Detailed Description
To make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some, but not all embodiments of the present invention. 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.
A first part: elucidating the specific technical scheme
In order to introduce a phosphorus-containing group into a collagen molecule, the phosphorus-containing group plays an important role in the function of collagen. Referring to fig. 1, the present invention provides a method for preparing an alkyl phosphono modified collagen-based material, comprising: preparing a collagen solution; performing an alkyl phosphono modification reaction on collagen in the collagen solution; diluting a reaction product (namely reaction liquid after reaction) and then dialyzing; taking the dialysate and casting the dialysate into a mold; and (4) carrying out freeze drying treatment to obtain the alkyl phosphono modified collagen-based material.
Optionally, the dialysis bag can be a cellulose membrane dialysis bag, and the residual molecular weight of the dialysis bag is 8-14KDa, optionally 10KDa and 12 KDa. Small molecule molecules, typically less than 8000, can be removed.
Optionally, the freeze-drying treatment comprises pre-freezing at-20 deg.C for 4 hr, and then freezing at-80 deg.C for 24 hr to obtain solid collagen material for storage.
As an alternative embodiment of the collagen solution.
Optionally, the collagen solution comprises: a collagen acid solution or a collagen aqueous solution; wherein the concentration of collagen is 5-50mg/mL, optionally 15mg/mL, 25mg/mL, 40 mg/mL. And the collagen acid solution is a solution of collagen dissolved in acetic acid or hydrochloric acid; wherein the concentration of acetic acid or hydrochloric acid is 0.01-0.1mol/L, optionally 0.04mol/L, 0.06mol/L, and 0.08 mol/L.
Alternatively, the collagen may be, for example, but not limited to, collagen extracted from animal tissue or recombinant collagen prepared by biomedical engineering techniques.
The collagen solution of the embodiment can be carried out in a certain range according to the reaction conditions, the collagen solution is a reaction medium, and different concentrations of the collagen solution can correspond to different reaction conditions, so that the reaction effect is improved.
As an alternative embodiment of the alkylphosphonyl modification reaction.
The alkylphosphonyl modification reaction comprises: mixing the collagen solution with a 4- (dialkylphosphono) butanoic acid solution; adding a cross-linking agent, heating and stirring at constant temperature to react the collagen with the 4- (dialkyl phosphonyl) butyric acid. The reaction formula of the alkyl phosphonyl group modification reaction is
Wherein Col-is collagen, and R is linear chain or branched chain alkyl with carbon atom number of 1-20. Specifically, the mass ratio of the collagen to the 4- (dialkylphosphono) butyric acid is 1: (0.05-5), which can be selected from (1: 0.1) or (1: 1) or (1: 3); the molar ratio of 4- (dialkylphosphine) butyric acid to 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1: (1-1.5), optionally (1: 1.2) or (1: 1.4), collagen-based materials of different degrees of modification can be prepared.
Optionally, the cross-linking agent comprises: 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in a molar ratio of 1: (1-1.5), optionally 1: 1.2 or 1: 1.3. the cross-linking agent is easy to dissolve in water, has low toxicity, is easy to remove in the post-treatment process, and can ensure that the reaction is more complete.
Optionally, the alkyl substituent of the 4- (dialkylphosphono) butanoic acid includes at least one of a linear or branched alkyl group having 1 to 20 carbon atoms, such as, but not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, and the like.
Alternatively, the 4- (dialkylphosphono) butanoic acid solution is an aqueous 4- (dialkylphosphono) butanoic acid solution, wherein the concentration of 4- (dialkylphosphono) butanoic acid is 0.01 to 1mol/L, alternatively 0.05mol/L, 0.1mol/L,0.5mol/L, and is generally adjusted according to the modification degree requirement and the reaction concentration.
Optionally, the heating temperature of the constant-temperature heating and stirring is 25-60 ℃, optionally 35 ℃, 45 ℃ and 50 ℃, and the heating time is 12-72 hours, so that the reaction can be stably performed, the heating temperature is too low to cause incomplete reaction, and the heating temperature is too high to cause side reaction, even collagen denaturation.
The alkylphosphonyl modification reaction of the embodiment is to react collagen with 4- (dialkylphosphono) butyric acid under the action of a cross-linking agent, so that a phosphorus-containing group is introduced into a collagen molecule, which can play an important role in the function of collagen, for example, the activity of most enzymes in a living body is regulated by means of phosphorylation and dephosphorylation, and the activity of the enzymes can be regulated by introducing a phosphono group.
In addition, the aqueous solution of each substance in the present case may be an aqueous solution of each substance dissolved in deionized water.
Further, the invention provides an alkyl phosphono modified collagen, the molecular structural formula of which is:
col-is collagen, and R is C1-20 linear or branched alkyl.
Further, the present invention provides an alkylphosphonyl modified collagen-based material prepared by the preparation method as described above.
Further, the invention also provides an application of the alkyl phosphono modified collagen-based material in wound repair.
Optionally, the wound surface is such as, but not limited to, a scald wound surface, a burn wound surface, a damaged skin, and the like.
