CN109879984B - Vinyl etherification modified sodium alginate and preparation method and application thereof - Google Patents
Vinyl etherification modified sodium alginate and preparation method and application thereof Download PDFInfo
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Abstract
本发明公开了一种乙烯基醚化改性海藻酸钠，通过海藻酸钠接枝10～30％的乙烯基醚基团制备得到。所述乙烯基醚化改性海藻酸钠是由海藻酸钠与含有乙烯基醚基团、且能与海藻酸钠中所含羧基官能团进行酰胺化反应的化合物反应制备得到。本发明的乙烯基醚化改性海藻酸钠遇水或生理盐水在短时间内通过氢键作用形成水凝胶，且无溶胀解离现象。本发明的乙烯基醚化改性海藻酸钠具有很好的生物相容性和生物可降解性；引入了碳碳双键，提供了新的反应位点；可应用于快速止血材料的制备与研究。
The invention discloses a vinyl etherified modified sodium alginate, which is prepared by grafting 10-30% vinyl ether groups of the sodium alginate. The vinyl etherified modified sodium alginate is prepared by reacting sodium alginate with a compound containing vinyl ether groups and capable of undergoing amidation reaction with the carboxyl functional groups contained in the sodium alginate. The vinyl etherified modified sodium alginate of the present invention forms a hydrogel through hydrogen bonding in a short time when it encounters water or physiological saline, and has no swelling and dissociation phenomenon. The vinyl etherified modified sodium alginate of the invention has good biocompatibility and biodegradability; carbon-carbon double bond is introduced to provide a new reaction site; it can be used in the preparation of rapid hemostatic materials and Research.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to vinyl etherification modified sodium alginate as well as a preparation method and application thereof.
Background
Hemostats refer to various preparations capable of assisting the body to complete the hemostasis of wounds, and are widely applied to the trauma treatment and surgical operation process. In general, a hemostatic agent possesses at least any one of three characteristics: 1. the ability to enhance the blood's auto-coagulation; 2. the ability to absorb large amounts of blood; 3. excellent ability to adhere to wounds. The polysaccharide hydrogel with high water content and high air permeability can effectively absorb blood of wounds, has the advantages of adhesion resistance, degradability, biocompatibility and the like, and can be effectively applied to hemostatic materials.
The sodium alginate is natural marine polysaccharide extracted from brown algae or gulfweed, the molecule of the natural marine polysaccharide is formed by connecting two structural units of beta-D-mannuronic acid (G) and alpha-L-guluronic acid (M), and can form three different repeating units of MM, MG and GG, as shown in figure 6 (figure 6 is a structural schematic diagram of the sodium alginate), and the sodium alginate (Pawar S N, Edgar KJ. Alginate derivative: A review of chemistry, properties and applications [ J ] Biomaterials,2012, 33(11): 3279-.
Sodium alginate can react with Ca2+Is crosslinked to form a polymer havingCalcium alginate hydrogel with good biocompatibility. However, the calcium alginate hydrogel can generate ion migration in normal saline, so that the gel structure is dissociated, and a severe swelling phenomenon is generated. In addition, calcium alginate hydrogel in PBS buffer solution can generate calcium phosphate precipitation, which leads to the hydrogel thoroughly disintegrating. In order to overcome the defects of the common ionic calcium alginate hydrogel, research and development of modified sodium alginate have been paid attention to by researchers. The chemical modification of the sodium alginate mainly comprises carboxyl modification and hydroxyl modification, and aims to introduce a new functional group into the sodium alginate and generate a new reaction site so as to prepare the novel covalent crosslinking hydrogel.
Disclosure of Invention
The first purpose of the invention is to provide a vinyl etherification modified sodium alginate.
The second purpose of the invention is to provide a preparation method of the vinyl etherification modified sodium alginate.
The third purpose of the invention is to provide the application of the vinyl etherification modified sodium alginate as a rapid hemostatic material.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the first aspect of the invention provides vinyl etherification modified sodium alginate, which is obtained by grafting 10-30% of vinyl ether (CH) with sodium alginate (hereinafter abbreviated as SA)2A group CH-O-).
The vinyl etherification modified sodium alginate is prepared by the reaction of sodium alginate and a compound which contains vinyl ether groups and can perform amidation reaction with carboxyl functional groups contained in the sodium alginate, and the reaction equation is as follows:
the compound which contains vinyl ether groups and can perform amidation reaction with carboxyl functional groups contained in sodium alginate is 3-aminoethanol vinyl ether.
