SR-B1 targeting polypeptide and application thereof in preparation of antitumor drugs

Abstract

The invention relates to an SR-B1 targeting polypeptide and application thereof in preparation of antitumor drugs, and belongs to the technical field of biological drug manufacturing. In order to solve the problem that an existing SR-B1 inhibitor cannot be clinically applied due to toxicity and potential risks, the invention provides an SR-B1 targeting polypeptide and application thereof in preparation of antitumor drugs. The SR-B1 targeting polypeptide comprises an SR-B1 targeting peptide fragment, a self-assembly peptide fragment and a hydrophobic molecule which are connected in sequence, can specifically recognize overexpressed SR-B1 in tumor cells, directly blocks cholesterol uptake of the tumor cells through effective combination with SR-B1, reduces the cholesterol content in the tumor cells, and inhibits tumor progression. The SR-B1 targeting polypeptide can form nanofibers in a tumor microenvironment, the residence time in tumor tissues is prolonged, and the inhibition effect is achieved for a long time. The SR-B1 targeting polypeptide has a wide clinical application prospect due to high tissue specificity and biological safety.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceutical manufacturing, and in particular relates to an SR-B1 targeting polypeptide and its application in the preparation of anti-tumor drugs. Background Art

[0002] Cholesterol plays a key role in the formation and progression of various cancers. It can directly activate oncogenic signaling pathways and a cascade of downstream signaling molecules. Activation of these signaling pathways leads to dysregulation of numerous gene expression, promoting the transcription and translation of genes associated with cell proliferation, survival, and angiogenesis, while inhibiting the expression of genes associated with apoptosis, thereby conferring advantages on cancer cells in terms of growth, survival, and angiogenesis. This series of changes fuels tumor progression, enhancing malignancy and invasive and metastatic capabilities, and significantly impacting patient disease progression and prognosis.

[0003] Existing cholesterol inhibitors, such as statins, reduce cholesterol synthesis by inhibiting a key enzyme in the cholesterol synthesis process (3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR). However, studies have found that primary clear cell renal cell carcinoma (ccRCC) has a unique cholesterol acquisition pathway. Unlike normal cells, renal cancer cells do not synthesize cholesterol themselves, but rather take up cholesterol from plasma lipoproteins in a receptor-mediated manner. This unique metabolic pattern means that statins, which inhibit cholesterol synthesis in renal cancer cells, cannot effectively reach the key target of cholesterol supply in renal cancer cells, making it difficult to significantly inhibit or interfere with the growth, proliferation, or survival of renal cancer cells.

[0004] SR-B1, or scavenger receptor type 1, is a cholesterol transporter whose primary function is to mediate intercellular cholesterol transport, particularly in the uptake of high-density lipoprotein cholesterol (HDL-C). As a small molecule inhibitor, BLT-1 inhibits cellular uptake of HDL-C by selectively blocking the function of SR-B1, making it of great significance in studying SR-B1-mediated cholesterol metabolism, such as in cancer and atherosclerosis. However, its strong hepatotoxicity, reproductive toxicity, and potential risks to adrenal and cardiovascular function limit its potential for clinical translation. Therefore, when developing new therapeutic strategies targeting cholesterol metabolic pathways in specific cancers, it is necessary to comprehensively consider the efficacy and safety of the drug. Summary of the Invention

[0005] To address the problem that existing SR-B1 inhibitors cannot be used clinically due to toxicity and potential risks, the present invention provides an SR-B1 targeting polypeptide and its use in the preparation of anti-tumor drugs. The polypeptide can specifically target tumors with high SR-B1 expression, inhibit the uptake of cholesterol by tumor cells, achieve long-term effective inhibition of tumor cells, and have a high degree of biosafety.

[0006] The technical solution of the present invention:

[0007] An SR-B1 targeting polypeptide comprises an SR-B1 targeting peptide segment, a self-assembling peptide segment and a hydrophobic molecule connected in sequence; the amino acid sequence of the SR-B1 targeting peptide segment is shown in SEQ ID No. 1, and the amino acid sequence of the self-assembling peptide segment is shown in SEQ ID No. 2; the hydrophobic molecule is a hydrophobic hexaalkyl compound.

[0008] Furthermore, the molecular structure of the SR-B1 targeting polypeptide is shown in Formula I:

[0009]

[0010] Formula I.

