CN114805631A - High-activity hyaluronic acid-polypeptide conjugate as well as preparation method and application thereof - Google Patents
High-activity hyaluronic acid-polypeptide conjugate as well as preparation method and application thereof Download PDFInfo
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- 230000000694 effects Effects 0.000 title claims abstract description 24
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical class CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 64
- 238000006243 chemical reaction Methods 0.000 claims abstract description 37
- 239000002363 auxin Substances 0.000 claims abstract description 34
- 229930192334 Auxin Natural products 0.000 claims abstract description 31
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims abstract description 31
- VYSYZMNJHYOXGN-UHFFFAOYSA-N ethyl n-aminocarbamate Chemical compound CCOC(=O)NN VYSYZMNJHYOXGN-UHFFFAOYSA-N 0.000 claims abstract description 20
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 19
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 19
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 claims abstract description 18
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- 239000000243 solution Substances 0.000 claims description 48
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 30
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 29
- 238000000108 ultra-filtration Methods 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 18
- 229920002385 Sodium hyaluronate Polymers 0.000 claims description 12
- 229940010747 sodium hyaluronate Drugs 0.000 claims description 12
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 12
- 238000010791 quenching Methods 0.000 claims description 10
- 230000000171 quenching effect Effects 0.000 claims description 10
- 230000035484 reaction time Effects 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 6
- 239000008351 acetate buffer Substances 0.000 claims description 4
- 239000000178 monomer Substances 0.000 claims description 4
- 239000002537 cosmetic Substances 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 239000012279 sodium borohydride Substances 0.000 claims description 3
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 3
- MNWBNISUBARLIT-UHFFFAOYSA-N sodium cyanide Chemical compound [Na+].N#[C-] MNWBNISUBARLIT-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 230000002500 effect on skin Effects 0.000 abstract 1
- 238000006467 substitution reaction Methods 0.000 description 11
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- 238000002983 circular dichroism Methods 0.000 description 4
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
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- 210000004207 dermis Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000007800 oxidant agent Substances 0.000 description 3
- 230000037303 wrinkles Effects 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- 206010003645 Atopy Diseases 0.000 description 2
- 102000002265 Human Growth Hormone Human genes 0.000 description 2
- 108010000521 Human Growth Hormone Proteins 0.000 description 2
- 239000000854 Human Growth Hormone Substances 0.000 description 2
- 102000003839 Human Proteins Human genes 0.000 description 2
- 108090000144 Human Proteins Proteins 0.000 description 2
- 230000003712 anti-aging effect Effects 0.000 description 2
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- 239000007974 sodium acetate buffer Substances 0.000 description 2
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
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- 239000012153 distilled water Substances 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- YSUPRNQWQGBZND-UHFFFAOYSA-N ethyl formate hydrazine Chemical compound C(=O)OCC.NN YSUPRNQWQGBZND-UHFFFAOYSA-N 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
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- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- DKACXUFSLUYRFU-UHFFFAOYSA-N tert-butyl n-aminocarbamate Chemical compound CC(C)(C)OC(=O)NN DKACXUFSLUYRFU-UHFFFAOYSA-N 0.000 description 1
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 230000037317 transdermal delivery Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0072—Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
- A61K8/735—Mucopolysaccharides, e.g. hyaluronic acid; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
Abstract
The invention discloses a high-activity hyaluronic acid-polypeptide conjugate and a preparation method and application thereof. The preparation method of the high-activity hyaluronic acid-polypeptide conjugate comprises the following steps: preparing aldehyde-modified hyaluronic acid; dissolving aldehyde-modified hyaluronic acid in a buffer solution, adding auxin and sodium cyanoborohydride, and reacting; adding ethyl carbazate for reaction; finally obtaining hyaluronic acid-polypeptide conjugate; the technical scheme of the invention can perfectly connect the auxin to the hyaluronic acid, and the combination rate is about 70%; the secondary structure of auxin in the hyaluronic acid-polypeptide conjugate of the invention is not changed; and in skin tissue testing, the hyaluronic acid-polypeptide conjugate can be effectively delivered to the dermal layer.
Description
Technical Field
The invention relates to the technical field of conjugates of hyaluronic acid, in particular to a high-activity hyaluronic acid-polypeptide conjugate and a preparation method and application thereof.
