Preparation of peptide nanotube-cyclodiguanylate complex for tumor treatmentTechnical Field
The invention relates to a preparation method for compositing peptide nanotubes and phenylboronic acid peptide nanotubes with cyclodiguanylic acid respectively. The peptide nanotube material can be used for delivering cyclodiguanylate, can enhance the immune treatment effect of cyclodiguanylate, and has good application prospect in the field of immune treatment.
Background
Cyclic dinucleotides, such as cyclic diguanylate (c-di-GMP), cyclic di-AMP (c-di-AMP), and cyclic guanylate (cGAMP), have been shown to be highly potent pathogen-associated molecular pattern molecules, and are natural agonists of the interferon stimulating gene (STING). When the cyclic dinucleotide binds to the interferon-stimulated gene, the interferon-stimulated gene is transferred from the endoplasmic reticulum to the golgi apparatus, thereby recruiting protein kinase, and then the interferon-stimulated gene is phosphorylated, and further activates nuclear transcription factor and interferon regulatory factor 3, inducing the production of Type I IFN and other pro-inflammatory cytokines, thereby activating antigen presenting cells, promoting infiltration of tumor-infiltrating lymphocytes in the tumor microenvironment. Thus, cyclic dinucleotides can be used as a promising and effective adjuvant to trigger the connection of innate immunity and acquired immunity, thereby allowing successful cancer immunotherapy. However, electronegativity, hydrophilicity and instability of cyclic dinucleotides have hindered their further use in tumour immunotherapy. The phosphate group on the cyclic dinucleotide limits its ability to enter the cytosol for binding to the interferon-stimulated gene. Phosphodiesterases can degrade cyclic dinucleotides, making their biological half-life low, and thus bioavailability low.
It has been reported that the short peptide KL-7, which is β -amyloid fibril-pie-generated, has amphiphilic ability, allowing self-assembly into amphiphilic Peptide Nanotubes (PNT) through antiparallel β -sheet through electrostatic, pi-pi stacking and hydrophobic interactions at neutral ph=7.0 (small, 2019, 1900157). Peptide nanotubes have previously been shown to have good biocompatibility, a good nanocarrier. The elongated nanotube structure has a high aspect ratio compared to the spherical nanostructure, and can prolong blood circulation and residence time in tumor (biomaterials.2018, 178, 570e 582). In the tumor microenvironment, various tumor cells overexpress Sialic Acid (SA) due to aberrant glycosylation, which is considered a marker for cancer progression and poor prognosis. The phenylboronic acid and sialic acid are specifically identified, and the phenylboronic acid and sialic acid have higher selectivity and affinity, are not influenced by physiology and metabolism, and show good physiological stability. PNT and 4-carboxyphenylboronic acid are used as raw materials, a phenylboronic acid modified peptide (PB-KL-7) is synthesized through an amide reaction, and then the phenylboronic acid modified peptide nanotube (PPNT) is obtained through self-assembly. The complex of c-di-GMP-PNT and c-di-GMP-PPNT is obtained by using PNT and PPNT as delivery vehicles of c-di-GMP. Because PNT and PPNT can reduce the contact probability of c-di-GMP and phosphodiesterase, the degradation rate of c-di-GMP in vivo is further reduced, the bioavailability of c-di-GMP is effectively improved, the secretion of Type I IFN and IL-6 inflammatory factors can be promoted in vivo, and the immunity and anti-tumor effect is enhanced.
Disclosure of Invention
Aiming at the defects of poor membrane permeability, low bioavailability and the like of c-di-GMP serving as an immunotherapy adjuvant, the invention aims to provide a preparation method of a c-di-GMP-PNT and c-di-GMP-PPNT compound with low manufacturing cost, simple operation and low cytotoxicity so as to improve the activity of the c-di-GMP on tumor immunotherapy.
