CN113621149A

CN113621149A - 3D printing antibacterial hydrogel dressing and preparation method thereof

Info

Publication number: CN113621149A
Application number: CN202110964983.0A
Authority: CN
Inventors: 任学宏; 杨振铭; 刘颖; 刘禹
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-08-20
Filing date: 2021-08-20
Publication date: 2021-11-09
Anticipated expiration: 2041-08-20
Also published as: CN113621149B

Abstract

The invention discloses a 3D printing antibacterial hydrogel dressing and a preparation method thereof, and belongs to the field of biological materials. The preparation method of the 3D printing antibacterial hydrogel dressing comprises the following steps: (1) preparing halamine modified cerium dioxide nanoparticles; (2) uniformly mixing GelMA, xanthan gum, CMC, a photoinitiator and water according to a mass ratio to obtain hydrogel ink; (3) adding the halamine modified cerium dioxide nanoparticles obtained in the step (1) into the ink obtained in the step (2), and uniformly mixing to obtain the 3D printing antibacterial hydrogel ink; (4) and (4) preparing the antibacterial dressing from the 3D printing antibacterial hydrogel ink obtained in the step (3) by adopting a direct-writing 3D printer. The 3D printing antibacterial dressing disclosed by the invention has the advantages of better printing precision and mechanical property, stronger antibacterial property, ideal biocompatibility and good application prospect in the aspect of skin wound healing.

Description

3D printing antibacterial hydrogel dressing and preparation method thereof

Technical Field

The invention relates to a 3D printing antibacterial hydrogel dressing and a preparation method thereof, and belongs to the field of biological materials.

Background

Skin tissue is one of the important components of the human body, accounts for 15 percent of the weight of the adult on average, plays a role in isolating the internal tissue of the human body from the external environment, and loses the protection effect when being damaged. Skin infection is a common threat to skin health, and wound healing and skin health are threatened by problems of wound infection resistance and the like caused by antibiotic abuse phenomena. Therefore, there is an urgent need to develop a highly effective antibacterial wound dressing having broad-spectrum antibacterial properties.

Most antibacterial agents such as nano silver, cationic polypeptides, biguanide antibacterial agents have cytotoxicity or environmental problems to some extent. For example, the long-term use of the nano-silver antibacterial agent can cause silver deposition in human bodies, has certain potential safety hazard, can cause biological accumulation phenomenon in the environment and destroy ecological balance. Cationic polypeptides and biguanide antibacterial agents have the defects of cytotoxicity and easy degradation, and the wide application of the cationic polypeptides and the biguanide antibacterial agents is severely limited. The haloamine antibacterial agent is widely applied to various industries all the time by virtue of the spectrum antibacterial characteristic, but the development of the haloamine antibacterial agent is limited to a certain extent by the characteristic of ultraviolet resistance.

In recent years, many scholars have prepared the halamine-metal oxide hybrid with better ultraviolet stability by combining the halamine antibacterial agent with metal oxides, such as zinc oxide and titanium dioxide, and all show better antibacterial performance. However, there has been no report on the combination of ceria with a halamine compound to date, and the main reason for this is that ceria has relatively low photocatalytic activity and generates relatively less active oxygen under uv light conditions, and thus has relatively weak antibacterial activity, compared to zinc oxide and titania.

The nature of the matrix plays a key role in wound healing, and traditional dressings such as cotton and linen have not been able to meet the complex and varied clinical application requirements. More and more biopolymers, including chitosan, gelatin and polycaprolactone, are used as alternative materials for wound dressings. Among them, gelatin has good biocompatibility, but poor mechanical properties, limiting its application in tissue engineering and wound healing.

During wound healing, a moist environment and proper gas exchange are important to promote wound repair. But conventional hydrogel wound dressings cannot meet both requirements.

The wound dressing is a common material for acute and chronic wound healing treatment, mainly plays a role in protecting a wound, provides a sanitary and clean healing environment for the wound and promotes the rapid healing of the wound, and has been widely applied in clinical practice. Although traditional wound auxiliary materials such as gauze and the like can provide a certain protection effect, the traditional wound auxiliary materials have relatively single functions and do not have the effects of resisting bacteria, promoting wound healing and the like, so that a novel multifunctional wound dressing needs to be developed to meet complex conditions and various requirements in clinical application.

Disclosure of Invention

[ problem ] to

The current antibacterial dressing has the problems that the efficient spectrum antibacterial performance and the good biocompatibility are difficult to be considered, meanwhile, the dry dressing brings good air permeability, meanwhile, the wet environment which is favorable for wound healing cannot be provided, and the wet hydrogel cannot realize gas exchange between the wound and the outside, so that the wound is not favorable for rapid healing of the wound.

[ solution ]

In order to solve at least one problem, the invention uses the halamine modified cerium dioxide nano-particles as an antibacterial component, and combines methacrylic acid acylated gelatin (GelMA), sodium carboxymethyl cellulose (CMC) and xanthan gum to prepare the 3D printing antibacterial dressing by a photo-crosslinking method.