The molecular weight of the sodium alginate is 50-500 kDa.
The second aspect of the invention provides a preparation method of the vinyl etherification modified sodium alginate, which comprises the following steps:
dissolving a mixture of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and hydroxysuccinimide (NHS) in water to obtain a catalyst solution, adding the catalyst solution into a Sodium Alginate (SA) aqueous solution with the concentration of 1% and the pH value of 4-7, stirring and activating for 0.5-3 h at the temperature of 15-30 ℃, adding 3-aminoethanol vinyl ether (APVE), stirring for 1-30 h (preferably 10-20 h) at the temperature of 15-30 ℃, pouring a reaction solution into ethanol to generate flocculent precipitate, filtering, washing the obtained filter cake with ethanol and diethyl ether in sequence, and then vacuumizing and drying the filter cake to obtain the vinyl etherification modified sodium alginate;
the molar ratio of the sodium carboxylate group in the sodium alginate to the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the hydroxysuccinimide is 1: (0.5-5): (0.5 to 5); the molar ratio of the sodium carboxylate group in the sodium alginate to the 3-aminoethanol vinyl ether is 1: (0.5-5).
The method for calculating the molar quantity of the sodium carboxylate of the sodium alginate comprises the following steps: the molar quantity of the sodium alginate sugar units is obtained by dividing the mass of the sodium alginate by the molar mass of the sugar units, and each sugar unit of the sodium alginate has a sodium carboxylate group, so the molar quantity of the sugar units is the molar quantity of the sodium alginate sodium carboxylate groups.
The third aspect of the invention provides vinyl etherification modified sodium alginate crosslinked hydrogel which is formed by vinyl etherification modified sodium alginate in water.
The vinyl etherification modified sodium alginate is dissolved in deionized water or physiological saline to form hydrogel within 1 s-5 min.
The fourth aspect of the invention provides an application of the vinyl etherification modified sodium alginate as a rapid hemostatic material.
Due to the adoption of the technical scheme, the invention has the following advantages and beneficial effects:
the vinyl etherification modified sodium alginate forms hydrogel through the action of hydrogen bonds in water or normal saline within a short time (1 s-5 min), and does not swell and dissociate, so the vinyl etherification modified sodium alginate has good biocompatibility and biodegradability; the vinyl etherification modified sodium alginate of the invention introduces carbon-carbon double bonds, and provides a new reaction site; can be applied to the preparation and research of the rapid hemostatic material.
The vinyl etherification modified sodium alginate has the characteristic of quickly forming hydrogel in a short time (about 10 s), can be used as a quick hemostatic material, and does not swell and dissociate in normal saline. In addition, the vinyl etherification modified sodium alginate with excellent biocompatibility can be used for crosslinking with other functionalized micromolecules and macromolecules to prepare and synthesize multifunctional biomedical materials, and has wide application prospect.
Drawings
FIG. 1 is a Raman spectrum of vinyl etherification modified sodium alginate prepared in example 1; wherein (a) is raw material sodium alginate, and (b) is vinyl etherification modified sodium alginate prepared in example 1.
FIG. 2 is an infrared spectrum of acidified vinyl ether modified sodium alginate prepared in example 2; wherein (a) is acidified sodium alginate as a raw material, and (b) is acidified vinyl ether modified sodium alginate prepared in example 2.
FIG. 3 is a graph showing the rheological behavior of a hydrogel formed from the vinyl ether modified sodium alginate prepared in example 1 at a temperature of 25 ℃.
FIG. 4 is a graph showing the rheological behavior of a hydrogel formed from the vinyl ether modified sodium alginate prepared in example 1 at a temperature of 37 ℃.
FIG. 5 is a bar graph of the cytotoxicity of hydrogel formed by vinyl etherification modified sodium alginate with different grafting ratios on mouse embryonic fibroblasts (NIH3T 3).
FIG. 6 is a schematic diagram of the structure of sodium alginate.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below in connection with preferred embodiments. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
The reagents and materials used in the examples of the invention were as follows: sodium Alginate (SA), chemical pure (200mPas), celosia glauca group ltd; 3-aminopropanol vinyl ether (APVE), analytically pure, Changzhou Cyclolingchemical industries, Inc.; 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), analytically pure, Shanghai Allantin reagent, Inc.; n-hydroxysuccinimide (NHS), chemically pure, shanghai alatin reagent, ltd; absolute ethanol, analytically pure, shanghai tatatake technologies ltd; anhydrous ether, analytical grade, shanghai tatatake technologies ltd.
