CN113512094A - Covalent photo-crosslinked polypeptide and collagen bionic material formed by covalent photo-crosslinked polypeptide self-assembly - Google Patents
Covalent photo-crosslinked polypeptide and collagen bionic material formed by covalent photo-crosslinked polypeptide self-assembly Download PDFInfo
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- CN113512094A CN113512094A CN202110729121.XA CN202110729121A CN113512094A CN 113512094 A CN113512094 A CN 113512094A CN 202110729121 A CN202110729121 A CN 202110729121A CN 113512094 A CN113512094 A CN 113512094A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- 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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- 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
- A61L27/50—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 belongs to the technical field of collagen bionic materials, and particularly relates to a covalent photo-crosslinking polypeptide and a collagen bionic material formed by self-assembling the covalent photo-crosslinking polypeptide. The covalent photo-crosslinking polypeptide is a polypeptide sequence capable of forming a triple helix structure, and comprises T1 and T2 domains containing continuous Tyr at two ends, N1 and N2 domains containing repeated Gly-Xaa-Yaa and/or Gly-Xaa-Xaa, and an M domain containing Tyr between N1 and N2 domains. The covalent photo-crosslinking polypeptide is self-assembled under photocatalysis to form a bionic material which can simulate the structure and the function of natural collagen; the polypeptide fragment with biological function can be conveniently introduced into the M domain, the self-assembly is not influenced, and the polypeptide self-assembly strategy has wide applicability; the prepared bionic material has good biocompatibility, can be used as a scaffold material for cell growth, and has wide application prospect in the fields of tissue engineering and regenerative medicine.
Description
Technical Field
The invention belongs to the technical field of collagen bionic materials, and particularly relates to a covalent photo-crosslinking polypeptide and a collagen bionic material formed by self-assembling the covalent photo-crosslinking polypeptide.
Background
Collagen, a major component of extracellular matrix, is widely used in regenerative medicine and tissue engineering fields due to its unique structure and good biological properties. However, the natural collagen has the problems of difficult control of extraction quality, virus hidden danger, immunogenicity and the like, and the application of the natural collagen material is greatly limited. Therefore, constructing a new strategy to mimic the structure and function of natural collagen is a research hotspot in the field of biomaterials. The collagen polypeptide with a specially designed sequence on the molecular level has the advantages of easy synthesis and modification, good biocompatibility, no virus transmission hidden danger and the like, and is more and more concerned in the field of collagen protein bionics.
Various non-covalent interacting collagen polypeptide self-assembly strategies have been developed to construct biomimetic materials of collagen, including pi-pi stacking, metal ion-ligand interactions, cation-pi interactions, amphiphilic polypeptides, and the like. Pi-pi stacking generally requires the introduction of unnatural amino acids, and metal ion-ligand interactions and cation-pi interactions require metal ion-dependent actuation. Meanwhile, self-assembly of amphiphilic polypeptide is mostly mediated by adding hydrophobic synthetic polymer material. The non-natural amino acids, metal ions, and synthetic polymer materials used in these strategies may have potential risks of biocompatibility and degradability. Moreover, when the environment of the non-covalent collagen polypeptide assembly changes, the poor transformation of the micro/nano structure is easy to occur, the stability and the biological function of the non-covalent collagen polypeptide assembly are greatly influenced, and the clinical application of the non-covalent collagen polypeptide material is limited.
In contrast to non-covalent self-assembly strategies, covalent self-assembly systems can result in extremely stable assembly structures by forming irreversible bonds. Covalent cross-linking of proteins is currently carried out mainly by chemical cross-linking agents, but the selectivity of the cross-linking agents is poor and there is a risk of residual toxicity. Disulfide bond formation between cysteines has also been used to mediate polypeptide self-assembly, however disulfide bond formation is spontaneous, making its assembly system difficult to control. Therefore, there is a need to develop a new covalent self-assembly strategy to construct collagen biomimetic materials.
The inventor unexpectedly finds a collagen bionic material formed by self-assembling covalent photocrosslinking polypeptide and covalent photocrosslinking polypeptide. The covalent photo-crosslinking polypeptide is a polypeptide sequence capable of forming a triple helix structure, and comprises T1 and T2 domains containing continuous Tyr at two ends, N1 and N2 domains containing repeated Gly-Xaa-Yaa and/or Gly-Xaa-Xaa, and an M domain containing Tyr between N1 and N2 domains. The middle domain M comprises at least one Gly-Tyr-Yaa sequence. The covalent photo-crosslinking polypeptide is self-assembled under photocatalysis to form a bionic material which can simulate the structure and the function of natural collagen. The intermediate domain M can be conveniently introduced into polypeptide fragments with biological functions, does not influence the self-assembly, and has wide applicability and flexibility; the prepared bionic material has good biocompatibility, can be used as a scaffold material for cell growth, and has wide application prospect in the fields of tissue engineering and regenerative medicine.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a covalent photocrosslinking polypeptide and a collagen bionic material formed by the self-assembly of the covalent photocrosslinking polypeptide. The covalent photo-crosslinking polypeptide is self-assembled under photocatalysis to form a bionic material which can simulate the structure and the function of natural collagen. The collagen bionic material has good biocompatibility and high stability, and can be used as a scaffold material for cell growth to promote cell adhesion.
