Biocompatible self-assembly oncolytic peptide, bioactive solution and application of biocompatible self-assembly oncolytic peptide

Abstract

The invention provides a biocompatible self-assembly oncolytic peptide, a bioactive solution and application of the biocompatible self-assembly oncolytic peptide and the bioactive solution, and belongs to the technical field of biological medicines.The biocompatible self-assembly oncolytic peptide comprises an end-capping group, a phosphorylated gelling factor and a functional sequence which are sequentially connected; the amino acid sequence of the phosphorylated gelling factor is GDFDFDpY, and the functional sequence of the phosphorylated gelling factor is RRDKRFYVVMWKDK. The biocompatible self-assembly oncolytic peptide provided by the invention is dephosphorylated under the action of ALP, generates in-situ self-assembly, changes a secondary structure and Zeta potential, and is physically combined with a cancer cell membrane through spiral structure insertion and electrostatic interaction, so that cell death is caused in a mode of damaging a cell membrane.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a biocompatible self-assembling oncolytic peptide, a bioactive solution and applications thereof. Background Art

[0002] Oncolytic therapy is considered a major milestone in the development of immunotherapy. In recent years, it has made rapid progress both domestically and internationally. It relies on oncolytic viruses or oncolytic peptides to target and lyse tumor cells, inducing immunogenic cell death (ICD) in tumor cells and effectively enhancing epitope amplification of tumor antigens, thereby activating the body's anti-tumor immune response. However, the application of oncolytic viruses still faces significant challenges due to complex engineering processes, strong autoimmunogenicity, lack of safety validation, lengthy purification times, and high production costs.

[0003] In contrast, oncolytic peptides are a new class of oncolytic drugs, offering advantages such as clear structure, simple synthesis, and low autoimmunogenicity. They are primarily derived from natural antimicrobial peptides or designed de novo with antimicrobial peptide inspiration, and typically possess a cationic amphiphilic structure. Their molecular mechanism relies on electrostatic interactions to bind to negatively charged tumor cell membranes, followed by intercalation into the cancer cell membranes and lysis, thereby exerting potent oncolytic activity. Most reported oncolytic peptide drugs, including the first candidate oncopeptide molecule, LTX-315, currently in Phase II clinical trials, can only be administered via intratumoral injection. This is because, while the exposed negatively charged phosphatidylserine and increased expression of anionic components on the outer leaflet of the membrane facilitate oncolytic peptide recognition of tumor cells, nonselective cell killing is unavoidable during systemic administration, not to mention poor in vivo bioactivity and stability, and severe hemolysis. Therefore, there is an urgent need to develop safe and effective oncolytic peptide drugs suitable for systemic delivery in this field. Summary of the Invention

[0004] The purpose of the present invention is to provide a biocompatible self-assembling oncolytic peptide, a bioactive solution and its application; the biocompatible self-assembling oncolytic peptide provided by the present invention can be dephosphorylated by alkaline phosphatase ALP, undergo in situ self-assembly, change the secondary structure and zeta potential, and physically bind to the cancer cell membrane through helical structure insertion and electrostatic interaction, thereby destroying the cell membrane and causing cell death.

[0005] A biocompatible self-assembling oncolytic peptide comprises a sequentially connected blocking group, a phosphorylated colloid factor and a functional sequence; the amino acid sequence of the phosphorylated colloid factor is G D F D F D pY, the functional sequence is RR D KRFYVVMWK D K.

[0006] Preferably, the blocking group is β-naphthylacetic acid (Nap).

[0007] Preferably, the amino acid sequence of the phosphorylated collagen-forming factor is in D configuration.

[0008] Further preferably, the structural formula of the anti-tumor polypeptide is as shown in Formula I:

[0009] Formula I.

[0010] The present invention provides a bioactive solution comprising the biocompatible self-assembling oncolytic peptide.

[0011] Preferably, the bioactive solution uses phosphate buffered saline (PBS) as a solvent.

[0012] Preferably, the pH of the PBS is 7.2-7.4.

