H2O and H2O.

[0067] Figure 2 shows the characterization of G5 and G5C9; in Figure 2, A is the 1H NMR spectrum of G5 NHAC-PBA in D2O, with characteristic peaks highlighted in the red box; Figure 2, B is the synthetic route of G5C9; Figure 2, C is the UV spectral scan of C9, C9-FITC, G5 NHAC-PBA, and G5C9-FITC; Figure 2, D is the TEM image and size distribution histogram of G5C9.

[0068] Figure 3 shows the in vitro cytotoxicity assessment of G5, C9, and G5C9. In Figure 3A, two glioblastoma cell lines (U87MG and U251MG) and the human microvascular endothelial cell line HUVEC were treated with different concentrations of G5, C9, and G5C9 for 24 h and 48 h, respectively, and cell viability was determined by the MTT assay. In Figure 3B, U251MG and U87MG cells were treated with different concentrations of C9 and G5C9 (μg / mL) for 14 days, and colonies were stained with crystal violet. The figure shows representative images of the colonies and a bar chart for quantitative analysis. In Figure 3C, the hemolytic activity of G5, C9, and G5C9 against human erythrocytes was shown. Triton X 100 (0.1%) served as a positive control, and PBS served as a negative control. Statistical differences between each group and the negative control group were calculated. Data are expressed as mean ± SEM, n = 3. The differences were statistically significant: **p<0.01, ****p<0.0001, and the mean squares (NS) were not significant.

[0069] Figure 4 shows the uptake of C9-FITC and G5C9-FITC by U251MG and U87MG cells. In Figure 4A, flow cytometry plots and MFI statistics of U251MG and U87MG cells after 3 h and 6 h of administration of 100 μg / mL FITC-labeled C9 / G5C9. In Figure 4B, flow cytometry plots and MFI statistics of U251MG and U87MG cells after 6 h of administration of 100 μg / mL FITC-labeled G5C9 and 10 μg / mL CPZ, respectively. In Figure 4C, flow cytometry plots and MFI statistics of U251MG and U87MG cells after 6 h of administration of 100 μg / mL FITC-labeled G5C9 and 0.04 U Neu. Data are expressed as mean ± SD, n = 3. Statistically significant differences were defined as: **p < 0.01, ****p < 0.0001, NS (not significant).

[0070] Figure 5 shows a confocal microscopy image of the co-localization of C9-FITC / G5C9-FITC (green fluorescence) with subcellular features of the cell membrane / lysosome (red fluorescence); the image shows U251MG cells after incubation with C9-FITC and G5C9-FITC for 2 h. The cell nuclei were stained with Hoechst 33342 (blue fluorescence), and the scale bar is 20 μm.

[0071] Figure 6 shows the apoptotic cells of U251MG and U87MG cells after 48 h of treatment with C9 and G5C9, and the data were visualized by flow cytometry after annexin V-FITC / PI staining.

[0072] Figure 7 shows the apoptosis and cell cycle arrest induced by G5C9 in U251MG and U87MG cells. In Figure 7, A represents the quantitative distribution of early apoptotic cells, late apoptotic cells, and total apoptotic cells; B represents the cell cycle distribution of U251MG and U87MG cells after 24 hours of treatment with C9 and G5C9, as detected by flow cytometry; and C represents the distribution of cells in the G0 / G1, S, and G2 / M phases (bar charts). Data are expressed as mean ± SD, n = 3. Statistical significance: *****p < 0.01, p < 0.001, ****p < 0.0001, NS, not significant.

[0073] Figure 8 shows the inhibition of U251MG and U87MG cell migration by G5C9. In Figure 8, A represents the Transwell assay of U251MG and U87MG cells after 24 hours of treatment with C9 and G5C9, showing the migrating cells. Figure 8 B represents the quantitative analysis of migrating cells. Data are expressed as mean ± SD, n = 3. Statistical significance: *****p < 0.01, p < 0.001, ****p < 0.0001, NS, not significant.

[0074] Figure 9 shows the antiproliferative activity of C9 and G5C9 in vivo; where A in Figure 9 represents U251MG cells (1×10⁻⁶). 7 Subcutaneous injection was administered to mice to form tumor xenografts. Mice were injected with 20 mg / kg C9 / G5C9 or PBS around the tumor every other day for 30 consecutive days, and tumor volume (mm²) was measured every other day. 3 Tumor specimens were obtained from three groups of mice after sacrifice over 30 consecutive days. Scale bar: 1 cm; Figure 9B shows the tumor volume measured during the experiment in the three groups of mice; Figure 9C shows the body weight of mice in each group over 30 days. Data are expressed as mean ± SD, n = 5. Statistical significance is expressed as p < 0.0001, NS, not significant.

[0075] Figure 10 shows the antiproliferative activity of C9 and G5C9 in vivo; in Figure 10, A represents representative images of three groups of tumor sections H&E staining (top) and Ki-67 immunohistochemical analysis (bottom). Scale bar: 275 μm; Figure 10, B shows the Ki-67 index of the three groups analyzed using ImageJ software. Data are expressed as mean ± SD, n = 5. Statistical significance is expressed as p < 0.0001, NS, not significant.