The purpose of the invention is realized by the following technical scheme:
in a first aspect, the present invention provides a covalent photocrosslinked polypeptide, which is a polypeptide sequence capable of forming a triple helix conformation, the polypeptide sequence having the structure shown in formula (i):
T1-N1-M-N2-T2 is shown as formula (I);
wherein said M comprises at least 1 Gly-Tyr-Yaa, and/or Gly-Xaa-Tyr;
said N1 comprises at least 3 repeats of Gly-Xaa-Yaa, and/or Gly-Xaa-Xaa;
said N2 comprises at least 3 repeats of Gly-Xaa-Yaa, and/or Gly-Xaa-Xaa;
said T1 comprises at least 2 contiguous tyrs;
said T2 comprises at least 2 contiguous tyrs;
xaa and Yaa are any different amino acids.
Preferably, the covalent photocrosslinked polypeptide further comprises a polypeptide fragment having a biologically active function.
Preferably, the polypeptide fragment having a biologically active function comprises the sequence shown as follows:
Gly-Phe-Hyp-Gly-Glu-Arg；
and/or Gly-Arg-Hyp-Gly-Glu-Arg;
and/or Gly-Leu-Hyp-Gly-Glu-Arg;
and/or Gly-Met-Hyp-Gly-Glu-Arg;
and/or Gly-Ala-Hyp-Gly-Glu-Arg;
and/or Gly-Leu-Lys-Gly-Glu-Asn;
and/or Gly-Leu-Hyp-Gly-Glu-Asn;
and/or Gly-Val-Met-Gly-Phe-Hyp;
and/or Gly-Pro-Leu-Gly-Ile-Ala-Gly-Ile-Thr-Gly-Ala-Arg;
and/or Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val;
and/or Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-Hyp-Gly-Ile-Leu;
and/or Gly-Pro-Gln-Gy-Leu-Ala-Gly-Gln-Arg-Gly-Ile-Val;
and/or Gly-Arg-Hyp-Gly-Lys-Arg-Gly-Lys-Gln-Gly-Gln-Lys;
and/or Gly-Leu-Hyp-Gly-Gln-Arg-Gly-Glu-Arg.
Preferably, the polypeptide fragment having a biologically active function is Gly-Phe-Hyp-Gly-Glu-Arg.
Preferably, the Xaa is Pro; the Yaa is Hyp.
Preferably, the N1 is the same as N2.
Preferably, the N1 and N2 are (Gly-Pro-Hyp)nOr (Gly-Pro-Pro)nAnd n is any integer greater than or equal to 3.
Preferably, n is any integer from 3 to 5, including 3, or 4, or 5.
Preferably, the T1 and T2 are the same.
Preferably, both T1 and T2 are (Tyr)mAnd m is any integer greater than or equal to 2.
Preferably, said m is 2, or 3, or 4, or 5.
Preferably, the sequence of the covalent photo-cross-linking polypeptide is as follows:
(Tyr)2(Gly-Pro-Hyp)3(Gly-Tyr-Hyp)(Gly-Pro-Hyp)3(Tyr)2；
(Tyr)2(Gly-Pro-Hyp)4(Gly-Tyr-Hyp)(Gly-Pro-Hyp)4(Tyr)2；
(Tyr)2(Gly-Pro-Hyp)5(Gly-Tyr-Hyp)(Gly-Pro-Hyp)5(Tyr)2；
(Tyr)5(Gly-Pro-Hyp)4(Gly-Tyr-Hyp)(Gly-Pro-Hyp)4(Tyr)5；
(Tyr)2(Gly-Pro-Hyp)4(Gly-Tyr-Hyp)(Gly-Pro-Hyp)(Gly-Phe-Hyp-Gly-Glu-Arg)(Gly-Pro-Hyp)4(Tyr)2；
(Tyr)2(Gly-Pro-Hyp)4(Gly-Tyr-Hyp)(Gly-Pro-Hyp)(Gly-Tyr-Hyp)(Gly-Pro-Hyp)4(Tyr)2。
in a second aspect, the present invention provides an application of the covalent photocrosslinking polypeptide described in the first aspect in preparing a collagen biomimetic material.
In a third aspect, the present invention provides a collagen biomimetic material formed by self-assembly of the covalent photocrosslinked polypeptide described in the first aspect.
The invention has the beneficial effects that:
(1) the invention provides a novel covalent photo-crosslinking polypeptide self-assembly system;
(2) the covalent photo-crosslinking polypeptide is self-assembled to form a bionic material which can simulate the structure and the function of natural collagen under photocatalysis, and a new polypeptide design strategy is provided for the bionic collagen;
(3) compared with non-covalent self-assembly polypeptide, the covalent photo-crosslinking polypeptide has ultrahigh stability and is not influenced by external pH or solvent;
(4) the covalent photo-crosslinking polypeptide is completely composed of natural amino acids, has good biocompatibility, can promote cell adhesion and has wide clinical application potential;
(5) the intermediate structural domain M of the covalent photo-crosslinking polypeptide is convenient to introduce other amino acid sequences, has wide flexibility and applicability, is easy to add sequences with biological functions, and endows the polypeptide self-assembly material with good biological functions;
(6) the covalent photo-crosslinking polypeptide biomimetic material has the advantages of simple preparation method of the self-assembly material, mild conditions and wide application prospect in the fields of tissue engineering and regenerative medicine.
Drawings
FIG. 1 is a circular dichroism chromatogram of a covalent photo-crosslinking polypeptide according to examples 1 to 6 of the present invention;
FIG. 2 is an SEM image of a covalently photocrosslinked polypeptide assembly material of examples 1-6 of the present invention;
FIG. 3 is an SEM image of the covalently photocrosslinked polypeptide assembly material of example 2 of the present invention at different pH and different solvents;
FIG. 4 is a cytotoxicity map, a cell adhesion map and an immunofluorescence imaging map of the collagen biomimetic material based on HeLa cells and a cell adhesion map and an immunofluorescence imaging map based on HFF-1 cells according to the embodiment 6 of the present invention;
FIG. 5 is a circular dichroism diagram of polypeptides of the invention according to comparative examples 1 to 6;
FIG. 6 SEM pictures of polypeptide assembly materials according to comparative examples 1-6 of the present invention.
