Preparation method and application of tumor targeting peptide / cell penetrating peptide-SN38 conjugate

Abstract

The invention provides a preparation method and application of a tumor targeting peptide / cell penetrating peptide-SN38 conjugate, and belongs to the technical field of polypeptide preparation and biological medicine. The invention provides a series of novel tumor targeting peptide / cell penetrating peptide-SN38 conjugates, and provides an efficient synthesis method of polypeptide solid phase coupling SN38. The novel tumor targeting peptide / cell penetrating peptide-SN38 conjugate prepared by the invention can effectively solve a series of defects of poor cell selectivity, poor water solubility, poor uptake and the like of SN38, shows a relatively strong effect of inhibiting tumor cell proliferation, further improves the anti-tumor application potential of SN38, and realizes a safer and more effective cancer treatment function.

Description

Technical Field

[0001] This invention belongs to the field of peptide preparation and biomedical technology, specifically relating to a method for preparing and applying a tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Cancer is one of the leading causes of death worldwide, and its incidence continues to rise. Chemotherapy, as a conventional treatment, relies heavily on small-molecule cytotoxic drugs. However, these drugs generally suffer from key drawbacks such as poor targeting, low water solubility, and a narrow therapeutic window. While killing tumor cells, they also damage normal tissues, leading to dose-limiting adverse reactions such as bone marrow suppression and gastrointestinal toxicity, affecting efficacy and patients' quality of life. Therefore, developing novel drug delivery systems that can selectively deliver drugs, improve solubility, and reduce systemic toxicity is a critical issue that urgently needs to be addressed in current anti-tumor drug development.

[0004] 7-Ethyl-10-hydroxycamptothecin (SN38) belongs to the camptothecin class of small-molecule antitumor drugs. SN38 inhibits the function of topoisomerase I, leading to the accumulation of DNA strand breaks and triggering a widespread DNA damage response. This, in turn, prevents DNA replication and transcription, ultimately inducing tumor cell apoptosis, making it a highly effective inhibitor of topoisomerase I. SN38 is the main active metabolite of irinotecan and has been shown to be effective against various tumor cells, including colorectal cancer, small cell lung cancer, and lymphoma. Its bioactivity is 100–1000 times that of irinotecan, and it does not require hepatic carboxylesterase for metabolism, reducing individual drug variability. Despite its advantages of strong antitumor activity and broad-spectrum anticancer activity, SN38 has drawbacks such as poor water solubility and significant toxic side effects. First, SN38 is highly hydrophobic, with low solubility in water, resulting in poor bioavailability. Second, SN38 is rapidly metabolized in vivo with a short half-life. Finally, SN38 lacks tumor targeting; after systemic administration, it is widely distributed in normal tissues and organs, causing severe toxic side effects such as bone marrow suppression, diarrhea, and mucosal inflammation, limiting its further clinical application. In recent years, peptide-drug conjugates (PDCs) have attracted widespread attention as an emerging targeted delivery strategy. PDCs consist of three parts: a targeting peptide, a cleavable linker, and an active drug molecule. This design achieves specific drug accumulation in the lesion area through peptide guidance, thereby significantly reducing toxic side effects on normal tissues. Furthermore, PDCs have advantages such as small molecular weight, strong tissue penetration, relatively simple synthesis processes, and low immunogenicity, making them particularly suitable for the treatment of solid tumors.

