Tumor targeting peptide and application

Abstract

The invention relates to a tumor targeting peptide and application thereof, in particular to a tumor targeting peptide with an amino acid sequence of COOH-Thr-Leu-Gly-Ser-NH2 (TLGS) and application of the tumor targeting peptide in anti-tumor research, and belongs to the technical field of biomedicine. The tumor targeting peptide can specifically target human cervical cancer cells and has high affinity, and a peptide chain is non-toxic, free of hemolysis and good in biological safety. The tumor targeting peptide can be prepared through chemical synthesis or gene engineering, and can be grafted to an anti-tumor system in various forms, including but not limited to nanoparticles, anti-tumor drugs, microspheres or drug carriers and the like. The targeting peptide is combined with an anti-tumor drug or a developing agent, so that targeting delivery or imaging of the drug can be realized, the targeting property is remarkably improved, side effects are reduced, and a new technical means is provided for precise medical treatment of tumors.

Description

Technical Field

[0001] The present invention relates to a tumor targeting peptide and application thereof, more specifically to a polypeptide capable of specifically targeting human cervical cancer cells and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Cervical cancer is one of the most common malignant tumors in women worldwide, with high morbidity and mortality, especially in developing countries. Currently, the main treatments for cervical cancer include surgical resection, radiotherapy and chemotherapy. However, these traditional treatments have obvious limitations: surgical resection is only suitable for early-stage patients and may cause postoperative complications; although radiotherapy and chemotherapy have certain effects on patients in the middle and late stages, they lack specificity and are prone to damage normal tissues, leading to serious side effects such as bone marrow suppression, gastrointestinal reactions and immune system damage.

[0003] In recent years, targeted therapy, as an emerging treatment strategy, has attracted widespread attention due to its high specificity and low toxicity. Targeted therapy specifically recognizes markers on the surface of tumor cells and accurately delivers therapeutic drugs to the tumor site, thereby minimizing damage to normal tissues. Peptide targeting molecules have become a hot topic in targeted drug research due to their small molecular weight, strong penetration, low immunogenicity, and ease of chemical modification.

[0004] Therefore, developing a cervical cancer targeting peptide with high targeting, high stability and multifunctionality is of great significance for improving the diagnostic accuracy and treatment effect of cervical cancer. The present invention aims to provide a new cervical cancer targeting peptide to solve the above technical problems and provide a new solution for the precision medicine of cervical cancer. Summary of the invention

[0005] The purpose of the present invention is to provide a tumor targeting peptide, which has the advantages of high specificity, good biocompatibility, short peptide chain sequence, simple synthesis, low cost, etc., and provides support for chemotherapy drugs and anti-tumor nanosystems in the field of cervical cancer treatment.

[0006] In order to achieve the purpose of the invention, this application adopts the following technical solutions:

[0007] A tumor targeting peptide, wherein the sequence of the tumor targeting peptide is COOH-Thr-Leu-Gly-Ser-NH2 (TLGS).

[0008] The tumor targeting peptide has specific targeting ability for cervical cancer cells, can selectively target tumor cells in vitro, and can accurately target tumor tissues in mice and accumulate in the surrounding areas.

[0009] The application of the tumor targeting peptide in tumor targeting therapy and imaging.

[0010] The application described is as follows: The tumor targeting peptide is covalently grafted to the micelle monomer PEG-PCL to prepare a tumor targeting carrier, named TLGS-PEG-PCL (Pep-PP). The Nile Red dye (Nile Red) is loaded to form a targeted tumor imaging platform, named Nile Red@TLGS-PEG-PCL (NR@Pep-PP). The anti-tumor drug cisplatin (Pt) is loaded to form a targeted anti-tumor targeting platform, named cisplatin@TLGS-PEG-PCL (Pt@Pep-PP).

