CN116763715A - Preparation method and application of paeonol-soluble transdermal microneedle for treating psoriasis - Google Patents

Abstract

A preparation method of paeonol soluble transdermal microneedle for treating psoriasis comprises S1, preparing paeonol hydrophilic micelle; s2, preparing a microneedle matrix solution; s3, preparing a microneedle backing layer solution; s4, bubble removal is carried out on the microneedle substrate solution and the microneedle backing layer solution; s5, pouring the microneedle matrix solution into a microneedle mould to fill up the surface, vacuumizing, centrifuging, scraping the colloid with bubbles, pouring the microneedle matrix solution into the microneedle mould to fill up the surface, and repeatedly vacuumizing and centrifuging until pinholes are filled up; s6, pouring the solution of the microneedle backing layer into a microneedle mould, centrifuging to enable the bottom of the microneedle mould to be flat and bubble-free, drying, and demoulding the microneedle. The paeonol transdermal patch can directly deliver to the epidermis layer or the upper dermis layer by penetrating the superficial epidermis barrier while avoiding contact with important nerves and capillaries in the epidermis, improves the stability and transdermal absorption efficiency of the medicine, and achieves the aim of accurately treating psoriasis by paeonol, thereby having remarkable social and economic benefits.

Description

Preparation method and application of paeonol-soluble transdermal microneedle for treating psoriasis

Technical Field

The invention relates to the technical field of pharmaceutical preparations, in particular to a preparation method and application of paeonol soluble transdermal microneedles for treating psoriasis.

Background

Psoriasis is a common immune-mediated chronic inflammatory skin disease, and causes psychological and life quality injuries to patients equivalent to cancers, myocardial infarction and depression, and statistics data show that global psoriasis patients reach 6000 ten thousand people and still increase year by year. Psoriasis has strong hereditary susceptibility and autoimmune pathogenic characteristics, and is clinically manifested in that keratinocyte hyperproliferation leads to epidermis thickening, obvious invasive erythema appears on the skin, the plaques can be mutually combined to cover a large area of skin, the psoriasis frequently appears on the scalp, trunk and limbs of a human body, and a large number of silver scales appear at itching plaques. In addition, psoriasis is not just a skin disease, and patients often have diseases such as psoriatic arthritis, cardiovascular diseases, metabolic syndrome, coronary artery diseases, type 2 diabetes and the like, and the prevalence rate gradually increases with age.

Conventional treatment of psoriasis is mainly divided into two types, one is oral methotrexate, cyclosporin A and Wu Sinu monoclonal antibodies and other drugs which are low in cost and good in benefit and are widely used, however, adverse reactions of nausea, vomiting and liver injury of patients are often caused by large-dose use, and furthermore, repeated attacks of psoriasis further increase the use dosage of the drugs and the generation of toxic and side effects. In addition, the topical administration is a traditional topical administration mode, such as ointment, liniment and vitamin D3, and although the topical administration can effectively reduce toxic and side effects, the proliferation of the skin horny layer reduces the skin permeability of the smeared medicine, the medicine absorption rate is low, and the treatment effect is reduced. Paeonol is the main phenolic component of cortex moutan, and has antioxidant, antiinflammatory, heart protecting and neuroprotective effects. Recent researches show that paeonol has good treatment effect on psoriasis, however, the paeonol has poor water solubility, and the first pass effect of human body self causes the systemic circulation dosage to be reduced after the medicine is orally taken, and the skin horny layer thickening caused by the psoriasis is smeared by using an ointment for external application, so that the skin permeation efficiency of the medicine is greatly reduced. Therefore, a new paeonol administration mode is needed to be used for improving the drug utilization rate after skin horny layer hyperplasia and reducing toxic and side effects.