[0076] Figure 11 shows RNA sequencing analysis of G5C9-treated and untreated U251MG cells. In Figure 11, A shows the transcriptome profile of U251MG cells treated with 100 μg / mL G5C9 for 24 h, with the overall FPKM clustering results clustered using log10(FPKM+1) values. Red indicates upregulated genes, and green indicates downregulated genes. Figure 11, B is a volcano plot of RNA sequences from the G5C9-treated and control groups in U251MG cells, showing significantly upregulated or downregulated genes. Figure 11, C shows KEGG analysis of G5C9-treated and untreated U251MG cells. Figure 11, D shows Western blotting detection of PI3K / AKT and mTOR signaling pathway-related proteins in the G5C9-treated and control groups.

[0077] Figure 12 shows the effects of G5C9 on TFEB nuclear translocation, lysosomal biogenesis, and autophagic cell death. In Figure 12, A shows the intracellular fluorescence localization of TFEB-GFP after treatment with 100 μg / mL G5C9 (blue: nucleus, green: TFEB, scale bar: 20 μm); B shows the effect of G5C9 (100 μg / mL, 4 h) on TFEB nuclear translocation in U251MG cells as detected by Western blotting (changes in Flag tag expression represent changes in TFEB expression); C shows the fluorescence intensity of lysosomes as detected by flow cytometry after treatment with 100 μg / mL G5C9 (right bar represents the MFI of lysosomes); D shows the fluorescence microscopy image of U251MG autophagosomes generated after 24 h of treatment with 100 μg / mL G5C9 using the MDC method (scale bar: 275 μm); E shows the fluorescence intensity of 100 μg / mL G5C9. Western blot analysis of key autophagy markers in U251MG cells after 24 h of G5C9 treatment was performed to determine the expression levels of LC3B I / LC3B II. Data are expressed as mean ± SD, n = 3. Statistical significance is indicated by **p < 0.01, ***p < 0.001, and ****p < 0.0001. Detailed Implementation

[0078] The present invention will be further described in detail below through specific embodiments.

[0079] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0080] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0081] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0082] Example

[0083] Experimental methods

[0084] (1) Cell lines and cell culture

[0085] Human malignant glioblastoma cell lines U251MG and U87MG, and human umbilical vein endothelial cell line HUVEC were used. All cell lines were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) and incubated at 37°C and 5% CO2. Cells were subjected to [further treatment / treatment] before use in subsequent experiments. The Lonza mycoplasma detection kit was used to detect mycoplasma.

[0086] (2) Discovery, identification and secondary structure determination of novel scorpion venom polypeptide Ctri9495

[0087] The inventors had previously constructed a cDNA library of the venom glands of the Tibetan hog scorpion *Chaerilus tricostatus* (He D, Cao Z, Zhang R, Li W. Molecular Cloning and Functional Identification of the Antimicrobial Peptide Gene Ctri9594 from the Venom of the Scorpion *Chaerilus tricostatus*. Antibiotics (Basel). 2021 Jul 23; 10(8):896). Colonies were randomly selected from the library and cultured overnight in liquid medium containing 35 μg / mL chloramphenicol. CDNAs of 500 bp were screened using M13 positive and M13 negative primers of the pDNA-LIB library vector. The approximately 500 bp plasmid pDNR-LIB was sequenced using an ABI 3730XL sequencer (Thermo Fisher Scientific, USA) with universal M13 positive primers. Open reading frames (ORFs) and amino acid sequences were predicted using Gene Runner software (version 6.5.52). Signal peptide sequences were analyzed using the Signal 5.0 server (https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / ; accessed 24 / 09 / 24), and the same sequences were searched in the NCBI BLAST database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi; accessed 24 / 09 / 24).

[0088] The peptides were chemically synthesized and identified by Nanjing Genscript Biotech Co., Ltd. The purity of the synthesized peptides was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The average molecular weight of the synthesized peptides was determined by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The conditions and methods for RP-HPLC and MALDI-TOF-MS were based on existing techniques (He D, Cao Z, Zhang R, Li W. Molecular Cloning and Functional Identification of the Antimicrobial Peptide Gene Ctri9594 from the Venom of the Scorpion Chaerilus tricostatus. Antibiotics (Basel). 2021 Jul 23; 10(8):896). The solubility of the peptides was tested in ultrapure water, 1x DPBS (pH = 7.1), and DMSO.

[0089] The chemical structure of the peptide was generated by Chemdraw software (version 22.0.0, Perkinelemer, USA); the three-dimensional structure model was predicted by the Heliquest online web server (http: / / heliquest.ipmc.cnrs.fr / cgi-bin / ComputParamsV2.py; accessed: 24 / 09 / 24); and the PEP-FOLD3 web server (https: / / bioserv.rpbs.univ-paris-diderot.fr / services / PEP-FOLD3 / ; accessed: 15 / 10 / 24).

[0090] Circular dichroism (CD) analysis was performed using a JASCO J815 spectrophotometer (JASCO Inc., Easton, Maryland, USA) to determine the secondary structure of the synthetic peptides. The collected results were analyzed using the K2D3 online analysis server (https: / / cbdm-01.zdv.uni-mainz.de / ~andrade / k2d3 / ; accessed 24 / 09 / 24).

[0091] (3) Synthesis and characterization of PBA-modified fifth-generation polyamidoamine dendritic macromolecules (G5 PMAMA-NHAC-PBA)

[0092] To improve targeted delivery capabilities, PBA molecules were coupled to the surface of acetylated G5 PAMAM. Specifically, a DMSO solution of G5 PAMAM-NHAC and a DMSO solution of 4-(bromoethyl)phenylboronic acid (PBA) were added dropwise at a molar ratio of 2:3. The mixture was stirred at 1500 rpm for 24 h, then purified using a 1400 MW CO2 ultrafiltration membrane and dialyzed with water. The dialysate was lyophilized, yielding the product G5 PAMAM-NHAC-PBA (hereinafter referred to as G5).

