Magnetic heat responsive nanoparticles, and methods of making and using the same

Abstract

The present application relates to a kind of magnetocaloric response nanoparticles and its preparation method and application, belong to the field of biological medical technology.The present application provides a kind of preparation method of magnetocaloric response nanoparticles, specifically the mixture of iron trichloride hexahydrate, polyacrylic acid, urea, ethylene glycol and water, after stirring reaction, iron oxide nanocluster is obtained by centrifugation;Iron oxide nanocluster and dopamine are mixed in water and reacted, and then centrifuged to obtain the reactant;The reactant is mixed with water and mixed with the solution of baphlavin, incubated and centrifuged to obtain magnetocaloric response nanoparticles.The present application couples the magnetic nanomaterial with high magnetocaloric conversion efficiency and natural product active monomer, constructs a kind of magnetocaloric response nanoparticles, can produce heat under the condition of alternating magnetic field, improves bactericidal effect, also provides additional anti-inflammatory and immunoregulatory activity, to promote the rapid healing of infected wound.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a magnetocaloric responsive nanoparticle, its preparation method, and its application. Background Technology

[0002] Infected wound healing remains a significant global public health problem, posing a substantial medical and economic burden. Deep tissue injuries are particularly dangerous, often developing into chronic, intractable ulcers due to bacterial colonization, excessive inflammation, and dysregulation of repair, leading to a higher risk of death. Biofilms are organized aggregates of microorganisms. Bacteria irreversibly attach to the surface of inert or active entities, multiply, differentiate, and secrete polysaccharide matrixes that encapsulate the bacterial community, forming a membrane-like bacterial aggregate. Antibiotics are the traditional treatment for bacterial infections; however, due to antibiotic overuse, traditional antibiotic therapy is increasingly plagued by drug resistance.

[0003] Physical therapies, such as thermotherapy (photothermal therapy, magnetothermal therapy, etc.), exhibit broad-spectrum antibacterial activity against various pathogens (even antibiotic-resistant bacteria and bacteria in biofilms), with short treatment times and negligible bacterial resistance, showing increasing potential in treating bacterial infections. Among them, magnetothermal therapy mediated by magnetic nanomaterials has received widespread attention in promoting the healing of infected wounds. The heat generation principle of magnetothermal therapy mediated by magnetic nanomaterials is to convert the electromagnetic energy of an external magnetic field into heat energy of the particles themselves and their surrounding environment through magnetic relaxation (relaxation loss) or hysteresis loss. However, considering that the healing of infected wounds is a complex pathophysiological process that requires close coordination of antibacterial activity, inflammation control, cell migration, and angiogenesis, and that existing magnetothermal healing-promoting drugs (excipients) still have problems such as poor heat generation, resulting in poor antibacterial effects; weak anti-inflammatory and angiogenesis-promoting abilities, which are not conducive to wound healing.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a magnetothermal responsive nanoparticle, its preparation method, and its application, so as to solve the problem of poor heat generation and antibacterial effects of existing magnetothermal healing-promoting drugs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for preparing magnetocalorically responsive nanoparticles, comprising the following steps: (1) Mix ferric chloride hexahydrate, polyacrylic acid, urea, ethylene glycol and water, stir for 55-65 min to obtain a mixed solution, react at 160-200℃ for 5-7 h, and centrifuge to purify to obtain iron oxide nanoclusters; (2) Iron oxide nanoclusters and dopamine were mixed and dissolved in water, and reacted at a temperature of 35~55℃ for 3~5h. The precipitate was collected by centrifugation to obtain the reactants. (3) Mix the reactants with water to obtain a reactant solution, mix the reactant solution with shikonin solution, incubate for 10-14 hours, and centrifuge to collect the precipitate to obtain magnetothermal response nanoparticles.