The first object of the invention is to provide a method for preparing 3D printing antibacterial ink, which comprises the following steps:

(1) preparation of halamine modified ceria nanoparticles

Adding CeO into water₂-NPs (nanoparticles) and 3-Aminopropyltriethoxysilane (APS) to obtain APS-modified CeO₂(CeO₂/APS) solution; then, dissolving 5, 5-dimethylhydantoin and sodium hydroxide in an ethanol/water solution for reaction, and removing the solvent after the reaction is finished to obtain a mixed solution; then pouring epoxy chloropropane and ethanol into the mixed solution for reaction to obtain 3-epoxy propyl-5, 5-dimethyl hydantoin (GH); subsequently, GH and CeO were mixed in water₂Performing mixed reaction on the/APS solution, and extracting, washing and chlorinating the obtained suspension after the reaction is finished to obtain halamine modified cerium dioxide nano particles;

(2) preparation of the ink

Uniformly mixing GelMA, xanthan gum, CMC, a photoinitiator and water according to a mass ratio to obtain an ink solution;

(3) preparation of 3D printing antibacterial hydrogel ink

And (3) adding the halamine modified cerium dioxide nanoparticles obtained in the step (1) into the ink solution obtained in the step (2), and uniformly mixing to obtain the 3D printing antibacterial ink.

In one embodiment of the present invention, the CeO in the step (1)₂-the preparation of NPs is: mixing cerium nitrate (0.1mol) and urinePlain (0.3mol) was completely dissolved in dimethylformamide (DMF, 100mL) to give a mixture; then stirring the mixture for 2h at 120 ℃, and obtaining CeO after washing and filtering₂-NPs。

In one embodiment of the present invention, the APS-modified CeO of step (1)₂(CeO₂The preparation method of the/APS) solution comprises the following steps: CeO was added to 100mL of water₂NPs (10g) and APS (2g), pH adjusted to 4 with dilute sulfuric acid and stirring continuously at 80 ℃ for 5h to give APS-modified CeO₂(CeO₂/APS) solution.

In one embodiment of the present invention, the preparation method of 3-glycidyl-5, 5-dimethylhydantoin (GH) in step (1) comprises: 5, 5-dimethylhydantoin (12.8g) and sodium hydroxide (4g) were stirred in ethanol (100mL) at room temperature for 30min, then ethanol and water were removed by vacuum drying for 2 days to give a mixture; then, epichlorohydrin (9.3g) and ethanol (100mL) were poured into the above mixture, and stirring was continued at 80 ℃ for 2 hours, and the resulting sodium chloride was removed by filtration to give 3-glycidyl-5, 5-dimethylhydantoin (GH).

In one embodiment of the present invention, the 3-glycidyl-5, 5-dimethylhydantoin GH and CeO in step (1)₂The reaction conditions of the/APS solution are 80-90 ℃ for 20-25h, more preferably 85 ℃ for 24 h.

In one embodiment of the invention, the extraction in step (1) is a soxhlet extraction with a mixture of ethanol and water in a weight ratio of 1: 1.

In one embodiment of the present invention, the chlorination in step (1) is to disperse the washed nanoparticles in 10% sodium hypochlorite solution and stir at room temperature for 2h to obtain the halamine modified ceria nanoparticles (CeO)₂APSGH-Cl nanoparticles).

In one embodiment of the present invention, the mass ratio of GelMA, xanthan gum, CMC, photoinitiator and water in step (2) is 10-20: 2: 8: 0.5: 100.

in one embodiment of the present invention, the photoinitiator in step (2) comprises 2-hydroxy-2-methyl-1- [4- (2-hydroxyethoxy) phenyl ] -1-propanone (Irgacure 2959), and phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate (LAP) photoinitiator.

In one embodiment of the present invention, the preparation method of GelMA in step (2) comprises:

dissolving 10g of gelatin in 100mL of water, swelling for 1 hour, then dropwise adding 6mL of methacrylic anhydride into the solution at 50 ℃, diluting the solution with 400mL of water after reacting for 3 hours to terminate the reaction, dialyzing for 3 days at 50 ℃ by using a dialysis bag with the cut-off molecular weight of 8000-15000, replacing water every 6 hours, and finally freeze-drying the solution for 2 days to obtain the methacrylic acidylated gelatin GelMA.

In one embodiment of the present invention, the mass concentration of the halamine-modified ceria nanoparticles in the hydrogel in step (3) is 3 to 6%.

The second purpose of the invention is to obtain the 3D printing antibacterial ink prepared by the method.

A third object of the present invention is to provide a method for preparing a 3D-printed antibacterial hydrogel dressing, comprising the steps of:

the 3D printing antibacterial ink obtained by the invention is used for manufacturing an antibacterial dressing by adopting a direct-writing 3D printer.

