WO2014206043A1

WO2014206043A1 - Application method of cyclodextrin multi-aldehyde cross-linking agent

Info

Publication number: WO2014206043A1
Application number: PCT/CN2013/090019
Authority: WO
Inventors: 刘卅; 蔡洁; 任力; 王迎军
Original assignee: 华南理工大学
Priority date: 2013-06-28
Filing date: 2013-12-20
Publication date: 2014-12-31
Also published as: CN103342824B; CN103342824A

Abstract

Provided is an application method of a cyclodextrin multi-aldehyde cross-linking agent. In the cyclodextrin multi-aldehyde cross-linking agent, there are 2-3 acid groups per mole on average, which are used to modify collagen, the modification method comprising: mixing the aqueous solution of the cyclodextrin multi-aldehyde cross-linking agent and the collagen aqueous solution, freezing the obtained mixed solution in a mold to prepare modified collagen gel, taking out of the modified collagen gel after the mold is unfrozen at room temperature, and drying the modified collagen gel to obtain porous spongy modified collagen.

Description

Application method of cyclodextrin polyaldehyde crosslinking agent

Technical field

The present invention relates to a cyclodextrin and a collagen crosslinker, and in particular to a method for applying a cyclodextrin polyaldehyde crosslinker. Background technology

Collagen is a biological natural high molecular substance, English name collages collagen exists in the book

In all cell animals, it is the most abundant type of protein in the body, present in almost all tissues, and is an extracellular matrix component. Collagen has a special three-stranded helical peptide chain structure that imparts many good biological and chemical properties to collagen. Collagen has good biocompatibility, biodegradability, low antigenicity; has cell adaptation, cell proliferation and promotes platelet aggregation; has good solubility, non-toxicity, unique fiber formation characteristics ; and easy to modify and a wide range of sources. Therefore, as a very important biological material, collagen is widely used in wound dressings, artificial skin, beauty, drug release, tissue engineering scaffolds and cell culture. The value of collagen is receiving more and more attention.

However, the pure collagen extracted from animal tissues has low mechanical strength, and the tissue repairing material cannot withstand the mechanical environment in the body, and the degradation rate in the body is too fast and the thermal stability is insufficient. Therefore, people have modified collagen by various methods, including physical crosslinking, chemical crosslinking and the like. Among them, more is chemical cross-linking. The main crosslinking agents in chemical crosslinking are: glutaraldehyde, carbodiimide (EDC), epoxy compounds, genipin, glyoxylic acid, sulfur compounds, hexamethylene diisocyanate, acyl azide and diphenyl. Phosphate, dialdehyde starch, etc. The above chemical cross-linking agents can effectively improve the mechanical strength and stability of collagen, etc., but also all have some disadvantages through cross-linking, for example, some may cause cytotoxicity, some may cause color of collagen materials, Some have calcification potential, Some do not significantly improve some of the disadvantages of collagen materials.

Cyclodextrin is a cyclic oligosaccharide composed of 6~8 D-(+) glucopyranose units through α-1, 4-glycosidic linkages, corresponding to α, β, Υcyclodextrin, cyclodextrin The model of the molecule is like a cone-shaped cylinder with an inner hollow to the top. In the ring structure of the cyclodextrin molecule, the polar hydroxyl group is located at the edge of the cyclodextrin unit, and the secondary and primary polar hydroxyl groups protrude from the edge, so that hydrophilic groups are distributed both above and below, and are hydrophilic. . Inside the cyclodextrin cavity, a sugar-oxygen bridge atom is arranged. Since the non-bonded electrons of the oxygen atom are directed to the center, the electron density in the cavity is high; the hydrogen atoms on the glucopyranose ring C-3, C-5 are located in the cavity and cover the oxygen atom of the glycoside. The interior of the cavity becomes a hydrophobic space. The hydrophobic cavity can form a clathrate by hydrophobic interaction, hydrogen bonding, van der Waals force, etc., and thus exhibits special adsorption properties. Cyclodextrins form stable inclusion complexes with many organic compounds, halogens and certain inorganic compounds. It can enclose certain compounds or certain groups of compounds like a molecular bag to improve the water solubility, stability and other physical and chemical properties of the encapsulated material. Cyclodextrins can act as sustained release agents for the substances to be included, and they have no significant toxic side effects to humans. The advantages of cyclodextrin make it widely available in all areas of biomedical science. Summary of the invention