The microneedle drug delivery system utilizes a transdermal drug delivery mode to deliver drugs through the skin, overcomes the problems of low drug utilization rate, poor patient compliance and the like of the traditional drug delivery mode, gradually releases the drugs along with the extension of time, and has the advantages of flexibility, time-space selectivity, weak invasiveness and the like. The microneedle patch is formed by arranging a plurality of micron-sized tiny needlepoints on a backing layer in an array mode, the size, the length and the shape of the microneedle can be flexibly adjusted according to treatment requirements, the general length of a needle body is 10-2000 mu m, the effectiveness and painless minimally invasive performance of an invasive needle head injector are considered, and a micron-sized drug administration channel is formed by penetrating a shallow epidermis barrier and avoiding contact with nerves and capillaries in epidermis, so that the drug is directly delivered to the epidermis layer or an upper dermis layer, and the possibility of cross contamination of the drug can be furthest reduced. Therefore, the development of the paeonol soluble transdermal microneedle for treating psoriasis is used for improving transdermal administration efficiency and relieving toxicity and side effects caused by patient treatment, and is a technical problem which needs to be solved urgently at present.

Disclosure of Invention

Aiming at the situation, the invention aims to overcome the defects of the prior art, and the invention aims to provide a preparation method and application of paeonol soluble transdermal microneedles for treating psoriasis, which can effectively solve the problems of low transdermal administration efficiency and strong toxicity and side effects of medicaments for treating psoriasis.

In order to achieve the above purpose, the technical scheme of the invention is that the preparation method of the paeonol soluble transdermal microneedle for treating psoriasis comprises the following steps:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at a solute mass ratio of 1:2 at room temperature, steaming for 15-25min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ to preheat for 4-5 min, adding the preheated deionized water into the film for ultrasonic treatment for 4-6 min, centrifuging for 4-6 min at 2800-3200rpm, and removing excessive drugs with poor solubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with molecular weight of 5 ten thousand into paeonol hydrophilic micelle with concentration of 2 mL of 3 mg/mL, stirring uniformly, and performing ultrasonic treatment for 10-12 min to completely dissolve the paeonol hydrophilic micelle to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 10-12 min to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 2800-3200rpm for 2-5min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 1-2min, placing the mould in a sleeve at 4000-4500rpm for centrifugation for 8-12 min, scraping the colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, vacuumizing repeatedly, and centrifuging until pinholes are filled;

s6, pouring the solution 230 mg of the backing layer of the micro needle after bubble removal into a micro needle mould, centrifuging at 1800-2200rpm for 2-4min to enable the bottom of the micro needle mould to be flat and bubble-free, drying at 32-37 ℃ for 70-75h, and demoulding the micro needle to obtain the paeonol soluble transdermal micro needle for treating psoriasis.

The paeonol soluble transdermal microneedle for treating psoriasis is applied to the preparation of medicaments for treating psoriasis.

According to the paeonol soluble transdermal microneedle for treating psoriasis, disclosed by the invention, the paeonol hydrophilic micelle is prepared into the microneedle patch, and the microneedle patch can directly deliver paeonol to the epidermis layer or the upper dermis layer by penetrating the superficial epidermis barrier and avoiding contact with important nerves and capillaries in the epidermis to form a micron-sized administration channel, so that the stability and transdermal absorption efficiency of a medicament are improved, the compliance of a patient on the medicament is improved, the aim of accurately treating psoriasis by paeonol is fulfilled, and the social and economic benefits are remarkable.

Drawings

FIG. 1 is a graph showing the particle size distribution of paeonol hydrophilic micelles according to the invention.

FIG. 2 is a TEM image of paeonol hydrophilic micelles of the present invention.

FIG. 3 is an infrared spectrum of a paeonol hydrophilic micelle of the present invention.

FIG. 4 is a graph showing the results of in vitro hemolysis experiments of paeonol hydrophilic micelles of the invention.

FIG. 5 is a graph showing the results of the cytotoxicity test of paeonol hydrophilic micelles of the invention on HUVEC cells.

FIG. 6 is a bright field diagram of a paeonol-soluble transdermal microneedle of the present invention.

FIG. 7 is an overall bright field diagram of doxorubicin microneedles of the present invention.

