CN107417801B - Injectable hydrogel and preparation method and application thereof - Google Patents
Injectable hydrogel and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses an injectable hydrogel and a preparation method and application thereof, wherein the preparation method comprises the following steps: suspending 10-20 g of chitosan in 50-100 ml of isopropanol, adding 20-40 ml of 5-15 mol/L NaOH aqueous solution, stirring for 20-40 min at room temperature, dropwise adding 20-30 g of monochloroacetic acid, heating to 55-65 ℃, stirring for 2-4 h, filtering, washing and drying to obtain carboxymethyl chitosan; dropwise adding a sodium periodate aqueous solution into a xanthan gum aqueous solution according to the molar ratio of 1: 1-3 of the xanthan gum to the sodium periodate, stirring and reacting for 2-4 h in a dark room, adding ethylene glycol, stirring for 1-2 h, and dialyzing and purifying the reaction solution to obtain a hydroformylation xanthan gum; the hydroformylated xanthan gum reacts with carboxymethyl chitosan to form injectable hydrogels. The injectable hydrogel synthesized by modifying the functional groups by using the natural polysaccharide is safe and non-toxic, low in cost and remarkable in enzymolysis resistance.
Description
Technical Field
The invention relates to the field of injectable hydrogel, in particular to injectable hydrogel and a preparation method and application thereof.
Background
In clinical work, commercial porcine fibrin glue is generally used for plugging and treating intestinal fistula, so that part of intestinal fistula patients can recover the continuity of intestinal tracts through conservative treatment, and the intestinal fistula operation treatment is avoided. However, the fibrin glue is expensive because it is derived from organisms, and in addition, the biological material has poor resistance to enzymatic hydrolysis, so that it is completely digested by intestinal fluid quickly during the application process, and the treatment effect of some patients is unsatisfactory.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the above-mentioned deficiencies of the prior art, the present invention aims to provide an injectable hydrogel, a preparation method and applications thereof, and aims to solve the problems of high price and poor enzymatic hydrolysis resistance of the existing fibrin glue.
The technical scheme of the invention is as follows:
the invention provides a preparation method of an injectable hydrogel, which comprises the following steps:
step A, suspending 10-20 g of chitosan in 50-100 ml of isopropanol, adding 20-40 ml of 5-15 mol/L NaOH aqueous solution, stirring at room temperature for 20-40 min, dropwise adding 20-30 g of monochloroacetic acid, heating to 55-65 ℃, stirring for 2-4 h, filtering, washing and drying to obtain carboxymethyl chitosan;
b, according to the molar ratio of the xanthan gum to the sodium periodate of 1: 1-3, dropwise adding a sodium periodate aqueous solution into the xanthan gum aqueous solution, stirring and reacting for 2-4 h in a dark room, adding ethylene glycol, continuously stirring for 1-2 h, and then dialyzing and purifying the reaction solution to obtain the hydroformylation xanthan gum;
and C, performing a cross-linking reaction on the hydroformylation xanthan gum and the carboxymethyl chitosan to generate the injectable hydrogel.
The preparation method of the injectable hydrogel comprises the following steps of filtering, washing and drying in the step A: and after filtering, washing the filtered residue for 3-5 times by using a methanol/water mixed solution with the volume ratio of 70-90%, then washing for 2-4 times by using ethanol, and finally drying in vacuum to obtain the carboxymethyl chitosan.
In the step B, the mass volume ratio of the xanthan gum aqueous solution is 0.5-2.0%, and the mass volume ratio of the sodium periodate aqueous solution is 5-20%.
In the step B, when the reaction solution is dialyzed and purified, a dialysis bag is used for dialyzing and purifying the reaction solution with distilled water for 2-4 days, and the water is renewed at least 5 times every day.
The invention provides an injectable hydrogel prepared by the preparation method.
The invention also provides an injectable hydrogel, which comprises the hydroformylation xanthan gum and carboxymethyl chitosan, wherein the hydroformylation xanthan gum has a structural formula as follows:
n is an integer greater than 1.