In order to solve the above deficiencies of the prior art, an object of the present invention is to provide a method for applying a cyclodextrin polyaldehyde cross-linking agent, wherein a cyclodextrin polyaldehyde cross-linking agent is used for modifying collagen. The cyclodextrin polyaldehyde cross-linking agent does not produce cytotoxicity while improving the mechanical strength and stability of collagen.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for applying a cyclodextrin polyaldehyde crosslinking agent, wherein the cyclodextrin polyaldehyde crosslinking agent is used for modifying a collagen; wherein the chemical structure of the cyclodextrin polyaldehyde crosslinking agent is:

Preferably, the cyclodextrin polyaldehyde crosslinker is used for the preparation of porous sponge-like modified collagen. Preferably, the preparation method of the porous sponge-like modified collagen comprises the following steps:

(1) mixing a cyclodextrin polyaldehyde crosslinking agent aqueous solution with an aqueous collagen solution, and reacting at 2 to 6 ° C for 20 to 30 hours to obtain a modified collagen solution;

(2) The modified collagen solution obtained in the step (1) is kept at 35 to 38 ° C for 15 to 20 minutes, then the modified collagen solution is transferred to a mold, and the mold is frozen at -15 to 25 ° C. ~50h, preparing a modified collagen gel;

(3) The mold was taken out at room temperature for 4 to 6 hours, and the modified collagen gel was taken out and dried to obtain the porous sponge-like modified collagen.

Preferably, the mass concentration of the aqueous solution of the cyclodextrin polyaldehyde crosslinking agent in the step (1) is 0.075% to 0.6%, and the mass concentration of the aqueous collagen solution is 0.6%.

Preferably, the reaction temperature in the step (1) is 4 °C.

Preferably, the modified collagen solution in step (2) is incubated in a 37 ° C water bath at a freezing temperature of -20 ° C. Preferably, the drying method described in the step (3) is freeze drying.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

(1) The present invention uses a cyclodextrin polyaldehyde having an aldehyde group of 2 or 3 as a crosslinking agent; since the cyclodextrin polyaldehyde crosslinking agent has the advantages of non-toxicity and good crosslinking effect, it is particularly suitable for Modification of collagen. The cyclodextrin polyaldehyde having 2 or 3 aldehyde groups is used for the modification of collagen, and the modified collagen has high cross-linking efficiency, and the modified collagen material has greatly improved thermal stability and markedly enhanced degradation resistance.

(2) The present invention cross-links cyclodextrin polyaldehyde with collagen, which can not only make up for some shortcomings of collagen, improve the performance of collagen materials, but also avoid the introduction of cytotoxic chemical cross-linking agents such as glutaraldehyde. It not only maintains some of the original properties of collagen, but also introduces some excellent properties of cyclodextrin, such as special rigid structure, inclusion coordination, molecular recognition and assembly, which greatly expands the application range of collagen. DRAWINGS

Figure 1 is a schematic diagram of a synthetic route of a cyclodextrin polyaldehyde; Figure 2 is a schematic diagram of cyclodextrin polyaldehyde cross-linked collagen;

Figure 3 is an infrared spectrum (FTIR) of β-cyclodextrin and β-cyclodextrin polyaldehyde;

Figure 4 is a nuclear magnetic resonance spectrum (1H-NMR) of β-cyclodextrin polyaldehyde;

Figure 5 is an infrared spectrum (FTIR) of β-cyclodextrin polyaldehyde and collagen before and after cross-linking;

Figure 6 is a graph showing the degree of crosslinking of β-cyclodextrin polyaldehyde crosslinked collagen;

Figure 7 is a DSC chart before and after cross-linking of β-cyclodextrin polyaldehyde with collagen;

Figure 8 is a graph showing the mechanical properties of β-cyclodextrin polyaldehyde before and after cross-linking with collagen;

Figure 9 is a graph showing the degree of enzymatic hydrolysis before and after cross-linking of β-cyclodextrin polyaldehyde with collagen. detailed description

The present invention will be described in detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

The principle of the present invention is shown in Figures 1 and 2, wherein cyclodextrin is oxidized to a cyclodextrin polyaldehyde derivative, i.e., a polyfunctional compound, as a crosslinking agent by using 2-iodobenzoic acid (hydrazine). Then, the aldehyde group of the cyclodextrin polyaldehyde is reacted with the active amino group on the collagen to form a Schiff base to crosslink the modified collagen, so that the mechanical properties, thermal stability and degradation resistance of the collagen are greatly improved. The present invention is suitable for α-cyclodextrin, β-cyclodextrin and cyclodextrin.

