CN115043908B - Hydroxyapatite targeting peptide and application thereof - Google Patents

Hydroxyapatite targeting peptide and application thereof Download PDF

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CN115043908B
CN115043908B CN202210660345.4A CN202210660345A CN115043908B CN 115043908 B CN115043908 B CN 115043908B CN 202210660345 A CN202210660345 A CN 202210660345A CN 115043908 B CN115043908 B CN 115043908B
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hydroxyapatite
targeting peptide
hap
phage
scaffold
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CN115043908A (en
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毛传斌
李龑
杨明英
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Zhejiang University ZJU
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/42—Phosphorus; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/12—Phosphorus-containing materials, e.g. apatite
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3637—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the origin of the biological material other than human or animal, e.g. plant extracts, algae
    • A—HUMAN NECESSITIES
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    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3847—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
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    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
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Abstract

The invention discloses a screening and application of a hydroxyapatite targeting peptide. The invention obtains the hydroxyapatite targeting peptide which can be specifically identified with the hydroxyapatite, and then prepares the phage carrying the hydroxyapatite targeting peptide by a freezing casting method to obtain the 3D scaffold which is used for culturing mesenchymal stem cells. The phage carrying the hydroxyapatite targeting peptide can not only specifically identify hydroxyapatite powder but also identify HAP nanorods, successfully obtain a new HAP identification structural domain, is beneficial to proliferation of mesenchymal stem cells on a scaffold, and provides data support for bone tissue regeneration.