The invention also provides an injectable hydrogel, wherein the injectable hydrogel has a structural formula as follows:
wherein m and n are integers more than 1.
The invention also provides application of the injectable hydrogel, and the injectable hydrogel is applied to preparation of a material for intestinal fistula plugging.
Has the advantages that: the invention provides an injectable hydrogel and a preparation method and application thereof.
Drawings
FIG. 1 is a reaction equation for the synthesis of a hydroformylated xanthan gum of the present invention, wherein a is xanthan gum, b is a hydroformylated xanthan gum, and n is an integer greater than 1.
FIG. 2 is a reaction equation for synthesizing carboxymethyl chitosan according to the present invention, in which c is chitosan, d is carboxymethyl chitosan, and m is an integer greater than 1.
Fig. 3 is a reaction equation for synthesizing the injectable hydrogel of the present invention, wherein e is the injectable hydrogel, and m and n are integers greater than 1.
Fig. 4 is a graph of the results of infrared spectroscopy of the carboxymethyl chitosan, the hydroformylated xanthan gum, and the injectable hydrogel of the present invention.
Fig. 5 is a graph showing the results of in vitro toxicity tests of the injectable hydrogel of the present invention.
Fig. 6 is a graph showing the results of the enzymolysis resistance test of the injectable hydrogel of the present invention, wherein P <0.001 indicates that there is a significant statistical difference.
Detailed Description
The invention provides an injectable hydrogel and a preparation method and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and more clear. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The method for preparing the injectable hydrogel according to the preferred embodiment of the present invention comprises the steps of:
s100, suspending 10-20 g of Chitosan (CS) in 50-100 ml of isopropanol, adding 20-40 ml of 5-15 mol/L NaOH aqueous solution, stirring at room temperature for 20-40 min, dropwise adding 20-30 g of monochloroacetic acid, heating to 55-65 ℃, stirring for 2-4 h, filtering, washing and drying to obtain carboxymethyl chitosan (NOCC);
s200, xanthan gum and sodium periodate (NaIO)4) The molar ratio of the sodium periodate to the xanthan gum is 1: 1-3, dropwise adding a sodium periodate aqueous solution into a xanthan gum aqueous solution, stirring and reacting for 2-4 h in a dark room, adding ethylene glycol, continuously stirring for 1-2 h, and then dialyzing and purifying the reaction solution to obtain a hydroformylation xanthan gum;
s300, performing a cross-linking reaction on the hydroformylation xanthan gum and the carboxymethyl chitosan to generate an injectable hydrogel, namely the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel.
Further, in this embodiment, in the step S100, a reaction equation for synthesizing carboxymethyl chitosan is shown in fig. 2, and the steps of filtering, washing, and drying specifically include: after filtration, washing the filtered residue with a methanol/water mixed solution with a volume ratio of 70-90% (v/v) for 3-5 times, then washing with ethanol for 2-4 times, and finally drying in vacuum to obtain the carboxymethyl chitosan.
Preferably, when step S100 is performed, 10g of chitosan may be suspended in 75ml of isopropanol and then stirred at room temperature; equally dividing 25ml of 10mol/L NaOH aqueous solution into 5 parts, and sequentially adding the 5 parts of NaOH aqueous solution into the stirred slurry at intervals of 5 min; then, the obtained slurry was stirred for 30min, then 20g of monochloroacetic acid was added dropwise, heated to 60 ℃ and stirred at that temperature for 3 h; subsequently, the reaction mixture was filtered, and the filtered residue was thoroughly washed 3 times with an 80% (v/v) methanol/water mixture, 2 times with ethanol, and dried under vacuum to give the final product NOCC having a degree of substitution of 85% and showing good hydrophilicity.
