Disubstituted clustered peptide with helical structure, preparation method thereof and application of disubstituted clustered peptide in cryoprotectant

Abstract

The invention relates to a disubstituted clustered peptide with a helical structure and a preparation method and application of the disubstituted clustered peptide in a cryoprotectant, the disubstituted clustered peptide has the following structural general formula: # imgabs0 #, m: n = (1: 9)-(5: 5), the degree of polymerization (m + n) is 10-200, and m and n are integers; the disubstituted cluster peptide can be obtained through ring opening polymerization of N-substituted-amino acid-N-carboxylic acid anhydride five-membered ring monomers, the preparation method is simple, the polymerization activity is controllable, and the molecular weight of the prepared cluster peptide is controllable and narrow in distribution; more importantly, steric hindrance generated by a chiral center on a main chain of the disubstituted cluster peptide and a disubstituent enables the disubstituted cluster peptide to form a stable helical structure, and the disubstituted cluster peptide shows an excellent ice recrystallization inhibition effect in an ice recrystallization inhibition activity experiment, does not have hemolytic activity, is low in cytotoxicity, can effectively improve the survival rate of cells after cryopreservation, and has a good application prospect. Good application prospects are realized in the aspect of cell cryopreservation agents.

Description

Technical Field

[0001] The present invention relates to the field of polymer chemistry and cell cryopreservation technology, and in particular to a disubstituted polypeptide with a helical structure, a preparation method thereof, and application thereof in a cryoprotectant. Background Art

[0002] Living cells play a key role in biomedical applications, including cell therapy, tissue engineering, and drug screening. However, when cells are removed from their natural physiological environment, they rapidly lose vitality and undergo irreversible death. Although some cell types can be expanded in vitro and maintain functional activity, this process not only requires a large investment of time and resources, but also causes cells to gradually age and eventually lose activity during serial passage. Therefore, the development of reliable long-term preservation technologies is crucial to maintain the functional integrity of cells, which is of great significance for the advancement of cell-based medicine.

[0003] Cryopreservation technology is a technology that uses low temperatures (usually -80°C to -196°C) to inhibit biological or chemical activities, thereby preserving biological samples (such as cells, tissues, organs, embryos, etc.) for a long time. Cryoprotectants, as a key component of cryopreservation technology, mainly inhibit the formation of ice crystals to reduce the mechanical and osmotic damage to cells by ice crystals, thereby maintaining the structural integrity and functionality of cells during long-term cryopreservation. Dimethyl sulfoxide (DMSO) is a traditional cryoprotectant commonly used for cell cryopreservation, but it has certain toxicity. Therefore, the development of new cryoprotectants with low toxicity and high efficiency has become a key technical bottleneck that needs to be broken through in this field.

[0004] A large number of synthetic polymers have been reported as cryopreservatives, including peptides, polyvinyl alcohol (PVA), and zwitterionic polymers. Among these, polyvinyl alcohol is considered to have the highest ice-recrystallization inhibitory activity among synthetic polymers. While the ice-recrystallization inhibitory activity of most synthetic polymers is far lower than that of PVA, PVA's molecular weight dependence and potential metabolic issues limit its further application.

[0005] Based on this, there is an urgent need for a cryoprotectant with high cryoprotectant activity, low toxicity and good biocompatibility to improve the freezing survival rate of cells. Summary of the Invention

[0006] To solve the above problems, the present invention provides a disubstituted polypeptide with a helical structure, a preparation method thereof, and an application in a cryoprotectant. The chiral center on the main chain of the disubstituted polypeptide and the steric hindrance generated by the disubstituted groups enable it to form a stable helical structure, achieve the separation of hydrophilic and hydrophobic regions, thereby effectively inhibiting the growth of ice crystals. In addition, this type of polypeptide has low cytotoxicity and no hemolytic activity, can effectively improve the survival rate of cells after freezing, and has good application prospects in cell cryopreservatives.

[0007] Specifically, the following technical solutions are provided:

[0008] The first aspect of the present invention provides a disubstituted polypeptide having a helical structure, wherein the disubstituted polypeptide has the following general structural formula:

[0009]

[0010] wherein m:n=(1:9)-(5:5), the degree of polymerization (m+n) is 10-200, and m and n are integers;

[0011] R1 and R3 are independently selected from one of C1-C4 alkyl, C1-C2 hydroxyalkyl, C1-C2 fatty acid, and -RS(O)-R', R is a C1-C2 divalent alkyl, and R' is a methyl group;

[0012] R2 and R4 are independently selected from one of C1-C4 alkyl groups.