[0013] Preferably, the mass-to-volume ratio of the biocompatible self-assembling oncolytic peptide to the buffer solution is 2 mg:0.5-1 mL.

[0014] The present invention provides the use of the biocompatible self-assembling oncolytic peptide and the bioactive solution in the preparation of anti-tumor immune drugs.

[0015] Preferably, the anti-tumor immune drugs include anti-breast cancer, anti-colon cancer and anti-melanoma drugs.

[0016] Compared with existing technologies, the present invention offers the following advantages: The biocompatible, self-assembling oncolytic peptide provided by the present invention is dephosphorylated by extracellular overexpressed ALP, undergoing in situ self-assembly, converting the original β-sheet secondary structure into an α-helix and increasing the zeta potential. Subsequently, through helical insertion and electrostatic interactions, it physically binds to the cancer cell membrane, disrupting the cell membrane and causing cell death. Furthermore, the oncolytic peptide has no killing effect on normal cells that lowly express ALP, demonstrating excellent biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 High-resolution mass spectrum (top) and liquid chromatography (LC) at an absorption wavelength of 254 nm (bottom) of the biocompatible self-assembling oncolytic peptide pD2RP synthesized in Example 1;

[0018] Figure 2 The high-resolution mass spectrum (top) and liquid chromatography (LC) at an absorption wavelength of 254 nm (bottom) of the peptide D2RP synthesized in Comparative Example 1 are shown;

[0019] Figure 3The high-resolution mass spectrum (top) and liquid chromatography (LC) at an absorption wavelength of 254 nm of the peptide D2RpP synthesized in Comparative Example 2 are shown below.

[0020] Figure 4 is the structural formula of the polypeptide synthesized in Comparative Example 1 and Comparative Example 2;

[0021] Figure 5 The microscopic morphology of pD2RP in ultrapure water, 1×PBS, and after ALP catalysis in Experimental Example 1;

[0022] Figure 6 Circular dichroism spectra of pD2RP before and after ALP catalysis in Experimental Example 2;

[0023] Figure 7 is the Zeta potential of pD2RP before and after ALP catalysis in Experimental Example 3;

[0024] Figure 8 This is a biological scanning electron micrograph of 4T1 cells after incubation with empty culture medium and pD2RP in Experimental Example 4;

[0025] Figure 9 This is a real-time imaging image of propidium iodide when pD2RP is incubated with 4T1 cells in Experimental Example 5;

[0026] Figure 10 The cell survival rate curves after incubating 4T1, MC38, B16-F10 and L929 cells with different concentrations of pD2RP, D2RP and D2RpP in Experimental Example 6 are shown. DETAILED DESCRIPTION

[0027] The present invention provides a biocompatible self-assembling oncolytic peptide, comprising a blocking group, a phosphorylated colloid factor and a functional sequence connected in sequence; the amino acid sequence of the phosphorylated colloid factor is G D F D F D pY, the functional sequence is RR D KRFYVVMWK D K.

[0028] In the present invention, the blocking group is preferably β-naphthylacetic acid (Nap); the amino acid sequence of the phosphorylated colloid factor is in D configuration; the first tyrosine at the N-terminus of the biocompatible self-assembling oncolytic peptide has an alkaline phosphatase reaction site; further preferably, the structural formula of the biocompatible self-assembling oncolytic peptide is as shown in Formula I:

[0029] Formula I.

[0030] The present invention has no particular limitation on the preparation method of the biocompatible self-assembling oncolytic peptide, and conventional preparation methods in the art may be used. In the specific implementation of the present invention, the Fmoc-short peptide solid phase synthesis method is preferably used.

[0031] The present invention provides a bioactive solution comprising the biocompatible self-assembling oncolytic peptide. Preferably, the bioactive solution uses PBS as a solvent; the pH of the PBS is preferably 7.2 to 7.4, more preferably 7.3; in the present invention, the mass-to-volume ratio of the biocompatible self-assembling oncolytic peptide to the buffer is preferably 2 mg:0.5 to 1.0 mL, more preferably 0.2 mg:0.9 to 1.1 mL. In the present invention, the bioactive solution is preferably obtained by mixing the biocompatible self-assembling oncolytic peptide with the PBS.