[0005] To address the insufficient targeting of SN38, this study selected a peptide with a clear affinity for tumor receptors as the targeting unit. Octreotide is a somatostatin analog, an 8-amino acid cyclic peptide containing a pair of intramolecular disulfide bonds, which specifically binds to somatostatin receptor 2 (SSTR2), which is highly expressed in various tumors, including neuroendocrine tumors. Octreotide is structurally stable and possesses strong biological activities, such as effectively inhibiting the secretion of excessive hormones (e.g., insulin, glucagon, growth hormone) by tumor cells, inhibiting cell proliferation, and anti-angiogenesis. Luteinizing hormone-releasing hormone (LHRH), also commonly known as gonadotropin-releasing hormone (GnRH), controls the synthesis and release of sex hormones under physiological conditions. LHRH can target the luteinizing hormone-releasing hormone receptor (LHRH-R), which belongs to the GPCR family and is significantly overexpressed on the surface of various hormone-dependent tumor cells, especially in malignant tumors such as breast cancer, prostate cancer, and ovarian cancer. The LHRH signaling pathway is abnormally activated in the tumor microenvironment, thereby participating in the regulation of tumor cell growth, survival, and invasion through downstream G protein-mediated intracellular signal transduction. Therefore, the LHRH system is not only an important neuroendocrine regulator but also a molecular target of great interest in tumor-targeted therapy research. By conjugating SN38 with such targeting peptides, the recognition and endocytosis of drugs on specific tumor cells can be significantly enhanced, increasing local drug concentrations within the tumor. Furthermore, this invention introduces the cell-penetrating peptide CPP12, a polypeptide sequence with highly efficient membrane-penetrating capabilities, which promotes transmembrane transport of the conjugate and increases intracellular drug accumulation, thus overcoming the limitation in efficacy caused by insufficient SN38 uptake.

[0006] Currently, the development of SN38 as a highly efficient topoisomerase I inhibitor primarily relies on liquid-phase synthesis strategies, which suffer from cumbersome procedures and limited efficiency. This invention innovatively employs a solid-phase synthesis method, achieving precise and efficient conjugation of SN38 with peptides, significantly improving synthetic controllability and reproducibility. Addressing the inherent bottlenecks of SN38, such as low solubility, weak membrane penetration, and insufficient targeting, this invention successfully conjugates SN38 for the first time with targeting peptides (Octreotide, LHRH) and cell-penetrating peptides (CPP12), constructing a series of novel PDCs. This strategy not only effectively improves the drug's water solubility and biomembrane permeability but also, through a dual mechanism of active targeting and enhanced cellular uptake, lays a crucial foundation for overcoming the delivery barriers of SN38 and improving its tumor selectivity. Summary of the Invention

[0007] This invention aims to design and synthesize a series of PDCs based on SN38, addressing the problems of poor water solubility, low cell penetration, and poor tumor tissue targeting of SN38. This invention covalently conjugates the targeting peptide LHRH, Octreotide, and the penetrating peptide CPP12 with SN38 to construct a series of SN38-based PDCs. In vitro experiments show that the peptide SN38 conjugates have a strong ability to inhibit tumor cell proliferation, and the modified conjugated peptides can significantly improve the antitumor selectivity of SN38. Solubility experiments show that the modified conjugated peptides improve the solubility of SN38, solving the problem of poor SN38 solubility. Cell uptake experiments show that the conjugates, under the action of the targeting peptides or penetrating peptides, promote SN38 entry into cells, improving the cells' ability to take up SN38. Scratch assays show that the conjugated peptides can persistently and efficiently inhibit the migration ability of tumor cells. Furthermore, unlike traditional liquid-phase methods for preparing SN38-based peptide conjugates, the "one-step" solid-phase conjugation strategy established in this invention simplifies the synthesis process of peptide-SN38 conjugates, providing an efficient and rapid synthesis method for PDCs molecules loaded with SN38. Therefore, the construction, synthesis, and application of the tumor-targeting peptide / cell-penetrating peptide-SN38 conjugates of this invention have good practical application value.

[0008] Specifically, the present invention is achieved through the following technical solution: In a first aspect, the present invention provides a tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate, the tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate comprising the following amino acid residue sequence: This invention provides a series of SN38-based peptide drug conjugates, which significantly improve the solubility, selectivity, cellular uptake, and anti-cell migration ability of SN38. The aforementioned cell-penetrating peptide-SN38 conjugates greatly enhance the solubility, cellular uptake, and anti-cell migration ability of SN38.

