Preparation method and application of hyaluronic acid microneedle with copper ions doped with Prussian blue

Abstract

The invention discloses a preparation method and application of a copper ion doped Prussian blue hyaluronic acid microneedle, and relates to the technical field of biological antibacterial materials, the preparation method comprises the following steps: S1, preparing copper ion doped Prussian blue CuPB: dissolving K4 [Fe (CN) 4] and PVP in an ethanol-water mixed solution, and adjusting the pH value to 2.0 with hydrochloric acid to obtain a mixture 1; dissolving CuNO3 in an ethanol-water mixed solution to obtain a mixture 2; adding the mixture 2 into the mixture 1, magnetically stirring for 2 hours, then introducing a FeCl3 aqueous solution, reacting for 6 hours, centrifuging at 10000 * g for 15 minutes, collecting precipitates, washing with ethanol and deionized water for 3 times respectively, and drying at 60 DEG C for 24 hours to obtain CuPB nanoparticles; s2, preparing the hyaluronic acid microneedle with the copper ions doped with the Prussian blue. The CuPB-HA (at) MNs disclosed by the invention can promote cell proliferation and repair while quickly responding to sterilization.

Description

Technical Field

[0001] The present invention relates to the technical field of biological antibacterial materials, and in particular to a preparation method and application of copper ion-doped Prussian blue hyaluronic acid microneedles. Background Art

[0002] Acne is a common chronic inflammatory skin disease that affects more than 80% of the world's population and originates from the sebaceous glands. Propionibacterium acnes , P. acnes ) colonization and inflammatory cascade have been identified as key factors in the occurrence and progression of acne lesions. The key to treatment is to control bacterial proliferation and reduce sebum secretion. Current treatment strategies mainly rely on antibiotic intervention and local ointment application. However, frequent use of antibiotics can easily lead to bacterial resistance, and local application of emulsions is difficult to reach the lesions directly. Studies have shown that Staphylococcus aureus ( Staphylococcus aureus, S. aureus ) is another skin commensal bacteria that resides in sebum-rich areas and may also contribute to the pathogenesis of acne when it proliferates abnormally in these areas. This discovery has prompted clinical treatments to simultaneously address multiple goals: effectively inhibiting the proliferation of Propionibacterium acnes and Staphylococcus aureus, significantly reducing inflammation, and sustainably regulating sebum secretion. Consequently, the development of novel non-antibiotic therapeutic agents has become a key research direction in the field of acne treatment.

[0003] As a non-drug treatment, phototherapy is gaining increasing attention in the clinical treatment of acne due to its limited adverse reactions and lack of drug resistance. Generally speaking, red light can inhibit sebum secretion and enhance collagen deposition. Furthermore, red light (620-750 nm) can penetrate to a depth of 5 mm, reaching the dermis and even the subcutaneous tissue, making it more effective for deep inflammation and repair. Combining it with appropriate photosensitizers can achieve synergistic therapeutic effects through multiple mechanisms. Prussian blue (PB) is a coordination polymer with a three-dimensional network structure that exhibits significant light absorption in the red wavelength range and excellent biocompatibility. However, its effective antibacterial effect requires a high dose, which can be reduced through modification. Copper ions and their complexes have been shown to have broad-spectrum antimicrobial properties and have also been shown to play an important role in promoting cell migration, collagen deposition, and tissue regeneration. However, the combination of these two light sources for the treatment of acne has not yet been studied. Summary of the Invention

[0004] In response to the above problems, the present invention provides a preparation method and application of copper ion-doped Prussian blue hyaluronic acid microneedles, which can effectively inhibit bacterial growth and promote acne tissue repair under red light.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a method for preparing copper ion-doped Prussian blue hyaluronic acid microneedles, which specifically comprises the following steps: S1: Preparation of copper ion-doped Prussian blue (CuPB): K4[Fe(CN)4] and polyvinylpyrrolidone (PVP) were dissolved in an ethanol-water mixture, and the pH value was adjusted to 2.0 with hydrochloric acid to obtain a mixture 1; Dissolve CuNO3 in an ethanol-water mixed solution to obtain mixture 2; Mixture 2 was added to mixture 1 and magnetically stirred for 2 h. Then, an aqueous FeCl3 solution was slowly added dropwise in a water bath at 80°C. After reacting for 6 h, the mixture was centrifuged at 10,000 × g for 15 min. The precipitate was collected and washed three times with ethanol and deionized water, respectively, and dried at 60°C for 24 h to obtain CuPB nanoparticles. S2: Preparation of copper ion-doped Prussian blue hyaluronic acid microneedles: Dissolve CuPB in PBS, sonicate for 30 minutes, add hyaluronic acid HA powder, and stir to mix evenly to form a CuPB-HA hydrogel; drop the uniform CuPB-HA hydrogel on a polydimethylsiloxane (PDMS) template, evacuate in a vacuum drying oven for 10 minutes to form a needle tip, remove surface bubbles, and then add an appropriate amount of gel to form a backing. After drying at room temperature for 24 hours, gently demold the template to obtain a microneedle.

