CN115006546A - Disulfide heterojunction material for promoting wound healing and preparation method thereof - Google Patents
Disulfide heterojunction material for promoting wound healing and preparation method thereof Download PDFInfo
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- C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
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Abstract
The invention discloses a double-sulfur heterojunction material for promoting wound healing and a preparation method thereof, wherein the double-sulfur heterojunction material comprises bismuth sulfide, copper sulfide, polydopamine and oxidase; the preparation method mainly comprises the three steps of preparing bismuth sulfide, preparing a bismuth sulfide copper sulfide heterojunction and loading oxidase on the bismuth sulfide copper sulfide heterojunction. The invention provides a disulfide heterojunction material, under near-infrared irradiation, local hyperpyrexia can be generated, the permeability of bacterial cell membranes is changed, and bacterial organelles or cytoplasm matrixes leak in a short time, so that bacteria die, and the action time of the process is short, so that the adaptation time of the bacteria is short, and drug resistance is not easy to generate; meanwhile, a large amount of active oxygen can be generated under the irradiation of near infrared light, pathogenic bacteria can be effectively killed, and a good antibacterial effect is achieved.
Description
Technical Field
The invention relates to the field of medical materials, in particular to a disulfide heterojunction material for promoting wound healing and a preparation method thereof.
Background
The skin is the largest organ of the human body, has the functions of protecting, excreting, regulating the body temperature and the like, and is the first defense line for preventing the invasion of pathogenic microorganisms such as bacteria, viruses and the like. Once the skin has a wound, wound infection may occur and healing may be difficult for a long time. Currently, debridement and antibiotic combination therapy are mainly used for clinically treating wound infection. However, excessive use of antibiotics may lead to the development of resistant bacteria and the possibility of recurrence of wound infection, resulting in difficult wound healing and endangering human health. Therefore, the preparation of the material which can effectively kill bacteria, does not generate drug resistance and can promote wound healing has important significance.
Hydrogen sulfide has been reported to promote angiogenesis and endothelial cell growth and migration. At present, inorganic sodium sulfide and sodium hydrosulfide are mainly used for preparing hydrogen sulfide, but the sodium sulfide and the sodium hydrosulfide have strong biological toxicity and no bacteriostatic action. Therefore, it is very important to select a hydrogen sulfide source which has good biocompatibility and antibacterial effect. Bismuth sulfide is a sea urchin-shaped metal sulfide, has more photocatalytic sites and good biocompatibility, has been applied to the fields of photocatalysis and the like, but has poor photo-thermal performance and low antibacterial capability of single bismuth sulfide. Copper sulfide is spherical metal sulfide, has good photo-thermal and photodynamic properties, can generate local hyperpyrexia and a large amount of active oxygen under the irradiation of near infrared light, and has proved to have remarkable effect of killing bacteria, but the single copper sulfide has stronger biological toxicity and larger damage to normal cells.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide the disulfide heterojunction material for promoting wound healing and the preparation method thereof.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a disulfide heterojunction material for promoting wound healing comprises bismuth sulfide, copper sulfide, polydopamine, and oxidase.
Further, the oxidase is lactate oxidase or glucose oxidase.
A preparation method of a disulfide heterojunction material for promoting wound healing comprises the following steps:
step 1: preparation of bismuth sulfide
Mixing a certain amount of bismuth nitrate pentahydrate and L-cysteine, and adding deionized water for dissolving; putting the solution into a hydrothermal kettle for reaction, taking out the solution after the reaction, centrifuging the solution to obtain a precipitate, and drying the precipitate to obtain bismuth sulfide;
step 2: preparation of bismuth sulfide copper sulfide heterojunction
S21: copper chloride and bismuth sulfide are mixed according to the mass ratio of bismuth element to copper element of 1: 0.5-1, adding water for dissolving, and then adding a surfactant;
s22: adjusting the pH value of the solution to 9.0 by using a sodium hydroxide solution, adding a reducing agent while stirring, and reacting for 5 minutes;
s23: slowly adding a sodium sulfide solution into the solution obtained in the step S22, carrying out hydrothermal reaction to obtain a turbid solution, centrifuging, and drying the precipitate to obtain a bismuth sulfide copper sulfide heterojunction, namely the disulfide metal heterojunction powder;
and step 3: loading of oxidase onto bismuth sulfide copper sulfide heterojunction
S31: weighing a certain amount of dopamine hydrochloride to dissolve in a Tris solution (with the pH value of 8.6);
s32: immersing the disulfide metal heterojunction into the solution prepared by S31, placing the disulfide metal heterojunction into a constant-temperature shaking table for reaction, centrifuging to remove supernatant after the reaction is finished, and keeping the precipitate;
s33: dripping oxidase into the precipitate obtained in the step S32, and then carrying out freeze drying to obtain a disulfide heterojunction material; the oxidase is lactate oxidase or glucose oxidase.