[0009] A second aspect of this invention provides a method for preparing the aforementioned tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate, employing a one-step solid-phase synthesis method. Compared to the traditional liquid-phase synthesis method for SN38, this simplifies the synthesis process of the peptide-SN38 conjugate, achieving efficient and rapid synthesis of PDC molecules loaded with SN38. The preparation method includes synthesizing the LHRH-SN38 conjugate peptide (…) using solid-phase peptide synthesis technology. Figure 1 Octreotide-SN38 conjugated peptide ( Figure 2 ) and CPP12-SN38 conjugated peptide ( Figure 3This invention provides a series of efficient synthetic methods for peptide-drug conjugates, specifically involving targeted linkage strategies between three different types of peptides and the antitumor active molecule SN38. In addition to direct coupling with SN38, for the linear targeting peptide LHRH, SN38 is directly covalently coupled to it via a linker group r4, achieving efficient one-step synthesis of the corresponding conjugate. For the cyclic peptide Octreotide, conjugates with different linkage modes are constructed by covalently linking SN38 to the peptide via linker group r4 and the hydrophilic linker unit AEEA. For the cell-penetrating peptide CPP12, AEEA is used as the linker group to couple with SN38. After coupling with SN38, the cyclic peptides Octreotide and CPP12 are further stabilized into cyclic structures on a solid-phase synthesis resin through disulfide bonds and amide cyclization. These methods are simple to operate and highly efficient, providing a systematic and modular synthetic scheme for preparing peptide-SN38 conjugates with targeted delivery capabilities.

[0010] Specifically, the above-mentioned peptides were synthesized using a solid-phase peptide synthesis method (Fmoc-SPPS) based on 9-fluorene methoxycarbonyl. Peptides OS-1~4 were synthesized using Wang resin, and peptides LS-2~3 and CS-1~2 were synthesized using Rink-amide-MBHA resin.

[0011] A third aspect of the present invention provides the use of the above-mentioned tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate in the preparation of a drug delivery system.

[0012] Preferably, the drug delivery system is a somatostatin receptor and luteinizing hormone-releasing hormone receptor targeted drug delivery system.

[0013] A fourth aspect of the present invention provides the use of the above-mentioned tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate in a medicament for the prevention and treatment of tumor-related diseases.

[0014] Preferably, the tumor includes solid tumors and non-solid tumors, such as small cell lung cancer, meningioma, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, breast cancer, melanoma, leukemia, lymphoma, and glioblastoma.

[0015] More preferably, the cancer or tumor is a human cancer or tumor that highly expresses somatostatin receptor or luteinizing hormone-releasing hormone receptor.

[0016] The beneficial technical effects of the above technical solution are as follows: 1. The tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate synthesized in this invention is the first to achieve solid-phase conjugation of LHRH, Octreotide, and CPP12 with SN38. Previous studies have generally employed liquid-phase conjugation strategies when synthesizing peptide-SN38 conjugates. This study attempts to construct peptide-SN38 conjugates using an integrated strategy of solid-phase peptide synthesis and in-situ drug conjugation.

[0017] 2. The above technical solution provides a series of SN-38-based peptide drug conjugates. These conjugates exhibit high antitumor activity and can improve the selectivity of SN38 for both tumor and normal cells. Specifically, the IC50 of each peptide over 48 hours... 50 (like Figure 12The following values ​​were observed in MDA-MB-231 cells: 6.06±0.52 μM (OS-1), 3.70±0.26 μM (OS-2), 10.06±0.93 μM (OS-3), 8.90±0.85 μM (OS-4), 2.70±0.19 μM (LS-2), 4.70±0.35 μM (LS-3), 3.56±0.30 μM (CS-1), and 5.43±0.63 μM (CS-2); in MCF-7 cells: 1.24±0.14 μM (OS-1), 0.33±0.02 μM (OS-2), 6.32±0.72 μM (OS-3), 3.57±0.23 μM (OS-4), and 0.50±0.05 μM (OS-2). μM (LS-2), 1.96±0.18 μM (LS-3), 3.06±0.26 μM (CS-1), 4.67±0.52 μM (CS-2); in HepG2 cells, 2.63±0.21 μM (OS-1), 1.28±0.16 μM (OS-2), 5.08±0.48 μM (OS-3), 4.09±0.42 μM (OS-4), 4.27±0.56 μM (LS-2), 6.32±0.62 μM (LS-3), 3.93±0.29 μM (CS-1), 4.57±0.61 μM (CS-2); in HUVEC cells, 64.39±4.58 μM (OS-1), 26.35±2.21 μM (CS-2). μM (OS-2), >100 μM (OS-3), >100 μM (OS-4), 26. 91±2. 65 μM (LS-2), 73.66±5. 32 μM (LS-3), 4. 04±0. 43 μM (CS-1), 5. 38±0.49 μM (CS-2); finally in L929 cells, 40.62±3.69 μM (OS-1), 20.79±2.34 μM (OS-2), >100 μM (OS-3), >100 μM (OS-4), 16.59±1.19 μM (LS-2), 47.08±3.96 μM (LS-3), 4.95±0.32 μM (CS-1), 6.69±0.65 μM (CS-2). Compared with SN38, the Octreotide-SN38 conjugate and the LHRH-SN38 conjugate showed significant differences in cytotoxicity against normal cells and tumor cells, indicating that their selectivity in inhibiting tumor cell proliferation was significantly improved.