Detailed Description
The technical solution of the present invention is further described with reference to the following specific examples, but the scope of the present invention is not limited to the following.
The sequences of the covalent photocrosslinking polypeptides involved in the following examples are shown in table 1 below, but are not limited to the sequences described in table 1. Wherein, the GPO sequence is the (Gly-Pro-Hyp) sequence described in the application; the Y is Tyr; the GYO is (Gly-Tyr-Hyp); the GFOGER is (Gly-Phe-Hyp-Gly-Glu-Arg). And the covalent photocrosslinking polypeptide sequences are synthesized by a classical solid-phase synthesis method, wherein the solid-phase synthesis method specifically comprises the following steps:
(1) 100mg Rink ammonia resin was added to a reactor with sieve plate and the resin was swollen with 5mL of dichloromethane;
(2) removing the Fmoc protecting group at the N terminal from a 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by a color reaction;
(3) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBT (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, adding DIEA (6eq) dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 h.
(4) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by color reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 5min, 5min and 15min, respectively. Washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;
(5) repeating steps (3) and (4) until the target peptide sequence is completed.
(6) The N-terminus was blocked with 25% acetic anhydride (DMF) for 25 min.
(7) And (3) cutting the target peptide: at room temperature with TFA (trifluoroacetic acid): triisopropylsilane: H2The resin was shaken for 3h with O95: 2.5:2.5 to complete the cleavage of the peptide. And (3) precipitating with glacial ethyl ether, centrifuging and washing to finally obtain the target polypeptide shown in the table 1.
TABLE 1 sequence Listing of covalently photocrosslinked Polypeptides
Defining:
unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Representative exemplary methods and compositions are described in the following examples, although any methods and compositions similar or equivalent to those described herein can also be used to effect self-assembly of collagen polypeptides. And it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention and the appended claims.
It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a collagen polypeptide" includes a plurality of such collagen polypeptides, reference to "the collagen polypeptide" is a reference to one or more collagen polypeptides and equivalents thereof known to those skilled in the art, and so forth.
"comprising" means "including but not limited to," and is not intended to exclude, for example, other components, integers, and the like. In particular, where the statement "comprises a Gly-Pro-Hyp sequence of at least 3 repeats" is explicitly contemplated to include the listed items (unless a limiting term is included such as "consisting of … …"), this is meant to not be intended to exclude other components, integers, not listed in the recited step.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no such stated or intervening value in the stated range, to the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included in the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.
It is appreciated that certain features are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
It will be apparent to those skilled in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has independent components and features that can be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method may be performed in the order of events recited or in any other order that is logically possible.
The term "collagen" is the major component of the connective tissue of animals, and is the most abundant and widely distributed functional protein in mammals, and is a trimer structure composed of three peptide chains, including types I, II, III, V and XI collagen.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to two or more amino acid residues linked to each other by peptide bonds or modified peptide bonds. The term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers that contain modified residues and non-naturally occurring amino acid polymers.
The term "heat-altered temperature" refers to the temperature at which half of the triple helix structure of a collagen polypeptide is converted to random coil. The heat-altered temperature can be measured by a known structure analysis method such as Circular Dichroism (CD), but is not limited thereto, and can be measured by other known methods. It is well known to those skilled in the art that collagen polypeptides having a heat-altered temperature all have a triple helix structure.
The term "biomimetic material" refers to a material developed to mimic various characteristics or characteristics of an organism. An artificial material designed and manufactured by following the operation mode of a living system and the structural rule of a biomaterial is generally called a biomimetic material, and the biomimetic material described in this specification has the structural characteristics of collagen.
Example 1 covalent PhotoCross-linking Polypeptides YY (GPO)3GYO(GPO)3Characterization of the Properties of YY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The results are shown in FIG. 1, a, covalently photocrosslinking the polypeptide YY (GPO) at 225nm3GYO(GPO)3The CD value of YY changes along with the temperature and shows Z-shaped changes, which shows that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 25 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM ammonium persulfate aqueous solution and 0.2mL, 1mg/mL covalent photocrosslinkable polypeptide YY (GPO) dissolved in buffer pH103GYO(GPO)3YY mixing. And then exposing the collagen bionic material to a white light lamp for 6 minutes, centrifuging to obtain a precipitate, and repeatedly centrifuging and washing for 3 times by using water to obtain the collagen bionic material.
3. SEM characterization of self-assembled materials
And (2) dripping 20 mu L of the collagen bionic material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the collagen bionic material is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. The results are shown in FIG. 2, a, covalently photocrosslinked polypeptide YY (GPO)3GYO(GPO)3YY self-assembles to form a fibrillar network structure resembling native collagen.
Example 2 covalent PhotoCross-linking Polypeptides YY (GPO)4GYO(GPO)4Characterization of the Properties of YY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The results are shown in FIG. 1 b, covalently photocrosslinking the polypeptide YY (GPO) at 225nm4GYO(GPO)4The CD value of YY changes along with the temperature and shows Z-shaped changes, which shows that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 47 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM ammonium persulfate aqueous solution and 0.2mL, 1mg/mL covalent photocrosslinkable polypeptide YY (GPO) dissolved in buffer pH104GYO(GPO)4YY mixing. And then exposing the collagen bionic material to a white light lamp for 6 minutes, centrifuging to obtain a precipitate, and repeatedly centrifuging and washing for 3 times by using water to obtain the collagen bionic material.
