Copper oxide nano-enzyme microneedle with active oxygen scavenging capacity as well as preparation method and application of copper oxide nano-enzyme microneedle

Abstract

The invention discloses a copper oxide nano-enzyme microneedle with active oxygen scavenging capacity as well as a preparation method and application of the copper oxide nano-enzyme microneedle. The preparation method of the nano-enzyme microneedle comprises the following steps: dissolving a copper salt in deionized water, adding L-ascorbic acid into the solution, heating and stirring the mixed solution, after the reaction is finished, centrifuging to remove larger precipitates, taking supernate, dialyzing to remove unreacted micromolecules, and freeze-drying to obtain CuxO. The preparation method comprises the following steps: dispersing CuxO in a sodium hyaluronate solution, adding the dispersion liquid into a microneedle mold, then putting the microneedle mold into a vacuum drying oven, vacuumizing, scraping off the redundant solution, then using the sodium hyaluronate solution as a backing, drying at normal temperature, and demolding to obtain the CuxO microneedle. The copper oxide nano-enzyme (CuxO) prepared by the method has good dispersity, and the particle size is about 4.6 nm. The obtained CuxO microneedle can be used for treating androgenetic alopecia and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of hair loss treatment, specifically relating to a copper oxide nanoenzyme microneedle with reactive oxygen species scavenging ability, its preparation method, and its application. Background Technology

[0002] Androgenetic alopecia is a progressive hair loss disorder caused by abnormal androgen metabolism. High levels of oxidative stress are observed around the hair follicles of patients with androgenetic alopecia. Excessive reactive oxygen species (ROS) cause irreversible oxidative damage, leading to disrupted cell signaling, inducing inflammation around the hair follicles, and accelerating the progression of androgenetic alopecia. Eliminating ROS around the hair follicles and restoring the follicular microenvironment is a potential treatment strategy for androgenetic alopecia.

[0003] Copper, an essential element for the human body, is a component or activator of many enzymes, such as superoxide dismutase (SOD), which helps scavenge superoxide anion free radicals in the body and reduce oxidative stress damage. Copper-based antioxidant nanomaterials, due to their excellent reactive oxygen species scavenging capabilities, have been widely developed for the treatment of diseases such as enteritis, chronic wounds, and Parkinson's syndrome. Copper also plays an important role in hair health; it is crucial for amino oxidases required for the oxidation of thiol groups to disulfide crosslinking. Copper deficiency can lead to gray hair and reduced keratin fiber strength. Therefore, copper oxide nanozymes are a promising material for treating hair loss with clinical applications. However, due to the skin barrier function of the stratum corneum, inorganic nanomaterials have difficulty penetrating the area around hair follicles. Summary of the Invention

[0004] In response to the aforementioned problems, current status, and future prospects, this invention provides a copper oxide nanoenzyme microneedle with reactive oxygen species scavenging ability, its preparation method, and its application. This copper oxide nanoenzyme microneedle has reactive oxygen species scavenging ability, requires a lower dosing frequency compared to conventional therapeutic drugs, and has good biocompatibility. It can be used for the health care and treatment of androgenetic alopecia-related diseases and has broad application prospects in the biomedical field.

[0005] This invention first provides a method for preparing copper oxide nanoenzyme microneedles with reactive oxygen species scavenging ability, which includes the following steps:

[0006] (1) Dissolve copper salt in deionized water, add L-ascorbic acid to adjust the pH of the solution to alkaline, heat and stir the mixture to react, centrifuge to collect the supernatant, dialyze, and freeze-dry to obtain copper nanozyme Cu. x O;

[0007] (2)Cu xO was dispersed in a sodium hyaluronate solution. After the dispersion was mixed, it was added to a microneedle mold. Vacuum was applied to remove air bubbles from the mold, and excess dispersion was scraped off. Sodium hyaluronate solution was then used as a backing. After drying at room temperature, the product was demolded to obtain Cu. x O nanozyme microneedles.

[0008] As a preferred embodiment of the present invention, the copper salt is a soluble copper salt, such as copper chloride, copper sulfate, or copper nitrate, preferably CuCl2. In the mixture solution of step (1), the concentration of the copper salt is 1-100 mM, the concentration of L-ascorbic acid is 1-1000 mM, and the pH is 7.1-12.0. Preferably, the final concentration of the copper salt solution is 10 mM. The final concentration of L-ascorbic acid is 100 mM.