[0093] To characterize G5, its nuclear magnetic resonance spectrometer was used to record G5's nuclear magnetic resonance (NMR) data. 1 ¹H NMR spectra. Ultraviolet-visible (UV-Vis) spectra were generated using a NanoDrop 8000UV-vis spectrophotometer (Thermo Fisher Scientific, USA). Particle size and zeta potential were determined using a dynamic light scattering (DLS) instrument (Zs90, Malvern, UK).

[0094] (4) Preparation and characterization of the G5C9 complex

[0095] To prepare the C9 peptide and G5 complex (denoted as G5C9), G5 and C9 peptides were mixed in PBS buffer (pH 7.4, 10 mM) at a mass ratio of 1:4 and stirred, and reacted at room temperature for 4 h. After dialyzing the solution through a 10000 MWCO dialysis membrane for 2 days, the product G5C9 was obtained by lyophilization.

[0096] Particle size and zeta potential analysis were performed as described in (2). 10 μL of G5C9 was coated onto a copper mesh surface with an ultrathin carbon film and allowed to stand for 3–5 min before transmission electron microscopy (TEM) imaging. Excess liquid was then removed with filter paper, and the sample was dried at room temperature. Image acquisition and analysis were performed using a transmission electron microscope (Tecnai G2 Spirit, Field Electron and Ion Company, USA) with an accelerating voltage of 60 kV and a current of 3.5 μA.

[0097] (5) Cytotoxicity test

[0098] Cell viability was determined using the MTT assay: The cytotoxicity of G5, C9, and G5C9 against two glioma cell lines was measured using the MTT assay. The procedure was roughly as follows: After cell counting, cells were seeded in 96-well plates at 8000 cells per well, with three replicates per cell line. After overnight incubation, different concentrations of C9 and G5C9 were added. 24 and 48 hours after drug administration, 10 μL of MTT reagent was added to each well. After 4 hours, the liquid in the wells was removed, DMSO was added, and the plates were shaken for 10 minutes. The OD value was measured at 570 nm using a microplate reader.

[0099] Hemolytic activity assay: Clinically collected human whole blood was used for hemolytic activity assay. After adding an anticoagulant, the blood was allowed to stand. For the experiment, the lower layer of red blood cells was collected, washed three times with phosphate-buffered saline (PBS), and a 4% (v / v) red blood cell suspension was prepared. Different concentrations of peptides and PDPC were then incubated with 200 μL of the red blood cell suspension at 37°C for 2 h. Subsequently, 100 μL of the supernatant from each sample was transferred to a new 96-well plate, and the absorbance was measured at 570 nm using a microplate reader.

[0100] (6) Cloning experiment

[0101] U251MG and U87MG cells were seeded at a density of 200 cells / well in 6-well plates and allowed to adhere overnight. Afterward, the cells were treated with different concentrations of C9 and G5C9. After 12 days, the cells were washed with PBS, fixed with 4% paraformaldehyde for 30 min, and stained with 1% crystal violet. ImageJ software was used for colony imaging and quantification.

[0102] (7) Cell uptake experiment

[0103] Cell uptake of C9 and G5C9 was detected using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, USA). Cells were seeded at a density of 3 × 10⁶ cells / year. 5 Cells were incubated overnight at 37°C in 6-well plates. The original culture medium was then replaced with 100 μg / mL C9-FITC and G5C9-FITC dissolved in the culture medium. After incubation at 37°C for 3 h and 6 h, cells were washed three times with cold PBS and finally analyzed by flow cytometry using FlowJo software (version 10.8.1, FlowJo LLC, USA).

[0104] (8) Confocal imaging

[0105] Cells were seeded in Beyotime 35mm confocal culture dishes (Beyotime, China) at a density of 2 × 10⁶ cells / mL. 5The cells were incubated overnight at 37°C. Then, the original culture medium was replaced with 100 μg / mL C9-FITC and G5C9-FITC dissolved in the culture medium. After incubation at 37°C for 2 h, the cells were stained with Hoechst 33342 (Beyotime, China), Lyso-tracker Red (Beyotime, China), and CellMask (Thermo Fisher Scientific, USA). The cells were then washed three times with cold PBS and finally analyzed using a SpinSR 10 rotating disk confocal microscope (Olympus Life Science, Japan).

[0106] (9) Determination of lysosomal biogenesis

[0107] U251MG and U87MG cells were seeded in 12-well plates. After overnight incubation, cells were treated with C9 and G5C9 for 3 hours, followed by staining with Lyso-tracker Red (Beyotime, China). Cells were then washed three times with cold PBS, and collected for flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter Life Sciences, Indianapolis, USA). Fluorescence intensity analysis was performed using FlowJo software (version 10.8.1; FlowJo LLC, USA).

[0108] (10) Apoptosis and cell cycle detection

[0109] The effects of C9 and G5C9 treatments on cell apoptosis after 24 and 48 hours were determined using the Alexa Fluor 488 Annexin V / PI apoptosis assay kit and flow cytometry. The effects of C9 and G5C9 treatments on cell cycle progression after 24 and 48 hours were also determined by PI staining and flow cytometry.