[0013] Furthermore, R1 and R3 are independently selected from one of C1-C4 alkyl groups.

[0014] More preferably, R1, R2, R3, and R4 are all methyl groups.

[0015] Furthermore, m:n = (1:9)-(2:8), for example, 1:9, 1.5:9.5, 2:8, etc., including but not limited to the ratios listed above; in some preferred embodiments of the present invention, the degree of polymerization (m+n) is 25-200, for example, 25, 50, 75, 100, 125, 150, 175, 200, etc., including but not limited to the values ​​listed above.

[0016] The second aspect of the present invention provides a method for preparing the disubstituted polypeptide according to the first aspect, comprising the following steps:

[0017] The monomer represented by Formula I, the monomer represented by Formula II, and benzylamine are subjected to a ring-opening polymerization reaction in the presence of an organic acid and a solvent to obtain the disubstituted polypeptide; wherein the molar ratio of the monomer represented by Formula I to the monomer represented by Formula II is (1:9)-(5:5);

[0018] The structures of formula I and formula II are shown below:

[0019]

[0020] wherein R1 and R3 are independently selected from one of a C1-C4 alkyl group, a C1-C2 hydroxyalkyl group, a C1-C2 fatty acid, and -RS(O)-R', R is a C1-C2 divalent alkyl group, and R' is a methyl group;

[0021] R2 and R4 are independently selected from one of C1-C4 alkyl groups.

[0022] Furthermore, the organic acid is selected from one or more of acetic acid, formic acid, propionic acid, and benzoic acid, and the organic solvent is selected from one or more of chloroform, 1,2-dichloroethane, tetrahydrofuran, toluene, trifluorotoluene, fluorobenzene, and N,N-dimethylformamide.

[0023] Furthermore, the ratio of the total molar amount of the monomer represented by formula I and the monomer represented by formula II to the molar amount of the benzylamine is (10-200):1, and the molar ratio of the monomer to the initiator benzylamine is adjusted to obtain polymers with different degrees of polymerization.

[0024] Furthermore, the ring-opening polymerization reaction is carried out at a temperature of 25-80° C. and for a time of 0.5-48 h.

[0025] Furthermore, when at least one of R1, R2, R3, and R4 contains a hydroxyl group or a carboxyl group, the corresponding monomer is first protected and then deprotected after the ring-opening polymerization.

[0026] Furthermore, after the reaction is complete, the solvent is removed, and the residue is dissolved in a small amount of dichloromethane, and then an excess of diethyl ether is added to precipitate a solid to obtain the disubstituted polypeptide.

[0027] The third aspect of the present invention provides a use of the disubstituted polypeptide described in the first aspect in a cryoprotectant.

[0028] Furthermore, the disubstituted polypeptide is m:n=(1:9)-(2:8), the degree of polymerization (m+n) is 25-200; the ice recrystallization inhibition activity of the disubstituted polypeptide at low concentration (0.5 mg / mL) is comparable to that of PVA.

[0029] In this article, unless otherwise specified, "degree of polymerization" refers to the average degree of polymerization. For example, the prepared product contains disubstituted peptides with degrees of polymerization of 22, 24, 26, 28, etc., and the average degree of polymerization is obtained by statistical calculation.

[0030] Compared with the prior art, the present invention has the following beneficial effects:

[0031] The disubstituted polypeptide provided by the present invention possesses a stable helical structure and, at varying molecular weights, exhibits similar ice recrystallization inhibition effects as polyvinyl alcohol. Furthermore, the disubstituted polypeptide, with a protein-like backbone structure, exhibits excellent biocompatibility, lacks hemolytic activity, and exhibits low cytotoxicity, suggesting promising applications as a cell cryopreservative.

[0032] 2. The above-mentioned disubstituted polypeptides can be obtained by ring-opening polymerization of N-substituted-amino acid-N-carboxylic acid anhydride five-membered ring monomers. The preparation method is simple and the polymerization activity is controllable. The prepared polypeptides have controllable molecular weight and narrow distribution.

[0033] 3. The disubstituted polypeptide described in the present invention can be used as a cell cryopreservative and can significantly improve the freezing survival rate of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 H NMR spectra (left) and C NMR spectra (right) of dimethylated peptides with different m:n ratios, 300 MHz, CD2Cl2;

[0035] Figure 2 Middle: (a) Gel permeation chromatography (GPC) of dimethylated peptides with different m:n ratios (polymerization degree m+n is 25); (b) GPC of dimethylated peptides (PNMA55) with different polymerization degrees (m:n=5:5); (c) GPC of dimethylated peptides (PNMA28) with different polymerization degrees (m:n=2:8); (d) Mass spectrometry of PNMA28 (polymerization degree m+n is 25).