[0009] As a preferred embodiment of the present invention, in step (1), the reaction temperature of the mixture for heating and stirring is 5-100°C, and the reaction time is 1-24 hours. More preferably, the reaction temperature is 80°C, and the reaction time is 12 hours.

[0010] As a preferred embodiment of the present invention, in step (1), after the reaction is completed, the mixed solution is centrifuged at a speed ≥1000g for 5-20 minutes; the molecular weight cutoff of the dialysis bag is 3-20kDa, and the dialysis time is 12-96 hours. More preferably, the centrifugation speed is 6500g, the centrifugation time is 15 minutes; the molecular weight cutoff of the dialysis bag is 10kDa, and the dialysis time is 48 hours.

[0011] As a preferred embodiment of the present invention, in step (2), Cu is used to disperse Cu x The sodium hyaluronate solution used in O is exactly the same as the sodium hyaluronate solution used as a backing; wherein the sodium hyaluronate has a molecular weight of 1-100 kDa and a final concentration of 0.05-1 g / mL; more preferably, the sodium hyaluronate used has a molecular weight of 10 kDa and a final concentration of 0.3 g / mL.

[0012] This invention also provides a copper oxide nanoenzyme microneedle (also known as Cu) with reactive oxygen species scavenging ability prepared by the above method. x O nanozyme microneedles or Cu x O microneedles). In one embodiment of the present invention, each Cu... x The O nanozyme microneedles consist of a 20×20 needle array, each needle being conical with a tip height of 600 μm, a tip width of 300 μm, and a spacing of 400 μm between each needle. Each Cu sheet... x The Cu content in the O nanozyme microneedles is 1-1000 ng. Preferably, each Cu nanoneedle contains 1-1000 ng. x The Cu content in the O microneedles is 13 ng.

[0013] The present invention also provides the application of the above-mentioned copper oxide nanoenzyme microneedles with active oxygen scavenging ability in the preparation of drugs or preparations for treating hair loss, preferably the application in the preparation of drugs or preparations for treating androgenetic alopecia.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] (1) Copper nanoenzyme material is used to treat androgenetic alopecia by clearing reactive oxygen species and regulating the ecological niche around hair follicles. Compared with finasteride hormone drugs, it has fewer side effects and a lower dosing frequency compared with minoxidil.

[0016] (2) Soluble microneedle patches, as a transdermal drug delivery method, can penetrate the epidermis and deliver drugs in situ, while avoiding systemic side effects. Sodium hyaluronate, as a component inherent in the skin, is a common microneedle material that dissolves rapidly after entering the skin, and its degradation products are very safe. This invention uses sodium hyaluronate microneedles to deliver Cu. x O nanozymes overcome the problem that inorganic nanomaterials are difficult to penetrate into the hair follicle area. Attached Figure Description

[0017] Figure 1 Cu x Characterization of O nanozymes. (a) Cu x TEM image of O nanozyme. (b) Cu x Statistical distribution of O nanozyme particle size. (c)Cu x X-ray energy spectrum of O nanozyme.

[0018] Figure 2 Cu x In vitro reactive oxygen species scavenging capacity of O nanozymes. (a) TMB+·OH and Cu x (b) UV absorption spectrum of O nanozyme after incubation. - With Cu x UV absorption spectrum of O nanozyme after incubation. (c)Cu x UV absorption spectrum of O nanozyme after co-incubation with DPPH.

[0019] Figure 3 Cu x Characterization of O microneedles. (a)Cu x Optical image of O microneedles. (b)Cu x SEM images of O microneedles. (c) Blank microneedles and Cu x Compression force versus displacement curves of O-microneedles.

[0020] Figure 4 Cu x Safety evaluation of O and reactive oxygen species scavenging capacity at the cellular level. (a) Cu at different concentrationsx (b) Cell viability of dermal papilla cells treated with O. (b) Cell viability of dermal papilla cells after H2O2 stimulation. x Changes in reactive oxygen species (ROS) in dermal papilla cells under O pretreatment (green: DCFH-DA probe; blue: Hoechst 33342). (c) Statistical analysis of ROS fluorescence intensity values ​​in cells of each treatment group. ***P<0.001.

[0021] Figure 5 Cu x Evaluation of the efficacy of O-microneedling in treating androgenetic alopecia. (a) Representative images of the treatment effects in each treatment group on days 0, 10, 14, 21, and 28. (b) Statistical analysis of the regenerated hair coverage on the backs of mice in each treatment group on day 28. (c) Statistical analysis of hair length in mice in each treatment group on day 28. *p<0.05, **p<0.01, ***p<0.001.