The invention provides a preparation method of c-di-GMP-PNT and c-di-GMP-PPNT, which comprises the following steps:
(1) Synthesis of KL-7 peptide the Fmoc solid phase peptide method was used to synthesize the KL-7 (Ac-KLAVFFAL-NH2) peptide segment. The N end of the KL-7 peptide is subjected to a blocking reaction, 0.2 mmol KL-7 is used as a raw material, a reaction solvent is a mixed solution of acetic anhydride-N, N-diisopropylethylamine-N, N-dimethylformamide, the volume ratio is 5:1:50, the dosage is 6 mL-7 mL, the reaction temperature is room temperature, the reaction time is 15 min-25 min, the KL-7 peptide is subjected to cleavage deprotection, the blocked 0.2 mmol KL-7 is used as a raw material, the reaction solvent is a mixed solution of trifluoroacetic acid-benzyl sulfide-dimercaptoethane-anisole, the volume ratio is 90:5:3:2, the dosage is 6mL, the reaction temperature is room temperature, and the reaction time is 1.5 h-2.5 h. The lysate was precipitated with diethyl ether in an amount of 40 mL.
(2) PNT KL-7 (15.6 mg, 0.018 mmol) was prepared, the reaction solvent was hexafluoroisopropanol, the amount was 2 mL-3 mL, the reaction time was 25-35 min, and the reaction temperature was 0oC-3o C. Then, HIFP was removed under a nitrogen atmosphere to obtain a polypeptide film attached to the wall of the centrifuge tube. The polypeptide film is dissolved, the solvent is acetonitrile water solution, the volume ratio is 2:3, the dosage is 5 mL, the reaction temperature is 37o ℃, and the reaction time is 24 h.
(3) The preparation PPNT comprises the steps of preparing a mixed solution of KL-7, 4-carboxyphenylboronic acid, HOBT and HCTU in a nitrogen atmosphere, wherein the mass ratio of the KL-7 to the 4-carboxyphenylboronic acid to the HOBT to the HCTU is 1:10:12:12, the reaction solvent is N-methylmorpholine-N, N-dimethylformamide, the volume ratio is 1:19, the dosage is 3 mL-4 mL, the reaction temperature is room temperature, and the reaction time is 24 h.
(4) The preparation of the C-di-GMP-PNT compound comprises the steps of using PNT as a carrier, compounding the C-di-GMP with the PNT through electrostatic action, wherein the mass ratio of the C-di-GMP to the PNT is 1:1-1:2, the purified water dosage is 0.1-1 mL, and after 37o C water bath standing and incubation for 24 h, dispersing the mixture in an ultrasonic manner for 1 h-3 h.
(5) The preparation of the C-di-GMP-PPNT compound comprises the steps of using PPNT as a carrier, compounding the C-di-GMP with PPNT through electrostatic action, wherein the mass ratio of the C-di-GMP to PPNT is 1:1-1:2, using 0.1-1 mL of purified water, standing in a 37o C water bath, incubating for 24h, and then dispersing 1 h-3 h by ultrasonic.
TABLE 1 average particle size, PDI distribution, zeta surface potential of c-di-GMP-PNT and c-di-GMP-PPNT complexes after aqueous dispersion
| Cyclic diguanylate-peptides nanotube composites | Average particle diameter 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 |
Drawings
Table 1 shows the average particle diameters, PDI values, and Zeta potentials of PNT, PPNT, c-di-GMP-PNT and c-di-GMP-PPNT measured by DLS.
Detailed description of the preferred embodiments
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
Unless otherwise indicated, all chemical reagents used in the examples were conventional commercial reagents, and the technical means used in the examples were conventional means well known to those skilled in the art.
Example 1. Preparation of c-di-GMP-PNT complex:
the mass ratio of C-di-GMP to PNT is 1:1.4, the purified water dosage is 0.14 mL, and after standing and incubating for 24 h under the condition of 37: 37o C constant temperature water bath, the ultrasonic dispersion is carried out for 2: 2 h.
Example 2 preparation of c-di-GMP-PPNT Complex:
the mass ratio of C-di-GMP to PPNT is 1:1.4, the purified water dosage is 0.15: 0.15 mL, and after standing and incubating for 24: 24 h under the condition of 37 ℃ ando ℃ constant temperature water bath, the solution is dispersed by ultrasound for 2: 2 h.
Example 3 determination of particle size and surface potential:
The particle size and surface potential and particle size distribution of the c-di-GMP-PNT (0.40 mM c-di-GMP, 0.56 mM PNT), c-di-GMP-PPNT (0.40 mM c-di-GMP, 0.56 mM PPNT) dispersions were determined using a Zetasizer Nano ZS laser particle sizer. Test conditions are a test temperature of 25o C, a He-Ne laser source, a wavelength 633 nm, and an angle 173 ofo. Three measurements were made for each sample and an average was taken. The average particle size, PDI distribution, zeta surface potential of the c-di-GMP-PNT and c-di-GMP-PPNT complexes after water dispersion are shown in Table 1.