[0110] (11) Cell migration experiment

[0111] Cell migration assays were performed in 6.5 mm transwell chambers. Cells (8 × 10⁶ cells per well) 4Cells were treated with C9 and G5C9 for 24 h, then resuspended in serum-free medium in the upper half of the transwell chamber and fresh, intact growth medium in the lower half. Cells were incubated at 37°C for 24 h, allowing migration from the upper to the lower part of the chamber. The transwell membrane was fixed with pre-cooled methanol for 30 min. Cells remaining on the upper part of the membrane were gently removed with a cotton swab, and the cells were stained with crystal violet for 15 min. Cells that had migrated into the transwell chamber were imaged using an EVOS M7000 microscope (Thermo Fisher Scientific, USA) and quantified using ImageJ software.

[0112] (12) Mouse xenograft tumor model

[0113] All animal research was conducted in accordance with the regulations of the Animal Research Ethics Committee of the University of Macau and in compliance with all relevant ethical guidelines (Approval No.: UMARE-050-2023). 100 μL containing 1×10 7 A suspension of U251MG cells (50% PBS and 50% Matrigel) was injected subcutaneously into 7-week-old female thymic nude mice to establish a xenograft mouse model. Ten days later, when the tumor volume reached approximately 100 mm², [the xenograft was observed]. 3 Mice were randomly divided into three groups: 1) Control group: each mouse received 100 μL of PBS peritumoral space every other day; 2) C9 treatment group: each mouse received 100 μL of C9 solution (20 mg / kg) peritumoral space every other day; 3) G5C9 treatment group: each mouse received 100 μL of G5C9 solution (20 mg / kg) every other day. C9 and G5C9 were dissolved in dimethyl sulfoxide (DMSO) and diluted in PBS. Body weight and tumor volume were measured every other day. Tumor volume (mm²) 3 The result was calculated using the formula (length × width²) / 2. Mice were sacrificed 30 days after drug treatment, and tumors were collected.

[0114] (13) Immunohistochemistry

[0115] Collected tumors were fixed with 4% paraformaldehyde for 48 hours at room temperature, embedded in paraffin blocks, and sectioned to a thickness of 5 μm. Tissue sections were dewaxed, boiled in citrate buffer for 5 min, and antigens were extracted. After blocking endogenous peroxidase activity and nonspecific antibody binding, the sections were incubated overnight at 4°C with primary antibody (1:2000). Immunoreactivity was detected using a DAB kit according to the manufacturer's instructions (Proteintech, #PK10006). Sections were lightly reverse-stained with hematoxylin. Color images of the immunohistochemical staining were obtained using an EVOS M7000 optical microscope.

[0116] (14) mRNA extraction, RNA sequencing and analysis

[0117] use Total RNA was extracted from U251MG cells treated with G5C9 (100 μg / mL) for 24 h using Reagent (Thermo Fisher Scientific, USA). RNA samples were then sent to Novogene Biotech Ltd. (Tianjin, China) for Illumina Hiseq PE150 sequencing and preliminary RNA-seq data analysis.

[0118] (15) Ectopic gene overexpression and transient transfection

[0119] 3×Flag-TFEB-GFP expression plasmid was purchased from Heyuan Biotechnology Co., Ltd. (Shanghai, China). Before transient transfection, U251MG cells were injected at 5×10⁻⁶ cells / year. 5 Cells were seeded at a density of 1×10⁶ cells per well in 6-well plates or 1×10⁶ cells per well. 5 Cells were seeded at the desired density in Beyotime 35mm confocal dishes (Beyotime, China). After overnight incubation, cells were transfected in Opti-MEM medium with Lipofectamine 3000 (Thermo Fisher Scientific, USA) and 3×Flag-TFEB-GFP plasmid. Six hours after transfection, the medium was replaced with fresh, intact medium. Cells were then incubated for 48 hours, followed by fresh medium for subsequent experiments.

[0120] (16) Autophagosome monoamine cadaverine (MDC) staining

[0121] Autophagic vesicles in autophagic cells were detected using an MDC staining kit (Beyotime, China). MDCs can specifically label autophagosomes and produce fluorescence through ion trapping and specific binding to membrane lipids. 2 × 10⁻⁶ 5 U251MG cells were seeded in 6-well plates and incubated overnight before treatment. After administration of different concentrations of G5C9, cells were stained with MDC in the dark at 37°C for 30 min and washed three times with PBS buffer. Fluorescence was observed using an EVOS M7000 microscope (Thermo Fisher Scientific, USA).

[0122] (17) Immunoblotting

[0123] Cells treated with C9 or G5C9 were lysed using frozen RIPA lysis buffer containing a protease inhibitor (Complete EDTA-free, #10634200, Roche) and a phosphatase inhibitor (PhosSTOP, #04906837001, Roche), and centrifuged at 16000g for 25 min at 4°C. The supernatant was collected, and protein levels were determined using the Pierce BCA Protein Assay Kit (#23225). All samples were dissolved in lithium dodecyl sulfate sample buffer and heated at 95°C for 10 min. Equal volumes of protein samples were separated on 10%–15% SDS-PAGE gels and then transferred to nitrocellulose membranes. The membranes were blocked in TBST with 5% skim milk at room temperature for 1 h, incubated overnight with primary antibody at 4°C, and then incubated with the corresponding secondary antibody at room temperature for 1 h. Finally, the bands were visualized using the ChemiDoc MP imaging system (Bio-Rad) by adding chemiluminescent HRP substrate reagent (1:1) (Immobilon Western, WBKLS0500, Millipore).