[0011] The beneficial effects of the present invention are:

[0012] The tumor targeting peptide prepared by the present invention can specifically identify cervical cancer cells and show excellent targeting ability both in vivo and in vitro environments; combined with nanocarriers, anti-tumor drugs or fluorescent probes, it can realize the integration of tumor diagnosis, treatment and drug delivery; after coupling with nanodrug delivery carriers and anti-tumor drugs, it can be injected through the tail vein to significantly improve the tumor inhibition effect of drug-loaded nanocarriers in vivo or in vitro; combined with fluorescent probes, it can realize accurate imaging of tumor sites, providing an important basis for the diagnosis and treatment of diseases; at the same time, the tumor targeting peptide has good biocompatibility, and by coupling with nanocarriers or drugs, their systemic toxicity can be significantly reduced, making them safer. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a graph showing the in vitro targeting laser confocal microscopy results of the fluorescent molecule-labeled tumor targeting peptide obtained in Example 1.

[0014] Figure 2 This is a diagram showing the in vitro targeting flow cytometry results of the fluorescent molecule-labeled tumor targeting peptide obtained in Example 1.

[0015] Figure 3 This is a graph showing the MTT toxicity results of the tumor targeting vector TLGS-PEG-PCL (Pep-PP) obtained in Example 1.

[0016] Figure 4 This is a live-dead staining image of the tumor targeting vector TLGS-PEG-PCL (Pep-PP) obtained in Example 1.

[0017] Figure 5 This is a graph showing the hemolysis rate of the tumor targeting vector TLGS-PEG-PCL (Pep-PP) obtained in Example 1.

[0018] Figure 6 This is the SEM result of red blood cells after hemolysis of the tumor targeting carrier TLGS-PEG-PCL (Pep-PP) obtained in Example 1.

[0019] Figure 7 This is the in vivo imaging result of the targeted tumor imaging platform NR@Pep-PP obtained in Example 1.

[0020] Figure 8 This is a diagram of the in vivo distribution imaging results of the targeted tumor imaging platform NR@Pep-PP obtained in Example 1.

[0021] Fig. 9 This is a graph showing the MTT toxicity results of the targeted anti-tumor platform Pt@Pep-PP obtained in Example 1. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with specific embodiments. In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market, and the methods in the following embodiments, unless otherwise specified, are conventional methods in the art, and the details and forms of the technical solutions of the present invention can be modified and replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

[0023] Embodiment 1:

[0024] Tumor targeting peptide preparation

[0025] The polypeptide was obtained by Fmoc solid phase synthesis. Dichlorotrityl chloride resin was swelled with DCM, and an appropriate amount of amino acid and DIEA were added to react for 2 hours, and the unreacted groups were blocked by methanol. The Fmoc protecting group was removed using a 20% piperidine solution, and then the corresponding amino acid, PyBoP, HoBt and DIEA were added in sequence. After reacting for 2 hours, the Fmoc protecting group was removed using a 20% piperidine solution, and the above steps were repeated until the last amino acid was connected. Finally, a cutting agent (TFA: water: triisopropylsilane = 95:2.5:2.5) was used for cutting, and the polypeptide was precipitated in ice ether, and finally freeze-dried in a freeze dryer for use.

[0026] Preparation of tumor-targeting peptides labeled with fluorescent molecules

[0027] After the peptide synthesis was completed, the peptide and fluorescein isothiocyanate were dissolved in DMF, an appropriate amount of DIEA was added, mixed, and reacted at room temperature for 12 hours.

[0028] In vitro specificity of tumor targeting peptides

[0029] The fluorescently labeled tumor targeting peptide prepared in this example was co-incubated with HeLa cells and NIH-3T3 cells, and the samples were subjected to in vitro targeting performance tests using a laser confocal microscope at different times. The results showed that the fluorescently labeled tumor targeting peptide could effectively aggregate around the HeLa cell membrane, and had specificity and targeting.

[0030] The fluorescently labeled tumor targeting peptide prepared in this example was co-incubated with HeLa cells, and the in vitro targeting performance was tested by flow cytometry at different times. The results showed that the fluorescently labeled tumor targeting peptide could rapidly aggregate in HeLa cells.