[0006] Furthermore, in step S1, the dosage ratio of K4[Fe(CN)4], PVP, and mixed solution is 0.4mmol:3g:20mL; the dosage ratio of CuNO3 and mixed solution is 0.01~0.04mmol:10mL; the dosage ratio of K4[Fe(CN)4], CuNO3 and FeCl3 aqueous solution is 2:0.1~0.4:2.

[0007] Furthermore, in step S1, the volume ratio of ethanol to water in the mixed solution is 1:1; and the molar concentration of hydrochloric acid is 12 mol / L.

[0008] Furthermore, in step S2, the usage ratio of CuPB, PBS, and HA powder is 100 mg:1 mL:120 mg.

[0009] The present invention also provides a use of the copper ion-doped Prussian blue hyaluronic acid microneedles prepared by the above preparation method in preparing a drug for treating acne.

[0010] Furthermore, the microneedles can effectively inhibit bacterial growth and promote tissue repair in acne-affected areas under red light.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a method for preparing copper ion-doped Prussian blue hyaluronic acid microneedles. 2+ Doping can enhance the photocatalytic activity and antibacterial properties of PB, promote cell migration, and stimulate repair. CuPB-HA@MNs not only rapidly kill bacteria but also promote cell proliferation and repair. Animal experiments have shown that seven days after microneedle application, tissue in the insertion area was significantly restored to normal shape, with significant collagen deposition. The preparation method of the present invention is simple and easy to implement, requiring minimal equipment investment. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is the overall technical roadmap of the present invention; Figure 2 The present invention is (a) PB, (b) Cu5PB, (c) Cu 10 PB and (d)Cu 20 HRTEM image of PB; Figure 3 The present invention PB, Cu5PB, Cu 10 PB and Cu 20 XRD pattern of PB; Figure 4 The present invention PB, Cu5PB, Cu 10 PB and Cu 20 FTIR spectrum of PB; Figure 5 (a) Cu 10 Full XPS spectrum of PB; (b) XPS spectrum of Fe before PB illumination 2+ Peak separation; (c) Cu 10 XPS Fe before PB irradiation 2+ Peak separation; Figure 6 The present invention PB, Cu5PB, Cu 10 PB and Cu 20 PB at (a) 0.3W / cm 2 and (b) 0.2 W / cm 2 The photothermal heating curve below; Figure 7 The present invention PB, Cu5PB, Cu 10 PB and Cu 20 PB at 0.3W / cm 2 ROS production under light, (a) 1 O2, (b) O2 - , (c) ·OH; Figure 8 The Cu prepared by the template method of the present invention10 Optical microscopy images of PB-HA@MNs; Figure 9 The HA@MNs and Cu 10 Mechanical experimental diagram of PB-HA@MNs; Figure 10 This is the antibacterial experimental coating of PB-HA@MNs and CuPB-HA@MNs of the present invention against Propionibacterium acnes; Figure 11 The antibacterial rate statistics of PB-HA@MNs and CuPB-HA@MNs of the present invention against Propionibacterium acnes; Figure 12 This is the antibacterial experimental coating of PB-HA@MNs and CuPB-HA@MNs of the present invention against Staphylococcus aureus; Figure 13 The antibacterial rate statistics of PB-HA@MNs and CuPB-HA@MNs of the present invention against Staphylococcus aureus; Figure 14 The present invention PB and Cu 10 PB ion release statistics under dark and light conditions; Figure 15 This is a staining image of the fibroblast cytoskeleton by PB-HA@MNs, CuPB-HA@MNs and HA@MNs of the present invention; Figure 16 This is the cell migration experiment of PB-HA@MNs, CuPB-HA@MNs and HA@MNs of the present invention on fibroblasts; Figure 17 The present invention Cu 10 PB-HA@MNs and Cu 10 Masson staining analysis of acne tissue of PB-HA@MNs+light; Figure 18 The present invention Cu 10 PB-HA@MNs and Cu 10 Schematic diagram of immunohistochemistry (TNF-α, IL-1β and MMP-2) staining of PB-HA@MNs+light; Figure 19 The present invention Cu 10 PB-HA@MNs and Cu 10 Quantitative analysis results of immunohistochemistry (TNF-α, IL-1β and MMP-2) of PB-HA@MNs+light; Figure 20 The present invention Cu 10 PB-HA@MNs and Cu 10HE staining of the heart, liver, spleen, lung, and kidney of PB-HA@MNs+light. DETAILED DESCRIPTION

