CN110408057B - DHPMC blending crosslinking modified collagen suitable for biomedicine and preparation method thereof - Google Patents

DHPMC blending crosslinking modified collagen suitable for biomedicine and preparation method thereof Download PDF

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CN110408057B
CN110408057B CN201910641001.7A CN201910641001A CN110408057B CN 110408057 B CN110408057 B CN 110408057B CN 201910641001 A CN201910641001 A CN 201910641001A CN 110408057 B CN110408057 B CN 110408057B
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李国英
陈春秀
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Abstract

The invention provides a DHPMC blending crosslinking modified collagen suitable for biomedicine and a preparation method thereof, and the DHPMC blending crosslinking modified collagen mainly comprises the following raw materials: 100 parts of collagen and 40-200 parts of dialdehyde hydroxypropyl methyl cellulose, wherein the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 19-35%, and the relative viscosity is reduced by 23.57-70.47% when the T is 25 +/-0.01 ℃. According to the invention, the collagen is modified by blending and crosslinking DHPMC, and the prepared modified collagen product has higher thermal stability and stronger enzyme degradation resistance brought by covalent bonds formed by chemical crosslinking, and retains higher biocompatibility brought by physical blending.

Description

DHPMC blending crosslinking modified collagen suitable for biomedicine and preparation method thereof
Technical Field
The invention relates to the technical field of collagen-based biomedical materials, in particular to DHPMC blending crosslinking modified collagen suitable for biomedicine and a preparation method thereof, and more particularly relates to modified collagen prepared by crosslinking modification of modified dialdehyde hydroxypropyl methyl cellulose (DHPMC) and collagen and a preparation method thereof.
Background
Collagen is fibrous structural protein with the largest content in animals, is an important component of a cell matrix, and is an excellent natural high molecular substance. The three-strand helical structure of the collagen has good biocompatibility, biodegradability, film-forming property and mechanical property, and can be widely applied to the fields of food industry, biomedicine, cosmetics and the like (Lizhou English, the biological property of collagen [ J ]. Chinese leather, 2002,31(21): 20-21). But because the thermal stability and the mechanical strength of the collagen are low, the collagen is easy to be degraded by organisms, the viscosity is high, the film forming capability is not strong, and the like, the application of the collagen in the field of biological medicine materials is limited. Therefore, the collagen material to be used is usually modified by physical or chemical methods to enhance the usability of the collagen material.
Common methods for modifying collagen include: (1) modified by physical crosslinking or crosslinking by chemical crosslinking agents; (2) modified by blending with other natural or synthetic polymers, and the like.
First, the crosslinking modification method refers to a method of improving the mechanical properties and thermal stability of collagen fibers by forming covalent bonds to connect collagen molecules. Among them, physical crosslinking and chemical crosslinking are common crosslinking modification methods. Physical crosslinking methods such as ultraviolet irradiation, radiation irradiation, etc. can enhance the performance of the collagen membrane and avoid the addition of toxic substances. However, the obtained collagen product has the disadvantages of low crosslinking degree and poor uniformity. The chemical crosslinking method such as carbodiimide, glutaraldehyde, etc. can obtain uniform and stable crosslinked products, is favorable for adjusting and controlling the crosslinking degree, mechanical property and biocompatibility, but also has the defects of introducing exogenous toxic reagents and difficult removal of residual reagents. It is described in the literature that crosslinking of collagen fibers with glutaraldehyde vapor enhances the thermal stability, tensile strength and breaking strength of collagen fibers (structure and properties of collagen fibers of type I, Zhang Hao, Ding Chang Kun, glutaraldehyde crosslinked and modified mouse tail type [ J ]. research and development, 2016,39(02): 26-29). But glutaraldehyde has certain cytotoxicity and calcification and is easily hydrolyzed to form free small molecular aldehyde (Zhang Yunfeng, Du Guting, modification method of collagen summary [ J ] chemical technology and development, 2015,44(06): 46-50). Therefore, modified collagen products obtained by using only physical crosslinking or chemical crosslinking have not been generally widely used in the field of biomedical materials.
Secondly, blending modification of collagen and natural polymers is also a common method for improving the performance of collagen. It should be noted that blending modification refers to a process of mixing two or more polymers, and holding the components in each other to form a macroscopically uniform substance. Factors influencing blending modification mainly include compatibility, poor compatibility and poor interface bonding; good compatibility, tight interface bonding and capability of integrating the excellent performances of all the components. Generally, the more closely the components of the blend are polar, compositional, viscosity, molecular weight, and surface tension, the more compatible the resulting blend will be. Therefore, natural polymers used are mainly, as most representative examples, natural polysaccharides such as starch, carboxymethyl cellulose and sodium alginate, in order to form good compatibility with collagen. According to the published literature, hydroxypropyl methylcellulose is blended with type I collagen (CuicuiDing, Min Zhang, Guoying Li. preparation and characterization of collagen/hydroxypropyl methyl cellulose [ HPMC) blend film [ J ]. Carbohydrate polymers.119(2015)194-201), so that the thermal stability, mechanical action and hydrophilicity of the cross-linked modified collagen product are improved by virtue of secondary bonds such as intermolecular hydrogen bonds and mutual entanglement. And the natural polymer modifier used in the method can not introduce exogenous toxicity generally and basically keep the original activity of the collagen, and is more suitable for applying the obtained modified collagen product as a biomedical material.