[0018] 3. The above technical solution can improve the water solubility of SN38. The introduction of hydrophilic amino acids and hydrophilic linkers into the tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate greatly improves the water solubility of SN38, further expanding its wide clinical application.

[0019] 4. The above technical solution can improve the penetration of SN38 into tumor cells. This invention uses targeting peptides Octreotide and LHRH, as well as cell-penetrating peptide CPP12, conjugated with SN38, and introduces hydrophilic groups such as AEEA and r4. Compared to free SN38, the tumor-targeting peptide-SN38 conjugate and the cell-penetrating peptide-SN38 can more effectively carry SN38 into cells to exert its effect in a shorter time.

[0020] 5. The above technical solution can enhance the anti-migration effect of SN38 on tumor cells. Tumor migration is an important cause of death in cancer patients. The tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate exhibits a stronger ability to inhibit tumor cell migration than SN38 at different time points, further enhancing the clinical application potential of SN38.

[0021] In summary, this invention designs and synthesizes a series of novel conjugates of tumor-targeting peptides / cell-penetrating peptides-SN38. These conjugates overcome the problems of poor selectivity, low targeting, and poor water solubility of SN38, exhibiting significant advantages in anti-tumor applications. This study establishes a one-step strategy for synthesizing peptide-SN38 conjugates, achieving for the first time the direct covalent coupling of Octreotide, LHRH, and CPP12 with SN38 on a solid phase, simplifying the synthetic route. This method has strong application value. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0023] Figure 1 This is a schematic diagram of the solid-phase polypeptide synthesis method for the LHRH-SN38 series conjugates of the present invention; Figure 2 This is a schematic diagram of the solid-phase polypeptide synthesis method for the Octreotide-SN38 series conjugates of the present invention; Figure 3 This is a schematic diagram of the solid-phase polypeptide synthesis method for the CPP12-SN38 series conjugates of the present invention; Figure 4 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of LS-2 of this invention are shown below. Figure 5The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of LS-3 of this invention are shown below. Figure 6 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of OS-1 of this invention are shown below. Figure 7 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of OS-2 of this invention are shown below. Figure 8 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of OS-3 of this invention are shown below. Figure 9 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrometry of OS-4 of this invention are shown below. Figure 10 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of CS-1 of this invention are shown below. Figure 11 The chemical structural formula, primary amino acid sequence, analytical reversed-phase high-performance liquid chromatogram, and mass spectrum of CS-2 of this invention are shown below. Figure 12 This invention provides an intracellular evaluation method for tumor-targeting peptide / cell-penetrating peptide-SN38 conjugates. 50 (μM); Figure 13 The solubility of the tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate of the present invention in water; Figure 14 This is a diagram showing the uptake of tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate by MCF-7 cells; Figure 15 This invention demonstrates the inhibitory effect of the tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate on the migration of MCF-7 and HepG2 tumor cells. Figure 16 The modified version of SN38 of this invention 1 H NMR and mass spectra.

