阅读说明：本技术 一种乙烯基胶原蛋白自组装微球的制备方法 (Preparation method of vinyl collagen self-assembled microspheres ) 是由 徐娜 罗资金 任龙芳 邢燕梅 冉旭东 于 2021-01-04 设计创作，主要内容包括：本发明公开一种乙烯基胶原蛋白自组装微球的制备方法,具体按照以下步骤实施：步骤1,利用甲基丙烯酸酐对废弃皮胶原蛋白进行改性,得到乙烯基胶原蛋白；步骤2,配置乙烯基胶原蛋白水溶液和植物单宁水溶液；步骤3,乙烯基胶原蛋白自组装微球的制备。本发明一种乙烯基胶原蛋白自组装微球的制备方法,即实现了皮革废弃物胶原蛋白资源化利用,同时又将不饱和双键接枝到微球上,增加了微球的化学可修饰性。(The invention discloses a preparation method of a vinyl collagen self-assembly microsphere, which is implemented according to the following steps: step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen; step 2, preparing a vinyl collagen aqueous solution and a plant tannin aqueous solution; and 3, preparing the vinyl collagen self-assembly microspheres. The preparation method of the vinyl collagen self-assembly microspheres realizes resource utilization of the collagen of the leather waste, and simultaneously grafts unsaturated double bonds onto the microspheres, thereby increasing the chemical modifiability of the microspheres.)

1. A preparation method of a vinyl collagen self-assembly microsphere is characterized by comprising the following steps:

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

step 2, preparing a vinyl collagen aqueous solution and a plant tannin aqueous solution;

and 3, preparing the vinyl collagen self-assembly microspheres.

2. The method for preparing the self-assembled vinyl collagen microspheres according to claim 1, wherein the specific implementation process of the step 1 is as follows:

adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature is stabilized for 20min, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 12-24 h, and placing the dried sample into a dryer for later use;

wherein 10ml of phosphate buffer solution is required for 1g of collagen.

3. The method for preparing self-assembled vinyl collagen microspheres according to claim 2, wherein the pH of the phosphate buffer solution in step 1 is 7.4.

4. The method for preparing the vinyl collagen self-assembled microspheres according to claim 1, wherein in the step 2, the concentration of the vinyl collagen aqueous solution is 2-4 mg/ml; 1-4 mg/ml of the plant tannin water solution.

5. The method for preparing self-assembled microspheres of vinyl collagen according to claim 1, wherein the specific implementation process of step 3 is as follows:

adding a certain amount of the plant tannin solution prepared in the step 2 into the vinyl collagen solution, magnetically stirring at a constant temperature of 20 ℃ and a rotating speed of 250rpm/min for 20min, centrifuging, and freeze-drying to obtain pure vinyl collagen self-assembled microspheres; wherein the mass ratio of the vinyl collagen to the vegetable tannin in the reaction solution is 1: 0.25 to 2.

Technical Field

The invention relates to the technical field of natural high polymer materials, in particular to a preparation method of a vinyl collagen self-assembled microsphere.

Background

More than 80% of leather solid waste is collagen, and the collagen with different molecular weights and structures can be extracted by processing defective leather materials, leftover materials and the like generated in the leather making process.

Self-assembly is a technique in which the basic building blocks spontaneously adopt an ordered structure. During the self-assembly process, the basic building blocks spontaneously organize or aggregate into a stable structure with a certain regular geometric appearance under the interaction based on non-covalent bonds.

The polymer microsphere has the special characteristics of shape specificity (spherical shape), good embedding property, small size, large specific surface area, strong adsorbability, surface enrichment of functional groups and the like, so that the polymer microsphere is widely applied to multiple fields such as biomedicine, coating and food and becomes one of the hotspots of current scientific research. The vinyl collagen self-assembly microspheres are prepared from modified collagen by a self-assembly method, and the preparation method is simple and environment-friendly, short in reaction time and simple in reaction conditions, can realize reutilization of waste collagen, and changes waste into valuable. Meanwhile, unsaturated double bonds are introduced on the surface of the microsphere, so that other functional groups can be conveniently modified on the microsphere in the follow-up process, and the functionalization of the microsphere is realized.

Disclosure of Invention

The invention aims to provide a preparation method of vinyl collagen self-assembly microspheres, which realizes resource utilization of leather waste collagen, and simultaneously grafts unsaturated double bonds onto the microspheres, thereby increasing the chemical modifiability of the microspheres.

The invention adopts the technical scheme that a preparation method of the vinyl collagen self-assembly microspheres is implemented according to the following steps:

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

step 2, preparing a vinyl collagen aqueous solution and a plant tannin aqueous solution;

and 3, preparing the vinyl collagen self-assembly microspheres.