Further, in this embodiment, a reaction equation of reacting xanthan gum with sodium periodate to generate hydroformylation xanthan gum is shown in fig. 1, in the step S200, according to a molar ratio of the xanthan gum to the sodium periodate of 1: 1-3, a sodium periodate aqueous solution is added dropwise to the xanthan gum aqueous solution, and the mixture is stirred and reacted for 2-4 hours in a dark room, so as to cleave carbon-carbon bonds of substrate groups and generate reactive aldehyde groups in xanthan gum units of a molecular chain, thereby obtaining the hydroformylation xanthan gum (Xan-CHO).
Further, in this embodiment, in the step S200, the mass-to-volume ratio (w/v) of the xanthan gum aqueous solution is 0.5 to 2.0%, for example, may be 0.5%, 0.6%, 1.0%, 2.0%; the mass-to-volume ratio (w/v) of the sodium periodate aqueous solution is 5 to 20%, and may be, for example, 5%, 8%, 15%, or 20%.
Further, in this embodiment, in the step S200, the purpose of adding ethylene glycol and continuing stirring for 1 to 2 hours is to neutralize unreacted sodium periodate, wherein 1 to 3ml of ethylene glycol may be added, for example, 1ml, 2ml, or 3ml may be added.
Further, in this embodiment, in the step S200, when the reaction solution is dialyzed and purified, the purified reaction solution is dialyzed with distilled water using a dialysis bag for 2 to 4 days, and the water is renewed at least 5 times per day to obtain the Xan-CHO product. For example, the water may be dialyzed for 2 days with 8 updates per day, or may be dialyzed for 3 days with 5 updates per day. The dialysis bag preferably uses MWCO 12000-14000. Preferably, the Xan-CHO product is freeze dried in a freeze dryer and then stored in sealed plastic bags at 4 ℃.
Preferably, when step S200 is performed, 0.6% xanthan gum aqueous solution and 8% NaIO are prepared4Aqueous solution, 80ml of aqueous xanthan gum solution was poured into a beaker and 2ml of NaIO was added dropwise4Aqueous solution (xanthan gum and NaIO)4The molar ratio is 1: 1.5); then, the mixed solution is continuously stirred for 3 hours in a dark room; 1ml of ethylene glycol was added to neutralize the unreacted NaIO4The reaction was stirred for another 1h and the solution was purified with distilled water using dialysis bags for 3 days with 5 water updates per day during dialysis; the Xan-CHO product was finally freeze dried in a freeze dryer and stored in sealed plastic bags at 4 ℃.
Further, in this embodiment, when the step S300 is implemented, the hydroformylation xanthan gum and carboxymethyl chitosan are dissolved in water according to a predetermined ratio (weight percentage) to form the injectable hydrogel by gelation in a well plate, and the reaction equation of the reaction between Xan-CHO and NOCC to form the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel is shown in fig. 3, wherein the predetermined ratio of Xan-CHO to NOCC is 1%: (0.1 to 0.5%), for example, 1%: 0.1%, 1%: 0.33%, 1%: 0.5 percent.
The embodiment of the invention also provides an injectable hydrogel which is prepared by adopting the preparation method. The injectable hydrogel prepared by the invention, namely the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel, comprises the hydroformylation xanthan gum and carboxymethyl chitosan, wherein the structures of the hydroformylation xanthan gum and the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel are respectively as follows:
The embodiment of the invention also provides application of the injectable hydrogel, and the injectable hydrogel is applied to preparation of a material for intestinal fistula plugging.
The injectable hydrogel synthesized by modifying the functional groups by using the natural polysaccharide is safe and non-toxic, has low cost and remarkable enzymolysis resistance, and has more remarkable superior properties than fibrin glue.
The invention is illustrated in detail below with specific examples:
example 1
1. Synthesis of hydroformylation xanthan gum: preparation of 0.6% (w/v) aqueous Xanthan Gum solution and 8% (w/v) NaIO4The aqueous solution, then 80ml of aqueous xanthan gum solution was poured into a beaker and 2ml of NaIO was added dropwise4Aqueous solution (xanthan gum and NaIO)4In a molar ratio of 1: 1.5); then, the mixed solution is continuously stirred for 3 hours in a dark room; then 1ml of ethylene glycol was added to neutralize the unreacted NaIO4The reaction was stirred for another 1h and the solution was purified with distilled water using dialysis bags for 3 days with 5 water updates per day during dialysis; finally, the Xan-CHO product was freeze dried in a freeze dryer and then stored in sealed plastic bags at 4 ℃.
