CN117229423B - Polypeptide nano material for binding collagen and preparation method and application thereof - Google Patents
Polypeptide nano material for binding collagen and preparation method and application thereof Download PDFInfo
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- 102000008186 Collagen Human genes 0.000 title claims abstract description 98
- 108010035532 Collagen Proteins 0.000 title claims abstract description 98
- 229920001436 collagen Polymers 0.000 title claims abstract description 98
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 64
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 58
- 229920001184 polypeptide Polymers 0.000 title claims abstract description 56
- 239000002086 nanomaterial Substances 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 55
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 22
- 239000011347 resin Substances 0.000 claims description 22
- 229920005989 resin Polymers 0.000 claims description 22
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 19
- 238000001514 detection method Methods 0.000 claims description 19
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 16
- 230000008878 coupling Effects 0.000 claims description 16
- 238000010168 coupling process Methods 0.000 claims description 16
- 238000005859 coupling reaction Methods 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 14
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 claims description 12
- SJRJJKPEHAURKC-UHFFFAOYSA-N N-Methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 claims description 12
- 229940024606 amino acid Drugs 0.000 claims description 12
- 150000001413 amino acids Chemical class 0.000 claims description 12
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 claims description 12
- 239000001257 hydrogen Substances 0.000 claims description 12
- 229910052739 hydrogen Inorganic materials 0.000 claims description 12
- DBTMQODRSDEGRZ-UHFFFAOYSA-N 9h-fluoren-9-ylmethyl n-(2-oxoethyl)carbamate Chemical compound C1=CC=C2C(COC(=O)NCC=O)C3=CC=CC=C3C2=C1 DBTMQODRSDEGRZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000010511 deprotection reaction Methods 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 230000015572 biosynthetic process Effects 0.000 claims description 9
- 239000007822 coupling agent Substances 0.000 claims description 9
- 238000007789 sealing Methods 0.000 claims description 9
- 210000002966 serum Anatomy 0.000 claims description 9
- 230000008685 targeting Effects 0.000 claims description 9
- 238000003786 synthesis reaction Methods 0.000 claims description 8
- 239000003795 chemical substances by application Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 238000009835 boiling Methods 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 125000006239 protecting group Chemical group 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 230000008961 swelling Effects 0.000 claims description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 6
- 239000006166 lysate Substances 0.000 claims description 5
- 238000010532 solid phase synthesis reaction Methods 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 238000010438 heat treatment Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- VSTXCZGEEVFJES-UHFFFAOYSA-N 1-cycloundecyl-1,5-diazacycloundec-5-ene Chemical compound C1CCCCCC(CCCC1)N1CCCCCC=NCCC1 VSTXCZGEEVFJES-UHFFFAOYSA-N 0.000 claims description 3
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 239000003517 fume Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 claims description 3
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 3
- 239000012498 ultrapure water Substances 0.000 claims description 3
- 238000001291 vacuum drying Methods 0.000 claims description 3
- -1 benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate Chemical compound 0.000 claims description 2
- 238000007664 blowing Methods 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims description 2
- 125000004122 cyclic group Chemical group 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 229960002989 glutamic acid Drugs 0.000 claims description 2
- 239000011259 mixed solution Substances 0.000 claims description 2
- 230000007017 scission Effects 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims description 2
- 239000012351 deprotecting agent Substances 0.000 claims 1
- 238000005303 weighing Methods 0.000 claims 1
- 230000003993 interaction Effects 0.000 abstract description 15
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 210000001772 blood platelet Anatomy 0.000 description 32
- 108010047303 von Willebrand Factor Proteins 0.000 description 31
- 102100036537 von Willebrand factor Human genes 0.000 description 31
- 229960001134 von willebrand factor Drugs 0.000 description 31
- 125000003275 alpha amino acid group Chemical group 0.000 description 7
- 230000002401 inhibitory effect Effects 0.000 description 6
- 108010044426 integrins Proteins 0.000 description 6
