CN106117346B - Lanthanide metal-mediated collagen polypeptide and preparation method of biomimetic material thereof - Google Patents
Lanthanide metal-mediated collagen polypeptide and preparation method of biomimetic material thereof Download PDFInfo
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- CN106117346B CN106117346B CN201610479023.4A CN201610479023A CN106117346B CN 106117346 B CN106117346 B CN 106117346B CN 201610479023 A CN201610479023 A CN 201610479023A CN 106117346 B CN106117346 B CN 106117346B
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/18—Metal complexes
- C09K2211/182—Metal complexes of the rare earth metals, i.e. Sc, Y or lanthanide
Abstract
The invention discloses a lanthanide metal mediated collagen polypeptide and a preparation method of a biomimetic material thereof, which comprises the following steps of (1) designing and synthesizing the biomimetic collagen polypeptide, wherein the collagen polypeptide with a specific amino acid sequence comprises ① and contains more (Gly-Pro-Hyp) n or (Gly-Pro-Pro) n repeated sequences in the middle to help the polypeptide to form a collagen classical triple helix structure, ② contains aspartic acid or glutamic acid at two ends to realize coordination with lanthanide metal, and (2) preparing the collagen biomimetic luminescent nano material.
Description
Technical Field
The invention particularly relates to a lanthanide metal-mediated collagen polypeptide and a preparation method of a bionic material thereof, belonging to the technical field of preparation of biological nano materials.
Background
Collagen, a major component of the extracellular matrix, has gained widespread attention and use in regenerative medicine and tissue engineering due to its good biological properties and structural characteristics. Collagen has a perfect (Gly-X-Y) n repeating amino acid sequence and a classical triple helix structure. The molecular scaffold formed by the collagen fibers can provide complete structure and better mechanical strength for mammals. Therefore, the design of the collagen biomimetic polypeptide has many potential applications in the construction of novel biological functional materials.
Research on biomimetic collagen polypeptides has found various strategies to prepare collagen polypeptide nanomaterials, including: cysteine-modified polypeptides, electrostatic interactions, pi-pi stacking interactions, cation-pi interactions, metal coordination, amphiphilic polypeptides, nucleation and hydrophobic interactions, and the like. Various collagen polypeptides are utilized to successfully synthesize highly ordered structures such as microfilaments, meshes, microtubes, hollow spheres, nanodiscs, nanosheets and the like. Because the collagen polypeptide has good biocompatibility and degradation performance, the nano material constructed by the collagen polypeptide self-assembly method has huge application prospect in the fields of drug controlled release, tissue engineering scaffold material, biomineralization and the like.
Lanthanide metal ions as photoluminescent materials have the advantages of long luminescence lifetime, low toxicity, linear emission, high photochemical stability and the like, and thus are widely applied in the fields of medical diagnosis and cell imaging. The solid-phase synthesis method has the advantages of no need of separating and purifying intermediates in the reaction process, high yield, easy process detection and the like. The invention relates to a lanthanide metal-mediated self-assembled collagen polypeptide and a preparation method of a bionic luminescent material thereof. The specially designed collagen polypeptides can be self-assembled to form a nano material with a controllable structure under the mediation of different lanthanide metal ions and under a very mild condition; moreover, the nano material has good and controllable luminescent property. The collagen bionic luminescent material formed by self-assembling the collagen polypeptide and the lanthanide metal ions has great potential in the aspects of cell imaging, medical diagnosis, cell culture luminescent scaffolds and the like.
Disclosure of Invention
The invention aims to provide a lanthanide metal-mediated collagen polypeptide and a preparation method of a biomimetic material thereof aiming at the defects in the prior art, and the specially designed collagen polypeptide is self-assembled under the mediation of lanthanide metal ions and very mild conditions to form a nano material with a controllable structure and good luminescence property.