[0024] First, SN38 was modified to synthesize Boc-SN38-COOH. SN38 (500 mg, 1.27 mmol) and Boc anhydride (382 μL, 1.66 mmol) were mixed in anhydrous DCM, and an anhydrous amount of pyridine was added. The mixture was stirred overnight at room temperature. After the reaction was complete, the reaction mixture was washed successively with 0.5% sodium bicarbonate solution and 0.1 mol / L hydrochloric acid. The solvent was removed by rotary evaporation to obtain the crude Boc-SN38 product. This crude product was dissolved in anhydrous DCM with succinic anhydride (300 mg, 3 mmol), and DBU (460 μL, 3 mmol) and a small amount of DMAP were added. The mixture was reacted at 28 °C for 8 h. The reaction solution was quenched with 20 mL of double-distilled water and acidified with 1% hydrochloric acid aqueous solution. After filtration and vacuum drying, Boc-SN38-COOH was obtained. NMR and mass spectra are shown below. Figure 16 As shown.

[0025] In this embodiment, all target compounds were prepared using solid-phase peptide synthesis technology based on 9-fluorene methoxycarbonyl (Fmoc-SPPS). Wang resin (degree of substitution 0.33 mmol / g) was preferred for synthesizing peptides OS-1~4; Rink-AmideMBHA resin (degree of substitution 0.38 mmol / g) was preferred for synthesizing peptides LS-2~3 and CS-1~2. The preferred scale of peptide synthesis was 0.10 mmol. The basic peptide synthesis procedure is as follows: Figure 2 , Figure 3 and Figure 4 As shown.

[0026] Peptide solid-phase synthesis: Weigh Rink-amide-MBHA resin or Wang resin into a synthesis tube. Perform standard washing on the resin (DMF wash twice, DCM wash twice, DMF wash once, DCM wash once, DMF wash twice). After draining the liquid from the synthesis tube, add DMF and soak for 2 hours. After draining again, add a mixed solution of DMF and DCM (4:1). v : v The synthesis tube was placed in a constant temperature shaker at 28°C for 1 hour, the solution was dried, and a standard wash was performed to complete the activation of the resin.

[0027] The method for removing the Fmoc protecting group is as follows: Add 2-3 mL of 20% piperidine solution (using DMF as solvent) to the synthesis tube, and react twice in a constant temperature shaker at 28℃. The first reaction is 5 min, followed by two washes with DMF, and a small amount of resin is taken using a capillary tube for Kaiser test. The second reaction is 10 min, followed by one standard wash.

[0028] The amino acid condensation reaction method was as follows: each amino acid was condensed twice at 28 °C. When using Wang resin, the condensation reaction time for the first amino acid was 6 h and 10 h, and the reactant ratio was amino acid:DIC:Oxyma:DAMP = 3 equivalents:6 equivalents:3 equivalents:0.5 equivalents. For other amino acids and using Rink-amide-MBHA resin, the condensation was performed twice, with condensation times of 30 min and 40 min, respectively. After the reaction, a standard wash was performed once.

[0029] In the synthesis of octreotide, the thiol group of the cysteine ​​side chain is protected with an Acm group. The linear peptide obtained in the above steps was cyclized at 28°C under solid-phase conditions. This process was carried out in two steps, with reaction times of 1 h and 1.5 h, respectively. The molar ratio of amino acid residues to cyclizing reagent Tl(TFA)3 in the cyclization reaction was 3:2.34. The specific operation was as follows: Tl(TFA)3 was dissolved in 2.5 mL of DMF solution containing 125 μL of anisole. The system was first cooled in an ice bath for 5 min, and then transferred to 28°C for cyclization.

[0030] For the cyclization of the CPP12 amide ring, Pd(PPh3)4 reagent was first added to remove the OAll carboxyl protecting group on glutamic acid. Then, 20% piperidine solution was added to remove the Fmoc protecting group and expose the amino group. The cyclization reagent ratio was HATU:HOAT = 2.4 equivalents: 2.4 equivalents. The reaction was carried out twice in a constant temperature shaker at 45℃, the first time for 120 min and the second time for 120 min.

[0031] Solid-phase synthesis of peptide-SN38: The reactant ratio for the synthesis of Boc-SN38-COOH was Boc-SN38-COOH:HATU:HOAT:DIEA = 2 equivalents: 1.8 equivalents: 2 equivalents: 4 equivalents. The reaction was carried out twice at 28 °C, the first time for 60 min and the second time for 90 min.