Background
Transdermal delivery systems deliver the active ingredient through the skin, and have the advantages of being painless, self-administering, and disposable at any time.
Korean laser et al provided a method for preparing a hyaluronic acid-protein conjugate, which first converts sodium hyaluronate into aldehyde-modified hyaluronic acid by an oxidation method, then reacts the aldehyde-modified hyaluronic acid with protein in a buffer solution, and then reacts with ethyl carbazate by adding sodium cyanoborohydride. The degree of substitution cannot be stably maintained at a fixed value; the hyaluronic acid-auxin conjugate prepared by this method has a low yield, and a large amount of auxin remains unreacted in the reaction system.
In view of the above, how to improve the substitution stability of the hyaluronic acid conjugate is a problem to be solved by those skilled in the art.
Disclosure of Invention
The main purpose of the embodiments of the present invention is to provide a high-activity hyaluronic acid-polypeptide conjugate, and a preparation method and application thereof, aiming at improving the substitution stability of the hyaluronic acid-polypeptide conjugate.
The technical solution of the present invention for solving the above technical problems is to provide a method for preparing a high-activity hyaluronic acid-polypeptide conjugate, which comprises the steps of:
preparing aldehyde-modified hyaluronic acid;
dissolving aldehyde-modified hyaluronic acid in a buffer solution, adding auxin and sodium cyanoborohydride, and reacting;
adding ethyl carbazate for reaction;
obtaining the hyaluronic acid-polypeptide conjugate.
In one embodiment of the present invention, the buffer solution is an acetate buffer solution;
and/or the pH value of the buffer solution is 5-6.
In an embodiment of the present invention, in the step of dissolving the aldehyde-modified hyaluronic acid in a buffer solution, adding auxin and sodium cyanoborohydride, and reacting, a molar ratio of the aldehyde-modified hyaluronic acid to the auxin is 1: 1-1: 10.
And/or the molar ratio of the aldehyde-modified hyaluronic acid to the sodium cyanoborohydride is 1: 3-1: 7;
and/or after the auxin and the cyano sodium borohydride are added, the reaction time is 20-24 hours under the condition of keeping out of the sun.
In an embodiment of the invention, in the step of adding ethyl carbazate for reaction, the molar ratio of the aldehyde-modified hyaluronic acid to the ethyl carbazate is 1: 3-1: 7;
and/or the reaction time is 20-24 h after the ethyl carbazate is added.
In an embodiment of the present invention, the step of adding ethyl carbazate to perform the reaction further includes:
carrying out ultrafiltration on the solution after reaction;
freezing the washed solution;
wherein the washing solution used in the ultrafiltration is a PB solution.
In one embodiment of the present invention, the step of preparing aldehyde-modified hyaluronic acid comprises:
adding sodium periodate into the dissolved sodium hyaluronate solution for reaction;
adding ethylene glycol to carry out quenching reaction;
aldehyde-modified hyaluronic acid is obtained.
In an embodiment of the present invention, the step of adding sodium periodate to the dissolved sodium hyaluronate solution to perform a reaction includes:
and adding the sodium periodate into the dissolved sodium hyaluronate solution, and stirring for 2-4 hours under the condition of keeping out of the sun.
In an embodiment of the present invention, the step of adding ethylene glycol to perform quenching reaction includes:
adding the ethylene glycol, and quenching for 1-2 h;
after the reaction was stopped, the solution was ultrafiltered.
The invention also provides a high-activity hyaluronic acid-polypeptide conjugate which is prepared by adopting the preparation method of the high-activity hyaluronic acid-polypeptide conjugate.