[0032] The present invention provides the use of the biocompatible self-assembling oncolytic peptide and the bioactive solution in the preparation of anti-tumor immune drugs. In the present invention, the dosage form of the anti-tumor immune drug is preferably an injectable preparation; the anti-tumor immune drug preferably includes anti-breast cancer, anti-colon cancer, and anti-melanoma drugs.

[0033] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] Peptide Nap-G D F D F D pY-RR D KRFYVVMWK D Synthesis of K

[0037] The peptide obtained was abbreviated as pD2RP using the classic Fmoc-short peptide solid phase synthesis method. The specific steps are:

[0038] 1) Weigh 0.5 mmol of 2-Cl-Trt resin into a solid phase synthesis tube, add 15 mL of dichloromethane (DCM), and shake on a shaker for 10 minutes to allow the 2-Cl-Trt resin to fully swell;

[0039] 2) Use an ear bulb to completely squeeze out DCM from the solid phase synthesizer containing 2-Cl-Trt resin;

[0040] 3) Dissolve 0.75 mmol of Fmoc-protected D-lysine (Fmoc-D-Lys(Boc)-OH) in 10 mL of DCM, add 1.5 mmol of N,N-diisopropylethylamine (DIEA), mix well, add to the above solid phase synthesizer, and place on a shaker at room temperature for 1 hour;

[0041] 4) Blocking: Remove the reaction solution from the solid phase synthesis tube with an ear bulb, then wash with 10 mL of DCM for 1 minute each time, for a total of 3 washes. Add 16 mL of a solution with a volume ratio of DCM:DIEA:methanol of 17:1:2 and react at room temperature for 15 minutes;

[0042] 5) Use an ear bulb to remove the reaction solution from the solid-phase synthesis tube, first wash with DCM, using 10 mL of DCM each time for 1 minute, for a total of 3 washes, then wash with N,N-dimethylformamide (DMF), using 10 mL of DMF each time for 1 minute, for a total of 3 washes, add 15 mL of DMF containing 20% ​​piperidine (volume ratio), react for 30 minutes, then wash with DMF, using 10 mL of DMF each time for 1 minute, for a total of 5 washes;

[0043] 6) Weigh 1 mmol of the second Fmoc-protected L-lysine (Fmoc-Lys(Boc)-OH), 1 mmol of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and 2 mmol of DIEA, dissolve them in 10 mL of DMF, add the dissolved amino acid solution to the above solid-phase synthesis tube, and react for 1 hour;

[0044] 7) Repeat steps 5) and 6) to add amino acids and blocking groups Nap in sequence; then wash with DMF 5 times and DCM 5 times before proceeding to the next step;

[0045] 8) Prepare 10 mL of a cutting solution consisting of 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIS), and 2.5% H2O (volume ratio) and add it to the solid phase synthesizer. Allow to react at room temperature for 1 hour. Cut the product from the 2-Cl-Trt resin and remove the solvent using a rotary evaporator to obtain a crude product. This crude product is then separated and purified using semi-preparative HPLC to obtain the polypeptide of Example 1. The polypeptide obtained in Example 1 was detected by high-resolution mass spectrometry and HPLC. The results are shown in FIG. Figure 1 The structural formula of the polypeptide of Example 1 is shown in Formula I.

[0046] Comparative Example 1

[0047] Peptide Nap-G D F DF D Y-RR D KRFYVVMWK D Synthesis of K

[0048] The polypeptide of Comparative Example 1 was prepared according to the Fmoc-short peptide solid phase synthesis method of Example 1. The obtained polypeptide is referred to as D2RP. When synthesizing the polypeptide of Comparative Example 1, the phosphate group is missing compared with the method of Example 1. The polypeptide obtained in Comparative Example 1 was tested by high resolution mass spectrometry and high performance liquid chromatography. The results are shown in FIG. Figure 2 , see the structural formula Figure 4 .