[0031] Preparation of tumor targeting carrier TLGS-PEG-PCL (Pep-PP)

[0032] The tumor targeting peptide, EDC and NHS were dissolved in water, the pH was adjusted to 6, mixed well, and stirred for 30 min. 2 -PEG-PCL, adjust the pH to 7-8 with triethylamine, and stir at room temperature overnight. 2 The molar ratio of -PEG-PCL to polypeptide was about 1:20. After the reaction was completed, the product was dialyzed with water for 3 times and freeze-dried to obtain the monomer product. The above product was dissolved in DMSO, stirred for half an hour, and then PBS solution was added dropwise, stirred overnight, and dialyzed to obtain the tumor targeting vector.

[0033] Cytotoxicity of tumor-targeting vectors

[0034] The tumor targeting vector prepared in this example was evaluated for its biological toxicity by MTT method, live-death detection and other means. Pep-PP was co-incubated with cells for 24 hours. After the incubation, its MTT toxicity was detected by microplate reader, and the live-death of cells was observed by inverted fluorescence microscope. The results showed that the tumor targeting vector prepared in this example had no cytotoxicity, high cell survival rate and good biocompatibility.

[0035] Hemolytic activity of tumor targeting vectors

[0036] The tumor targeting vector prepared in this example was co-incubated with fresh blood, and after centrifugation to extract the upper serum, the hemolysis rate was detected by an ELISA instrument, and the morphology of red blood cells after co-incubation was observed by scanning electron microscopy. The results showed that the tumor targeting vector prepared in this example had no hemolytic activity and had good biocompatibility.

[0037] Preparation of Nile Red@TLGS-PEG-PCL (NR@Pep-PP) as a targeted tumor imaging platform

[0038] TLGS-PEG-PCL monomer and Nile red were dissolved in DMSO, stirred for half an hour in the dark, and then PBS solution was added dropwise, stirred overnight, and the tumor imaging platform NR@Pep-PP was obtained after dialysis.

[0039] In vivo imaging of the targeted tumor imaging platform NR@Pep-PP

[0040] The tumor imaging platform NR@Pep-PP prepared in this example was injected into the HeLa tumor-bearing nude mouse model by tail vein injection, and the small animal was imaged in vivo according to a certain time gradient to detect its in vivo targeting ability. At the same time, the mice were euthanized after 30 hours, and their internal organs were dissected and imaged. The results showed that the tumor imaging platform obtained in this example can quickly and accurately target the surrounding of tumor tissue in mice.

[0041] Preparation of Targeted Antitumor Platform Cisplatin@TLGS-PEG-PCL (Pt@Pep-PP)

[0042] TLGS-PEG-PCL monomer and cisplatin were dissolved in DMSO, stirred for half an hour in the dark, and then PBS solution was added dropwise. The mixture was stirred overnight and the anti-tumor platform Pt@Pep-PP was obtained after dialysis.

[0043] Toxicity of the targeted anti-tumor platform Pt@Pep-PP

[0044] The anti-tumor platform prepared in this example was evaluated for its biological toxicity using the MTT method. Pt@Pep-PP was incubated with cells for 4 hours. After the incubation, the MTT toxicity was detected using an ELISA instrument. The results showed that the anti-tumor platform prepared in this example had an excellent anti-tumor effect.

[0045] Figure 1 The results of in vitro targeting laser confocal microscopy of the fluorescent molecule-labeled tumor targeting peptide obtained in Example 1 are shown. Among them, TLGS-FITC is the fluorescent molecule-labeled tumor targeting peptide obtained in Example 1, and VILP-FITC is a negative control group with clear no targeting performance. The results show that the fluorescently labeled tumor targeting peptide can effectively aggregate around the HeLa cell membrane, with specificity and targeting.