Further, the centrifugation time in step 1 and step 2 is 10 minutes, and the rotation speed is 8000 rpm.
Further, 480mg of bismuth nitrate pentahydrate, 280mg of L-cysteine, 10mL of deionized water and 150 ℃ of a hydrothermal reaction kettle are added in the step 1, and the reaction is carried out for 12 hours.
Further, the dosage of the copper chloride in the step 2 is 0.5mM, and the amount of water for dissolving is 25 mL; the amount of sodium hydroxide was 1mM and the amount of sodium sulfide solution was 0.5 mM.
Further, the surfactant in the step 2 is PVP, and the adding amount is 0.24 g; the reducing agent was hydrazine hydrate and was added in an amount of 64. mu.L.
Further, the temperature of the hydrothermal reaction in the step 2 is 75 ℃, and the reaction lasts for 2 hours.
Further, the pH value of the Tris solution in the step 3 is 8.6, the concentration of the dopamine hydrochloride solution is 2mg/mL, and the concentration of the oxidase dropwise added is 1 mg/mL.
Further, the temperature of the constant temperature shaking table in the step 3 is controlled at 37 ℃, and the reaction time is 24 hours.
In the process of preparing the bismuth sulfide copper sulfide heterojunction, cuprous oxide microspheres are firstly generated on the surface of bismuth sulfide by adjusting pH and adding hydrazine hydrate, then sodium sulfide is slowly added, when sulfur ions attack copper ions, oxygen ions overflow the microsphere structure, the oxygen ion overflow speed is higher in the process, and thus a hollow nano microsphere structure is generated. By controlling the reaction time, copper sulfide and cuprous sulfide microspheres with different mass ratios can be generated on the surface of the bismuth sulfide, and the photo-thermal and photo-dynamic performances are enhanced due to simple substance junctions formed between the copper sulfide and the cuprous sulfide. In addition, the copper sulfide and the cuprous sulfide on the surface of the bismuth sulfide are of a microsphere structure, so that the bismuth sulfide and copper sulfide heterojunction has a larger specific surface area, and the catalytic activity is enhanced.
According to the invention, a one-pot method is adopted to synthesize the bismuth sulfide copper sulfide heterojunction in situ, heterojunction powder is soaked in a polydopamine solution to improve the biocompatibility of the heterojunction and change the surface potential, then, an oxidase (lactate oxidase or glucose oxidase) solution is dripped into the dopamine-coated heterojunction, and the oxidase is loaded on the surface of the heterojunction through electrostatic interaction, so that the double-sulfur heterojunction material with obvious antibacterial effect and obvious effect of promoting wound healing is obtained.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention provides a disulfide heterojunction material, under near-infrared light irradiation, local hyperpyrexia can be generated, the permeability of bacterial cell membranes is changed, and bacterial organelles or cytoplasm matrixes leak in a short time, so that bacteria die; meanwhile, a large amount of active oxygen can be generated under the irradiation of near infrared light, pathogenic bacteria can be effectively killed, and a good antibacterial effect is achieved.
(2) When the disulfide heterojunction material prepared by the invention is used, hydrogen sulfide can be generated in an infected microenvironment, so that the generation of blood vessels at wounds and the proliferation and migration of endothelial cells are promoted, and the rapid healing of the wounds is effectively promoted.
(3) The bismuth sulfide copper sulfide heterojunction provided by the invention is loaded with oxidase (lactate oxidase or glucose oxidase), so that the generation of hydrogen peroxide can be promoted, and the hydrogen peroxide can generate a large amount of active oxygen under the catalysis of the double-sulfur heterojunction, so that bacteria can be killed more effectively.