- 102000006495 integrins Human genes 0.000 description 6
- 239000002105 nanoparticle Substances 0.000 description 6
- 102100038394 Platelet glycoprotein VI Human genes 0.000 description 5
- 101710194982 Platelet glycoprotein VI Proteins 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000000329 molecular dynamics simulation Methods 0.000 description 4
- 108020003175 receptors Proteins 0.000 description 4
- 102000005962 receptors Human genes 0.000 description 4
- 230000003143 atherosclerotic effect Effects 0.000 description 3
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- 210000004623 platelet-rich plasma Anatomy 0.000 description 3
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
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- 238000001338 self-assembly Methods 0.000 description 2
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- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- OTKXCALUHMPIGM-FQEVSTJZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 OTKXCALUHMPIGM-FQEVSTJZSA-N 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 208000037260 Atherosclerotic Plaque Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108010017642 Integrin alpha2beta1 Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000004630 atomic force microscopy Methods 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
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- 230000000694 effects Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000000879 optical micrograph Methods 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 229940043267 rhodamine b Drugs 0.000 description 1
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- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention belongs to the technical field of nano material preparation, and particularly relates to a polypeptide nano material for combining collagen, and a preparation method and application thereof. The polypeptide nanomaterial has a structure shown in a formula I;formula I. The beneficial effects of the invention are as follows: by adopting the technical scheme, the polypeptide nanomaterial provided by the invention has strong interaction with collagen, and can form steric hindrance at the collagen, so that the adhesion of platelets on the collagen is inhibited, and the polypeptide nanomaterial can inhibit the adhesion of 75% of platelets on the collagen.
Description
Technical Field
The invention belongs to the technical field of nano material preparation, and particularly relates to a polypeptide nano material for combining collagen, and a preparation method and application thereof.
Background
There is growing evidence that platelets play an important role in atherosclerotic lesions. The massive aggregation of platelets at atherosclerotic plaques leads to thrombus formation and thus promotes the development of atherosclerotic disease. Inhibiting adhesion of blood platelet at collagen of atherosclerosis lesion has important significance in delaying disease development. At the site of atherosclerotic lesions, collagen exposure at the vessel wall is caused by damage to the vessel wall. Platelet surface receptor protein vi binds to collagen and activates platelets, and the activated platelet surface produces new receptor proteins, such as integrin α2β1, which can bind directly to collagen. In addition, the newly generated receptor protein glycoprotein Ib-IX-V of activated platelets binds to collagen by virtue of von Willebrand factor (vWF) released by inflammatory endothelial cells. The A3 domain of vWF will bind to collagen first and vWF will be deployed under the action of blood flow leaving the A1 domain exposed and the receptor proteins, the lb-ix-v and A1 domain, on the platelet surface will bind to allow the platelets to adhere to collagen. Currently, drugs that inhibit platelet aggregation at collagen are common, and drugs that inhibit platelet adhesion to collagen directly from the root have been studied relatively rarely.
Disclosure of Invention
The invention discloses a polypeptide nanomaterial for combining collagen and a preparation method and application thereof, which are used for solving any one of the problems in the prior art and potential problems.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows: a polypeptide nanomaterial for binding collagen, the polypeptide nanomaterial having a structure of formula i:
i
Wherein R is 1 Is a polypeptide sequence with multiple intramolecular hydrogen bonds, R 2 For binding to collagen targeting peptides, R 1 And R is R 2 Is connected with each other through an amide bond.
Further, the R 1 The structure is as follows:
。
further, the R 2 The structure of the collagen-binding targeting peptide is:
、
or (b)
。
Another object of the present invention is to provide a method for preparing the above-mentioned polypeptide nanomaterial for binding collagen, which is a solid-phase synthesis method of polypeptide.