[0036] Figure 3 Circular dichroism spectra of dimethylated peptides with different m:n ratios (polymerization degree m+n is 25) in aqueous solution;

[0037] Figure 4 The figure is a comparison of ice recrystallization inhibition activities of different m:n dimethyl-peptides (polymerization degree m+n is 25) and polyvinyl alcohol;

[0038] Figure 5 This is a comparison of the ice recrystallization inhibitory activities of dimethyl-peptide PNMA28 with different polymerization degrees;

[0039] Figure 6 The graph shows the hemolytic activity and cytotoxicity test of dimethyl-peptide PNMA28 (polymerization degree m+n is 25) at different concentrations;

[0040] Figure 7 This is a test chart of the cryopreservation ability of dimethylated peptide PNMA28 (polymerization degree m+n is 25) on different cells at different concentrations. DETAILED DESCRIPTION

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used herein in the specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the relevant listed items. The term "includes" or "comprising" described in the present invention means that in addition to the components described, it may also include or contain other components. The term "includes" or "comprising" described in the present invention may also be replaced by the closed form "for" or "consisting of..."

[0042] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0043] Example 1

[0044] This example involves the preparation of dimethylated peptides with different m:n ratios and different degrees of polymerization. The synthetic routes are as follows:

[0045]

[0046] The specific operations are as follows:

[0047] Synthesized according to the preparation method in the literature (Macromolecules 2023, 56, 8899–8911) N Me-(L)-Ala-NCA and N Me-(D)-Ala-NCA.

[0048] Will N Me-(L)-Ala-NCA (258.2 mg, 2.1 mmol) and N Me-(D)-Ala-NCA (258.2 mg, 2.1 mmol) was dissolved in 10.0 mL of chloroform. Then, a 2.46 mol / L acetic acid solution in chloroform (458.0 μL, 1.13 mmol) and a 0.14 mol / L benzylamine solution in chloroform (1.58 mL, 0.22 mmol) were added, and the reaction mixture was stirred at 50° C. The monomer conversion was monitored by nuclear magnetic resonance spectroscopy. When the monomer conversion was greater than 99%, the solvent was removed, the residue was redissolved in a small amount of dichloromethane, and an excess of ether was added to precipitate the polymer. The solid was collected, washed thoroughly with ether, and dried in vacuo to obtain a white solid dimethylated peptide (m:n=5:5, 210.3 mg, 48.5% yield), which was named PNMA55-25.

[0049] (1) By adjusting the molar ratio of L-configuration and D-configuration monomers, dimethylated peptides with different m:n ratios were prepared. The relevant parameters are summarized in Table 1.

[0050] Table 1

[0051]

[0052]

[0053] The nuclear magnetic resonance hydrogen and carbon spectra of dimethylated peptides with different m:n ratios are shown in Figure 2. Figure 1 As shown, Figure 2 (a) is the GPC diagram of dimethyl poly peptides with different m:n ratios. It can be seen from the figure that the molecular weight distribution of dimethyl poly peptides with different m:n ratios prepared above is narrow, and the macromolecular mass spectrometry characterization of PNMA28-25 is performed. The characterization results are as follows: Figure 2 As shown in (d), its molecular weight is close to the theoretical molecular weight.

[0054] In addition, the circular dichroism spectra of the dimethylated peptides with different m:n ratios were characterized (the circular dichroism spectra of different polymers in aqueous solution were tested at a concentration of 0.2 mg / mL). Figure 3 As shown in Figure 3, when m:n gradually changes from 5:5 to 1:9, the average residue ellipticity and helicity in the circular dichroism spectra of the corresponding dimethylated peptides gradually increase.

[0055] (2) By adjusting the molar ratio of monomer to benzylamine, PNMA55 with different degrees of polymerization were prepared. The relevant parameters are summarized in Table 2.

[0056] Table 2

[0057] Dimethylated peptide The molar ratio of monomer (total molar amount of two monomers) to benzylamine PNMA55-10 10:1 PNMA55-20 20:1 PNMA55-30 30:1 PNMA55-40 40:1 PNMA55-50 50:1 PNMA55-100 100:1 PNMA55-200 200:1

[0058] GPC diagrams of PNMA55 with different degrees of polymerization are shown in Figure 2. Figure 2 As shown in (b), it can be seen from the figure that the molecular weight distribution of PNMA55 with different polymerization degrees prepared is narrow.