[0049] Comparative Example 2

[0050] Peptide Nap-G D F D F D Y-RR D KFvYVVMWK D Synthesis of K

[0051] The polypeptide of Comparative Example 2 was prepared according to the Fmoc-short peptide solid phase synthesis method of Example 1. The obtained polypeptide is referred to as D2RpP. When synthesizing the polypeptide of Comparative Example 2, the phosphate group is located on the tyrosine of the functional sequence compared with the method of Example 1. The polypeptide obtained in Comparative Example 2 was detected by high-resolution mass spectrometry and high-performance liquid chromatography. The results are shown in FIG. Figure 3 , see the structural formula Figure 4 .

[0052] Experimental Example 1

[0053] Micromorphology characterization

[0054] Weigh 4 mg of the peptide prepared in Example 3, add 500 μL of ultrapure water to dissolve it, then add 500 μL of 2×PBS (pH = 7.4), mix well, and then add ALP (50 U mL -1 ), placed in a 37°C thermostat and reacted for 12 hours. Transmission electron microscope samples were prepared before and after adding PBS and ALP, and their micromorphology was observed. The results are shown in Figure 5 The biocompatible self-assembling oncolytic peptide pD2RP prepared in Example 1 has no obvious assembly structure when dissolved in pure water. After adding PBS, it forms worm-like nanofibers. After adding ALP catalysis, the morphology changes and a dense nanofiber network is formed.

[0055] Experimental Example 2

[0056] Secondary structure determination by circular dichroism

[0057] Weigh 4 mg of the peptide prepared in Example 1 and dissolve it in 400 μL of ultrapure water. Take 200 μL of the solution and add it to 200 μL of 2×PBS (pH=7.4) to prepare a 2 mM 1×PBS solution. Then take 100 μL of the 1×PBS solution and add 50 UmL-1 of ALP to carry out enzyme catalysis at 37°C for 12 hours. Take 5 μL of the catalyzed solution and dilute it in 495 μL of 1×PBS. Then add the sample to a cuvette dedicated to a circular dichroism spectrometer and test it using a circular dichroism spectrometer (BioLogic MOS-450). Figure 6 As shown in Figure 3, it can be seen that before ALP catalysis, pD2RP is mainly in an antiparallel β-sheet conformation, which becomes dominant in α-helix after ALP catalysis.

[0058] Experimental Example 3

[0059] Zeta potential measurement

[0060] Prepare 600 μL of 0.5 mM pD2RP in 1×PBS before and after ALP enzyme catalysis according to the method of Experimental Example 2. Add the solution to the test sample cell and remove the bubbles. Keep the sample cell dry and perform the test. The instrument automatically measures three times. The results are as follows: Figure 7 As shown in Figure 3, the Zeta potential of pD2RP before enzyme catalysis is 14.3 mV, while that after dephosphorylation is 16.7 mV, which is a significant difference.

[0061] Experimental Example 4

[0062] Observation of cell surface nanofibers

[0063] According to 2×10 5 Mouse breast cancer cells 4T1 were plated on a 12-well plate at a density of 1 mL / well. After the cells adhered overnight, 1 mL of culture medium containing 40 μM pD2RP was replaced and incubated for another 6 hours. The control group was incubated with empty culture medium. The culture medium was then removed, and glutaraldehyde fixative for electron microscopy was added and fixed at 4°C for 12 hours. The cells were washed with PBS and dried for biological scanning electron microscopy testing. Figure 8 As shown, a large number of nanofiber clusters appeared on the cell surface after incubation with pD2RP, while the cell surface of the Control group was clean and smooth.

