CN111471193B - Dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel and preparation method thereof - Google Patents
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Abstract
The invention discloses dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel and a preparation method thereof. Firstly, carrying out cholesterol group hydrophobic modification on polysaccharide, then specifically oxidizing the polysaccharide by utilizing sodium periodate to prepare an amphiphilic polysaccharide derivative with a dialdehyde structure, and then self-assembling the amphiphilic polysaccharide derivative in an aqueous solution to form dialdehyde polysaccharide nano-particles with a core-shell structure; then mixing and stirring 3-6% of dialdehyde polysaccharide nano-particle solution and 0.4-0.9% of collagen solution according to the volume ratio of 1 (0.5-1.5), and standing at 30-37 ℃ for 24h to prepare the dialdehyde polysaccharide nano-particle crosslinked collagen hydrogel. The invention has the advantages that: the green and harmless dialdehyde polysaccharide nano-particles are used as a cross-linking agent, and the prepared hydrogel has uniform aperture, good mechanical property and good biocompatibility, and has good application prospect in the field of tissue engineering scaffold materials.
Description
Technical Field
The invention relates to the technical field of hydrogel, in particular to dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel which has uniform pore diameter, good mechanical property and good biocompatibility and can be applied to the fields of medical dressings, drug release and tissue scaffolds, and a preparation method of the hydrogel.
Background
Collagen (Collagen) is the most abundant protein in animals, about one third of the protein in the body, and is widely present in the tissues of skin, cartilage, tendon, visceral intercellular substance and ligament of mammals. The glue has excellent performances of low immunogenicity, good biocompatibility, biodegradability and the like, so that the glue can be widely applied to the fields of biological materials such as wound dressings, drug delivery carriers, tissue engineering, cosmetics and the like. Collagen of 27 different structures has now been found, of which type I collagen is the one found in all types of collagen with the highest content (about 90% of the total collagen in the organism), the most widespread, the most studied and the most widely used.
Under physiological conditions, collagen can self-assemble to form collagen fibers, which can further form a three-dimensional network structure and bind water filled in the collagen fibers, so that the solution is converted into hydrogel. The highly hydrated network-like structure of collagen hydrogels behaves like natural tissue and is biodegradable. Therefore, the application of collagen hydrogel in the field of tissue engineering has attracted a great deal of attention. Researchers hope that collagen hydrogel can be used as a temporary substitute material for extracellular matrix to realize in vitro cell culture and tissue regeneration. Therefore, the collagen hydrogel is often used as a tissue filler, a scaffold material and a drug carrier and widely applied to the biomedical fields of tissue engineering, wound dressing, drug sustained release and the like. However, the application of pure collagen hydrogels is often limited by their poor mechanical properties, high swelling capacity, and excessively fast biodegradation rate. The chemical crosslinking modification can enable the interior of the collagen hydrogel to form a compact space network structure, thereby effectively improving the defects. Thus, glutaraldehyde, polyphenols and oxidized polysaccharides are generally used as cross-linking agents to immobilize collagen to overcome the above disadvantages. However, due to rapid crosslinking reaction, electrostatic interaction, hydrogen bonding, etc., local flocculation inevitably occurs once collagen is contacted with a crosslinking agent, which affects the uniform spatial network structure inside the collagen hydrogel, and further affects the physical and chemical properties of the hydrogel. Based on the above, the invention provides a collagen hydrogel which is prepared by using novel dialdehyde polysaccharide nanoparticles as a cross-linking agent and chemically cross-linking and modifying collagen and has the advantages of high mechanical strength, low swelling degree and good biocompatibility.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel and a preparation method thereof, and solves the defects in the prior art.
In order to realize the purpose, the technical scheme adopted by the invention is as follows:
a dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel, comprising: the collagen hydrogel is prepared by mixing and stirring uniformly a collagen aqueous solution with the mass fraction of 0.4-0.9% prepared from type I collagen and a dialdehyde polysaccharide nano-particle aqueous solution with the mass fraction of 3-6% prepared from dialdehyde polysaccharide nano-particles according to the mass ratio of 1 (0.5-1.5), and based on the chemical crosslinking reaction activity of Schiff base.
