WO2020211504A1 - Hydrogel polypeptidique et son procédé de préparation - Google Patents

Hydrogel polypeptidique et son procédé de préparation Download PDF

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Publication number
WO2020211504A1
WO2020211504A1 PCT/CN2020/073061 CN2020073061W WO2020211504A1 WO 2020211504 A1 WO2020211504 A1 WO 2020211504A1 CN 2020073061 W CN2020073061 W CN 2020073061W WO 2020211504 A1 WO2020211504 A1 WO 2020211504A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
hydrogel
preparation
self
peptide
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Application number
PCT/CN2020/073061
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English (en)
Chinese (zh)
Inventor
汪少芸
陈惠敏
何庆燕
Original Assignee
福州大学
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Publication date
Application filed by 福州大学 filed Critical 福州大学
Publication of WO2020211504A1 publication Critical patent/WO2020211504A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/02—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques
    • C08J3/03—Making solutions, dispersions, lattices or gels by other methods than by solution, emulsion or suspension polymerisation techniques in aqueous media
    • C08J3/075—Macromolecular gels
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2389/00—Characterised by the use of proteins; Derivatives thereof

Definitions

  • the invention belongs to the technical field of biological materials, and specifically relates to a polypeptide hydrogel and a preparation method thereof.
  • Self-assembled peptide hydrogels are widely used in tissue engineering, drug delivery, cell culture and other fields due to their good biocompatibility, reversibility and degradability.
  • the types of peptides that can self-assemble into hydrogels include amino acid paired peptides, ⁇ -hairpin peptides, Fluorenylmethoxycarbonyl (Fmoc) peptides and amphiphilic peptides, but these peptides that can self-assemble into hydrogels have longer sequences. Often contain synthetic non-degradable end groups. The self-assembly of natural oligopeptides (4-8 amino acids) has received less research and attention.
  • Engineering peptide-based hydrogels introduce functional molecules into some natural peptide fragments to make the hydrogel have certain specific functions, but most of the engineering peptide-based hydrogels are now designed based on the original self-assembled peptide system The design is limited by the above modification, so it is necessary and meaningful to develop a new self-assembled peptide system.
  • the purpose of the present invention is to overcome the existing research ideas and methods of polypeptide hydrogels at home and abroad in view of the shortcomings of the prior art, and provide a polypeptide hydrogel and a preparation method thereof.
  • the polypeptide sequence of the polypeptide hydrogel is short in length, the gel forming method is simple and rapid, and the obtained polypeptide hydrogel has good biocompatibility.
  • a self-assembled polypeptide to form a hydrogel its amino acid sequence is: Glu-Ile-Trp-Leu-Lys.
  • a polypeptide hydrogel is a hydrogel formed by adjusting the pH and concentration of a polypeptide solution formed by self-assembled polypeptides to form a hydrogel.
  • a method for preparing a polypeptide hydrogel the specific steps are: the polypeptides that self-assemble to form a gel are prepared into a polypeptide solution of a certain concentration in a PBS buffer of a certain pH, vortexed until the polypeptide powder is dissolved, and allowed to stand at room temperature for condensation Gelatinize.
  • the pH range of the PBS buffer in the above preparation method is: pH 5-8.
  • the pH of the PBS buffer in the above preparation method is 7.
  • the concentration range of the polypeptide solution in the above preparation method is: 7.27-14.54 mM.
  • the standing time in the above preparation method is: 30 min.
  • the significant advantages of the present invention are: a polypeptide hydrogel provided by the present invention and a preparation method thereof, the polypeptide sequence of the polypeptide self-assembled to form the hydrogel is short; the preparation method of the polypeptide hydrogel is simple and rapid; the obtained polypeptide
  • the hydrogel has good biocompatibility and reversibility; it provides flexibility for the chemical design of preparing peptide-based hydrogels, provides simplicity for peptide synthesis, and facilitates the development and utilization of peptide-based hydrogel materials.
  • Figure 1 shows the gelation phase diagram under different peptide concentrations and different pH conditions.
  • Sol peptide solution
  • Gel peptide hydrogel
  • Precipate peptide precipitation.