[0013] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0014] Unless otherwise specified, the instruments, reagents, and materials used in the following examples are all conventional instruments, reagents, and materials available in the prior art and can be obtained through regular commercial channels. The experimental methods and detection methods used in the following examples are all conventional experimental methods and detection methods available in the prior art, unless otherwise specified.

[0015] Example 1 The present invention provides a method for preparing copper ion-doped Prussian blue hyaluronic acid microneedles, such as Figure 1 , specifically including the following steps: S1: Preparation of copper ion-doped Prussian blue (CuPB): 0.8 mmol K4[Fe(CN)4] and 6 g PVP were dissolved in 40 mL of a 1:1 ethanol-water mixture, and the pH was adjusted to 2.0 with hydrochloric acid (12 mol / L) to obtain mixture 1; 0.04 mmol CuNO3 was dissolved in 40 mL of ethanol and water (1:1) mixed solution to obtain mixture 2; Mixture 2 was added to mixture 1 and magnetically stirred for 2 h. Then, a 0.8 mmol FeCl3 aqueous solution was slowly added dropwise in an 80°C water bath. After reacting for 6 h, the mixture was centrifuged at 10,000 × g for 15 min. The precipitate was collected and washed three times with ethanol and deionized water, respectively, and dried at 60°C for 24 h to obtain CuPB nanoparticles (denoted as Cu5PB according to the copper doping ratio). S2: Preparation of copper ion-doped Prussian blue hyaluronic acid microneedles: Dissolve 100 mg of Cupb in 1 mL of PBS, sonicate for 30 minutes, add 120 mg of HA powder, and stir to mix evenly to form a Cupb-HA hydrogel; drop the uniform Cupb-HA hydrogel on the PDMS template, evacuate in a vacuum drying oven for 10 minutes to form a needle tip, remove surface bubbles, and then add an appropriate amount of gel to form a backing. After drying at room temperature for 24 hours, gently demold the template to obtain microneedles (CuPB-HA@MNs).

[0016] Example 2 The difference from Example 1 is that: In step S1, the amount of CuNO3 is 0.08 mmol, which is recorded as Cu10 PB.

[0017] Example 3 The difference from Example 1 is that: In step S1, the amount of CuNO3 is 0.16 mmol, recorded as Cu 20 PB.

[0018] Comparative Example 1 Preparation of Prussian Blue PB: 0.8 mmol K4[Fe(CN)4] and 6 g PVP were dissolved in 40 mL of ethanol / water mixed solution, and the pH value was adjusted to 2.0 with hydrochloric acid; 0.8 mmol FeCl3 aqueous solution was added dropwise under continuous stirring in an 80°C water bath. After reacting for 6 hours, the mixture was centrifuged at 10,000 × g for 15 minutes, and the precipitate was collected and washed three times with ethanol and deionized water, respectively, and dried at 60°C for 24 hours to obtain PB nanoparticles.

[0019] Comparative Example 2 Preparation of HA@MNs: Weigh 120 mg of HA powder and add it to 1 mL of PBS, stir and mix evenly to form an HA hydrogel; drop the uniform HA hydrogel on the PDMS template, evacuate in a vacuum drying oven for 10 minutes to form a needle tip, remove surface bubbles, and then add an appropriate amount of gel to form a backing. After drying at room temperature for 24 hours, gently demold the template to obtain HA@MNs.

[0020] Comparative Example 3 Preparation of PB-HA@MNs: Dissolve 100 mg of PB in 1 mL of PBS, sonicate for 30 minutes, add 120 mg of HA powder, and stir to mix evenly to form a PB-HA hydrogel; drop the uniform PB-HA hydrogel on the PDMS template, evacuate in a vacuum drying oven for 10 minutes to form a needle tip, remove surface bubbles, and then add an appropriate amount of gel to form a backing. After drying at room temperature for 24 hours, gently demold to obtain PB-HA@MNs.

[0021] Experimental Example 1 Material Characterization like Figure 2 As shown, HRTEM images correspond to (a) PB, (b) Cu5PB, (c) Cu 10 PB and (d)Cu 20 PB, it can be observed that the overall particle size of PB after doping is reduced, and the edges and corners are relatively rounded.