[0011] Furthermore, after the SR-B1 targeting polypeptide was suspended in pure water, its hydrophobic ends aggregated together due to hydrophobic interaction to form nano-microsphere micelles, and the particle size of the nano-microsphere micelles was 30.46 nm±2.81 nm.

[0012] A use of the SR-B1 targeting polypeptide provided by the present invention in the preparation of an anti-tumor drug.

[0013] Furthermore, the tumor is a tumor with specific high expression of SR-B1.

[0014] Furthermore, the tumor with specific high expression of SR-B1 is renal clear cell carcinoma, renal papillary cell carcinoma, melanoma, colorectal cancer or esophageal squamous cell carcinoma.

[0015] Furthermore, the tumor is renal clear cell carcinoma.

[0016] Furthermore, the anti-tumor drug has SR-B1 targeting polypeptide as the only active ingredient or one of the active ingredients.

[0017] Furthermore, the anti-tumor drug is a parenteral dosage form, specifically an injectable dosage form.

[0018] Furthermore, the anti-tumor drug can specifically target and bind to SR-B1, thereby blocking the uptake of cholesterol by tumor cells.

[0019] Furthermore, the anti-tumor drug can self-assemble to form a nanofiber structure, thereby prolonging the time the drug remains in the tumor site.

[0020] Beneficial effects of the present invention:

[0021] The SR-B1 targeting polypeptide provided by the present invention can specifically and accurately identify SR-B1 overexpressed in tumor cells, and directly block the key pathway of cholesterol uptake by tumor cells by effectively binding to SR-B1, effectively reducing the cholesterol content in tumor cells and inhibiting tumor progression.

[0022] The SR-B1 targeting peptide provided by the present invention is embedded with self-assembling peptide segments that undergo conformational changes within the tumor microenvironment and self-assemble into nanofibers. Due to its unique nanofiber retention mechanism, the SR-B1 targeting peptide's retention time within tumor tissue is significantly prolonged, effectively preventing the rapid clearance of the peptide. This allows the SR-B1 targeting peptide to continuously inhibit tumor cells' cholesterol uptake, thereby producing a long-term, effective inhibitory effect on tumor cell proliferation, migration, and invasion.

[0023] The SR-B1-targeting peptide provided by the present invention has high tissue specificity, enhancing local drug concentration in tumors while reducing toxicity to normal tissues. Experiments in mouse models have confirmed that the SR-B1-targeting peptide has no significant toxicity or adverse effects on major mouse organs, demonstrating its high biosafety and promising broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the molecular structure of the SR-B1 targeting polypeptide FKC and the control polypeptide FKC-C in Example 1;

[0025] Figure 2 : This is the ESI-MS mass spectrum of the SR-B1 targeting peptide FKC and the control peptide FKC-C in Example 1;

[0026] Figure 3 1 is a high performance liquid chromatogram of the SR-B1 targeting polypeptide FKC and the control polypeptide FKC-C in Example 1;

[0027] Figure 4 This is a comparison chart of the particle size changes of the SR-B1 targeting peptide FKC and the control peptide FKC-C before and after co-incubation with the SR-B1 solution in Example 2;

[0028] Figure 5 This is a comparison of the ThT fluorescence intensities of the SR-B1 targeting peptide FKC and the control peptide FKC-C before and after co-incubation with the SR-B1 solution in Example 2;

[0029] Figure 6CLSM fluorescence images of primary ccRCC cells after co-incubation of the SR-B1 targeting peptide FKC and the control peptide FKC-C in Example 3, scale: 20 μm;

[0030] Figure 7 This is a comparison of the fluorescence intensity of the SR-B1 targeting peptide FKC and the control peptide FKC-C after co-incubation with primary ccRCC cells in Example 3, and the fluorescence intensity of the positive control SR-B1;

[0031] Figure 8 These are scanning electron microscopic images of primary ccRCC cells treated with the SR-B1 targeting peptide FKC and the control peptide FKC-C in Example 4;

[0032] Figure 9 CLSM fluorescence images of primary ccRCC cells treated with the SR-B1 targeting peptide FKC and the control peptide FKC-C in Example 4 for 24 hours, 48 ​​hours, and 72 hours, respectively. Scale bar: 20 μm.