[0124] (18) Statistical analysis

[0125] All experiments were performed at least twice. Data are expressed as mean ± standard deviation (SD). GraphPad Prism 8.0 software was used for correlation tests, and statistical significance was considered between the control and experimental groups. Multiple t-tests were used for one-way or two-way ANOVA, with p < 0.05 indicating statistical significance.

[0126] Experimental results

[0127] (1) Isolation and identification of novel scorpion venom polypeptide Ctri9495

[0128] The precursor nucleotide sequence of the novel peptide Ctri9495 (SEQ ID NO:1) was cloned and identified from a constructed cDNA library of the venom gland of the Tibetan three-ridged scorpion (Chaerilus tricostatus). As shown in Figure 1, the open reading frame encoding the mature peptide, consisting of 65 amino acids, is listed. The signal peptide region consists of 23 amino acids, followed by a 10-amino acid mature peptide. The C-terminal propeptide consists of 32 amino acid residues. Subsequently, a typical glycine-lysine-arginine residue (GKR) cleavage site appears at the N-terminus of the C-terminal propeptide. In this example, a novel hydrophobic decapeptide (FLGGLLSSIF (SEQ ID NO:2)) with logP = 3.1 was isolated from the Tibetan three-ridged scorpion *Chaerilus tricostatus* and named Ctri9495 (denoted as C9). Using the Heliquest and PEP-FOLD 3 online servers, the secondary structure of C9 was predicted to be a typical amphiphilic molecule with a helical structure (Figure 1, B and C). The chemical structure of C9 is shown in Figure 1, D. The physicochemical properties of C9 are shown in Table 1. Peptide C9 exhibits high hydrophobicity (1.040) and hydrophobic moment (0.568), and is insoluble in water. This phenotype was confirmed in solubility tests (Table 2).

[0129] Table 1 Physicochemical properties of polypeptide C9

[0130] Table 2 Solubility test of C9 in different solvents

[0131] *1×DPBS: Phosphate buffer solution containing potassium chloride (KCl), potassium dihydrogen phosphate (KH2PO4), sodium chloride (NaCl), and disodium hydrate (Na2HPO4-7H2O).

[0132] (2) Synthesis and identification of G5 PAMAM-NHAC-PBA and G5C9

[0133] To improve targeted delivery, PBA molecules were coupled to the surface of G5 PAMAM. Based on the integral of the relevant characteristic peaks in the 1H NMR spectrum (Figure 2A), the characteristic peak of the acetyl group was 1.9 ppm, and the average number of PBA molecules (7.4 to 7.7 ppm) conjugated to each G5 PAMAM dendrimer (2.2 to 3.5 ppm) was 12.73. The synthetic route for G5C9 is shown in Figure 2B. The C-terminus of C9 is linked to the acetyl group of G5 PAMAM. The synthesized complexes G5C9 and G5C9-FITC were characterized by 1H NMR and UV spectroscopy. Aliphatic peaks appeared at 1.9 ppm, 2.2 ppm, 2.4 ppm, 2.6 ppm, 2.9 ppm, 3.1 ppm, 3.2 ppm, and 3.3 ppm. Aromatic peaks were reached at 6.9 ppm, 7.3 ppm, and 7.7 ppm. For ultraviolet spectra, FITC has a characteristic absorbance peak at around 500 nm, while G5 PAMAM and peptides do not (C in Figure 2).

[0134] The average particle sizes of G5 PAMAM-NHAC and G5C9 were measured to be 7.57 ± 1.77 nm and 16.66 ± 2.39 nm, respectively, using dynamic light scattering (FLRS) (Table 3). Both materials exhibited good polymer dispersion index (PDI), indicating that the connection with C9 slightly increased the particle size of G5. The Zeta potentials of G5 PAMAM-NHAC and G5C9 were 6.39 ± 1.25 mV and 7.01 ± 0.77 mV, respectively. The transmission electron microscopy (TEM) image of D in Figure 2 also confirmed that G5C9 is spherical with an average diameter of 19.93 nm.

[0135] Table 3. Particle size, potential, and PDI value of G5 and G5C9

[0136] (3) In vitro cytotoxicity observation of G5, C9 and G5C9

[0137] The cytotoxicity of G5, C9, and G5C9 against two human glioblastoma cell lines, U251MG and U87MG, the human umbilical vein endothelial cell line HUVEC, and human erythrocytes was investigated using the MTT assay (Figure 3A). The results showed that at concentrations ranging from 12.5 μg / mL to 100 μg / mL, G5 exhibited no cytotoxicity against either of the two glioblastoma cell lines and HUVEC (not used in subsequent bioactivity experiments); when the treatment time was 48 h, peptide C9 showed only weak antiproliferative activity against U251MG cells. However, the complex G5C9 exhibited significant cytotoxicity against glioblastoma cells within 48 h, while HUVEC cells showed a survival rate >80%, demonstrating cancer cell selectivity. Furthermore, in this embodiment, hiatus used a colony formation assay to test the proliferation-inhibiting effects of peptide C9 and the complex G5C9 in these two human glioblastoma cell lines. As shown in Figure 3B, the 50 μg / mL complex LG5C9 inhibited colony formation in U251MG and U87MG cells, with inhibitory activities of 56.7% and 61.3%, respectively. Even at a high concentration of 100 μg / mL, peptide C9 did not inhibit colony formation in either cell line. Notably, compared to the 0.1% Triton X-100 positive control, none of the three treatments showed any hemolytic activity, with a hemolysis rate <8% (Figure 3C).