3. SEM characterization of self-assembled materials
And (2) dripping 20 mu L of the collagen bionic material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the collagen bionic material is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. The result is shown in FIG. 2 b, which shows covalent photocrosslinking of the polypeptide YY (GPO)4GYO(GPO)4YY self-assembles to form a fibrillar network structure resembling native collagen.
Example 3 covalent PhotoCross-linking Polypeptides YY (GPO)5GYO(GPO)5Characterization of the Properties of YY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The results are shown in FIG. 1 c, covalently photocrosslinking the polypeptide YY (GPO) at 225nm5GYO(GPO)5The CD value of YY changes along with the temperature and shows Z-shaped changes, which shows that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 57 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM ammonium persulfate aqueous solution and 0.2mL, 1mg/mL covalent photocrosslinkable polypeptide YY (GPO) dissolved in buffer pH105GYO(GPO)5YY mixing. And then exposing the collagen bionic material to a white light lamp for 6 minutes, centrifuging to obtain a precipitate, and repeatedly centrifuging and washing for 3 times by using water to obtain the collagen bionic material.
3. SEM characterization of self-assembled materials
And (2) dripping 20 mu L of the collagen bionic material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the collagen bionic material is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. Results are shown in FIG. 2, c, covalently photocrosslinked polypeptide YY (GPO)5GYO(GPO)5YY self-assembles to form a fibrillar network structure resembling native collagen.
Example 4 covalent PhotoCross-linking Polypeptides YY (GPO)4GYOGPOGYO(GPO)4Characterization of the Properties of YY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The result is shown in FIG. 1 as d, covalently photocrosslinking the polypeptide YY (GPO) at 225nm4GYOGPOGYO(GPO)4The CD value of YY changes along with the temperature and shows Z-shaped changes, which shows that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 52 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM ammonium persulfate aqueous solution and 0.2mL, 1mg/mL covalent photocrosslinkable polypeptide YY (GPO) dissolved in buffer pH104GYOGPOGYO(GPO)4YY mixing. And then exposing the collagen bionic material to a white light lamp for 6 minutes, centrifuging to obtain a precipitate, and repeatedly centrifuging and washing for 3 times by using water to obtain the collagen bionic material.
3. SEM characterization of self-assembled materials
And (2) dripping 20 mu L of the collagen bionic material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the collagen bionic material is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. The result is shown in FIG. 2 as d, covalently photocrosslinking the polypeptide YY (GPO)4GYOGPOGYO(GPO)4YY self-assembles to form a fibrillar network structure resembling native collagen.
4. Stability study of samples at different pH and in different organic solvents
Crosslinking of the polypeptide YY (GPO) by covalent light4GYOGPOGYO(GPO)4YY was used as an example to study the effect of pH on the material. Glue to be assembledAnd (3) soaking the collagen bionic material in aqueous solutions with the pH values of 4, 6, 8 and 10, culturing for 24 hours, and observing the change of the appearance of the collagen bionic material. Also covalently photocrosslinking the polypeptide YY (GPO)4GYOGPOGYO(GPO)4YY is taken as an example, and the influence of different organic solvents on the material is researched. And (3) soaking the assembled collagen bionic material in acetonitrile, tetrahydrofuran, dichloromethane and xylene, culturing for 24 hours, and observing the change of the material appearance.
The experimental results of the scanning electron microscope are shown in fig. 3. Wherein a is pH4, b is pH6, c is pH8, d is pH10, e is acetonitrile, f is tetrahydrofuran, g is dichloromethane, and h is xylene. Under the conditions of different pH values and different organic solvents, the shape of the collagen bionic material is not changed, which shows that the collagen bionic material has good stability in different pH values and various organic solvents.
Example 5 covalent photo-crosslinking of polypeptide Y5(GPO)4GYO(GPO)4Y5Characterization of properties of
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4-70 ℃, and the temperature rise rate is 10 ℃/h. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The result is shown in FIG. 1 as e, at 225nm, covalently photocrosslinking the polypeptide Y5(GPO)4GYO(GPO)4Y5The CD value of the polypeptide changes along with the temperature and shows Z-shaped change, which indicates that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 41 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl20.6mL of aqueous solution, 10mM ammonium persulfate aqueous solution and 0.2mL, 1mg/mL of covalent photocrosslinking polypeptide Y dissolved in pH10 buffer5(GPO)4GYO(GPO)4Y5And (4) mixing. And then exposing the collagen bionic material to a white light lamp for 6 minutes, centrifuging to obtain a precipitate, and repeatedly centrifuging and washing for 3 times by using water to obtain the collagen bionic material.
3. SEM characterization of self-assembled materials
And (2) dripping 20 mu L of the collagen bionic material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the collagen bionic material is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. The result is shown in FIG. 2 as e, covalently photocrosslinking polypeptide Y5(GPO)4GYO(GPO)4Y5Self-assembly forms a fibrous network structure resembling native collagen.
Example 6 covalent PhotoCross-linking Polypeptides YY (GPO)4GYOGPOGFOGER(GPO)4Characterization of the Properties of YY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The result is shown in FIG. 1 at f, covalently photocrosslinking the polypeptide YY (GPO) at 225nm4GYOGPOGFOGER(GPO)4The CD value of YY (GYOGFOGER) changes along with the temperature and shows Z-shaped change, which indicates that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 28 ℃.