Example 4 determination of encapsulation efficiency:
According to the preparation steps (4) and (5), the prepared c-di-GMP-PNT (0.52 mM c-di-GMP, 0.73 mM PNT) solution and c-di-GMP-PPNT (0.69 mM c-di-GMP, 0.98 mM PPNT) solution are placed in a high-speed centrifuge, 20min is centrifuged at the rotating speed of 10000 rpm, supernatant is collected and detected by HPLC, peak areas are recorded, the measurement is repeated three times, an average value is taken, the content of the c-di-GMP is calculated in a standard curve, and the encapsulation rate is calculated according to the formula (1), so that the encapsulation rates of the c-di-GMP-PNT compound and the c-di-GMP-PPNT compound are respectively 51.64% and 10.44%, and PNT and PPNT can be effective loads of c-di-GMP.
Example 5 determination of cumulative drug release rate:
According to the above preparation steps (4) and (5), 0.14 mL C-di-GMP-PNT complex solution (0.52 mM C-di-GMP, 0.73 mM PNT) and 0.15 mL C-di-GMP-PPNT complex solution (0.47 mM C-di-GMP, 0.66 mM PPNT) were placed in dialysis bags of 3500 Da and sealed with dialysis clips, respectively, and then placed in PBS solution (80 mL) at ph=7.4, and slowly stirred with a magnetic stirrer (37o C, 120 rpm). At certain time intervals (0 h, 0.5 h, 1 h, 1.5h, 2 h..24 h), 1 mL PBS dialysis solutions were taken separately for UV-visible detection of 255 nm and 1 mL fresh PBS solution was added. The cumulative release rate of c-di-GMP was calculated according to formula (2), and at 24 h, the cumulative release rate of c-di-GMP-PNT was 98.8% and the cumulative release rate of c-di-GMP-PPNT was 69.1%.
Wherein, Cn represents the concentration of C-di-GMP in the dialysis fluid at the nth sampling, mug/mL, V represents the total volume of the dialysis fluid, mL, Ci represents the concentration of C-di-GMP in the dialysis fluid at the ith sampling, mug/mL, vs represents the volume of the dialysis fluid taken out each time, C0 represents the concentration of C-di-GMP in the drug-loaded micelle, mug/mL, and V0 represents the volume of the loaded C-di-GMP micelle.
Example 6. Proinflammatory Activity assay of c-di-GMP-PNT and c-di-GMP-PPNT for stimulation of RAW 264.7 cells, respectively:
RAW 264.7 cells were inoculated into 12-well plates, cultured to a cell density of 80% -90%, 0.03 mL c-di-GMP-PNT and 0.04 mL c-di-GMP-PPNT were respectively taken in DMEM medium with serum of 1.2 mL to obtain c-di-GMP-PNT complex (0.02 mM c-di-GMP, 0.02mM PNT) and c-di-GMP-PPNT complex (0.02 mM c-di-GMP, 0.02mM PPNT), the above complex solution was added to 12-well plates inoculated with cells, a control group was added with fresh serum DMEM medium without drug, and after incubation in a 37oC,5%CO2 constant temperature incubator of 6h, 12 h, 24h, cell supernatants were collected. Finally, the IFN- β, IL-6 content of the cell supernatants was assayed according to the procedure of the Elisa kit (Elabscience, E-EL-M0033 c). The results show that over time, the c-di-GMP-PNT and c-di-GMP-PPNT complexes stimulated RAW 264.7 cells to produce IFN- β, IL-6 inflammatory factor content increased significantly compared to free c-di-GMP.
The above examples are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solution of the present invention should fall within the scope of protection defined by the claims of the present invention without departing from the spirit of the design of the present invention.
Furthermore, no device or method is required to address each and every problem sought to be solved by the present invention, for it to be encompassed by the present claims. Furthermore, no element, component, or method step in the present disclosure is intended to be dedicated to the public regardless of whether the element, component, or method step is explicitly recited in the claims. It will be apparent to those of ordinary skill in the art that various changes and modifications in form, reagents and synthetic details may be made therein without departing from the spirit and scope of the invention as set forth in the appended claims.