[0033] Figure 10 Fluorescence images of the mouse model inoculated with FKC and FKC-C at different administration times in Example 5;

[0034] Figure 11 This is a comparison of fluorescence signals at different administration times in the mouse model inoculated with FKC and FKC-C in Example 5;

[0035] Figure 12 Fluorescent images of the main organs of the mouse model inoculated with FKC and FKC-C in Example 5;

[0036] Figure 13 This is a comparative diagram of the distribution of FKC and FKC-C in the major organs of mice inoculated with FKC and FKC-C in Example 5;

[0037] Figure 14 These are microscopic images of tumor tissues of mice inoculated with FKC and FKC-C in Example 5. The low-power scale bar is 1 μm, and the high-power scale bar is 500 nm.

[0038] Figure 15 Fluorescence images of primary ccRCC cells in different treatment groups in Example 6;

[0039] Figure 16 This is a comparison of the fluorescence intensity of NBD cholesterol in primary ccRCC cells in different treatment groups in Example 6;

[0040] Figure 17 This is a comparison of cholesterol levels in primary ccRCC cells in different treatment groups in Example 6;

[0041] Figure 18 This is a comparison of cell viability of primary ccRCC cells in different treatment groups in Example 7;

[0042] Figure 19 These are the staining images of migrating and invading primary ccRCC cells in different treatment groups in Example 8;

[0043] Figure 20 This is a comparison of the migration numbers of primary ccRCC cells in different treatment groups in Example 8;

[0044] Figure 21 This is a comparison of the invasion numbers of primary ccRCC cells in different treatment groups in Example 8;

[0045] Figure 22 This is a comparison of tumor volumes in mice in different treatment groups in Example 9;

[0046] Figure 23 The following are photos of actual tumors in mice of different treatment groups and a comparison of tumor weights in Example 9;

[0047] Figure 24 This is a comparison of the cholesterol content in tumor tissues of mice in different treatment groups in Example 10;

[0048] Figure 25 These are H&E staining images of the heart, liver, spleen, lung, and kidney of mice in different treatment groups in Example 11. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0050] Example 1

[0051] This embodiment provides an SR-B1 targeting polypeptide FKC and a preparation method thereof.

[0052] In this embodiment, the SR-B1 targeting polypeptide FKC is composed of a SR-B1 targeting peptide segment, a self-assembling peptide segment, and a hydrophobic molecule connected in sequence. The molecular structure diagram is shown in FIG. Figure 1As shown; wherein the amino acid sequence of the SR-B1 targeting peptide is shown as SEQ ID No. 1, specifically FAEKFKEAVKDYFAKFWD; the amino acid sequence of the self-assembling peptide is shown as SEQ ID No. 2, specifically KLVFF, and the hydrophobic molecule is a hydrophobic hexaalkyl compound (C6), specifically a carboxylic acid, carboxylate or ester compound carrying a hexaalkyl chain. The hydrophobic hexaalkyl compound used in this embodiment is hexanoic acid.

[0053] The SR-B1 targeting peptide FKC uses the SR-B1 targeting peptide segment as the target head, specifically binding to the SR-B1 protein overexpressed in tumor cells to achieve SR-B1 targeted recognition; the self-assembling peptide segment is derived from β-amyloid protein, and due to the interaction of hydrogen bonds, it can self-assemble into water-insoluble nanofibers with a β-sheet secondary structure, thereby achieving long-term retention of the SR-B1 targeting peptide in tumor tissue; the hydrophobic hexaalkyl compound can maintain the hydrophilic and hydrophobic balance of the SR-B1 targeting peptide.

[0054] The SR-B1 targeting peptide FKC provided in this example was synthesized by conventional solid-phase peptide synthesis method in the art from C-terminus to

[0055] The N-terminus is linked sequentially to the hydrophobic hexaalkyl compound C6, the self-assembling peptide, and the required amino acids in the SR-B1 targeting peptide sequence through an amide bond to obtain an artificially synthesized SR-B1 targeting polypeptide FKC, the molecular structure of which is shown in Formula I:

[0056]

[0057] Formula I.

[0058] In this example, the SR-B1 targeting peptide FKC was suspended in pure water at a concentration of 20 μmol / L. Its hydrophobic ends aggregated due to hydrophobic interactions to form nanosphere micelles with a particle size of 30.46 nm ± 2.81 nm. Following injection, the nanosphere micelles are easily absorbed through the membrane, increasing the chances of peptide binding to the target and achieving more efficient targeting.