[0007] Preferably, the mass ratio of ferric chloride hexahydrate, polyacrylic acid and urea in step (1) is 52~56:23~27:110~130; The mass-to-volume ratio of ferric chloride hexahydrate to ethylene glycol is 52-56 mg: 2 mL; The volume ratio of ethylene glycol to water is 2:0.10~0.20.

[0008] Preferably, the mass ratio of iron oxide nanoclusters to dopamine in step (2) is 1~3:1.

[0009] Preferably, the mass-to-volume ratio of the reactant to water in step (3) is 100-200 mg: 3-5 mL; The solvent of the shikonin solution is dimethyl sulfoxide, and the concentration of the shikonin solution is 1~3 mg / mL; The volume ratio of the reactant solution to the shikonin solution is 3-5:1.

[0010] Preferably, the incubation speed in step (3) is 100~200 rpm; the incubation temperature is 35~39℃.

[0011] This invention provides magnetocaloric responsive nanoparticles prepared by the aforementioned preparation method.

[0012] This invention provides the application of the aforementioned magnetocaloric responsive nanoparticles in the preparation of drugs for wound repair.

[0013] This invention provides a composite hydrogel for wound repair, the composite hydrogel comprising the following components in parts by weight: Polyvinyl alcohol 230-250 parts, gelatin 50-70 parts, magnetothermal responsive nanoparticles 300-600 parts, borax aqueous solution 100-1000 parts, and water 3000-5000 parts; The concentration of the borax aqueous solution is 1~3wt%; The magnetocaloric responsive nanoparticles are the magnetocaloric responsive nanoparticles described above.

[0014] This invention provides a method for preparing the composite hydrogel, which involves mixing polyvinyl alcohol, gelatin, magnetothermal responsive nanoparticles, borax, and water.

[0015] This invention provides the application of the composite hydrogel described above or the composite hydrogel prepared by the method described above in wound repair.

[0016] The present invention has the following technical effects and advantages: This invention constructs an iron oxide nanocluster with high saturation magnetization, and then modifies the surface of the iron oxide nanocluster with dopamine to give it amino groups, enabling it to react with shikonin to form magnetocalorically responsive nanoparticles loaded with shikonin. Shikonin itself has antibacterial effects; loading it onto the iron oxide nanocluster significantly enhances its antibacterial efficacy. Furthermore, under an alternating magnetic field, it generates heat, further improving the bactericidal effect, and provides additional anti-inflammatory and immunomodulatory activities, thereby promoting rapid healing of infected wounds. Attached Figure Description

[0017] Figure 1 Microscopic observation results of magnetocaloric responsive nanoparticles; Figure 2 These are the experimental results regarding the properties of the composite hydrogel; Figure 3 Bacterial culture results for different treatment groups; Figure 4 Results of bacterial biofilm culture staining in different treatment groups; Figure 5 Thermal imaging results of skin defects in mice; Figure 6 Results of plate application of wound exudate at different time points; Figure 7 The results show the blood flow in the wound area of ​​mice in different treatment groups. Detailed Implementation

[0018] This invention provides a method for preparing magnetocalorically responsive nanoparticles, comprising the following steps: (1) Mix ferric chloride hexahydrate, polyacrylic acid, urea, ethylene glycol and water, stir for 55-65 min to obtain a mixed solution, react at 160-200℃ for 5-7 h, and centrifuge to purify to obtain iron oxide nanoclusters; The stirring time is preferably 60 min; the reaction temperature is preferably 180℃; and the reaction time is preferably 6 h. (2) Iron oxide nanoclusters and dopamine were mixed and dissolved in water, and reacted at a temperature of 35~55℃ for 3~5h. The precipitate was collected by centrifugation to obtain the reactants. The preferred temperature for the reaction is 45°C; the preferred reaction time is 4 hours. (3) Mix the reactants with water to obtain a reactant solution, mix the reactant solution with shikonin solution, incubate for 10-14 h, and centrifuge to collect the precipitate to obtain magnetothermal response nanoparticles; The incubation time is preferably 12 hours.