Although the above documents disclose that blending to form intermolecular hydrogen bonds can enhance the relevant properties of collagen, the enhanced properties are not sufficient to fully satisfy the requirements of the biomedical material field which is rapidly developed at present, and the collagen product is required to have higher thermal stability and enzyme degradation resistance in the relevant fields such as tissue engineering scaffolds in biomedicine.
In order to integrate the advantages of the above-mentioned collagen modification methods, in recent years, covalent crosslinking between collagen and natural polymers has been a popular collagen modification method, in which dialdehyde cellulose is mixed with collagen, and then crosslinked to form a dialdehyde cellulose/collagen composite film by intermolecular Schiff base bond formation. The composite membrane has good thermal stability, enzyme degradation resistance and biocompatibility, does not introduce exogenous toxicity, and can be used as a scaffold material in tissue engineering (Yongmei Cheng, joining Lu, Shilin Liu, et al. the preparation, chromatography and evaluation of regenerated cellulose/collagen composite hydrogel [ J ].107(2014) 57-64). Besides covalent bonds, secondary bonds such as hydrogen bonds are formed between molecules, which is beneficial to improving the thermal stability of collagen. (Baifaithful but yearly but Weihua, development of dialdehyde carboxymethyl cellulose-collagen composite hemostatic material [ J ].2018,32(10): 3628-3633).
However, in the course of research, the inventors of the present invention have found that in the above-mentioned collagen modification method for forming covalent crosslinks, dialdehyde cellulose, which is obtained by oxidatively modifying cellulose to have a high aldehyde content, is generally selected as a crosslinking agent in the published technical literature to crosslink and modify collagen, so as to increase the aldehyde content, thereby achieving the purpose of enhancing the crosslinking effect. The relevant literature indicates that the course of the cellulose oxidation reaction is generally accompanied by its degradation reaction. By improving the oxidation degree, the high aldehyde group content of the cellulose is achieved, the severe degradation of the cellulose is inevitably caused, and the molecular weight and the viscosity are obviously reduced. Although Schiff base bond covalent crosslinking can be formed among molecules of the blend, the compatibility of the cellulose with high aldehyde group content and the collagen obtained after modification is greatly reduced compared with the compatibility of the cellulose and the collagen before modification, the performance advantages brought by the physical blending of the collagen and natural polymers are lost, and the difference between the viscosity of the cellulose with high aldehyde group content and the viscosity of the collagen after modification is mainly reflected. Therefore, the application and popularization of the modified collagen products prepared by the method are greatly limited.
Therefore, a modified collagen and a preparation method thereof are needed, which have both higher thermal stability and stronger enzyme degradation resistance brought by covalent bond formation through chemical crosslinking and retain higher biocompatibility brought by physical blending, and are very beneficial to the application and development of modified collagen products in the biomedical field.
Disclosure of Invention
The invention provides a DHPMC blending crosslinking modified collagen suitable for biomedicine and a preparation method thereof, wherein the modified dialdehyde hydroxypropyl methyl cellulose (DHPMC) has proper aldehyde content and relative molecular mass by controlling local selective oxidation of the hydroxypropyl methyl cellulose (HMPC), and the modified dialdehyde hydroxypropyl methyl cellulose (DHPMC) is used as a crosslinking agent to obtain the modified collagen which simultaneously has higher thermal stability and stronger enzyme degradation resistance brought by forming covalent bonds by chemical crosslinking and retains higher biocompatibility brought by physical blending. In order to achieve the purpose, the invention adopts the technical scheme formed by the following technical measures.
A DHPMC blending crosslinking modified collagen suitable for biomedicine mainly comprises the following raw materials in parts by weight:
100 parts of collagen, namely 100 parts of collagen,
40-200 parts of dialdehyde hydroxypropyl methyl cellulose;
the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 19-35%, and the relative viscosity is reduced by 23.57-70.47% at 25 +/-0.01 ℃. Since the relative molecular mass can be indirectly characterized by the relative viscosity, the relative molecular mass in the present invention is characterized by the relative viscosity.
Since the collagen is usually in a solid state or a liquid state as a solution according to the preparation purpose of the cross-linking modification, in order to facilitate the cross-linking modification reaction between the collagen and the dialdehyde hydroxypropyl methylcellulose, one skilled in the art can select a suitable solution of the dialdehyde hydroxypropyl methylcellulose according to the general knowledge in the art, and select a suitable concentration of the collagen solution when the selected collagen material is in a non-solid state, so as to facilitate the normal progress of the cross-linking modification reaction. In addition, the weight parts of the collagen and the dialdehyde hydroxypropyl methyl cellulose in the raw materials refer to dry weight.
It is worth noting that the hydroxypropyl methylcellulose, when selectively oxidized by sodium periodate, forms two aldehyde substituents, primarily by breaking bonds with hydroxyl groups on both carbons at the 2,3 positions of its sugar ring. In order to better illustrate the invention, the invention provides a preparation method for controlling the selective oxidation of hydroxypropyl methyl cellulose to obtain dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 19-35% and the relative viscosity reduced by 23.57-70.47% at 25 +/-0.01 ℃, which comprises the following specific steps:
preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 1-3%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: (0.04-0.20) adding sodium periodate into the solution, mixing, adjusting the pH to 2-6, reacting at 30-50 ℃, and stirring for reaction for 3-5 hours under the condition of shading; and after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose.