[0022] Figure 6 The quality of newly generated hair regrowth in each group on day 28. (a) SEM images of newly generated hair in each group. (b) Statistics on the diameter of newly generated hair in each group. **p<0.01, ***p<0.001.

[0023] Figure 7 Ki67 and DHE staining of skin in each treatment group on day 14. (a) Representative fluorescence images of Ki67 in the dorsal skin of mice in each group (green: Ki67; blue: DAPI). (b) Ki67 intensity statistics for each group. (c) Representative fluorescence images of DHE in the dorsal skin of mice in each group (red: DHE; blue: DAPI). (d) DHE intensity statistics for each group. **p<0.01, ***p<0.001. Detailed Implementation

[0024] The present invention will be further described and illustrated below with reference to specific embodiments. The embodiments described are merely examples of the content of this disclosure and do not constitute a limitation on the scope. In this invention, the technical features of each embodiment can be freely combined without conflict.

[0025] Example 1 Cu x Preparation and characterization of O

[0026] A CuCl2 solution (containing 67.22 mg of CuCl2 at a concentration of 10 mM) was stirred for 10 minutes in a magnetically stirred oil bath at 80°C. 880.6 mg of L-ascorbic acid was dissolved in deionized water to prepare a 100 mM solution, which was then slowly added to the CuCl2 solution using a dropper. The pH of the mixed solution was adjusted to 8.0-9.0 using NaOH solution, and the reaction was carried out at 80°C with stirring for 12 hours. After the reaction, the solution was centrifuged at 6500 g for 15 minutes to remove larger precipitates. The supernatant was dialyzed against a dialysis bag with a molecular weight cutoff of 10 kDa for 48 hours to remove unreacted small molecules. Cu was obtained by lyophilization and concentration. x O nanozymes. Figure 1 a shows Cu x The morphology and good dispersibility of O nanozymes. Cu x The particle size distribution histogram of O nanozymes is shown below. Figure 1 As shown in b, the average particle size is 4.56 ± 1.42 nm (n = 200). The X-ray energy dispersive spectroscopy results also show that Cu... x O nanozymes include Cu and O elements, such as Figure 1 As shown in c.

[0027] Cu x O nanozymes reacted with TMB+·OH and NBT+·O2, respectively. - Co-incubation with DPPH radicals to detect Cu x The reactive oxygen species scavenging capacity of O nanozymes in vitro. The UV absorption spectrum after the reaction is as follows: Figure 2 As shown in ab in the figure. With Cu x As the concentration of O nanozyme increased, the characteristic absorption peak intensities of the three free radical indicators all decreased significantly, thus the prepared Cu x O nanozymes can significantly scavenge free radicals and exert good antioxidant capacity in vitro.

[0028] Example 2 Cu x Preparation and characterization of O microneedles

[0029] Sodium hyaluronate (molecular weight 10,000, 0.3 g / mL, dissolved in ultrapure water) and Cu x After mixing the O nanozyme, it was added to the polydimethylsiloxane microneedle mold. The mold was placed in a vacuum drying oven and evacuated for 5 minutes. Excess solution was scraped off, and sodium hyaluronate solution (10,000 molecular weight, 30% (w / v), dissolved in ultrapure water) was added as a backing. After drying at room temperature for 24 hours, the mold was removed. Optical and SEM images of the microneedles are shown below. Figure 3 ab in the preparation of Cu x O microneedles (Cu) x O-MNs are morphologically complete and conical. For example... Figure 3As shown in Figure c, when the compression displacement reaches 400 μm, the maximum pressure that each blank microneedle can withstand is 2.09 N, while Cu... x Each O-MNs needle can withstand a maximum pressure of 1.67N, allowing for effective skin penetration. Each Cu... x The O microneedle patch contains 13±2ng of Cu.

[0030] Example 3: Verification of Cu on hair papilla cells x Safety and antioxidant capacity of O nanozymes

[0031] Hair papilla cells were divided into 1×10 4 The cells were seeded at a density of cells / well in a 96-well plate. After the cells were fully adhered, they were then seeded according to Cu... x Cells were treated with Cu at concentrations of 100 ng / mL, 200 ng / mL, 300 ng / mL, 400 ng / mL, and 500 ng / mL. After 24 hours of culture, 15 μL of 5 mg / mL MTT solution was added to each well and incubated for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to each well. After thorough dissolution, the absorbance was measured at 490 nm using a microplate reader to calculate cell viability. Figure 4 As shown in figure a, the survival rate of dermal papilla cells co-cultured with Cu at concentrations below 300 ng / mL for 24 hours exceeded 82.95%, indicating that Cu... x O nanozymes have good biosafety.