The method for synthesizing the cyclodextrin polyaldehyde crosslinking agent of the present invention comprises the following steps:

(1) 0.5 mmol of the purified cyclodextrin is added to 5 to 8 ml of dimethyl sulfoxide, and the cyclodextrin is completely dissolved to obtain a mixture;

(2) adding 2.2 to 2.8 equivalents of 2-iodobenzoic acid to the mixture obtained in the step (1), and reacting for 30 to 48 hours while maintaining stirring at 25 to 30 ° C, and then filtering the reaction solution. Keep the filtrate;

(3) Drop the filtrate obtained in the step (2) into acetone, and filter and retain the precipitate;

(4) The precipitate obtained in the step (3) is dried under vacuum at 30 to 40 ° C for 24 hours, and then dissolved in deionized water;

(5) The solution obtained in the step (4) is filtered, the filtrate is retained, and the filtrate is freeze-dried for 48 hours to obtain The cyclodextrin polyaldehyde crosslinker.

Example 1

(1) Preparation of cyclodextrin polyaldehyde crosslinker:

0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.4 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 25 ° C for 48 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 h, the dried sample was finally dissolved in 50 ml of deionized water, and the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a pale yellow sample.

The yield of β-cyclodextrin polyaldehyde was 90%.

(2) Modified collagen:

The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. Different concentrations of cyclodextrin polyaldehyde aqueous solution were modified as follows:

(1) uniformly mixing the cyclodextrin polyaldehyde aqueous solution with a collagen aqueous solution having a mass concentration of 0.6%, and reacting in a refrigerator at 4 ° C for 24 hours to obtain a modified collagen solution;

(2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 18 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;

(3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen.

(III) The β-cyclodextrin polyaldehyde and the porous sponge-like modified collagen product of Example 1 were subjected to characterization by freeze-drying.

FT-IR analysis was performed on a NICOLET 760-type infrared spectrophotometer, and the sample preparation was performed by KBr tableting. The NMR analysis was performed using a Bruker 400 MHz nuclear magnetic resonance apparatus, and the solvent was deuterated DMSO. DSC analysis of β-cyclodextrin polyaldehyde modified collagen DIAMOND DSC type differential produced by American company Scanning calorimeter, accurately weigh 5~10mg sample in DSC坩埚, use air as reference, heat from 35 °C to 210 °C, heating rate is 5 °C/min, nitrogen flow in sample chamber It is 20 mL/min. The cross-linking degree of β-cyclodextrin polyaldehyde-modified collagen was determined by the ninhydrin method using a glycine solution as a standard curve, and the UV absorbance value was measured at 570 nm using a LAMBDA950 ultraviolet spectrophotometer. The content of aldehyde group in the synthesized β-cyclodextrin polyaldehyde derivative is determined by the "alkali consumption method", and the specific steps are as follows: Accurately weigh a certain amount of β-cyclodextrin polyaldehyde derivative in a 250 ml Erlenmeyer flask Accurately add O.Olmol/L sodium hydroxide solution 30, and then place the conical flask in a 70 ° C water bath for 4 min, then quickly rinse it with tap water to cool it, then add 0.01 mol/L H ₂ S0 ₄ solution. 20 ml and 30 ml of distilled water, add phenolphthalein indicator, shake well to make it evenly mixed, and titrate to the end point. Calculated as follows:

Number of aldehyde groups per mole of β-cyclodextrin polyaldehyde = ( CiVi-2C ₂ V2 ) I ( G/1131 )

Where: CI - concentration of NaOH solution, mol / L;

VI - the volume of the NaOH solution consumed, L;

C2——concentration of H2S04 solution, mol/L;

V2 - the volume of the H2S04 solution consumed, L;

G——sample quality, g _;

Molecular mass of 1131-cyclodextrin polyaldehyde, g/mol.

