CN111363168A

CN111363168A - Mixed gel with anticoagulation effect, preparation method and application thereof

Info

Publication number: CN111363168A
Application number: CN202010157098.7A
Authority: CN
Inventors: 赵安莎; 高缘; 丁艺蕾; 杨苹
Original assignee: Southwest Jiaotong University
Current assignee: Southwest Jiaotong University
Priority date: 2020-03-09
Filing date: 2020-03-09
Publication date: 2020-07-03

Abstract

The invention discloses a mixed gel with an anticoagulation effect, a preparation method and an application thereof, and relates to the technical field of 3D printing. The mixed gel with the anticoagulation effect comprises a photocuring polymer formed by methacrylic anhydride modified gelatin, wherein the photocuring polymer is covalently connected with functionalized anticoagulation molecules, and the functionalized anticoagulation molecules are products obtained by amide chemical reaction and covalent crosslinking of heparin and alginate, so that the mixed gel with the slow release effect and the anticoagulation effect is formed and can be applied to photocuring 3D printing. The method for preparing the mixed gel with the anticoagulation function comprises the steps of carrying out photocuring reaction on methacrylic anhydride modified gelatin, alginate and heparin, and then carrying out covalent crosslinking reaction, wherein the finally formed photocuring polymer contains heparin and alginate, and the anticoagulant function is achieved, the heparin can be slowly released in the application process, and the problem of burst release is solved to a great extent.

Description

Mixed gel with anticoagulation effect, preparation method and application thereof

Technical Field

The invention relates to the technical field of 3D printing, and particularly relates to a mixed gel with an anticoagulation effect, and a preparation method and application thereof.

Background

The 3D printing technology (Three-dimensional printing) is a novel rapid prototyping technology, which has been used in a plurality of fields such as daily machine-shaping, aerospace, biomedical engineering, etc., and at present, 3D has a plurality of prototyping modes, but the basic principle is 'layer-by-layer accumulation, layer-by-layer accumulation manufacturing'.

3D printing is taken as a hot forming means of life, production and scientific research at present, and has a plurality of forming modes, and the defects brought by the forming modes are that the forming materials are few, and the forming modes are fixed; and for the biomedical engineering field, the main material applied to the 3D printing mode is generally mixed with some proteins or printed together with cells, and the main material does not have an anticoagulation effect.

In view of this, the present application is presented.

Disclosure of Invention

The invention aims to provide a mixed gel with an anticoagulation effect, and aims to provide a mixed gel system with a slow-release effect.

The invention also aims to provide a preparation method of the mixed gel with the anticoagulation effect, and the prepared mixed gel has the heparin slow-release effect.

The third purpose of the invention is to provide the application of the mixed gel in photocuring 3D printing.

The technical problem to be solved by the invention is realized by adopting the following technical scheme.

The invention provides a mixed gel with an anticoagulation effect, which comprises a photocuring polymer formed by methacrylic anhydride modified gelatin, wherein functional anticoagulation molecules are introduced into the photocuring polymer, and the functional anticoagulation molecules are mainly products obtained by amide chemical reaction and covalent crosslinking of heparin and alginate.

The invention also provides a preparation method of the mixed gel with the anticoagulation effect, which comprises the following steps:

carrying out photocuring reaction on methacrylic anhydride modified gelatin, alginate and heparin, and then carrying out covalent crosslinking.

The invention also provides application of the mixed gel in photocuring 3D printing.

The embodiment of the invention provides a mixed gel with an anticoagulation effect, which has the following beneficial effects: the functionalized anticoagulant molecules are introduced into a photocuring polymer formed by methacrylic anhydride modified gelatin, and mainly are products obtained by amide chemical reaction and covalent crosslinking of heparin and alginate, so that mixed gel with a slow release effect and an anticoagulation effect is formed, and the mixed gel can be applied to photocuring 3D printing.