[0059] This example also provides a non-conformable self-assembly control polypeptide FKC-C, which is composed of a SR-B1 targeting peptide segment, a non-conformable peptide segment, and a hydrophobic molecule connected in sequence. The molecular structure diagram is shown in FIG. Figure 1 As shown; the amino acid sequence of the SR-B1 targeting peptide is shown as SEQ ID No. 1, specifically FAEKFKEAVKDYFAKFWD; the amino acid sequence of the invariant peptide is shown as SEQ ID No. 3, specifically KAAGG, and the hydrophobic molecule is a hydrophobic hexaalkyl compound (C6), specifically a carboxylic acid, carboxylate or ester compound containing a hexaalkyl chain.

[0060] The control polypeptide FKC-C is synthesized by conventional solid-phase peptide synthesis methods in the art. The hydrophobic hexaalkyl compound C6 and the required amino acids in the constant conformation peptide segment and the SR-B1 targeting peptide segment are sequentially linked from the C-terminus to the N-terminus through amide bonds to obtain an artificially synthesized control polypeptide FKC-C. Its molecular structure is shown in Formula II:

[0061]

[0062] Formula II.

[0063] When the control polypeptide FKC-C was suspended in pure water at a concentration of 20 μmol / L, its hydrophobic ends aggregated together due to hydrophobic interaction to form nano-microsphere micelles, whose particle size was similar to that of FKC nano-microsphere micelles.

[0064] Electrospray ionization mass spectrometry (ESI-MS) analysis of the SR-B1 targeting peptide FKC and the control peptide FKC-C was performed. Figure 2 As shown, the molecular weight of the SR-B1 targeting peptide FKC is 3390.01, and the molecular weight of the control peptide FKC-C is 3011.45; the detected molecular weight corresponds to the theoretical molecular weight, proving that the mass spectrometry detection results of the samples are correct.

[0065] The SR-B1 targeting peptide FKC and the control peptide FKC-C (20×10 -6 M) was subjected to high performance liquid chromatography analysis, and the results were as follows Figure 3 As shown, there is only one main peak in the HPLC graph, the purity of the SR-B1 targeting peptide FKC is greater than 95%, and the purity of the control peptide FKC-C is greater than 95%.

[0066] Example 2

[0067] This example demonstrates that the SR-B1 targeting polypeptide has the ability to undergo conformational change and self-assemble into water-insoluble nanofibers after co-incubation with SR-B1.

[0068] The SR-B1 targeting peptide FKC and the control peptide FKC-C obtained in Example 1 were incubated with the SR-B1 solution for 1 hour, and dynamic light scattering (DLS) analysis was performed on the peptide samples before and after incubation. The results were as follows: Figure 4 As shown in the figure, the particle size of FKC increased significantly after co-incubation with SR-B1, indicating that FKC underwent allosteric behavior after binding to SR-B1 protein; while the particle size did not change significantly after co-incubation with FKC-C, indicating that FKC-C did not undergo structural changes.

[0069] Thioflavin T (ThT) was added to the systems obtained by co-incubating PBS solution, SR-B1+PBS solution, FKC, and FKC-C with SR-B1 for 0 h and 1 h, respectively. The resulting mixtures were co-incubated for 1 h, and then the fluorescence intensity of each mixture was measured using a fluorescence microplate reader. The results are shown in Figure 2. Figure 5 As shown in the figure, the fluorescence intensity of the mixture obtained by co-incubation of FKC and SR-B1 was significantly higher than that of PBS solution, FKC before incubation, FKC-C, and SR-B1. This indicates that FKC undergoes allosteric behavior after binding to SR-B1 protein, self-assembling to form a β-sheet structure, and the fluorescence intensity is significantly enhanced after the ThT reagent binds to the β-sheet structure. However, the fluorescence intensity of the mixture obtained after co-incubation of FKC-C did not change compared with FKC-C before incubation, indicating that FKC-C did not undergo allosteric behavior or self-assembly.

[0070] Example 3

[0071] This example demonstrates that the SR-B1 targeting polypeptide has a targeting effect on tumor cells with high expression of SR-B1.

[0072] The SR-B1-targeting peptide FKC and the control peptide FKC-C obtained in Example 1 were each labeled with cyanine 7 dye (Cy7). Primary clear cell renal cell carcinoma (ccRCC) cells were incubated with Cy7-labeled FKC and FKC-C for 1 hour. Immunofluorescence detection of SR-B1 was then performed as a positive control, and CLSM fluorescence images were acquired to assess the colocalization of FKC and FKC-C with SR-B1.