[0064] Experimental Example 5

[0065] Live cell imaging to detect cell membrane rupture

[0066] According to 8×10 3Mouse breast cancer 4T1 cells were plated in a 96-well plate at a density of 1 / well. After the cells adhered overnight, a 2 mM stock solution of pD2RP in 1× PBS was prepared according to the method in Experimental Example 2. This was then diluted in a 1× propidium iodide (PI) solution to create a 1× PI solution containing 40 μM peptide. An inverted fluorescence microscope (Leica, DMi8S) was used to observe the nuclear entry of PI. The parameters were pre-adjusted, and after the culture medium in the plate was poured out, the PI solution containing the peptide was quickly added, and imaging was then started. Figure 9 As shown in the figure, at the 15th minute, a large number of red fluorescent bright spots appeared, indicating that the cell membrane ruptured and PI entered the cell nucleus.

[0067] Experimental Example 6

[0068] Cytotoxicity

[0069] We measured the toxic effects of the polypeptide pD2RP in Example 1, the polypeptide D2RP in Comparative Example 1, and the polypeptide D2RpP in Comparative Example 2 on mouse breast cancer cells 4T1, mouse colon cancer cells MC38, mouse melanoma cells B16-F10, and mouse fibroblasts L929, respectively. The four types of cells were plated in 96-well plates at a density of 8×103, and the polypeptides were diluted twice to set up 9 concentration gradients. After overnight attachment, the cells were incubated with different polypeptides for 48 hours. The toxicity of the polypeptides to different cells was evaluated by the MTT method. After incubation with the polypeptides, thiazolyl blue (at a concentration of 0.5 mg / mL) was added for 4 hours, and then dimethyl sulfoxide (DMSO) was added to determine the absorbance at 490 nm. The results are shown as follows: Figure 10 The peptide D2RP from Comparative Example 1 was highly cytotoxic. Because it lacks a phosphate group, its toxicity against the four cell types was essentially the same. pD2RP and D2RpP showed similar cell-killing abilities to D2RP in 4T1 and MC38 cells, which both highly express ALP, while exhibiting very low toxicity in B16-F10 and L929 cells, which both lowly express ALP, demonstrating excellent biocompatibility.

[0070] The above experimental examples demonstrate that the biocompatible, self-assembling oncolytic peptide provided by the present invention, through the action of ALP overexpressed outside tumor cells, undergoes dephosphorylation and self-assembly in situ, altering its secondary structure and zeta potential. This allows it to physically bind to cancer cell membranes, disrupting them and causing cell death. Furthermore, the oncolytic peptide has no killing effect on normal cells that lowly express ALP, demonstrating excellent biocompatibility.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A biocompatible self-assembling oncolytic peptide, characterized in that: It includes a blocking group, a phosphorylated colloid factor and a functional sequence connected in sequence; the amino acid sequence of the phosphorylated colloid factor is G D F D F D pY, the functional sequence is RR D KRFYVVMWK D K.

2. The biocompatible self-assembling oncolytic peptide according to claim 1, characterized in that The blocking group is β-naphthylacetic acid (Nap).

3. The compatible self-assembling oncolytic peptide according to claim 2, characterized in that: The amino acid sequence of the phosphorylated collagen-forming factor is in D configuration.

4. The biocompatible self-assembling oncolytic peptide according to claim 3, characterized in that The structural formula of the polypeptide that induces immunogenic death of tumor cells is shown in Formula I: Formula I.

5. A biologically active solution, characterized in that The invention comprises the biocompatible self-assembling oncolytic peptide according to any one of claims 1 to 4.

6. The bioactive solution according to claim 5, characterized in that The biologically active solution uses PBS as a solvent.

7. The bioactive solution according to claim 6, characterized in that The pH of the PBS is 7.2-7.

4.

8. The bioactive solution according to any one of claims 5 to 7, characterized in that The mass volume ratio of the biocompatible self-assembling oncolytic peptide to the buffer solution is 2 mg:0.5~1 mL.

9. Use of the biocompatible self-assembling oncolytic peptide according to any one of claims 1 to 4 and the bioactive solution according to any one of claims 5 to 7 in the preparation of anti-tumor immunotherapy drugs.

10. The use according to claim 9, characterized in that The anti-tumor immune drugs include anti-breast cancer, anti-colon cancer and anti-melanoma drugs.

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