The invention also discloses a preparation method of the dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel, which comprises the following steps:
(1) preparing an aqueous collagen solution: dissolving I type collagen in 0.25M acetic acid aqueous solution to prepare collagen aqueous solution with the mass fraction of 0.4-0.9%, and then slowly adjusting the pH value of the collagen aqueous solution to be neutral under the ice-bath condition by using 2M sodium hydroxide solution;
(2) preparing dialdehyde polysaccharide nanoparticle solution: dissolving 1-5 parts by mass of polysaccharide in 50 parts by mass of molten imidazole under the condition of stirring in an oil bath at 100 ℃, and stabilizing for 30min to prepare a 1-10% clear and transparent polysaccharide solution; adding cholesteryl chloroformate (the molar ratio of the cholesteryl chloroformate to a polysaccharide molecular dehydration unit (AGU) is (0.005-0.05): 1) into the system, stirring for 1-3 h at 90-110 ℃, precipitating with excessive ethanol after the reaction is finished, filtering and washing for 4-6 times, and drying at 40-70 ℃ to obtain the amphiphilic polysaccharide derivative; dispersing amphiphilic polysaccharide derivatives in water to obtain a polysaccharide derivative solution with the mass concentration of 1% -10%, adding sodium periodate (the molar ratio of sodium periodate to AGU is 0.1-1.0), reacting at 30-45 ℃ in the dark for 2-5 h, dialyzing after the reaction is finished, and freeze-drying to prepare the cholesterol grafted amphiphilic dialdehyde polysaccharide derivative; directly dissolving the amphiphilic dialdehyde polysaccharide derivative into deionized water to carry out self-assembly to obtain dialdehyde polysaccharide nano-particle aqueous solution with the mass fraction of 3-6%;
(3) preparation of collagen-based hydrogel: and (2) taking the collagen aqueous solution with the mass fraction of 0.4-0.9%, adding the dialdehyde polysaccharide nano-particle aqueous solution with the mass fraction of 3-6% under the ice bath condition, mixing and stirring uniformly according to the mass ratio of 1 (0.5-1.5), and standing at 30-37 ℃ for 24h to prepare the polysaccharide nano-crosslinked collagen hydrogel.
Preferably, the polysaccharide is any one or a mixture of starch, hydroxyethyl starch, sodium alginate, xanthan gum, pullulan, dextran, chitosan, guar gum and carrageenan.
Compared with the prior art, the invention has the advantages that:
(1) the collagen hydrogel is prepared by crosslinking and modifying collagen through the dialdehyde polysaccharide nano particles, so that the problems of collagen flocculation caused by rapid reaction, electrostatic interaction and the like of a crosslinking agent and the collagen can be effectively overcome, the problems of cytotoxicity and the like caused by free precipitation of a micromolecular aldehyde crosslinking agent can be avoided, and the application requirement in the field of biological materials is met.
(2) According to the dialdehyde polysaccharide nano-particle crosslinked collagen hydrogel provided by the invention, aldehyde groups of dialdehyde polysaccharide nano-particles can react with amino groups of collagen to perform Schiff base reaction so as to chemically crosslink and modify the collagen, so that a compact space network structure is formed; more importantly, the dialdehyde polysaccharide nano particles can be used as a nano particle filler to further improve the mechanical strength, the swelling degree, the biodegradation performance and the like of the collagen hydrogel.
(3) The coating of the micromolecule medicine can be realized in the process of forming the dialdehyde polysaccharide nano-particles through molecular self-assembly, and further the dialdehyde polysaccharide nano-particles are endowed with the function of directional administration of the cross-linked collagen hydrogel.
