Preparation of a Peptide Nanotube-Cyclic Diguanylate Complex for Tumor Therapy

Abstract

The present invention discloses the preparation of a peptide nanotube-cyclic diguanosine monophosphate complex for tumor treatment, which method improves the problems of poor membrane permeability and low bioavailability of cyclic diguanosine monophosphate (c-di-GMP). Peptide nanotubes (PNT) and phenylboronic acid peptide nanotubes (PPNT) are used to deliver c-di-GMP, which can enhance the immune anti-tumor effect of c-di-GMP in vivo.

Description

Technical Field

[0001] The present invention relates to a preparation method for the complex of peptide nanotubes, phenylboronic acid peptide nanotubes and cyclic diguanosine monophosphate respectively. The peptide nanotube material can be used for delivering cyclic diguanosine monophosphate, enhancing the immunotherapeutic effect of cyclic diguanosine monophosphate, and having good application prospects in the field of immunotherapy. Background Art

[0002] Cyclic dinucleotides, such as cyclic diguanosine monophosphate (c-di-GMP), cyclic diadenosine monophosphate (c-di-AMP) and cyclic guanosine-adenosine monophosphate (cGAMP), etc., have been proven to be highly efficient pathogen-associated molecular pattern molecules and natural agonists of the stimulator of interferon genes (STING). When cyclic dinucleotides bind to the stimulator of interferon genes, the stimulator of interferon genes transfers from the endoplasmic reticulum to the Golgi apparatus, and then recruits protein kinases. Then, the stimulator of interferon genes is phosphorylated, and further activates nuclear transcription factors and interferon regulatory factor 3, inducing the production of Type I IFN and other pro-inflammatory cytokines, thereby activating antigen-presenting cells and promoting the infiltration of tumor-infiltrating lymphocytes in the tumor microenvironment. Therefore, cyclic dinucleotides can be used as a promising effective adjuvant to trigger the connection between innate immunity and acquired immunity, thus making cancer immunotherapy successful. However, the negative charge, hydrophilicity and instability of cyclic dinucleotides hinder their further application in tumor immunotherapy. The phosphate group on cyclic dinucleotides limits its ability to enter the cytoplasm and bind to the stimulator of interferon genes. Phosphodiesterase can degrade cyclic dinucleotides, resulting in a low biological half-life and thus low bioavailability.

[0003] It is reported that the short peptide KL-7 derived from β-amyloid fibrils has amphiphilic ability, allowing it to self-assemble into amphiphilic peptide nanotubes (PNT) through antiparallel β-sheets at neutral pH = 7.0 via electrostatic, π-π stacking, and hydrophobic interactions (Small. 2019, 1900157). Previously, peptide nanotubes have been shown to have good biocompatibility and are good nanocarriers. Compared with spherical nanostructures, the elongated nanotube structure has a high aspect ratio, which can prolong blood circulation and residence time in tumors (Biomaterials. 2018, 178, 570e582). In the tumor microenvironment, various tumor cells overexpress sialic acid (SA) due to abnormal glycosylation, which is considered a marker of cancer development and poor prognosis. The specific recognition of phenylboronic acid and sialic acid has higher selectivity and affinity, and is not affected by physiology and metabolism, showing good physiological stability. Using PNT and 4-carboxyphenylboronic acid as raw materials, a phenylboronic acid-modified peptide segment (PB-KL-7) was synthesized through an amide reaction, and then self-assembled to obtain phenylboronic acid-modified peptide nanotubes (PPNT). Using PNT and PPNT as delivery carriers for c-di-GMP, c-di-GMP-PNT and c-di-GMP-PPNT complexes were obtained. Since PNT and PPNT can reduce the chance of c-di-GMP contacting phosphodiesterase, thereby reducing the degradation rate of c-di-GMP in vivo, effectively improving the bioavailability of c-di-GMP, and promoting the secretion of Type I IFN and IL-6 inflammatory factors in vivo, enhancing the immune anti-tumor effect. Summary of the Invention

[0004] Aiming at the defects of c-di-GMP as an immunotherapy adjuvant, such as poor membrane permeability and low bioavailability, the purpose of the present invention is to provide a preparation method of c-di-GMP-PNT and c-di-GMP-PPNT complexes with low production cost, simple operation, and low cytotoxicity, so as to improve the activity of c-di-GMP in tumor immunotherapy.

