A self-assembling polypeptide and its application

Abstract

The present invention belongs to the field of biomedicine technology, relates to medical hydrogels, and specifically to a self-assembling polypeptide and its application. The amino acid sequence is shown in SEQ ID NO.1. The self-assembling polypeptide provided by the present invention can not only maintain a solution state under conditions of high pH, ​​but also its acidity is more easily neutralized, thereby avoiding the impact on the pH of body fluids in the body, but also has less cytotoxicity and good cell compatibility, and at the same time has better mechanical properties.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, relates to medical hydrogel, and specifically relates to a self-assembling polypeptide and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Ionic-complementary self-assembling peptides (ISAPs) are biomaterials composed of polypeptides, and their gelation mechanism is physical self-assembly, hereinafter referred to as ISAPs or singular ISAP or polypeptide. ISAPs have a special amino acid sequence arrangement, usually composed of repeated positively charged amino acids, hydrophobic amino acids, negatively charged amino acids, and hydrophobic amino acids. For example, the classic sequence AC-RADARADARADARADA-NH2 (RADA16) is composed of polypeptides. ISAPs and their mechanical strength are determined by the sequence. The use of more hydrophobic amino acids can increase the mechanical strength of the self-assembling polypeptide gel, but it will also bring problems such as difficulty in synthesis, difficulty in purification, insolubility, and increase in the viscosity of the polypeptide solution. In the previous research of the present invention, a PP-type self-assembling polypeptide is provided. By adding proline P at both ends of the self-assembling polypeptide, not only its liquid-solid transition ability is increased, but also the difficulty of synthesis and purification is reduced, and the viscosity of the polypeptide solution is reduced. Such as AC-PRVDVRVDP-NH2 (PRVDP9).

[0004] In order to use the self-assembling polypeptide as a medical hydrogel for hemostasis, closure or embolization, or as a cell culture scaffold, it is necessary to maintain the solution state before the trigger factor is stimulated so as to facilitate the use of a catheter or the like to be transported to the wound surface or to facilitate its full mixing with the cells. However, the inventors have found that both PRVDP9 and RADA16 need to be kept in a solution state under low pH conditions (usually pH <3). If the pH value of the ISAP solution increases, the solution will become a gel or the peptide will precipitate. When it is used as a cell culture scaffold, the pH value of its solution is too low, which is not conducive to cell culture. The application scope of the assembled polypeptide as a medical hydrogel for hemostasis, closure or embolization includes surgical incision closure, hemostasis, closure of lung air leakage, closure of the meningeal dura mater, closure of vascular anastomosis and intravascular interventional embolization, etc., which need to be performed in vivo, and the pH of body fluids in vivo is relatively stable. When it is used as a medical hydrogel for hemostasis, closure or embolization, if its pH value is too low, its acidity is difficult to be neutralized, thereby affecting the pH of body fluids in vivo, which is not conducive to in vivo application. Summary of the invention

[0005] In order to address the deficiencies of the prior art, the purpose of the present invention is to provide a self-assembling polypeptide and its application, the solution prepared by which can not only maintain a solution state under conditions of high pH, ​​but also its acidity is more easily neutralized, thereby avoiding affecting the pH of body fluids in the body.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In one aspect, a self-assembling polypeptide has an amino acid sequence as shown in SEQ ID NO.1.

[0008] First, studies on several polypeptides have shown that the self-assembling polypeptides are easily soluble in water and can form good gels when mixed with DMEM culture medium, PBS buffer solution, or body fluids such as blood.

[0009] Secondly, under the concentration condition of 1% w / v (g / mL, the same below), the pH condition of the liquid-solid phase transition of the self-assembling polypeptide solution is about 6, which is closer to the pH of body fluids.

[0010] Again, it was unexpectedly discovered through research that cells can maintain good proliferation on the gel formed by the self-assembling polypeptide, indicating that the self-assembling polypeptide has good cell compatibility and biocompatibility.