The invention also provides a preparation method of the mixed gel with the anticoagulation effect, which comprises the steps of carrying out photocuring reaction on methacrylic anhydride modified gelatin, alginate and heparin, and then carrying out covalent crosslinking reaction, wherein the finally formed photocuring polymer contains heparin and alginate, so that the anticoagulation effect is realized, the heparin can be slowly released in the application process, and the burst release problem is solved to a great extent.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram of the synthesis of methacrylamido gelatin;

FIG. 2 shows a photo-curing mechanism of GelMA;

FIG. 3 is a flow chart of the preparation of GelMA-A-H mixed gel;

FIG. 4 is a hydrogen nuclear magnetic resonance spectrum of GelMA;

FIG. 5 is an infrared spectrum of GelMA-A-H;

FIG. 6 is an XPS spectrum of GelMA, GelMA/A/H and GelMA-A-H gels;

FIG. 7 is a high resolution spectrum of S for GelMA, GelMA/A/H, and GelMA-A-H gels;

figure 8 is a release profile of heparin.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The mixed gel with anticoagulation function provided by the embodiment of the invention, and the preparation method and the application thereof are specifically explained below.

The embodiment of the invention provides a preparation method of mixed gel with an anticoagulation effect, which comprises the following steps:

s1 preparation of methacrylic anhydride modified gelatin (GelMA)

The gelatin contains a cell adhesion peptide sequence-RGD capable of promoting cell adhesion, has good biocompatibility, and can be well applied to the field of biomedical engineering. Specifically, referring to fig. 1, the methacrylic anhydride modified gelatin is obtained by reacting pig skin gelatin with methacrylic anhydride, the raw material is easy to obtain, and the prepared gelatin material has excellent biocompatibility.

In other embodiments, commercially available methacrylic anhydride modified gelatin may also be used.

Preferably, the method for preparing the methacrylic anhydride modified gelatin comprises the following steps: mixing pigskin gelatin and phosphate buffer solution at the temperature of 50-70 ℃ to obtain reaction solution, dropwise adding methacrylic anhydride into the reaction solution to react at the temperature of 50-60 ℃ for 2-4h, then adding phosphate buffer solution to dilute the solution to stop the reaction, dialyzing and centrifugally separating the diluted solution to obtain reaction clear liquid, and finally drying the reaction clear liquid to obtain the methacrylic anhydride modified gelatin.

The methacrylic anhydride is added in a dropping manner, the dropping speed of the methacrylic anhydride is 0.4-0.6mL/min, and the reaction is prevented from being too fast; impurities are removed through subsequent dialysis and centrifugal separation, macromolecular impurities are mainly removed through dialysis, and white flocculent precipitates are removed through centrifugal separation to obtain clear liquid. The reaction clear solution is preferably dried by freeze drying, and the properties of the gel can be maximally not damaged.

In a preferred embodiment, the dialysis process is carried out by putting the diluted solution in a dialysis bag for 6 to 8 days, changing the solution every 5 to 7 hours and mechanically stirring, and unreacted gelatin raw material can be effectively removed by dialysis for a long time. The centrifugation process is to place the solution in the dialysis bag into a centrifuge tube and centrifuge at 2000-3000rpm for 3-8 min.

Preparation of S2 GelMA-A-H gel precursor liquid

Mixing and dissolving methacrylic anhydride modified gelatin, alginate, heparin and photoinitiator to obtain the body fluid before gelation. The inventor introduces alginate and heparin at the same time, so that the technical problem of heparin burst release is solved, a polymer formed after methacrylic anhydride modified gelatin is subjected to photocuring is positioned between the alginate and the heparin and can also play a role of a bridge, and the heparin can be slowly released in application due to the existence of amido bond (-CO-NH-).

In some preferred embodiments, the preparation process of the body fluid before gelation is to dissolve alginate and heparin in phosphate buffered saline solution to obtain a first mixed solution, mix and dissolve the first mixed solution and methacrylic anhydride modified gelatin at a temperature of 35-40 ℃ to obtain a second mixed solution, cool the second mixed solution to room temperature, and mix and dissolve the second mixed solution and the photoinitiator. Alginate and heparin are relatively insoluble and are preferably dissolved first.

In other embodiments, the order of mixing may not be limited, but it is ensured that the materials are all sufficiently dissolved.