The reaction principle of the selective oxidation of the hydroxypropyl methylcellulose by sodium periodate is shown as follows:
Figure BDA0002131859940000041
wherein the radical R is CH3、CH2CHOHCH3Or H.
Further, purifying and drying, specifically, performing rotary evaporation for 3-5 times by using absolute ethyl alcohol and deionized water respectively, and placing the materials in an oven for drying.
Wherein the degree of oxidation is determined by the hydroxylamine hydrochloride method. The method specifically comprises the following steps:
0.1g of the oxidized product was dissolved in 30ml of deionized water, and the pH was adjusted to 4.5 to prepare solution A. 0.43g of hydroxylamine hydrochloride was weighed out and dissolved in 20ml of deionized water, and the pH was adjusted to 4.5 to prepare a solution B. Mixing the solution A and the solution B, and stirring and reacting for 4 hours at the temperature of 40 ℃. Titration with 0.1mol/l NaOH was then carried out, the volume used was recorded and the degree of oxidation OD (equation 1) was calculated as follows.
Figure BDA0002131859940000042
In the formula:
c: the concentration of NaOH used during titration, mol/L;
v: the volume of NaOH used in titration, ml;
m: the mass of the oxidation product weighed, g.
Wherein the relative viscosity is measured by the Ubbelohde viscosity method. The method specifically comprises the following steps:
5mg/ml of dilute oxidation product solutions under different oxidation conditions were prepared. Firstly, placing the pure solvent in a constant-temperature water bath at 25 +/-0.01 ℃ for 30min, and then accurately measuring the flowing-out time of the pure solvent in a capillary tube by using a stopwatch, and recording the flowing-out time as t0(ii) a Then measure by the same methodThe flowing time of the collagen diluted solution with different concentrations in the capillary is determined and is recorded as t1. The elution times of the pure solvent and of the diluted collagen solution at each concentration were determined in five replicates and averaged. The relative viscosity (formula 2) was calculated as follows:
Figure BDA0002131859940000051
wherein, the collagen is of animal origin, generally, those skilled in the art can select a suitable collagen source according to practical application, and in order to better illustrate the excellent technical effect of the present invention, the technical effect data measured in the present invention are all modified collagen products obtained according to the present invention by using collagen extracted from cowhide.
In general, other additives known in the prior art, such as polyethylene glycol, glycerol, etc., can be added by those skilled in the art according to the actual application requirements. However, it is a prerequisite that these processing aids do not adversely affect the achievement of the objects of the present invention and the achievement of the advantageous effects of the present invention.
Preferably, in order to make the obtained cross-linked and modified collagen product have excellent performances in three aspects of thermal stability, enzymatic degradation resistance and biocompatibility, the raw materials mainly comprise the following components in parts by weight:
100 parts of collagen, namely 100 parts of collagen,
100-140 parts of dialdehyde hydroxypropyl methyl cellulose;
the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 20-30%, and the relative viscosity is reduced by 23.57-45.66% at 25 +/-0.01 ℃.
The preparation method of the DHPMC blending crosslinking modified collagen suitable for biomedicine comprises the following steps:
(1) stock preparation
The raw materials mainly comprise the following components in parts by weight:
100 parts of collagen, namely 100 parts of collagen,
40-200 parts of dialdehyde hydroxypropyl methyl cellulose;
the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 19-35%, and the relative viscosity is reduced by 23.57-70.47% at 25 +/-0.01 ℃.
(2) Modification of crosslinking
Preparing the prepared dialdehyde hydroxypropyl methyl cellulose in the step (1) into dialdehyde hydroxypropyl methyl cellulose solution with the pH value of 3-7, adding collagen into the solution, mixing, adjusting the pH value to 3-7, and reacting at the temperature of 0-35 ℃ for 24-72h to obtain the DHPMC blending crosslinking modified collagen product.
Preferably, in order to make the cross-linking between the dialdehyde hydroxypropyl methylcellulose and the collagen more effective, the dialdehyde hydroxypropyl methylcellulose is configured into a dialdehyde hydroxypropyl methylcellulose solution with the pH value of 3-7, wherein the mass concentration of the dialdehyde hydroxypropyl methylcellulose solution is 5-20%, and according to the common knowledge in the art, a weak acid buffer solution which does not react with the dialdehyde hydroxypropyl methylcellulose or influence the reaction of the dialdehyde hydroxypropyl methylcellulose and the collagen is generally selected to be configured, such as an acetic acid-sodium acetate buffer solution with the pH value of 4.
Generally, the collagen is added to the solution and mixed, and those skilled in the art can select an appropriate adding manner according to different collagens, for example, the collagen is prepared into a solution or is added after being dissolved, or a solid collagen film is directly soaked in a dialdehyde hydroxypropyl methyl cellulose solution, and the adding manner is a conventional processing manner in the field. Secondly, if the collagen solution is added into the solution and mixed, the blending and crosslinking conditions, such as pH, temperature, concentration, can be changed according to the final product application by those skilled in the art, so that the product form can be gel, film, etc.