[0019] In this invention, the mass ratio of ferric chloride hexahydrate, polyacrylic acid and urea in step (1) is 52~56:23~27:110~130, preferably 54:25:120; The mass-to-volume ratio of ferric chloride hexahydrate to ethylene glycol is 52-56 mg:2 mL, preferably 54 mg:2 mL; The volume ratio of ethylene glycol to water is 2:0.10~0.20, preferably 2:0.15.

[0020] In this invention, the mass ratio of iron oxide nanoclusters to dopamine in step (2) is 1 to 3:1, preferably 2:1.

[0021] In this invention, the mass-to-volume ratio of the reactant to water in step (3) is 100-200 mg: 3-5 mL, preferably 150 mg: 4 mL; The solvent of the shikonin solution is dimethyl sulfoxide, and the concentration of the shikonin solution is 1~3 mg / mL, preferably 2 mg / mL; The volume ratio of the reactant solution to the shikonin solution is 3-5:1, preferably 4:1.

[0022] In this invention, the incubation speed in step (3) is 100~200 rpm, preferably 150 rpm; the incubation temperature is 35~39℃, preferably 37℃.

[0023] This invention provides magnetocaloric responsive nanoparticles prepared by the aforementioned preparation method.

[0024] This invention provides the application of the aforementioned magnetocaloric responsive nanoparticles in the preparation of drugs for wound repair.

[0025] This invention provides a composite hydrogel for wound repair, the composite hydrogel comprising the following components in parts by weight: Polyvinyl alcohol 230-250 parts, gelatin 50-70 parts, magnetothermal responsive nanoparticles 300-600 parts, borax aqueous solution 100-1000 parts, and water 3000-5000 parts; The preferred mass fraction of the polyvinyl alcohol is 240 parts; The preferred mass fraction of the gelatin is 60 parts; The preferred mass fraction of the magnetocaloric responsive nanoparticles is 450 parts; The preferred mass fraction of the borax aqueous solution is 500 parts; The preferred mass fraction of the water is 4000 parts; The concentration of the borax aqueous solution is 1-3 wt%, preferably 2 wt%; The magnetocaloric responsive nanoparticles are the magnetocaloric responsive nanoparticles described above.

[0026] This invention provides a method for preparing the composite hydrogel, which involves mixing polyvinyl alcohol, gelatin, magnetothermal responsive nanoparticles, borax, and water.

[0027] This invention provides the application of the composite hydrogel described above or the composite hydrogel prepared by the method described above in wound repair.

[0028] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0029] Example 1

[0030] A method for preparing magnetocalorically responsive nanoparticles includes the following steps: (1) 540 mg of ferric chloride hexahydrate was dissolved in 20 mL of ethylene glycol under magnetic stirring. Then, 250 mg of polyacrylic acid, 1200 mg of urea and 1.5 mL of deionized water were added and stirred for 60 min to obtain a bright yellow mixed solution. The mixed solution was placed in a polytetrafluoroethylene-lined stainless steel autoclave and reacted at 180 °C for 6 h. The iron oxide nanoclusters were obtained by centrifugation and purification. (2) Mix 100 mg of iron oxide nanoclusters and 50 mg of dopamine in water and react at 45 °C for 4 h to obtain a reaction solution. Centrifuge to collect the precipitate to obtain the reactant. (3) Mix 150 mg of reactant with 4 mL of water to obtain reactant solution. Mix reactant solution with 1 mL of 2 mg / L shikonin solution (solvent is dimethyl sulfoxide). Incubate for 12 h at 150 rpm and 37 °C. Centrifuge to collect precipitate and obtain magnetothermal response nanoparticles.

