Magnetic hydrogel material with sterilization and temperature-sensitive shrinkage performance as well as preparation method and application of magnetic hydrogel material

Abstract

The invention belongs to the technical field of hydrogel, and particularly relates to a magnetic hydrogel material with sterilization and temperature-sensitive shrinkage performance as well as a preparation method and application of the magnetic hydrogel material. According to the magnetic hydrogel material, PNIPAm copolymerized PAm double-network hydrogel is used as a matrix, nano clay is grafted on the matrix to form copolymerized double-network hydrogel with temperature-sensitive shrinkage performance, and magnetic substances and tannic acid are doped in the copolymerized double-network hydrogel with the temperature-sensitive shrinkage performance. The magnetic hydrogel material with the sterilization and temperature-sensitive shrinkage performance is obtained. The PNIPAm copolymerized PAm dual-network hydrogel is prepared from isopropyl acrylamide and acrylamide through a free radical polymerization reaction. The magnetic hydrogel material disclosed by the invention has sterilization and mechanical loading properties, can efficiently kill staphylococcus aureus and escherichia coli, and effectively promotes the repair of infectious diabetes wounds through efficient sterilization and continuous mechanical loading on the wounds.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogels, and particularly relates to a magnetic hydrogel material with both bactericidal and thermosensitive shrinkage properties, and a preparation method and application thereof. Background Art

[0002] Diabetes, as a global chronic metabolic disorder disease, the number of its patients is expected to reach 643 million by 2030 and 783 million by 2045. Diabetic dermopathy is one of the most common complications of diabetes, with an estimated prevalence of 51.1% to 97.0%. Even minor skin injuries can lead to the formation of infectious diabetic wounds in diabetic patients. These wounds are prone to infection and the formation of biofilms, resulting in the obstruction of the healing process. Due to the complex wound microenvironment, the treatment of infectious diabetic wounds remains a major clinical challenge.

[0003] Although some progress has been made in the treatment of chronic diabetic wounds, problems such as low bioavailability, complex disease microenvironment, and insufficient drug delivery efficiency still limit the treatment effect. Therefore, combination therapies that can coordinate multiple functions of anti-infection effect, good biocompatibility, and promoting wound repair have shown the prospect of accelerating the repair of infectious diabetic wounds. Given the rising multi-drug resistance and refractory pathogenic biofilms, conventional antibiotics have proven to have limited therapeutic effects clinically.

[0004] In addition to the magnetic field and magnetic suction characteristics, magnetic materials also have a magnetothermal effect, that is, the temperature of a magnet or paramagnet changes with the change of the magnetic field strength. Generally, under adiabatic conditions, magnetization will cause the temperature to rise, while demagnetization will cause the temperature to drop. Using the magnetothermal effect, we can adjust the magnetic field strength to raise the temperature of the magnetic material to the required temperature. Research shows that acting at 50 °C for 20 minutes can effectively sterilize; in addition, thermosensitive hydrogels are widely used in the biomedical field based on their temperature-responsive characteristics. And wound healing is closely related to skin contraction, and surgical suture and skin stretching techniques also promote wound repair by applying external force to cause wound contraction.

[0005] Therefore, there is an urgent need for a hydrogel material with both bactericidal and thermosensitive shrinkage properties to exert the combined effects of anti-infection and promoting wound repair through the thermosensitive shrinkage of the material to achieve mechanical loading. Summary of the Invention

[0006] The purpose of the present invention is to provide a magnetic hydrogel material with both bactericidal and thermosensitive shrinkage properties, and a preparation method and application thereof.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a magnetic hydrogel material with both bactericidal and thermosensitive shrinkage properties. Using a PNIPAm copolymerized PAm double-network hydrogel as the matrix, nano-clay is grafted onto the matrix to form a copolymerized double-network hydrogel with thermosensitive shrinkage properties, and magnetic substances and tannic acid are doped into the copolymerized double-network hydrogel with thermosensitive shrinkage properties to obtain the magnetic hydrogel material; The PNIPAm copolymerized PAm double-network hydrogel is formed by free radical polymerization of isopropylacrylamide and acrylamide.