After three repeated experiments, it was concluded that the average number of aldehyde groups per mole of β-cyclodextrin polyaldehyde was about 2.5. 3 is a FTIR diagram of β-cyclodextrin polyaldehyde and β-cyclodextrin of the present embodiment, and it can be clearly seen from the two spectra that the β-cyclodextrin polyaldehyde has a newly formed peak at 1733.93 cnr ¹ This peak is a characteristic peak of the aldehyde group, indicating that an aldehyde group is formed in the synthesized product. In addition, in the figure, 3376 cnr ¹ is the stretching vibration peak of -OH, 2926 cnr ¹ is the -CH ₂ stretching vibration peak, 1158 cnr ¹ is the COC stretching vibration peak, and 1081 cnr ¹ is the C-0-H bending vibration peak, 1030 ( ^1_ ¹ is ( -0 and 0 -0 coupled vibration peak, 707 cnr ¹ is the ring vibration peak. It can be seen that the absorption peaks of these β-cyclodextrin polyaldehydes are not significantly different from the infrared spectrum of β-cyclodextrin. This indicates that the oxidation of β-cyclodextrin with 2-iodobenzoic acid (ΙΒΧ) to synthesize polyaldehyde derivatives does not destroy the specific structure of β-cyclodextrin, which allows the synthesis of β-cyclodextrin polyaldehyde derivatives to remain. Many excellent properties of β-cyclodextrin. Figure 4 is a NMR spectrum of the β-cyclodextrin polyaldehyde of the present example, and it can be seen from the spectrum that δ 9.70 (s, 1H), 9.53 (s, 1H), 5.68 (s, 14H), 4.94 ( s, 2H), 4.84 (s, 5H), 4.43 (s, 4H), 3.64 (s, 24H), 3.56 (s, 14H where δ 9.70 (s, IH), 9.53 (s, IH) is aldehyde The characteristic peak of hydrogen, that is, the product formed by the present invention does have the formation of an aldehyde group, and the ratio of the area of δ 4.94 to the peak area of δ 4.84 is 2: 5 to 3:

Between 4, this indicates that the β-cyclodextrin polyaldehyde synthesized in the present invention has between 2 and 3 aldehyde groups per mole of aldehyde groups, and has 2 to 3 reactive groups, which is suitable for use as a crosslinking agent.

5 is an FTIR chart of the β-cyclodextrin polyaldehyde aqueous solution having a mass concentration of 0.45% before and after crosslinking with collagen in the present embodiment, and 33 1 8 CHT ¹ is an amide oxime band peak (Ν-Η stretching vibration:), 3071 cnr ¹ is the amide B band peak (N-H stretching vibration:), 1655 cnr ¹ is the amide I band peak (C= 0 stretching vibration:), 1549 cnr ¹ is the amide II band peak (N-H bending vibration), from the figure It can be seen that the position and intensity of the amide I band associated with the collagen triple helix conformation are substantially unchanged, and there is still a high absorption peak, indicating that the triple helix structure of the collagen after cross-linking is not destroyed. The absorption peak intensity of the amide B band and the amide I band decreased after cross-linking, that is, the amino group content decreased, indicating that β-cyclodextrin dialdehyde was cross-linked with collagen. In addition, since the C=N stretching vibration peak of the Schiff base is about 1660 cm" ^{1, it} is blocked by the peak of the amide I band of the collagen, so no new absorption peak is found in the infrared spectrum.

Figure 6 is a graph showing the degree of crosslinking of the β-cyclodextrin polyaldehyde crosslinked collagen of the present embodiment, wherein Col-C-1 to Col-C-5 are different concentrations of β-cyclodextrin polyaldehyde crosslinked collagen The degree of cross-linking, the concentration of β-cyclodextrin polyaldehyde is 0.075%, 0.15%, 0.30%, 0.45% and 0.60%, respectively. As the concentration of β-cyclodextrin polyaldehyde increases, the degree of crosslinking increases. , the highest reached about 91%. When the concentration of the cross-linking agent is low, the degree of cross-linking increases rapidly as the concentration of the cross-linking agent increases, and when the cross-linking agent concentration reaches 0.45%, the concentration of the cross-linking agent is increased, and the degree of cross-linking is The change is not large, mainly because when the concentration of the cross-linking agent exceeds 0.45%, the free amino group in the collagen is substantially completely reacted. Col-G-6 is the cross-linking degree of 0.25% glutaraldehyde cross-linked collagen, the degree of cross-linking is 81.26%, and the aldehyde content of 0.25% glutaraldehyde is about 0.3%. The aldehyde content in β-cyclodextrin polyaldehyde About 4 times, but the degree of cross-linking is not as high as 0.3% β-cyclodextrin polyaldehyde cross-linked collagen, so β-cyclodextrin polyaldehyde is a cross-linking efficiency. Agent.

