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

Abstract

The present application relates to a kind of SR-B1 targeting polypeptide and its application in the preparation of antitumor drugs, belong to biological medicine manufacturing technical field.To solve the problem that existing SR-B1 inhibitor cannot be applied in clinical application due to toxicity and potential risk, the present application provides a kind of SR-B1 targeting polypeptide and its application in the preparation of antitumor drugs.SR-B1 targeting polypeptide includes SR-B1 targeting peptide segment, self-assembly peptide segment and hydrophobic molecule connected in turn, can specifically recognize overexpressed SR-B1 in tumor cell, directly block tumor cell uptake cholesterol by effectively combining with SR-B1, reduce the cholesterol content in tumor cell, inhibit tumor progression.SR-B1 targeting polypeptide can form nanofiber in tumor microenvironment, prolong the residence time in tumor tissue, long-term inhibition effect is exerted.SR-B1 targeting polypeptide has wide clinical application prospect due to high tissue specificity and biological safety.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing technology, and particularly relates to an SR-B1 targeting polypeptide and its application in the preparation of antitumor drugs. Background Technology

[0002] Cholesterol plays a crucial role in the formation and development of various cancers, directly activating oncogenic signaling pathways and their downstream cascade reactions. Activation of these pathways leads to abnormal regulation of numerous gene expression, promoting the transcription and translation of genes related to cell proliferation, survival, and angiogenesis, while inhibiting the expression of apoptosis-related genes. This gives cancer cells an advantage in growth, survival, and angiogenesis. This series of changes drives tumor development, enhancing its malignancy and metastatic potential, severely impacting patient disease progression and prognosis.

[0003] Existing cholesterol inhibitors, such as statins, reduce cholesterol synthesis by inhibiting a key enzyme in the process (3-hydroxy-3-methylglutaryl-CoA reductase, HMGCR). However, research has revealed that primary clear cell renal cell carcinoma (ccRCC) has a unique cholesterol acquisition pathway. Unlike normal cells, cholesterol in renal cell carcinoma cells is not synthesized by the cells themselves, but rather taken up from plasma lipoproteins via receptor-mediated processes. This unique metabolic pattern means that the inhibitory effects of statins on cholesterol synthesis cannot effectively target the critical cholesterol supply sites of renal cell carcinoma cells, thus failing to significantly inhibit or interfere with the growth, proliferation, or survival of these cells.

[0004] SR-B1, or type B scavenger receptor 1, is a cholesterol transporter whose main function is to mediate cholesterol transport between cells, particularly in the uptake of high-density lipoprotein (HDL) cholesterol. BLT-1, as a small molecule inhibitor, selectively blocks SR-B1 function to inhibit cellular uptake of HDL cholesterol, and is 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 clinical translational potential. Therefore, when developing novel therapeutic strategies targeting specific cancer cholesterol metabolism pathways, it is necessary to comprehensively consider both the efficacy and safety of the drugs. Summary of the Invention

[0005] To address the problem that existing SR-B1 inhibitors cannot be used clinically due to toxicity and potential risks, this invention provides an SR-B1-targeting polypeptide and its application in the preparation of anti-tumor drugs. This 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 has high biocompatibility.

[0006] The technical solution of the present invention:

[0007] An SR-B1 targeting polypeptide comprises an SR-B1 targeting peptide, a self-assembled 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, and the amino acid sequence of the self-assembled peptide 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 peptide is shown in Formula I:

[0009]

[0010] Formula I.

[0011] Furthermore, when the SR-B1 targeting peptide is suspended in pure water, its hydrophobic ends aggregate together due to hydrophobic interactions to form nanosphere micelles with a particle size of 30.46 nm ± 2.81 nm.

[0012] The application of an SR-B1 targeting polypeptide provided by the present invention in the preparation of antitumor drugs.

[0013] Furthermore, the tumor is a tumor that specifically highly expresses SR-B1.

[0014] Furthermore, the tumors that specifically highly express SR-B1 are clear cell renal cell carcinoma, papillary renal cell carcinoma, melanoma, colorectal cancer, or esophageal squamous cell carcinoma.

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

[0016] Furthermore, the antitumor drug uses SR-B1 targeting peptide as the sole active ingredient or one of its active ingredients.

[0017] Furthermore, the antitumor drug is a non-gastrointestinal dosage form, specifically an injectable dosage form.