[0005] The present invention provides a preparation method of c-di-GMP-PNT and c-di-GMP-PPNT, which includes the following steps:

[0006] (1) Synthesis of KL-7 peptide: The KL-7 (Ac-KLAVFFAL-NH2) peptide segment was synthesized by the Fmoc solid-phase peptide method. For the N-terminal blocking reaction of the KL-7 peptide segment, 0.2 mmol of KL-7 was used as the raw material, and the reaction solvent was a mixed solution of acetic anhydride - N,N-diisopropylethylamine - N,N-dimethylformamide with a volume ratio of 5:1:50 and a dosage of 6 mL - 7 mL. The reaction temperature was room temperature, and the reaction time was 15 min - 25 min. For the cleavage and deprotection of the KL-7 peptide segment, the above-blocked 0.2 mmol of KL-7 was used as the raw material, and the reaction solvent was a mixed solution of trifluoroacetic acid - benzyl methyl sulfide - dimercaptoethane - anisole with a volume ratio of 90:5:3:2 and a dosage of 6 mL. The reaction temperature was room temperature, and the reaction time was 1.5 h - 2.5 h. Diethyl ether was used to precipitate the cleavage solution, with a dosage of 40 mL.

[0007] (2) Preparation of PNT: KL-7 (15.6 mg, 0.018 mmol), the reaction solvent was hexafluoroisopropanol, with a dosage of 2 mL - 3 mL, the reaction time was 25 - 35 min, and the reaction temperature was 0 o C-3 o C. Then, under a nitrogen atmosphere, HIFP was removed to obtain a polypeptide film attached to the centrifuge tube wall. The polypeptide film was dissolved, and the solvent was an acetonitrile aqueous solution with a volume ratio of 2:3 and a dosage of 5 mL. The reaction temperature was 37 o C, and the reaction time was 24 h.

[0008] (3) Preparation of PPNT: Under a nitrogen atmosphere, the molar ratio of KL-7, 4-carboxyphenylboronic acid, HOBT, and HCTU was 1:10:12:12. The reaction solvent was a mixed solution of N-methylmorpholine - N,N-dimethylformamide with a volume ratio of 1:19 and a dosage of 3 mL - 4 mL. The reaction temperature was room temperature, and the reaction time was 24 h.

[0009] (4) Preparation of c-di-GMP-PNT complex: Using PNT as the carrier, c-di-GMP was complexed with PNT through electrostatic interaction; the molar ratio of c-di-GMP to PNT was 1:1 - 1:2, the dosage of purified water was 0.1 - 1 mL, and after static incubation in a 37 o C water bath for 24 h, it was then ultrasonically dispersed for 1 h - 3 h.

[0010] (5) Preparation of c-di-GMP-PPNT complex: Using PPNT as the carrier, c-di-GMP was complexed with PPNT through electrostatic interaction, the molar ratio of c-di-GMP to PPNT was 1:1 - 1:2, the dosage of purified water was 0.1 - 1 mL, 37o After static incubation in a water bath at 24 h, ultrasonically disperse for 1 h - 3 h.

[0011] Table 1 Average particle size, PDI distribution, and Zeta surface potential of c-di-GMP-PNT and c-di-GMP-PPNT complexes after aqueous dispersion

[0012] Cyclic diguanosine monophosphate-peptide nanotube complex Average particle size nm PDI Zeta surface potential mV c-di-GMP-PNT 211.45±2.80 0.212 +33.0±0.90 c-di-GMP-PPNT 1980±5.20 0.155 +2.27±0.04 Description of the drawings

[0013] Table 1 shows the average particle size, PDI value, and Zeta potential of PNT, PPNT, c-di-GMP-PNT, and c-di-GMP-PPNT measured by DLS. Specific implementation method

[0014] The following examples are used to illustrate the present invention but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the methods, steps, or conditions of the present invention all fall within the scope of the present invention.