[0031] Example 2

[0032] A composite hydrogel for wound repair

[0033] Formulation: 240 mg polyvinyl alcohol, 60 mg gelatin, 450 mg magnetocaloric responsive nanoparticles prepared in Example 1, 300 mg of 2 wt% borax solution and 3000 mg of water; Preparation method: 240 mg of polyvinyl alcohol and 60 mg of gelatin were dissolved in 3000 mg of water, and then 450 mg of the magnetothermal responsive nanoparticles prepared in Example 1 were added. 300 mg of 2 wt% borax solution was added for crosslinking to obtain a composite hydrogel.

[0034] Experimental Example 1: Physicochemical Properties of Magnetothermic Nanoparticles

[0035] 1. Structural characteristics

[0036] The magnetocaloric responsive nanoparticles prepared in Example 1 were observed under a microscope. The microscopic observation results of the magnetocaloric responsive nanoparticles are as follows: Figure 1 As shown.

[0037] according to Figure 1 It can be seen that the magnetocaloric responsive nanoparticles prepared in Example 1 have a clustered structure.

[0038] 2. Physicochemical properties

[0039] The saturation magnetization of the magnetocaloric responsive nanoparticles prepared in Example 1 was measured using a superconducting quantum interference device (SQUID). The results showed that the saturation magnetization of the magnetocaloric responsive nanoparticles prepared in Example 1 was 60 emu / g.

[0040] Experimental Example 2: Properties of Composite Hydrogels

[0041] The composite hydrogel was placed in an alternating magnetic field for 10 minutes, and its temperature was observed using a thermal imaging instrument. The temperature of the composite hydrogel was recorded every 1 minute. The experimental results of the properties of the composite hydrogel are as follows: Figure 2 As shown, Figure A represents a thermal image of the composite hydrogel at different times, and Figure B represents a temperature graph of the composite hydrogel at different times.

[0042] according to Figure 2 It can be seen that the composite hydrogel prepared in Example 2 has a good heating effect, and can be heated to above 50°C within 10 minutes.

[0043] Experiment Example 3: Antibacterial Effect Experiment

[0044] 1. Strains

[0045] Activated *Escherichia coli* and *Staphylococcus aureus* were inoculated separately into LB liquid medium. The medium was incubated at 37°C and 200 rpm on a shaker until it reached a density of 1 × 10⁻⁶. 7 CFU (OD) 600 =0.1), to obtain Escherichia coli and Staphylococcus aureus bacterial solutions, respectively.

[0046] 2. Experimental Design

[0047] (1) Colony plating: The obtained bacterial solution was added to sterilized 1.5 mL EP tubes. Six treatments were set up for each type of bacteria: blank control group (Control), shikonin group (SK), blank gel group (Blank gel), iron oxide nanocluster gel group (Mag gel), composite hydrogel group (MagSK gel), and composite hydrogel + alternating magnetic field group (MagSK). The control group (gel+B) was prepared with added physiological saline. The shikonin group was prepared with the same mass (20 μg) of shikonin as the composite gel. The blank gel group was prepared with 100 mg of blank gel (prepared in the same way as the composite hydrogel in Example 2, except that it did not include magnetothermal responsive nanoparticles). The iron oxide nanocluster gel group was prepared with 100 mg of iron oxide nanocluster gel (prepared in the same way as the composite hydrogel in Example 2, except that the iron oxide nanoclusters prepared in step (1) of Example 1 were used to replace the magnetothermal responsive nanoparticles in the composite hydrogel in Example 2). The composite hydrogel group was prepared with 100 mg of the composite hydrogel prepared in Example 2. The composite hydrogel + alternating magnetic field group was prepared with 100 mg of the composite hydrogel prepared in Example 2 and treated with an alternating magnetic field. Each treatment was repeated 3 times. After treatment, the different treatment groups were diluted and plated. After dilution, they were incubated at 37°C for 12 h. Then the number of colonies in the agarose plates of each treatment group was observed, and the bacterial concentration after the original treatment was calculated. The bacterial culture results of different treatment groups are as follows: Figure 3 As shown.