Further, the polypeptide solid-phase synthesis method specifically comprises the following steps:
s1) swelling resin: swelling Fmoc-Gly-Wang resin with N, N-dimethylformamide for 4-6h;
s2) deprotection: after the methylene dichloride and the N, N-dimethylformamide solvent are alternately and repeatedly washed for a plurality of times, 7-8mL of deprotection agent is added, and the shaking table is used for shaking for 15-20min, so that Fmoc groups of amino acid are completely removed;
s3) detection: alternately flushing the centrifugal tube with dichloromethane and N, N-dimethylformamide for multiple times, adding a Carisser detection reagent and a small amount of Fmoc-Gly-Wang resin into a 1.5mL centrifugal tube, and heating the centrifugal tube in boiling water for 1-2min; if the Fmoc-Gly-Wang resin particles turn purple, indicating that Fmoc protecting groups are removed, and repeating the step S2) if the purple is shallow or the resin does not turn purple until the Fmoc is completely removed;
s4) coupling: fmoc-O-tertiary butyl-L-glutamic acid and benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate which are 10 times equivalent to Fmoc-Gly-Wang resin are weighed in a 15mL centrifuge tube, added into a 8-9mL shaking table for pre-reaction for 10-15min, added into a polypeptide synthesis tube and placed on the shaking table for reaction for 1-2h;
s5) detection: alternately flushing with dichloromethane and N, N-dimethylformamide for multiple times, adding a card detection reagent and a small amount of resin into a 1.5mL centrifuge tube, and heating the centrifuge tube in boiling water for 1-2min; if Fmoc-Gly-Wang resin color is not changed, the amino acid coupling is proved to be successful, and if the color is changed to purple, S4) is repeated;
s6) cyclic coupling: repeating S2), S3), S4) and S5), completing the coupling of all amino acids, and removing Fmoc protecting groups by using a deprotection agent after detecting that the last amino acid is completely coupled;
s7) detection: repeating S2);
s8) coupling dodecanoic acid: drying decanoic acid-COOH in a vacuum drying oven in the absence of light, adding a coupling agent, pre-reacting on a shaking table for 10-15min, pouring into a polypeptide synthesis tube, adding sealing films on the upper and lower openings of the polypeptide tube, wrapping with tinfoil paper, placing into a bag, and reacting on the shaking table for 1d;
s9) detection: repeating S5);
s10) shrinkage: shrinking resin in methanol for 15-20min, and transferring the shrunk resin into a serum bottle;
s11) cleavage: adding 3mL of lysate into serum bottle containing resin, magnetically stirring in ice bath for 3-4 hr, suction filtering, rinsing serum bottle with trifluoroacetic acid, blowing trifluoroacetic acid to residual small amount of liquid with nitrogen, adding glacial ethyl ether, transferring polypeptide into centrifuge tube at 4deg.C at 8000 r.min -1 Centrifuging, repeatedly washing with glacial ethyl ether for multiple times, transferring the polypeptide to a 1.5mL centrifuge tube, sealing with a sealing film, placing in a fume hood overnight to volatilize the ethyl ether, and storing at-20deg.C.
Further, the deprotection agent is a mixed solution of N, N-dimethylformamide and 1, 8-diazabicyclo undec-7-ene, and the mass ratio of the N, N-dimethylformamide to the 1, 8-diazabicyclo undec-7-ene is 98:2.
Further, the coupling agent is a mixture of N, N-dimethylformamide and N-methylmorpholine, and the mass ratio between the N, N-dimethylformamide and the N-methylmorpholine is 19:1.
Further, the lysate comprises: 2.5% of ultrapure water, 2.5% of triisopropylsilane and the balance of trifluoroacetic acid.
Use of said polypeptide nanomaterial for binding collagen in a product for inhibiting platelet binding to collagen.
The beneficial effects of the invention are as follows: by adopting the technical scheme, the polypeptide nanomaterial provided by the invention has strong interaction with collagen, and can form steric hindrance at the collagen, so that the adhesion of platelets on the collagen is inhibited. Among them, BPa and collagen have the strongest interaction and are used as polypeptide nanomaterials for inhibiting adhesion of platelets at collagen. BPa nanomaterial inhibits 75% of platelets from adhering to collagen.