Figure 7 is a graph showing the mass concentration of 0.45% β-cyclodextrin polyaldehyde aqueous solution in the present embodiment before and after cross-linking with collagen In the DSC chart, the heat denaturation temperature is the temperature at which the triple helix structure unique to collagen is completely destroyed. As can be seen from the figure, the thermal denaturation temperature of pure collagen is 71.4 °C. The heat-denatured temperature of the collagen modified by β-cyclodextrin polyaldehyde crosslinking was 88.3 ° C, which was 16.9 ° C higher than that of pure collagen. The heat-denatured temperature of the collagen modified by crosslinking with glutaraldehyde was 82.3 ° C, which was 10.9 ° C higher than that of pure collagen. It can be seen that β-cyclodextrin polyaldehyde cross-linked collagen can significantly improve the thermal stability of collagen, even more effective than glutaraldehyde-modified collagen, which again proves that collagen and β-cyclodextrin dialdehyde occur. Cross-linking. The increase of the interaction between collagen molecules makes the activity of the peptide chain constrained, and the difficulty of phase change increases. The heat energy and temperature required for denaturation increase, and the thermal stability of the material increases, which can expand the application range of the material.

Fig. 8 is a graph showing the mechanical properties of the β-cyclodextrin polyaldehyde and collagen before and after cross-linking of the present embodiment. The collagen hydrogel is mainly subjected to compressive stress in many fields of application, and thus the collagen hydrogel was subjected to a compression test. A cylindrical collagen gel (about 6 mm in diameter and 15 mm in height) was compression-tested at 1 mm/min using a biomechanical testing machine, and linear fitting was performed using the initial straight line portion of the obtained stress-strain curve. For compression modulus. It can be seen from the figure that the compressive modulus of pure collagen is only about 0.6 KPa. As the concentration of the cyclodextrin polyaldehyde crosslinking agent increases, the compressive strength increases continuously, reaching a maximum of 3.522 KPa, which is better than pure collagen. Increased nearly 6 times. It can be seen that the compressive strength of the cyclodextrin polyaldehyde is significantly improved after cross-linking, and the mechanical properties are ten-day J FJinU.

9 is a graph showing the enzymatic stability of the β-cyclodextrin polyaldehyde and collagen before and after cross-linking of the present embodiment, and the relative enzymatic hydrolysis degree of the pure collagen is determined to be 100%, as shown in the figure, along with the cross-linking agent When the concentration increases, the degree of enzymatic hydrolysis of the modified collagen material decreases in turn. The higher the concentration of the cross-linking agent, the smaller the degree of enzymatic hydrolysis, indicating that the higher the cross-linking agent concentration, the better the cross-linking effect, and the more resistant the collagen material has. Strong. When the concentration of the new cross-linking agent is 0.6%, the relative enzymatic hydrolysis degree is 28.68%, and the degree of enzymatic hydrolysis is significantly lower than that of pure collagen, which indicates that the novel cross-linking is very effective in improving the bio-stability of collagen and its degradation rate. Can be greatly reduced. At the same time, it indicates that the new cross-linking agent cross-links with collagen, forming a series of chemical bonds in the molecule and between molecules, and even forming a network cross-linking between the molecules, thereby making it more stable. It can be seen from the above figure that the relative enzymatic hydrolysis degree of the collagen material cross-linked with glutaraldehyde is about 30.05%, and it is known that the anti-enzymatic ability of the collagen material modified by the novel cross-linking agent is slightly better than that of glutaraldehyde. A brief description of the new cross-linking agent can effectively improve the anti-enzymatic ability of collagen, It slows down the degradation time of collagen, which is mainly due to the inability of the enzyme to fully contact the collagen molecules due to cross-linking. The degradation of the enzyme can only be carried out on the surface of the collagen material, thus slowing down the degradation of collagen. Example 2

(1) Preparation of cyclodextrin polyaldehyde crosslinker:

0.5 mmol of the purified Y-cyclodextrin was weighed into a 50 ml flask, and 5 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.2 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, the mixture was reacted under magnetic stirring at 25 ° C for 30 hours. After the reaction was completed, the mixture was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.

The cyclodextrin polyaldehyde yield was determined to be 87%.

(2) Modified collagen:

(2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 20 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;

(3) After the mold was left at room temperature for 6 hours, the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 3 0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 8 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.8 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, the mixture was reacted under magnetic stirring at 25 ° C for 30 hours. After the reaction was completed, the mixture was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.