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

[0019] Furthermore, the antitumor drug can self-assemble into a nanofiber structure, prolonging the time the drug remains at the tumor site.

[0020] The beneficial effects of this invention are:

[0021] The SR-B1 targeting peptide provided by this invention can specifically and accurately identify SR-B1 overexpressed in tumor cells. By effectively binding to SR-B1, it directly blocks the key pathway of cholesterol uptake by tumor cells, effectively reduces the cholesterol content in tumor cells, and inhibits tumor progression.

[0022] The SR-B1 targeting peptide provided by this invention contains self-assembling peptide segments that can undergo allosteric changes in the tumor microenvironment and self-assemble into nanofibers. Based on the unique nanofiber retention mechanism, the retention time of the SR-B1 targeting peptide in tumor tissue is significantly prolonged, effectively avoiding the problem of rapid peptide clearance. This allows the SR-B1 targeting peptide to continuously inhibit the uptake of cholesterol by tumor cells, thereby producing a long-term and effective inhibitory effect on the proliferation, migration, and invasion of tumor cells.

[0023] The SR-B1 targeting peptide provided by this invention exhibits high tissue specificity, enhancing local drug concentration in tumors while reducing toxicity to normal tissues. Mouse model experiments have demonstrated that the SR-B1 targeting peptide does not produce significant toxicity or adverse effects on major organs in mice, exhibiting high biocompatibility and thus possessing broad clinical application prospects. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the molecular structures of the SR-B1 targeting peptide FKC and the control peptide FKC-C in Example 1;

[0025] Figure 2 The images show the ESI-MS mass spectra of the SR-B1 targeting peptide FKC and the control peptide FKC-C in Example 1.

[0026] Figure 3 The high-performance liquid chromatograms of the SR-B1 targeting peptide FKC and the control peptide FKC-C in Example 1 are shown.

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

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

[0029] Figure 6The images show CLSM fluorescence images of primary ccRCC cells after co-incubation of SR-B1 targeting peptide FKC and control peptide FKC-C in Example 3. Scale bar: 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 micrographs of primary ccRCC cells after treatment with the SR-B1 targeting peptide FKC and the control peptide FKC-C, respectively, in Example 4.

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

[0033] Figure 10 These are fluorescence images of mouse models 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 mouse models in Example 5 inoculated with FKC and FKC-C.

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

[0036] Figure 13 This is a comparative diagram showing 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 Microscopic images of mouse tumor tissues inoculated with FKC and FKC-C in Example 5, with a scale bar of 1 μm under low magnification and 500 nm under high magnification;

[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 of different treatment groups in Example 6;

[0040] Figure 17 This is a comparison of cholesterol content in primary ccRCC cells from 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 stained images of primary ccRCC cells that migrated and invaded in different treatment groups in Example 8;

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

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

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

[0046] Figure 23 These are photographs of tumors in mice from different treatment groups and a comparison of tumor weights in Example 9.

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

[0048] Figure 25 Images of H&E staining of the heart, liver, spleen, lungs and kidneys of mice in different treatment groups in Example 11. Detailed Implementation

[0049] The technical solution of the present invention will be further described below with reference to embodiments, but it is not limited thereto. Any modifications or equivalent substitutions to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be covered within the protection scope of the present invention. In the following embodiments, the process equipment or apparatus not specifically specified are all conventional equipment or apparatus in the art. Unless otherwise specified, the raw materials used in the embodiments of the present invention are all commercially available; unless otherwise specified, the technical means used in the embodiments 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 its preparation method.

[0052] In this embodiment, the SR-B1 targeting peptide FKC is composed of an SR-B1 targeting peptide segment, a self-assembled peptide segment, and a hydrophobic molecule connected sequentially. A schematic diagram of the molecular structure is shown below. Figure 1As shown in the figure; the amino acid sequence of the SR-B1 targeting peptide is shown in SEQ ID No. 1, specifically FAEKFKEAVKDYFAKFWD; the amino acid sequence of the self-assembled peptide is shown in SEQ ID No. 2, specifically KLVFF; 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 as a target to specifically bind to the overexpressed SR-B1 protein in tumor cells, achieving SR-B1 targeted recognition. The self-assembled peptide is derived from β-amyloid protein and can self-assemble into water-insoluble nanofibers with β-sheet secondary structures due to hydrogen bond interactions, enabling the SR-B1 targeting peptide to remain in tumor tissue for a long time. The hydrophobic hexaalkyl compound can maintain the hydrophilic-hydrophobic balance of the SR-B1 targeting peptide.