(4) The preparation method adopts a one-pot method, avoids the separation process in the post-treatment process and the purification process of the intermediate compound, greatly improves the reaction efficiency, saves the preparation time and simplifies the preparation operation.
Drawings
FIG. 1 is an SEM representation of bismuth sulfide, copper sulfide and bismuth sulfide copper sulfide heterojunctions according to the present invention;
FIG. 2 is a photothermal image of a dual-sulfur heterojunction material of the invention;
FIG. 3 is a photo-thermal stability analysis of a bismuth sulfide copper sulfide heterojunction of the present invention;
FIG. 4 is a graph of the photodynamic performance of a dual sulfur heterojunction material of the present invention;
FIG. 5 is a graph of the antibacterial performance of the dual-sulfur heterojunction material of the present invention;
FIG. 6 is an electron microscope scanning image of the morphology of bacteria treated with the disulfide heterojunction material of the present invention;
FIG. 7 is a graph of wound healing promotion performance of the disulfide heterojunction material of the invention.
Detailed Description
The present invention will be further described with reference to the following description and examples, which include but are not limited to the following examples.
The invention provides a double-sulfur heterojunction material for promoting wound healing, which comprises bismuth sulfide, copper sulfide, polydopamine solution and loaded enzyme, wherein the loaded enzyme is lactate oxidase or glucose oxidase.
Example 1
The embodiment provides a preparation method of a double-sulfur heterojunction material for promoting wound healing, which comprises the following steps:
step 1: preparation of bismuth sulfide
Mixing 480mg of pentahydrate bismuth nitrate and 280mg of L-cysteine, adding 10mL of deionized water for dissolving, transferring the solution into a hydrothermal kettle at 150 ℃ for reaction for 12 hours, taking out after the reaction is finished, centrifuging for 10 minutes at 8000rpm, retaining the precipitate, and drying to obtain bismuth sulfide.
Step 2: preparation of bismuth sulfide copper sulfide heterojunction
S21: 0.5mM of copper chloride and bismuth sulfide are added according to the mass ratio of bismuth element to copper element of 1: 1, mixing, adding 25mL of water for dissolving, and adding 0.24g of PVP (polyvinyl pyrrolidone) as a surfactant;
s22: adjusting the pH to 9.0 with 1mM sodium hydroxide solution, adding 64 μ L hydrazine hydrate while stirring, and reacting for 5 minutes;
s23: and (4) slowly adding 0.5mM sodium sulfide solution into the solution obtained in the step S22, reacting for 2h at 75 ℃ to generate copper sulfide on the surface of the bismuth sulfide in situ to obtain turbid solution, centrifuging for 10 min at 8000rpm to obtain precipitate, and drying to obtain bismuth sulfide copper sulfide heterojunction powder. (as shown in FIG. 1, wherein A is bismuth sulfide, B is copper sulfide, and C is SEM representation of a disulfide metal heterojunction.)
And step 3: preparation of disulfide biological heterojunction material
S31: weighing a certain amount of dopamine hydrochloride to dissolve in a Tris solution with the pH value of 8.6 to obtain a dopamine hydrochloride solution with the concentration of 2 mg/mL;
s32: and (3) immersing a proper amount of bismuth sulfide and copper sulfide heterojunction powder into the solution prepared in the step S31, placing the solution into a constant-temperature shaking table at 37 ℃ for reaction for 24 hours, centrifuging after the reaction is finished, keeping a precipitate, dripping 1mg/mL of lactate oxidase solution into the solution, and freeze-drying to obtain the disulfide heterojunction material capable of promoting wound healing.
The prepared disulfide heterojunction material is subjected to photo-thermal performance experiments, photodynamic performance experiments, bacteriostatic experiments, bacterial morphology tests and animal experiments.
Comparative example: preparing copper sulfide: 0.5mM CuCl was taken 2 ·2H 2 Adding 25mL of water into O for dissolving, adding 0.24g of PVP as a surfactant, adjusting the pH to 9.0 by using 1mM of sodium hydroxide solution, adding 64 mu L of hydrazine hydrate as a reducing agent while stirring, slowly adding sodium sulfide solution (0.5mM) after reacting for 5 minutes, reacting for 2 hours at 75 ℃ so as to generate copper sulfide on the surface of bismuth sulfide in situ, centrifuging the obtained turbid solution (8000rpm for 10 minutes), and drying the obtained precipitate to obtain the copper sulfide.