The final yield of cyclodextrin polyaldehyde was 91%.

(2) Modified collagen:

(3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 4

(1) Preparation of cyclodextrin polyaldehyde crosslinker:

0.5 mmol of the purified α-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.6 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 28 ° C for 30 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. Dry for 24h, and finally use the dried sample Dissolve in 50 ml of deionized water, remove impurities by filtration, and freeze the filtrate to obtain the final sample.

After testing, the final yield of cyclodextrin polyaldehyde reached 90%.

(2) Modified collagen:

(2) The modified collagen solution obtained in the step (1) was placed in a 37 ° C water bath for 15 min, and the modified collagen solution was dropped into a mold of a certain shape with a pipette, and then the mold was placed in a refrigerator at -20 ° C. Frozen for 48h, preparing a modified collagen gel;

(3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 5

(1) Preparation of cyclodextrin polyaldehyde crosslinker:

0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.4 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 30 ° C for 48 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.

The final yield of cyclodextrin polyaldehyde was 92%.

(2) Modified collagen:

The cyclodextrin polyaldehyde crosslinking agent prepared by the step (1) is formulated into five cyclodextrin polyaldehyde aqueous solutions having a mass concentration of 0.075%, 0.15%, 0.30%, 0.45%, and 0.60%, and then the five kinds are used. different density The cyclodextrin polyaldehyde aqueous solution was modified with collagen as follows:

(3) The mold was taken out at room temperature for 6 hours, and the modified collagen gel was taken out, and then the modified collagen gel was dried by a freeze dryer to obtain a porous sponge-like modified collagen. Example 6

(1) Preparation of cyclodextrin polyaldehyde crosslinker:

0.5 mmol of the purified β-cyclodextrin was weighed into a 50 ml flask, and 7 ml of a polar aprotic solvent dimethyl sulfoxide (DMSO) was added to the flask, and the cyclodextrin was magnetically stirred until the dissolution was completed, and 2.6 was added thereto. Equivalent to the mild oxidant 2-iodobenzoic acid (IBX). Then, it was reacted under magnetic stirring at 30 ° C for 48 hours. After the reaction was completed, it was filtered, and the filtrate was dropped into 150 ml of acetone, and the precipitated material was filtered to precipitate, and the precipitate was vacuumed at 35 ° C in a vacuum drying oven. After drying for 24 hours, the dried sample was finally dissolved in 50 ml of deionized water, the impurities were removed by filtration, and the filtrate was freeze-dried to obtain a final sample.

The final yield of cyclodextrin polyaldehyde was 93%.

(2) Modified collagen:

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and modifications may be made without departing from the spirit and scope of the invention. Simplifications, which are equivalent replacement means, are included in the scope of the present invention.

Claims

claims

1. An application method of cyclodextrin polyaldehyde cross-linking agent, characterized in that the cyclodextrin poly-aldehyde cross-linking agent is used to modify collagen; wherein the cyclodextrin poly-aldehyde cross-linking agent is The chemical structure is:

2. The application method according to claim 1, characterized in that the cyclodextrin polyaldehyde cross-linking agent is used for the preparation of porous sponge-like modified collagen.

3. The application method according to claim 2, characterized in that the preparation method of the porous sponge-like modified collagen includes the following steps:

(1) Mix the cyclodextrin polyaldehyde cross-linking agent aqueous solution and the collagen aqueous solution, and react at 2 to 6°C for 20 to 30 hours to obtain the modified collagen solution;

(2) Insulate the modified collagen solution obtained in step (1) at 35~38°C for 15~20 minutes, then transfer the modified collagen solution to the mold, and then freeze the mold at -15~-25°C for 40 ~50h, prepare modified collagen gel;

(3) Place the mold at room temperature for 4 to 6 hours, take out the modified collagen gel, and dry it to obtain porous sponge-like modified collagen.

4. The application method according to claim 3, characterized in that, in step (1), the mass concentration of the cyclodextrin polyaldehyde cross-linking agent aqueous solution is 0.075%~0.6%, and the mass concentration of the collagen aqueous solution is 0.6%.

5. The application method according to claim 3, characterized in that the reaction temperature in step (1) is 4°C.

6. The application method according to claim 3, characterized in that in step (2), the modified collagen solution is insulated in a 37°C water bath, and the freezing temperature is -20°C.

7. The application method according to claim 3, characterized in that the drying method described in step (3) is freeze drying.