Drawings
Fig. 1 is an SEM photograph of dialdehyde starch-cholesterol nanoparticles:
(A) and (B) photographs of different degrees of oxidation for the same amount of cholesterol grafted (0.01mol/mol AGU): (A) degree of oxidation of 46%, (B) degree of oxidation of 87%; (C) and (D) photographs of different amounts of cholesterol grafted for the same degree of oxidation (46%): (C)0.005mol/mol AGU, (D)0.02mol/mol AGU;
FIG. 2 is an SEM photograph of different hydrogels after lyophilization; (A) pure collagen hydrogel (Col), (B) oxidized starch cross-linked modified collagen hydrogel (OS/Col), (C) dialdehyde starch-cholesterol nanoparticle cross-linked modified collagen hydrogel (ocnps/Col);
FIG. 3 is a graph of stress-strain curves and compressive failure strength in compression mode for different hydrogel samples; (A) a stress-strain curve (B) compression failure strength comparison graph;
FIG. 4 is a schematic diagram showing the cell viability of L929 cells cultured in different hydrogel leaching solutions for 24h and 72 h;
FIG. 5 is a bright field micrograph and a fluorescence micrograph of L929 cells stained with calcein cell stain after 24h incubation on different hydraulic surfaces, with the bright field micrograph on the left and the fluorescence micrograph on the right.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail by referring to the following examples.
The dialdehyde polysaccharide nanoparticle cross-linking agent is prepared by a simple, convenient, green and efficient method. In molten imidazole, the polysaccharide-cholesterol polymer is prepared by esterification reaction of acyl chloride group of cholesterol chloroformate as hydrophobic agent and hydroxyl group on the polysaccharide molecular skeleton chain. Molten imidazole can rapidly and completely dissolve polysaccharides, providing a homogeneous reaction medium, which is an important condition for producing polysaccharide esters of higher purity. Imidazole is used as a solvent in a reaction system and also used as a reaction medium to participate in the formation of an imidazole-cholesterol compound intermediate, and the intermediate is favorable for the next step of rapid and complete reaction with polysaccharide molecules, so that the reaction rate is greatly improved. In addition, imidazole is used as a basic substance to bind with hydrochloric acid, which is a by-product obtained from the reaction, to promote the forward progress of the esterification reaction. Only about one percent of hydroxyl groups of polysaccharide molecules are occupied in the esterification reaction, so that enough ortho-dihydroxy groups are remained for the subsequent sodium periodate oxidation reaction to normally carry out. The sodium periodate can specifically oxidize ortho-dihydroxy in the polysaccharide-cholesterol polymer into aldehyde group to prepare the dialdehyde polysaccharide-cholesterol polymer. By a direct dissolution method, the amphiphilic polymer dialdehyde polysaccharide-cholesterol polymer is self-assembled to form dialdehyde polysaccharide nano-particles on the premise of not introducing any organic solvent and ensuring the green and safe product. The intramolecular or intermolecular attraction between the hydrophobic segments of cholesterol facilitates the self-assembly process of the amphiphilic polymer. The cholesterol of the hydrophobic segment tends to gather at the core of the particle, while the hydrophilic dialdehyde polysaccharide chain forms the shell of the particle, and the hydrophilic shell plays a role in protecting the inner core and enhances the water solubility of the dialdehyde polysaccharide nano particle. The dialdehyde polysaccharide nano-particles not only can chemically cross-link and modify collagen, but also can further improve the physical and chemical properties of the collagen hydrogel due to the characteristics of nano-scale size and high specific surface area. In addition, in the process of forming dialdehyde polysaccharide nano-particles by molecular self-assembly, the coating of small molecular drugs can be realized, and the hydrogel prepared by crosslinking collagen has the function of directional administration. Therefore, the application of the prepared collagen hydrogel in the fields of biological materials and the like by taking the dialdehyde polysaccharide nano particles as the cross-linking agent has advantages.