[0011] From this, it was found through experiments that the gel formed by the above self-assembling polypeptide has better mechanical properties compared with the PP-type self-assembling polypeptide and the classic RADA16 studied previously.

[0012] In another aspect, a polypeptide composition comprises the self-assembling polypeptide and a pharmaceutical excipient.

[0013] In a third aspect, a polypeptide preparation contains the self-assembling polypeptide described above, and the preparation is in the form of a powder or a solution.

[0014] A fourth aspect is a use of the self-assembling polypeptide, polypeptide composition or polypeptide preparation in the preparation of a medical hydrogel.

[0015] A fifth aspect is a use of the self-assembling polypeptide, polypeptide composition or polypeptide preparation in cultured cells.

[0016] A sixth aspect is a use of the self-assembling polypeptide, polypeptide composition or polypeptide preparation in the preparation of a cell culture scaffold.

[0017] The beneficial effects of the present invention are:

[0018] 1. The self-assembling polypeptide provided by the present invention can maintain a solution state at a concentration of 1% w / v and a pH value of <6.0.

[0019] 2. When the 2.5% w / v peptide solution formed by the self-assembling polypeptide provided by the present invention is mixed with DMEM at a volume ratio of 1:1, the pH value can reach about 6, indicating that it is more easily neutralized by the cell culture medium.

[0020] 3. The peptide gel formed by the self-assembling polypeptide provided by the present invention can maintain good cell proliferation, has less cytotoxicity and good cell compatibility.

[0021] 4. The self-assembling polypeptide provided by the present invention has good liquid-solid phase transition ability and better mechanical properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0023] Figure 1 : is a high performance liquid chromatogram of PRRVDP13 prepared in an embodiment of the present invention;

[0024] Figure 2 is a mass spectrum of PRRVDP13 prepared in an embodiment of the present invention;

[0025] Figure 3 This is a graph showing the pH value of a test in which the polypeptide solution and Dulbecco's Modified Eagle Medium (DMEM) cell culture medium were mixed in a certain volume ratio in an embodiment of the present invention, n=6,

[0026] ***P < 0.001;

[0027] Figure 4 These are pictures of the survival of cells cultured on different polypeptide surfaces in the embodiments of the present invention, A is RADA16, B is PRVDP9, C is PRRVDP13, and D is a control (a culture plate without polypeptide);

[0028] Figure 5 This is a bar graph of the results of cytotoxicity detection in the examples of the present invention, n=5, ***p<0.001;

[0029] Figure 6The PRRVDP13 polypeptide solution in the embodiment of the present invention can be mixed with the DMEM medium in equal volumes. A is a cell suspension added to a 2.5% w / v PRRVDP13 polypeptide solution. Cell photograph, B is a cell photograph of a 2.5% w / v PRRVDP13 polypeptide solution added to a cell suspension, C is a cell suspension added to a 1.25% w / v PRRVDP13 polypeptide solution. Cell photograph, D is a cell photograph of a 1.25% w / v PRRVDP13 polypeptide solution added to a cell suspension, E is a fluorescent staining picture of a cell suspension added to a 2.5% w / v PRRVDP13 polypeptide solution (Calcein-AM, PI (Biyuntian, C2015S)), 10x objective (scale is 100 μm), F is a cell suspension added to 2.5% w / v Fluorescence staining image of PRRVDP13 peptide solution (Calcein-AM, PI (Biyuntian, C2015S)), 5x objective (scale bar is 200μm);

[0030] Figure 7 It is a characterization diagram of the mechanical properties of the polypeptides in the embodiments of the present invention, A is the storage modulus and energy loss modulus of RADA16, B is the storage modulus and energy loss modulus of PRVDP9, C is the storage modulus and energy loss modulus of PRRVDP13, D is the shear viscosity of RADA16, E is the shear viscosity of PRVDP9, and F is the shear viscosity of PRRVDP13.