[0054] The SR-B1 targeting peptide FKC provided in this embodiment was synthesized using a conventional solid-phase peptide synthesis method in the art, from the C-terminus to...

[0055] The N-terminus is sequentially linked via an amide bond to a hydrophobic hexaalkyl compound C6 and the required amino acids from the self-assembled peptide and SR-B1 targeting peptide sequences, yielding the artificially synthesized SR-B1 targeting peptide FKC, whose molecular structure is shown in Formula I:

[0056]

[0057] Formula I.

[0058] In this embodiment, 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, forming nanosphere micelles with a particle size of 30.46 nm ± 2.81 nm. After injection, the nanosphere micelles are easily absorbed through the membrane, increasing the chance of the peptide binding to the target and achieving more efficient targeting.

[0059] This embodiment also provides a control polypeptide FKC-C that is immutable and self-assembled, composed of an SR-B1 targeting peptide, an immutable peptide, and a hydrophobic molecule connected sequentially. A schematic diagram of its molecular structure is shown below. Figure 1 As shown in SEQ ID No. 1, the amino acid sequence of the SR-B1 targeting peptide is FAEKFKEAVKDYFAKFWD; the amino acid sequence of the immutable peptide is SEQ ID No. 3, KAAGG; the hydrophobic molecule is a hydrophobic hexaalkyl compound (C6), specifically a carboxylic acid, carboxylate, or ester compound containing a hexaalkyl chain.

[0060] The control peptide FKC-C was synthesized using conventional solid-phase peptide synthesis methods in the art. A hydrophobic hexaalkyl compound C6 and an immutable peptide segment, along with the required amino acids from the SR-B1 targeting peptide sequence, were sequentially linked from the C-terminus to the N-terminus via amide bonds to obtain the artificially synthesized control peptide FKC-C. Its molecular structure is shown in Formula II.

[0061]

[0062] Formula II.

[0063] When the control peptide FKC-C was suspended in pure water at a concentration of 20 μmol / L, its hydrophobic ends aggregated together due to hydrophobic interactions to form nanosphere micelles, and their particle size was similar to that of FKC nanosphere micelles.

[0064] Electrospray ionization mass spectrometry (ESI-MS) analysis was performed on the SR-B1 targeting peptide FKC and the control peptide FKC-C. The results are as follows: Figure 2 As shown, the molecular weight of the SR-B1 targeting peptide FKC is 3390.01, while 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 sample are correct.

[0065] SR-B1 targeting peptide FKC and control peptide FKC-C (20×10 -6 M) was analyzed by high-performance liquid chromatography, and the results are as follows: Figure 3 As shown, there is only one main peak in the high performance liquid chromatogram, indicating that the purity of the SR-B1 targeting peptide FKC is >95%, and the purity of the control peptide FKC-C is >95%.

[0066] Example 2

[0067] This embodiment confirms that SR-B1 targeting peptides, when co-incubated with SR-B1, exhibit allosteric transformation and self-assembly into water-insoluble nanofibers.

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

[0069] Thiamine T (ThT) was added to PBS solution, SR-B1+PBS solution, FKC, and FKC-C co-incubated with SR-B1 for 0 h and 1 h, respectively. The resulting mixtures were then co-incubated for 1 h, and the fluorescence intensity of each mixture was measured using a fluorescence microplate reader. The results are as follows: Figure 5 As shown, the fluorescence intensity of the mixture obtained by co-incubating 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 produce a β-sheet structure. 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-incubating FKC-C did not change compared to FKC-C before incubation, indicating that FKC-C did not undergo allosteric behavior or self-assembly.

[0070] Example 3

[0071] This embodiment confirms that the SR-B1 targeting peptide has a targeting effect on tumor cells with high SR-B1 expression.

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

[0073] like Figure 6 As shown in the CLSM fluorescence image, the positions of Cy7-labeled FKC and FKC-C coincide with the position 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 embodiment confirms that the SR-B1 targeting peptide in FKC and FKC-C can specifically bind to the overexpressed SR-B1 protein in tumor cells, thereby achieving SR-B1 targeted recognition.

[0075] Example 4

[0076] This embodiment confirms that the SR-B1 targeting peptide can form nanofiber structures on the cell membrane of tumor cells and remain there for a long time.

