Bioactive peptide for promoting corneal re-epithelialization, preparation method, hydrogel preparation and application

Abstract

The application relates to a corneal re-epithelialization-promoting self-assembled bioactive peptide, a preparation method and a hydrogel preparation and application thereof. The self-assembled bioactive peptide can be synthesized in one step through a solid-phase synthesis method, and has the advantages of high yield and simple and rapid preparation. The hydrogel preparation made of the self-assembled bioactive peptide has the advantages of simple preparation, rapid gel formation, efficient promotion of corneal re-epithelialization from the aspects of morphology and structure, improvement of in-vivo therapeutic effect against corneal damage, and the like, and is not easily degraded by proteases in tears, and has good chemical stability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fibronectin mimetic peptides, in particular to a self-assembled bioactive peptide for promoting corneal re-epithelialization, a preparation method, a hydrogel preparation and an application thereof. BACKGROUND

[0002] Fibronectin, also known as fibronectin, is a glycoprotein that exists in large quantities in the extracellular matrix and also exists in plasma. As an adhesion protein, fibronectin plays an indispensable role in cell adhesion, migration, growth and differentiation through interaction with integrin receptors. Studies have shown that fibronectin has at least two cell adhesion sites, including arginine-glycine-aspartic acid (RGD) sequence and proline-histidine-serine-arginine-asparagine (PHSRN) sequence, wherein PHSRN is a synergistic ligand that enhances the cell adhesion ability of RGD. Nishida et al. found that exogenous PHSRN can effectively enhance the motility of corneal epithelial cells, thereby stimulating the migration of corneal epithelial cells in a concentration-dependent manner, which strongly proves that PHSRN peptide mimics the biological activity of fibronectin and is a promising therapeutic agent for promoting corneal wound healing; however, this water-soluble PHSRN is easily degraded by proteases in tears after local instillation, which seriously hinders its clinical application. Therefore, only by improving the chemical stability of PHSRN can this mimetic peptide sequence be widely applied and achieve its great clinical value.

[0003] In recent years, polypeptides and their derivatives have been attracting increasing attention in the fields of nanomedicine, tissue engineering and tissue regeneration due to their good in vivo biocompatibility and safety. Self-assembling polypeptides can self-assemble under a variety of stimuli to form different nanostructures (such as nanotubes and nanofibers), and can greatly prolong the pre-corneal retention time after local instillation, thereby improving their bioavailability. SUMMARY

[0004] In order to solve the defects and deficiencies of the existing mimetic peptide sequence (PHSRN) with low chemical stability, the present application provides a self-assembled bioactive peptide for promoting corneal re-epithelialization, a preparation method, a hydrogel preparation and an application thereof.

[0005] The technical solution adopted by the present application is that the self-assembled bioactive peptide for promoting corneal re-epithelialization comprises or consists of the following items:

[0006] (1) a self-assembled bioactive peptide Nap-FPHSRN obtained by linking a self-assembling polypeptide module Nap-Phe and a fibronectin mimetic peptide sequence (PHSRN) through an amide bond;

[0007] (2) The self-assembling bioactive peptide Nap-FFPHSRN was obtained by linking the self-assembling peptide module Nap-Phe-Phe with the fibronectin mimetic peptide sequence (PHSRN) through an amide bond;

[0008] (3) The self-assembling bioactive peptide Nap-FFFPHSRN was obtained by linking the self-assembling polypeptide module Nap-Phe-Phe-Phe with the fibronectin mimetic peptide sequence (PHSRN) through amide bonds.

[0009] The amino acid sequence of the self-assembling bioactive peptide Nap-FPHSRN is SEQ ID NO: 1.

[0010] The amino acid sequence of the self-assembling bioactive peptide Nap-FFPHSRN is SEQ ID NO: 2.

[0011] The amino acid sequence of the self-assembling bioactive peptide Nap-FFFPHSRN is SEQ ID NO: 3.

[0012] The polypeptide sequences of the self-assembling bioactive peptides Nap-FPHSRN, Nap-FFPHSRN, and Nap-FFFPHSRN are as follows: dinaphthylacetic acid-phenylalanine-proline-histidine-serine-arginine-asparagine, dinaphthylacetic acid-phenylalanine-phenylalanine-proline-histidine-serine-arginine-asparagine, and dinaphthylacetic acid-phenylalanine-phenylalanine-phenylalanine-proline-histidine-serine-arginine-asparagine, and the structural formulas include:

[0013]

[0014] A method for preparing a self-assembling bioactive peptide that promotes corneal re-epithelialization is characterized in that the self-assembling bioactive peptide is synthesized by a solid-phase synthesis method, and the specific steps are as follows: taking a dichloro resin and placing it in a solid-phase reactor, adding dichloromethane for swelling, and sequentially adding asparagine, arginine, serine, histidine, proline, phenylalanine, phenylalanine and dinaphthylacetic acid; or sequentially adding asparagine, arginine, serine, histidine, proline, phenylalanine, phenylalanine and dinaphthylacetic acid; or sequentially adding asparagine, arginine, serine, histidine, proline, phenylalanine, phenylalanine, phenylalanine and dinaphthylacetic acid, and extending the peptide chain until the final product, the self-assembling bioactive peptide, is obtained.