The invention also provides application of the high-activity hyaluronic acid-polypeptide conjugate prepared by the preparation method of the high-activity hyaluronic acid-polypeptide conjugate in preparing cosmetic, wrinkle removing and plastic preparations.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method shortens the reaction time by changing the dosage of the oxidant, stabilizes the substitution degree of the aldehyde-modified hyaluronic acid at a fixed value, and reduces the production cost;
(2) the hyaluronic acid-polypeptide conjugate prepared by the implementation steps of the invention explores the functional high molecular structure of the characterization hyaluronic acid-auxin conjugate;
(3) in the hyaluronic acid-polypeptide conjugate prepared by the implementation steps of the invention, the auxin is perfectly connected to the hyaluronic acid, the binding rate is about 70%, and the auxin reacts to the maximum extent, so that the production cost is saved;
(4) and the secondary structure of auxin in the hyaluronic acid-polypeptide conjugate is not changed;
(5) through tests on skin tissues, the hyaluronic acid-polypeptide conjugate can be effectively delivered to the dermis layer, and has a good anti-aging effect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a scheme showing the synthesis scheme of the high-activity hyaluronic acid-polypeptide conjugate according to the present invention;
FIG. 2 is a scheme showing the synthesis scheme of aldehyde-modified hyaluronic acid and the aldehyde-modified hyaluronic acid substitution assay according to the present invention;
FIG. 3 is a graph showing the measurement of the degree of substitution of aldehyde-modified hyaluronic acid 1 H-NMR；
FIG. 4 is a GPC analysis chart of the auxin used in example 1;
FIG. 5 is a GPC analysis chart of the HA-hGH conjugate prepared in example 1;
FIG. 6 is a graph showing the number of hGH molecules per HA contained in the HA-hGH conjugate obtained in example 1 and the biological binding rate based on the charged amount of hGH;
FIG. 7 is a graph of comparative Circular Dichroism (CD) analysis of hGH and HA-hGH conjugate obtained in example 1;
FIG. 8 is a comparative analysis chart of transdermal administration activity of HA-hGH conjugate obtained in example 1.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
In the present invention, unless otherwise expressly stated or limited, the terms "connected," "secured," and the like are to be construed broadly, and for example, "secured" may be a fixed connection, a removable connection, or an integral part; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
In addition, the technical solutions in the embodiments of the present invention may be combined with each other, but it must be based on the realization of those skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination of technical solutions should not be considered to exist, and is not within the protection scope of the present invention.
The invention provides a high-activity hyaluronic acid-polypeptide conjugate, a preparation method and application thereof, aiming at improving the substitution stability of the hyaluronic acid-polypeptide conjugate.
The high-activity hyaluronic acid-polypeptide conjugate, the preparation method and the application thereof proposed by the present invention will be described in the following specific examples:
in the technical scheme of the embodiment, the preparation method of the high-activity hyaluronic acid-polypeptide conjugate comprises the following steps:
s10: preparing aldehyde-modified hyaluronic acid;
s20: dissolving aldehyde-modified hyaluronic acid in a buffer solution, adding auxin and sodium cyanoborohydride, and reacting;
s30: adding ethyl carbazate for reaction;
s40: obtaining the hyaluronic acid-polypeptide conjugate.
In step S20, the aldehyde-modified hyaluronic acid is dissolved in a buffer solution (pH 5 to 6) under dark conditions as shown in fig. 1, and then auxin and sodium cyanoborohydride are added to the solution to react at room temperature (18 to 30 ℃) for 20 to 24 hours.
In step S30, as shown in the second part of fig. 1, ethyl carbazate is added to the solution after the reaction in step S20, and the reaction is performed for 24 hours to block unreacted aldehyde groups, which can improve the stability of the product and prevent non-atopic reaction of the hyaluronic acid-polypeptide conjugate with human proteins.
In one embodiment of the present invention, the buffer solution is an acetate buffer solution; and/or the pH value of the buffer solution is 5-6.
It can be understood that the buffer solution in step S20 is acetate buffer solution, and the pH of the buffer solution can be set to 5-6, and when the pH is in the range of 5-6, the protein can be prevented from being denatured, and the reaction between amino acid (e.g. lysine) and aldehyde group can be prevented, so as to obtain the protein with the maximum activity.
In one embodiment of the present invention, in the step of S20, the molar ratio of the aldehyde-modified hyaluronic acid to the auxin is 1:1 to 1: 10.
And/or the molar ratio of the aldehyde-modified hyaluronic acid to the sodium cyanoborohydride is 1: 3-1: 7;
and/or after the auxin and the cyano sodium borohydride are added, the reaction time is 20-24 hours under the condition of keeping out of the sun.