The invention takes natural biological macromolecules as raw materials, firstly, cholesterol is chemically grafted on polysaccharide molecular chains in fused imidazole, and the polysaccharide-cholesterol polymer is prepared. Then, the polysaccharide is specifically oxidized by sodium periodate to prepare the dialdehyde polysaccharide-cholesterol polymer. Then, the amphiphilic polymer dialdehyde polysaccharide-cholesterol polymer is used for self-assembly to form dialdehyde polysaccharide nano-particles. The particle size of the dialdehyde polysaccharide nano-particles is controlled by adjusting the introduction amount of cholesterol and aldehyde groups in the dialdehyde polysaccharide-cholesterol polymer. Based on the chemical crosslinking reactivity of Schiff base between aldehyde group of dialdehyde polysaccharide nano-particles and amino group of collagen molecules, dialdehyde polysaccharide nano-particle crosslinked collagen hydrogel with excellent physicochemical property and good biocompatibility is prepared.
Example 1
(1) Preparing an aqueous collagen solution: dissolving collagen in 0.25M acetic acid aqueous solution to prepare collagen acetic acid aqueous solution with mass fraction of 0.6%, and slowly adjusting pH to be neutral by 2M sodium hydroxide solution under ice bath condition to obtain collagen aqueous solution.
(2) Preparing dialdehyde starch nanoparticle solution: under the condition of oil bath stirring at 100 ℃, 5 parts by mass of soluble starch is dissolved in 50 parts by mass of molten imidazole and is stabilized for 30min to prepare a 10% clear and transparent starch solution. Adding cholesteryl chloroformate (wherein the molar ratio of cholesteryl chloroformate to starch molecule dehydration unit (AGU) is 0.02:1) into the system, stirring at 100 deg.C for 1h, precipitating with excessive ethanol after reaction, filtering, washing for 5 times, and oven drying at 60 deg.C to obtain amphiphilic starch derivative. Dispersing the amphiphilic starch derivative in water to obtain a polysaccharide derivative solution with the mass concentration of 8%, adding sodium periodate (wherein the molar ratio of the sodium periodate to AGU is 0.5:1), reacting at 37 ℃ in the dark for 4 hours, dialyzing and freeze-drying after the reaction is finished to prepare the cholesterol grafted amphiphilic dialdehyde starch derivative, and directly dissolving the amphiphilic dialdehyde starch derivative in deionized water to self-assemble to obtain the dialdehyde starch nanoparticle aqueous solution with the mass fraction of 4%.
(3) Preparation of collagen-based hydrogel: taking the neutral collagen solution with the mass fraction of 0.6% after the pH is adjusted, adding the dialdehyde starch nanoparticle aqueous solution with the mass fraction of 4% under the ice bath condition, mixing and stirring uniformly according to the mass ratio of 1:1.2, and incubating for 24h at 30 ℃ to obtain the dialdehyde starch nano cross-linked collagen hydrogel.
Example 2
(1) Preparing an aqueous collagen solution: dissolving collagen in 0.25M acetic acid aqueous solution to prepare collagen acetic acid aqueous solution with the mass fraction of 0.5%, and slowly adjusting the pH to be neutral by using 2M sodium hydroxide solution under the ice-bath condition to obtain the collagen aqueous solution.
(2) Preparing dialdehyde xanthan gum nanoparticle solution: under the condition of stirring in an oil bath at 100 ℃, 4 parts by mass of soluble starch is dissolved in 50 parts by mass of molten imidazole and is stabilized for 30min to prepare a clear and transparent xanthan gum solution with the concentration of 8%. Adding cholesteryl chloroformate (wherein the molar ratio of cholesteryl chloroformate to xanthan gum molecular dehydration unit (AGU) is 0.04:1) into the system, stirring at 90 deg.C for 2.5h, precipitating with excessive ethanol after reaction, filtering, washing for 5 times, and oven drying at 40 deg.C to obtain amphiphilic xanthan gum derivative. Dispersing amphiphilic xanthan gum derivatives in water by ultrasonic to obtain a xanthan gum derivative solution with the mass concentration of 6%, adding sodium periodate (wherein the molar ratio of the sodium periodate to AGU is 0.4:1), reacting for 4 hours at 40 ℃ in a dark place, dialyzing and freeze-drying after the reaction is finished to prepare the cholesterol grafted amphiphilic dialdehyde xanthan gum derivatives, dispersing the amphiphilic dialdehyde xanthan gum derivatives in deionized water by using a probe type ultrasonic instrument, and self-assembling to obtain the dialdehyde xanthan gum nanoparticle aqueous solution with the mass fraction of 4%.