Through the preparation steps, the finally obtained cross-linked and modified collagen product has good biodegradability, biocompatibility and low cytotoxicity, compared with unmodified collagen sponge, the thermal stability is improved by 68.9 ℃, and the enzymolysis resistance is enhanced by 39% under the condition of 6h of pancreatic enzyme degradation; compared with the unmodified cellulose, the dialdehyde hydroxypropyl methyl cellulose prepared according to the preferred technical scheme of the invention has the lowest loss of 23.57 percent.
The cross-linked and modified collagen product obtained by the invention can be used as a biomedical material and used in the fields of drug controlled release, biological stents, hemostatic materials and the like. For example, the modified collagen can be used as a hemostatic sponge, a substitute material of extracellular matrix, a slow-release drug carrier and the like.
The technical principle of the invention is as follows:
the invention selects the dialdehyde hydroxypropyl methyl cellulose modified by oxidation as the cross-linking agent, firstly, the HPMC has low price, good degradability, good dispersibility and film forming property and good compatibility with natural high molecular compounds. In addition, although different HPMC sugar rings have different substituents at the 2 and 3 positions and different degrees of substitution, the different HPMC sugar rings have different relative physical properties, but contain a large number of hydroxyl groups, and the hydroxyl active groups are mainly directed to the substituents at the 2 and 3 positions of the sugar rings. It is easy to know that when sodium periodate is selectively oxidized, the bonds with hydroxyl groups on the 2 and 3-position carbons are mainly broken to form two aldehyde groups. For different types of HPMC, the oxidation product DHPMC has similar physicochemical properties, such as aldehyde group content and relative viscosity change trend, if the same sodium periodate oxidation conditions are used. If other oxidants are used, the content and position of aldehyde groups in the final product of the oxidized HPMC are uncertain easily, the reaction is difficult to control, more side reactions are generated, and the repeatability of the experiment is poor.
Secondly, the modified dialdehyde hydroxypropyl methyl cellulose has active groups of hydroxyl and aldehyde groups simultaneously, so that hydrogen bond bonds belonging to physical combination and Schiff base bonds belonging to chemical combination are formed respectively when the modified dialdehyde hydroxypropyl methyl cellulose is crosslinked and blended with collagen, so that the crosslinking modification method is not limited to a single physical or chemical crosslinking mode any more, and the obtained crosslinking modified collagen product has better functionality.
In addition, on the premise of ensuring that the modified HPMC and collagen are subjected to cross-linking blending to form physical bonding and chemical bonding, in order to reserve the excellent characteristics of the HPMC before modification and the superior functions of the HPMC before modification and the collagen when blended as far as possible, the technical scheme of the invention overcomes the technical prejudice of pursuing high aldehyde content in the existing published documents, and the dialdehyde hydroxypropyl methyl cellulose with proper aldehyde content and relative molecular mass is selected as a cross-linking agent, so that the compatibility of the HPMC after oxidative modification and the collagen after blending and cross-linking is enhanced, the film-forming performance of the collagen after modification is enhanced, the product performance meets the biomedical material standard, and the method is widely applied to the related fields.
The invention has the following beneficial technical effects:
1. according to the invention, the modified collagen is blended and crosslinked through DHPMC, the prepared modified collagen product has the characteristics of higher thermal stability and stronger enzyme degradation resistance brought by forming covalent bonds through chemical crosslinking, higher biocompatibility brought by physical blending is retained, no exogenous toxicity is introduced, the activity of the collagen is basically maintained, and the modified collagen product has the characteristics of reproducibility, biodegradability, low cytotoxicity, good water solubility and the like, has a wider applicable pH range, meets the biomedical material standard, and can be widely applied to related fields.
2. The cross-linked and modified collagen product has good biodegradability, biocompatibility and low cytotoxicity, compared with unmodified collagen sponge, the thermal stability is improved by 68.9 ℃, the enzymolysis resistance is enhanced by 39% under the condition of 6h of pancreatin degradation, and the loss range of relative viscosity is 23.57-70.47% compared with that of unmodified cellulose.
3. In the preferred technical scheme of the invention, when the hydroxypropyl methyl cellulose is selectively oxidized, sodium periodate is selectively and locally oxidized in an oxidation mode, and side reactions in the selective oxidation reaction are few, so that the obtained product has a certain amount of aldehyde groups but cannot be greatly degraded due to oxidation, and the excellent performance of the product is maintained.
Drawings
FIG. 1 is an infrared spectrum of dialdehyde hydroxypropyl methyl cellulose synthesized in different ratios of sodium periodate to hydroxypropyl methyl cellulose in examples 1-2 of the present invention. In the figure, the lines are shown in different ratios of sodium periodate to hydroxypropyl methylcellulose in examples 1-2, and the HPMC line is the infrared spectrum of hydroxypropyl methylcellulose.
FIG. 2 is a DSC of a collagen sponge obtained by cross-linking and modifying collagen with dialdehyde hydroxypropyl methylcellulose in examples 1-2 of the present invention. In the figure, the temperature identified by each line is the thermal stability temperature (Td) of the cross-linked modified product of example 1-2, and pure collagen is a blank of spring collagen sponge that has not been modified with dialdehyde hydroxypropyl methylcellulose.
Detailed Description
The invention is further illustrated by the following examples in conjunction with the accompanying drawings. It should be noted that the examples given are not to be construed as limiting the scope of the invention, and that those skilled in the art, on the basis of the teachings of the present invention, will be able to make numerous insubstantial modifications and adaptations of the invention without departing from its scope.