A second part: some examples are given below
Example 1
10g of collagen derived from tendon bovis Seu Bubali was dissolved in 1000mL of an acetic acid solution having a concentration of 0.1mol/L to prepare a collagen solution having a mass concentration of 10mg/mL, and an aqueous solution of 4- (diethylphosphono) butyric acid (CAS: 38694-48-3) having a molar concentration of 0.05mol/L was prepared, 100mL of each of the collagen solutions was mixed with the 4- (diethylphosphono) butyric acid solution, and 10mL of an aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride having a molar concentration of 0.5mol/L and 10mL of an aqueous solution of N-hydroxysuccinimide having a molar concentration of 0.5mol/L were added. The mixed solution is heated and stirred for 24 hours at the temperature of 40 ℃, then 200mL of deionized water is added for dilution, and the diluted solution is placed in a 10KDa dialysis bag for dialysis for 72 hours, and the water is replaced every 24 hours. The dialyzed solution was cast into molds and pre-frozen at-20 ℃ for 4 hours and then frozen at-80 ℃ for 24 hours. And finally, freeze-drying to obtain the alkyl phosphono modified collagen-based material.
Example 2
20g of recombinant human collagen fermented by yeast is dissolved in 1000mL of deionized water to prepare a collagen aqueous solution with the mass concentration of 20mg/mL and a 4- (diethylphosphono) butyric acid aqueous solution with the molar concentration of 0.06mol/L, 100mL of each collagen aqueous solution is mixed with the 4- (diethylphosphono) butyric acid (CAS: 38694-48-3) solution, 18mL of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution with the molar concentration of 0.5mol/L and 18mL of N-hydroxysuccinimide aqueous solution with the molar concentration of 0.5mol/L are added, and the mixed solution is heated and stirred at 40 ℃ for 24 hours. Then 200mL of deionized water was added for dilution, and the diluted solution was placed in a 10kDa dialysis bag for dialysis for 72 hours, and water was changed every 24 hours. And casting the dialyzed solution into a mold, pre-freezing for 4 hours at-20 ℃, then freezing for 24 hours at-80 ℃, and freeze-drying to obtain the alkyl phosphono modified collagen-based material.
Example 3
5g of recombinant human collagen fermented by yeast is dissolved in 1000mL of deionized water to prepare an aqueous collagen solution with a mass concentration of 5mg/mL and an aqueous 4- (diethylphosphono) butyric acid solution with a molar concentration of 0.05mol/L, 100mL of each aqueous collagen solution is mixed with the 4- (diethylphosphono) butyric acid (CAS: 38694-48-3) solution, 12.5mL of an aqueous 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution with a molar concentration of 0.5mol/L and 12.5mL of an aqueous N-hydroxysuccinimide solution with a molar concentration of 0.5mol/L are added, and the mixed solution is heated and stirred at 40 ℃ for 24 hours. Then 200mL of deionized water was added for dilution, and the diluted solution was placed in a 10kDa dialysis bag for dialysis for 72 hours, and water was changed every 24 hours. And casting the dialyzed solution into a mold, pre-freezing for 4 hours at-20 ℃, then freezing for 24 hours at-80 ℃, and freeze-drying to obtain the alkyl phosphono modified collagen-based material.
Example 4
50g of recombinant human collagen fermented by yeast is dissolved in 1000mL of deionized water to prepare an aqueous collagen solution with a mass concentration of 50mg/mL and an aqueous 4- (diethylphosphono) butyric acid solution with a molar concentration of 0.03mol/L, 100mL of each aqueous collagen solution is mixed with the 4- (diethylphosphono) butyric acid (CAS: 38694-48-3) solution, 6mL of an aqueous 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution with a molar concentration of 0.5mol/L and 6mL of an aqueous N-hydroxysuccinimide solution with a molar concentration of 0.5mol/L are added, and the mixed solution is heated and stirred at 40 ℃ for 24 hours. Then 200mL of deionized water was added for dilution, and the diluted solution was placed in a 10kDa dialysis bag for dialysis for 72 hours, and water was changed every 24 hours. And casting the dialyzed solution into a mold, pre-freezing for 4 hours at-20 ℃, then freezing for 24 hours at-80 ℃, and freeze-drying to obtain the alkyl phosphono modified collagen-based material.
Example 5
40g of recombinant human collagen fermented by yeast is dissolved in 1000mL of deionized water to prepare an aqueous collagen solution with a mass concentration of 40mg/mL and an aqueous 4- (diethylphosphono) butyric acid solution with a molar concentration of 0.012mol/L, 100mL of each aqueous collagen solution is mixed with the 4- (diethylphosphono) butyric acid (CAS: 38694-48-3) solution, 2.4mL of an aqueous 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride solution with a molar concentration of 0.5mol/L and 2.4mL of an aqueous N-hydroxysuccinimide solution with a molar concentration of 0.5mol/L are added, and the mixed solution is heated and stirred at 40 ℃ for 24 hours. Then 200mL of deionized water was added for dilution, and the diluted solution was placed in a 10kDa dialysis bag for dialysis for 72 hours, and water was changed every 24 hours. And casting the dialyzed solution into a mold, pre-freezing for 4 hours at-20 ℃, then freezing for 24 hours at-80 ℃, and freeze-drying to obtain the alkyl phosphono modified collagen-based material.
In conclusion, the alkylphosphono modified collagen-based material, the preparation method, the modified collagen and the application of the invention adopt a chemical method to carry out alkylphosphono grafting modification on the collagen, introduce phosphorus-containing groups into collagen molecules and introduce important phosphorus elements in a living body into the collagen, and provide a functionalized phosphorus-containing collagen-based wound repair material with excellent physicochemical property, good biocompatibility and controllable molding, which has wide clinical application prospect, simple preparation process and industrial production.
In light of the foregoing description of the preferred embodiments of the present invention, it is to be understood that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.