[0015] The asparagine, arginine, serine, histidine, proline and phenylalanine are all protected by Fmoc.

[0016] A hydrogel preparation prepared by using self-assembling bioactive peptides is prepared by the following steps: dispersing the self-assembling bioactive peptides in a phosphate buffer solution, adding a weak alkaline aqueous solution, ultrasonically dissolving, and standing to obtain the self-assembling bioactive peptide hydrogel preparation.

[0017] The weak base aqueous solution is one or more of a carbonate solution, a phosphate solution, an acetate solution, and a borate solution. Preferably, the weak base aqueous solution is a sodium carbonate aqueous solution.

[0018] The weight percentage of the self-assembling bioactive peptide in the hydrogel preparation is 0.5% to 2%.

[0019] The pH value of the phosphate buffer solution is 7.4.

[0020] Application of a hydrogel preparation in preparing corneal wound healing eye drops

[0021] The present invention provides a self-assembling bioactive peptide that promotes corneal re-epithelialization, a preparation method, a hydrogel formulation, and applications thereof. The self-assembling bioactive peptide can be synthesized in a single step via solid-phase synthesis, resulting in high yield and simple and rapid preparation. The hydrogel formulation made from the self-assembling bioactive peptide has the advantages of simple preparation, rapid gelation, and effectively promotes corneal re-epithelialization from a morphological and structural perspective, improving the in vivo efficacy of clinical treatments against corneal damage. Furthermore, the hydrogel formulation is not easily degraded by proteases in tears and exhibits good chemical stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the structural formula of the self-assembled bioactive peptide (taking Nap-FFPHSRN as an example).

[0023] Figure 2 This is the NMR spectrum of the self-assembled bioactive peptide (taking Nap-FFPHSRN as an example).

[0024] Figure 3 This is the mass spectrum of the self-assembled bioactive peptide (taking Nap-FFPHSRN as an example).

[0025] Figure 4 Transmission electron microscopy image of self-assembled bioactive peptide hydrogel (taking Nap-FFPHSRN as an example).

[0026] Figure 5 This is the in vitro chemical stability curve of the self-assembled bioactive peptide. DETAILED DESCRIPTION

[0027] The present invention is further described below with reference to the accompanying drawings and the following embodiments. It should be understood that the accompanying drawings and the following embodiments are only used to illustrate the present invention, rather than to limit the present invention.

[0028] Example 1 Synthesis of self-assembling bioactive peptides (taking Nap-FFPHSRN as an example):

[0029] 1 g of dichlororesin (degree of substitution 1-1.1 mmol) was placed in a solid phase reactor, 5 mL of dichloromethane was added to swell for 15 minutes, and 1.5 mmol of Fmoc-protected asparagine (N), 1.5 mmol of Fmoc-protected arginine (R), 1.5 mmol of Fmoc-protected serine (S), 1.5 mmol of Fmoc-protected histidine (H), 1.5 mmol of Fmoc-protected proline (P), 1.5 mmol of Fmoc-protected phenylalanine (F), 1.5 mmol of Fmoc-protected phenylalanine (F) and 1.5 mmol of dinaphthylacetic acid (Nap) were added in sequence to extend the peptide chain until the final product Nap-FFPHSRN was obtained. The NMR spectrum of Nap-FFPHSRN is shown in FIG. Figure 2 As shown, the mass spectrum of Nap-FFPHSRN is as follows Figure 3 As shown, the molecular formula of the product can be verified by NMR and mass spectrometry.

[0030] Example 2: 0.5% wt Nap-FFPHSRN hydrogel was prepared using phosphate buffer solution as solvent:

[0031] 5 mg of Nap-FFPHSRN was accurately weighed and dispersed in 0.9 mL of phosphate buffer solution. 0.1 mL of sodium carbonate aqueous solution (1 equivalent to Nap-FFPHSRN) was added, and the mixture was dissolved by ultrasonication. The mixture was allowed to stand to obtain a 0.5% wt Nap-FFPHSRN hydrogel system.

[0032] Example 3: 1% wt Nap-FFPHSRN hydrogel was prepared using phosphate buffer solution as solvent:

[0033] 10 mg of Nap-FFPHSRN was accurately weighed and dispersed in 0.9 mL of phosphate buffer solution. 0.1 mL of sodium carbonate aqueous solution (1 equivalent to Nap-FFPHSRN) was added, and the mixture was dissolved by ultrasonication. The mixture was allowed to stand to obtain a 1% wt Nap-FFPHSRN hydrogel system.

[0034] Example 4: 2% wt Nap-FFPHSRN hydrogel was prepared using phosphate buffer solution as solvent:

[0035] 20 mg of Nap-FFPHSRN was accurately weighed and dispersed in 0.9 mL of phosphate buffer solution. 0.1 mL of sodium carbonate aqueous solution (1 equivalent to Nap-FFPHSRN) was added, and the mixture was dissolved by ultrasonication. The mixture was allowed to stand to obtain a 2% wt Nap-FFPHSRN hydrogel system.