Preferably, the concentration of the methacrylic anhydride modified gelatin in the phosphate buffered saline solution is 0.1-0.6g/mL, and controlling the concentration of the methacrylic anhydride modified gelatin is beneficial to obtain a uniform gel material, and can also affect the slow release performance of the gel material if the concentration is too high and too viscous to be dissolved.

Specifically, the photoinitiator is selected from at least one of 1- [4- (2-hydroxyethoxy) -phenyl ] -2-hydroxy-2-methyl-1-acetone), 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone and lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP); lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate is preferred. The LAP as a photosensitizer can enable the photocuring process to be faster, and the prepared gel material has better biocompatibility.

Specifically, the alginate is selected from any one of sodium alginate, oxidized sodium alginate and polyaldehyde sodium alginate. The alginate or the derivatives thereof are easily obtained materials, and the addition of the alginate effectively solves the problem of burst release of heparin.

Preparation of S3 GelMA-A-H hydrogel

And (2) carrying out a photocuring reaction on the body fluid before the gel under the irradiation of light, wherein the photocuring reaction is to react the body fluid before the gel for 3-6s under the irradiation of ultraviolet light, and the wavelength of the ultraviolet light is 400-410 nm. The gel polymer is formed by reaction under the action of a photoinitiator through short-time illumination.

Preferably, the mass ratio of the photoinitiator to the methacrylic anhydride modified gelatin is 0.03-0.5:1, and the dosage of the photoinitiator is controlled within the range, so that the photocuring reaction can be rapidly initiated.

In some preferred embodiments, the method further comprises the following steps: immersing the gel material obtained by the photo-curing reaction in an amino compound (such as hexamethylene diamine or ethylene diamine) solution, and reacting for 20-30h under acidic conditions. The inventors have found that the reaction needs to be carried out under acidic conditions, and that the reaction of the hexamethylenediamine solution with the gel material is preferably carried out at a pH of 5 to 7, and that the gel material cannot be formed if the reaction is carried out under alkaline conditions. Referring to fig. 2-3, after the photocuring is completed, the amino compound serves as a bridge to connect the heparin and the alginate, and then the structure formed by the amino compound, the heparin and the alginate is connected with the gel polymer through a hydrogen bond.

In some preferred embodiments, the concentration of hexamethylenediamine in the hexamethylenediamine solution is 0.1 to 0.35g/mL, and the hexamethylenediamine solution is a mixture of hexamethylenediamine and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-N-hydroxysuccinimide (NHS). EDC-NHS plays a role of an activator, can promote the reaction of hexamethylenediamine with heparin and alginate, and the hexamethylenediamine plays a role of a bridge to connect the heparin and the alginate after the reaction. In other embodiments, other active agents may be employed, not to mention here.

In other embodiments, the soaking may be performed only with EDC-NHS solution without introducing hexamethylenediamine, and the heparin and alginate may be reacted with the amino groups present in the gel material, but the introduction amount of heparin may be affected due to insufficient amino group content in the gel material after curing.

Further, the preparation method comprises filtering the gel crude material after the reaction is finished under the acidic condition, and washing the gel crude material for multiple times by using phosphate buffer solution and normal saline to remove impurities on the gel crude material.

The introduction of the hexamethylenediamine and the EDC-NHS solution requires strict control of the reaction steps, the hexamethylenediamine is introduced after the photocuring, and if the hexamethylenediamine and the EDC-NHS solution are introduced, the body fluid before the gelation is cured, and the method cannot be applied to 3D printing photocuring materials.

The embodiment of the invention provides a mixed gel with an anticoagulation effect, which comprises a photocuring polymer formed by methacrylic anhydride modified gelatin, wherein functionalized anticoagulation molecules are covalently connected to the photocuring polymer, and the functionalized anticoagulation molecules comprise heparin and alginate. According to the invention, heparin and alginate are introduced into a photocuring polymer formed by methacrylic anhydride modified gelatin to prepare a gel material with an anticoagulation effect, and the heparin greatly reduces the burst release condition and can be applied to photocuring 3D printing.