[0022] like Figure 3 As shown, XRD shows that Cu 2+ The successful grafting does not change the original structure, Cu5PB, Cu 10 PB and Cu 20 The PB peaks generally decreased, indicating that the particle size was reduced.

[0023] like Figure 4 As shown in Figure 3, there is a small peak at 2027 nm for CuPB relative to PB, which is presumably due to the formation of Cu-CN bonds caused by copper doping.

[0024] like Figure 5 As shown, from Cu 10 The XPS total peak spectrum of PB shows Cu 2+ The successful doping ( Figure 5 a), from PB and Cu 10 High-resolution XPS peak distribution of Fe2P in PB 2+ The reduction of divalent Cu 2+ Doping ( Figure 5 bc).

[0025] Figure 6 For PB and CuPB at 0.3W / cm 2 and 0.2W / cm 2 The photothermal heating curve under the same concentration shows that with the increase of power, the photothermal performance of CuPB and PB is enhanced ( Figure 6 a). Under the same power and concentration, CuPB has better photothermal performance than PB ( Figure 6 b), among which Cu 10 PB has the best photothermal performance, which indicates that Cu 2+ Enhanced the photothermal effect of PB.

[0026] like Figure 7 As shown, PB and CuPB at 0.3W / cm 2 The relative production of ROS under light of 660 nm showed that both PB and CuPB produced three kinds of reactive oxygen species. CuPB generally produced more reactive oxygen species than PB, among which Cu 10 PB produces the most ROS, indicating that Cu 2+ Enhanced the photodynamic performance of PB ( Figure 7 ac); Figure 8 Cu prepared by template method 10 Optical microscope image of PB-HA@MNs shows that the microneedles have a 15×15 needle tip distribution, the overall color is blue, the needle body is conical, and the needle tip is visible, indicating the successful preparation of the microneedle patch.

[0027] Experimental Example 2 Performance Test like Figure 9 As shown, mechanical experiments show that both HA@MNs and Cu 10 PB-HA@MNs have good mechanical properties, among which Cu 10PB-HA@MNs has better mechanical properties, and the force is enough to pierce the skin.

[0028] like Figure 10 As shown in the figure, the antibacterial schematic diagram of PB-HA@MNs and CuPB-HA@MNs against Propionibacterium acnes shows that the damage of the microneedles to the bacteria is limited under dark conditions, while under light conditions, the number of bacteria is greatly reduced, and the antibacterial performance is enhanced with the increase of copper doping amount. It is speculated that the damage to bacteria is caused by the synergistic effect of copper ions and photothermal / photodynamic effects.

[0029] like Figure 11 As shown in the figure, the antibacterial statistical results of PB-HA@MNs and Cupb-HA@MNs against Propionibacterium acnes showed that the antibacterial rate of microneedles against bacteria gradually increased with the doping of copper under dark conditions, and the bactericidal effect of Cupb-HA@MNs was higher than that of PB-HA@MNs under light conditions. 10 The killing effect of PB-HA@MNs on bacteria was as high as 99.6%.

[0030] like Figure 12 As shown in the figure, the antibacterial schematic diagram of PB-HA@MNs and CuPB-HA@MNs against Staphylococcus aureus shows that the damage of the microneedles to bacteria is limited under dark conditions, while under light conditions, the number of bacteria is greatly reduced, and the antibacterial performance is enhanced with the increase of copper doping amount. It is speculated that the damage to bacteria is caused by the synergistic effect of copper ions and photothermal / photodynamic forces.

[0031] like Figure 13 As shown in the figure, the antibacterial statistical results of PB-HA@MNs and Cupb-HA@MNs against Staphylococcus aureus showed that the killing effect of microneedles on bacteria under dark conditions increased with the increase of copper doping, indicating that copper has a killing effect on bacteria under dark conditions. The bactericidal effect of Cupb-HA@MNs under light conditions was higher than that of PB-HA@MNs, among which Cu 10 The killing effect of PB-HA@MNs on bacteria is as high as 99.5%.

[0032] like Figure 14 As shown, PB and Cu 10 The comparison of ion release before and after PB irradiation shows that a large amount of ions are released after irradiation, which can promote the killing effect on bacteria.

[0033] like Figure 15 As shown, the results of cytotoxicity and proliferation experiments showed that PB-HA@MNs and CuPB-HA@MNs had a significant effect on promoting cell proliferation and had low cytotoxicity; like Figure 16As shown in the figure, the cell migration experiment shows that HA@MNs has a certain effect of promoting migration, and the migration effect becomes more obvious with the increase of copper doping amount.