[0008] The magnetic hydrogel material of the present invention improves the lower critical solution temperature (LCST) of the poly(N-isopropylacrylamide) (PNIPAm) hydrogel by doping nano-clay and using a PNIPAm copolymerized PAm double-network hydrogel, makes the material have a magnetic thermal effect by introducing magnetite, and enhances the bactericidal effect of the material by introducing tannic acid. The combined action of each component enables the magnetic hydrogel material to provide stable mechanical loading simultaneously at a relatively high bactericidal temperature, thereby realizing the combined strategy of coupling hyperthermia bactericidal and mechanical promotion of healing.

[0009] Further, the magnetic substance is magnetite.

[0010] In a second aspect, the present invention provides a preparation method of the magnetic hydrogel material, including the following steps: Dissolve isopropylacrylamide, catalyst, acrylamide, crosslinking agent, nano-clay, magnetic substance and tannic acid in water to make a uniform precursor solution; Under the atmosphere of a protective gas, add an initiator and an accelerator to the precursor solution and mix evenly, and polymerize at 4 - 7 °C for 12 - 16 h to obtain the magnetic hydrogel material.

[0011] Further, in the precursor solution, the mass concentrations of isopropylacrylamide, catalyst, acrylamide, crosslinking agent, nano-clay, magnetic substance and tannic acid are 8% - 12%, 0.2% - 0.5%, 1% - 3%, 0.1% - 0.5%, 3%, 15%, 1% in sequence.

[0012] Further, the catalyst is sodium pyrophosphate, the crosslinking agent is N,N'-methylenebisacrylamide, the initiator is tetramethylethylenediamine, and the accelerator is an ammonium persulfate solution with a mass concentration of 28% - 32%.

[0013] Further, the volume ratio of water, initiator and accelerator is 1000:1 - 2.5:4 - 6.

[0014] In a third aspect, the present invention provides the application of the magnetic hydrogel material in the preparation of bactericidal products.

[0015] Furthermore, the sterilization includes killing Staphylococcus aureus and killing Escherichia coli.

[0016] Fourthly, the present invention provides an application of the magnetic hydrogel material in preparing a dressing for promoting the repair of infectious diabetic wounds.

[0017] Fifthly, the present invention provides a dressing for promoting the repair of infectious diabetic wounds, and the dressing includes the magnetic hydrogel material.

[0018] Compared with the prior art, the present invention has the following beneficial effects: By utilizing the magnetothermal effect, the present invention prepares a magnetic hydrogel material with both sterilization and mechanical loading functions. The experimental results show that this material can efficiently kill Staphylococcus aureus and Escherichia coli, and effectively promote the repair of infectious diabetic wounds through sterilization and continuous mechanical loading on the wound surface. Description of the Drawings

[0019] Figure 1 Rheological characterizations of the nanofluid with 1% (NiLa 1 ), 2% (NiLa 2 ), and 3% (NiLa 3 ) mass concentration in the precursor solution.

[0020] Figure 2 Stress-strain curves and rheological characterizations of the material system with sequential addition of Fe 3 O 4 and TA, where A is the stress-strain curve of the material and B is the rheological characterization of the material.

[0021] Figure 3 SEM images of the MHd material network structure, where A is the SEM image at 100× magnification; B is the SEM image at 200× magnification; C is the SEM image at 4000× magnification.

[0022] Figure 4 Tensile test diagrams of the MHd material, where A is the actual tensile test diagram and B is the stress-strain curve.

[0023] Figure 5 Real-time temperature infrared imaging detection of the magnetothermal effect generated by the MHd material under high-frequency alternating magnetic fields.

[0024] Figure 6 Intuitive display of the area change of the MHd material after thermosensitive shrinkage.

[0025] Figure 7 Percentage of the area change of the MHd material after thermosensitive shrinkage.

[0026] Figure 8The bactericidal effects of the MHd material against Staphylococcus aureus and Escherichia coli.

[0027] Figure 9 Biocompatibility detection of the MHd material.