In one embodiment of the present invention, the printing comprises the steps of:

the 3D printing antibacterial ink disclosed by the invention is stored in an injector, air bubbles are eliminated through centrifugation, then the 3D printing antibacterial ink is pneumatically extruded through a nozzle on a 3D printer to prepare a grid-shaped dressing, and then the 3D printing antibacterial hydrogel dressing is formed by irradiating the printing dressing with ultraviolet light.

In one embodiment of the present invention, the printing includes the following specific steps:

the 3D-printed antibacterial hydrogel ink of the present invention was stored in a syringe, centrifuged at 5000rpm for 10min to eliminate air bubbles, and then extruded through a nozzle (diameter 400 μm) on a 3D printer to prepare a mesh-shaped dressing (20.0 × 20.0mm), and subsequently, the printed dressing was irradiated with ultraviolet light (10w) at a distance of 10cm for 15min to form a 3D-printed antibacterial hydrogel dressing.

The four purposes of the invention are that the 3D printing antibacterial hydrogel dressing prepared by the method is prepared.

A fifth object of the present invention is to provide a band-aid comprising the 3D-printed antimicrobial hydrogel dressing according to the present invention.

The sixth purpose of the invention is to apply the 3D printing antibacterial hydrogel dressing or the 3D printing antibacterial ink in the field of biomedicine.

[ advantageous effects ]

(1) The 3D printing antibacterial dressing has better printing precision and mechanical property, and has better swelling property compared with the common hydrogel dressing; the antibacterial agent has stronger antibacterial performance, and can respectively reach 99.6 percent and 99.8 percent of antibacterial rate after contacting escherichia coli and staphylococcus aureus for 30 min. On the other hand, the prepared 3D printing hydrogel dressing has ideal biocompatibility through biocompatibility test and discovery, and the hydrogel dressing can effectively promote the healing of mouse wounds and accelerate the regeneration of dermis and epidermis tissues in the wound healing process in the mouse skin wound modeling process, and is a hydrogel dressing with good application prospect in the aspect of skin wound healing.

(2) According to the invention, firstly, the halamine grafted and modified cerium dioxide nanoparticles improve the ultraviolet stability of the halamine compound, and effectively avoid the condition that the halamine structure is damaged by ultraviolet light in the photocrosslinking process.

Drawings

Fig. 1 is a schematic diagram of a preparation method of a 3D-printed antibacterial hydrogel dressing.

Fig. 2 is a schematic diagram of a method of preparing halamine-modified ceria nanoparticles.

FIG. 3 is a graph showing the results of measurement of characterization of each of the substances obtained in the preparation process of example 1, wherein (a) CeO₂、CeO₂/APS、CeO₂APSGH and CeO₂FT-IR spectra of/APSGH-Cl NPs; (b) CeO (CeO)₂And CeO₂XPS spectra of/APSGH-Cl NPs; (c) CeO (CeO)₂And CeO₂XPS surface element distribution of APSGH-Cl; (d) CeO (CeO)₂APSGH-Cl XPS peak spectrum; (e) CeO (CeO)₂TEM image ofImage and particle size distribution; (f) CeO (CeO)₂TEM image and particle size distribution of/APSGH-Cl.

Fig. 4 is a comparison of the post-uv active chlorine content of the pure halamine antimicrobial dressing of comparative example 1 and the antimicrobial dressing of halamine modified ceria nanoparticles of example 2.

Fig. 5 shows the results of the contact antimicrobial test of the 3D-printed antimicrobial hydrogel dressings obtained by the inks of the blank group, the control example 2, the control example 3 and the example 2.

Fig. 6 is a result of a contact antibacterial bacteriostatic rate test of 3D printed antibacterial hydrogel dressings obtained by the inks of comparative example 2, comparative example 3, and example 2; wherein (a) is an inhibitory effect on Escherichia coli; (b) is used for inhibiting Staphylococcus aureus.

Fig. 7 is a result of contact antibacterial zone inhibition of 3D-printed antibacterial hydrogel dressings obtained with the inks of comparative example 2, comparative example 3, and example 2; wherein (a) is the inhibition zone for Escherichia coli; (b) is the bacteriostatic circle for staphylococcus aureus.

Fig. 8 is a comparison of swelling performance of a 3D-printed hydrogel dressing and a conventional hydrogel dressing of comparative example 4.

Fig. 9 is a biocompatibility test of the control, control example 2(GCX), 3D printed antimicrobial hydrogel dressing of example 2, wherein (a) is an optical microscope photograph of cells after being cultured in a solution containing dressing leachate for 4, 8, 24h, respectively; (b) the cell survival rate was counted.

FIG. 10 is a graph of the wound healing promotion effect of the gauze group, control example 2(GCX), 3D printed antimicrobial hydrogel dressing of example 2, wherein (a) gauze, GCX-CeO were used₂the/APSGH-Cl dressing bandaged

wounds

1, 2,4, 8 and 12d, and the control group did not carry out any treatment; (b) wound area closure rate and (c) the time to complete closure of the wound required for various treatments; (d) (i) control group (without any treatment), (ii) gauze group, (iii) GCX group and (iv) GCX-CeO₂H of the/APSGH-Cl group&E stained images, solid and dashed arrows indicate epidermis and dermis, respectively (scale bar 50 μm); day 8 (e) thickness of epidermis and (f) dermis; p is less than or equal to 0.05 and p is less than or equal to 0.01.