[0046] Figure 2 This is a diagram showing the in vitro targeting flow cytometry results of the fluorescent molecule-labeled tumor targeting peptide obtained in Example 1.

[0047] Among them, TLGS-FITC is the tumor targeting peptide labeled with fluorescent molecules obtained in Example 1, and VILP-FITC is a negative control group with clear no targeting performance. The results show that the fluorescently labeled tumor targeting peptide can quickly aggregate in HeLa cells, and has specificity and targeting.

[0048] Figure 3 This is a graph showing the MTT toxicity results of the tumor targeting vector Pep-PP obtained in Example 1. The results show that the tumor targeting vector prepared in this example has no cytotoxicity, a high cell survival rate, and good biocompatibility.

[0049] Figure 4This is a live-dead staining image of the tumor targeting vector TLGS-PEG-PCL (Pep-PP) obtained in Example 1. The results show that the tumor targeting vector prepared in this example has no cytotoxicity, a high cell survival rate, and good biocompatibility.

[0050] Figure 5 This is a graph showing the hemolysis rate of the tumor targeting carrier Pep-PP obtained in Example 1. The results show that the hemolytic activity of Pep-PP at different concentrations is all below 2%, and has good biocompatibility.

[0051] Figure 6 This is a SEM result of hemolyzed red blood cells of the tumor targeting carrier TLGS-PEG-PCL (Pep-PP) obtained in Example 1. The results show that the morphology of the red blood cells after co-incubation is a normal round cake-shaped concave shape, indicating that it is non-hemolytic and has good biocompatibility.

[0052] Figure 7 This is the in vivo imaging result of the targeted tumor imaging platform NR@Pep-PP obtained in Example 1. The results show that NR@Pep-PP can quickly gather around the tumor and has tumor targeting properties.

[0053] Figure 8 This is the in vivo distribution imaging result diagram of the targeted tumor imaging platform NR@Pep-PP obtained in Example 1. The results show that NR@Pep-PP accumulates in large quantities in tumor tissues, and there is a certain amount of liver metabolism and a small amount of kidney metabolism.

[0054] Fig. 9 This is the MTT toxicity result of the targeted anti-tumor platform Pt@Pep-PP obtained in Example 1. The results show that compared with the non-targeted anti-tumor platform Pt@PP and the free anti-tumor drug Pt, Pt@Pep-PP has excellent anti-tumor ability.

Claims

1. A tumor targeting peptide, characterized in that: The sequence of the peptide is COOH-Thr-Leu-Gly-Ser-NH2 (TLGS), and the peptide can specifically bind to human cervical cancer cells.

2. The tumor targeting peptide according to claim 1, characterized in that The peptide includes but is not limited to a linear peptide chain, a circular peptide chain and a structure in which a branched chain contains the sequence, wherein the peptide structure retains the ability to target cervical cancer cells.

3. The tumor targeting peptide according to claim 1, characterized in that The peptide includes derivatives, modifications or variants thereof, wherein the structure retains the ability to target cervical cancer cells.

4. The tumor targeting peptide according to claim 3, characterized in that The modification includes but is not limited to PEGylation, fluorescent labeling, biotinylation or glycosylation.

5. The tumor targeting peptide according to claim 1, characterized in that The cervical cancer includes HeLa, SiHa, C33A, CaSki, HT-3 and other cell lines.

6. A pharmaceutical composition comprising the tumor targeting peptide according to any one of claims 1 to 4, characterized in that: The drug combination includes a drug carrier or an auxiliary material.

7. Use of the pharmaceutical composition according to claim 6 in the preparation of a drug for treating or diagnosing tumors.

8. A drug delivery system, characterized in that The invention comprises any one of the tumor targeting peptides according to claims 1 to 4 and a drug carrier, wherein the drug carrier comprises at least one of micelles, liposomes, microspheres, polymers or nanoparticles.

9. The drug delivery system according to claim 8, characterized in that Used to deliver drugs or imaging agents to tumor sites.

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