(1) Photothermal performance test
Experiment 1: the bismuth sulfide copper sulfide heterojunction prepared in the example, the bismuth sulfide copper sulfide heterojunction loaded with lactate oxidase, and the copper sulfide in the comparative example were placed in 48-well plates, and a Phosphate Buffered Saline (PBS) without adding any material was used as a control, and 500 μ L of PBS was added to each sample well plateUsing 808nm near infrared light (1.5W/cm) 2 ) Each sample well plate was irradiated and the temperature change was captured every 30 seconds using a FLIR infrared detector. The results are shown in FIG. 2, where PBS represents phosphate buffer, BC represents bismuth sulfide copper sulfide heterojunction, and BCPL represents lactate oxidase-loaded disulfide heterojunction.
Experiment 2: the bismuth sulfide copper sulfide heterojunction prepared in the embodiment is placed in a 48-hole plate, 500 mu L PBS is added into the sample hole plate, the sample hole plate is irradiated by 808nm near infrared light for 15 minutes, then the infrared irradiation lamp is turned off and cooled for 15 minutes, a FIRT infrared detector is used for capturing temperature change every 30 seconds, the cycle operation is carried out for three times, data are recorded, and the photo-thermal stability of the sample is analyzed, and the result is shown in figure 3.
To summarize: from fig. 2, it can be known that copper sulfide, bismuth sulfide and a disulfide heterojunction loading lactate oxidase can achieve local hyperpyrexia under near-infrared light irradiation, wherein the temperature of copper sulfide rises faster in a shorter time, so that skin is burned, the temperature of bismuth sulfide rises slower, and the rising speed of the disulfide heterojunction loading lactate oxidase is between the two, so that the temperature is easier to control; as can be seen from fig. 3, the bismuth sulfide copper sulfide heterojunction has good photo-thermal cycling capability, provides the possibility of recycling, promotes environmental friendliness, and is also beneficial to photo-thermal antibacterial stability.
(2) Experiment of photodynamic Properties
The bismuth sulfide, bismuth sulfide copper sulfide heterojunction prepared in the example, and the lactate oxidase-loaded disulfide heterojunction and the copper sulfide in the comparative example were placed in a 48-well plate, in which Phosphate Buffered Saline (PBS) without a material added thereto was used as a control, 0.5mL of 100 μ g/mL Methylene Blue (MB) was used as an active oxygen (· OH) marker, and each group was placed in near-infrared light (1.5W/cm) under dark conditions 2 ) The sample was irradiated for 15 minutes, and 100. mu.L of the solution was measured for absorbance in a microplate reader at intervals of 5 minutes, and the results of the measurement are shown in FIG. 4.
To summarize: the disulfide heterojunction material loading the lactate oxidase degrades most methylene blue, has the strongest capacity of generating hydroxyl radicals by combining with oxygen-containing substances, and can achieve excellent antibacterial effect.
(3) Experiment for inhibiting bacteria
Experiment 1: the bismuth sulfide, bismuth sulfide copper sulfide heterojunction and lactate oxidase-loaded disulfide heterojunction prepared in this example and copper sulfide in the comparative example were placed in 48-well plates, in which Phosphate Buffered Saline (PBS) without material was used as a control, and 100 μ L of liquid medium and 100 μ L of 106CFU/mL staphylococcus aureus (s.aureus) were added to each sample well, and 3 parallel experimental groups were set up. Culturing the components under 808nm laser irradiation and dark condition for 20 min, uniformly coating the bacteria liquid on a solid culture medium, culturing at 37 ℃ for 24h, and observing the sterilization effect of various materials.
Experiment 2: 106CFU/mL Staphylococcus aureus (S.aureus) in experiment 1 above was changed to 10 6 Experiment 1 was repeated with CFU/mL E.coli (E.coli).
The results of experiment 1 and experiment 2 are shown in fig. 5, in which NIR (+) indicated near-infrared light irradiation and NIR (-) indicated no near-infrared light irradiation.