FIG. 11 shows gauze, medical sponge and sponge3D printing antimicrobial hydrogel dressing (GCX-CeO) of example 2₂APSGH-Cl) and the results of the hemolysis and coagulation tests of the conventional hydrogel dressing of control example 4; wherein (a) is a result of a hemolytic performance test; (b) blood coagulation index test results; (c) red blood cell adsorption SEM images.

Detailed Description

The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.

The test method comprises the following steps:

1. and (3) testing antibacterial performance:

the dressing was tested for antibacterial activity using the modified AATCC 100-2004 method. The bacteria used in the experiment were gram-negative Staphylococcus aureus (ATCC 6538) and gram-positive Escherichia coli (ATCC 8099). The pipette gun pipetted 25. mu.L of the bacterial suspension into the center of the sample (2.54X 2.54cm) and covered another piece of sample, and the sterilized weight was pressed on top in order to bring the sample into full contact with the bacteria. After 10, 30, 60min of contact, the sample was clamped into a centrifuge tube containing 5mL of sterile sodium thiosulfate solution to quench the active chlorine in the sample. After vortexing for 2min, the appropriate amount of solution was diluted 10, 100, 1000 times with phosphate buffer solution in sequence, and 100 μ L each was inoculated onto an agar plate. Then transferred to a 37 ℃ incubator, incubated for 24h, taken out to record the colony number and calculate the sterilization result. In addition, the inhibition zone of the dressing was tested by the agar diffusion method. Both the S.aureus and E.coli suspensions were diluted to about 10⁶CFU/mL, 100. mu.L each was distributed evenly onto the surface of the agar plate using a spreading rod. And then tabletting the sample into a circular sample with the diameter of 1.5cm and the thickness of 0.1cm, attaching the circular sample to the center of an agar plate, transferring the circular sample to an incubator, incubating the circular sample at 37 ℃ for 24 hours, and recording the situation of the inhibition zone.

2. Animal modeling

BALB/c mice are selected for animal modeling to investigate the effect of the developed wound dressing on the wound healing process. BALB/c mice of 20 males, 4-5 weeks old (25-30g) were divided into 4 groups, and 5 mice were housed in cages each. The adaptation period was ended 5 days after check-in for modeling. The mice were pre-anesthetized with 3% isoflurane by an animal anesthesia respirator, removed after the mice were anesthetized, and continuously anesthetized with 2.5% isoflurane. And then, a skin sampler with the diameter of 0.7cm is used for punching and modeling the back of the mouse, the skin injury depth is controlled to be full-skin injury, medical gauze/sample dressing is matched with a breathable medical adhesive tape for wound dressing, and after that, the wound surface is opened every other day for photographing record. And randomly selecting two tissues of the whole epidermis from each group at day 8 to perform HE section staining, shooting by using an optical microscope, and recording the thickness and the appearance of the epithelium and the dermis.

3. Testing of available chlorine content:

the chlorine content of the dressing was determined by iodometry/thiosulfate titration. A sample (size: 2.54X 2.54cm) was put into 20mL of ionized water, 0.3g of potassium iodide and 2mL of a 1% starch solution by mass were added, and after stirring at room temperature for 30min, the solution became dark blue, and was titrated with a sodium thiosulfate solution until the blue color disappeared. The chlorine content of the dressing was calculated using the following formula (1):

where N is the equivalent concentration (equiv/L) of the sodium thiosulfate solution used for titration, V is the volume (mL) of the sodium thiosulfate solution consumed, m is the mass (g) of the sample, and each sample was measured three times and the average value was taken.

4. Measurement of swelling ratio:

the swelling of the dressing in PBS was determined by weight gain. The sample was soaked in PBS, the sample was removed at a predetermined time point, and the filter paper was immediately weighed (W) to remove excess liquid on the surface_s). The samples were measured three times each and calculated by the following formula (2):

wherein, W_dDenotes the dry weight of the sample, W_sRepresents the mass of the sample after swelling.

5. Cell viability assay:

in order to determine the biocompatibility of the prepared sample, human lung adenocarcinoma cells (A549) are selected for in vitro cytotoxicity test, and the specific operation steps are as follows: the cells were cultured in DMEM (high glucose) medium containing 10% fetal bovine serum, and the samples were soaked in the medium for 24 hours to prepare an extract. The cells were seeded at a density of 10⁴Per mL in 5% CO₂And culturing for 24 hours in an incubator at the temperature of 37 ℃. The DMEM medium was replaced with sample extract (100. mu.L/well) for the experimental group and the medium for the control group. And continuously culturing for 24 and 48 hours, and then determining the cell survival rate by adopting a CCK-8 method. At predetermined time points 50. mu.L of CCK-8 solution was added to each well and incubation continued for 2h in the dark. The OD value was measured using a spectrophotometer, and the cell survival rate was calculated as compared with the control group. And observing the cell morphology by using an optical microscope, and comparing the cell morphology difference among different groups.