In some preferred embodiments, the mass ratio of the photo-curable polymer to the heparin to the alginate is 10:0.5-2: 0.5-2; preferably 10:0.8-1.5:0.8-1.5, such as 10:1: 1. The dosage of the three components is preferably controlled within the range, so that the molding effect can be ensured, and the anticoagulant effect is good.

In some preferred embodiments, the functionalized anticoagulant molecules further comprise an amino compound covalently linked to heparin and alginate, respectively, and the specific components of the amino compound may be as described above with reference to the preparation method. Heparin plays a main role in the functionalized anticoagulant molecules, and the introduction of other molecules plays a role in the slow release effect of the heparin.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

This example provides a method for preparing a mixed gel with anticoagulation effect, which includes the following steps:

(1) synthesis of methacrylamido gelatin (GelMA)

Weigh 5g of pigskin gelatin and mix into 50mL PBS (phosphate buffered saline 10% w/v) and stir at 60 ℃ until completely dissolved, add 1mL MA (methacrylic anhydride, C)₈H₁₀O₃154.16Da) was added to the current solution at a rate of 0.5mL/min and the reaction was stirred at 50 ℃ for 3 h. Then 4 times volume of 50 ℃ PBS solution is added and continuously stirred to stop the reaction, the diluted solution is placed in a 14kDa dialysis bag for dialysis (7d), liquid changing and stirring are continuously carried out during the dialysis, the solution in the dialysis bag is taken out after seven days and is moved to a centrifuge tube, the centrifugation is carried out for 5min at 2500rpm, and clear liquid is taken out and is frozen and dried to obtain the white foam-shaped modified gelatin.

(2) Preparation of GelMA-A-H gel precursor liquid

To 1mL of PBS solution (10% w/v) was added sodium alginate 0.01g, heparin 0.01g, and sonicated until completely dissolved. To this system was added 0.1g of GelMA gelatin at 37 deg.C and shaken until completely dissolved. After the solution was cooled to room temperature, 0.003g of photoinitiator LAP was added to the system, shaken continuously until completely dissolved, and stored away from light.

(3) Preparation of GelMA-A-H hydrogel

And taking the prepared gel precursor liquid and placing the gel precursor liquid under the ultraviolet light of 405nm for 5s of irradiation. EDC/NHS solution was prepared, followed by the addition of a small amount of hexamethylenediamine to give a solution of 0.36mg/mL hexamethylenediamine, and the pH was adjusted to acidic conditions (pH of about 6). Adding the EDC/NHS solution into the solidified gel container until the gel is completely immersed, and storing for 24h at room temperature. The EDC/NHS solution was discarded and washed three times with PBS solution and physiological saline to remove the residual EDC/NHS.

Example 2

(1) synthesis of methacrylamido gelatin (GelMA)

Weigh 5g of pigskin gelatin and mix into 50mL PBS (phosphate buffered saline 10% w/v) and stir at 50 ℃ until completely dissolved, add 1mL MA (methacrylic anhydride, C)₈H₁₀O₃154.16Da) was added to the current solution at a rate of 0.4mL/min and the reaction was stirred at 50 ℃ for 4 h. Then 4 times volume of 50 ℃ PBS solution is added and continuously stirred to stop the reaction, the diluted solution is placed in a 14kDa dialysis bag for dialysis (6d), liquid changing and stirring are continuously carried out in the process, the solution in the dialysis bag is taken out after six days and is moved to a centrifuge tube, the solution is centrifuged at 2000rpm for 8min, and clear liquid is taken out and is frozen and dried to obtain the white foam-shaped modified gelatin.

(2) Preparation of GelMA-A-H gel precursor liquid

To 1mL of PBS solution (10% w/v) were added 0.01g of oxidized sodium alginate (A), 0.01g of heparin (H), and sonicated until complete dissolution. To this system was added 0.6g of GelMA gelatin at 35 ℃ and shaken until completely dissolved. After the solution was cooled to room temperature, 0.3g of photoinitiator 1- [4- (2-hydroxyethoxy) -phenyl ] -2-hydroxy-2-methyl-1-propanone was added to the system), shaken until completely dissolved and stored away from light.