[0031] Figure 8 These are pictures of the liquid (left) and solid (right) states of the polypeptide PRRVDP13 in an embodiment of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0034] In view of the problem that the existing self-assembling polypeptides have low acidity and are difficult to be neutralized, which is not conducive to cell culture and in vivo use, the present invention provides a self-assembling polypeptide and its application.

[0035] A typical embodiment of the present invention provides a self-assembling polypeptide, whose amino acid sequence is shown in SEQ ID NO.1.

[0036] In some embodiments, the amino group at the N-terminus is modified to an acetamide group (CH3CONH-).

[0037] In some embodiments, the carboxyl group at the C-terminus is modified to an amide group (-CONH2).

[0038] Another embodiment of the present invention provides a polypeptide composition, comprising the above-mentioned self-assembling polypeptide and pharmaceutical excipients.

[0039] The pharmaceutical excipients described in the present invention include, for example, pure water, alkaline solutions containing NaHCO3 or NaOH, buffer solutions, osmotic pressure-adjusted solutions containing sucrose or mannitol, cell culture media, and the like.

[0040] A third embodiment of the present invention provides a polypeptide preparation, which contains the self-assembling polypeptide described above, and the preparation is in the form of a powder or a solution.

[0041] When the powder is used, it is added to a solution such as pure water, an alkaline solution containing NaHCO3 or NaOH, a buffer solution, pure water, an alkaline solution containing NaHCO3 or NaOH, a cell culture medium, etc. to dissolve.

[0042] In some embodiments, the concentration of the self-assembling polypeptide in the solution is 0.1-5% w / v; preferably 0.1-1.25% w / v; preferably 1.25-5% w / v.

[0043] In some embodiments, in the solution, the concentration of the self-assembling polypeptide is 1% w / v, and the pH value is less than 6.29; preferably 2.50 to 3.50; preferably 5.50 to 6.28.

[0044] A fourth embodiment of the present invention provides a use of the self-assembling polypeptide, polypeptide composition or polypeptide preparation in preparing a medical hydrogel. Specifically, the medical hydrogel is used for hemostasis and / or sealing body fluids.

[0045] A fifth embodiment of the present invention provides a use of the above self-assembling polypeptide, polypeptide composition or polypeptide preparation in cultured cells.

[0046] A sixth embodiment of the present invention provides a use of the self-assembling polypeptide, polypeptide composition or polypeptide preparation in preparing a cell culture scaffold.

[0047] A seventh embodiment of the present invention provides a method for hemostasis, wherein a solution containing the self-assembling polypeptide is applied to a bleeding site of a wound.

[0048] An eighth embodiment of the present invention provides a method for culturing cells, comprising mixing a cell suspension with the self-assembling polypeptide to form a gel, and then adding a culture medium for culturing.

[0049] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0050] Example

[0051] Synthesis of PRRVDP13 (AC-PRVDVRVRVRVDP-NH2, as shown in SEQ ID NO.1):

[0052] 1. Materials

[0053] Fmoc-Pro-OH (N-fluorenylmethoxycarbonyl-proline), Fmoc-Val-OH (N-fluorenylmethoxycarbonyl-valine), Fmoc-Arg(pbf)-OH (N-fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-arginine), and Fmoc-Asp(OtBu)-OH (N-fluorenylmethoxycarbonyl-4-tert-butyl ester-aspartic acid) were purchased from Shanghai Jier Biochemical Co., Ltd., RinkAmide-AM Resin (resin) was purchased from Xi'an Lanxiao Technology New Materials Co., Ltd., DIC (N,N'-diisopropylcarbodiimide), HOBT (1-hydroxybenzotriazole), and DIEA (N,N-diisopropylethylamine) were purchased from Suzhou Haofan Biotechnology Co., Ltd. PIPE (piperidine), acetic anhydride, DMF (N,N-dimethylformamide), TFA (trifluoroacetic acid), methyl tert-butyl ether, methanol and DCM (dichloromethane) were purchased from Sinopharm Chemical Reagent Co., Ltd.