It is understood that, in a solution of aldehyde-modified hyaluronic acid, auxin is added to react with the aldehyde-modified hyaluronic acid, and during the reaction, the protein N-terminus of the aldehyde-modified hyaluronic acid and the auxin reacts to prepare a hyaluronic acid-protein conjugate; the added sodium cyanoborohydride can be used as a reducing agent and can reduce carbon-nitrogen double bonds generated after the aldehyde modified hyaluronic acid reacts with the N-terminal of the protein.
In a possible embodiment, the molar ratio of aldehyde-modified hyaluronic acid to auxin is 1:1 to 1:10, preferably the molar ratio of aldehyde-modified hyaluronic acid to auxin is 1: 1.
In one possible embodiment, the molar ratio of aldehyde-modified hyaluronic acid to sodium cyanoborohydride is 1:3 to 1:7, and preferably the molar ratio of aldehyde-modified hyaluronic acid to sodium cyanoborohydride is 1: 5.
In a feasible embodiment, after the auxin and the sodium cyanoborohydride are added, the reaction time is 20-24 h (preferably 24h) under the condition of avoiding light.
In a feasible embodiment, 1 time of auxin (molar ratio) and 5 times of sodium cyanoborohydride (molar ratio) are added into aldehyde modified hyaluronic acid, and the mixture is reacted for 24 hours under the condition of avoiding light.
In an embodiment of the invention, in the step S30, the molar ratio of the aldehyde-modified hyaluronic acid to the ethyl carbazate is 1:3 to 1: 7;
and/or the reaction time is 20-24 h after the ethyl carbazate is added.
Understandably, the molar ratio of the aldehyde-modified hyaluronic acid to the ethyl carbazate is 1:3 to 1:7, preferably, the molar ratio of the aldehyde-modified hyaluronic acid to the ethyl carbazate is 1: 5; after the ethyl carbazate is added, the reaction time is 20-24 h, preferably 24 h; the ethyl carbazate can be used for blocking unreacted aldehyde groups, improving the stability of the product and preventing the non-atopic reaction of the hyaluronic acid-polypeptide conjugate and human protein.
In an embodiment of the present invention, after the step of S30, the method further includes:
carrying out ultrafiltration on the solution after reaction;
freezing the washed solution;
wherein the washing solution used in the ultrafiltration is a PB solution.
It will be appreciated that after completion of step S30, the reacted solution is subjected to ultrafiltration, which results in a solution of hyaluronic acid-polypeptide conjugate, which is frozen for 1 day and the final product is lyophilized. Wherein, a 50K ultrafiltration membrane is adopted for ultrafiltration, a flushing fluid is a PB solution with the thickness of 5mm,
in an embodiment of the present invention, the step S10 includes:
s11: adding sodium periodate into the dissolved sodium hyaluronate solution for reaction;
s12: adding ethylene glycol to carry out quenching reaction;
s13: aldehyde-modified hyaluronic acid is obtained.
Understandably, dissolving sodium hyaluronate in water to prepare a sodium hyaluronate solution, adding sodium periodate into the solution, stirring for 2h at room temperature (18-30 ℃) in the absence of light, then adding excessive glycol for quenching reaction for 1h, performing ultrafiltration for 4h by using a 5K ultrafiltration membrane, and performing ultrafiltration to obtain aldehyde-modified hyaluronic acid. Wherein, the sodium periodate oxidant can form ring opening at the fixed position of hyaluronic acid, so that the reaction can be better controlled.
In an embodiment of the present invention, the step of S11 includes:
s111: and adding the sodium periodate into the dissolved sodium hyaluronate solution, and stirring for 2-4 hours under the condition of keeping out of the sun.
Understandably, the molar ratio of the hyaluronic acid monomer to the sodium periodate is 1: 1-1: 2, and preferably, the molar ratio of the hyaluronic acid monomer to the sodium periodate is 1: 2; the use of sodium periodate oxidizing agent allows the formation of open loops at fixed positions of hyaluronic acid, allowing a better control of the reaction.
In an embodiment of the present invention, the step of S12 includes:
s121: adding the ethylene glycol, and quenching for 1-2 h;
s122: after the reaction was stopped, the solution was ultrafiltered.
It is to be understood that, in step S121, ultrafiltration is performed using distilled water, thereby purifying the product.
The invention also provides a high-activity hyaluronic acid-polypeptide conjugate which is prepared by adopting the preparation method of the high-activity hyaluronic acid-polypeptide conjugate.