[0138] (4) Cellular uptake and intracellular colocalization of C9 and G5C9

[0139] Flow cytometry was used to detect the uptake of C9-FITC and the complex G5C9-FITC by U251MG and U87MG cells. As shown in Figures 4A and 4B, after 3 and 6 hours of incubation with C9-FITC, the mean fluorescence intensity (MFI) of the two cell lines was comparable to that of the control group, indicating that the C9 peptide was not taken up by the cells even after 6 hours. In stark contrast, after 3 hours of incubation with G5C9-FITC, the MFI of both cell lines increased significantly, being 20 to 30 times higher than the background fluorescence of C9-FITC.

[0140] Because the cellular uptake of the complex G5C9-FITC is very high, while C9-FITC uptake is insufficient, the inventors hypothesized that G5C9-FITC enters cells via receptor-mediated endocytosis. Phenothiazine chlorpromazine (CPZ) is a widely used endocytosis inhibitor. Flow cytometry was used to detect the cellular uptake of G5C9-FITC with and without CPZ. After CPZ treatment, the MFI of cells was significantly reduced, indicating that the uptake of G5C9-FITC by cells was blocked, confirming that G5C9-FITC enters cells via endocytosis (Figure 4, C and D).

[0141] As described above, the G5 PAMAM used to construct G5C9 was modified with a terminal PBA group. PBA has been shown to selectively recognize sialic acid overexpressed on the surface of cancer cells. In this embodiment, neuraminidase (Neu) was used to remove sialic acid. The results, as shown in Figure 4, indicate that the high cellular uptake of G5C9 is dependent on the binding of the PBA group to sialic acid. When sialic acid was removed by Neu, the MFI of the cells decreased sharply.

[0142] Furthermore, the inventors investigated the intracellular localization of C9-FITC and G5C9-FITC in U251MG cells (Figure 5). After incubation with C9-FITC for 2 hours, the plasma membrane of U251MG cells showed strong colocalization of the green fluorescence signal of C9-FITC with the red fluorescent CellMask dye marker. In contrast, the plasma membrane of U251MG cells treated with G5C9-FITC did not show any green fluorescence, but punctate green fluorescence signals were detected in the perinuclear region of the cytoplasm. To precisely locate the organelle subcellular localization of G5C9-FITC, the inventors used the lysosomal dye Lyso-tracker red to track lysosomes of U251MG cells after incubation with G5C9-FITC for 2 hours. The colocalization of lysosomal red fluorescence and the green fluorescence of G5C9-FITC confirmed that the uptake of G5C9-FITC occurs through endocytosis, thus G5C9-FITC targets the perinuclear lysosomal compartment.

[0143] (5) G5C9 induces cell cycle arrest, apoptosis, and inhibits cell migration in glioblastoma cells.

[0144] To verify whether peptide C9 and the complex G5C9 can induce apoptosis, this study analyzed the proportion of apoptotic cells after Annexin V-FITC and PI staining. The results, shown in Figures 6 and 7A, indicate that after treatment with 50 μg / mL G5C9 for 48 h, the total number of apoptotic cells in U251MG and U87MG cells increased to 15.1% and 9.3%, respectively, with most apoptotic cells in the late apoptotic stage. After treatment with 50 μg / mL C9, the number of apoptotic cells in both cell lines was only 5%, indicating that the C9 peptide had little effect on inducing apoptosis.

[0145] The cell cycle distribution of U251MG and U87MG cells treated with C9 and G5C9 was further investigated. The results are shown in Figures 7B and C. After 24 hours of G5C9 treatment, the number of U251MG and U87MG cells in the G0 / G1 phase significantly increased. After treatment with 50 μg / mL and 100 μg / mL G5C9, the percentage of U251MG cells in the G0 / G1 phase increased from 40.9% to 67.6% and 56.5%, respectively, while the percentage of U87MG cells increased from 45.5% to 59.3% and 73.6%, respectively. Due to the increase in G0 / G1 phase cells, the number of U251MG cells in the G2 phase decreased from 39.7% to 19.6% and 14.2%, respectively. Compared with the control group, the number of U251MG and U87MG cells treated with C9 did not change significantly at each stage, indicating that the C9 peptide did not penetrate the cells and had no effect on cell cycle progression.

[0146] Furthermore, the number of migrating U251MG and U87MG cells was observed using a transwell migration assay after 24 h of treatment with C9 and G5C9. Treatment with 50 μg / mL and 100 μg / mL G5C9 reduced the number of migrating U251MG cells to 80% and 70% of the control group, respectively. This phenomenon was more pronounced in U87MG cells; after treatment with 50 μg / mL and 100 μg / mL G5C9, the migration rates of U87MG cells were 60% and 40% of the control cells, respectively. Therefore, G5C9 inhibited the migration of both glioma cell types in a dose-dependent manner, while C9 showed no migration-inhibiting effect (Figure 8, A and B).

[0147] (6) G5C9 inhibits the proliferation of U251MG cells in vivo.