Example 1
Weighing 2g of Sodium Alginate (SA) with the molecular weight of 50kDa, dissolving the Sodium Alginate (SA) in 200mL of dilute hydrochloric acid aqueous solution with the pH value of 4 to prepare 1% sodium alginate aqueous solution, dissolving 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (0.96g) and hydroxysuccinimide (NHS) (0.58g) in 5mL of deionized water to prepare catalyst solution, quickly adding the catalyst solution into the SA aqueous solution after full dissolution, stirring for 30min at the temperature of 15 ℃, adding 0.58mL of 3-aminoethanol vinyl ether (APVE), continuously stirring and reacting for 5h at the temperature of 15 ℃, settling by using 800mL of ethanol, performing suction filtration to collect precipitates, washing the precipitates by using 150mL of ethanol for three times respectively, then washing the precipitates by using 150mL of ether for three times, and drying the precipitates for 8h in vacuum to obtain vinyl etherified modified sodium alginate (abbreviated as SA-VE-1), the vinyl ether group grafting rate was calculated by elemental analysis to be 15%.
The graft ratio of the vinyl ether group of SA-VE was calculated from the following formula:
in the formula, N% is the nitrogen content in the SA-VE sample tested by element analysis, and m is the mass of the SA-VE sample.
The Raman spectrum of the vinyl etherification modified sodium alginate SA-VE-1 is shown in figure 1, and figure 1 is the Raman spectrum of the vinyl etherification modified sodium alginate prepared in example 1; wherein (a) is raw material sodium alginate, and (b) is vinyl etherification modified sodium alginate prepared in example 1. As can be seen from the graph, when the Raman spectra of the SA raw material shown in FIG. 1(a) and SA-VE-1 shown in FIG. 1(b) are compared, the wave number of SA-VE-1 is 1620cm-1A distinct carbon-carbon double bond absorption peak is generated, which indicates the successful grafting of the vinyl ether group.
Cutting SA-VE-1 into uniform short fibers, weighing 0.05g of SA-VE-1 into a sample bottle, adding 5mL of deionized water, and generating hydrogel in 10s, which is abbreviated as hydrogel-1. 5mL of 3mol/L hydrogen bond disruptor NaSCN solution is added into a hydrogel-1 system for forming gel, and the result shows that the hydrogel-1 can be rapidly and completely dissociated, which indicates that the intermolecular hydrogen bond interaction is the main reason for forming the hydrogel by SA-VE.
The storage modulus and loss modulus of hydrogel-1 are shown in fig. 3 and 4, and fig. 3 is a rheological behavior diagram of hydrogel formed by vinyl etherification modified sodium alginate prepared in example 1, and the temperature is 25 ℃; FIG. 4 is a graph showing the rheological behavior of a hydrogel formed from the vinyl ether modified sodium alginate prepared in example 1 at a temperature of 37 ℃. In the figure, G 'represents the storage modulus and G' represents the loss modulus. As can be seen from FIGS. 3 and 4, the storage modulus of hydrogel-1 was always higher than the loss modulus, indicating the successful formation of a gel network structure with a certain strength. Wherein the storage modulus is maintained at 100-1000Pa, and the loss modulus is maintained at 10-1000 Pa; comparing the rheological behavior of hydrogel-1 at 25 deg.C (FIG. 3) with that of hydrogel-1 at 37 deg.C (FIG. 4), it can be seen that there is a certain decrease in the strength of hydrogel-1 as the temperature increases from 25 deg.C to 37 deg.C, which can also prove that the hydrogel system is composed of hydrogen bonds.