Drawings
FIG. 1 is a statistical plot of the number of hydrogen bonds between collagen and integrin alpha 2I domain/GPVI/vWF A3 domain of example 2;
FIG. 2 is a statistical plot of the interaction between collagen and integrin alpha 2I domain/GPVI/vWF A3 domain of example 2;
FIG. 3 is a graph of the break force between collagen and integrin alpha 2I domain/GPVI/vWF A3 domain of example 2;
FIG. 4 is a graph showing the adhesion profile between collagen and integrin alpha 2I domain/GPVI/vWF A3 domain of example 2;
FIG. 5 is a statistical plot of the number of hydrogen bonds between collagen and vWF A3 domain/amino acid sequences in example 3;
FIG. 6 is a graph showing the statistics of interactions between collagen and vWF A3 domain/amino acid sequences in example 3;
FIG. 7 is a statistical chart showing the number of hydrogen bonds between collagen and BPa/BPb/BPc in example 4;
FIG. 8 is a statistical plot of the interaction energy between collagen and BPa/BPb/BPc in example 4;
FIG. 9 is a graph showing the breaking force between collagen and BPa/BPb/BPc in example 4;
FIG. 10 is a graph showing the adhesion force distribution between collagen and BPa/BPb/BPc in example 4;
FIG. 11 is a graph showing the fluorescence intensity of BPa/BPb/BPc attached to collagen in example 4;
FIG. 12 is a graph showing the concentration statistics of BPa/BPb/BPc attached to collagen in example 4;
FIG. 13 is a statistical plot of the number of hydrogen bonds between collagen and vWF A3 domains in the control and experimental groups of example 5;
FIG. 14 is a statistical plot of the interaction between collagen and vWF A3 domains in the control and experimental groups of example 5;
FIG. 15 is a graph of the solvent accessible surface area of vWF A3 domain in the control and experimental groups of example 5;
FIG. 16 is a graph of root mean square error of vWF A3 domain in the control and experimental groups of example 5;
FIG. 17 is an optical micrograph of platelets attached to collagen in the control and experimental groups of example 5;
FIG. 18 is a statistical chart of the number of platelets adhered to collagen in the control and experimental groups of example 5;
FIG. 19 is an ultraviolet spectrum of platelets adhered to collagen in the control and experimental groups of example 5.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The corresponding materials and starting materials in the examples described below are available from other commercial sources without specific reference thereto.
The invention relates to a polypeptide nanomaterial for binding collagen, which has a structure shown in a formula I:
i
Wherein R is 1 Is self-assembled unit, is a polypeptide sequence with multiple hydrogen bonds in molecule, R 2 Targeting peptide for binding collagen, dodecanoic acid and R 1 Is connected by an amide bond, R 1 And R is R 2 Is connected with each other through an amide bond.
The polypeptide nanomaterial for combining collagen provided by the invention comprises a self-assembly unit with dodecanoic acid and strong hydrogen bond acting force and a targeting peptide for combining collagen, wherein the polypeptide nanomaterial can be self-assembled to form nano particles and realize the function of targeting collagen.
Preferably, the donor of the dodecanoic acid in the polypeptide nanomaterial for inhibiting adhesion of platelets to collagen is a compound having a structure shown in formula ii:
II type
The self-assembly with strong hydrogen bonding forcesUnit R 1 The structure is as follows:
the collagen-binding targeting peptide R 2 The structure of (2) is as follows:
or (b)
Or (b)
。
Preferably, the polypeptide nanomaterial for inhibiting adhesion of platelets to collagen described by formula i is of the structure of formula a, formula b and formula c:
a, a
B
Formula c.
Example 1
This example provides a polypeptide nanomaterial for binding collagen having the structure shown below (formulas a, b, and c):
a, a
B
C, a step of
That is, in the structure shown in formula I, R 1 Is of the structure GNNQQNY, R 2 Is of the structure SITTIDV (hereinafter referred to as BPa), VDVMQRE (hereinafter referred to as BPb), or YLTSEMH (hereinafter referred to as BPc).