[0073] like Figure 6 As shown in the CLSM fluorescence images, the locations of FKC and FKC-C labeled with Cy7 are consistent with the locations of SR-B1 in tumor cells, indicating that both FKC and FKC-C can precisely target SR-B1 in tumor cells. Figure 7 The fluorescence intensity comparison shows that the fluorescence intensity of FKC and FKC-C is comparable to that of SR-B1, indicating that both FKC and FKC-C have good targeting properties and high binding efficiency with SR-B1, reducing the risk of off-target effects.

[0074] Based on this, this example confirms that the SR-B1 targeting peptide in FKC and FKC-C, as a target head, can specifically bind to the SR-B1 protein overexpressed in tumor cells, thereby achieving targeted recognition of SR-B1.

[0075] Example 4

[0076] This example demonstrates that the SR-B1 targeting polypeptide has the ability to form nanofiber structures on the cell membranes of tumor cells and retain them for a long time.

[0077] The SR-B1 targeting peptide FKC and the control peptide FKC-C obtained in Example 1 were incubated with primary ccRCC cells for 1 hour, respectively. Subsequently, the cells were rinsed with phosphate-buffered saline (PBS) and placed in a 4% paraformaldehyde solution and fixed at room temperature for 4 hours. Next, the cells were dehydrated with ethanol at concentrations of 30%, 50%, 70%, 90%, and 100%, followed by critical point drying with isoamyl acetate. Finally, the morphological changes of primary ccRCC cells were analyzed using a scanning electron microscope, and the results are shown in Figure 2. Figure 8 As shown in the figure, obvious reticular nanofiber structures were formed on the cell membrane surface of primary ccRCC cells treated with FKC, while no reticular nanofiber structures were observed on the cell membrane surface of cells treated with FKC-C.

[0078] The SR-B1 targeting peptide FKC and the control peptide FKC-C obtained in Example 1 were labeled with cyanine 7 dye (Cy7). Primary clear cell renal cell carcinoma (ccRCC) cells were seeded in culture dishes and incubated with Cy7-labeled FKC and FKC-C for 1 hour, respectively. The cells were then washed three times with phosphate buffered saline (PBS) and cultured with fresh medium. Confocal laser scanning microscopy (CLSM) was used to image the cells at 24 hours, 48 ​​hours, and 72 hours to evaluate the intracellular retention of FKC and FKC-C. The results are shown in Figure 2. Figure 9 As shown in the results, FKC can be retained in primary ccRCC cells for a long time, and the retention effect is significantly better than that of FKC-C.

[0079] This shows that the SR-B1 targeting peptide FKC, driven by the self-assembling peptide segment, can form a nanofiber structure on the cell membrane of tumor cells, thereby prolonging the retention time.

[0080] Example 5

[0081] This example demonstrates through animal model experiments that the SR-B1 targeting polypeptide has the ability to target ccRCC tumors in vivo and maintain long-term retention.

[0082] In this example, Balb / c nude mice were used as experimental animals, and renal cancer cell line 786-O was inoculated into the right buttocks of the mice. 3 When the 400×10⁻ 6 M) These mice were administered Cy7-labeled SR-B1-targeting peptide FKC and control peptide FKC-C obtained in Example 1. Fluorescence imaging was performed using a small animal in vivo imaging system 1, 6, 12, 24, 48, and 72 hours after injection. Subsequently, the mice were sacrificed and the biodistribution of FKC and FKC-C in major organs was assessed. The results are shown in Figure 2. Figure 10-13As shown in the figure, FKC is mainly distributed in the tumor site, indicating that it has good SR-B1 targeting in vivo, and FKC can remain in the tumor site for a long time in the body, while FKC-C can target the tumor site but cannot remain in the tumor site for a long time.

[0083] Tumor tissue sections were observed using biological transmission electron microscopy (Bio-TEM). Figure 14 As shown, no morphological changes were observed in tumor tissues treated with FKC-C, whereas nanofiber formation was clearly detected in tumor tissues treated with FKC, further confirming that the metabolic delaying ability of FKC originated from self-assembled nanofibers.

[0084] Example 6

[0085] This example demonstrates that the SR-B1 targeting polypeptide has the effect of inhibiting the uptake of cholesterol by tumor cells and reducing the intracellular cholesterol content.