Description

Hydroxyapatite targeting peptide and application thereof
Technical Field
The invention belongs to a polypeptide in the field of nano medicine and application thereof, and relates to a hydroxyapatite targeting peptide and application of a 3D scaffold prepared by the same in mesenchymal stem cell culture.
Background
Natural bone consists of an organic matrix containing type I collagen and inorganic Hydroxyapatite (HAP) crystals and non-collagenous proteins (NCP). In bone organic matrix, type 1 collagen accounts for about 90%, while NCPs include Osteocalcin (OCN), osteopontin (OPN), osteonectin (ON), bone Sialoprotein (BSP), decorin, and biglycan, accounting for about 10% of the protein (BP) content of the total bone. HAP nanocrystals in bone are formed by biomineralization, in which minerals nucleate and assemble in an organized manner under the control of biomolecules in an organic matrix. In bone biomineralization, HAP nanocrystals nucleate and assemble within collagen fibrils, and their nanostructures (e.g., orientation, shape, and organization) are well controlled. In recent years, different groups have new insights into the mechanisms of bone biomineralization. However, there are still unresolved problems, in particular which domains of bone proteins initiate HAP nucleation and bind HAP. Phage will be used as a platform to screen HAP binding peptides on bone proteins and analyze HAP nucleation and binding to the corresponding protein domains.
The most common strategies for studying protein-HAP interactions are HAP nucleation testing, solid state Nuclear Magnetic Resonance (NMR) and bioinformatic simulation. These methods, although established for over a decade, are inefficient. Recently, phage display has been successfully applied to the study of protein-protein interactions to identify protein-ligand interaction sites. However, understanding of the basic mechanism by which phage-derived crystal-binding peptides bind to their target crystals remains limited.
Disclosure of Invention
The invention aims at overcoming the defects of the prior art and providing a hydroxyapatite targeting peptide and application thereof, wherein the phage carrying the hydroxyapatite targeting peptide can specifically identify hydroxyapatite powder and HAP nanorods.
The invention aims at realizing the following technical scheme:
1. a hydroxyapatite targeting peptide has an amino acid sequence of SEQ ID No.1, and specifically is DSSTPSST, namely Asp-Ser-Ser-Thr-Pro-Ser-Ser-Thr.
2. Application of hydroxyapatite targeting peptide in preparing bone repair medicine and material.
The target peptide of the invention is used for bone repair by adsorbing hydroxyapatite.
3. A 3D scaffold, an in vivo biological scaffold for bone repair comprising a bacteriophage having the hydroxyapatite targeting peptide of claim 1.
4. The application of the 3D bracket is applied to the culture of mesenchymal stem cells, and bone repair is realized through the culture of the mesenchymal stem cells.
The mesenchymal stem cells were isolated from the thigh tibia of 334 rat from Fisher company.
The present invention combines phage display technology with computational modeling for the first time to study protein-HAP crystal interactions to identify potential HAP nucleation/binding domains on bone proteins. The invention successfully obtains a new HAP recognition domain and is beneficial to proliferation of mesenchymal stem cells on a bracket,
in the implementation of the invention, the hydroxyapatite targeting peptide is compared with various bone proteins by using comparison software to obtain conserved domains, and the functions and contributions of the conserved domains in HAP binding and nucleation are analyzed.
The hydroxyapatite in particular was HAP powder from Sigma.
The invention obtains the hydroxyapatite targeting peptide which can be specifically identified with the Hydroxyapatite (HAP), then prepares the phage carrying the hydroxyapatite targeting peptide and the HAP nano rod through a freeze casting method to obtain the DT phage-HAP scaffold with the 3-bit structure, and cultures Mesenchymal Stem Cells (MSCs) by utilizing the scaffold. Data support is provided for bone tissue regeneration.
The beneficial effects of the invention are as follows:
the invention screens and obtains polypeptide DT which can be specifically combined with HAP protein; the invention discovers a novel HAP binding domain and prepares a novel 3D scaffold material for bone repair.
Drawings
FIG. 1 is a graph of ELISA results for high frequency phage carrying targeting peptide of sequence SEQ ID NO.1 with HAP;
in FIG. 2, (A) is a TEM image of DT phage, and (B-D) is a TEM image of three phages (DT phage, EV phage (carrying another high frequency polypeptide: ESSATQHV) and wild phage) aligned with HAP nanoparticles, respectively.
FIG. 3 is a TEM image and a physical image of a 3D scaffold prepared from DT phage;
FIG. 4 is a statistical plot of MSCs cell proliferation in DT phage 3D scaffolds.
Detailed Description
The present invention will be described in further detail by way of examples, which are illustrative of the present invention and are not intended to limit the present invention thereto.
Embodiments of the invention are as follows:
1. and (3) experimental verification:
1.1, the phage carrying the targeting peptide with the sequence shown as SEQ ID NO.1 and phage carrying other polypeptide sequences are subjected to DNA sequencing, and the result shows that the phage carrying the targeting peptide with the sequence shown as SEQ ID NO.1 has higher frequency.
1.2 phage ELISA experiments and RELIC/MATCH experiments are respectively carried out on phage carrying targeting peptide with sequence shown in SEQ ID NO.1 and phage carrying other polypeptide sequences and HAP particles. The results show that phage HAP protein of the invention carrying targeting peptide with sequence shown as SEQ ID NO.1 has the third highest affinity (FIG. 1).
1.3 alignment experiments were performed on high frequency phage (carrying specific sequence polypeptides) and HAP nanorods, which indicated that DT phage and HAP had the best affinity (fig. 2).
Taken together, targeting polypeptide DT with very high affinity for HAP was obtained by phage panning.
2. Domain and proliferation:
2.1, screening domains: phage and bone proteins (tooth matrix protein, type I collagen, osteocalcin, bone regulatory protein, bone adhesion protein and bone saliva representative) with high occurrence frequency of DNA sequencing in example 1 are fitted and aligned by RELIC/MATCH and RELIC/MOTIF software to obtain the most similar conserved domains so as to obtain domains of various bone proteins, and the repeatability of subsequent industrial implementation can be ensured.
2.2 obtaining fd phage capable of displaying HAP binding peptide by PEG purification amplification and purification, final concentration of phage solution was about 4.5X10 13 pfu/mL. 20mg HAP nanorods in 100. Mu.L were sonicated for 30min, mixed with 500. Mu.L of selected fd phage, and immediately frozen in liquid nitrogen. The water in the frozen mixture was sublimated at-40 ℃ for 12h in a freeze-dryer to form a DT phage-HAP scaffold with a mass ratio of 35% (fig. 3). HAP scaffolds were implanted into 96 wells and subjected to uv sterilization for 48 h. 4X 10 of the additive is added to each hole 3 The MSCs were cultured for 72h and then incubated with MTT reagent. The absorbance at 490nm was measured by an ultraviolet spectrophotometer to investigate the proliferation of cells. The results showed that the DT phage-HAP scaffold was effective in promoting proliferation of MSCs (FIG. 4).
In conclusion, the invention successfully assembles the DT phage and the HAP nanorods into the 3D scaffold by using a freeze casting method, and can effectively promote proliferation of MSCs.
The amino acid sequence related to the invention is as follows:
SEQ ID No.1；
name: amino acid sequence of hydroxyapatite targeting peptide
The source is as follows: artificial sequence (Artificial Sequence)
DSSTPSST。
Sequence listing
<110> university of Zhejiang
<120> a hydroxyapatite targeting peptide and use
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Asp Ser Ser Thr Pro Ser Ser Thr
1 5

Claims (4)

1. A hydroxyapatite targeting peptide, characterized in that the amino acid sequence of the targeting peptide is SEQ ID No.1.
2. Use of a hydroxyapatite targeting peptide according to claim 1, in the preparation of a bone repair material.
3. A 3D scaffold comprising a bacteriophage comprising the hydroxyapatite targeting peptide of claim 1.
4. Use of a 3D scaffold according to claim 3, in the culture of mesenchymal stem cells.
CN202210660345.4A 2022-06-10 2022-06-10 Hydroxyapatite targeting peptide and application thereof Active CN115043908B (en)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007038683A2 (en) * 2005-09-28 2007-04-05 The Regents Of The University Of California Calcium binding peptides
JP2009197023A (en) * 2000-11-14 2009-09-03 Nektar Therapeutics Al Corp Hydroxyapatite-targeting poly(ethylene glycol) and related polymers
CN106906187A (en) * 2017-03-17 2017-06-30 浙江大学 A kind of bi-functional bacteriophage and purposes

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009197023A (en) * 2000-11-14 2009-09-03 Nektar Therapeutics Al Corp Hydroxyapatite-targeting poly(ethylene glycol) and related polymers
WO2007038683A2 (en) * 2005-09-28 2007-04-05 The Regents Of The University Of California Calcium binding peptides
CN106906187A (en) * 2017-03-17 2017-06-30 浙江大学 A kind of bi-functional bacteriophage and purposes

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