It can be understood that the hyaluronic acid-polypeptide conjugate prepared by the above method is a novel functional polymer structure, and the auxin can be perfectly linked to the hyaluronic acid, and the binding rate is about 70%, so that the auxin is maximally reacted, thereby saving the production cost.
The invention also provides application of the high-activity hyaluronic acid-polypeptide conjugate prepared by the preparation method of the high-activity hyaluronic acid-polypeptide conjugate in preparing cosmetic, wrinkle removing and reshaping preparations.
Understandably, the hyaluronic acid-auxin conjugate prepared by the method has good anti-aging effect when being applied to human skin through a model; can be used for preparing skin caring, wrinkle removing, and shaping preparations.
Example 1:
as shown in the first part of fig. 2, aldehyde-modified hyaluronic acid (HA-ALD) was prepared: weighing 5.00g of hyaluronic acid, adding into a 1000ml flask, adding 500ml of water, stirring until dissolving, and preparing into 10mg/ml solution;
under the condition of keeping out of the sun, adding sodium periodate which is 2 times of the hyaluronic acid monomer, and quickly stirring for reaction for 2 hours;
adding 5g of ethylene glycol to carry out quenching reaction for 1 h;
after the reaction is stopped, performing ultrafiltration for 4 hours by using a 5k ultrafiltration membrane to obtain aldehyde-modified hyaluronic acid after ultrafiltration; wherein, as shown in the second part of FIG. 2, the measurement of the degree of substitution of aldehyde-modified hyaluronic acid: 0.50g of hyaluronic acid modified with aldehyde obtained by ultrafiltration was weighed out, and 100ml of acetic acid-sodium acetate buffer solution (100mM, pH 5.2) was added to prepare a solution of 5 mg/ml. 0.38g of sodium cyanoborohydride which is 5 times the amount of hyaluronic acid and 0.83g of tert-butyl carbazate are added to react for 24 hours in a dark place. The resulting product was packed into a dialysis membrane (10K) and dialyzed against a large amount of water for one day. After dialysis, the mixture was lyophilized for one day. Measuring 1H-NMR of the lyophilized product, and judging the substitution degree by nuclear magnetic results, wherein the part with chemical shift of 2ppm is an acetyl methyl peak as shown in FIG. 3; the substitution degree of the tert-butyloxycarbonyl peak at 1.4ppm is 10%, and the yield of the aldehyde-modified hyaluronic acid obtained after ultrafiltration is more than 85%.
As shown in fig. 1, a hyaluronic acid-polypeptide conjugate was prepared: weighing 1g of the aldehyde-modified hyaluronic acid prepared above, adding 100mm acetic acid-sodium acetate buffer (pH 5.5) to prepare a solution of 5 mg/ml;
adding auxin stock solution with the molar ratio of 1 time of that of aldehyde modified hyaluronic acid, adding sodium cyanoborohydride with the molar ratio of 5 times of that of aldehyde modified hyaluronic acid, and reacting for 24 hours in the dark;
then adding 5 times of hydrazine ethyl formate in molar ratio of aldehyde modified hyaluronic acid, and reacting for 24 hours;
performing ultrafiltration on the product obtained after the reaction by using an ultrafiltration membrane (50K) for 4 hours; ultrafiltering to obtain hyaluronic acid-polypeptide (HA-hGH) conjugate solution, lyophilizing the solution for 24h, and obtaining the final product.
As shown in FIG. 4, the retention time of human growth hormone was measured to be 41.340min, and the HA-hGH conjugate was measured, and as shown in FIG. 5, the retention time of HA-hGH conjugate was found to be 24.700 min; by comparing the retention time of human auxin in FIG. 4 with that of the product HA-hGH conjugate obtained from the present application in FIG. 5, it can be seen that the HA-hGH conjugate is 16min shorter than the retention time of human auxin, indicating that the auxin (hGH) HAs been attached to the Hyaluronic Acid (HA).
As shown in FIG. 6, the biological binding rate of HA-hGH conjugate was deduced from the peak area of hGH peak in GPC chart of the product of this example. According to calculation, when the feeding molar ratio of the hGH to the HA is 1, 3 and 6 respectively, the corresponding biological binding rate is about 70%.