[0086] In this example, NBD cholesterol (a fluorescent cholesterol derivative) was used to evaluate the cholesterol uptake ability of primary ccRCC cells. Primary ccRCC cells were seeded in culture dishes and incubated with the SR-B1 targeting peptide FKC (20×10 -6 M) or control peptide FKC-C (20×10 -6 M) were co-incubated for 1 hour, with the SR-B1 inhibitor BLT-1 as a positive control. Subsequently, the culture medium was replaced with fresh culture medium containing NBD cholesterol, and the cells were cultured for another 72 hours. After washing the cells with phosphate-buffered saline (PBS), the intracellular NBD fluorescence signal and fluorescence intensity were detected using a fluorescence microscope and a fluorescence microplate reader, respectively. The results are shown in Figure 2. Figure 15 As shown, only weak green NBD fluorescence signals were observed in primary ccRCC cells treated with FKC, demonstrating that FKC (20 × 10 -6 M) can significantly inhibit the uptake of cholesterol by cells. Figure 16 As shown in the results, the cholesterol uptake in primary ccRCC cells after FKC treatment was minimal and less than that in BLT-1, indicating that FKC is better than the existing SR-B1 inhibitor BLT-1 in inhibiting cholesterol uptake in tumor cells.

[0087] Primary ccRCC cells were incubated with the SR-B1 targeting peptide FKC (20×10 -6 M) and control peptide FKC-C (20×10 -6M) were co-incubated for 1 hour. The SR-B1 inhibitor BLT-1 was used as a positive control. The cells were washed with phosphate-buffered saline (PBS) and the medium was replaced with fresh medium. The cells were then cultured for an additional 72 hours, and intracellular cholesterol was extracted to determine the effects of FKC-C or FKC on cholesterol metabolism in ccRCC cells. Figure 17 As shown, FKC (20×10 -6 The cholesterol content in primary ccRCC cells treated with FKC-C and BLT-1 was significantly decreased, and the effect was better than that of FKC-C and BLT-1.

[0088] Example 7

[0089] This example demonstrates that the SR-B1 targeting polypeptide has a killing effect on tumor cells.

[0090] Primary ccRCC cells were incubated with the SR-B1 targeting peptide FKC (20×10 -6 M) and control peptide FKC-C (20×10 -6 M) for 1 hour. The SR-B1 inhibitor BLT-1 was used as a positive control. Cells were then cultured in fresh medium for an additional 24, 48, and 72 hours. Subsequently, 10 µL of CCK-8 solution was added to the cells and incubated for 4 hours. Finally, absorbance was measured and cell viability was calculated.

[0091] The results are as follows Figure 18 As shown, FKC (20×10 -6 The viability of primary ccRCC cells treated with SR-B1 was significantly decreased, indicating that the proliferation ability of primary ccRCC cells was significantly inhibited by the SR-B1 targeting peptide FKC, and the effect was better than that of FKC-C and BLT-1.

[0092] Example 8

[0093] This example demonstrates that the SR-B1 targeting polypeptide has an inhibitory effect on the migration and invasion of tumor cells.

[0094] The SR-B1 targeting polypeptide FKC (20×10 -6 M) and control peptide FKC-C (20×10 -6Primary ccRCC cells treated with α-actin (α-actin) (M) were seeded into the upper chamber of a Transwell chamber (8μm pore size). The SR-B1 inhibitor BLT-1 was used as a positive control. Complete medium containing 10% fetal bovine serum (FBS) was added to the lower chamber of a 24-well plate. The cells were cultured for 72 hours before performing a cell migration assay. For the cell invasion assay, Matrigel was first applied to the upper chamber, and complete medium containing 10% fetal bovine serum (FBS) was then added to the lower chamber of the 24-well plate. The cells were cultured for 72 hours. Subsequently, the cells on the bottom surface of the lower chamber were fixed with 4% paraformaldehyde and stained with 0.2% crystal violet. Finally, the stained cells were observed using a light microscope.

[0095] The results are as follows Figures 19-21 As shown, FKC (20×10 -6 M) significantly reduced the migration and invasion abilities of primary ccRCC cells to 41.85±4.87 and 40.39±4.45, respectively, compared to the PBS control group. This demonstrates that the SR-B1-targeting peptide FKC effectively inhibits the migration and invasion abilities of primary ccRCC cells.