(3) Preparation of GelMA-A-H hydrogel

And taking the prepared gel precursor liquid and placing the gel precursor liquid under 400nm ultraviolet light for irradiating for 3 s. EDC/NHS solution was prepared, followed by addition of a small amount of ethylenediamine to give a concentration of 0.1mg/mL of ethylenediamine in the solution, and adjustment of the pH to acidic conditions (pH of about 5). Adding the EDC/NHS solution into the solidified gel container until the gel is completely immersed, and storing for 20h at room temperature. The EDC/NHS solution was discarded and washed three times with PBS solution and physiological saline to remove the residual EDC/NHS.

Example 3

(1) synthesis of methacrylamido gelatin (GelMA)

Weigh 5g of pigskin gelatin and mix into 50mL PBS (phosphate buffered saline 10% w/v) and stir at 70 ℃ until completely dissolved, add 1mL MA (methacrylic anhydride, C)₈H₁₀O₃154.16Da) was added to the current solution at a rate of 0.6mL/min and the reaction was stirred at 60 ℃ for 2 h. Then 4 times volume of 50 ℃ PBS solution is added and continuously stirred to stop the reaction, the diluted solution is placed in a 14kDa dialysis bag for dialysis (8d), liquid changing and stirring are continuously carried out in the meantime, the solution in the dialysis bag is taken out after eight days and is moved to a centrifuge tube, the solution is centrifuged at 2000rpm for 8min, and clear liquid is taken out and is frozen and dried to obtain the white foam-shaped modified gelatin.

(2) Preparation of GelMA-A-H gel precursor liquid

To 1mL of PBS solution (10% w/v) was added sodium alginate 0.01g, heparin 0.01g, and sonicated until completely dissolved. To this system was added 0.1g of GelMA gelatin at 40 ℃ and shaken until completely dissolved. After the solution was cooled to room temperature, 0.003g of photoinitiator LAP was added to the system, shaken continuously until completely dissolved, and stored away from light.

(3) Preparation of GelMA-A-H hydrogel

The prepared gel precursor solution is taken and placed under the ultraviolet light of 410nm for 6 s. EDC/NHS solution was prepared, followed by the addition of a small amount of hexamethylenediamine to give a solution of 0.35mg/mL hexamethylenediamine, and the pH was adjusted to acidic conditions (pH of about 7). Adding the EDC/NHS solution into the solidified gel container until the gel is completely immersed, and storing for 30h at room temperature. The EDC/NHS solution was discarded and washed three times with PBS solution and physiological saline to remove the residual EDC/NHS.

Example 4

This example provides a method for preparing a mixed gel with anticoagulation, which comprises the following steps: the procedure for introducing no hexamethylenediamine into the system, i.e. (3), is as follows: and taking the prepared gel precursor liquid and placing the gel precursor liquid under the ultraviolet light of 405nm for 5s of irradiation. EDC/NHS solution was prepared and the pH was adjusted to acidic conditions (pH about 6). Adding the EDC/NHS solution into the solidified gel container until the gel is completely immersed, and storing for 24h at room temperature. The EDC/NHS solution was discarded and washed three times with PBS solution and physiological saline to remove the residual EDC/NHS.

Comparative example 1

This comparative example provides a method of preparing a mixed gel, which differs from example 1 substantially without covalent crosslinking, i.e. (3) as follows: and (3) taking the prepared gel precursor solution, placing the gel precursor solution under 405nm ultraviolet light for irradiating for 5s, filtering, and washing with PBS solution and physiological saline for three times respectively, wherein G/A/H is recorded.

Comparative example 2

The present comparative example provides a method of preparing a hybrid gel that is directly photocured using GelMA, comprising the steps of:

weigh 5g of pigskin gelatin and mix into 50mL PBS (phosphate buffered saline 10% w/v) and stir at 50 ℃ until completely dissolved, add 1mL MA (methacrylic anhydride, C)₈H₁₀O₃154.16Da) was added to the current solution at a rate of 0.4mL/min and the reaction was stirred at 40 ℃ for 4 h. Then 4 volumes of 50 ℃ PBS solution were added and stirred continuously to stop the reaction, the diluted solution was put into a 14kDa dialysis bag for dialysis (6d), during which time the solution was changed and stirred continuously, six days later the solution in the dialysis bag was taken out and transferred to a centrifuge tube, centrifuged at 2000rpm for 8min, and the clear solution was taken out and freeze-dried to obtain white foamed modified gelatin (GelMA). GelMA 10% and LAP 0.3% as a pre-gel solution (dissolved in PBS) andand (3) curing the mixture under 405nm ultraviolet light for 3-6 s. After the gel is solidified, the gel material is freeze-dried in a freeze dryer.