[0032] Peptide cleavage and purification: Add peptide cleavage reagent to the synthesized peptide in a ratio of TFA:TIPS:H2O = 95:2.5:2.5 (TFA:TIPS:H2O = 95:2.5:2.5). v : v : v All peptides were reacted in a constant temperature shaker at 28℃ for 2 h. Then, the peptide solution was bubbled with high-purity nitrogen to 2-3 ml, and anhydrous diethyl ether pre-chilled was added. The mixture was centrifuged for 5 min (3000 r), the supernatant was discarded, and the reaction was repeated twice more with anhydrous diethyl ether added again. After drying, the crude peptide sample was obtained.

[0033] The crude peptide samples were first dissolved in a water / acetonitrile mixed solvent containing 0.1% TFA, followed by analytical characterization and preparative purification. The purity and molecular weight of the peptides were determined by analytical reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). Purification was performed on a semi-preparative RP-HPLC system. After semi-preparative chromatographic separation, the purity of the peptides was higher than 95%. Finally, the target peptide solution was freeze-dried to obtain a solid powder, which was then sealed and stored at -20 °C for later use.

[0034] The chemical structural formulas, primary amino acid sequences, analytical reversed-phase high-performance liquid chromatography (RP-HPLC) chromatograms, and ESI-MS mass spectra of the eight polypeptides obtained in this invention are shown below. Figures 4-11 As shown.

[0035] Cell viability was determined using the Methylthiazolyldiphenyl-tetrazolium bromide (MTT) colorimetric assay. HepG2, MCF-7, MDA-MB-231, HUVEC, and L929 cells in logarithmic growth phase were seeded at 6 × 10³ cells per well in 96-well plates and cultured overnight at 37°C with 5% CO2 to allow cell adhesion. Peptide samples were dissolved in DMSO to prepare a 10 mmol / L stock solution, which was then diluted with serum-free DMEM or RPMI-1640 medium to obtain a series of working solutions. Working solutions of 0.03, 1, 3, 10, 30, and 100 μmol / L were added to each well at 50 μL, and cells were cultured for 48 h. After culture, 15 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated for 4 h. Discard the supernatant, add 150 μL of DMSO to each well, and incubate for 30 min to fully dissolve the formazan crystals. After vortexing and mixing, measure the absorbance of each well at 492 nm using a microplate reader. All experiments were independently repeated three times.

[0036] like Figure 12 As shown, the conjugates all exhibited strong inhibitory effects on tumor cell proliferation, with IC50 concentrations of [missing information]. 50 The values ​​were all less than 7 μM. The IC50 values ​​of LS-2 were measured in three types of tumor cells. 50 The values ​​were all lower than LS-3, indicating stronger antiproliferative activity. This enhanced activity may be related to the four arginine residues introduced into the LS-2 structure, which improve biological activity by enhancing molecular solubility. Furthermore, in LHRH-highly expressed MCF-7 and MDA-MB-231 cells, the conjugate's IC50 was significantly higher than that of LS-3. 50All values ​​were lower than those in HepG2 cells with low LHRH expression; compared with normal cells, the inhibitory efficiency against tumor cells was increased by at least 4 times. These results indicate that conjugating SN38 with an LHRH-targeting peptide significantly reduces toxicity to normal cells and enhances selective killing of tumor cells. Furthermore, the modification of the arginine residue in the structure further enhances the antitumor effect of the conjugate.

[0037] Octreotide-SN38 conjugates OS-1, OS-2, OS-3, and OS-4 exhibited significantly enhanced selectivity in MCF-7 tumor cells, with inhibitory activity more than 40-fold higher compared to normal cells. The IC50 values ​​of the four conjugates in MDA-MB-231, MCF-7, and HepG2 tumor cells were compared. 50 The study found that OS-2 with four arginine residues exhibited the strongest inhibitory effect on cell proliferation. Compared to unmodified OS-3, OS-2 showed approximately 3-fold, 20-fold, and 5-fold increased activity against the three cell types, respectively, which may be attributed to increased solubility and thus improved cellular uptake efficiency. Furthermore, OS-1 containing an AEEA linker and acetylated OS-4 also showed slightly stronger activity than OS-3, presumably related to improved solubility or molecular stability.