[0036] Example 5 In vitro chemical stability of self-assembled bioactive peptides:

[0037] In 10 mL glass bottles, 1.6 mM PHSRN and self-assembled bioactive peptides (Nap- FPHSRN, Nap-FFPHSRN, Nap-FFFPHSRN) phosphate buffer solutions with a volume of 1 mL were prepared, respectively. Then, 5 x 10 -4 wt% protease K phosphate buffer solution with a volume of 1 mL was added to each self-assembled bioactive peptide solution, respectively, and incubated in a 37 °C air bath. Samples were taken at 0 min, 5 min, 30 min, 2 h, 4 h, 8 h and 12 h time points for quantitative detection of the content of self-assembled bioactive peptides therein. As shown in Figure 2, the presence of PHSRN was not detected after 5 min, while the chemical stability of self-assembled bioactive peptides (Nap-FPHSRN, Nap-FFPHSRN, Nap-FFFPHSRN) was significantly improved. After 4 h of co-incubation, the remaining contents of Nap-FPHSRN and Nap-FFPHSRN were 7.31 ± 0.64% and 16.77 ± 3.76%, respectively; while even after 12 h of co-incubation, the remaining content of Nap-FFFPHSRN remained at 33.76 ± 4.96%. Figure 5

[0038] Those skilled in the art should know that although the present application has been described in the above specific embodiments, the inventive idea of the present application is not limited to this application, and any modification using the inventive idea of the present application will be included in the scope of protection of the present patent.

[0039] The above is only the preferred embodiment of the present application, and the protection scope of the present application is not limited to the above examples. Any technical solution falling within the scope of the inventive idea of the present application shall be included in the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principle of the present application shall also be considered as falling within the protection scope of the present application. SEQUENCE LISTING <110> Wenzhou Medical University Affiliated Eye Optometry Hospital <120> Self-assembled bioactive peptides for promoting corneal re-epithelialization, preparation method, hydrogel preparation and application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence​ <400> 1 Xaa Phe Pro His Ser Arg Asn 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <400> 2 Xaa Phe Phe Pro His Ser Arg Asn 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <400> 3 Xaa Phe Phe Phe Pro His Ser Arg Asn 1 5

Claims

1. A self-assembling bioactive peptide that promotes corneal re-epithelialization, characterized in that: Contains or consists of: (1) The self-assembling bioactive peptide Nap-FPHSRN is obtained by linking the self-assembling peptide module Nap-Phe with the fibronectin mimetic peptide sequence PHSRN through an amide bond; (2) The self-assembling bioactive peptide Nap-FFPHSRN is obtained by linking the self-assembling peptide module Nap-Phe-Phe with the fibronectin mimetic peptide sequence PHSRN through an amide bond; (3) The self-assembling bioactive peptide Nap-FFFPHSRN was obtained by linking the self-assembling peptide module Nap-Phe-Phe-Phe with the fibronectin mimetic peptide sequence PHSRN through an amide bond; The polypeptides of the self-assembling bioactive peptides Nap-FPHSRN, Nap-FFPHSRN and Nap-FFFPHSRN are: dinaphthylacetic acid-phenylalanine-proline-histidine-serine-arginine-asparagine, dinaphthylacetic acid-phenylalanine-phenylalanine-proline-histidine-serine-arginine-asparagine and dinaphthylacetic acid-phenylalanine-phenylalanine-phenylalanine-proline-histidine-serine-arginine-asparagine.

2. A method for preparing the self-assembling bioactive peptide for promoting corneal re-epithelialization according to claim 1, characterized in that: The self-assembling bioactive peptide is synthesized by a solid-phase synthesis method, and the specific steps are as follows: taking a dichloro resin and placing it in a solid-phase reactor, adding dichloromethane to swell it, and sequentially adding asparagine, arginine, serine, histidine, proline, phenylalanine, phenylalanine and dinaphthylacetic acid; or sequentially adding asparagine, arginine, serine, histidine, proline, phenylalanine, phenylalanine and dinaphthylacetic acid; or sequentially adding asparagine, arginine, serine, histidine, proline, phenylalanine, phenylalanine, phenylalanine and dinaphthylacetic acid, and extending the peptide chain until the final product, the self-assembling bioactive peptide, is obtained.

3. The preparation method according to claim 2, characterized in that The asparagine, arginine, serine, histidine, proline and phenylalanine are all protected by Fmoc.

4. A hydrogel preparation prepared using the self-assembling bioactive peptide according to claim 1, characterized in that: The hydrogel preparation is prepared by the following steps: dispersing the self-assembling bioactive peptide in a phosphate buffer solution, adding a weak alkaline aqueous solution, ultrasonically dissolving, and standing to obtain the self-assembling bioactive peptide hydrogel preparation.

5. The hydrogel preparation according to claim 4, characterized in that The weak alkaline aqueous solution is one or more of carbonate solution, phosphate solution, acetate solution and borate solution.

6. The hydrogel preparation according to claim 4, characterized in that The weight percentage of the self-assembling bioactive peptide in the hydrogel preparation is 0.5% to 2%.

7. Use of the hydrogel preparation according to claim 4 in preparing corneal wound healing eye drops.

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