It is to be noted that 1) the degree of oxidation of the dialdehyde hydroxypropylmethylcellulose prepared in the following examples and comparative examples was measured by the hydroxylamine hydrochloride method. 0.1g of the oxidized product was dissolved in 30ml of deionized water and the pH was adjusted to 4.5. 0.43g of hydroxylamine hydrochloride was weighed out and dissolved in 20ml of deionized water, and the pH was adjusted to 4.5. The two solutions were mixed and reacted at 40 ℃ with stirring for 4 h. Titration with 0.1mol/l NaOH was then carried out, the volume used was recorded and the degree of oxidation OD (equation 1) was calculated as follows.
Figure BDA0002131859940000081
In the formula:
c: the concentration of NaOH used during titration, mol/L;
v: the volume of NaOH used in titration, ml;
m: the mass of the oxidation product weighed, g.
The relative viscosity of the dialdehyde hydroxypropyl methylcellulose prepared in the following examples and comparative examples was measured by the Ubbelohde viscosity method. Preparing 5mg/ml of different oxygenA dilute solution of the oxidation product under digestion conditions. Firstly, placing the pure solvent in a constant-temperature water bath at 25 +/-0.01 ℃ for 30min, and then accurately measuring the flowing-out time of the pure solvent in a capillary tube by using a stopwatch, and recording the flowing-out time as t0(ii) a The flowing time of the diluted collagen solution with different concentrations in the capillary is measured by the same method and is marked as t1. The elution times of the pure solvent and of the diluted collagen solution at each concentration were determined in five replicates and averaged. The relative viscosity (formula 2) was calculated as follows:
Figure BDA0002131859940000091
2) the thermal denaturation temperature, the degree of crosslinking and the enzymatic degradation performance of the dialdehyde hydroxypropyl methyl cellulose crosslinking modified collagen prepared in the following examples and comparative examples are tested by the following methods and by using the existing equipment.
The thermal denaturation temperature of the cross-linked modified collagen sponge was measured using a differential scanning calorimeter.
Measuring the crosslinking degree of the dialdehyde hydroxypropyl methyl cellulose crosslinking modified collagen: weighing 0.15g of modified collagen solution, mixing with 2ml of borax buffer solution with the pH value of 10, immediately adding 2ml of newly-prepared 0.1% (V/V) 2,4, 6-trinitrobenzene sulfonic acid (TNBS) solution, stirring and mixing by vortex oscillation, and reacting in a constant temperature water bath kettle at 50 ℃ in a dark place for 60 min. After the reaction is finished, 4ml of 6M hydrochloric acid solution is immediately added into the solution, the solution is evenly shaken and then placed into a constant temperature water bath kettle at 60 ℃ to continue the reaction for 90min until the solution is clear. After cooling at room temperature, the crosslinking number of the solution was determined using an ultraviolet-visible spectrophotometer at 340 nm. The measurement was repeated 3 times, and the average value was taken (formula 2).
Figure BDA0002131859940000092
In the formula:
A0: absorbance of native collagen;
A1: and the absorbance of the collagen after the cross-linking modification of the dialdehyde hydroxypropyl methyl cellulose.
Determining the enzyme degradation resistance of the dialdehyde hydroxypropyl methyl cellulose crosslinking modified collagen: cross-linking modified collagen (mass m) after freeze-drying0) The cells were immersed in phosphate buffer (pH 7.4) containing 0.02% pancreatin and shaken at 37 ℃. Respectively performing enzymolysis for 6h, taking out the rest collagen sponge sample after enzyme treatment, washing with deionized water for several times, drying in a 70 deg.C oven, cooling in a drier after 3h, and weighing m1The enzymatic degradation rate of the crosslinked and modified collagen was calculated (formula 3).
Figure BDA0002131859940000093
In the formula:
m0: sample mass before enzymolysis, g;
m1: mass of sample after enzymolysis, g.
The collagen samples used in examples 1-2 and comparative example 1 described below were collagen extracted from bovine skin.
Example 1
(1) Preparation of dialdehyde hydroxypropyl methyl cellulose
Preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 2%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: adding sodium periodate into the solution according to the proportion of 0.04, mixing, then adjusting the pH to 3, adjusting the reaction temperature to 35 ℃, and stirring and reacting for 4 hours under the dark condition; after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 19.18 percent and the relative viscosity reduced by 23.57 percent when the T is 25 +/-0.01 ℃.
(2) Modification of crosslinking
Mixing 40 parts by weight of dialdehyde hydroxypropyl methyl cellulose prepared in the step (1) with 800 parts by weight of acetic acid-sodium acetate (pH 3.98) to prepare dialdehyde hydroxypropyl methyl cellulose solution, weighing 100 parts by weight of collagen sample, dissolving 8000 parts by weight of acetic acid-sodium acetate (pH 3.98) to prepare collagen solution, mixing the two solutions, stirring and reacting at 4 ℃ for 24 hours, and freeze-drying to obtain the collagen product modified by crosslinking of the modified HPMC.
Through determination, the thermal denaturation temperature of the prepared collagen product cross-linked and modified by utilizing the modified HPMC is 59.2 ℃; the degree of crosslinking was 42.16%; the pancreatic enzyme degradation resistant rate after 6 hours is 46.41 percent.