[0038] CS-1 and CS-2 exhibited some antitumor activity, but their activity was significantly weaker than that of the free SN38 molecule. This difference may be attributed to the incomplete release of SN38 from the conjugate structure. Notably, in all tested cell lines, CS-1 showed slightly higher activity than CS-2, which may be attributed to the introduction of the linker AEEA, which improved the molecule's water solubility and thus its cellular uptake. Furthermore, due to the lack of target specificity of the cell-penetrating peptide CPP12, CS-1 and CS-2 exhibited comparable cytotoxicity in both tumor and normal cells, with IC50 values ​​of [missing information]. 50 The values ​​showed no significant difference.

[0039] Example 3 Solubility Test: The absorbance of SN38, LS-2, OS-2, CS-1, and CS-2 in PBS was measured using a UV-2600 UV-Vis spectrophotometer at 25℃. Since the absorbance of saturated solutions of LS-2, OS-2, CS-1, and CS-2 was too high and exceeded the instrument's detection range, appropriate dilution was necessary. Solutions of SN38, LS-2, OS-2, CS-1, and CS-2 at different concentrations in PBS (100, 50, 25, 12.5, and 6.25 μM) were prepared, and standard curves for each compound were plotted. Subsequently, saturated solutions of each sample in PBS were prepared. To control the absorbance within the linear range of the standard curve, the saturated solutions of LS-2, OS-2, CS-1, and CS-2 were diluted 100-fold before measurement. The obtained absorbance values ​​were substituted into the corresponding standard curve equations to calculate the concentrations, and the data were linearly fitted using GraphPad Prism 10.0 software. All experiments were independently repeated three times.

[0040] Full-wavelength scanning confirmed that SN38 has a maximum absorption wavelength at 365 nm, consistent with the excitation wavelength reported in the literature. Subsequent concentration-absorbance standard curves showed that SN38 and its four conjugates exhibited good linearity at this wavelength, with coefficients of determination (R²) all greater than 0.99. Solubility measurements showed that the solubility of free SN38 in PBS was only 15.6 μM. After peptide conjugation, the solubilities of LS-2, OS-2, CS-1, and CS-2 increased to approximately 4800 μM, 4490 μM, 3740 μM, and 3410 μM, respectively, representing increases of 219–308 times compared to SN38. Figure 13 Among them, CS-1, which incorporates the AEEA linker, showed slightly higher solubility than CS-2. These results indicate that peptide conjugation strategies can significantly enhance the solubility of SN38, providing experimental evidence for the design and optimization of the drug delivery system.

[0041] MCF-7 cells were spaced at 1 × 10⁶ cells per well. 4 Cells were seeded at a density of [number] cells per well in 24-well plates and cultured overnight. First, the nuclei were stained with SYBR Green I for 15 min. After removing the stain, medium containing the specified concentration of peptide (1 mL / well) was added, and the cells were cultured for another 48 h. After incubation, the medium was discarded, the cells were washed three times with PBS, and then replaced with phenol red-free medium. Images were then captured under a confocal microscope, and cell uptake was quantitatively analyzed using ImageJ software. All experiments were independently repeated three times.

[0042] The results are as follows Figure 14As shown, after 12 h of treatment, the cell uptake rates of LS-2 and LS-3 were 50.4% and 40.1%, respectively, both significantly higher than the 4.6% in the SN38 group. Among them, the uptake rate of LS-2, which is linked to four arginine residues, was also significantly higher than that of LS-3, approximately 11 times that of SN38. Figure 14 B, 14F, 14D). The uptake rates of peptides OS-1 to OS-4 were 44.5%, 55.6%, 31.3%, and 34.4%, respectively, all significantly higher than SN38; especially OS-2, which contains four arginine residues, had an uptake rate approximately 12 times that of SN38. Figure 14 A, 14E, 14D). CS-1 and CS-2 exhibited strong blue fluorescence signals within cells, with uptake rates of 80.9% and 70.8%, respectively, and there was no statistically significant difference between the two. Figure 14 C, 14G). The CS-1 uptake rate with the AEEA linker was approximately 17.6 times that of SN38 (C, 14G). Figure 14 (C, 14D). The above results demonstrate that conjugating SN38 with cell-penetrating peptides and targeting peptides can significantly promote their internalization within cells. This strategy provides a useful reference for improving the intracellular delivery efficiency of small molecule drugs.