2. Preparation of self-assembling materials
0.4mL, 1mM Ru (bpy)3Cl2Aqueous solution, 1.2mL, 10mM ammonium persulfate aqueous solution and 0.4mL, 2mg/mL covalent photocrosslinking polypeptide GYOGFOGER dissolved in buffer pH 10. And then exposing the collagen bionic material to a white light lamp for 6 minutes, centrifuging to obtain a precipitate, and repeatedly centrifuging and washing for 3 times by using water to obtain the collagen bionic material.
3. SEM characterization of self-assembled materials
And (2) dripping 20 mu L of the collagen bionic material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the collagen bionic material is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. The result is shown in f in fig. 2, the covalent photo-crosslinking polypeptide GyOGFOGER self-assembles to form a fiber network structure similar to natural collagen.
4. Cell experiments
4.1 cytotoxicity assay
The cytotoxicity of the collagen biomimetic material on HeLa cells is determined by a CCK-8 method. HeLa cells (DMEM medium containing 10% fetal bovine serum and 1% double antibody) in 5% CO2The temperature was set at 37 ℃ in the incubator (see the temperature). HeLa cell suspension (5X 10 per well) in a volume of 100. mu.L3Density of individual cells) were seeded in 96 wells and incubated for 24 hours (37 ℃, 5% CO)2) To ensure complete adherence. Then, 100. mu.L of each suspension of GyOGFOGER assemblies at different initial concentrations (0.05, 0.1, 0.15, and 0.2mg/mL) was added to 96-well plates, and 100. mu.L of DMEM medium was added to control wells, and incubation was continued at 37 ℃ for 24 hours. Then, 100. mu.L of 10% Cell Counting Kit-8(CCK-8) was added to each well, cultured in a Cell incubator, and applied to a Tecan Infinite F200/M200 multi-functional microplate reader (Tecan,
switzerland) measured their OD at 450nm, and the culture was stopped when only the wells containing no material reached an OD of around 1 for a suitable time. The activity of HeLa cells was calculated as the average absorbance of three measurements per condition.
The result of the cytotoxicity experiment is shown in a in figure 4, the GyOGFOGER assembly shows the cell survival rate similar to that of the collagen bionic material without the material under different concentrations, and the collagen bionic material has good biocompatibility.
4.2 cell adhesion experiments
The 96 microwell plates (untreated tissue) were coated with type I collagen, BSA, gyogfog assemblies, respectively. BSA served as control. The coated wells were washed three times with PBS buffer (10mM) before adding HeLa cells. Then, 100. mu.L (1X 10) was taken6cells/mL, serum-free medium) HeLa cell suspension was seeded in 96-well plates and incubated at 37 ℃ for 4 hours, and finally the unattached HeLa cells were washed with 10mM PBS buffer. The ability of the material to adhere to cells was compared by total deoxyribonucleic acid (DNA) quantification (Hoechst 33258, Solarbio) of the adhered cells. The method comprises the following steps: adherent cells in the microplate were lysed using repeated freeze-thaw, Hoechst 33258 dye was added to the cell lysate to a final concentration of 5 μ g/mL, and the mixture was incubated in the dark for 1 hour, and finally their fluorescence intensity at an emission wavelength of 465mm was measured using a microplate reader (tecamin transmit M200) at an excitation wavelength of 360 mm. Triplicate determinations were made.
The results of the cell adhesion experiments are shown in b of FIG. 4, wherein the adhesion value of HeLa cells and native collagen is 100% of the reference value, the adhesion ability of HeLa cells and BSA is 18.7% of that of native collagen, and the adhesion ability of HeLa cells and BSA are similar to that of collagen biomimetic material YY (GPO)4GYOGPOGFOGER(GPO)4The adhesion capacity of YY was 87.8%. The result shows that the BSA has poor adhesion capability to the HeLa cells, and the collagen bionic material has good adhesion capability to the HeLa cells.
Meanwhile, the adhesion condition of the HFF-1 cells on the surfaces of the three materials is also studied, and the result is shown in c in fig. 4, wherein the adhesion percentage of the HFF-1 cells to BSA is 19.4%, and the adhesion percentage of the HFF-1 cells to the collagen bionic material is about 83.2%, which indicates that the BSA has poor adhesion capability to the HFF-1 cells, and the collagen bionic material has good adhesion capability to the HFF-1 cells.
5. Immunofluorescence assay
5.1 HeLa cell immunofluorescence assay
The 24-well plates (tissue untreated) were coated with Bovine Serum Albumin (BSA) and GYOGFOGER assemblies, respectively. The 24-well plates were washed three times with 10mM PBS buffer (10mM, pH 7.2-7.4) before adding HeLa cells. Then 600 cells/mm2HeLa cell suspension at a density was added to the culture dish and incubated at 37 ℃ for 4 hours. The adhered HeLa cells were fixed with 4.0% formaldehyde solution for 10 minutes, permeabilized with 0.1% Tritom X-100 solution for 5 minutes, and blocked with 1% BSA in PBS buffer (10mM, pH 7.2-7.4) at room temperature for 0.5 hour. The fixed HeLa cells were incubated with phalloidin-tetramethylrhodamine isothiocyanate (Solepao, China) (1mL, 100mM in PBS) (10mM, pH 7.2-7.4) for 1 hour in the dark, followed by addition of Hoechst 33258(Sigma-Aldrich) (1mL, 5. mu.g/mL in ultrapure water) and incubation at 37 ℃ for 10 minutes to stain the cell actin and cell nucleus, respectively. Imaging results were obtained using a Leica (Leica Microsystems imc., Wetzlar, germany) fluorescence microscope.