Example 2
(1) Preparation of dialdehyde hydroxypropyl methyl cellulose
Preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 2%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: adding sodium periodate into the solution in a proportion of 0.20, mixing, then adjusting the pH to 4, adjusting the reaction temperature to 35 ℃, and stirring and reacting for 4 hours under the dark condition; after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 31.05 percent and the relative viscosity reduced by 70.47 percent when the T is 25 +/-0.01 ℃.
(2) Modification of crosslinking
Mixing 200 parts by weight of dialdehyde hydroxypropyl methyl cellulose prepared in the step (1) with 8000 parts by weight of acetic acid-sodium acetate (pH 3.98) buffer solution to prepare dialdehyde hydroxypropyl methyl cellulose solution, weighing 100 parts by weight of collagen sample, dissolving with 8000 parts by weight of acetic acid-sodium acetate (pH 3.98) buffer solution to prepare collagen solution, mixing the two solutions, stirring and reacting at 4 ℃ for 24h, and freeze-drying to obtain the collagen product modified by crosslinking of the modified HPMC.
Through determination, the thermal denaturation temperature of the prepared collagen product cross-linked and modified by utilizing the modified HPMC is 60.4 ℃; the degree of crosslinking was 56.37%; the pancreatin degradation resistant rate after 6 hours is 38.76 percent.
As shown in FIG. 1 in the drawings of the specification, it can be seen that 1732cm-1And 780cm-1The characteristic absorption peak of aldehyde group (-CHO) appears. The former belongs to the telescopic vibration of free aldehyde group, and the latter belongs to the vibration of hemiacetal. With the increase of the dosage of the sodium periodate, the peak of aldehyde group is gradually increased, which shows that the content of aldehyde group is gradually increased. Fig. 2 shows that when dialdehyde hydroxypropyl methylcellulose is used for blending modification with collagen, the thermal stability of the collagen can be improved.
In conclusion, the higher the aldehyde group content of the dialdehyde hydroxypropyl methyl cellulose is, the lower the relative viscosity is, and the biocompatibility is poorer when the dialdehyde hydroxypropyl methyl cellulose is subjected to crosslinking modification with collagen; however, the lower the aldehyde group content, the lower the degree of crosslinking when the collagen is subjected to crosslinking modification, and the lower the heat denaturation and enzyme degradation resistance. Therefore, the dialdehyde hydroxypropyl methyl cellulose after modification needs to be selected to have proper aldehyde content and relative molecular mass, so as to obtain the modified collagen with higher thermal stability and stronger enzyme degradation resistance and higher biocompatibility.
Example 3
(1) Preparation of dialdehyde hydroxypropyl methyl cellulose
Preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 2%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: adding sodium periodate into the solution according to the proportion of 0.08, mixing, then adjusting the pH to 3, adjusting the reaction temperature to 35 ℃, and stirring and reacting for 4 hours under the dark condition; after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 21.67 percent and the relative viscosity reduced by 27.05 percent when the T is 25 +/-0.01 ℃.
(2) Modification of crosslinking
Mixing 120 parts by weight of dialdehyde hydroxypropyl methyl cellulose prepared in the step (1) with 2892 parts by weight of deionized water to prepare dialdehyde hydroxypropyl methyl cellulose solution, weighing 100 parts by weight of collagen sample, dissolving with 8000 parts by weight of 0.5mol/L acetic acid to prepare collagen solution, mixing the two solutions, adjusting the pH of the mixed solution to 5, stirring and reacting at 4 ℃ for 24h, and freeze-drying to obtain the collagen product modified by crosslinking of the modified HPMC.
Through determination, the thermal denaturation temperature of the prepared collagen product cross-linked and modified by utilizing the modified HPMC is 68.9 ℃; the degree of crosslinking was 65.54%; the pancreatin degradation resistant rate is 32.15% in 6 h.
Example 4
(1) Preparation of dialdehyde hydroxypropyl methyl cellulose
Preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 3%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: adding sodium periodate into the solution according to the proportion of 0.08, mixing, then adjusting the pH to 4, adjusting the reaction temperature to 40 ℃, and stirring and reacting for 4 hours under the dark condition; after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 19.97 percent and the relative viscosity reduced by 26.31 percent when the T is 25 +/-0.01 ℃.
(2) Modification of crosslinking
Mixing 140 parts by weight of dialdehyde hydroxypropyl methyl cellulose prepared in the step (1) with 4480 parts by weight of acetic acid-sodium acetate (pH 3.98) to prepare dialdehyde hydroxypropyl methyl cellulose solution, weighing 100 parts by weight of collagen sample, dissolving with 8000 parts by weight of acetic acid-sodium acetate (pH 3.98) to prepare collagen solution, mixing the two solutions, stirring and reacting at 4 ℃ for 24 hours, and freeze-drying to obtain the collagen product modified by crosslinking of the modified HPMC.
Through determination, the thermal denaturation temperature of the prepared collagen product cross-linked and modified by utilizing the modified HPMC is 67.6 ℃; the degree of crosslinking was 63.21%; the pancreatic enzyme degradation resistant rate after 6 hours is 35.44 percent.
Example 5
(1) Preparation of dialdehyde hydroxypropyl methyl cellulose
Preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 3%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: adding sodium periodate into the solution according to the proportion of 0.12, mixing, then adjusting the pH value to 5, adjusting the reaction temperature to 35 ℃, and stirring and reacting for 4 hours under the dark condition; after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 25.39% and the relative viscosity reduced by 44.47% when the T is 25 +/-0.01 ℃.