(3) Preparation of collagen-based hydrogel: and (3) taking the neutral collagen solution with the mass fraction of 0.5% after the pH is adjusted, adding a dialdehyde xanthan gum nanoparticle aqueous solution with the mass fraction of 4% under an ice bath condition, mixing and stirring uniformly according to the mass ratio of 1:1, and incubating for 24h at 37 ℃ to obtain the dialdehyde xanthan gum nano cross-linked collagen hydrogel.
Example 3
(1) Preparing an aqueous collagen solution: dissolving collagen in 0.25M acetic acid aqueous solution to prepare collagen acetic acid aqueous solution with mass fraction of 0.6%, and slowly adjusting pH to be neutral by 2M sodium hydroxide solution under ice bath condition to obtain collagen aqueous solution.
(2) Preparing a dialdehyde alginic acid nanoparticle solution: under the condition of stirring in an oil bath at 100 ℃, 3 parts by mass of soluble starch is dissolved in 50 parts by mass of molten imidazole and is stabilized for 30min to prepare a 6% clear and transparent sodium alginate solution. Adding cholesteryl chloroformate (wherein the molar ratio of cholesteryl chloroformate to sodium alginate molecular dehydration unit (AGU) is 0.01:1) into the system, stirring at 100 ℃ for 1.5h, precipitating with excessive ethanol after the reaction is finished, centrifuging to remove supernatant, washing for 5 times, centrifuging, precipitating, and drying at 40 ℃ to obtain the amphiphilic sodium alginate derivative. Dispersing the amphiphilic sodium alginate derivative in a water/ethanol mixed solution to obtain a polysaccharide derivative solution with the mass concentration of 4%, adding sodium periodate (wherein the molar ratio of the sodium periodate to AGU is 1:1), reacting for 4 hours at 40 ℃ in the dark, dialyzing and freeze-drying after the reaction is finished to obtain the cholesterol grafted amphiphilic dialdehyde sodium alginate derivative, and directly dissolving the amphiphilic dialdehyde sodium alginate derivative in deionized water to prepare a dialdehyde starch nanoparticle aqueous solution with the mass fraction of 2% through ultrasonic self-assembly.
(3) Preparation of collagen-based hydrogel: taking the neutral collagen solution with the mass fraction of 0.6% after the pH is adjusted, adding the dialdehyde starch nanoparticle aqueous solution with the mass fraction of 2% under the ice bath condition, mixing and stirring uniformly according to the mass ratio of 1:1, and incubating for 24h at 37 ℃ to obtain the dialdehyde starch nano cross-linked collagen hydrogel.
The present invention provides the following experimental data, all of which are experimental results obtained with starch as a representative polysaccharide:
as shown in fig. 1, the amphiphilic polymer dialdehyde polysaccharide-cholesterol polymer can be self-assembled in an aqueous solution to form dialdehyde polysaccharide nanoparticles, and the aldehyde content and the cholesterol grafting amount of the dialdehyde polysaccharide-cholesterol polymer determine the particle size and morphology of the dialdehyde polysaccharide nanoparticles.
As shown in FIG. 2, OCNPs/Col have more pore structures than the OS/Col and Col hydrogels, and pore size uniformity is the best of the three. The more uniform pore size distribution is advantageous for uniform cell growth and also for counteracting the pressure to be applied, thereby exhibiting higher mechanical strength.
As shown in FIG. 3, the OCNPs/Col hydrogel has a mechanical strength greatly improved compared with the OS/Col and Col hydrogels, and the compressive failure strength of the OCNPs/Col hydrogel can reach 70.8kPa, which is improved by about 26kPa compared with the OS/Col hydrogel.