[0148] The complex G5C9 inhibited the survival of glioblastoma cells in vitro, while peptide C9 did not show any inhibitory effect on proliferation. To determine whether G5C9 also possesses antitumor activity in vivo, this example established a tumor xenograft model by injecting U251MG cells into nude mice. When the volume of the xenograft tumor increased to 100 mm... 3 Mice were randomly divided into three groups and received peritumoral injections of 20 mg / kg C9, 20 mg / kg G5C9, or PBS every other day for 30 days. Tumor size and body weight were measured before each administration, until the end of the experiment on day 30. The specific experimental procedure is as follows:

[0149] The results showed that the G5C9 complex significantly inhibited xenograft tumor growth; compared with the control group, G5C9 inhibited tumor growth by 69.3%. In contrast, peptide C9 had no inhibitory effect on tumor cell proliferation, consistent with the conclusions of in vitro experiments (Figure 9, A and B). Furthermore, there was no significant difference in body weight among the three groups of experimental mice (Figure 9, C). These results indicate that the G5C9 complex strongly inhibits the growth of U251MG xenograft tumors and is non-toxic to nude mice, while peptide C9 has no antitumor activity.

[0150] After mice were euthanized, tumors were dissected and collected, fixed with paraformaldehyde, and prepared into sections. Hematoxylin and eosin (H&E) staining was used to observe morphological changes in the tumor tissue. H&E staining showed that hematoxylin staining of the nuclear chromatin was blue-purple, and eosin staining of the cytoplasm was red. Tumor sections from the control group and the peptide C9 treatment group showed generally enlarged tumor cells and nuclei. The tumor tissue structure was intact and the shape was regular. No necrotic areas were observed in either group. In contrast, tumor sections from the complex G5C9 treatment group showed nuclear agglutination and a large number of necrotic cells (Figure 10A). To evaluate the antiproliferative effects of different treatment groups at the tissue level, Ki-67 antibody staining was performed on the tumor sections. Ki-67 is a nuclear protein expressed during the active phase of the cell cycle and is a marker of cell proliferation and an indicator of high-grade tumors. As shown in Figure 10B, compared with the control group and the C9 treatment group, the number of Ki-67 positive spots in the G5C9 treatment group sections was significantly reduced.

[0151] (7) G5C9 inhibits cell proliferation associated with PI3K-AKT, mTOR signaling pathways and lysosomal bioactivity.

[0152] To investigate how G5C9 inhibits the growth of glioblastoma cells, the inventors performed RNA sequencing analysis on G5C9-treated and untreated U251MG cells and analyzed the differences in gene expression between the two groups. The results showed that 595 genes were significantly upregulated and 1084 genes were significantly downregulated, with a log2 fold greater than 1 or less than -1 (p < 0.05) (Figure 11, A and B). Next, to determine which biological pathways were most affected by G5C9 treatment, the inventors performed KEGG pathway enrichment analysis on the differentially expressed genes. The results showed enrichment of the PI3K / AKT and mTOR signaling pathways. Furthermore, KEGG analysis also enriched amino acid and lysosomal biosynthesis (Figure 11, C), indicating that G5C9 activity is closely related to lysosomal biological function. Correspondingly, G5C9 significantly downregulated the expression levels of key proteins in the PI3K-AKT and mTOR signaling pathways (Figure 11, D).

[0153] (8) G5C9 induces autophagic U251MG cell death by promoting nuclear translocation of transcription factor EB (TFEB).

[0154] RNA sequencing results suggest that the biological functions of G5C9 are closely related to lysosomal functions. Mammalian target of rapamycin 1 (mTORC1), located on the lysosomal membrane, is a central serine / threonine protein kinase with nutrient, bioenergy, and redox sensor functions. mTORC1 controls protein synthesis in response to cellular stress signals, thereby promoting cell growth by regulating anabolism and catabolism. This study further investigated the relationship between G5C9 and mTORC1. Western blotting results showed that G5C9 significantly downregulated the phosphorylation level of the mTORC1 substrate p70S6 kinase (p70S6K). TFEB is a downstream target of mTORC1 and plays a crucial role in regulating the expression of genes related to autophagy and lysosomal biogenesis. Under normal conditions, phosphorylated TFEB (pTFEB) is located in the cytoplasm and on the lysosomal surface, interacting with mTORC1. However, drug inhibition of mTORC1, cellular starvation, and lysosomal disruption activate TFEB and promote its nuclear translocation in its unphosphorylated form. Therefore, the inventors tested the transcriptional activity of TFEB after G5C9 treatment.

[0155] U251MG cells were transiently transfected with a 3×Flag-TFEB plasmid labeled with GFP, followed by G5C9 treatment. In the control group, TFEB fluorescence was distributed in the cytoplasm. In contrast, after G5C9 treatment, TFEB fluorescence gradually accumulated in the nucleus within 4 h, indicating that G5C9 treatment promoted the nuclear translocation of TFEB (Figure 12A).