To summarize: the double-sulfur heterojunction loading the lactate oxidase kills most of staphylococcus aureus and escherichia coli under the irradiation of near-infrared light, so that the problem of wound infection can be effectively solved.
(4) Bacterial morphology testing
Experiment 1: the bismuth sulfide, bismuth sulfide copper sulfide heterojunction prepared in this example, bismuth sulfide copper sulfide heterojunction loaded with lactate oxidase, and copper sulfide in comparative example were placed in a 48-well plate, wherein Phosphate Buffered Saline (PBS) without material was used as a control, and 100 μ L of liquid medium and 100 μ L of 106CFU/mL staphylococcus aureus (s.aureus) were added to each sample well, and 3 parallel experimental groups were set up. After culturing the components under 808nm laser irradiation and in the dark for 20 minutes, centrifuging the sample, taking out the sample, placing the sample in 500 mu L of 2.5% glutaraldehyde, storing the sample at 4 ℃ for 12 hours, dehydrating the sample by gradient ethanol (30%, 50%, 70%, 90% and 100% v/v) for 10 minutes in each gradient, and observing the bacterial morphology by a scanning electron microscope after the sample is naturally air-dried.
Experiment 2: 106CFU/mL of the above-described golden grape of experiment 1 was addedThe coccus (S.aureus) was changed to 10 6 Experiment 1 was repeated with CFU/mL E.coli (E.coli).
To summarize: the experimental result is shown in fig. 6, the lactate oxidase-loaded disulfide heterojunction can destroy the integrity of a bacterial membrane, and cause the leakage of bacterial organelles or cytoplasmic matrixes, so that bacteria die, the action time is short, the action effect is remarkable, and therefore, the adaptation time of the bacteria is short, and the drug resistance is not easy to generate.
(5) Animal experiments
Establishing a wound infection model: the mice were grouped and numbered, depilated on the back, anesthetized by injecting 5% chloral hydrate (0.1mL/10g) into the abdominal cavity, a full-thickness skin wound of about 1 cm in diameter was created 2cm outside the spinal column of the mice, and 10. mu.L of Staphylococcus aureus (1X 10) solution was added dropwise to the mice using a pipette gun 8 CFU/mL), and finally, a special component free woundplast or sterile gauze wrapped to prevent the mouse from licking or scratching the wound.
Wound treatment analysis: infection model a photograph of the wound was taken 24 hours after establishment and the length and width of the wound was measured with a ruler. Placing materials (bismuth sulfide, bismuth sulfide copper sulfide heterojunction, lactate oxidase-loaded double-sulfur heterojunction, and PBS) at wound according to the components, wherein the receiving intensity of all illumination groups is 1.5W/cm 2 808nm for 10 minutes. The wound was exposed to air after the light was applied. Wounds were recorded every two days for the following eight days,
the results are shown in FIG. 7, where Bi 2 S 3 + represents irradiation of bismuth sulfide and near infrared light, BC + represents irradiation of bismuth sulfide and copper sulfide heterojunction and near infrared light, and BCPL + represents irradiation of double-sulfur heterojunction loaded with lactate oxidase and near infrared light; bi 2 S 3 And BC and BCPL mean that no near-infrared light irradiation is performed.
To summarize: the wound recovery speed is fastest and the wound area is smallest after the treatment of the lactate oxidase-loaded double-sulfur heterojunction material and the illumination. In contrast, the mice wounds also had pus exudation and significant inflammation in the PBS-treated placebo group. Under the synergistic effect of photo-heat and photodynamic, the material prepared by the embodiment has a remarkable antibacterial effect, the inflammatory reaction of the wound is reduced, and meanwhile, the generated hydrogen sulfide promotes angiogenesis and endothelial cell proliferation and migration, so that the wound healing is rapidly promoted.
Example 2
Based on the preparation method of the biological heterojunction material for promoting wound healing provided in example 1, in step 2, the 0.5mM copper chloride and bismuth sulfide can be further added according to the mass ratio of bismuth element to copper element of 1: 0.75, and then 25mL of water was added to dissolve.