[0028] Figure 10 To observe the promotion of wound healing in diabetic mice infected with Staphylococcus aureus by the MHd material. A is a schematic diagram of the animal experiment, and B shows the wound healing conditions at different time points. Specific implementation manners

[0029] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well-known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0030] Example 1: Preparation of a magnetic hydrogel (MHd) material with both bactericidal and thermosensitive shrinkage properties.

[0031] Prepare PNIPAm-PAm / Fe 3 O 4 / Clay / TA magnetic hydrogel by a one-pot method of free radical polymerization. Dissolve N-isopropylacrylamide (NIPAm), sodium pyrophosphate (TSSP), acrylamide (Am), N,N'-methylenebisacrylamide (MBAA), nanoclay (Clay), iron oxide (Fe 3 O 4 ), and tannic acid (TA) in 20 ml of deionized water in sequence to make a uniform precursor solution. The concentrations of the components in the precursor solution are shown in Table 1. After purging the precursor solution with nitrogen in an ice-water bath for 30 min, add N,N,N',N'-tetramethylethylenediamine (TEMED) (30 μL) and 30% ammonium persulfate (APS) (100 μL), mix well, and then transfer the hydrogel precursor solution into a mold. After polymerization at 4°C for 12 hours, the prepared sample is obtained.

[0032] Table 1: Composition of the magnetic hydrogel material For a mechanically active dressing based on a thermosensitive hydrogel network, an intervention temperature that is too much higher than the LCST will cause the material itself to rapidly shrink and detach from the adhered skin tissue, so that the mechanical stimulus generated by the material contraction cannot be transmitted to the wound surface. The LCST of PNIPAM-based hydrogels is about 30 - 32 °C, and too high an intervention temperature will cause it to rapidly lose water and thus be difficult to adhere to the skin tissue. However, diabetic wounds are often accompanied by infections. To combine thermosensitive contraction and hyperthermia antibacterial, when choosing a higher intervention temperature (i.e., a temperature sufficient to kill bacteria), it is still necessary to stably transmit the mechanical force generated by the material contraction to the wound edge tissue by adhesion. It is often necessary to make necessary adjustments to the LCST of the thermosensitive hydrogel. In this example, the LCST of the PNIPAm-based hydrogel is increased by doping with nanoclay and using a PNIPAm copolymerized PAm double-network hydrogel, in order to enable the mechanically active dressing to provide stable mechanical stimuli at a higher bactericidal temperature, thereby realizing a combined therapy of coupling hyperthermia bactericidal and mechanical promotion of wound healing. In this example, the LCST is first increased by doping with nanoclay, with the mass concentration of nanoclay in the pre-liquid as the single factor, and the mass concentrations of the other components fixed as NIPAm (10.5%), TSSP (0.35%), Am (2%), MBAA (0.25%), Fe 3 O 4 (15%), TA (1%), to verify the enhancing effect of doping nanoclay on the LCST of the MHd. As the doping ratio of nanoclay increases from 1%, 2% to 3%, the LSCT increases from 33 °C (1%) to 38 °C ( Figure 1 ), and due to its special lamellar structure, the nanoclay provides more cross-linking sites for the hydrogel network, reducing the use of the cross-linking agent (MBAA). As the content of nanoclay increases, the storage modulus of the hydrogel is also improved.

[0033] To endow the material with a magnetic hyperthermia effect, we introduce magnetite into the system; to enhance the bactericidal effect of the material, tannic acid is added. The mechanical properties of the material are evaluated by a tensile-strain experiment and the Young's modulus is calculated.

[0034] The test groups are set as follows: (1) Hd group: the components and mass concentrations in the pre-liquid are NIPAm 10.5%, TSSP 0.35%, Am 2%, MBAA 0.25%, Clay 3%; (2) Hd / Fe 3 O 4 group: the components and mass concentrations in the pre-liquid are NIPAm 10.5%, TSSP 0.35%, Am 2%, MBAA 0.25%, Clay 3%, Fe 3 O 4 15%; (3) Hd / Fe 3 O4 / TA group: The components and mass concentrations in the precursor solution are 10.5% NIPAm, 0.35% TSSP, 2% Am, 0.25% MBAA, 3% Clay, 15% Fe 3 O 4 15%, and 1% TA. The preparation method is as described above.