[0156] Immunoblotting confirmed the accumulation of TFEB in the cell nucleus and the corresponding decrease in TFEB in the cytoplasm (Figure 12, B). Since nuclear translocation TFEB reactivates lysosomal biogenesis and the expression of autophagy-related genes, we stained lysosomes with LysoTracker red and used flow cytometry to detect lysosomal biogenesis in U251MG cells treated with G5C9. After 4 hours of G5C9 treatment, the fluorescence intensity of lysosomes in U251MG cells increased threefold compared to the control group, confirming a significant increase in the number of lysosomes in U251MG cells after G5C9 treatment (Figure 12, C). Simultaneously, MDC staining was used to detect autophagic U251MG cell death. As shown in Figure 12, D, the fluorescence signal of autophagosomes stained with MDC increased in a concentration-dependent manner after G5C9 treatment. Western blot further confirmed the occurrence of autophagy. After G5C9 treatment, the LC3B II protein level increased, and the LC3B II / LC3B I ratio increased, indicating the conversion of LC3B I to LC3B II. Meanwhile, after autophagy occurs, the autophagy substrate P62 is cleared and its expression is reduced (E in Figure 12).

[0157] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A scorpion venom polypeptide, characterized in that, The amino acid sequence of the scorpion venom polypeptide is FLGGLLSSIF.

2. A biomaterial related to the scorpion venom polypeptide of claim 1, wherein the biomaterial comprises any one of 1) to 12): 1) A nucleic acid molecule encoding the scorpion venom polypeptide of claim 1; 2) An expression cassette containing the nucleic acid molecule described in 1); 3) A carrier containing the nucleic acid molecule described in 1); 4) A carrier containing the expression box described in 2); 5) Transgenic cell lines containing the nucleic acid molecules described in 1); 6) A transgenic cell line containing the expression cassette described in 2); 7) A transgenic cell line containing the vector described in 3); 8) A transgenic cell line containing the vector described in 4); 9) Recombinant microorganisms containing the nucleic acid molecules described in 1); 10) Recombinant microorganisms containing the expression cassette described in 2); 11) Recombinant microorganisms containing the vector described in 3); 12) Recombinant microorganisms containing the vector described in 4).

3. The use of the scorpion venom polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, or the biomaterial of claim 2, in at least one of (a1) to (a4): (a1) Induces tumor cell apoptosis; (a2) Preparation of products that induce tumor cell apoptosis; (a3) Inhibits tumor cell proliferation; (a4) Prepare products that inhibit tumor proliferation.

4. A nanoparticle, characterized in that, The nanoparticles are prepared from the scorpion venom polypeptide and drug delivery carrier described in claim 1; Preferably, the drug delivery carrier comprises PAMAM dendrimers; Preferably, the PAMAM dendrimer comprises acetylated fifth-generation PAMAM dendrimer, namely G5PAMAM-NHAC dendrimer; Preferably, the dendritic macromolecules of PAMAM are terminally modified with 4-(bromoethyl)phenylboronic acid.

5. The nanoparticles according to claim 4, characterized in that, The C-terminal carboxyl group of the scorpion venom polypeptide is linked to the terminal amino group of the PAMAM dendritic macromolecule.

6. The method for preparing the nanoparticles according to claim 5, comprising the following steps: Nanoparticles are obtained by mixing scorpion venom peptides with a drug delivery carrier in a buffer solution.

7. The preparation method according to claim 6, characterized in that, The mass ratio of the scorpion venom polypeptide to the drug delivery carrier is 1:(2 to 5); Preferably, the reaction is carried out at room temperature for 3 to 6 hours; Preferably, the buffer solution comprises PBS buffer.

8. The use of the nanoparticles according to any one of claims 4-5 in at least one of (b1) to (b13): (b1) Preparation of antitumor drugs; (b2) Inhibits the proliferation of tumor cells; (b3) Prepare products that inhibit the proliferation of tumor cells; (b4) Induces tumor cell apoptosis; (b5) Preparation of products that induce tumor cell apoptosis; (b6) Inhibits tumor cell migration; (b7) Prepare products that inhibit tumor cell migration; (b8) Arresting the tumor cell cycle; (b9) Prepare products that arrest the tumor cell cycle; (b10) Inhibits phosphorylation of p70S6K; (b11) Prepare a product that inhibits the phosphorylation of p70S6K; (b12) Promotes nuclear translocation of transcription factor EB (TFEB); (b13) Prepare products that promote nuclear translocation of transcription factor EB (TFEB).

9. The application according to claim 8, characterized in that, The tumors described in (b1) to (b9) include at least one of lung cancer, liver cancer, colon cancer, esophageal cancer, breast cancer, pancreatic cancer, and glioblastoma.

10. A product comprising the nanoparticles according to any one of claims 4-5.

11. A method for inducing tumor cell apoptosis, inhibiting tumor cell proliferation, inhibiting tumor cell migration, or arresting the tumor cell cycle, said method comprising administering to a subject in need a therapeutically effective amount of the scorpion venom polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the biomaterial of claim 2, or the nanoparticles of any one of claims 4-5.

12. The method according to claim 11, characterized in that, The tumor cells include at least one of lung cancer cells, liver cancer cells, colon cancer cells, esophageal cancer cells, breast cancer cells, pancreatic cancer cells, and glioblastoma cells.

13. A method for inhibiting the phosphorylation of p70S6K, the method comprising administering to a subject in need a therapeutically effective amount of the scorpion venom polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the biomaterial of claim 2, or the nanoparticles of any one of claims 4-5.

14. A method for promoting nuclear translocation of transcription factor EB (TFEB), the method comprising administering to a subject in need a therapeutically effective amount of the scorpion venom polypeptide of claim 1 or a pharmaceutically acceptable salt thereof, the biomaterial of claim 2, or any nanoparticle of claims 4-5.

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