[0034] like Figure 17 As shown, Cu 10 PB-HA@MNs has the effect of promoting collagen deposition, and the effect of promoting collagen deposition is more obvious under light conditions.

[0035] like Figure 18 As shown, immunohistochemical analysis showed that Cu 10 The PB-HA@MNs+light group had the best anti-inflammatory effect and significantly inhibited the key pro-inflammatory factors TNF-α, IL-1β, and MMP-2 involved in acne infection.

[0036] like Figure 19 As shown, quantitative analysis of immunohistochemistry revealed that Cu 10 PB-HA@MNs and Cu 10 The PB-HA@MNs+light group had significant anti-inflammatory effects, and Cu 10 PB-HA@MNs+light has the most obvious anti-inflammatory effect.

[0037] like Figure 20 As shown in the biosafety experiments, no obvious organ damage, histological abnormalities or lesions were found in any group. 10 PB-HA@MNs treatment did not affect the physiological conditions of mice.

[0038] In summary, Cu 2+ The doping of Cu increases the absorption of visible light, and the enhanced plasma resonance effect significantly improves the photothermal performance. 10 PB has the best photothermal performance. And the Cu doping makes Cu 10 PB generates the largest amount of reactive oxygen species, and the enhanced photothermal, photodynamic mechanism and ion release synergistically enhance the antibacterial effect, with an antibacterial rate of over 99% against both bacteria. At the same time, the released copper and iron ions can also promote the proliferation and migration of fibroblasts, thereby promoting wound healing and repair. The hyaluronic acid wrapping not only enhances the biocompatibility of the material, but also helps to control the slow release of ions. In the mouse acne model, Cu 10 PB-HA@MNs exhibited rapid antibacterial and anti-inflammatory properties and significantly promoted tissue regeneration and repair.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing copper ion-doped Prussian blue hyaluronic acid microneedles, characterized by: The specific steps include: S1: Preparation of copper ion-doped Prussian blue (CuPB): K4[Fe(CN)4] and polyvinylpyrrolidone were dissolved in an ethanol-water mixture, and the pH value was adjusted to 2.0 with hydrochloric acid to obtain a mixture 1; Dissolve CuNO3 in an ethanol-water mixed solution to obtain mixture 2; Mixture 2 was added to mixture 1 and magnetically stirred for 2 h. Then, an aqueous FeCl3 solution was slowly added dropwise in a water bath at 80°C. After reacting for 6 h, the mixture was centrifuged at 10,000 × g for 15 min. The precipitate was collected and washed three times with ethanol and deionized water, respectively, and dried at 60°C for 24 h to obtain CuPB nanoparticles. S2: Preparation of copper ion-doped Prussian blue hyaluronic acid microneedles: Dissolve CuPB in PBS, sonicate for 30 minutes, add hyaluronic acid HA powder, and stir to mix evenly to form a CuPB-HA hydrogel; drop the uniform CuPB-HA hydrogel on the polydimethylsiloxane template, evacuate in a vacuum drying oven for 10 minutes to form a needle tip, remove the surface bubbles, and then add an appropriate amount of gel to form a backing. After drying at room temperature for 24 hours, gently demold to obtain the microneedle.

2. The method for preparing copper ion-doped Prussian blue hyaluronic acid microneedles according to claim 1, characterized in that: In step S1, the dosage ratio of K4[Fe(CN)4], PVP, and mixed solution is 0.4mmol:3g:20mL; the dosage ratio of CuNO3 and mixed solution is 0.01~0.04mmol:10mL; the dosage ratio of K4[Fe(CN)4], CuNO3, and FeCl3 aqueous solution is 2:0.1~0.4:

2.

3. The method for preparing copper ion-doped Prussian blue hyaluronic acid microneedles according to claim 1, characterized in that: In step S1, the volume ratio of ethanol to water in the mixed solution is 1:1; and the molar concentration of hydrochloric acid is 12 mol / L.

4. The method for preparing copper ion-doped Prussian blue hyaluronic acid microneedles according to claim 1, characterized in that: In step S2, the usage ratio of CuPB, PBS, and HA powder is 100 mg:1 mL:120 mg.

5. Use of the copper ion-doped Prussian blue hyaluronic acid microneedles prepared by the preparation method according to any one of claims 1 to 4 in the preparation of a drug for treating acne.

6. The use according to claim 5, characterized in that: The microneedles can effectively inhibit bacterial growth and promote tissue repair of acne-affected areas under red light.

Images