[0015] Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0016] Example 1. Preparation of c-di-GMP-PNT complex:

[0017] With the molar ratio of c-di-GMP to PNT being 1:1.4 and the amount of purified water being 0.14 mL, under the condition of a constant temperature water bath at 37 o C, after static incubation for 24 h, ultrasonically disperse for 2 h.

[0018] Example 2. Preparation of c-di-GMP-PPNT complex:

[0019] With the molar ratio of c-di-GMP to PPNT being 1:1.4 and the amount of purified water being 0.15 mL, under the condition of a constant temperature water bath at 37 o C, after static incubation for 24 h, ultrasonically disperse for 2 h.

[0020] Example 3. Measurement of particle size and surface potential:

[0021] Use a Zetasizer Nano ZS laser particle size analyzer to measure the particle size, surface potential, and particle size distribution of the c-di-GMP-PNT (0.40 mM c-di-GMP, 0.56 mM PNT) and c-di-GMP-PPNT (0.40 mM c-di-GMP, 0.56 mM PPNT) dispersion systems. Test conditions: The test temperature is 25o C, a He-Ne laser source with a wavelength of 633 nm and an emission angle of 173 o Each sample was measured three times and the average value was taken. The average particle size, PDI distribution, and Zeta surface potential of the c-di-GMP-PNT and c-di-GMP-PPNT complexes after being dispersed in water are listed in Table 1.

[0022] Example 4. Determination of encapsulation efficiency:

[0023] According to the above preparation steps (4) and (5), the prepared c-di-GMP-PNT (0.52 mM c-di-GMP, 0.73 mM PNT) and c-di-GMP-PPNT (0.69 mM c-di-GMP, 0.98 mM PPNT) solutions were placed in a high-speed centrifuge and centrifuged at a speed of 10,000 rpm for 20 min. The supernatant was collected and detected by HPLC. The peak area was recorded, and the measurement was repeated three times. The average value was taken and substituted into the standard curve to calculate the content of c-di-GMP. The encapsulation efficiency was calculated according to formula (1), and the encapsulation efficiencies of the c-di-GMP-PNT complex and the c-di-GMP-PPNT complex were 51.64% and 10.44% respectively, indicating that PNT and PPNT can effectively load c-di-GMP.

[0024]

[0025] Example 5. Determination of the cumulative drug release rate:

[0026] According to the above preparation steps (4) and (5), 0.14 mL of the c-di-GMP-PNT complex solution (0.52 mM c-di-GMP, 0.73 mM PNT) and 0.15 mL of the c-di-GMP-PPNT complex solution (0.47 mM c-di-GMP, 0.66 mM PPNT) were respectively placed in dialysis bags with a molecular weight cut-off of 3500 Da and sealed with dialysis clips. Then they were put into PBS solution (80 mL) with pH = 7.4 and slowly stirred with a magnetic stirrer (37 o °C, 120 rpm). At certain time intervals (0 h, 0.5 h, 1 h, 1.5 h, 2 h... 24 h), 1 mL of the PBS dialysis solution was taken for UV-visible detection at 255 nm, and 1 mL of fresh PBS solution was added. The cumulative release rate of c-di-GMP was calculated according to formula (2). At 24 h, the cumulative release rate of c-di-GMP-PNT was 98.8%; the cumulative release rate of c-di-GMP-PPNT was 69.1%.

[0027]

[0028] Among them, C n represents the concentration of c-di-GMP in the dialysate at the nth sampling, μg / mL; V is the total volume of the dialysate, mL; C i represents the concentration of c-di-GMP in the dialysate at the ith sampling, μg / mL; Vs is the volume of the dialysate taken each time, mL; C0 is the concentration of c-di-GMP in the drug-loaded micelles, μg / mL; V0 is the volume of the micelles loaded with c-di-GMP, mL.