The degree of oxidation of Xan-CHO (proportion of xanthogenic repeat units) was determined by hydroxylamine hydrochloride titration. 5ml of xanthan gum CHO aqueous solution (0.2% w/v) was dissolved in 15ml of hydroxylamine hydrochloride solution (2.3% w/v), the pH was recorded by a pH meter of 1, and then the mixed solution was stirred for 24 hours, and the pH was measured at 2. The relevant reactions and calculation formulas are as follows:
Xan-(CHO)n+nH2N·HCl＝Xan-(CH＝N-OH)n+nH2O+nHCl (1)
C1(H+)＝10-pH1 (2)
C2(H+)＝10-pH2 (3)
ΔC＝C2(H+)-C1(H+) (4)
degree of oxidation (%) ═ 993 × Δ C × 20 × 10-3/(2×W) (5)
In formula (5), 993 is the molecular weight of the Xan-CHO repeat unit in g/mol. 20 represents the total volume of the reaction solution (15ml +5 ml). W refers to the weight of Xan-CHO in g. The calculated degree of oxidation of the prepared Xan-CHO was 44.1%.
2. Synthesis of carboxymethyl chitosan: suspending 10g of chitosan in 75ml of isopropanol, stirring at room temperature, equally dividing 25ml of 10mol/L NaOH aqueous solution into 5 parts, and sequentially adding the 5 parts of NaOH aqueous solution into the stirred slurry at intervals of 5 min; then, stirring the obtained slurry for 30min, dropwise adding 20g of monochloroacetic acid, heating to 60 ℃, and stirring for 3 h; subsequently, after the reaction mixture was filtered, the filter residue was washed thoroughly 3 times with an 80% (v/v) methanol/water mixture, circulated 2 times with ethanol and finally dried under vacuum to give carboxymethyl chitosan.
3. Synthesis of the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel: mixing the hydroformylation xanthan gum with carboxymethyl chitosan according to the weight percentage of 1%: dissolving 0.33% of the xanthan gum in water, and gelatinizing in a pore plate to generate the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel, namely the injectable hydrogel.
Infrared spectrum detection: and detecting the infrared spectrum characteristics of the chitosan, the carboxymethyl chitosan, the xanthan gum, the hydroformylation xanthan gum and the final crosslinked product hydroformylation xanthan gum/carboxymethyl chitosan hydrogel by using FTIR infrared spectroscopy. A Nicolet-6700 spectrometer was used at room temperature. The powder of each polymer was ground and placed on a dry KBr dish at 4cm-1The spectra were recorded in 32 scans. The results of the infrared spectroscopy are shown in fig. 4, which confirms the successful preparation of carboxymethyl chitosan and the hydroformylated xanthan gum by FTIR as shown in fig. 4. Both chitosan and carboxymethyl chitosan showed 1340cm-1Characteristic absorption bands (C-C-H and O-C-H oscillations) of nearby polysaccharide structures and 1100cm-1(C-O oscillation). Compared with chitosan, carboxymethyl chitosan is at 1610cm due to asymmetric and symmetric stretching vibration of COO-group-1And 1432cm-1Has a characteristic absorption band. In xanthan and hydroformylated xanthan, absorption bands typical of polysaccharide structures are also observed. In addition, at 1740cm-1Point is the C ═ O group stretching vibration, which is more pronounced in the hydroformylated xanthan than in the xanthan. The above results indicate that hydroformylation of xanthan gum and carboxymethylation of chitosan were successful.