(2) Modification of crosslinking
Mixing 100 parts by weight of the dialdehyde hydroxypropyl methyl cellulose prepared in the step (1) with 20000 parts by weight of deionized water to prepare a dialdehyde hydroxypropyl methyl cellulose solution, weighing 100 parts by weight of a collagen sample, dissolving with 8000 parts by weight of 0.5mol/L acetic acid to prepare a collagen solution, mixing the two solutions, stirring and reacting for 2 hours at the pH of 7 and the temperature of 4 ℃, and freeze-drying to obtain the collagen product modified by crosslinking of the modified HPMC.
Through determination, the thermal denaturation temperature of the prepared collagen product cross-linked and modified by utilizing the modified HPMC is 61.05 ℃; the degree of crosslinking was 60.24%; the pancreatin degradation resistance rate of 6h is 36.64%. As known from the relevant documents, the optimal reaction pH value of glutaraldehyde and collagen is 6-8. Under the condition, the crosslinking reaction of Schiff base bonds formed by aldehyde groups in dialdehyde cellulose and amino groups in collagen is facilitated, and the crosslinking degree is increased.
Example 6
(1) Preparation of dialdehyde hydroxypropyl methyl cellulose
Preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 1%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: adding sodium periodate into the solution in a proportion of 0.10, mixing, then adjusting the pH to 2, adjusting the reaction temperature to 30 ℃, and stirring and reacting for 5 hours under the dark condition; after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 23.53 percent and the relative viscosity reduced by 35.24 percent when the T is 25 +/-0.01 ℃.
(2) Modification of crosslinking
80 parts by weight of dialdehyde hydroxypropyl methyl cellulose prepared in the step (1) and 400 parts by weight of phosphate buffer solution (PBS, 10mmol/L NaH)2Pa/Na2HPa, 100mmol/L NaCl, pH 7.4) and prepared as a dialdehyde hydroxypropyl methylcellulose solution. Then, 100 parts of collagen sponge was weighed out, cut into pieces, and dissolved in 8000 parts of phosphate buffer (PBS, 10mmol/L NaH2Pa/Na2HPa, 100mmol/L NaCl, pH 7.4) at low temperature. Culturing the collagen solution at 37 deg.C for 3 hr to obtain pure collagen gel, and soaking the collagen gel in dialdehyde hydroxypropyl methylcellulose solution. Reacting for 24h at the temperature of 30 ℃ to obtain the collagen product modified by the modified HPMC through crosslinking.
Through determination, the thermal denaturation temperature of the prepared collagen product cross-linked and modified by utilizing the modified HPMC is 51.3 ℃; the degree of crosslinking was 32.16%; the pancreatin degradation resistance rate of 6h is 54.39%.
Example 7
(1) Preparation of dialdehyde hydroxypropyl methyl cellulose
Preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 3%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: adding sodium periodate into the solution in a proportion of 0.15, mixing, then adjusting the pH to 6, adjusting the reaction temperature to 50 ℃, and stirring and reacting for 3 hours under the dark condition; after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose with the oxidation degree of 26.74% and the relative viscosity reduced by 25.81% when the T is 25 +/-0.01 ℃.
(2) Modification of crosslinking
180 parts by weight of the dialdehyde hydroxypropylmethylcellulose obtained in step (1) were mixed with 820 parts by weight of a phosphate buffer solution (PBS, 10mmol/L NaH2Pa/Na2HPa, 100mmol/L NaCl, pH 7.4) to prepare a double weighing of 100 parts by weight of collagen sponge, which was cut into pieces and dissolved in 8000 parts by weight of a phosphate buffer solution (PBS, 10mmol/L NaH2Pa/Na2HPa, 100mmol/L NaCl, pH 7.4) at a low temperature. Culturing the collagen solution at 37 deg.C for 3 hr to obtain pure collagen gel, and soaking the collagen gel in dialdehyde hydroxypropyl methylcellulose solution. Reacting for 24 hours at the temperature of 37 ℃ to obtain the collagen product modified by the modified HPMC through crosslinking.
Through determination, the thermal denaturation temperature of the prepared collagen product cross-linked and modified by utilizing the modified HPMC is 63.9 ℃; the degree of crosslinking was 53.60%; the pancreatin degradation resistance rate of 6h is 35.87%.
Comparative example 1
Directly testing a collagen sponge sample, wherein the thermal denaturation temperature is 52.1 ℃; the degree of crosslinking is 0; the pancreatin degradation resistance rate of 6h is 98.66%.
The hydroxypropyl methylcellulose samples are directly tested, and the HPMC can be divided into four substitution types according to the difference of the content of methoxyl group and hydroxypropoxyl group in the structure of the HPMC, namely 1828, 2208, 2906 and 2910. Different HPMC models have different viscosities, and the viscosity of each model has a large fluctuation range, and is generally expressed by taking a middle value. For example, when the content of the methoxyl group of the HPMC is 27-30%, the content of the hydroxypropoxyl group is 4.0-7.5%, and the relative viscosity of the HPMC is 403% at the temperature of 25 +/-0.01 DEG C
Comparative example 2
100 parts of collagen sponge and 100 parts of HPMC powder were weighed and dissolved separately in 667 parts of 0.1mol/L acetic acid solution. And taking the prepared collagen and HPMC solution, blending according to the mass ratio of the collagen to the HPMC of 7/3, stirring and blending at 4 ℃, performing centrifugal deaeration after uniform stirring, and standing for 24 hours.