[0054] 2. Synthesis Method

[0055] The Fmoc solid phase synthesis method was used. First, 2.10 g of Rink Amide-AM resin was weighed and soaked in 20 mL of DCM for one hour, then the DCM was removed by filtration under reduced pressure and washed twice with DMF. Then 20 mL of 20% v / v PIPE / DMF solution was added to remove the Fmoc protecting group, and after 10 minutes, the solution was filtered under reduced pressure and the process was repeated once. The resin was then washed with DMF six times. The removal of the protecting group of the resin was detected using ninhydrin.

[0056] Then, weigh 3.04g Fmoc-Pro-OH and 1.34g HOBT, add 15 ml DMF to dissolve, add 1.25g DIC to activate, add to the reactor, and react for 2 hours. Take a small amount of resin to check whether the connection is complete. After the connection is complete, filter under reduced pressure and use DMF to wash the resin six times. Then add 20% v / v PIPE / DMF solution to remove the amino acid Fmoc protecting group.

[0057] Repeat the above steps for each amino acid from the C-terminus to the N-terminus. After all amino acids are connected, remove the protecting group of the last amino acid and wash the resin. Then add 5.7mL acetic anhydride, 10.5mL DIEA and 10mL DCM to react for 30 minutes to acetylate the N-terminus. After the reaction is completed, the resin is repeatedly rinsed with DMF, DCM and methanol and vacuum dried overnight.

[0058] Add the dried resin to 30 mL of pre-cooled cutting reagent and stir the reaction at room temperature for 2.5 hours. After the reaction is completed, reduce the pressure and filter, and slowly drip the filtrate into pre-cooled methyl tert-butyl ether to produce a white precipitate. Collect the precipitate by centrifugation and dry it in a vacuum to obtain a crude peptide powder. Use a high-performance liquid chromatography to purify the crude peptide powder and collect the target peak; or use a high-performance liquid chromatography to convert it into acetate and collect the target peak; or use a high-performance liquid chromatography to convert it into hydrochloride and collect the target peak. Finally, after concentration and lyophilization, the pure product, namely PRRVDP13, can reach a purity of 97%. Figures 1-2 shown.

[0059] According to the above synthesis method, by changing the amino acids and amino acid ratios, the following peptides were synthesized:

[0060] AC-RADARADARADARADA-NH2 (RADA16, as shown in SEQ ID NO. 2);

[0061] AC-PRVDVRVDP-NH2 (PRVDP9, as shown in SEQ ID NO. 3);

[0062] AC-KLNLKLNLNL-NH2 (as shown in SEQ ID NO.4);

[0063] AC-PRVDVRVKVRVDP-NH2 (as shown in SEQ ID NO.5);

[0064] AC-RADMSRADMS-NH2 (as shown in SEQ ID NO.6).

[0065] Unless otherwise specified, the solution in this example refers to an aqueous solution, and water is purified water or water for injection. This example is based on the AC-PRVDVRVDP-NH2 provided previously, and screened AC-KLNLKLNLNL-NH2, AC-PRVDVRVKVRVDP-NH2 and AC-RADMSRADMS-NH2. The above polypeptides were prepared into 1mL of 2.5% w / v solution, and 100μL of 10-fold concentration PBS buffer was added and mixed thoroughly so that the final solution was approximately 1x PBS. Or an equal volume of DMEM was added and mixed to trigger gelation. It was found that only AC-PRVDVRVKVRVDP-NH2 was easily dissolved and formed a good gel when mixed with DMEM or PBS. Based on this, similar sequences with liquid-solid phase transition ability were further designed.