In vitro toxicity detection: the safety of the hydrogels was evaluated by the leaching solution method. First, a Xan-CHO/NOCC hydrogel (1%: 0.33%) was prepared well in 24-well plates, followed by extraction with 10% FBS (fetal bovine serum) in DMEM (media) at 37 ℃ for 24h, followed by serial dilution of the stock solution to give different concentrations of leachate (100%, 50%, 25%, 12.5%); fibroblasts were co-cultured with the extract (200. mu.L) for 48h, then 10. mu.L of LCCK-8 was added to each well, incubated at 37 ℃ for 4h, and after homogenization of the solution, absorbance at 450nm was measured. In vitro toxicity test results are shown in fig. 5, and it can be found from fig. 5 that the proliferation of cells is not affected no matter what proportion of the leaching solution and the fresh culture medium is mixed, so that the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel prepared by the invention is safe and non-toxic.
And (3) digestive enzyme enzymolysis resistance test: three pieces of X-CHO/NOCC hydrogel (1%: 0.33%) and fibrin gel were prepared in 6-well plates and then immersed in fresh duodenal fluid at 37 ℃. The duodenal fluid is replaced every six hours. After 6h, 12h, 24h, 48h and 72h, the hydrogel masses were taken and immediately wiped off with tissue paper. The weight of the hydrogels was then measured before they were placed back in the 6-well plate. The results of digestive enzyme enzymolysis resistance tests are shown in fig. 6, and the results in fig. 6 show that the enzymolysis resistance of the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel prepared by the invention is particularly and obviously superior to that of the commercialized fibrin glue.
Example 2
1. Synthesis of hydroformylation xanthan gum: preparation of 0.5% (w/v) aqueous Xanthan Gum solution and 5% (w/v) NaIO4Aqueous solution, then the xanthan aqueous solution was poured into a beaker and NaIO was added dropwise4Aqueous solution (xanthan gum and NaIO)4In a 1:1 molar ratio); then, the mixed solution is continuously stirred for 2 hours in a dark room; then 1ml of ethylene glycol was added to neutralize the unreacted NaIO4The reaction was stirred for another 1h and the solution was purified with distilled water using dialysis bags for 2 days with 8 water updates per day during dialysis; finally, the Xan-CHO product was freeze dried in a freeze dryer and then stored in sealed plastic bags at 4 ℃.
2. Synthesis of carboxymethyl chitosan: suspending 10g of chitosan in 50ml of isopropanol, stirring at room temperature, equally dividing 20ml of 5mol/L NaOH aqueous solution into 5 parts, and sequentially adding the 5 parts of NaOH aqueous solution into the stirred slurry at intervals of 5 min; thereafter, the resulting slurry was stirred for 20min, 20g of monochloroacetic acid was added dropwise, and then heated to 55 ℃ and stirred for 2 h. Subsequently, after the reaction mixture was filtered, the filter residue was washed thoroughly 5 times with a 70% (v/v) methanol/water mixture, circulated 4 times with ethanol and finally dried under vacuum to give carboxymethyl chitosan.
3. Synthesis of the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel: mixing the hydroformylation xanthan gum with carboxymethyl chitosan according to the weight percentage of 1%: dissolving 0.33% of the mixture in water, and forming gel in a pore plate to generate the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel.
The hydroformylation xanthan gum/carboxymethyl chitosan hydrogel prepared in this example is subjected to the same infrared spectrum detection, in vitro toxicity detection and digestive enzyme enzymolysis resistance tests as those in example 1, and the result shows that the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel prepared in this example has the same non-toxicity and obvious enzymolysis resistance.
Example 3
1. Synthesis of hydroformylation xanthan gum: preparation of 2% (w/v) aqueous Xanthan Gum solution and 20% (w/v) NaIO4Aqueous solution, then the xanthan aqueous solution was poured into a beaker and NaIO was added dropwise4Aqueous solution (xanthan gum and NaIO)4In a molar ratio of 1: 3); then, the mixed solution is continuously stirred for 4 hours in a dark room; then 3ml of ethylene glycol was added to neutralize the unreacted NaIO4The reaction was stirred for an additional 2h and the solution was purified with distilled water using dialysis bags for 4 days with 5 water updates per day during dialysis; finally, the Xan-CHO product was freeze dried in a freeze dryer and then stored in sealed plastic bags at 4 ℃.