As can be seen from the fluorescence microscopy image, the HeLa cells on the BSA-coated surface were spherical, indicating that their cell adhesion ability was poor (the result is shown in d in fig. 4); and the HeLa cells on the surface of the 24-pore plate coated by the GyOGFOGER assembly are in a spread appearance (the result is shown as e-f in figure 4), which shows that the assembly material has good performance of promoting cell adhesion and well simulates the biological function of natural collagen.
5.2 HFF-1 cellular immunofluorescence assay
The 24-well plates (tissue untreated) were coated with Bovine Serum Albumin (BSA) and GYOGFOGER assemblies, respectively. Prior to the addition of HFF-1 cells, the 24-well plate was washed three times with 10mM PBS buffer (10mM, pH 7.2-7.4). Then 1200 cells/mm2A density of HFF-1 cell suspension was added to the dish and incubated at 37 ℃ for 12 hours. The adhered HFF-1 cells were fixed with 4.0% formaldehyde solution for 10 minutes, permeabilized with 0.1% Triton X-100 solution to HeLa cells for 5 minutes, and blocked with 1% BSA in PBS buffer (10mM, pH 7.2-7.4) at room temperature for 0.5 hour. Mixing fixed HFF-1 cells with phalloidin-tetramethyl rhodamine isothiocyanateAcid ester (Solebao, China) (1mL, 100mM PBS solution) (10mM, pH7.2-7.4) was incubated for 1 hour in the dark, then Hoechst 33258(Sigma-Aldrich) (1mL, 5. mu.g/mL in ultrapure water) was added and incubated at 37 ℃ for 10 minutes to stain the cell actin and cell nucleus, respectively. Results of imaging were obtained using a fluorescence microscope from come (Leica Microsystems inc., Wetzlar, Germany).
As can be seen from the fluorescence microscopy image, the BSA coated surface HFF-1 cells were spherical, indicating that their cell adhesion ability was poor (the results are shown in FIG. 4, g); and HFF-1 cells on the surface of the 24-pore plate coated by the GyOGFOGER assembly are in a spread shape (the result is shown as h-i in figure 4), which shows that the assembly material has good performance of promoting cell adhesion and well simulates the biological function of natural collagen.
Comparative example
The sequences of the polypeptides involved in the following comparative examples are shown in table 2 below. Wherein, the GPO sequence is the (Gly-Pro-Hyp) sequence described in the application; the Y is Tyr; the GYO is (Gly-Tyr-Hyp). And the polypeptide sequences are synthesized by a classical solid-phase synthesis method, which comprises the following steps:
(1) 100mg Rink ammonia resin was added to a reactor with sieve plate and the resin was swollen with 5mL of dichloromethane;
(2) removing the Fmoc protecting group at the N terminal from a 20% piperidine/N, N-Dimethylformamide (DMF) solution, and detecting complete removal of the protecting group by a color reaction;
(3) dissolving amino acid (4eq) with the N-terminal protected by Fmoc, HOBT (4eq) and HBTU (4eq) in DMF, activating at low temperature for 20min, adding DIEA (6eq) dropwise into the solution, mixing the solution, adding into a reactor, and reacting for 3 h.
(4) After the reaction, the reaction solution was taken out of the reactor, and the resin was washed 3 times with 5mL of DMF and DCM, respectively. The amino acid condensation was complete as detected by color reaction, and the resin was treated with 20% piperidine/DMF solution 3 times for 5min, 5min and 15min, respectively. Washing the resin with 5mL of DMF and DCM for 3 times respectively, and detecting complete removal of the protecting group through color reaction;
(5) repeating steps (3) and (4) until the target peptide sequence is completed.
(6) The N-terminus was blocked with 25% acetic anhydride (DMF) for 25 min.
(7) And (3) cutting the target peptide: at room temperature with TFA (trifluoroacetic acid): triisopropylsilane: H2The resin was shaken for 3h with O95: 2.5:2.5 to complete the cleavage of the peptide. And (3) precipitating with glacial ethyl ether, centrifuging and washing to finally obtain the target polypeptide shown in the table 2.
Table 2 sequence listing of polypeptides
Comparative example 1 characterization of Properties of the polypeptide YYGYOYY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 60 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
As a result, as shown in FIG. 5, the CD value of the polypeptide YYGYOYY at 225nm decreased linearly with the increase in temperature, indicating that the polypeptide could not form a triple helix structure.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM aqueous ammonium persulfate solution, and 0.2mL, 1mg/mL of the target polypeptide YYGYOYY dissolved in a buffer solution at pH10 were mixed. It was then exposed to a white light lamp for 6 minutes, centrifuged to give a precipitate, and then repeatedly centrifuged and washed with water 3 times to give a self-assembled material.
3. SEM characterization of self-assembled materials
And (3) dripping 20 mu L of the product on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the product is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. As a result, the polypeptide YYGYOYY self-assembles to form a spherical structure as shown in FIG. 6.
Comparative example 2 characterization of Properties of the polypeptide YYGPOGYOGPOYY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 60 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
As a result, as shown in b in FIG. 5, the CD value of the polypeptide YYGPOGYOGPOYY at 225nm decreased linearly with the increase in temperature, indicating that the polypeptide could not form a triple helix structure.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2An aqueous solution, 0.6mL, 10mM ammonium persulfate aqueous solution, and 0.2mL, 1mg/mL of the target polypeptide YYGPOGYOGPOYY dissolved in a buffer solution of pH10 were mixed. It was then exposed to a white light lamp for 6 minutes, centrifuged to give a precipitate, and then repeatedly centrifuged and washed with water 3 times to give a self-assembled material.