According to determination, after the HPMC is mixed with the collagen solution, the thermal denaturation temperature is 41.2 ℃; the crosslinking degree is 0, and the principle of the method adopted by the crosslinking degree test is that epsilon-amino which does not participate in the reaction in the collagen reacts with TNBS to generate a chromogenic substance. Since no-CHO or other active groups in HPMC react with epsilon-amino groups in collagen, the final absorbance of the reaction with TNBS is the same as that of pure collagen and TNBS, and the cross-linking degree is 0 as shown in formula 2.

Claims (10)

1. A DHPMC blending crosslinking modified collagen suitable for biomedicine is characterized by mainly comprising the following raw materials in parts by weight:
100 parts of collagen, namely 100 parts of collagen,
40-200 parts of dialdehyde hydroxypropyl methyl cellulose;
the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 19-35%, and the relative viscosity is reduced by 23.57-70.47% at the temperature T of 25 +/-0.01 ℃.
2. The DHPMC blended cross-linked modified collagen suitable for biomedicine according to claim 1, which is characterized in that said dialdehyde hydroxypropyl methyl cellulose is prepared by the following steps:
preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 1-3%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: (0.04-0.20) adding sodium periodate into the solution, mixing, adjusting the pH to 2-6, reacting at 30-50 ℃, and stirring for reaction for 3-5 hours under the condition of shading; and after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose.
3. The biomedical DHPMC blended cross-linked modified collagen according to claim 2, wherein: and purifying and drying, namely performing rotary evaporation for 3-5 times by using absolute ethyl alcohol and deionized water respectively, and placing the obtained product in an oven for drying.
4. The biomedical DHPMC blended cross-linked modified collagen according to claim 1, wherein said raw materials mainly comprise, in parts by weight:
100 parts of collagen, namely 100 parts of collagen,
100-140 parts of dialdehyde hydroxypropyl methyl cellulose;
the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 20-30%, and the relative viscosity is reduced by 23.57-45.66% at the temperature T of 25 +/-0.01 ℃.
5. A preparation method of DHPMC blending crosslinking modified collagen suitable for biomedicine is characterized by comprising the following steps:
(1) stock preparation
The raw materials mainly comprise the following components in parts by weight:
100 parts of collagen, namely 100 parts of collagen,
40-200 parts of dialdehyde hydroxypropyl methyl cellulose;
the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 19-35%, and the relative viscosity is reduced by 23.57-70.47% at the temperature of 25 +/-0.01 ℃ T;
(2) modification of crosslinking
Preparing the prepared dialdehyde hydroxypropyl methyl cellulose in the step (1) into dialdehyde hydroxypropyl methyl cellulose solution with the pH value of 3-7, adding collagen into the solution, mixing, adjusting the pH value to 3-7, and reacting at the temperature of 0-35 ℃ for 24-72h to obtain the DHPMC blending crosslinking modified collagen product.
6. The method for preparing DHPMC blended cross-linked modified collagen suitable for use in biomedicine according to claim 5, wherein: the dialdehyde hydroxypropyl methyl cellulose is prepared into a dialdehyde hydroxypropyl methyl cellulose solution with the pH value of 3-7, wherein the mass concentration of the dialdehyde hydroxypropyl methyl cellulose solution is 5-20%.
7. The method for preparing DHPMC blended and cross-linked modified collagen suitable for biomedicine according to claim 5, which is characterized in that the method for preparing dialdehyde hydroxypropyl methyl cellulose comprises the following steps:
preparing a hydroxypropyl methyl cellulose solution with the mass concentration of 1-3%, and mixing the hydroxypropyl methyl cellulose and sodium periodate according to the mass ratio of 1: (0.04-0.20) adding sodium periodate into the solution, mixing, adjusting the pH to 2-6, reacting at 30-50 ℃, and stirring for reaction for 3-5 hours under the condition of shading; and after the reaction time is up, purifying and drying to obtain the dialdehyde hydroxypropyl methyl cellulose.
8. The method for preparing DHPMC blended cross-linked modified collagen suitable for use in biomedicine according to claim 7, wherein: and purifying and drying, namely performing rotary evaporation for 3-5 times by using absolute ethyl alcohol and deionized water respectively, and placing the obtained product in an oven for drying.
9. The method for preparing the DHPMC blended cross-linked modified collagen suitable for biomedicine according to claim 5, characterized in that the raw materials mainly comprise, in parts by weight:
100 parts of collagen, namely 100 parts of collagen,
100-140 parts of dialdehyde hydroxypropyl methyl cellulose;
the dialdehyde hydroxypropyl methyl cellulose is obtained by selectively oxidizing hydroxypropyl methyl cellulose by sodium periodate, the oxidation degree is 20-30%, and the relative viscosity is reduced by 23.57-45.66% at the temperature T of 25 +/-0.01 ℃.
10. The use of the DHPMC blended cross-linked modified collagen suitable for use in biomedicine as claimed in claim 1 in the fields of controlled drug release, bioscaffolds, hemostatic materials.
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