The polypeptide nano material is prepared by a polypeptide solid-phase synthesis method, and comprises the following steps:
1. swelling resin: swelling Fmoc-Gly-Wang Resin with N, N-dimethylformamide for 4-6h;
2. deprotection: after 3 times of alternate and repeated washing with DCM and DMF solvents, 7-8mL of deprotection agent (DMF: DBU=98:2) is added, and the mixture is shaken on a shaker for 15min to completely remove Fmoc groups of amino acids;
3. and (3) detection: the tubes were alternately washed 3 times with DCM and DMF solvents, kaiser detection reagent and a small amount of resin were added to 1.5mL centrifuge tubes, and the tubes were heated in boiling water for 1min. If the resin particles turn purple, the Fmoc protecting group is indicated to be removed, and if the purple is shallow or the resin does not turn purple, the step 2 is repeated until the Fmoc is completely removed;
4. coupling: fmoc-Glu (OtBu) -OH and HBTU with 10 times equivalent weight of relative resin load are weighed into a 15mL centrifuge tube, 8-9mL of coupling agent (DMF: N-methylmorpholine=19:1) is added to the centrifuge tube for pre-reaction for 10min, then the coupling agent is added into a polypeptide synthesis tube, and the polypeptide synthesis tube is placed on the shaker for reaction for 1h;
5. and (3) detection: the tube was alternately washed 3 times with DCM and DMF, kaiser detection reagent and a small amount of resin were added to a 1.5mL centrifuge tube, and the tube was heated in boiling water for 1min. If the color of the resin is not changed, the amino acid coupling is proved to be successful, and if the color is changed into purple, the step 4 is repeated;
6. and (3) cycle coupling: repeating the steps 2, 3, 4 and 5, completing the coupling of all amino acids, and removing Fmoc protecting groups by using a deprotection agent after detecting the complete coupling of the last amino acid;
7. and (3) detection: repeating the second step;
8. coupling of dodecanoic acid: the decanoic acid-COOH is firstly placed in a vacuum drying box and dried for 10 hours in a dark place. Adding a coupling agent, pre-reacting for 10min on a shaking table, pouring into a polypeptide synthesis tube, adding sealing films at the upper and lower openings of the polypeptide tube, wrapping with tinfoil paper, placing into a bag, and reacting on the shaking table for 1d;
9. and (3) detection: repeating the fifth step;
10. shrinkage: shrinking the resin in methanol for 15min, and transferring the shrunk resin into a 10mL serum bottle;
11. cracking: 3mL of the lysate (2.5% ultrapure water+2.5% triisopropylsilane+95% trifluoroacetic acid) was added to the serum bottle containing the resin, magnetically stirred in an ice bath for 3 hours, suction-filtered, the serum bottle was rinsed with TFA, after which the TFA was purged with nitrogen to a small residual amount of liquid, and then iced diethyl ether was added, and the polypeptide was transferred to a centrifuge tube at 4℃at 8000 r.min -1 Centrifuging, repeatedly washing with glacial ethyl ether for three times, transferring the polypeptide to a 1.5mL centrifuge tube, sealing with a sealing film, placing in a fume hood overnight to volatilize the ethyl ether, and storing at-20deg.C.
Example 2
This example demonstrates the interaction between collagen and integrin alpha 2I domain/GPVI/vWF A3 domain by molecular dynamics simulation and atomic force microscopy. The number of hydrogen bonds formed between the collagen and vWF A3 domains (fig. 1), the total interaction energy (fig. 2), the breaking force (fig. 3) and the adhesion force (fig. 4) are all highest, followed by the collagen and integrin α2i domain, and finally by the collagen and GP vi. The results of this example demonstrate the strongest interaction between collagen and vWF A3 domain, further that platelets adhere to collagen primarily in dependence on vWF action.
Example 3
The present example uses molecular dynamics modeling to find amino acid sequences on vWF A3 domains that play a major role in collagen binding (SITTIDV, VDVMQRE, and yl tsemh). The number of bonds and interactions between collagen and the amino acid sequence we found are substantially identical to those between vWF A3 domains (fig. 5 and 6), indicating that the amino acid sequence we found is the major amino acid fragment on vWF A3 domains that binds to collagen.