[0035] The results are as Figure 2 shown. With the introduction of Fe 3 O 4 and TA, the mechanical properties of the hydrogel are significantly improved. As shown by rheological characterization, the introduction of Fe 3 O 4 and TA does not significantly change the LCST of the hydrogel.

[0036] Subsequently, MHd prepared from the Hd / Fe 3 O 4 / TA group was used as the sample for structural characterization and functional identification.

[0037] Example 2: Morphological and Structural Characterization of MHd Material The sample was freeze-dried to evaluate the microstructure of the hydrogel. The image of the sample was collected using a JSM-IT700HR scanning electron microscope produced by JEOL Ltd. (Japan). For each sample, the overall view of the sample was observed at low magnification first, and then the area to be observed was selected to collect pictures to observe the specific structure.

[0038] Its microscopic morphology was evaluated by scanning electron microscope ( Figure 3 ). It can be seen that the prepared MHd has a network structure with uniform pore size, and Fe 3 O 4 and nano-clay are uniformly dispersed in the network, ensuring that the material can have isotropic mechanical properties.

[0039] Example 3: Detection of Mechanical Properties of MHd Material Both ends of the prepared dumbbell-shaped (tensile specimen) magnetic hydrogel were fixed on the holders of an electronic universal testing machine (Shenzhen Sansi Zongheng Technology Co., Ltd., UTM6202). The initial position of the holders was adjusted so that the prepared sample was fully extended and without tension (as Figure 4 shown in A). After zeroing and calibrating the universal testing machine, the holders were moved at a constant speed of 5 mm / min to stretch the sample until the sample broke. The maximum stress at the time of sample breakage was recorded, and the stress-strain curve was plotted based on the tensile stress and the corresponding sample tensile length data (as Figure 4 shown in B), and the Young's modulus was determined through the stress-strain curve.

[0040] The Young's modulus value of the magnetic hydrogel material is 48.84 Kpa, indicating that the material has high mechanical properties and can meet subsequent mechanical loading.

[0041] Example 4: Detection of the magnetothermal properties of the MHd material Use the disk coil of a high-frequency induction heating machine (HNG-15KW, Huanneng Power Technology, Shenzhen) to provide an alternating magnetic field (AMF) to perform magnetic induction heating on the magnetic hydrogel material. Use an infrared thermal imager (K20, Hikmicro) to measure and record the real-time temperature of the material. The results show that the material can be heated to 50 °C and stably maintained for 20 minutes through the alternating magnetic field (as Figure 5 shown).

[0042] Example 5: Detection of the thermosensitive shrinkage properties of the MHd material Use the disk coil of a high-frequency induction heating machine (HNG-15KW, Huanneng Power Technology, Shenzhen) to provide an alternating magnetic field (AMF) to perform magnetic induction heating on the MHd, and keep the material temperature at 50 °C. Use a camera to record the change in the material area every 5 minutes, and calculate the shrinkage percentage of the material.

[0043] The results of calculating the percentage change in the thermosensitive shrinkage area of the MHd material show that the percentage of the shrinkage area of the material gradually increases with the extension of time when heated to 50 °C (as Figure 6 shown), and the percentage of the area shrinkage reaches 33% at 20 minutes (as Figure 7 shown), indicating that the material has good thermosensitive shrinkage properties.

[0044] Example 6: Detection of the antibacterial properties of the MHd material First, prepare 100 μL of sterile MHd material and place it in a 12-well plate. Subsequently, inoculate 20 μL of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) suspensions with a concentration of 1×10 8 CFU / mL on the surface of the MHd material. At the same time, set up a negative control group, that is, inoculate an equal amount (20 μL) of bacterial suspension under the condition of no hydrogel. The experimental group MHd (50 °C) and the control group are both placed in an alternating magnetic field for treatment, and the temperature of the MHd material is raised to 50 °C and maintained for 20 minutes through the magnetothermal effect; the experimental group MHd (-) is not treated with an alternating magnetic field. After the intervention is completed, collect the remaining bacterial solutions in each group for sufficient resuspension, and use the plate coating method for colony counting to evaluate the bacterial survival rate.