In order to achieve the purpose, the invention discloses the following technical scheme:
a preparation method of lanthanide metal mediated collagen polypeptide and biomimetic material thereof comprises the following steps:
(1) design and synthesis of bionic collagen polypeptide
The designed collagen polypeptide with a specific amino acid sequence comprises ①, a repeating sequence containing more (Gly-Pro-Hyp) n or (Gly-Pro-Pro) n in the middle to help the polypeptide to form a classic triple helix structure of the collagen, ②, two ends containing aspartic acid or glutamic acid to realize coordination with lanthanide metals, and the designed collagen polypeptide is synthesized by adopting an Fmoc solid phase synthesis method and a step-by-step synthesis method by using a manual synthesizer, wherein the step-by-step synthesis method comprises the steps of a, resin treatment swelling, b, activating amino acid, c, condensation reaction, d, α -amino deprotection, e, extending peptide resin, f, cutting peptide resin, g, and separation and purification of peptides.
(2) Preparation of collagen bionic luminous nano material
Preparing collagen polypeptide solution with concentration of 0.1-10.0mg/ml under the condition of 0.1M HEPES and pH7.0 buffer solution; preparing nitrate solution of different lanthanide metals with the concentration of 50 mM; preparing a mixed solution of lanthanide metal ions and collagen polypeptides under the condition that the molar ratio of the lanthanide metal ions to the collagen polypeptides is 1:1-15:1 respectively; placing the mixed solution in a thermostat at 4-70 deg.C for 1-96 hrs; carrying out solid-liquid separation on the reaction product at 10000rpm to obtain solid precipitate; and dispersing the solid by using ethanol, and then carrying out centrifugal purification for 3-5 times to obtain the luminescent nano material.
As a preferred technical scheme of the invention, the first amino acid at two ends of the collagen polypeptide in the step (1) is aspartic acid or glutamic acid, the second amino acid is aspartic acid, glutamic acid or tryptophan, and the middle sequence is GPO, GPP, GDP or GEP.
As a preferred technical solution of the present invention, in step (1), the lanthanide metal element is one or more of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, and lutetium.
As a preferred technical scheme of the invention, the resin used for the solid phase synthesis of the collagen polypeptide in the step (1) is dichloro resin, and the condensation system is an efficient HBTU/HOBt/DIEA condensation system.
As a preferred technical scheme of the invention, the concentration of the collagen polypeptide in the step (2) is 1.0-5.0mg/ml, and the molar ratio of the lanthanide metal ions to the collagen polypeptide is 1:1-9: 1.
As a preferred technical scheme of the invention, the mixed solution of lanthanide metal ions and collagen polypeptides in the step (2) is placed in a constant temperature box at the temperature of 4-45 ℃ for 6-64 hrs.
The invention discloses a preparation method of lanthanide metal mediated collagen polypeptide and bionic material thereof, which has the following advantages: the collagen polypeptide can be self-assembled to form a nano material with a controllable structure under the mediation of different lanthanide metal ions and under a very mild condition; the nano material has good and adjustable luminous performance; the collagen bionic luminescent material formed by self-assembling the collagen polypeptide and the lanthanide metal ions has great potential in the aspects of cell imaging, medical diagnosis, cell culture luminescent scaffolds and the like.
Drawings
FIG. 1 shows a collagen polypeptide DD (GPP)5GDP(GPP)6DD self-assembles under the mediation of lanthanide metal ions to form a scanning electron microscope picture of the nano material;
FIG. 2 shows a collagen polypeptide DD (GPP)5GDP(GPP)6DD self-assembles under the mediation of lanthanide metal ions to form a transmission electron microscope picture of the nano material;
FIG. 3 shows a collagen polypeptide DD (GPP)5GDP(GPP)6DD forms a fluorescence emission spectrogram of the nano material under the mediation of lanthanide metal ions by self-assembly;
FIG. 4 shows the collagen polypeptide DD (GPO)7DD self-assembles under the mediation of lanthanide metal ions to form a scanning electron microscope picture of the nano material;
FIG. 5 shows the collagen polypeptide DW (GPO)7Scanning electron microscopy of WD self-assembling under lanthanide metal ion mediation to form nanomaterials.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.
As shown in fig. 1-5, a method for preparing lanthanide metal-mediated collagen polypeptide and biomimetic material thereof comprises the following steps:
(1) design and synthesis of bionic collagen polypeptide
The designed collagen polypeptide with a specific amino acid sequence comprises ①, a repeating sequence containing more (Gly-Pro-Hyp) n or (Gly-Pro-Pro) n in the middle to help the polypeptide to form a classic triple helix structure of the collagen, ②, two ends containing aspartic acid or glutamic acid to realize coordination with lanthanide metals, and the designed collagen polypeptide is synthesized by adopting an Fmoc solid phase synthesis method and a step-by-step synthesis method by using a manual synthesizer, wherein the step-by-step synthesis method comprises the steps of a, resin treatment swelling, b, activating amino acid, c, condensation reaction, d, α -amino deprotection, e, extending peptide resin, f, cutting peptide resin, g, and separation and purification of peptides.