Example 4
In this example, three polypeptide nanomaterials, BPa, BPb and BPc, in example 1 were constructed as collagen-targeting nanoparticles using the amino acid sequence found in example 3, which has a strong interaction with collagen, as a targeting peptide for binding to collagen. The interactions between polypeptides and collagen were studied using molecular dynamics modeling and experimental methods. The number of hydrogen bonds formed between collagen and BPa, as shown in fig. 7, the total interaction energy, as shown in fig. 8, the breaking force, as shown in fig. 9, and the adhesion force, as shown in fig. 10, are all highest, followed by between collagen and BPc, and finally between collagen and BPb. In addition, after rhodamine B is modified on the three polypeptide molecules, the three polypeptides are respectively cultured together with collagen, and then the polypeptides which are not combined with the collagen are filtered out through a filter membrane. The fluorescence intensity of the polypeptide attached to the collagen was measured by a fluorescence spectrometer as shown in fig. 11, and the concentration of the polypeptide bound to the collagen was measured by an ultraviolet-visible absorption spectrometer as shown in fig. 12, and the result showed that BPa was the most attached to the collagen, BPc was the next most attached to the collagen, and BPb was the least attached to the collagen.
Example 5
The effect of BPa in inhibiting platelet adhesion to collagen was studied by molecular dynamics simulation and experimental methods in this example. In the simulations we constructed a control group containing only vWF A3 domain and collagen, while constructing an experimental group containing vWF A3 domain, collagen and BPa nanoparticles. The number of hydrogen bonds between collagen and vWF A3 domains was significantly reduced in the experimental group compared to the control group (fig. 13). The calculated total interaction energy between collagen and vWF A3 domain in the control group was also much greater than in the experimental group (fig. 14). In addition, the solvent accessible surface area of vWF A3 domains in the control and experimental groups was reduced from 890nm to 830nm and 880nm respectively (fig. 15). Further, the root mean square error of vWF A3 domains in the experimental group was greater than that in the control group (fig. 16), indicating that there was more vWF A3 domain and collagen binding in the control group, while BPa added to the experimental group blocked vWF A3 domain and collagen binding, and thus more vWF A3 domain was exposed to free movement in aqueous solution. These findings indicate that BPa nanoparticles can block vWF A3 domain binding to collagen.
In addition, experimental methods have also been used to demonstrate the above conclusions. We constructed gelatin micro-channels (5 mm inside diameter) and spread collagen on the inner walls of the channels. In the control group, only Platelet Rich Plasma (PRP) was injected into the channel by the microfluidic method, whereas in the experimental group, PRP and BPa nanoparticles were injected into the channel simultaneously by the microfluidic method. We then stained platelets within the channels that adhered to collagen by rayleigh's staining and observed the number of platelet adhesion in the channels of the control and experimental groups using an optical microscope. Platelets in the channel that adhere to collagen are then quantified using a platelet count method.
In the control group, more platelets adhered to collagen, whereas the experimental group to which BPa was added was observed, the number of platelets adhered to collagen was significantly reduced (fig. 17). The amount of platelets adhered to collagen in the control group and the experimental group was found to be 176×10, respectively 9 L and 40X 10 9 L (FIG. 18). In addition, UV-visible absorption spectroscopy was also used to study platelet adhesion at the collagen site, and the platelet-adhered collagen channels obtained in the control and experimental groups were centrifuged in normal saline. Shaking the centrifuged solution uniformly for ultraviolet spectrum detection. Platelets exhibit an absorption peak at a wavelength of 280nm (graph19 While the absorbance of platelets in the control group was much greater than that of the experimental group. Simulation and experimental results of this example demonstrate that BPa nanoparticles can effectively inhibit platelet adhesion at collagen.
While the foregoing embodiments have been described in detail in connection with the embodiments of the invention, it should be understood that the foregoing embodiments are merely illustrative of the invention and are not intended to limit the invention, and any modifications, additions, substitutions and the like made within the principles of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A polypeptide nanomaterial for binding collagen, the polypeptide nanomaterial having a structure of formula i:
i
Wherein R is 1 Is a polypeptide sequence with multiple intramolecular hydrogen bonds, R 2 For binding to collagen targeting peptides, R 1 And R is R 2 Are connected through an amide bond;
the R is 1 The structure is as follows:
；
the R is 2 The structure of the collagen-binding targeting peptide is:
。
2. a method for preparing the polypeptide nanomaterial of claim 1, wherein the method is a solid phase synthesis method of the polypeptide.