  • Figure 2 is a TEM image of a 14.54 mM, pH 7.0 peptide hydrogel, and the inset in the upper right corner is a macroscopic gelation image.
  • Figure 3 shows the storage modulus of 14.54 mM polypeptide hydrogel under different pH conditions.
  • Figure 4 is a graph showing the influence of 14.54 mM, pH 7.0 polypeptide hydrogel on HepG2 cell activity.
  • Control control group
  • EK experimental group.
  • a self-assembled polypeptide to form a hydrogel its amino acid sequence is: Glu-Ile-Trp-Leu-Lys.
  • the method for obtaining the polypeptide that self-assembles to form a hydrogel is: design a self-assembled polypeptide through theory, and use solid-phase synthesis technology for polypeptide synthesis.
  • a method for preparing polypeptide hydrogels the specific steps are: PBS buffers of different pH (pH3, pH4, pH5, pH6, pH7, pH8, pH9) are configured to different concentrations (7.27, 8.72, 10.18, 11.63, 13.08, 14.54mM) self-assembled peptide solution to form a hydrogel, vortex until the peptide powder is dissolved, and let it stand overnight at room temperature. Use an inverted test tube to determine whether the solution forms a peptide hydrogel. If the sample does not slip off in an inverted test tube, that is It is a peptide hydrogel.
  • the gelation phase diagram of the polypeptide hydrogel prepared in this example under different polypeptide concentrations and different pH conditions is shown in Figure 1.
  • the results from Fig. 1 show that the gel forming conditions of the polypeptide are in a "v" shape, that is, when the polypeptide concentration is low, the gelation pH range is narrow, and as the concentration increases, the gelation pH range increases. Therefore, in a wide range of pH 5-8, peptides can self-assemble to form hydrogels.
  • a polypeptide hydrogel the preparation method is as follows: Weigh the polypeptide powder assembled to form the hydrogel into a 2mL tube, add pH 7.0 PBS buffer to prepare a polypeptide solution with a concentration of 14.54 mM, and vortex to the powder Fully dissolve, stand at room temperature for 30 minutes to prepare polypeptide hydrogel. Take 10 ⁇ L of gel and drop it on the copper net. After 8 minutes, absorb the excess polypeptide hydrogel with filter paper. Next, take 10 ⁇ L of sodium phosphotungstate solution for negative staining. After 8 minutes, absorb the excess sodium phosphotungstate solution with filter paper and dry at room temperature. Overnight, observe its microstructure on a transmission electron microscope machine.
  • Figure 2 shows the TEM and macroscopic gel formation diagram of the 14.54 mM, pH 7.0 polypeptide hydrogel prepared in this example.
  • Figure 2 can visually see that the peptides self-assemble to form a solid-like hydrogel with a three-dimensional network structure, and the presence of nanofibers can be observed from the TEM image. This is because the pentapeptide has N'and C'at pH 7
  • the terminal amino groups and carboxyl groups are protonated, each with positive and negative charges, and self-assemble through electrostatic interaction to form nanofibers.
  • the fibers are further entangled to "lock" water molecules, and finally form a hydrogel with a three-dimensional network structure.
  • a polypeptide hydrogel the preparation method is as follows: Weigh the polypeptide powder self-assembled to form the hydrogel into a 2mL tube, add PBS buffers of different pH (pH5, pH6, pH7, pH8) to the concentration of 14.54 mM polypeptide solution, vortex until the powder is fully dissolved, and let it stand at room temperature for 30 minutes to form a polypeptide hydrogel.
  • PBS buffers of different pH (pH5, pH6, pH7, pH8)
  • Figure 3 shows the storage modulus diagram of the 14.54 mM polypeptide hydrogel in this example under different pH conditions.
  • Figure 3 The results show that when the pH is 7, its storage modulus reaches the maximum and has the best rheological properties.
  • a polypeptide hydrogel is prepared by weighing the polypeptide powder that has been assembled to form a hydrogel into a 2 mL tube, adding pH 7 PBS buffer to prepare a polypeptide solution with a concentration of 14.54 mM, and vortexing until the powder is fully dissolved Put 2 ⁇ L of the peptide hydrogel in a 96-well plate with HepG2 cells for culture. After 24 hours, add 20 ⁇ L MTT (5mg/mL) and continue to incubate for 4 hours. After aspirating the supernatant, add 150 ⁇ L of DMSO to each well, mix with a microplate shaker, and measure the absorbance at 570 nm.
  • the control group is a blank control that replaces 2 ⁇ L of peptide hydrogel with 2 ⁇ L of sterile water. Contains peptide hydrogel.
  • Figure 4 shows that the polypeptide hydrogel has no toxic side effects on HepG2 cells, and its activity can reach 100%, indicating that the polypeptide hydrogel has good biocompatibility.