[0077] The SR-B1 targeting peptide FKC and control peptide FKC-C obtained in Example 1 were incubated with primary ccRCC cells for 1 hour. Subsequently, the cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde solution at room temperature for 4 hours. Next, the cells were dehydrated sequentially with 30%, 50%, 70%, 90%, and 100% ethanol, followed by critical point drying with isoamyl acetate. Finally, the morphological changes of the primary ccRCC cells were analyzed using scanning electron microscopy, and the results are as follows: Figure 8 As shown, the cell membrane surface of primary ccRCC cells treated with FKC formed a distinct network of nanofibers, while no network of nanofibers was observed on the cell membrane surface of cells treated with FKC-C.

[0078] The SR-B1 targeting peptide FKC and 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 co-incubated with Cy7-labeled FKC and FKC-C for 1 hour. The cells were then washed three times with phosphate-buffered saline (PBS), and the culture medium was replaced with fresh medium for further culture. Cell imaging was performed using confocal laser scanning microscopy (CLSM) at 24, 48, and 72 hours to assess the retention of FKC and FKC-C within the cells. The results are as follows: Figure 9 As shown, FKC can remain in primary ccRCC cells for a long time, and the retention effect is significantly better than that of FKC-C.

[0079] This demonstrates that the SR-B1 targeting peptide FKC, driven by self-assembled peptides, can form nanofiber structures on the cell membrane of tumor cells, thereby prolonging its retention time.

[0080] Example 5

[0081] This embodiment demonstrates through animal model experiments that the SR-B1 targeting peptide can target ccRCC tumors in vivo and maintain their long-term presence.

[0082] In this embodiment, Balb / c nude mice were used as experimental animals. Renal cell carcinoma cells 786-O were inoculated into the right buttock of the mice. When the tumor volume reached 50 mm², 3 At that time, it was administered via tail vein injection (200 µL, 400 × 10⁻ 6 M) These mice were given the 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 at 1 h, 6 h, 12 h, 24 h, 48 h, and 72 h post-injection. Subsequently, the mice were sacrificed, and the biodistribution of FKC and FKC-C in major organs was assessed. Results are as follows: Figures 10-13As shown, FKC is mainly distributed in tumor sites, indicating that it has good SR-B1 targeting in vivo. Furthermore, FKC can be retained in tumor sites for a long time, while FKC-C, although it can target tumor sites, cannot be retained in tumor sites for a long time.

[0083] Tumor tissue sections were observed using a bio-transmission electron microscope (Bio-TEM), and the results are as follows: Figure 14 As shown, no morphological changes were observed in tumor tissue treated with FKC-C, while nanofiber formation was clearly detected in tumor tissue treated with FKC. This further confirms that the metabolic delay capability of FKC originates from self-assembled nanofibers.

[0084] Example 6

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

[0086] This example uses NBD cholesterol (a fluorescent cholesterol derivative) to evaluate the cholesterol uptake capacity of primary ccRCC cells. Primary ccRCC cells were seeded in culture dishes and treated with the SR-B1 targeting peptide FKC (20 × 10⁻⁶ cells) obtained in Example 1. -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 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 intensity were detected using fluorescence microscopy and a fluorescence microplate reader, respectively. Results are as follows: Figure 15 As shown, only a weak green NBD fluorescence signal was observed in primary ccRCC cells treated with FKC, proving that FKC (20 × 10⁻⁶) is insufficient for this purpose. -6 M) can significantly inhibit cellular cholesterol uptake. For example... Figure 16 As shown, primary ccRCC cells treated with FKC had the lowest cholesterol uptake, which was less than that of BLT-1, indicating that FKC's ability to inhibit cholesterol uptake by tumor cells is superior to that of the existing SR-B1 inhibitor BLT-1.

[0087] Primary ccRCC cells were compared with the SR-B1 targeting peptide FKC (20×10⁻⁶) obtained in Example 1. -6 M) and control peptide FKC-C (20×10) -6M) were co-incubated for 1 hour, with the SR-B1 inhibitor BLT-1 as a positive control. Cells were washed with phosphate-buffered saline (PBS), and the culture medium was replaced with fresh medium. Cells were then cultured for another 72 hours, and intracellular cholesterol was extracted to determine the effect of FKC-C or FKC on cholesterol metabolism in ccRCC cells. Results are as follows: Figure 17 As shown, using FKC (20×10 -6 Primary ccRCC cells treated with M) showed a significant decrease in intracellular cholesterol content, and the effect was better than that of FKC-C and BLT-1.