[0043] The effect of the peptide on MCF-7 was evaluated using a cell scratch assay. Figure 15 A) and HepG2 ( Figure 15 C) Effects on tumor cell migration ability. The two cell types were respectively planted at 1×10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 24-well plates and cultured overnight until cell confluence reached approximately 90%. Subsequently, two parallel scratches were created in each well using a sterile 200 μL pipette tip. After washing three times with PBS, the medium was replaced with serum-free medium containing different concentrations of peptides, and treated for 12 h or 24 h to observe its inhibitory effect on wound healing.

[0044] like Figure 15 As shown in Figure A, CS-1 and CS-2 exhibited similar anti-migration abilities at 12 h and 24 h time points. LS-2 and OS-2 also showed similar migration inhibition effects. It should be noted that CS-1, CS-2, LS-2, and OS-2 all showed significantly stronger inhibition of cell migration than the SN38 group and the DMSO control group, with relative migration rates of 43.36%, 40.65%, 23.27%, and 17.48%, respectively, compared to 72.73% and 84.68% in the SN38 group and the DMSO control group, respectively. Given that MCF-7 cells highly express SSTR2 and LHRH-R receptors, OS-2 and LS-2, which are linked to their corresponding targeting peptides, exhibited more significant anti-migration effects.

[0045] like Figure 15As shown in Figure C, compared with the DMSO control group, the migration ability of HepG2 cells treated with the conjugates was significantly inhibited. At 12 h and 24 h, CS-1, CS-2, LS-2, and OS-2 all showed similar migration inhibition effects. Of particular note, the inhibitory effects of the above four conjugates (relative migration rates of 41.69%, 41.35%, 39.45%, and 34.72%, respectively) were significantly stronger than those of the SN38 group (57.57%) and the DMSO control group (69.68%). In conclusion, after conjugation with cell-penetrating peptides and targeting peptides, the SN38 derivatives exhibit superior activity in inhibiting tumor cell migration.

[0046] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention. Although the specific embodiments of the present invention have been described above, they are not intended to limit the protection scope of the present invention. Those skilled in the art should understand that various modifications or variations that can be made by those skilled in the art without creative effort based on the technical solutions of the present invention are still within the protection scope of the present invention.

Claims

1. A class of tumor-targeting peptide / cell-penetrating peptide-SN38 conjugates, characterized in that, The tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate contains the following amino acid residue sequence. .

2. The method for preparing the tumor-targeting peptide-SN38 conjugate LHRH-SN38 conjugate, Octreotide-SN38 conjugate, and cell-penetrating peptide-SN38 conjugate CPP12-SN38 conjugate as described in claim 1, characterized in that, The preparation method is solid-phase synthesis of polypeptide-SN38 conjugate; Preferably, the polypeptide synthesis method is a solid-phase polypeptide synthesis method based on 9-fluorenemethyloxycarbonyl; on the solid phase, CPP12, Octreotide and LHRH are covalently coupled with SN38 derivatives protected by tert-butyloxycarbonyl and modified by succinic acid to construct conjugates.

3. The application of the tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate of claim 1 in the preparation of a drug delivery system; preferably, the drug delivery system is a system for targeted delivery of SN38 to somatostatin receptor and gonadotropin-releasing hormone receptor.

4. The application of the tumor-targeting peptide / cell-penetrating peptide-SN38 conjugate of claim 1 in a medicament for the prevention and treatment of tumor-related diseases; preferably, the tumor includes solid tumors and non-solid tumors, such as small cell lung cancer, meningioma, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, kidney cancer, bladder cancer, breast cancer, melanoma, leukemia, lymphoma, and glioblastoma; more preferably, the cancer or tumor is a human cancer or tumor that highly expresses somatostatin or luteinizing hormone-releasing hormone and their corresponding receptors.

5. A pharmaceutical preparation, characterized in that, The pharmaceutical formulation comprises the conjugate of claim 1 and pharmaceutically acceptable excipients and / or carriers, preferably, the carriers comprising buffers, solubilizers, fillers, disintegrants, diluents, binders, lubricants, emulsifiers, surfactants, colorants, suspending agents, and flavoring agents.

Images