3. SEM characterization of self-assembled materials
And (3) dripping 20 mu L of the material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the silicon chip is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. As a result, the polypeptide YYGPOGYOGPOYY self-assembles to form a spherical structure as shown in b in FIG. 6.
Comparative example 3 polypeptide YY (GPO)2GYO(GPO)2Property table of YYSign for
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 60 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The results are shown in FIG. 5 c, at 225nm, the polypeptide YY (GPO)2GYO(GPO)2The CD value of YY decreases linearly with increasing temperature, indicating that the polypeptide cannot form a triple helix structure.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM aqueous ammonium persulfate solution and 0.2mL, 1mg/mL of the polypeptide of interest YY (GPO) dissolved in buffer pH102GYO(GPO)2YY mixing. It was then exposed to a white light lamp for 6 minutes, centrifuged to give a precipitate, and then repeatedly centrifuged and washed with water 3 times to give a self-assembled material.
3. SEM characterization of self-assembled materials
And (3) dripping 20 mu L of the material on a silicon chip, placing the silicon chip in a constant temperature incubator at 25 ℃ until the silicon chip is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. The results are shown in FIG. 6 c, polypeptide YY (GPO)2GYO(GPO)2YY self-assembles to form spherical structures.
Comparative example 4 polypeptide (GPO)4GYO(GPO)4Characterization of properties of
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The results are shown in FIG. 5 at d, at 225nm, for the polypeptide (GPO)4GYO(GPO)4The CD value of the polypeptide changes along with the temperature and shows Z-shaped change, which indicates that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 51 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM ammonium persulfate aqueous solution and 0.2mL, 1mg/mL target polypeptide (GPO) dissolved in pH10 buffer4GYO(GPO)4. Subsequently, it was exposed to a white light lamp for 6 minutes and centrifuged, and as a result, as shown by d in fig. 6, no precipitate was generated and no assembly occurred.
Comparative example 5 polypeptide (GPO)2GYOGPOGYO(GPO)3Characterization of properties of
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The results are shown in FIG. 5, e, at 225nm, for the polypeptide (GPO)2GYOGPOGYO(GPO)3The CD value of the polypeptide shows Z-shaped change along with the change of temperature, which indicates that the polypeptide forms a stable triple helix structure,the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 27 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM ammonium persulfate aqueous solution and 0.2mL, 1mg/mL target polypeptide (GPO) dissolved in pH10 buffer2GYOGPOGYO(GPO)3And (4) mixing. Subsequently, it was exposed to a white light lamp for 6 minutes and centrifuged, and as a result, as shown by e in fig. 6, no precipitate was generated and no assembly occurred.
Comparative example 6 polypeptide YY (GPO)8Characterization of the Properties of YY
1. Circular dichroism spectrum
Circular Dichroism (CD) is obtained on a chiralscan CD chromatograph (Applied photophysics, England) equipped with a Peltier temperature controller. An aqueous solution of the polypeptide was prepared at a concentration of 300. mu.M at pH 10. The samples required equilibration at 4 ℃ for at least 24 hours before measurements were taken. Cuvettes with a path length of 1mm were used. The thermal change curve is obtained by monitoring the characteristic CD peak value at the wavelength of 225nm along with the change of temperature, the temperature change range is 4 ℃ to 80 ℃, and the temperature rise rate is 10 ℃/hour. And (4) carrying out first-order derivation on the thermal change curve, wherein the temperature corresponding to the extreme value is the thermal change temperature (Tm).
The results are shown in FIG. 5 at f, at 225nm, for the polypeptide YY (GPO)8The CD value of YY changes along with the temperature and shows Z-shaped changes, which shows that the polypeptide forms a stable triple helix structure, and the first derivative of the thermal change curve is obtained to obtain the thermal change temperature of the polypeptide of 50 ℃.
2. Preparation of self-assembling materials
0.2mL of 1mM Ru (bpy)3Cl2Aqueous solution, 0.6mL, 10mM aqueous ammonium persulfate solution and 0.2mL, 1mg/mL of the polypeptide of interest YY (GPO) dissolved in buffer pH108YY mixing. It was then exposed to a white light lamp for 6 minutes, centrifuged to give a precipitate, and then repeatedly centrifuged and washed with water 3 times to give a self-assembled material.
3. SEM characterization of self-assembled materials
Dropping 20 μ L of the above materials on a silicon wafer, and culturing at 25 deg.CAnd (3) cultivating the box until the sample is completely dried, carrying out gold spraying treatment on the dried sample, and carrying out scanning electron microscope characterization by using a Hitachi S-4800 scanning electron microscope (Hitachi Limited, Japan) with the operating voltage of 5.1-5.3 kV. The results are shown in FIG. 6, f, for the polypeptide YY (GPO)8YY self-assembles to form spherical structures.
The results of the assembly of the covalent photocrosslinking polypeptides and collagen polypeptides described in the above examples and comparative examples are shown in table 3:
TABLE 3 collagen polypeptide Assembly results and temperature Change by Heat
From the above results, it can be seen that the polypeptides 1, 2 and 3 of the examples have a triple helix structure, each of which has 2Y at both ends and one Y in the middle sequence, and are assembled to form a fiber network structure; the polypeptide 4 of example has a triple helix structure, with 2Y at each end, and two Y in the middle sequence, assembled to form a fiber network structure; the polypeptide 5 of the embodiment has a triple helix structure, two ends of the triple helix structure respectively contain 5Y, and a middle sequence of the triple helix structure contains two Y, and the triple helix structure is assembled to form a fiber network structure; example polypeptide 6 a polypeptide fragment gfiger having a cell adhesion function was introduced in the middle. After the polypeptide fragment GFOG ER is introduced, the assembly system can be self-assembled to form a fibrous collagen bionic material on the premise of ensuring that the intermediate sequence has a triple helix structure.