Comparative example 3

The comparative example provides a preparation method of sodium alginate gel, comprising the following steps: mixing and dissolving sodium Alginate and PBS buffer solution to prepare sodium Alginate solution with the mass fraction of 1%, soaking the sodium Alginate solution in calcium chloride with the mass fraction of 2% for 24 hours, filtering, washing and drying to obtain sodium Alginate gel which is marked as Alginate.

Comparative example 4

The comparative example provides a preparation method of sodium alginate-heparin gel, which comprises the following steps: mixing and dissolving sodium alginate, heparin and PBS buffer solution to obtain a precursor solution, preparing a solution with the mass fractions of sodium alginate and heparin being 1%, preparing EDC/NHS solution, adding a small amount of hexamethylenediamine to ensure that the concentration of the hexamethylenediamine in the solution is 0.35mg/mL, and adjusting the pH to an acidic condition (the pH is about 6). The precursor liquid was mixed with EDC/NHS solution and stored at room temperature for 24 h. The EDC/NHS solution was discarded and washed three times with PBS and physiological saline to remove residual EDC/NHS, labeled A-H.

Comparative example 5

This comparative example provides a method of preparing a mixed gel which differs from example 1 only in that: firstly, crosslinking and then photocuring are carried out, and the method comprises the following specific steps:

(1) synthesis of methacrylamido gelatin (GelMA)

Weigh 5g of pigskin gelatin and mix into 50mL PBS (phosphate buffered saline 10% w/v) and stir at 60 ℃ until completely dissolved, add 1mL MA (methacrylic anhydride, C)₈H₁₀O₃154.16Da) was added to the current solution at a rate of 0.5mL/min and the reaction was stirred at 50 ℃ for 3 h. Adding 4 times volume of 50 deg.C PBS solution, stirring to stop reaction, dialyzing the diluted solution in 14kDa dialysis bag (7d), changing solution and stirring, taking out the solution in dialysis bag after seven days, transferring to centrifuge tube, centrifuging at 2500rpm for 5min, taking out clear solution, and freeze drying to obtain white foamed modified gelatin。

(2) Preparation of GelMA-A-H gel precursor liquid

(3) Preparation of GelMA-A-H hydrogel

EDC/NHS solution was prepared, followed by the addition of a small amount of hexamethylenediamine to give a solution of 0.36mg/mL hexamethylenediamine, and the pH was adjusted to acidic conditions (pH of about 6). The EDC/NHS solution is mixed with GelMA-A-H gel precursor solution, and the gel is directly solidified, so that the gel can not be applied to 3D printing photocuring materials, because the state of the precursor liquid must be ensured in the process of photocuring the gel. The inventors adjusted the concentration of EDC/NHS solution and the gel cured within 10min even at very low concentrations, whereas the photo-curing 3D printing process could not be completed within 10min, which could not be applied in 3D printing of photo-curing materials.

Test example 1

A small amount of pure gelatin and GelMA lyophilized from example 1 were dissolved in deuterated water (D)₂O), and then transferred to a nuclear magnetic tube to be tested in a nuclear magnetic resonance instrument to obtain a nuclear magnetic resonance hydrogen spectrum, as shown in FIG. 4.

By comparing gelatin with GelMA with nmr hydrogen spectra (fig. 4), it was found that proton peaks-CH of the methacrylic group appear at δ ═ 5.33ppm and δ ═ 5.57ppm₂＝C(CH₃) Indicates that MA was successfully modified on the molecular chain of gelatin; the substitution degree was calculated on the nuclear magnetic spectrum by integrating the peaks at δ of 0.84ppm and δ of 5.57 ppm.