[0045] We measured the bactericidal performance of the MHd material in vitro. The results showed that in the negative control group (Control), colonies of both types of bacteria grew over the entire plate. In the MHd (-) group, only some bacteria were killed, while in the MHd (50°C) group, the killing rates of Staphylococcus aureus and Escherichia coli reached as high as 99%, and there were basically no surviving colonies on the plate (as Figure 8 shown). This indicates that by applying a high-frequency alternating magnetic field to heat the MHd material to 50°C, a very high synergistic bactericidal effect was produced by the magnetothermal effect and the explosive release of TA at 50°C, while the bactericidal effect relying solely on TA was very limited.

[0046] Example 7: Biocompatibility detection of the MHd material This experiment was detected by in vitro culturing of NIH-3T3 (mouse embryonic fibroblasts). The experiment was divided into a control group and an MHd material group. The MHd material prepared under sterile conditions was further sterilized by UV irradiation and then immersed in DMEM complete medium for more than one week to obtain DMEM complete medium containing the MHd material leachate. NIH-3T3 cells were inoculated in a 96-well plate, and the cells were divided into 2 groups: the control group was added with normal DMEM complete medium, and the MHd material group was added with DMEM complete medium containing the MHd material leachate. The NIH-3T3 cells were cultured for 24 hours, 48 hours, and 72 hours respectively, and CCK-8 reagent was added to the corresponding wells and incubated at 37°C for 2 hours. The cell viability was detected by measuring the absorbance value (OD450) at a wavelength of 450 nm.

[0047] Since the MHd material needs to be attached to the wound surface subsequently, therefore, we evaluated the biocompatibility of this material. The CCK8 detection results showed that at 24 h, 48 h, and 72 h of culture, the absorbance values (OD values) of the control group and the MHd material group were almost the same, without statistical difference, indicating that the MHd material had no effect on cell activity. And with the increase of the culture time, the absorbance value (OD value) of the cells at 72 h increased significantly, proving that the MHd material did not hinder the proliferation of cells ( Figure 9 ). The above results proved that the MHd material had good biocompatibility and could meet the requirements of subsequent in vivo animal experiments.

[0048] Example 8: In vivo animal detection of the MHd material promoting the healing of infected wounds in diabetic mice In animal experiments, a wound infection model of diabetic mice was established. All C57 mice (6 weeks old, male) were raised in a constant-temperature animal room for 1 week after purchase. Streptozotocin (STZ) was dissolved in citric acid buffer (0.1 M, pH 4.3) to prepare a 1% STZ solution, which was stored in the dark. One day before injection, the mice were fasted but given water. The mice were intraperitoneally injected with the STZ solution at a dose of 100 mg / kg body weight for 5 consecutive days. One week later, the blood glucose levels of the mice were measured. Diabetic mice with successful modeling were anesthetized, depilated, and disinfected, and a full-thickness skin defect wound with a diameter of 6 mm was created on the back. The prepared Staphylococcus aureus cultured to the logarithmic phase (10 8 CFU / mL, 20 µL) was dropped into the wound site to create an infected wound (recorded as -1 day). A 3M transparent film was attached to the wound, and an elastic bandage was used to fix it. The mice were returned to normal feeding. One day later, the mice were randomly divided into 2 groups: the control group and the MHd material group. In the MHd material group, the prepared MHd material was applied to the wound, placed in a high-frequency alternating magnetic field, and the temperature of the MHd material was observed in real time by infrared imaging. The MHd material was heated to 50 °C and maintained for 20 min for magnetic hyperthermia intervention; in the control group, the wound was covered with a vaseline gauze and treated with a high-frequency alternating magnetic field with the same parameters. After the intervention, a 3M transparent film was attached to the wound, and an elastic bandage was used to fix it. The MHd material was replaced and the intervention was repeated on the 3rd, 6th, 9th, and 12th days after the experiment. Before each intervention, a photo was taken to record and evaluate the wound healing situation.