[0088] Example 7

[0089] This embodiment confirms that the SR-B1 targeting peptide has a killing effect on tumor cells.

[0090] Primary ccRCC cells were compared with the SR-B1 targeting peptide FKC (20×10⁻⁶) obtained in Example 1. -6 M) and control peptide FKC-C (20×10) -6 (M) After co-incubating for 1 hour, with the SR-B1 inhibitor BLT-1 as a positive control, the cells were cultured in fresh medium for 24, 48, and 72 hours. Subsequently, 10 µL of CCK-8 solution was added to the cells, and co-incubation was performed for 4 hours. Finally, absorbance values ​​were measured and cell viability was calculated.

[0091] The results are as follows Figure 18 As shown, using FKC (20×10 -6 The viability of primary ccRCC cells treated with M) was significantly reduced, indicating that the proliferation capacity 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 embodiment confirms that the SR-B1 targeting peptide has an inhibitory effect on the migration and invasion of tumor cells.

[0094] The SR-B1 targeting peptide FKC (20×10) obtained in Example 1 was used. -6 M) and control peptide FKC-C (20×10) -6Primary ccRCC cells treated with M) were seeded into the upper chamber of a Transwell plate (8 μm pores). Using the SR-B1 inhibitor BLT-1 as a positive control, complete culture medium containing 10% fetal bovine serum (FBS) was added to the lower chamber of the 24-well plate. Cells were cultured for 72 hours for cell migration assays. For cell invasion assays, Matrigel was first applied to the upper chamber, and then complete culture medium containing 10% FBS was added to the lower chamber of the 24-well plate. Cells were cultured for 72 hours. Subsequently, 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 an optical microscope.

[0095] The results are as follows Figures 19-21 As shown, FKC (20×10 -6 The SR-B1 targeting peptide FKC 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 embodiment demonstrates through animal model experiments that the SR-B1 targeting peptide has the effect of inhibiting tumor progression in vivo.

[0098] This example established a human tumor xenograft (PDX) mouse model of clear cell renal cell carcinoma (ccRCC). Mice were divided into four groups and administered phosphate-buffered saline (PBS), sunitinib (orally administered at a dose of 20 mg / kg once daily 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⁻⁻¹). 6 Mice were treated with FKC-C and FKC every three days for a total of five administrations. Mouse weight and tumor volume were systematically monitored every three days. Mice were sacrificed after 21 days, and tumor tissue was collected and weighed.

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

[0100] Example 10

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

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

[0103] Example 11

[0104] This embodiment demonstrates the high biocompatibility of the SR-B1 targeting peptide through animal model experiments.

[0105] After euthanizing the mice in each group in Example 9, major organs, including the heart, liver, spleen, lungs, and kidneys, were extracted for H&E staining and biocompatibility analysis. The results are as follows: Figure 25 As shown, there were no morphological differences in the heart, liver, spleen, lungs, and kidneys among the mice in each group, indicating that the SR-B1 targeting peptide FKC and the control peptide FKC-C did not produce significant toxicity or adverse effects on the major organs of mice and have high biosafety.

Claims

1. An SR-B1 targeting polypeptide, characterized in that, The product comprises an SR-B1 targeting peptide, a self-assembled 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, and the amino acid sequence of the self-assembled peptide is shown in SEQ ID No. 2; the hydrophobic molecule is hexanoic acid; when the SR-B1 targeting polypeptide is suspended in pure water, its hydrophobic ends aggregate together due to hydrophobic interactions to form nanosphere micelles, the particle size of the nanosphere micelles is 30.46 nm ± 2.81 nm, and the molecular structure of the SR-B1 targeting polypeptide is shown in Formula I: ； Formula I.

2. The use of the SR-B1 targeting polypeptide as described in claim 1 in the preparation of antitumor drugs, characterized in that, The tumor is a clear cell renal cell carcinoma.

3. The application of the SR-B1 targeting polypeptide according to claim 2 in the preparation of antitumor drugs, characterized in that, The anti-tumor drug uses SR-B1 targeting peptide as the sole active ingredient or one of its active ingredients.

4. The application of the SR-B1 targeting polypeptide according to claim 3 in the preparation of antitumor drugs, characterized in that, The antitumor drug is a non-gastrointestinal dosage form, specifically an injectable dosage form.

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