Calculated degree of substitution:

test example 2

The gel materials prepared in example 1 and comparative examples 1-4 and KBr particles are mixed and ground into powder according to the mass ratio of 1:50, and the powder is tableted by a tabletting machine and then placed in an infrared spectrometer for testing.

As a result of the test in FIG. 5, GelMA cured only by light was 1654cm^-1(admin I)，1538cm^-1(adminII)，1453cm^-1(admin III) amide (-CONH-) and amino (-NH) belonging to gelatin appear²) (ii) a The covalently cross-linked G-A-H gel was at 1040cm compared to the covalently cross-linked sodium alginate gel G-A^-1Appear to belong to the heparin (-SO)₂H) The symmetric stretching vibration absorption peak; the same two groups of GelMA/A/H (example 4) and GelMA-A-H (example 1) were each 1041cm^-1And 1025cm^-1In the presence of-SO₂Absorption peak of H and GelMA-A-H group at 1544cm^-1(admin II) a more pronounced absorption peak of the amide (-CONH-) appears. It was demonstrated that covalent cross-linking did occur in the GelMA-A-H gel system.

Test example 3

GelMA (comparative example 2), GelMA/A/H (comparative example 1), GelMA-A-H (example 1) gels were prepared, pulverized and tableted as in test example 2. They were placed in an XPS instrument to test their XPS spectra for high resolution spectral element content of the S element. The results are shown in FIG. 6, FIG. 7 and Table 1.

The results showed that GelMA, GelMA/A/H and GelMA-A-H gels all contained S element, which is the S element of methionine constituting the gelatin in the composition. GelMA/A/H and GelMA-A-H have almost no difference in S content because the doped heparin content is consistent (about 1%), but the XPS spectrum of GelMA-A-H has a phenomenon that the peak value is obviously enlarged at 199.9eV, probably because the covalent crosslinking exists in GelMA-A-H to affect the XPS content.

TABLE 1 element content of GelMA, GelMA/A/H and GelMA-A-H gels

	O	C	N	S
					GelMA	22.48％	13.37％	63.66％	0.48％
GelMA/A/H	30.11％	9.48％	59.44％	0.98％
					GelMA-A-H	29.14％	10.86％	58.95％	1.05％

Test example 4

GelMA/A/H (comparative example 1) and GelMA-A-H (example 1) gels of the same volume are soaked in PBS of the same volume, static release of heparin is carried out at 37 ℃, the release time is 12 hours, 1 day, 3 days, 5 days, 7 days, 9 days, 11 days and 13 days respectively, liquid change and collection of release liquid are carried out every two days, and the accumulated release amount of the heparin in the release liquid is detected by a toluidine blue method. The results are shown in FIG. 8.

It can be seen from the figure that the cumulative release of heparin increases with time, and the release rate of the covalent crosslinking GelMA-A-H group samples is obviously lower than that of the non-covalent crosslinking GelMA/A/H group, and the covalent crosslinking group samples achieve the effect of releasing heparin more slowly and still have the trend of continuing to release after 13 days.

In summary, the mixed gel with anticoagulation provided by the invention is formed by covalently crosslinking heparin and alginate on a photocuring polymer formed by methacrylic anhydride modified gelatin, has a slow release effect and an anticoagulation effect, and can be applied to photocuring 3D printing.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Claims

1. The mixed gel with the anticoagulation effect is characterized by comprising a photocuring polymer formed by methacrylic anhydride modified gelatin, wherein functionalized anticoagulation molecules are introduced into the photocuring polymer, and the functionalized anticoagulation molecules are mainly products obtained by amide chemical reaction and covalent crosslinking of heparin and alginate.

2. The mixed gel with anticoagulant effect according to claim 1, wherein the mass ratio of the photocurable polymer, the heparin and the alginate is 10:0.5-2: 0.5-2; preferably 10:0.8-1.5: 0.8-1.5.

3. The mixed gel with anticoagulant effect according to claim 1 or 2, wherein said functionalized anticoagulant molecules further comprise an amino compound covalently linked to said heparin and said alginate, respectively;

preferably, the amino compound is selected from at least one of hexamethylenediamine and ethylenediamine.