Example 2
SA 2g having a molecular weight of 150kDa was weighed out and dissolved in 200mL of a dilute aqueous hydrochloric acid solution having a pH of 5 to prepare a 1% aqueous sodium alginate solution. Dissolving EDC (1.92g) and NHS (1.15g) in 10mL of deionized water to prepare a catalyst solution, quickly adding the catalyst solution into the SA aqueous solution after full dissolution, stirring for 1h at the temperature of 20 ℃, adding 1.13mL of APVE, continuously stirring and reacting for 10h at the temperature of 20 ℃, placing the reaction solution into a dialysis bag with MWCO of 7000 when the reaction is finished, dialyzing by using a dilute hydrochloric acid solution with the pH of 2, gradually forming a solidified gelatinous product in the dialysis bag after 24h, and freeze-drying the gelatinous product to obtain acidified Alg-VE; the SA solution was acidified by the same dialysis method to obtain Alg.
The purpose of acidifying the vinyl etherification product is to convert the-COO groups on the sugar units thereof-All converted to-COOH. The infrared spectrum is 1630-1610 cm-1Left and right, -COO-The absorption peaks of-CONH and C ═ C overlap, the composition of the grafted product cannot be judged, and the absorption peak of-COOH ranges from 1770 cm to 1720cm-1And the infrared absorption peak can be well distinguished from that of-CONH, so that the analysis is convenient.
The infrared spectrogram of the vinyl etherification modified sodium alginate after the acidification treatment is shown in figure 2; FIG. 2 is an infrared spectrum of acidified vinyl ether modified sodium alginate prepared in example 2; wherein (a) is acidified sodium alginate as a raw material, and (b) is acidified vinyl ether modified sodium alginate prepared in example 2. As can be seen by comparison, both curves have 1734cm-1a-COOH peak at position, indicating that all-COOH on the saccharide unit have been acidified; in addition, the vinyl etherified sodium alginate content is 1653cm-1The peak of-CONH characteristic appears, which proves the success of the amidation reaction.
Example 3
SA 2g having a molecular weight of 300kDa was weighed out and dissolved in 200mL of a dilute aqueous hydrochloric acid solution having a pH of 6 to prepare a 1% aqueous sodium alginate solution. EDC (3.84g) and NHS (2.3g) are dissolved in 20mL of deionized water to prepare a catalyst solution, the catalyst solution is quickly added into the SA water solution after being fully dissolved, the solution is stirred for 2h at the temperature of 25 ℃, then 2.26mL of APVE is added, the reaction is continuously stirred for 20h at the temperature of 25 ℃, 800mL of ethanol is used for settling, the precipitate is collected by suction filtration, 150mL of ethanol is respectively used for washing the precipitate for three times, then 150mL of diethyl ether is used for washing the precipitate for three times, the precipitate is dried for 8h in vacuum, vinyl etherification modified sodium alginate, namely SA-VE-2, and the grafting rate of the vinyl ether group is 20% through elemental analysis.
Cutting SA-VE-2 into uniform short fibers, weighing 0.05g of SA-VE-2 into a sample bottle, adding 5mL of deionized water, and generating hydrogel in 10s, which is abbreviated as hydrogel-2.
SA-VE-2 was cut into uniform short fibers, 0.1g was weighed into a rat tail at a wound depth of about 1/3 a, and the material was able to absorb the exuded blood to form a hydrogel and stop bleeding from the wound within 30 seconds.
Example 4
SA 2g having a molecular weight of 500kDa was weighed out and dissolved in 200mL of a dilute aqueous hydrochloric acid solution having a pH of 7 to prepare a 1% aqueous sodium alginate solution. EDC (9.6g) and NHS (5.75g) are dissolved in 20mL of deionized water to prepare a catalyst solution, the catalyst solution is quickly added into the SA water solution after being fully dissolved, the SA water solution is stirred for 3h at the temperature of 30 ℃, 5.65mL of APVE is added, the reaction is continuously stirred for 30h at the temperature of 30 ℃, 800mL of ethanol is used for settling, the precipitate is collected by suction filtration, 150mL of ethanol is respectively used for washing the precipitate for three times, then 150mL of diethyl ether is used for washing the precipitate for three times, the precipitate is dried for 8h in vacuum, the vinyl etherification modified sodium alginate, namely SA-VE-3, is obtained, and the grafting rate of the vinyl ether group is 25% through elemental analysis.
Cutting SA-VE-3 into uniform short fibers, weighing 0.05g of SA-VE-3 into a sample bottle, adding 5mL of deionized water, and generating hydrogel in 10s, which is abbreviated as hydrogel-3.
SA-VE-3 was cut into uniform short fibers, 0.2g was weighed into a rat tail at a wound depth of about 1/3 a, and the material was able to absorb the exuded blood to form a hydrogel and stop bleeding from the wound within 30 seconds.