[0066] AC-PRVDVRVRVRVDP-NH2(PRRVDP13). At a concentration of 1% w / v, the pH of the two peptide solutions can be pre-adjusted to a certain level without the peptide solutions turning into gels for several hours by adding 1% w / v NaHCO3 dropwise and shaking. However, when the concentration is 2% w / v, the peptide solution quickly turns into a gel when the pH value rises to about 3 by adding 1% w / v NaHCO3 dropwise and shaking. The classic sequence RADA16 and the original sequence PRVDP9 need to maintain their acidic conditions even at a lower concentration of 1% w / v to maintain the solution state. Otherwise, the solution turns into a gel and the color turns white as the pH value increases. The experimental results are shown in Table 1.

[0067] Table 1

[0068]

[0069] One potential application of ISAPs is to use them as scaffolds to support 3D cell culture. To verify whether they can be used as scaffolds for 3D cell culture, L929 cells (purchased from the Kunming Cell Bank of the Chinese Academy of Sciences) that have been cultured for 48-72 hours and are growing vigorously were digested and added to serum-containing DMEM medium and mixed and counted. 5 / mL cell suspension. The cell suspension and polypeptide are mixed in a certain ratio (60μL: 60μL), added to a 96-well culture plate, and after gelation, 120μL of serum-containing DMEM medium is added for culture. In this example, the ISAPs solution is mixed with a cell suspension or a cell culture medium to study biocompatibility.

[0070] As mentioned above, peptide solutions with relatively high concentrations (more than 2% w / v) cannot be pre-adjusted to a neutral pH before mixing with the cell suspension, and the acidity may be harmful to the cells. However, unexpectedly, the acidity of the 2.5% w / v peptide solution of PRRVDP13 was easily neutralized by the cell culture medium after mixing with DMEM, while the control solutions of PRVDP9 and RADA16 were relatively more difficult to neutralize. Figure 3 As shown, at the same volume ratio, the pH value of 2.5% w / v PRRVDP13 mixed with DMEM medium was significantly higher than that of 2.5% w / v PRVDP9 and RADA16, and the results were statistically significant. As a result, although the 2.5% concentration solution is acidic, PRRVDP13 may still be used as a cell culture scaffold. The experimental method is to mix 1.5mL of 2.5% w / v self-assembling polypeptide with 1.5mL of DMEM medium in a centrifuge tube, add culture medium after gelation, so that the total volume reaches 6mL, and the total ratio of polypeptide to culture medium is 1:3. Or mix 1.5mL of 2.5% self-assembling polypeptide with 1.5mL of DMEM medium to make the ratio of polypeptide to DMEM medium 1:1. Then detect the pH value of each group.

[0071] To test the biocompatibility of ISAP, 60 μL of 2.5% w / v peptide solution was mixed with an equal volume of DMEM medium in a cell culture plate to form a gel. 5 A suspension of L929 cells at 100 / mL was loaded onto the preformed gel. The cells maintained good proliferation on the peptide gel of PRRVDP13, but not on the peptide gels of other sequences, such as Figures 4-5 As shown, the results show that PRRVDP13 has very good cell compatibility, is very easy to operate, and can be used at relatively high concentrations. Cell proliferation detection was performed in a cell culture incubator with a 5% CO2 environment for about 48 hours, and the test method was CCK-8 kit (Biyuntian, C0038). For the peptide PRRVDP13, the cell suspension can be directly mixed with the peptide solution, and then the cells are evenly distributed in the gel space after the liquid-solid phase transition. The method is: take the above 1×10 5 60 μL of L929 cell suspension with a concentration of 100 μg / mL and 60 μL of PRRVDP13 peptide were added to a 96-well culture plate. After gelation, 120 μL of culture medium was added for culture. After culturing in a cell culture incubator with a 5% CO2 environment for 48 hours, the cells were observed under a microscope, or stained with Calcein-AM, PI (Biyuntian, C2015S) according to the instructions, and the cell survival status was observed under a fluorescence microscope. The results showed that most of the cells survived, such as Figure 6 shown.