(2) Preparation of collagen bionic luminous nano material
Preparing collagen polypeptide solution with concentration of 0.1-10.0mg/ml under the condition of 0.1M HEPES and pH7.0 buffer solution; preparing nitrate solution of different lanthanide metals with the concentration of 50 mM; preparing a mixed solution of lanthanide metal ions and collagen polypeptides under the condition that the molar ratio of the lanthanide metal ions to the collagen polypeptides is 1:1-15:1 respectively; placing the mixed solution in a thermostat at 4-70 deg.C for 1-96 hrs; carrying out solid-liquid separation on the reaction product at 10000rpm to obtain solid precipitate; and dispersing the solid by using ethanol, and then carrying out centrifugal purification for 3-5 times to obtain the luminescent nano material.
Wherein, the collagen polypeptide sequence in the step (1) is one or more of peptide sequences in a table 1 or a table 2, the first amino acid at two ends of the polypeptide is aspartic acid or glutamic acid, the second amino acid is aspartic acid, glutamic acid or tryptophan, and the middle sequence is GPO, GPP, GDP or GEP.
TABLE 1
TABLE 2
Wherein, the lanthanide metal element in the step (1) is one or more of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium and lutetium.
Wherein, the resin used in the solid phase synthesis of the collagen polypeptide in the step (1) is dichloro resin, and the condensation system is an efficient HBTU/HOBt/DIEA condensation system.
Wherein, the concentration of the collagen polypeptide in the step (2) is 1.0-5.0mg/ml, and the molar ratio of the lanthanide metal ions to the collagen polypeptide is 1:1-9: 1.
Wherein, the mixed solution of lanthanide metal ions and collagen polypeptides in the step (2) is placed in a constant temperature box at 4-45 ℃ for 6-64 hrs.
Example 1
Lanthanide metal mediated self-assembled collagen polypeptide DD (GPP)5GDP(GPP)6The DD and the bionic luminescent material thereof are prepared by the following steps:
(1) synthesis of collagen polypeptide DD (GPP) by solid phase Synthesis5GDP(GPP)6DD
Synthesizing collagen polypeptide DD (GPP) by Fmoc solid phase synthesis method and manual synthesizer step by step5GDP(GPP)6DD。
(2) Preparation of collagen bionic luminous nano material
Preparing a collagen polypeptide solution with the concentration of 5mg/ml under the condition of 0.1M HEPES and a buffer solution with the pH value of 7.0; preparing nitrate solution of different lanthanide metals with the concentration of 50 mM; preparing a mixed solution of lanthanide metal ions and collagen polypeptides under the condition that the molar ratio of the lanthanide metal ions to the collagen polypeptides is 2: 1; placing the mixed solution in a constant temperature box at 25 deg.C for 48 hrs; carrying out solid-liquid separation on the reaction product at 10000rpm to obtain solid precipitate; and dispersing the solid by using ethanol, and then carrying out centrifugal purification for 3-5 times to obtain the luminescent nano material.
Example 2
Lanthanide metal-mediated self-assembled collagen polypeptide DD (GPO)7The DD and the bionic luminescent material thereof are prepared by the following steps:
(1) synthesis of collagen polypeptide DD (GPO) by solid phase Synthesis7DD
Adopting Fmoc solid phase synthesis method, utilizing manual synthesizer to synthesize the collagen polypeptide DD (GPO) step by step7DD。
(2) Preparation of collagen bionic luminous nano material
Preparing a collagen polypeptide solution with the concentration of 3mg/ml under the condition of 0.1M HEPES and a buffer solution with the pH value of 7.0; preparing nitrate solution of different lanthanide metals with the concentration of 50 mM; preparing a mixed solution of lanthanide metal ions and collagen polypeptides under the condition that the molar ratio of the lanthanide metal ions to the collagen polypeptides is 1: 1; placing the mixed solution in a constant temperature box at 4 deg.C for 24 hrs; and carrying out solid-liquid separation on the reaction product at 10000rpm to obtain solid precipitate. And dispersing the solid by using ethanol, and then carrying out centrifugal purification for 3-5 times to obtain the luminescent nano material.