[0059] (3) By adjusting the molar ratio of L-configuration and D-configuration monomers and the molar ratio of monomers to benzylamine, PNMA28 with different degrees of polymerization were prepared. The relevant parameters are summarized in Table 3.

[0060] Table 3

[0061]

[0062] GPC diagrams of PNMA28 with different degrees of polymerization are shown in Figure 2. Figure 2 As shown in (c), it can be seen from the figure that the molecular weight distribution of PNMA28 with different polymerization degrees prepared is narrow.

[0063] Example 2

[0064] The ice recrystallization inhibitory activity of the dimethylated peptide prepared in Example 1 was investigated using a splat assay. The polymer samples tested included dimethylated peptides with varying m:n ratios (all with a degree of polymerization of 25), PNMA28 with varying degrees of polymerization, and PVA. The assay was performed using a polarizing optical microscope (RX50M, Sunny) and a low-temperature stage (LRT350) cooling system. The specific procedures were as follows:

[0065] Each polymer sample was dissolved in PBS buffer at a specified concentration. 10 μL of polymer solution was added dropwise to the surface of a low-temperature silicon wafer set at -60°C. The distance between the droplet and the silicon wafer was set to 1.0 m, and the droplet instantly froze to form a thin layer of solid ice film. The ice film was then heated to -6°C at a rate of 5°C / min and maintained at this temperature for 30 minutes. An image of the ice film was captured using a digital camera connected to a microscope to obtain the ice crystal size. Image processing was performed using ImageJ software: the ten largest ice crystals were selected from each image, and the maximum length of each ice crystal in any axis was recorded. This measurement process was repeated three independent experiments, and the average value was calculated to obtain the average maximum crystallite size (MLGS). Pure PBS solution was used as a negative control, and the relative MLGS (%) was calculated by normalizing the MLGS value of the polymer-containing sample with the PBS control value. The lower the relative MLGS value, the higher the ice recrystallization inhibition (IRI) activity.

[0066] The ice recrystallization inhibitory activity test results of dimethyl-peptides with different m:n ratios (all with a degree of polymerization of 25) are as follows: Figure 4 As shown, as the degree of helicity increases, its ability to inhibit ice recrystallization gradually increases. PNMA28 and PNMA19 exhibited excellent ice recrystallization inhibitory activity at low concentrations (0.5 mg / mL), comparable to that of polyvinyl alcohol. This is attributed to the fact that helical disubstituted peptides are more rigid than non-helical secondary structures, with more separated hydrophilic and hydrophobic surfaces, which enable better adsorption to ice crystal surfaces and thus inhibit their growth.

[0067] The ice recrystallization inhibition activity test results of PNMA28 with different polymerization degrees are as follows Figure 5 As shown in the figure, with the increase of polymerization degree, the ice recrystallization inhibitory activity of PNMA28 gradually increased and reached a relatively stable state. PNMA28 with an average polymerization degree of 25-200 showed excellent ice recrystallization inhibitory activity.

[0068] Example 3

[0069] Taking PNMA28-25 as an example, we further studied the biocompatibility of this type of dimethylated peptide and its effect on cell survival when used as a cell cryopreservation agent, as follows:

[0070] (1) Hemolytic activity test

[0071] 10% sheep red blood cells were centrifuged to remove serum and diluted to a specific concentration with PBS buffer, then stored in a refrigerator at 4°C. PNMA28-25 was diluted with PBS buffer to prepare a gradient dilution of PNMA28-25. The diluted peptide solution (50 μL) was mixed with the blood cell suspension (50 μL) in a 1.5 mL centrifuge tube and stored at 4°C for 6 days. After centrifugation at 1000 rpm for 10 minutes, the absorbance of the supernatant was measured at 450 nm using a microplate reader.

[0072]

[0073] The test results are as follows Figure 5 As shown, compared with the blank sample, PNMA28 had almost no hemolytic activity at a concentration of 1.0 mg / mL.

[0074] (2) Cytotoxicity test

[0075] A549 and 293T cells were cultured in high-glucose DMEM (10% fetal bovine serum) for 48 hours (37°C, 5% CO2). PNMA28-25 was diluted with the culture medium to prepare a serial dilution of the peptide. The cell culture medium was replaced with the diluted peptide solution and the cells were incubated in an incubator for 24 hours (37°C, 5% CO2). The cytotoxicity of the polymer was assessed by measuring cell viability.