[0049] As Figure 10 shown, the results of animal experiments showed that the wound healing situation in the MHd material group was better. The wound was completely healed on the 12th day, and the wound healing rate reached 98.4% on the 9th day; in contrast, the wound healing rate in the control group was only 75.5% on the 12th day; on the 3rd day, the wound healing rate in the MHd material group reached 76.7%, even exceeding the healing rate of the control group on the 12th day. This indicates that the MHd material can effectively promote the repair of diabetic infected wounds by combining the magnetic hyperthermia effect with the synergistic bactericidal performance of TA and providing continuous contractile force through thermosensitive contraction. In addition, it can be seen from the gross view of the wound that there are obvious differences in the wound state between the MHd material group and the control group. During the entire healing period, the wounds in the control group were light yellow and had more exudate. On the 3rd day, obvious bacterial biofilms could even be seen; while starting from the 3rd day (two days after the first intervention), in addition to the healing speed far exceeding that of the control group, the wound state in the MHd material group became light pink and had less exudate, indicating that the 50 °C intervention effectively reduced the bacterial load that inhibited wound healing. It can be seen that for infected wounds, reducing or solving the infection problem is crucial for improving the healing effect.

[0050] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, preferred embodiments of the present invention are described.

[0051] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.

Claims

1. A magnetic hydrogel material having both bactericidal and temperature-sensitive shrinkage properties, characterized in that: Taking PNIPAm copolymerized PAm double network hydrogel as a matrix, grafting nanoclay on the matrix to form a copolymerized double network hydrogel with temperature-sensitive shrinkage performance, and doping magnetic material and tannic acid in the copolymerized double network hydrogel with temperature-sensitive shrinkage performance to obtain the magnetic hydrogel material; The PNIPAm copolymerized PAm double network hydrogel is formed by free radical polymerization of isopropyl acrylamide and acrylamide.

2. The magnetic hydrogel material according to claim 1, characterized in that: The magnetic material is ferroferric oxide.

3. The method for preparing the magnetic hydrogel material according to any one of claims 1 to 2, characterized in that: The following steps are involved: dissolving isopropyl acrylamide, a catalyst, acrylamide, a cross-linking agent, nanoclay, a magnetic substance and tannic acid in water to prepare a uniform precursor solution; In a protective gas atmosphere, an initiator and an accelerator are added to the anterior body fluid and mixed evenly, and polymerized at 4-7° C. for 12-16 hours to obtain the magnetic hydrogel material.

4. The preparation method according to claim 3, characterized in that: In the precursor liquid, the mass concentrations of isopropyl acrylamide, catalyst, acrylamide, crosslinking agent, nanoclay, magnetic substance and tannic acid are 8%-12%, 0.2%-0.5%, 1%-3%, 0.1%-0.5%, 3%, 15% and 1% respectively.

5. The preparation method according to claim 3, characterized in that: The catalyst is sodium pyrophosphate, the cross-linking agent is N,N-methylenebisacrylamide, the initiator is tetramethylethylenediamine, and the accelerator is an ammonium persulfate solution with a mass concentration of 28% to 32%.

6. The preparation method according to claim 3, characterized in that: The volume ratio of the water, the initiator and the accelerator is 1000:1-2.5:4-6.

7. Use of the magnetic hydrogel material according to any one of claims 1 to 2 in the preparation of sterilization products.

8. The use according to claim 7, characterized in that: The sterilization includes killing Staphylococcus aureus and killing Escherichia coli.

9. Use of the magnetic hydrogel material according to any one of claims 1 to 2 in preparing a dressing for promoting the repair of infectious diabetic wounds.

10. A dressing for promoting the repair of infectious diabetic wounds, characterized in that: The dressing comprises the magnetic hydrogel material according to any one of claims 1 to 2.

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