As shown in FIG. 7, by determining the CD patterns of HA-hGH and hGH, it can be seen that the CD patterns of HA-hGH and hGH are substantially similar, indicating that the secondary structure of human growth hormone in the HA-hGH conjugate in this example is not changed.
As shown in fig. 8, the product HA-hGH conjugate was tested for transdermal administration; set up 5 sets of comparative tests, a: fluorescence microscopy of dorsal skin tissue of FITC-treated mice, B: fluorescence microscopy images of PBS (blank control) -treated mouse dorsal skin tissue, C: fluorescence microscopy images of dorsal skin tissue of hGH-FITC treated mice, D: fluorescence microscopy images of dorsal skin tissue of HA-FITC treated mice, E: fluorescence microscopy images of dorsal skin tissue of HA-hGH-FITC treated mice; it can be seen that hGH is not directly trans-epidermal, but the HA-hGH conjugate after attachment to HA can pass through the epidermis and be efficiently delivered to the dermis layer, i.e. the HA-hGH conjugate is efficiently delivered to the dermis layer.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (10)
1. A method for preparing a high-activity hyaluronic acid-polypeptide conjugate, which comprises the following steps:
preparing aldehyde-modified hyaluronic acid;
dissolving aldehyde-modified hyaluronic acid in a buffer solution, adding auxin and sodium cyanoborohydride, and reacting;
adding ethyl carbazate for reaction;
obtaining the hyaluronic acid-polypeptide conjugate.
2. The method of preparing the highly active hyaluronic acid-polypeptide conjugate according to claim 1, wherein the buffer solution is an acetate buffer solution;
and/or the pH value of the buffer solution is 5-6.
3. The method of preparing a highly active hyaluronic acid-polypeptide conjugate according to claim 1, wherein the step of dissolving the aldehyde-modified hyaluronic acid in a buffer solution, adding auxin and sodium cyanoborohydride, and reacting the mixture in a molar ratio of 1: 1-1: 10.
And/or the molar ratio of the aldehyde-modified hyaluronic acid to the sodium cyanoborohydride is 1: 3-1: 7;
and/or after the auxin and the cyano sodium borohydride are added, the reaction time is 20-24 hours under the condition of keeping out of the sun.
4. The method for preparing a highly active hyaluronic acid-polypeptide conjugate according to claim 1, wherein in the step of adding ethyl carbazate to perform the reaction, the molar ratio of the aldehyde-modified hyaluronic acid to the ethyl carbazate is 1:3 to 1: 7;
and/or the reaction time is 20-24 h after the ethyl carbazate is added.
5. The method of preparing a highly active hyaluronic acid-polypeptide conjugate according to claim 1, wherein the step of adding ethyl carbazate and performing the reaction further comprises:
carrying out ultrafiltration on the solution after reaction;
freezing the washed solution;
wherein the washing solution used in the ultrafiltration is a PB solution.
6. The method of preparing a highly active hyaluronic acid-polypeptide conjugate according to claim 1, wherein the step of preparing the aldehyde-modified hyaluronic acid comprises:
adding sodium periodate into the dissolved sodium hyaluronate solution for reaction;
adding ethylene glycol to carry out quenching reaction;
aldehyde-modified hyaluronic acid is obtained.
7. The method of preparing a highly active hyaluronic acid-polypeptide conjugate according to claim 6, wherein the step of adding sodium periodate to the dissolved sodium hyaluronate solution and reacting comprises:
adding sodium periodate into the dissolved sodium hyaluronate solution, and stirring for 2-4 hours in a dark condition;
the molar ratio of the hyaluronic acid monomer to the sodium periodate in the sodium hyaluronate solution is 1: 1-1: 2.
8. The method of preparing a highly active hyaluronic acid-polypeptide conjugate according to claim 6, wherein the step of adding ethylene glycol and performing a quenching reaction comprises:
adding the ethylene glycol, and quenching for 1-2 h;
after the reaction was stopped, the solution was ultrafiltered.
9. A high-activity hyaluronic acid-polypeptide conjugate prepared by the method for preparing the high-activity hyaluronic acid-polypeptide conjugate according to any one of claims 1 to 8.
10. Use of the highly active hyaluronic acid-polypeptide conjugate prepared by the method of any one of claims 1 to 8 for preparing a cosmetic, wrinkle-removing, or plastic preparation.
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