The present invention is also characterized in that,

the specific implementation process of the step 1 is as follows:

adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature is stabilized for 20min, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 12-24 h, and placing the dried sample into a dryer for later use;

wherein 10ml of phosphate buffer solution is required for 1g of collagen.

In step 1, the pH of the phosphate buffer solution was 7.4.

In the step 2, the concentration of the vinyl collagen aqueous solution is 2-4 mg/ml; 1-4 mg/ml of the plant tannin water solution.

The specific implementation process of the step 3 is as follows:

adding a certain amount of the plant tannin solution prepared in the step 2 into the vinyl collagen solution, magnetically stirring at a constant temperature of 20 ℃ and a rotating speed of 250rpm/min for 20min, centrifuging, and freeze-drying to obtain pure vinyl collagen self-assembled microspheres; wherein the mass ratio of the vinyl collagen to the vegetable tannin in the reaction solution is 1: 0.25 to 2.

The invention has the beneficial effects that:

(1) the method of the invention uses leather waste collagen as raw material to react, collagen is modified by methacrylic anhydride, and vinyl collagen self-assembly microspheres are prepared by a self-assembly method cross-linking method, which not only have the property of collagen microspheres, but also have better reaction activity because unsaturated double bonds are grafted on the microspheres due to the reaction with methacrylic anhydride, thereby enhancing chemical versatility and leading the microspheres to be modified with more groups to be functionalized.

(2) The method of the invention not only can change waste into valuable and improve economic value, but also opens up a new way for recycling the waste. Meanwhile, double bond functional groups are introduced to the collagen microspheres, so that the types of the functional groups on the molecular chains of the microspheres are enriched, and the repairability of the microspheres is enhanced.

Drawings

FIG. 1 is an SEM image of self-assembled vinyl collagen microspheres prepared in example 5;

FIG. 2 is a particle size distribution diagram of the self-assembled vinyl collagen microspheres prepared in example 5;

FIG. 3 is a schematic diagram of the chemical reaction process involved in step 1 of the process of the present invention;

FIG. 4 is a schematic diagram of the chemical reaction process involved in step 3 of the method of the present invention.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

The invention relates to a preparation method of a vinyl collagen self-assembly microsphere, which is implemented according to the following steps:

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

the specific implementation process of the step 1 is as follows:

adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature is stabilized for 20min, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 12-24 h, and placing the dried sample into a dryer for later use;

wherein, 10ml of phosphate buffer solution is needed for every 1g of collagen; the pH of the phosphate buffer solution was 7.4.

Step 2, preparing a vinyl collagen aqueous solution and a plant tannin aqueous solution, wherein the plant tannin is condensed plant tannin, specifically catechin, tannic acid or procyanidine;

the specific implementation process of the step 2 is as follows: respectively weighing a certain amount of vinyl collagen and plant tannin, then respectively adding the weighed vinyl collagen and plant tannin into two different beakers containing aqueous solutions, respectively magnetically stirring at constant temperature at normal temperature until the vinyl collagen and the plant tannin are completely dissolved to respectively obtain a vinyl collagen aqueous solution and a plant tannin aqueous solution; in the step 2, when the vinyl collagen solution and the plant tannin solution are prepared, stirring and dissolving assisting are needed under the condition of 20 ℃.

In the step 2, the concentration of the vinyl collagen aqueous solution is 2-4 mg/ml; 1-4 mg/ml of the plant tannin water solution.

And 3, preparing the vinyl collagen self-assembly microspheres.

The specific implementation process of the step 3 is as follows:

adding a certain amount of the plant tannin solution prepared in the step 2 into the vinyl collagen solution, magnetically stirring at a constant temperature of 20 ℃ and a rotating speed of 250rpm/min for 20min, centrifuging, and freeze-drying to obtain pure vinyl collagen self-assembled microspheres; wherein the mass ratio of the vinyl collagen to the vegetable tannin in the reaction solution is 1: 0.25 to 2.

The invention is described below in principle:

step 1, methacrylic anhydride modifies collagen to obtain vinyl collagen

In step 1, as shown in fig. 3, the preparation principle of the vinyl collagen is that amino groups on a molecular chain of the collagen and anhydride of methacrylic anhydride undergo nucleophilic substitution reaction, the amino groups are used as a nucleophile to attack carbon in one carboxyl group of the anhydride, and then the other carboxyl group leaves to form an amide bond;

in step 3, as shown in fig. 4, a hydrogen bond is easily formed between a carbonyl group (C ═ O) on a peptide bond in collagen and a phenolic hydroxyl group in the vegetable tannin. Meanwhile, hydrophobic amino acid side chains such as aromatic rings, pyrrole rings and aliphatic chains in collagen form hydrophobic regions in aqueous solution due to hydrophobic interaction. The plant tannin molecules containing hydrophobic groups enter a hydrophobic area through hydrophobic reaction, and then phenolic hydroxyl groups on the plant tannin are subjected to hydrogen bond bonding with polar groups at proper positions on a protein chain, so that the plant tannin-protein bonding is further enhanced.