Since most of natural collagen exists in the form of fibers, the covalent photocrosslinking polypeptide can be self-assembled to form a fiber structure similar to the natural collagen, and can better simulate the structure and function of the natural collagen.
In summary, only the covalent photocrosslinking polypeptides according to the invention: two terminal sequences T1 and T2, N1 and N2 sequences, and a middle sequence M; the middle sequence M is a Gly-Tyr-Hyp sequence capable of providing a middle cross-linking site; said N1 and N2 sequences comprise repeated Gly-Pro-Hyp sequences, and/or repeated Gly-Pro sequences, still more preferably said N1 and N2 comprise at least 3 repeated Gly-Pro-Hyp sequences; the two-terminal sequences T1 and T2 contain different numbers of tyrosines, and preferably, the two-terminal sequences T1 and T2 comprise at least 2 tyrosines and can self-assemble to form the collagen bionic material with a nanofiber structure.
However, although the examples of the present invention take N1 and N2 containing GPO repeat sequences as examples, according to the above results, on the basis of ensuring that the polypeptide sequence has a triple helix structure, the collagen polypeptides obtained by combining the two terminal sequences T and N1 and N2 and the intermediate sequence M of the present invention can self-assemble to form a collagen biomimetic material with a nanofiber structure. Thus, the N1 and N2 may also include GPP (Gly-Pro) repeats having triple helix forming structures and other polypeptide sequences capable of forming triple helix structures.
The above description is only for details of a specific exemplary embodiment of the present invention, and it is obvious to those skilled in the art that various modifications and changes may be made in the present invention in the practical application process according to specific preparation conditions, and the present invention is not limited thereto. All that comes within the spirit and principle of the invention is to be understood as being within the scope of the invention.
Claims (13)
1. A covalent photocrosslinked polypeptide, wherein said covalent photocrosslinked polypeptide is a polypeptide sequence capable of forming a triple helix conformation, and wherein said covalent photocrosslinked polypeptide has the structure of formula (i):
T1-N1-M-N2-T2 is shown as formula (I);
wherein said M comprises at least 1 Gly-Tyr-Yaa, and/or Gly-Xaa-Tyr,
said N1 comprises at least 3 repeats of Gly-Xaa-Yaa, and/or Gly-Xaa-Xaa;
said N2 comprises at least 3 repeats of Gly-Xaa-Yaa, and/or Gly-Xaa-Xaa;
said T1 comprises at least 2 contiguous tyrs;
said T2 comprises at least 2 contiguous tyrs;
xaa and Yaa are any different amino acids.
2. The covalent photocrosslinked polypeptide of claim 1, wherein M further comprises a polypeptide fragment having a biologically active function.
3. The covalent photocrosslinking polypeptide of claim 2, wherein the biologically active functional polypeptide fragment comprises a sequence as set forth in seq id no:
Gly-Phe-Hyp-Gly-Glu-Arg；
and/or Gly-Arg-Hyp-Gly-Glu-Arg;
and/or Gly-Leu-Hyp-Gly-Glu-Arg;
and/or Gly-Met-Hyp-Gly-Glu-Arg;
and/or Gly-Ala-Hyp-Gly-Glu-Arg;
and/or Gly-Leu-Lys-Gly-Glu-Asn;
and/or Gly-Leu-Hyp-Gly-Glu-Asn;
and/or Gly-Val-Met-Gly-Phe-Hyp;
and/or Gly-Pro-Leu-Gly-Ile-Ala-Gly-Ile-Thr-Gly-Ala-Arg;
and/or Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg-Gly-Val-Val;
and/or Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala-Hyp-Gly-Ile-Leu;
and/or Gly-Pro-Gln-Gy-Leu-Ala-Gly-Gln-Arg-Gly-Ile-Val;
and/or Gly-Arg-Hyp-Gly-Lys-Arg-Gly-Lys-Gln-Gly-Gln-Lys;
and/or Gly-Leu-Hyp-Gly-Gln-Arg-Gly-Glu-Arg.
4. The covalent photocrosslinked polypeptide of any one of claims 1-3 wherein Xaa is Pro; the Ya a is Hyp.
5. The covalent photocrosslinked polypeptide of claim 1, wherein N1 is the same as N2.
6. The covalent photocrosslinked polypeptide of claim 5, wherein N1 and N2 are (Gly-Pro-Hyp)nOr (Gly-Pro-Pro)nAnd n is any integer greater than or equal to 3.
7. The covalent photocrosslinked polypeptide of claim 6, wherein n is 3, or 4, or 5.
8. The covalent photocrosslinked polypeptide of claim 4, wherein T1 and T2 are the same.
9. The covalent photocrosslinked polypeptide of claim 8, wherein both T1 and T2 are (Tyr)mAnd m is any integer greater than or equal to 2.
10. The covalent photocrosslinked polypeptide of claim 9, wherein m is 2, or 3, or 4, or 5.
11. Use of a covalent photo-cross-linking polypeptide according to any of claims 1 to 10 in the preparation of a collagen biomimetic material.
12. A collagen biomimetic material formed by self-assembly of the covalent photocrosslinked polypeptide of any of claims 1-10.
13. Use of the collagen biomimetic material according to claim 12 for the preparation of implants, artificial skin, tissue engineering and cosmetology.
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