Example 3
Lanthanide metal mediated self-assembled collagen polypeptide DW (GPO)7WD and the preparation of the bionic luminescent material thereof are realized by the following steps:
(1) synthesis of collagen polypeptide DW (GPO) by solid phase Synthesis7WD
Adopting Fmoc solid phase synthesis method, utilizing manual synthesizer to synthesize the collagen polypeptide DW (GPO) step by step7WD。
(2) Preparation of collagen bionic luminous nano material
Preparing a collagen polypeptide solution with the concentration of 1mg/ml under the condition of 0.1M HEPES and a buffer solution with the pH value of 7.0; preparing nitrate solution of different lanthanide metals with the concentration of 50 mM; preparing a mixed solution of lanthanide metal ions and collagen polypeptides under the condition that the molar ratio of the lanthanide metal ions to the collagen polypeptides is 1: 1; placing the mixed solution in a constant temperature box at 25 deg.C for 24 hrs; carrying out solid-liquid separation on the reaction product at 10000rpm to obtain solid precipitate; and dispersing the solid by using ethanol, and then carrying out centrifugal purification for 3-5 times to obtain the luminescent nano material.
The foregoing is a detailed description of an exemplary embodiment of the invention. For those skilled in the art, the present invention may be modified and varied in practical use according to specific preparation conditions, and is not limited thereto. Any reference signs in the claims shall not be construed as limiting the claim concerned, insofar as they are within the spirit and principle of the invention.
Claims (3)
1. A preparation method of a lanthanide metal-mediated collagen polypeptide biomimetic material is characterized by comprising the following steps:
(1) design and synthesis of bionic collagen polypeptide
Designing a collagen polypeptide having an amino acid sequence as defined in ① DD (GPP)5GDP(GPP)6DD；②DD(GPO)7DD; or ③ DW (GPO)7WD is shown;
the designed collagen polypeptide is synthesized by adopting an Fmoc solid-phase synthesis method in a stepwise synthesis mode of a kinetic synthesizer, and comprises a resin treatment swelling step, a resin activation amino acid step, a condensation reaction step, a d and α -amino deprotection step, an extended peptide resin step, a peptide cutting resin step, a peptide separation and purification step and a peptide separation and purification step;
(2) preparation of collagen polypeptide bionic material
Preparing a collagen polypeptide solution with the concentration of 1.0-5.0mg/ml under the condition of 0.1M HEPES, pH7.0 buffer solution: preparing a nitrate solution of different lanthanide metals at a concentration of 50 mM: preparing a mixed solution of lanthanide metal ions and collagen polypeptide according to the molar ratio of the lanthanide metal ions to the collagen polypeptide of 1:1-2:1 respectively: placing the mixed solution in a thermostat at 4-25 deg.C for 24-48 hrs; carrying out solid-liquid separation on the reaction product at 10000rpm to obtain solid precipitate: and dispersing the solid by using ethanol, and then carrying out centrifugal purification for 3-5 times to obtain the luminescent nano material.
2. The method for preparing a lanthanide metal-mediated collagen polypeptide biomimetic material as recited in claim 1, wherein: in the step (2), the lanthanide metal element is one or more of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium and lutetium.
3. The method for preparing a lanthanide metal-mediated collagen polypeptide biomimetic material as recited in claim 1, wherein: the resin used in the solid phase synthesis of the collagen polypeptide in the step (1) is dichloro resin, and the condensation system is an efficient HBTU/HOBt/DIEA condensation system.
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| CN113416264B (en) * | 2021-06-16 | 2022-05-17 | 兰州大学 | Triblock polypeptide and collagen bionic material formed by self-assembly of triblock polypeptide |
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| Ln3+-Mediated Self-Assembly of a Collagen Peptide into Luminescent Banded Helical Nanoropes;Manman He et al.;《Chem. Eur. J》;20160205;第22卷(第6期);第1914页右栏倒数第1段和图1、第1915页左栏第3段 * |
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