[0032] Hair papilla cells were divided into 15×10 4 Cells were seeded into 35mm glass culture dishes. After complete cell adhesion, Cu solutions with concentrations of 50 ng / mL and 100 ng / mL were added. x Incubate with O solution for 12 hours, then wash three times with PBS (3 minutes each time). Add 650 μM H2O2 and incubate for 30 minutes to induce a cellular oxidative stress model, then wash three times with PBS (3 minutes each time). Add DCFH-DA probe (1:1000 dilution) and Hoechst33342 (1:500 dilution) to indicate reactive oxygen species levels in cells, incubate in the dark for 30 minutes, then wash three times with PBS (3 minutes each time). Observe and photograph using a laser confocal microscope. x Changes in reactive oxygen species in dermal papilla cells after pretreatment with O nanozymes and stimulation with H2O2, as shown in... Figure 4 As shown in bc. The green fluorescence of cells in the H2O2-treated group was significantly enhanced, as were the Cu2+ treatments at 50 ng / mL and 100 ng / mL. x In the group pretreated with O solution for 12 hours, the intensity of green fluorescence in dermal papilla cells was significantly reduced. This indicates that Cu... x O nanozymes can remove reactive oxygen species in cells at the cellular level, thus achieving the purpose of antioxidation.

[0033] Example 4: Cu in an animal model x O-micro's therapeutic effect on androgenetic alopecia

[0034] Six-week-old male C57BL / 6 mice were used and housed for one week to acclimatize. Hair was removed from the backs of the mice using depilatory cream, covering an area of ​​approximately 4 cm². 2 (2cm × 2cm). Mice were randomly divided into 4 groups: androgenetic alopecia model group, minoxidil group, blank microneedle group, and Cu group. x O Microneedling Group. Androgenetic alopecia model group: Topical testosterone applied, no other treatment received. Minoxidil group: Topical testosterone and 3% minoxidil treatment (100 μL / animal / day, continued until day 13). Blank microneedling group: Topical testosterone and blank microneedling treatment. Cu x O-microneedle group: Topical application of testosterone and Cu x Microneedle treatment. All groups received 0.5% testosterone solution (0.05 mL / cm²) for 28 consecutive days. 2 A male pattern baldness model was established. Testosterone was applied for 30 minutes, followed by minoxidil and microneedling administration. Microneedling administration was performed on days 1, 4, 7, 10, and 13, with the patch pressed for 3 minutes before removal. Back photographs of mice were taken on days 0, 10, 14, 21, and 28 to record hair growth in each group. On day 28, all mice were weighed and their weight recorded. Whole blood was collected from the orbital fossa, and the mice were euthanized by cervical dislocation. Back skin was obtained, fixed in 4% paraformaldehyde, and the hair was plucked with forceps. ImageJ software was used to analyze hair coverage, length, and diameter on day 28 for each group. The surface morphology of newly grown hair on day 28 was photographed using a scanning electron microscope. On day 14, one representative mouse from each group was selected, euthanized by cervical dislocation, and back skin was obtained. This skin was fixed in 4% paraformaldehyde, dehydrated in 15% sucrose solution, embedded in OCT, and sectioned. To measure hair follicle cell proliferation, skin sections were fixed, antigen retrieval was performed, serum blocking was applied, Ki67 primary antibody was blocked overnight, secondary antibody was incubated at room temperature for 50 minutes, cell nuclei were counterstained with DAPI for 8 minutes, and the sections were mounted with an anti-fluorescence quencher. Images were observed and acquired using a fluorescence microscope, and the positive rate of Ki67 was statistically analyzed using ImageJ software. To detect changes in reactive oxygen species (ROS) in the tissue surrounding hair follicles, sections were fixed, stained with the 1:1000 fluorescent probe DHE, cell nuclei were counterstained with DAPI for 8 minutes, and the sections were mounted with an anti-fluorescence quencher. Images were observed and acquired using a fluorescence microscope, and the relative fluorescence intensity of ROS in each group was statistically analyzed using ImageJ software.