[0072] like Figure 8As shown, the PRRVDP13 peptide solution exhibited good liquid-solid phase transition ability. The experimental method was to prepare the peptide into 1 mL of 2.5% w / v solution, add 100 μL of 10-fold concentrated PBS buffer, and mix thoroughly so that the final solution was approximately 1-fold PBS to trigger gelation. Gel strength is related to hemostatic ability and is usually expressed by storage modulus G'. Compared with RADA16 and PRVDP9, PRRVDP13 has better mechanical properties, such as Figure 7 A- Figure 7 C. The experimental method is as follows: 3 mL of 2.5% w / v peptide solution was prepared with injection water, 600 μL of 5-fold concentrated PBS buffer was added thereto to trigger gel formation, an appropriate amount of gel was added to the rheometer test bench, the stress of the rheometer was set to 1 Pa, the scanning frequency range was 0.1-10 Hz, and the storage modulus G' and loss modulus G" of the gel were measured. In order to detect the viscosity of the peptide solution, each peptide was prepared into a solution with a concentration of 2.5% w / v with injection water, and the rheometer was used at a shear rate of 0.001S -1 ~1000S -1 Peristaltic scanning was performed under the condition of , and the viscosity data of each polypeptide solution was obtained, as shown in Figure 7 D- Figure 7 F.

[0073] The PRRVDP13 peptide solution showed a good hemostatic effect. The animals (New Zealand rabbits) were anesthetized by injection of 2% sodium pentobarbital through the ear vein, fixed on the operating table in a supine position, the rabbit hair on the abdomen was cut off, disinfected, and a standard midline laparotomy was performed to free the liver, expose the left lobe of the liver, and place sterile gauze underneath. A wound with a length of 1.0 cm and a depth of 0.3 cm was made on the liver lobe. The exposed wound was wiped with gauze, and the test sample (about 1 mL) was injected deep into the wound until the entire wound site was covered. The hemostasis was observed and the hemostasis time was recorded. The results showed that a 2.5% w / v aqueous solution of the self-assembling polypeptide PRRVDP13, or a 1.25% w / v self-assembling polypeptide (the solution was 1 mg / mL of NaHCO3) achieved a complete hemostasis effect within 50 seconds and formed a solid gel. After 2 minutes of hemostasis, the polypeptide hydrogel on the surface was washed off with saline, and no new bleeding was formed. The normal saline control group rinsed the wound with normal saline. The bleeding time of the control group was more than 4 minutes.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A self-assembling polypeptide, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

2. The self-assembling polypeptide according to claim 1, characterized in that: The amino group at the N-terminus was modified to an acetamide group.

3. The self-assembling polypeptide according to claim 1, characterized in that: The carboxyl group at the C-terminus was modified to an amide group.

4. A polypeptide composition, characterized in that: The invention comprises the self-assembling polypeptide according to any one of claims 1 to 3 and pharmaceutical excipients.

5. A polypeptide preparation, characterized in that: The self-assembling polypeptide comprises any one of claims 1 to 3, and the preparation form is powder or solution.

6. The polypeptide preparation according to claim 5, characterized in that: In the solution, the concentration of the self-assembling polypeptide is 0.1-5% w / v.

7. The polypeptide preparation according to claim 5, characterized in that: In the solution, the concentration of the self-assembling polypeptide is 1% w / v, and the pH value is less than 6.

29.

8. Use of the self-assembling polypeptide according to any one of claims 1 to 3, the polypeptide composition according to claim 4, or the polypeptide preparation according to any one of claims 5 to 7 in the preparation of medical hydrogel.

9. Use of the self-assembling polypeptide according to any one of claims 1 to 3, the polypeptide composition according to claim 4, or the polypeptide preparation according to any one of claims 5 to 7 in cultured cells.

10. Use of the self-assembling polypeptide according to any one of claims 1 to 3, the polypeptide composition according to claim 4, or the polypeptide preparation according to any one of claims 5 to 7 in the preparation of a cell culture scaffold.

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