3. The preparation method according to claim 2, wherein the polypeptide solid-phase synthesis method specifically comprises the following steps:
s1) swelling resin: swelling Fmoc-Gly-Wang resin with N, N-dimethylformamide for 4-6h;
s2) deprotection: after the methylene dichloride and the N, N-dimethylformamide solvent are alternately and repeatedly washed for a plurality of times, 7-8mL of deprotection agent is added, and the shaking table is used for shaking for 15-20min, so that Fmoc groups of amino acid are completely removed;
s3) detection: alternately flushing the centrifugal tube with dichloromethane and N, N-dimethylformamide for multiple times, adding a Carisser detection reagent and a small amount of Fmoc-Gly-Wang resin into a 1.5mL centrifugal tube, and heating the centrifugal tube in boiling water for 1-2min; if the Fmoc-Gly-Wang resin particles turn purple, indicating that Fmoc protecting groups are removed, and repeating the step S2) if the purple is shallow or the resin does not turn purple until the Fmoc is completely removed;
s4) coupling: weighing Fmoc-O-tertiary butyl-L-glutamic acid and benzotriazole-N, N, N ', N' -tetramethylurea hexafluorophosphate which are 10 times equivalent relative to Fmoc-Gly-Wang resin load, adding 8-9mL of coupling agent into a 15mL centrifuge tube, pre-reacting for 10-15min on a shaking table, adding the coupling agent into a polypeptide synthesis tube, and placing the polypeptide synthesis tube on the shaking table for reacting for 1-2h;
s5) detection: alternately flushing with dichloromethane and N, N-dimethylformamide for multiple times, adding a card detection reagent and a small amount of resin into a 1.5mL centrifuge tube, and heating the centrifuge tube in boiling water for 1-2min; if Fmoc-Gly-Wang resin color is not changed, the amino acid coupling is proved to be successful, and if the color is changed to purple, S4) is repeated;
s6) cyclic coupling: repeating S2), S3), S4) and S5), completing the coupling of all amino acids, and removing Fmoc protecting groups by using a deprotection agent after detecting that the last amino acid is completely coupled;
s7) detection: repeating S2);
s8) coupling dodecanoic acid: drying decanoic acid-COOH in a vacuum drying oven in the absence of light, adding a coupling agent, pre-reacting on a shaking table for 10-15min, pouring into a polypeptide synthesis tube, adding sealing films on the upper and lower openings of the polypeptide tube, wrapping with tinfoil paper, placing into a bag, and reacting on the shaking table for 1d;
s9) detection: repeating S5);
s10) shrinkage: shrinking resin in methanol for 15-20min, and transferring the shrunk resin into a serum bottle;
s11) cleavage: adding 3mL of lysate into serum bottle containing resin, magnetically stirring in ice bath for 3-4 hr, suction filtering, rinsing serum bottle with trifluoroacetic acid, blowing trifluoroacetic acid to residual small amount of liquid with nitrogen, adding glacial ethyl ether, transferring polypeptide into centrifuge tube at 4deg.C at 8000 r.min -1 Centrifuging, repeatedly washing with glacial ethyl ether for multiple times, transferring the polypeptide to a 1.5mL centrifuge tube, sealing with a sealing film, placing in a fume hood overnight to volatilize the ethyl ether, and storing at-20deg.C.
4. A method according to claim 3, wherein the deprotecting agent is a mixed solution of N, N-dimethylformamide and 1, 8-diazabicyclo undec-7-ene in a mass ratio of 98:2.
5. A process according to claim 3, wherein the coupling agent is a mixture of N, N-dimethylformamide and N-methylmorpholine, the mass ratio therebetween being 19:1.
6. A method of preparing according to claim 3, wherein the lysate comprises: 2.5% of ultrapure water, 2.5% of triisopropylsilane and the balance of trifluoroacetic acid.
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