6. Testing hemolytic performance:

in order to examine the hemolytic performance of the dressing, mouse blood was selected to be subjected to hemolytic performance test, wherein a sample is lyophilized, then 15, 30, 45mg and 60 μ L of mouse blood are respectively added into 3mL of physiological saline, incubated for 3h at 37 ℃, and then centrifuged for 15min at 12000rpm, and then the absorbance at the wavelength of 540nm is tested by using ultraviolet-visible spectroscopy, and meanwhile, the physiological saline and deionized water components are respectively used as a negative control group and a positive control group, and the absorbance is calculated by the following formula (3) to obtain the hemolytic rate:

where Ap is the absorbance of the sample, and Ab and Aw are the absorbance at 540nm of the negative control and the positive control, respectively.

7. Blood coagulation performance test

A sample having a size of 10.0X 3.0mm was preheated at 37 ℃ for 2 hours, then 100. mu.L of whole mouse blood containing an anticoagulant was dropped into the center of the sample, then 10. mu.L of a calcium chloride solution (0.2mol/L) was immediately dropped, shaken at 30rpm in a constant temperature shaker at 37 ℃ for 10min, then the OD value of the solution at 540nm was measured, and the Blood Coagulation Index (BCI) was calculated by the following formula (4):

wherein BCI is the coagulation index of the sample, I_wAbsorbance at 540nm for the blank sample, I_sIs the absorbance of the test sample at 540 nm.

Meanwhile, in order to study the adhesion condition of erythrocytes, the above samples were subjected to crosslinking and dehydration treatment using 2.5% glutaraldehyde solution and gradient ethanol solution (25%, 50%, 75%, 85%, 90%, 95%, and 100%), and the adsorption condition of erythrocytes was observed using a Scanning Electron Microscope (SEM).

8. Morphology and composition characterization

The chemical structure of the synthesized samples was analyzed using infrared spectroscopy (FT-IR, Nicolet Avatar370, Thermo Fisher Scientific), X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Scientific), and observation and recording of the morphology of the samples were performed using transmission electron microscopy (TEM, Tecnai G20, FEI) and scanning electron microscopy (SEM, TM3030, hitachi), respectively.

9. UV stability test

To test the UV stability of the haloamine component in the sample, the sample was UV irradiated using a 10W UV lamp at a distance of 10cm from the sample, and the active chlorine content in the sample was determined and recorded at various time points.

Example 1

A method of making halamine-modified ceria nanoparticles, comprising the steps of:

(1) respectively dissolving 0.1mol of cerium nitrate and 0.3mol of urea in 100mLDMF solution, stirring until the cerium nitrate and the urea are completely dissolved, slowly dropwise adding the urea solution into the cerium nitrate solution at 120 ℃, reacting for 2 hours, generating cerium dioxide nano particles, centrifuging, filtering, washing with water for multiple times, drying, and grinding to obtain the cerium dioxide nano particles;

(2) CeO was added to 100mL of water₂NPs (10g) and APS (2g), pH adjusted to dilute sulfuric acid4, continuously stirring for 5 hours at the temperature of 80 ℃ to obtain APS modified CeO₂(CeO₂/APS) solution;

(3) stirring 5, 5-dimethylhydantoin (12.8g) and sodium hydroxide (4g) in ethanol (100mL) at room temperature for 30min, then removing ethanol and water by vacuum drying for 2 days to obtain a mixture; then, epichlorohydrin (9.3g) and ethanol (100mL) were poured into the above mixture, and the mixture was continuously stirred at 80 ℃ for 2 hours, and the resulting sodium chloride was removed by filtration to obtain 3-glycidyl-5, 5-dimethylhydantoin (GH);

(4) GH and CeO in water₂the/APS solution was mixed and kept at 85 ℃ for 24 hours, after which the resulting suspension was washed with a mixture of ethanol and water in a 1:1 weight ratio by Soxhlet extraction for 12 hours, yielding modified nanoparticles; then dispersing the modified nano particles in a 10% sodium hypochlorite solution, and stirring for 2 hours at room temperature to obtain CeO₂and/APSGH-Cl nano-particles, purifying with water and drying to obtain the halamine modified cerium dioxide nano-particles.

Performing infrared detection on each substance obtained in the preparation process, wherein the detection result is shown in figure 2:

as can be seen from fig. 3 (a): at 760cm^-1And 1600cm^-1A remarkable absorption peak of cerium dioxide appears at 3280cm^-1an-OH absorption peak appears nearby, which indicates that a large number of-OH groups exist on the surface of the prepared cerium dioxide nano-particle. In contrast, the-OH absorption peak of APS modified ceria was significantly reduced. In addition, after APS modification, at 1080cm^-1A new absorption peak appears, which corresponds to the main characteristic peak of Si-O. Furthermore, 852cm^-1The absorption of (B) can be attributed to the Ce-O-Si stretching vibration. It can be seen that APS is chemically bonded to the surface of ceria nanoparticles by covalent bond, and after further reaction with epoxy hydantoin, at 1705cm^-1A new absorption peak appears, which corresponds to the characteristic C ═ O peak on the hydantoin ring, and after chlorination, the corresponding peak is from 1705cm^-1Moved to 1710cm^-1This is due to the electron absorption effect of the chlorine atom.