Example 1

A preparation method of vinyl collagen self-assembly microspheres is implemented according to the following steps;

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

the specific implementation process of the step 1 is as follows:

adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature is stabilized for 20min, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 12h, and placing the dried sample into a dryer for later use;

wherein, 10ml of phosphate buffer solution is needed for every 1g of collagen; the pH of the phosphate buffer solution was 7.4.

Step 2, preparing a vinyl collagen aqueous solution and a catechin aqueous solution;

the specific implementation process of the step 2 is as follows: respectively weighing a certain amount of vinyl collagen and catechin, then respectively adding the weighed vinyl collagen and catechin into two different beakers containing aqueous solutions, respectively magnetically stirring at constant temperature at normal temperature until the vinyl collagen and the catechin are completely dissolved to respectively obtain a vinyl collagen aqueous solution and a catechin aqueous solution; in the step 2, when the vinyl collagen solution and the plant tannin solution are prepared, stirring and dissolving assisting are needed under the condition of 20 ℃.

In the step 2, the concentration of the vinyl collagen aqueous solution is 2 mg/ml; 1mg/ml of aqueous catechin solution.

And 3, preparing the vinyl collagen self-assembly microspheres.

The specific implementation process of the step 3 is as follows:

adding a certain amount of the catechin solution prepared in the step 2 into the vinyl collagen solution, magnetically stirring at a constant temperature of 20 ℃ and a rotation speed of 250rpm/min for 20min, centrifuging, and freeze-drying to obtain pure vinyl collagen self-assembled microspheres; wherein the mass ratio of the vinyl collagen to the catechin in the reaction solution is 1: 0.25.

example 2

A preparation method of vinyl collagen self-assembly microspheres is implemented according to the following steps;

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

the specific implementation process of the step 1 is as follows:

adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature is stabilized for 20min, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 24h, and placing the dried sample into a dryer for later use;

wherein, 10ml of phosphate buffer solution is needed for every 1g of collagen; the pH of the phosphate buffer solution was 7.4.

Step 2, preparing a vinyl collagen aqueous solution and a tannic acid aqueous solution;

the specific implementation process of the step 2 is as follows: respectively weighing a certain amount of vinyl collagen and tannic acid, then respectively adding the weighed vinyl collagen and tannic acid into two different beakers containing aqueous solutions, respectively magnetically stirring at constant temperature at normal temperature until the vinyl collagen and tannic acid are completely dissolved, and respectively obtaining a vinyl collagen aqueous solution and a tannic acid aqueous solution; in the step 2, the vinyl collagen solution and the tannic acid solution are prepared by stirring at the temperature of 20 ℃ to assist dissolution.

In the step 2, the concentration of the vinyl collagen aqueous solution is 4 mg/ml; 4mg/ml of aqueous tannic acid solution.

And 3, preparing the vinyl collagen self-assembly microspheres.

The specific implementation process of the step 3 is as follows:

adding a certain amount of the tannic acid solution prepared in the step 2 into the vinyl collagen solution, magnetically stirring at a constant temperature of 20 ℃ and a rotation speed of 250rpm/min for 20min, centrifuging, and freeze-drying to obtain pure vinyl collagen self-assembled microspheres; wherein the mass ratio of the vinyl collagen to the tannic acid in the reaction solution is 1: 2.

example 3

A preparation method of vinyl collagen self-assembly microspheres is implemented according to the following steps;

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

the specific implementation process of the step 1 is as follows:

adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature of 20min is stabilized, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 20h, and placing the dried sample into a dryer for later use;

wherein, 10ml of phosphate buffer solution is needed for every 1g of collagen; the pH of the phosphate buffer solution was 7.4.

Step 2, preparing a vinyl collagen aqueous solution and a protocyanine fixing aqueous solution;

the specific implementation process of the step 2 is as follows: respectively weighing a certain amount of vinyl collagen and protocyanine, then respectively adding the weighed vinyl collagen and protocyanine into two different beakers containing aqueous solutions, respectively magnetically stirring at constant temperature at normal temperature until the vinyl collagen and the protocyanine are completely dissolved, and respectively obtaining a vinyl collagen aqueous solution and a protocyanine aqueous solution; in the step 2, when preparing the vinyl collagen solution and the procyanidine solution, stirring and dissolving assistance are needed at the temperature of 20 ℃.

In the step 2, the concentration of the vinyl collagen aqueous solution is 3 mg/ml; 3mg/ml of an aqueous solution of procyanidin.

And 3, preparing the vinyl collagen self-assembly microspheres.