2. Synthesis of carboxymethyl chitosan: suspending 20g of chitosan in 100ml of isopropanol, stirring at room temperature, equally dividing 40ml of 15mol/L NaOH aqueous solution into 5 parts, and sequentially adding the 5 parts of NaOH aqueous solution into the stirred slurry at intervals of 5 min; thereafter, the resulting slurry was stirred for 40min, 30g of monochloroacetic acid was added dropwise, and then heated to 65 ℃ and stirred for 4 h. Subsequently, after the reaction mixture was filtered, the filter residue was washed thoroughly 3 times with a 90% (v/v) methanol/water mixture, circulated 2 times with ethanol and finally dried under vacuum to give carboxymethyl chitosan.
3. Synthesis of the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel: mixing the hydroformylation xanthan gum with carboxymethyl chitosan according to the weight percentage of 1%: dissolving 0.33% of the mixture in water, and forming gel in a pore plate to generate the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel.
The hydroformylation xanthan gum/carboxymethyl chitosan hydrogel prepared in this example is subjected to the same infrared spectrum detection, in vitro toxicity detection and digestive enzyme enzymolysis resistance tests as those in example 1, and the result shows that the hydroformylation xanthan gum/carboxymethyl chitosan hydrogel prepared in this example has the same non-toxicity and obvious enzymolysis resistance.
In conclusion, the invention provides the injectable hydrogel and the preparation method and the application thereof, and the injectable hydrogel synthesized by modifying the functional groups by using the natural polysaccharide is safe, non-toxic, low in cost and remarkable in enzymolysis resistance.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (5)
1. A method of preparing an injectable hydrogel, comprising:
step A, suspending 10-20 g of chitosan in 50-100 ml of isopropanol, adding 20-40 ml of 5-15 mol/L NaOH aqueous solution, stirring at room temperature for 20-40 min, dropwise adding 20-30 g of monochloroacetic acid, heating to 55-65 ℃, stirring for 2-4 h, filtering, washing and drying to obtain carboxymethyl chitosan;
b, according to the molar ratio of the xanthan gum to the sodium periodate of 1: 1-3, dropwise adding a sodium periodate aqueous solution into the xanthan gum aqueous solution, stirring and reacting for 2-4 h in a dark room, adding ethylene glycol, continuously stirring for 1-2 h, and then dialyzing and purifying the reaction solution to obtain the hydroformylation xanthan gum;
step C, performing a cross-linking reaction on the hydroformylation xanthan gum and the carboxymethyl chitosan to generate injectable hydrogel;
in the step A, the steps of filtering, washing and drying specifically comprise: after filtering, washing the filtered residue for 3-5 times by using a methanol/water mixed solution with the volume ratio of 70-90%, then washing for 2-4 times by using ethanol, and finally drying in vacuum to obtain the carboxymethyl chitosan;
in the step B, the mass volume ratio of the xanthan gum aqueous solution is 0.5-2.0%, and the mass volume ratio of the sodium periodate aqueous solution is 5-20%; and (3) dialyzing the purified reaction solution by using a dialysis bag and distilled water for 2-4 days, and updating water at least 5 times every day when the reaction solution is dialyzed and purified.
2. An injectable hydrogel produced by the production method according to claim 1.
3. The injectable hydrogel of claim 2 wherein the hydroformylated xanthan gum has the formula:
and n is an integer greater than 1.
4. The injectable hydrogel of claim 2, wherein the injectable hydrogel has the structural formula:
wherein m and n are integers more than 1.
5. Use of an injectable hydrogel according to any one of claims 2 to 4 for the preparation of a material for intestinal fistula closure.
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