[0048] (2) In vitro anti-biofilm: Staphylococcus aureus and Escherichia coli suspension (1×10⁻⁶) 7 CFU / mL) of bacterial suspension was inoculated into 96-well plates, with 200 µL of bacterial suspension in each well. The plates were incubated at 37°C for 48 hours to form a biofilm. Different treatments were applied to the biofilm-containing bacterial suspensions, followed by another 1 hour of incubation. The 96-well plates were then removed, and the supernatant containing the drug and bacteria that had not yet formed a biofilm was aspirated using an insulin syringe. The supernatant was discarded, and each well was fixed with an appropriate amount of methanol for 15 min. The methanol was then removed, and the plates were air-dried. 100 µL of 0.1% crystal violet solution was added to each well, and staining was performed at room temperature for 15 min. Each well was washed three times with phosphate-buffered saline (PBS) to remove unbound dye. The staining results were observed, and the staining results for different treatment groups are shown below. Figure 4 As shown.

[0049] according to Figure 3 and Figure 4 It is known that shikonin has antibacterial effects. The composite hydrogel prepared by combining iron oxide nanoclusters and shikonin significantly enhances the antibacterial effect of shikonin, and the antibacterial effect is even more pronounced under the action of an alternating magnetic field. This indicates that the composite hydrogel prepared by the method of this application has excellent antibacterial and anti-biofilm properties.

[0050] Experiment Example 5: Experiment on the Effect of Composite Hydrogels

[0051] 1. Construct a mouse model of full-thickness skin defect caused by Staphylococcus aureus infection.

[0052] The specific method was as follows: Kunming mice (7-8 weeks old, female) were used in this animal experiment. After hair removal from the back of each mouse, a circular full-thickness skin wound with a diameter of 8 mm was created using a skin biopsy device. Subsequently, 10 µL of Staphylococcus aureus (concentration of 1×10⁻⁶) was instilled into the wound. 7 Infection was caused by bacterial suspension (CFU / mL). The presence of bacterial exudate from the wound one day after infection indicates successful model construction.

[0053] 2. Experimental Design

[0054] The experiment was divided into a blank control group (Control), a 3M dressing group (3M), a blank hydrogel group (Blank gel), an iron oxide nanocluster gel group (Mag gel), a composite hydrogel group (MagSK gel), and a composite hydrogel + alternating magnetic field group (MagSK gel+B). Specifically, the control group received no wound treatment. In the 3M dressing group (3M), blank hydrogel group (Blank gel), iron oxide nanocluster gel group (Mag gel), and composite hydrogel group (MagSK gel), 3M dressing, blank hydrogel, iron oxide nanocluster gel, and the composite hydrogel prepared in Example 2 were respectively applied to the wound. In the composite hydrogel + alternating magnetic field group (MagSK gel+B), the composite hydrogel was applied to the wound, followed by treatment with an alternating magnetic field for 10 minutes. Skin infection was observed and photographed during the experiment. Mice were sacrificed at predetermined time points, and skin and major organs from each group were collected for immunofluorescence staining and histopathological analysis.

[0055] The temperature at the skin defect site in mice in the composite hydrogel + alternating magnetic field group was observed using a thermal imaging instrument. The thermal imaging results of the mouse skin defect site are as follows: Figure 5 As shown.

[0056] according to Figure 5 It can be seen that the composite hydrogel prepared by the method of this application has a good thermal effect.

[0057] Wound exudate was collected at different time points and plated to assess wound infection clearance. The results of plated exudate collection at different time points are as follows: Figure 6 As shown.

[0058] according to Figure 6 It can be seen that the composite hydrogel + alternating magnetic field treatment effectively removes bacteria accumulated at the wound site.

[0059] Blood flow in the wound area of ​​mice in different treatment groups was assessed using laser Doppler imaging. The results of blood flow measurements in the wound area of ​​mice in different treatment groups are as follows: Figure 7 As shown, Figure A represents a thermal imaging image of blood perfusion, and Figure B represents a bar chart of blood perfusion.