4. The mixed gel with anticoagulant effect according to claim 1 or 2, wherein the alginate is selected from any one of sodium alginate, oxidized sodium alginate and polyaldehyde sodium alginate.

5. A method for preparing a mixed gel with anticoagulant effect according to any one of claims 1 to 4, comprising the steps of:

and carrying out photocuring reaction on the methacrylic anhydride modified gelatin, the alginate and the heparin, and then carrying out covalent crosslinking.

6. The method of claim 5, comprising: mixing and dissolving the methacrylic anhydride modified gelatin, the alginate, the heparin and the photoinitiator to obtain a gel precursor fluid; carrying out photocuring reaction on the body fluid before the gel under illumination, and finally carrying out covalent crosslinking reaction on the gel after the curing reaction by using an activating agent solution;

preferably, the pre-gel body fluid is obtained by mixing and dissolving the components in phosphate buffered saline solution;

preferably, the mass ratio of the photoinitiator to the methacrylic anhydride modified gelatin is 0.03-0.5: 1;

preferably, in the preparation process of the body fluid before gelation, the alginate and the heparin are dissolved in the phosphate buffered saline solution to obtain a first mixed solution, then the first mixed solution and the methacrylic anhydride modified gelatin are mixed and dissolved at the temperature of 35-40 ℃ to obtain a second mixed solution, and then the second mixed solution is cooled to room temperature and then mixed and dissolved with the photoinitiator;

more preferably, the concentration of the methacrylic anhydride modified gelatin in the phosphate buffered saline solution is 0.1 to 0.6 g/mL;

the photoinitiator is selected from at least one of 1- [4- (2-hydroxyethoxy) -phenyl ] -2-hydroxy-2-methyl-1-acetone), 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone and lithium phenyl-2, 4, 6-trimethyl benzoyl phosphinate; lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate is preferred.

7. The preparation method according to claim 6, wherein the photo-curing reaction is to react the gel precursor liquid for 3-6s under the irradiation of ultraviolet light; preferably, the wavelength of the ultraviolet light is 400-410 nm.

8. The method of claim 6, wherein the covalent crosslinking reaction comprises the steps of: immersing the gel material obtained by the photocuring reaction in an amino compound solution, and reacting for 20-30h under an acidic condition;

preferably, the amino compound solution is a hexamethylenediamine solution, and the concentration of hexamethylenediamine in the hexamethylenediamine solution is 0.1-0.35 g/mL;

preferably, the pH of the reaction of the hexamethylenediamine solution with the gel material is 5-7;

preferably, the hexamethylenediamine solution is a mixed solution of hexamethylenediamine and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-N-hydroxysuccinimide;

more preferably, the preparation method further comprises filtering the gel crude material after the reaction is completed under acidic conditions, and then washing the gel crude material with phosphate buffered saline and physiological saline for a plurality of times.

9. The method according to claim 6, wherein the methacrylic anhydride-modified gelatin is obtained by reacting a porcine skin gelatin with methacrylic anhydride;

preferably, the preparation method of the methacrylic anhydride modified gelatin comprises the following steps: mixing the pigskin gelatin and phosphate buffer solution at the temperature of 50-70 ℃ to obtain a reaction solution, dropwise adding methacrylic anhydride into the reaction solution to react at the temperature of 50-60 ℃ for 2-4h, then adding phosphate buffer solution to dilute the solution to terminate the reaction, dialyzing and centrifugally separating the diluted solution to obtain a reaction clear solution, and finally drying the reaction clear solution to obtain the methacrylic anhydride modified gelatin;

more preferably, the dropping rate of the methacrylic anhydride is 0.4-0.6 mL/min;

more preferably, the dialysis process is to put the diluted solution into a dialysis bag for dialysis for 6-8 days, during which the solution is changed every 5-7 hours and mechanically stirred;

more preferably, the centrifugation process is to place the solution in the dialysis bag into a centrifuge tube and centrifuge at 2000-3000rpm for 3-8 min.

10. Use of the mixed gel with anticoagulation effect according to any one of claims 1-4 or the mixed gel prepared by the preparation method according to any one of claims 5-9 in photocuring 3D printing.