As can be seen from fig. 3 (b): the pure cerium dioxide surface only has C, O, Ce element characteristic peaks, and the N, Si and Cl element characteristic peaks appear after modification.

As can be seen from fig. 3 (c): pure CeO₂Nanoparticles and modified CeO₂The difference in surface elemental composition between/APSGH-Cl, which also confirms the successful modification of ceria nanoparticles by APS and GH.

As can be seen from fig. 3 (d): si^2pXPS spectra show that, in addition to Si-O-Si and Si-O-C, there is a new peak at 101.4eV, which may correspond to the formation of Si-O-Ce bonds.

As can be seen from fig. 3 (e) and (f): nearly half of CeO₂The particle size of the nanoparticles is less than 5nm, and it can be observed that the morphology change of the nanoparticles before and after modification is not obvious, and the particle size is slightly increased, which may be caused by surface grafting of APS and GH. Furthermore, CeO before and after modification₂The lattice spacing of the nanoparticles was about 0.31nm and 0.30nm, respectively, close to CeO₂Interplanar spacing values for the (111) faces of the nanostructures. The grafting modification of the cerium dioxide only occurs on the cerium surface, and the internal structure and the main performance of the nano cerium dioxide are not influenced.

Example 2

A method for preparing 3D printing antibacterial ink comprises the following steps:

GelMA, xanthan gum, CMC, a photoinitiator Irgacure 2959 and water are mixed according to a mass ratio of 15: 2: 8: 0.5: 100, mixing uniformly to obtain an ink solution; then, the halamine modified cerium dioxide nanoparticles obtained in example 1 were added to the ink solution and mixed uniformly to obtain the 3D printing antibacterial ink (GCX-CeO)₂APSGH-Cl); wherein the mass concentration of the halamine-modified ceria nanoparticles in the ink solution is 4%.

EXAMPLE 3 optimization of hydrogel Properties

Adjustment 1: optimization of xanthan gum

GelMA, xanthan gum, CMC, a photoinitiator Irgacure 2959 and water in a mass ratio of 15: 1: 8: 0.5: 100. 15: 3: 8: 0.5: 100 to obtain the ink solution.

3D printing is carried out on the obtained ink solution, and the parameters of the 3D printing are as follows:

the diameter of the needle head is 0.7mm, the pressure is 180-200 kPa, the printing speed is 2mm/s, the lifting height of each layer is 0.5mm, the printing size is 20.0 multiplied by 20.0mm, the distance is 1.0mm, and the obtained test result of the 3D printing model is as follows:

the 3D printing ink prepared from low-concentration (1% mass concentration of xanthan gum relative to water) xanthan gum has poor definition, and a 3D printing structure with high structure precision cannot be formed. While high concentration of xanthan gum (3% mass concentration of xanthan gum relative to water) can result in excessive solution viscosity, which affects print continuity. The xanthan gum with proper concentration (the mass concentration of the xanthan gum relative to the water is 2%) has relatively good 3D printing definition, and the printing process is stable and continuous.

And (3) adjustment 2:

GelMA, carrageenan, CMC, a photoinitiator Irgacure 2959 and water in a mass ratio of 15: 1: 8: 0.5: 100 to obtain the ink solution.

The resulting ink solution was subjected to 3D printing, with the following results:

in the high temperature (60 ℃) environment, the carrageenan can generate reversible gelation phenomenon at the high temperature (60 ℃) and has poor viscosity regulation effect, so that good definition cannot be obtained.

Comparative example 1

Adjusting the halamine modified ceria nanoparticles in example 2 to be γ - (β -hydroxy- γ -5, 5-dimethylhydantoinylamino) -propyltriethoxysilane (APSGH-Cl), and keeping the rest of the method consistent with example 2, to obtain 3D printing antibacterial ink;

the preparation method of APSGH-Cl comprises the following steps:

dissolving 12.8g of 5, 5-dimethylhydantoin and 4g of sodium hydroxide in 100mL of ethanol, stirring at room temperature for 30min, and then performing rotary evaporation and vacuum drying for two days to remove ethanol to obtain a mixture; and then 9.3g of epichlorohydrin is dissolved in 100mL of ethanol, added into the mixture, reacted for 2h at 80 ℃, filtered to remove generated sodium chloride, 2g of gamma-Aminopropyltriethoxysilane (APS) is added into the mixture, reacted for 24h at 80 ℃, evaporated to remove the solvent, chlorinated at 40 ℃ for 20min by using tert-butyl hypochlorite, evaporated again and removed to obtain APSGH-Cl.