Abstract

La présente invention concerne le domaine technique des matériaux biologiques, et concerne un polypeptide capable de s'auto-assembler et de former un hydrogel, un hydrogel de celui-ci et son procédé de préparation. La séquence d'acides aminés du polypeptide est Glu-Ile-Trp-Leu-Lys. La longueur de séquence du polypeptide est courte, l'hydrogel polypeptidique préparé présente une bonne biocompatibilité, et le processus de formation de gel est simple et rapide.
PCT/CN2020/073061 2019-04-18 2020-01-19 Hydrogel polypeptidique et son procédé de préparation WO2020211504A1 (fr)

Applications Claiming Priority (2)

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CN201910310852.3 2019-04-18
CN201910310852.3A CN110028551A (zh) 2019-04-18 2019-04-18 一种多肽水凝胶及其制备方法

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Publication number Priority date Publication date Assignee Title
CN110028551A (zh) * 2019-04-18 2019-07-19 福州大学 一种多肽水凝胶及其制备方法
CN113563421A (zh) * 2021-07-30 2021-10-29 福州大学 一种自组装多肽可逆水凝胶的制备方法
CN114634548A (zh) * 2021-10-08 2022-06-17 河南工业大学 一种混合水凝胶的制备方法及其在封装系统中的应用

Citations (7)

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WO2012078760A1 (fr) * 2010-12-08 2012-06-14 Advanced Technologies And Regenerative Medicine, Llc Hydrogels auto-assemblés à base d'amphiphiles peptidiques dicéphaliques
US20150374833A1 (en) * 2013-04-25 2015-12-31 Spiber Inc. Polypeptide hydrogel and method for producing same
CN107412150A (zh) * 2017-07-16 2017-12-01 常州大学 一种用于包裹药物的多肽水凝胶的制备方法
CN107529533A (zh) * 2017-09-07 2018-01-02 中国药科大学 一类可自组装成水凝胶的pH敏感多肽及其作为装载药物材料的应用
CN108853515A (zh) * 2018-06-28 2018-11-23 南开大学 短肽水凝胶的制备方法及应用、药物组合物
CN109157504A (zh) * 2018-08-15 2019-01-08 北京大学深圳研究生院 一种多肽水凝胶及其制备方法和应用
CN110028551A (zh) * 2019-04-18 2019-07-19 福州大学 一种多肽水凝胶及其制备方法

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CN108342373B (zh) * 2017-01-23 2022-11-01 天津科技大学 一种自组装纳米多肽纤维的制备方法及其应用
CN106821964A (zh) * 2017-02-09 2017-06-13 江苏食品药品职业技术学院 pH调控的短链多肽水凝胶及其制备和应用方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012078760A1 (fr) * 2010-12-08 2012-06-14 Advanced Technologies And Regenerative Medicine, Llc Hydrogels auto-assemblés à base d'amphiphiles peptidiques dicéphaliques
US20150374833A1 (en) * 2013-04-25 2015-12-31 Spiber Inc. Polypeptide hydrogel and method for producing same
CN107412150A (zh) * 2017-07-16 2017-12-01 常州大学 一种用于包裹药物的多肽水凝胶的制备方法
CN107529533A (zh) * 2017-09-07 2018-01-02 中国药科大学 一类可自组装成水凝胶的pH敏感多肽及其作为装载药物材料的应用
CN108853515A (zh) * 2018-06-28 2018-11-23 南开大学 短肽水凝胶的制备方法及应用、药物组合物
CN109157504A (zh) * 2018-08-15 2019-01-08 北京大学深圳研究生院 一种多肽水凝胶及其制备方法和应用
CN110028551A (zh) * 2019-04-18 2019-07-19 福州大学 一种多肽水凝胶及其制备方法

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