[0076] The test results are as follows Figure 6 As shown, at a concentration of 2 mg / mL, PNMA28-25 exhibited low cytotoxicity to both cell lines.

[0077] (3) Cell viability test

[0078] Cryopreservation experiments were conducted on sheep erythrocytes, A549 cells, and 293T cells using PNMA28-25 as a cryopreservative. Hydroxyethyl starch (HES) is commonly used for cryopreservation of blood cells, so PNMA28-25 was used together with HES as a cryopreservative to investigate whether the addition of PNMA28-25 could improve the survival rate of cells during cryopreservation.

[0079] For sheep red blood cells, the red blood cells were diluted with PBS to a certain concentration. A gradient dilution of the peptide cluster solution was prepared by diluting the PNMA28 solution with a PBS solution containing 215 mg / mL HES. The diluted peptide cluster solution (100 μL) was mixed with the sheep red blood cell suspension (100 μL), and the final red blood cell concentration was about 10%. The mixed solution was then immersed in liquid nitrogen and kept for 2 hours. Subsequently, the frozen red blood cell sample was quickly thawed in a 37°C water bath and the cell viability was tested.

[0080] For A549 and 293T cells, a gradient dilution of the PNMA28-25 peptide solution was prepared by diluting the peptide solution with culture medium containing 1% DMSO. The diluted peptide solution (100 μL) was mixed with the cell suspension (100 μL). The mixed cells were then placed in a gradient freezing box and stored at -80°C. Subsequently, the frozen cell samples were quickly thawed in a 37°C water bath and the cell viability was tested.

[0081] The test results are as follows Figure 7 As shown in the results, the addition of PNMA28-25 significantly increased cell survival. For red blood cells, when the concentration of PNMA28-25 was greater than 0.6 mg / mL, the recovery rate of red blood cells was significantly improved; for A549 and 293T cells, when the concentration of PNMA28-25 was greater than 1 mg / mL, the recovery rate of red blood cells was significantly improved. This shows that in the presence of this type of peptide, ice crystal growth is inhibited, thereby reducing the osmotic damage caused by the uncontrolled growth of extracellular ice crystals and the mechanical damage caused by ice crystal recrystallization during freezing and warming.

[0082] The above-described embodiments are merely preferred examples for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A double-substituted polypeptide having a helical structure, characterized in that: The disubstituted polypeptide has the following general structural formula: wherein m:n=(1:9)-(5:5), the degree of polymerization (m+n) is 10-200, and m and n are integers; R1 and R3 are independently selected from one of C1-C4 alkyl, C1-C2 hydroxyalkyl, C1-C2 fatty acid, and -RS(O)-R', R is a C1-C2 divalent alkyl, and R' is a methyl group; R2 and R4 are independently selected from one of C1-C4 alkyl groups.

2. The disubstituted polypeptide according to claim 1, characterized in that R1 and R3 are independently selected from one of C1-C4 alkyl groups.

3. The disubstituted polypeptide according to claim 2, characterized in that R1, R2, R3, and R4 are all methyl groups.

4. The disubstituted polypeptide according to claim 1 or 3, characterized in that m:n=(1:9)-(2:8).

5. The double-substituted clustered peptide according to claim 4, characterized in that The degree of polymerization (m+n) is 25-200.

6. A method for preparing the disubstituted polypeptide according to any one of claims 1 to 5, characterized in that: The following steps are involved: The monomer represented by Formula I, the monomer represented by Formula II, and benzylamine are subjected to a ring-opening polymerization reaction in the presence of an organic acid and a solvent to obtain the disubstituted polypeptide; wherein the molar ratio of the monomer represented by Formula I to the monomer represented by Formula II is (1:9)-(5:5); The structures of formula I and formula II are shown below:

7. The preparation method according to claim 6, characterized in that The organic acid is selected from one or more of acetic acid, formic acid, propionic acid, and benzoic acid, and the organic solvent is selected from one or more of chloroform, 1,2-dichloroethane, tetrahydrofuran, toluene, trifluorotoluene, fluorobenzene, and N,N-dimethylformamide.

8. The preparation method according to claim 6, characterized in that The ratio of the total molar amount of the monomer represented by formula I and the monomer represented by formula II to the molar amount of the benzylamine is (10-200):

1.

9. Use of the disubstituted polypeptide according to any one of claims 1 to 5 as a cryoprotectant.

10. The use according to claim 9, characterized in that The disubstituted polypeptide is m:n=(1:9)-(2:8), and the degree of polymerization (m+n) is 25-200.

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