Comparative example 2

The halamine modified ceria nanoparticles in example 2 were omitted and the rest was kept the same as example 2, to obtain a 3D printing antibacterial ink.

Comparative example 3

The halamine-modified ceria nanoparticles in example 2 were adjusted to CeO₂The nanoparticles, otherwise identical to those of example 2, yielded a 3D printing antimicrobial ink.

Comparative example 4

Firstly, dissolving 1.5g of GelMA in 10.0mL of water, adding 0.05g of photoinitiator Irgacure 2959 into the water, and obtaining a light yellow solution after the GelMA is dissolved; and then, simultaneously adding 0.2g of xanthan gum and 0.8g of CMC into the light yellow solution, fully stirring to obtain a uniform viscous solution, standing for 6 hours to fully dissolve the xanthan gum in a hydrated state, then transferring the solution into a transparent mold, performing radiation crosslinking at a position 10cm away from the hydrogel by using a 10w ultraviolet lamp, and stopping photocrosslinking after the solution is converted into the hydrogel with stable and elastic structure to obtain GelMA hydrogel (non-3D printing dressing).

Example 4

The 3D printing antibacterial inks obtained in example 2 and comparative examples 1, 2 and 3 were respectively stored in syringes, centrifuged at 5000rpm for 10min to eliminate air bubbles, and then pneumatically extruded through nozzles (diameter 400 μm) on a 3D printer to prepare a mesh-shaped dressing (20.0 × 20.0 mm). Subsequently, the printing dressing was irradiated with ultraviolet light (10w) at a distance of 10cm until a 3D-printed hydrogel dressing with structurally stable and certain elasticity was formed, the hydrogel dressing obtained with the ink of example 2 being abbreviated as GCX-CeO₂APSGH-Cl, the hydrogel dressing prepared by the ink of comparative example 1 is abbreviated as GCX-APSGH-Cl; the hydrogel prepared from the ink obtained in comparative example 2 is abbreviated as GCX; the hydrogel dressing obtained by the ink of comparative example 3 was abbreviated as GCX-CeO₂。

And (3) carrying out performance test on the obtained 3D printing antibacterial hydrogel dressing, wherein the test result is as follows:

FIG. 4 is a graph of the pure haloamine antimicrobial dressing of control example 1 (GCX-APSGH-Cl) and the haloamine modified ceria of example 2Antibacterial dressing of nanoparticles (GCX-CeO)₂APSGH-Cl) in UV light. As can be seen from fig. 4: compared with a pure halamine antibacterial agent, the halamine grafted modified cerium dioxide still retains 0.18% of active chlorine after being irradiated by ultraviolet light for 20min, and is obviously higher than that of a pure halamine dressing.

FIG. 5 shows blanks (without any treatment, bacteria naturally grow), control 2(GCX), and control 3 (GCX-CeO)₂)3D printed antimicrobial hydrogel dressing (GCX-CeO) obtained with the ink of example 2₂APSGH-Cl). FIG. 6 shows comparative example 2(GCX) and comparative example 3 (GCX-CeO)₂)3D printed antimicrobial hydrogel dressing (GCX-CeO) obtained with the ink of example 2₂APSGH-Cl) was tested. As can be seen from fig. 5 and 6: after the dressing containing the halamine modified cerium dioxide nanoparticles is contacted with escherichia coli and staphylococcus aureus for 30min, more than 99% of bacteria can be killed, the dressing has extremely strong antibacterial capacity, and wound infection can be effectively prevented.

FIG. 7 shows comparative example 2(GCX) and comparative example 3 (GCX-CeO)₂)3D printed antimicrobial hydrogel dressing (GCX-CeO) obtained with the ink of example 2₂APSGH-Cl); as can be seen from fig. 7: the hydrogel dressing introduced with the halamine modified cerium dioxide nanoparticles has no obvious inhibition zone, which shows that the main antibacterial component of the dressing is not easy to dissolve out of the dressing, and the antibacterial component can be effectively prevented from flowing into human body from wounds through blood in wound healing.

Figure 8 is a comparison of swelling performance of a 3D printed hydrogel dressing (GCX) and a non-3D printed dressing of comparative example 4; the 3D printing hydrogel dressing is prepared by omitting the addition of antibacterial agent halamine modified cerium dioxide nanoparticles on the basis of example 2. As can be seen from fig. 8: the swelling performance of the 3D printing dressing is more ideal, and the swelling efficiency of the dressing in the initial swelling stage is higher. On one hand, the CMC and the xanthan gum in the formula have higher concentration and good hydrophilicity, can absorb a large amount of water and keep good water-based property. On the other hand, with the help of the larger swelling space and contact area of the 3D scaffold, the 3D printed dressing can absorb moisture or wound exudate faster than a normal dressing.