The specific implementation process of the step 3 is as follows:

adding a certain amount of the protocyanine fixing solution prepared in the step 2 into the vinyl collagen solution, magnetically stirring for 20min at a constant temperature of 20 ℃ and at a rotating speed of 250rpm/min, centrifuging, and freeze-drying to obtain pure vinyl collagen self-assembled microspheres; wherein the mass ratio of the vinyl collagen to the procyanidine in the reaction solution is 1: 1.

example 4

A preparation method of vinyl collagen self-assembly microspheres is implemented according to the following steps;

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

the specific implementation process of the step 1 is as follows:

adding a certain amount of collagen and phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature of 20min is stabilized, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 20h, and placing the dried sample into a dryer for later use;

wherein, 10ml of phosphate buffer solution is needed for every 1g of collagen; the pH of the phosphate buffer solution was 7.4.

Step 2, preparing a vinyl collagen aqueous solution and a protocyanine fixing aqueous solution;

the specific implementation process of the step 2 is as follows: respectively weighing a certain amount of vinyl collagen and protocyanine, then respectively adding the weighed vinyl collagen and protocyanine into two different beakers containing aqueous solutions, respectively magnetically stirring at constant temperature at normal temperature until the vinyl collagen and the protocyanine are completely dissolved, and respectively obtaining a vinyl collagen aqueous solution and a protocyanine aqueous solution; in the step 2, when preparing the vinyl collagen solution and the procyanidine solution, stirring and dissolving assistance are needed at the temperature of 20 ℃.

In the step 2, the concentration of the vinyl collagen aqueous solution is 2.5 mg/ml; 2.5mg/ml of an aqueous solution of procyanidin.

And 3, preparing the vinyl collagen self-assembly microspheres.

The specific implementation process of the step 3 is as follows:

adding a certain amount of the protocyanine fixing solution prepared in the step 2 into the vinyl collagen solution, magnetically stirring for 20min at a constant temperature of 20 ℃ and at a rotating speed of 250rpm/min, centrifuging, and freeze-drying to obtain pure vinyl collagen self-assembled microspheres; wherein the mass ratio of the vinyl collagen to the procyanidine in the reaction solution is 1: 1.

example 5

A preparation method of vinyl collagen self-assembly microspheres is implemented according to the following steps;

step 1, modifying waste skin collagen by methacrylic anhydride to obtain vinyl collagen;

the specific implementation process of the step 1 is as follows:

adding 5g of collagen and 50mL of phosphoric acid buffer solution into a conical flask with a plug, then placing the conical flask with the plug into a constant-temperature magnetic stirrer with a water bath temperature of 70 ℃, stirring for 30min until the collagen is completely dissolved, then reducing the temperature of the constant-temperature magnetic stirrer to 50 ℃, slowly adding a certain amount of methacrylic anhydride into the conical flask after the temperature is stabilized for 20min, reacting for 2h at a rate of 1mL/min, pouring the fully-reacted solution into a dialysis bag with a molecular weight cutoff of 500D, then placing the dialysis bag into deionized water for dialysis for 24h, freeze-drying for 12-24 h, and placing the dried sample into a dryer for later use.

Wherein 10ml of phosphate buffer solution is required for 1g of collagen.

In step 1, the pH of the phosphate buffer solution was 7.4.

Step 2, preparing a vinyl collagen aqueous solution and a protocyanine fixing aqueous solution;

the specific implementation process of the step 2 is as follows: respectively weighing 0.06g of vinyl collagen and 0.03g of proanthocyanidin, and then respectively adding the weighed vinyl collagen and proanthocyanidin into two different beakers containing aqueous solutions, wherein the aqueous solutions in the two beakers are both 15 ml;

respectively stirring at constant temperature and magnetic force until the materials are completely dissolved to respectively obtain a vinyl collagen aqueous solution and a protocyanine fixed aqueous solution; in the step 2, when preparing the vinyl collagen solution and the procyanidine solution, stirring and dissolving assistance are needed at the temperature of 20 ℃.

And 3, preparing the vinyl collagen self-assembly microspheres.

The specific implementation process of the step 3 is as follows:

and (3) adding the protocyanine fixing solution prepared in the step (2) into the vinyl collagen solution, magnetically stirring at a constant temperature of 20 ℃ and at a rotating speed of 250rpm/min for 20min, centrifuging, and freeze-drying to obtain the pure vinyl collagen self-assembly microspheres.

Fig. 1 is an SEM image of the microspheres made in this case, and it can be seen that the microspheres are relatively uniform in size and the surface of the microspheres is relatively smooth. Fig. 2 is a distribution diagram of the particle size of the microspheres prepared in this example, and it can be seen that the particle size distribution of the microspheres is relatively uniform and mainly focuses on a certain range.