[0029] Example 6. Determination of the pro-inflammatory activities of c-di-GMP-PNT and c-di-GMP-PPNT in stimulating RAW 264.7 cells:

[0030] RAW 264.7 cells were seeded into a 12-well plate, and the cell density was cultured to 80%-90%. 0.03 mL of c-di-GMP-PNT and 0.04 mL of c-di-GMP-PPNT were taken respectively in 1.2 mL of serum-containing DMEM medium to obtain c-di-GMP-PNT complex (0.02 mM c-di-GMP, 0.02 mM PNT), c-di-GMP-PPNT complex (0.02 mM c-di-GMP, 0.02 mM PPNT). The above complex solutions were added to the 12-well plate seeded with cells. The control group was added with fresh serum-containing DMEM medium without drugs and placed in a 37 o °C, 5% CO2 constant temperature incubator. After incubation for 6 h, 12 h, and 24 h, the cell supernatants were collected. Finally, according to the operation steps of the Elisa kit (Elabscience, E-EL-M0033c), the contents of IFN-β and IL-6 in the cell supernatants were detected. The results showed that with the increase of time, compared with free c-di-GMP, the contents of IFN-β and IL-6 inflammatory factors produced by c-di-GMP-PNT and c-di-GMP-PPNT complexes in stimulating RAW 264.7 cells increased significantly.

[0031] The above examples only describe the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

[0032] Furthermore, there is no need for a certain device or method to express each problem solved by the present invention, as they are all included in the claims of the present invention. Additionally, whether all parts, components, or method steps in the disclosed facts of the present invention are explicitly recited in the claims, they do not contribute to the public. However, it is obvious to those of ordinary skill in the art that various changes and modifications can be made in form, reagents, and synthetic details without departing from the essence and scope of the present invention as set forth in the appended claims.

Claims

1. A method for preparing a peptide nanotube-cyclic diguanosine monophosphate complex, characterized in that, It includes the following steps: (1) Preparation of peptide nanotubes PNT and PPNT: Using Rink Amide resin as the solid-phase carrier, the Ac-KLVFFAL-NH2 peptide segment derived from β-amyloid fibrils, abbreviated as KL-7, was prepared; in an aqueous acetonitrile solution under the condition of pH = 7.0, through electrostatic, π-π stacking and hydrophobic interactions, after reverse parallel β-sheets, KL-7 self-assembled to form peptide nanotubes, abbreviated as PNT; KL-7, 4-carboxyphenylboronic acid, HOBT and HCTU were mixed in a molar ratio of 1:10:12:12, using a mixed solution of N-methylmorpholine and N,N-dimethylformamide with a volume ratio of 1:19 as the reaction solvent, with a dosage of 3 - 4 mL, reacting at room temperature for 20 - 24 h, and the phenylboronic acid-modified peptide segment, abbreviated as PB-KL-7, was synthesized. PB-KL-7 self-assembled to form peptide nanotubes, abbreviated as PPNT; (2) Preparation of c-di-GMP-PNT complex: Using PNT as the carrier, c-di-GMP was complexed with PNT through electrostatic interaction; the molar ratio of c-di-GMP to PNT was 1:1 - 1:2, with a dosage of 0.1 - 1 mL of purified water, after static incubation in a 37°C water bath for 24 h, and then ultrasonic dispersion for 1 - 3 h, an aqueous solution of c-di-GMP-PNT complex was obtained; (3) Preparation of c-di-GMP-PPNT complex: Using PPNT as the carrier, c-di-GMP was complexed with PPNT through electrostatic interaction, the molar ratio of c-di-GMP to PPNT was 1:1 - 1:2, with a dosage of 0.1 - 1 mL of purified water, after static incubation in a 37°C water bath for 24 h, and then ultrasonic dispersion for 1 - 3 h, an aqueous solution of c-di-GMP-PPNT complex was obtained.