Fig. 9 is a biocompatibility test of the 3D-printed antimicrobial hydrogel dressing of control group (cell natural growth), control example 2(GCX), example 2. As can be seen from fig. 9 (a): there appears to be no significant difference in cell morphology of a549 cells after 4, 8 and 24 hours incubation in leachate containing modified ceria nanoparticle dressing. Meanwhile, the cell viability test (fig. 9 (b)) shows that the cell viability of the dressing after 24 hours of culture is 91%, which indicates that the dressing has no significant cytotoxicity and good biocompatibility.

FIG. 10 shows blank groups (wound modeling without any treatment), gauze groups, control example 2(GCX), 3D printed antimicrobial hydrogel dressing of example 2 (GCX-CeO)₂APSGH-Cl) and the effect on wound healing, as can be seen from fig. 10 (a): the blank group had significant wounds after 12 days of healing, indicating that wounds without any treatment were difficult to heal. However, the wound size at 12 days was significantly smaller for the gauze group, which may be the result of the protective effect of the gauze on the wound. Meanwhile, the healing speed of the GCX and GCX-APSGH-Cl dressing-treated wounds is obviously higher than that of a control group and a gauze group, the wounds are obviously closed after 8 days of healing, and the wounds are almost completely healed on the 12 th day. As can be seen from fig. 10 (b): the wound size reduction rate of the control group after 12 days of healing was about 81%, which was mainly dependent on the self-contraction of the skin wound of the mice. The wound area reduction rate of the GCX-CeO2-APSGH-Cl group reached 98% on day 12. As can be seen from fig. 10 (c): further calculations of closure time confirmed that the untreated group was completely healed for about 16 days, which was about 4 days longer than the GCX and GCX-APSGH-Cl treated groups. This indicates that the wound treated with the prepared 3D printed antimicrobial dressing has an excellent wound healing promoting effect. As can be seen from fig. 10 (d): the control group found a thin epithelial layer and dermis 8 days after healing. As can be seen from fig. 10 (e) and (f): wounds treated with GCX and GCX-CeO2/APSGH-Cl dressings exhibited higher epidermal and dermal thicknesses, indicating better tissue regeneration

FIG. 11 shows gauze, medical sponge, 3D printed antimicrobial hydrogel dressing (GCX-CeO) of example 2₂APSGH-Cl) and control 4. As can be seen from fig. 11: the dressing of embodiment 2 can not destroy red blood cells, has better red blood cell and platelet adsorption performance because of more hydroxyl groups, can adsorb more red blood cells compared with common cotton gauze and gelatin sponge, and is favorable for hemostasis. In addition, the gaps formed by the 3D printing structure help the dressing to adsorb more red blood cells and platelets, and the positive effect on wound hemostasis is achieved.

Claims

1. A method for preparing 3D printing antibacterial ink is characterized by comprising the following steps:

(1) preparation of halamine modified ceria nanoparticles

Adding CeO into water₂Reaction of-NPs with APS to give CeO₂(ii) an APS solution; then, dissolving 5, 5-dimethylhydantoin and sodium hydroxide in an ethanol/water solution for reaction, and removing the solvent after the reaction is finished to obtain a mixed solution; then pouring epoxy chloropropane and ethanol into the mixed solution for reaction to obtain GH; subsequently, GH and CeO were mixed in water₂Performing mixed reaction on the/APS solution, and extracting, washing and chlorinating the obtained suspension after the reaction is finished to obtain halamine modified cerium dioxide nano particles;

(2) preparation of hydrogel inks

(3) preparation of 3D printing antibacterial hydrogel ink

2. The method of claim 1, wherein the mass concentration of the halamine-modified ceria nanoparticles of step (3) in the hydrogel is 3-6%.

3. The method according to claim 1 or 2, wherein the mass ratio of GelMA, xanthan gum, CMC, photoinitiator and water in the step (2) is 10-20: 2: 8: 0.5: 100.

4. the method of any one of claims 1 to 3, wherein GH and CeO are used in the step (1)₂The reaction condition of the/APS solution is that the reaction is carried out for 20-25h at 80-90 ℃.

5. 3D printing antibacterial ink prepared by the method of any one of claims 1 to 4.

6. A method for preparing a 3D printing antibacterial hydrogel dressing is characterized by comprising the following steps:

preparing the 3D printing antibacterial ink obtained in the claim 5 into an antibacterial dressing by using a direct-writing 3D printer.

7. The method according to claim 6, wherein said printing comprises in particular the steps of:

the 3D printed antimicrobial ink of claim 5 is stored in a syringe, air bubbles are eliminated, and then pneumatically extruded through a nozzle on a 3D printer to prepare a mesh-like dressing, and subsequently, the printed dressing is irradiated with ultraviolet light to form a 3D printed antimicrobial hydrogel dressing.

8. The 3D printed antimicrobial hydrogel dressing prepared by the method of claim 6 or 7.

9. A wound dressing comprising the 3D-printed antimicrobial hydrogel dressing of claim 8.

10. Use of the 3D-printed antimicrobial hydrogel dressing of claim 8 or the 3D-printed antimicrobial ink of claim 5 in the biomedical field.