[0096] Example 9

[0097] This example demonstrates through animal model experiments that the SR-B1 targeting polypeptide has the effect of inhibiting tumor progression in vivo.

[0098] In this example, a humanized renal clear cell carcinoma (ccRCC) xenograft (PDX) mouse model was established. The mice were divided into four groups and treated with phosphate buffered saline (PBS), sunitinib (administered orally at a dose of 20 mg / kg, once a day for a total of 15 doses), the SR-B1 targeting peptide FKC obtained in Example 1, and the control peptide FKC-C (200 μL, 400×10⁻ 6 M) were treated with FKC-C and FKC every three days for a total of five doses. Body weight and tumor volume were systematically monitored every three days. Mice were sacrificed after 21 days, and tumor tissue was weighed.

[0099] The results are as follows Figure 22 and Figure 23 As shown, the tumor volume of mice in the FKC-treated group grew slowly, and the tumor weight was significantly lower than that in the PBS control group and the FKC-C group after 21 days, which proved that the SR-B1 targeting peptide FKC significantly inhibited tumor proliferation.

[0100] Example 10

[0101] This example demonstrates through animal model experiments that the SR-B1 targeting polypeptide has the effect of inhibiting the uptake of cholesterol by tumor cells.

[0102] After the mice in each group in Example 9 were sacrificed, the tumor tissues were taken for cholesterol content determination. The results are as follows: Figure 24 As shown, the cholesterol content in the tumor tissue of the mice in the FKC-treated group decreased to 42.20±4.31% compared with that in the PBS control group, indicating that the SR-B1 targeting peptide effectively inhibited the uptake of cholesterol by renal clear cell carcinoma (ccRCC) cells in vivo.

[0103] Example 11

[0104] This example demonstrates through animal model experiments that the SR-B1 targeting polypeptide has a high degree of biosafety.

[0105] After the mice in each group in Example 9 were sacrificed, the main organs, including heart, liver, spleen, lung and kidney, were extracted and examined by H&E staining for biocompatibility analysis. Figure 25 As shown, there was no difference in the morphology of the heart, liver, spleen, lung and kidney among the mice in each group, indicating that the SR-B1 targeting peptide FKC and the control peptide FKC-C had no obvious toxicity or adverse effects on the major organs of mice and had a high degree of biosafety.

Claims

1. An SR-B1 targeting polypeptide, characterized in that It comprises an SR-B1 targeting peptide, a self-assembling peptide and a hydrophobic molecule connected in sequence; the amino acid sequence of the SR-B1 targeting peptide is shown in SEQ ID No. 1, the amino acid sequence of the self-assembling peptide is shown in SEQ ID No. 2; and the hydrophobic molecule is a hydrophobic hexaalkyl compound.

2. The SR-B1 targeting polypeptide according to claim 1, characterized in that The molecular structure of the SR-B1 targeting polypeptide is shown in Formula I: ； Formula I.

3. An SR-B1 targeting polypeptide according to claim 1 or 2, characterized in that After the SR-B1 targeting polypeptide is suspended in pure water, its hydrophobic ends aggregate together due to hydrophobic interaction to form nano-microsphere micelles. The particle size of the nano-microsphere micelles is 30.46 nm±2.81 nm.

4. Use of the SR-B1 targeting polypeptide according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

5. The use of the SR-B1 targeting polypeptide according to claim 4 in the preparation of anti-tumor drugs, characterized in that: The tumor is a tumor that specifically and highly expresses SR-B1.

6. Use of the SR-B1 targeting polypeptide according to claim 5 in the preparation of anti-tumor drugs, characterized in that: The tumor with specific high expression of SR-B1 is renal clear cell carcinoma, renal papillary cell carcinoma, melanoma, colorectal cancer or esophageal squamous cell carcinoma.

7. Use of the SR-B1 targeting polypeptide according to claim 6 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug uses the SR-B1 targeting polypeptide as the only active ingredient or one of the active ingredients.

8. Use of the SR-B1 targeting polypeptide according to claim 7 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug is in a parenteral administration form, specifically an injection administration form.

9. Use of the SR-B1 targeting polypeptide according to claim 8 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug can specifically target SR-B1 and bind to it, thereby blocking the uptake of cholesterol by tumor cells.

10. Use of the SR-B1 targeting polypeptide according to claim 9 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drug can self-assemble to form a nanofiber structure, thereby prolonging the time the drug stays in the tumor site.

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