[0035] Representative images of the treatment effects on androgenetic alopecia in each group at days 0, 10, 14, 21, and 28, along with the dorsal hair regrowth coverage and hair length of mice in each group on day 28, are shown below. Figure 5 As shown, the diameter of regenerated hair on the back of mice in each group on day 28 and the hair SEM images are as follows. Figure 6 As shown. Mice in the model group treated only with testosterone showed almost no hair growth on their backs, indicating the successful establishment of the androgenetic alopecia model. Minoxidil group and Cu x Mice in the O microneedling group differed from other groups in that a noticeable color change was observed on their backs starting from day 14, indicating that hair follicles were transitioning from the resting phase to the anagen phase. On day 28, Cu... x The microneedle group achieved a hair coverage rate of 74.0% and an average hair length of 5.9 mm, similar to the minoxidil group, but significantly higher than other groups. x The hair diameter in the O microneedle group was 27.7 μm, which was not significantly different from the minoxidil group but significantly higher than other groups. SEM images showed Cu x Mice in the O microneedle group had coarser fur and intact hair cuticles. This indicates that Cu... x Mice treated with O microneedles showed good hair growth and excellent quality of regenerated hair.

[0036] Representative fluorescence images and intensity statistics of Ki67 in the skin of each treatment group on day 14, and representative fluorescence images and intensity statistics of DHE in the dorsal skin of mice in each group are shown below. Figure 7 As shown. Cu x Ki67 expression in the microneedling group was significantly higher than that in the model group and the blank microneedling group, but there was no significant difference compared with the minoxidil group, indicating that the hair follicle cell proliferation level was comparable to that of minoxidil. The model group had the highest level of reactive oxygen species in its skin, consistent with the characteristics of androgenetic alopecia. Cu x The O microneedle group had the lowest level of reactive oxygen species, indicating that Cu x O-microneedling treatment can significantly reduce the level of reactive oxygen species in androgenetic alopecia and restore the homeostasis of the microenvironment around hair follicles.

[0037] Those skilled in the art will understand that the embodiments of the present invention are not limited to the above-described examples. The description in the specification is only for illustrating the basic principles of the present invention. Various changes and modifications can be made to the present invention without departing from its core spirit and scope, and all such changes and modifications fall within the protection scope of the present invention. The protection scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing copper oxide nanoenzyme microneedles with reactive oxygen species scavenging ability, characterized in that... Includes the following steps: (1) Dissolve copper salt in deionized water, add L-ascorbic acid to adjust the pH of the solution to alkaline, heat and stir the mixture to react, centrifuge to collect the supernatant, dialyze, and freeze-dry to obtain copper nanozyme Cu. x O; (2)Cu x O was dispersed in a sodium hyaluronate solution. After the dispersion was mixed, it was added to a microneedle mold. Vacuum was applied to remove air bubbles from the mold, and excess dispersion was scraped off. Sodium hyaluronate solution was then used as a backing. After drying at room temperature, the product was demolded to obtain Cu. x O nanozyme microneedles.

2. The preparation method according to claim 1, characterized in that, In the mixture solution of step (1), the concentration of copper salt is 1-100 mM, the concentration of L-ascorbic acid is 1-1000 mM, and the pH is 7.1-12.

0.

3. The preparation method according to claim 1, characterized in that, In the mixture solution of step (1), the concentration of copper salt solution is 10 mM, the concentration of L-ascorbic acid is 100 mM, and the pH is 8-9.

4. The preparation method according to claim 1, characterized in that, In step (1), the reaction temperature for heating and stirring is 5-100℃, and the reaction time is 1-24 hours.

5. The preparation method according to claim 1, characterized in that, In step (1), after the reaction is completed, the mixed solution is centrifuged at a speed of ≥1000g for 5-20 minutes; the molecular weight cutoff of the dialysis bag is 3-20kDa, and the dialysis time is 12-96 hours.

6. The preparation method according to claim 1, characterized in that, In step (2), the molecular weight of sodium hyaluronate is 1-100kDa, and the final concentration is 0.05-1g / mL.

7. A copper oxide nanoenzyme microneedle with reactive oxygen species scavenging ability prepared by the method according to any one of claims 1-6.

8. The use of the copper oxide nanoenzyme microneedles with reactive oxygen species scavenging ability as described in claim 7 in the preparation of drugs or formulations for treating hair loss.

9. The application according to claim 8, characterized in that, The hair loss described is androgenetic alopecia.

Images