As shown in fig. 4, the survival rate of the L929 cells after 24h and 72h of culture in different hydrogels was above 95%, and there was not much difference between the groups, indicating that the two modified collagen hydrogels were not substantially cytotoxic in the experimental concentration range.
As shown in FIG. 5, after the L929 cells and the OCNPs/Col hydrogel are co-cultured for 24h, the L929 cells grow and survive well on the hydrogel surface and are in a typical fusiform shape, which indicates that the OCNPs/Col hydrogel has good cell compatibility.
It will be appreciated by those of ordinary skill in the art that the examples described herein are intended to assist the reader in understanding the manner in which the invention is practiced, and it is to be understood that the scope of the invention is not limited to such specifically recited statements and examples. Those skilled in the art can make various other specific changes and combinations based on the teachings of the present invention without departing from the spirit of the invention, and these changes and combinations are within the scope of the invention.
Claims (2)
1. A preparation method of dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel is characterized by comprising the following steps: the dialdehyde polysaccharide nano-particle crosslinked collagen hydrogel is a collagen aqueous solution with the mass fraction of 0.4-0.9 percent and prepared from type I collagen and a dialdehyde polysaccharide nano-particle aqueous solution with the mass fraction of 3-6 percent and prepared from dialdehyde polysaccharide nano-particles, wherein the collagen aqueous solution and the dialdehyde polysaccharide nano-particle aqueous solution are mixed and stirred uniformly according to the mass ratio of 1 (0.5-1.5), and the collagen hydrogel is prepared based on the chemical crosslinking reaction activity of Schiff base.
The preparation method comprises the following steps:
(1) preparing an aqueous collagen solution: dissolving I type collagen in 0.25M acetic acid aqueous solution to prepare collagen aqueous solution with the mass fraction of 0.4-0.9%, and then slowly adjusting the pH value of the collagen aqueous solution to be neutral under the ice-bath condition by using 2M sodium hydroxide solution;
(2) preparing dialdehyde polysaccharide nanoparticle solution: dissolving 1-5 parts by mass of polysaccharide in 50 parts by mass of molten imidazole under the condition of stirring in an oil bath at 100 ℃, and stabilizing for 30min to prepare a 1-10% clear and transparent polysaccharide solution;
adding cholesteryl chloroformate into the system, wherein the molar ratio of the cholesteryl chloroformate to polysaccharide molecular dehydration units (AGU) is (0.005-0.05): 1;
stirring for 1-3 h at 90-110 ℃, precipitating with excessive ethanol after the reaction is finished, filtering and washing for 4-6 times, and drying at 40-70 ℃ to obtain an amphiphilic polysaccharide derivative;
dispersing an amphiphilic polysaccharide derivative in water to obtain a polysaccharide derivative solution with the mass concentration of 1% -10%, adding sodium periodate, wherein the molar ratio of the sodium periodate to a polysaccharide molecular dehydration unit (AGU) is 0.1-1.0, carrying out a dark reaction for 2-5 h at 30-45 ℃, and carrying out dialysis and freeze drying after the reaction is finished to obtain a cholesterol grafted amphiphilic dialdehyde polysaccharide derivative; directly dissolving the amphiphilic dialdehyde polysaccharide derivative into deionized water to carry out self-assembly to obtain dialdehyde polysaccharide nano-particle aqueous solution with the mass fraction of 3-6%;
(3) preparation of collagen-based hydrogel: and (2) taking the collagen aqueous solution with the mass fraction of 0.4-0.9%, adding the dialdehyde polysaccharide nano-particle aqueous solution with the mass fraction of 3-6% under the ice bath condition, mixing and stirring uniformly according to the mass ratio of 1 (0.5-1.5), and standing at 30-37 ℃ for 24h to prepare the polysaccharide nano-crosslinked collagen hydrogel.
2. The method for preparing dialdehyde polysaccharide nanoparticle crosslinked collagen hydrogel according to claim 1, which is characterized in that: the polysaccharide is any one or mixture of starch, sodium alginate, xanthan gum, pullulan, dextran, chitosan, guar gum and carrageenan.
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