Example 5
A mouse embryo fibroblast NIH3T3 is adopted to carry out cytotoxicity experiments on vinyl etherified sodium alginate SA-VE with different grafting rates, GBT 16886.5-2017 is referred, and an extraction solution method is adopted for the cytotoxicity test of the hydrogel. Three groups of samples (8 mg, SA-VE-1, SA-VE-2 and SA-VE-3 in examples 1, 3 and 4, respectively) were weighed out, and were soaked in DMEM medium in a sterilized centrifuge tube. And then adding 2mL of DMEM culture solution into each fully infiltrated sample, and standing for 24h to obtain an extract.
At the same time, the mouse embryo fibroblast NIH3T3 in logarithmic growth phase is digested by trypsin, and 5mL of DMEM culture solution is added to prepare 2X 105Perml cell suspension, 100. mu.L cell suspension per well (i.e., 5000 cells per well) was plated in 96-well plates in 5% CO2The cells were incubated in an incubator at 37 ℃ overnight for attachment, and the marginal wells were filled with sterile PBS buffer.
After the cells were fully attached to the wall, the culture medium in each well was aspirated, and 100. mu.L of the sample extract (four groups in total, 5 wells in parallel per group, and 20 wells in total, using pure DMEM culture medium as a blank control) was added thereto, and cultured for 24 hours. The sample leaching solution in each well was aspirated, 20. mu.L of 5mg/ml MTT solution was added, and incubation in the incubator was continued for 4 h. And (4) after incubation, removing supernatant by absorbing, adding 150 mu L of DMSO into each hole, and shaking the mixture in a shaking table at a low speed for 10min to fully dissolve the formazan crystals. The OD value in the Cell well plate at 490nm was measured using a microplate reader, and the Cell Viability (Cell Viability) was tested according to the following formula:
Cell Viability(％)＝(ODsample/ODcontrol)×100％
wherein, ODsampleRefers to the optical density, OD, of the sample set cell well platecontrolOptical density values for blank control cell well plates.
Data for cell viability are shown in table 1:
TABLE 1
| CellsSurvival rate% | |
| SA-VE-1 | 92.72 |
| SA-VE-2 | 101.97 |
| SA-VE-3 | 101.07 |
| |
100 |
The cytotoxicity of the material is characterized by the cell viability of the extract culture, with higher cell viability indicating less cytotoxicity of the material. The Cell Viability was calculated by the optical density value of each well plate measured by a microplate reader according to the above formula. The optical density values correspond to cell viability, with higher optical density values indicating higher cell viability. The cell survival rate of the blank control group pore plate is 100%, and the cell survival rate of the sample group pore plate is the ratio of the optical density value of the pore plate to the optical density value of the blank group.
FIG. 5 is a bar graph showing cytotoxicity of hydrogel formed by vinyl etherification modified sodium alginate with different grafting rates on mouse embryonic fibroblasts (NIH3T3), and it can be seen from FIG. 5 that SA-VE cells with different grafting rates have relative proliferation rates of more than 85%, which indicates that the material has no obvious inhibition effect on cells and the cytotoxicity is maintained at level 1 (for reference to GB/T14233.2-2005 for cytotoxicity classification standard of medical materials). In addition, the RGRs removed from the first set of SA-VE-1, SA-VE-2 and SA-VE-3 were all over 100%, indicating that the SA-VE material leachate was not only non-toxic to NIH3T3 cells, but also able to promote its proliferation.
From the above toxicity test, it can be seen that: the gel samples of SA-VE-1, SA-VE-2 and SA-VE-3 have no obvious inhibition effect on cells, the cytotoxicity is kept at level 1, and the gel samples have certain cell proliferation promoting effect. By combining the rat tail hemostasis experimental results in examples 3 and 4, it can be proved that the vinyl etherification modified sodium alginate SA-VE prepared by the method has a rapid hemostasis effect, and the material itself has good cell compatibility and does not generate toxicity, so that the method can be further applied to preparation and research of a rapid hemostasis material.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
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| CN105670310A (en) * | 2016-03-28 | 2016-06-15 | 东南大学 | Composite light-sensitive double-network hydrogel based on methyl vinyl ether maleic acid copolymer/alginic acid and preparation method of composite light-sensitive double-network hydrogel |
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