Example 3
Based on the preparation method of the biological heterojunction material for promoting wound healing provided in example 1, in step 2, the 0.5mM copper chloride and bismuth sulfide can be further added according to the mass ratio of bismuth element to copper element of 1: 0.5, and then 25mL of water was added to dissolve.
The above-mentioned embodiment is only one of the preferred embodiments of the present invention, and should not be used to limit the scope of the present invention, but all the insubstantial modifications or changes made within the spirit and scope of the main design of the present invention, which still solve the technical problems consistent with the present invention, should be included in the scope of the present invention.
Claims (10)
1. A disulfide heterojunction material for promoting wound healing, comprising bismuth sulfide, copper sulfide, polydopamine, and oxidase.
2. The disulfide heterojunction material of claim 1 wherein said oxidase is lactate oxidase or glucose oxidase.
3. A preparation method of a disulfide heterojunction material for promoting wound healing is characterized by comprising the following steps:
step 1: preparation of bismuth sulfide
Mixing a certain amount of bismuth nitrate pentahydrate and L-cysteine, and adding deionized water for dissolving; putting the solution into a hydrothermal kettle for reaction, taking out the solution after the reaction, centrifuging the solution to obtain a precipitate, and drying the precipitate to obtain bismuth sulfide;
step 2: preparation of bismuth sulfide copper sulfide heterojunction
S21: copper chloride and bismuth sulfide are mixed according to the mass ratio of bismuth element to copper element of 1: 0.5-1, adding water to dissolve, and adding a surfactant;
s22: adjusting the pH value of the solution to 9.0 by using a sodium hydroxide solution, adding a reducing agent while stirring, and reacting for 5 minutes;
s23: slowly adding a sodium sulfide solution into the solution obtained in the step S22, carrying out hydrothermal reaction to obtain a turbid solution, centrifuging, and drying the precipitate to obtain a bismuth sulfide and copper sulfide heterojunction, namely the disulfide metal heterojunction powder;
and 3, step 3: loading of oxidase onto bismuth sulfide copper sulfide heterojunction
S31: weighing a certain amount of dopamine hydrochloride to dissolve in a Tris solution;
s32: immersing the disulfide metal heterojunction into the solution prepared by S31, placing the disulfide metal heterojunction into a constant-temperature shaking table for reaction, centrifuging to remove supernatant after the reaction is finished, and keeping the precipitate;
s33: dripping oxidase into the precipitate obtained in the step S32, and then freeze-drying to obtain a disulfide heterojunction material; the oxidase is lactate oxidase or glucose oxidase.
4. The method for preparing a disulfide heterojunction material for promoting wound healing as claimed in claim 3, wherein the centrifugation time in step 1 and step 2 is 10 minutes, and the rotation speed is 8000 rpm.
5. The method for preparing a disulfide heterojunction material for promoting wound healing according to claim 3, wherein 480mg of bismuth nitrate pentahydrate and 280mg of L-cysteine are used in step 1, the volume of deionized water is 10mL, the temperature of a hydrothermal reaction kettle is 150 ℃, and the reaction is carried out for 12 h.
6. The method for preparing a disulfide heterojunction material for promoting wound healing according to claim 5, wherein in the step 2, the dosage of cupric chloride is 0.5mM, and the water for dissolution is 25 mL; the amount of sodium hydroxide was 1mM and the amount of sodium sulfide solution was 0.5 mM.
7. The method for preparing a disulfide heterojunction material for promoting wound healing according to claim 6, wherein the surfactant in step 2 is PVP and is added in an amount of 0.24 g; the reducing agent was hydrazine hydrate and was added in an amount of 64. mu.L.
8. The method for preparing a disulfide heterojunction material for promoting wound healing according to claim 3, wherein the hydrothermal reaction in step 2 is carried out at 75 ℃ for 2 h.
9. The method for preparing a disulfide heterojunction material for promoting wound healing according to claim 8, wherein the Tris solution in step 3 has a pH of 8.6, the dopamine hydrochloride solution has a concentration of 2mg/mL, and the oxidase is added dropwise at a concentration of 1 mg/mL.
10. The method for preparing a disulfide heterojunction material for promoting wound healing as claimed in claim 3, wherein the temperature of said constant temperature shaking table in step 3 is controlled at 37 ℃ and the reaction time is 24 h.
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