[0060] according to Figure 7 It can be seen that all groups showed a gradual increase in blood perfusion level over time. Quantitative assessment showed that the composite hydrogel + alternating magnetic field treatment group reached the highest perfusion level on day 7, exhibiting a large area of ​​high-intensity blood flow, which is consistent with the improvement in angiogenesis.

[0061] The experimental data obtained in summary show that the composite hydrogel + alternating magnetic field promotes microcirculation through thermal effects, improves the microenvironment of infected wounds, and thus promotes wound healing.

[0062] As shown in the above embodiments, this invention provides magnetocaloric responsive nanoparticles, their preparation method, and applications. This invention constructs an iron oxide nanocluster with high saturation magnetization, and then modifies the surface of the iron oxide nanocluster with dopamine to give it amino groups, enabling it to react with shikonin to form magnetocaloric responsive nanoparticles loaded with shikonin. Shikonin itself has antibacterial effects; loading it onto the iron oxide nanocluster significantly enhances its antibacterial effect. Furthermore, under alternating magnetic field conditions, it generates heat, improving the bactericidal effect, and also provides additional anti-inflammatory and immunomodulatory activities, thereby promoting rapid healing of infected wounds.

[0063] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing magnetocalorically responsive nanoparticles, characterized in that, Includes the following steps: (1) Mix ferric chloride hexahydrate, polyacrylic acid, urea, ethylene glycol and water, stir for 55-65 min to obtain a mixed solution, react at 160-200℃ for 5-7 h, and centrifuge to purify to obtain iron oxide nanoclusters; (2) Iron oxide nanoclusters and dopamine were mixed and dissolved in water, and reacted at a temperature of 35~55℃ for 3~5h. The precipitate was collected by centrifugation to obtain the reactants. (3) Mix the reactants with water to obtain a reactant solution, mix the reactant solution with shikonin solution, incubate for 10-14 hours, and centrifuge to collect the precipitate to obtain magnetothermal response nanoparticles.

2. The preparation method according to claim 1, characterized in that, The mass ratio of ferric chloride hexahydrate, polyacrylic acid and urea in step (1) is 52~56:23~27:110~130; The mass-to-volume ratio of ferric chloride hexahydrate to ethylene glycol is 52-56 mg: 2 mL; The volume ratio of ethylene glycol to water is 2:0.10~0.

20.

3. The preparation method according to claim 1, characterized in that, The mass ratio of iron oxide nanoclusters to dopamine in step (2) is 1~3:

1.

4. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of the reactant to water in step (3) is 100-200 mg: 3-5 mL; The solvent of the shikonin solution is dimethyl sulfoxide, and the concentration of the shikonin solution is 1~3 mg / mL; The volume ratio of the reactant solution to the shikonin solution is 3-5:

1.

5. The preparation method according to claim 1, characterized in that, The incubation speed in step (3) is 100~200 rpm; the incubation temperature is 35~39℃.

6. Magnetothermic responsive nanoparticles prepared by the preparation method according to any one of claims 1 to 5.

7. The use of the magnetocaloric responsive nanoparticles according to claim 6 in the preparation of a wound-healing drug.

8. A composite hydrogel for wound repair, characterized in that, The composite hydrogel comprises the following components in parts by weight: Polyvinyl alcohol 230-250 parts, gelatin 50-70 parts, magnetothermal responsive nanoparticles 300-600 parts, borax aqueous solution 100-1000 parts, and water 3000-5000 parts; The concentration of the borax aqueous solution is 1~3wt%; The magnetocaloric responsive nanoparticles are those described in claim 6.

9. The method for preparing the composite hydrogel according to claim 8, characterized in that, Simply mix polyvinyl alcohol, gelatin, magnetothermal responsive nanoparticles, borax, and water.

10. The application of the composite hydrogel of claim 8 or the composite hydrogel prepared by the preparation method of claim 9 in wound repair.

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