FIG. 8 is an overall fluorescence image of doxorubicin microneedles of the present invention.

FIG. 9 is a graph of the results of trypan blue microneedles and their transdermal solubilities according to the present invention; wherein 9A is a trypan blue soluble transdermal microneedle bright field map; 9B is a dissolution pattern of the ear of the penetrated mouse.

FIG. 10 is a fluorescence image of a near infrared dye IR820 soluble transdermal microneedle of the present invention.

FIG. 11 is a graph of fluorescence contrast of near infrared dye IR820 soluble transdermal microneedles of the present invention with paeonol microneedles.

FIG. 12 is a fluorescence image of near infrared dye IR820 soluble transdermal microneedle penetration into the ear of a mouse according to the present invention.

FIG. 13 is a graph showing experimental results of the in vivo therapeutic effect of paeonol-soluble transdermal microneedles of the present invention; 13A is an ear thickness quantization chart of the mouse model; 13B is a graph of the change in body weight of mice during treatment.

Description of the embodiments

The following describes in detail the embodiments of the present invention with reference to the drawings and examples.

Example 1

A method for preparing paeonol-soluble transdermal microneedles for treating psoriasis, comprising the following steps:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at the solute mass ratio of 1:2 at room temperature, steaming for 15min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ to preheat for 4min, adding the preheated deionized water into the film to carry out ultrasonic treatment for 4min, centrifuging at 2800rpm for 6 min, and removing the medicines with poor dissolubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with 5 ten thousand molecular weight into paeonol hydrophilic micelles in batches, uniformly stirring, and performing ultrasonic treatment for 10 min to completely dissolve the paeonol hydrophilic micelles to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 10 minutes to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 2800rpm for 5min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 1min, placing the mould in a sleeve at 4000rpm for centrifugation for 12 min, scraping the colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, and vacuumizing and centrifuging repeatedly until pinholes are filled;

s6, pouring the solution 230 mg of the micro-needle backing layer after bubble removal into a micro-needle mould, centrifuging at 1800rpm for 4min to enable the bottom of the micro-needle mould to be smooth and bubble-free, drying at 32 ℃ for 75h, and demoulding the micro-needle to obtain the paeonol soluble transdermal micro-needle for treating psoriasis.

Example 2

A method for preparing paeonol-soluble transdermal microneedles for treating psoriasis, comprising the following steps:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at the solute mass ratio of 1:2 at room temperature, steaming for 20min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ for preheating for 5min, adding the preheated deionized water into the film for ultrasonic treatment for 5min, centrifuging at 3000rpm for 5min, and removing the drugs with poor dissolubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with 5 ten thousand molecular weight into paeonol hydrophilic micelles in batches, uniformly stirring, and performing ultrasonic treatment for 10 min to completely dissolve the paeonol hydrophilic micelles to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 10 minutes to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 3000rpm for 3min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 2min, placing the mould in a sleeve 4200rpm for centrifugation for 10 min, scraping the colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, and vacuumizing and centrifuging repeatedly until pinholes are filled;

s6, pouring the solution 230 mg of the micro-needle backing layer after bubble removal into a micro-needle mould, centrifuging at 2000rpm for 3min to enable the bottom of the micro-needle mould to be smooth and bubble-free, drying at 35 ℃ for 72h, and demoulding the micro-needle to obtain the paeonol soluble transdermal micro-needle for treating psoriasis.

Example 3

A method for preparing paeonol-soluble transdermal microneedles for treating psoriasis, comprising the following steps:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at the solute mass ratio of 1:2 at room temperature, steaming for 25min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ to preheat for 5min, adding the preheated deionized water into the film to carry out ultrasonic treatment for 6 min, centrifuging at 3200rpm for 4min, and removing the medicines with poor solubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with 5 ten thousand molecular weight into paeonol hydrophilic micelles in batches, uniformly stirring, and performing ultrasonic treatment for 12 min to completely dissolve the paeonol hydrophilic micelles to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 12 minutes to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 3200rpm for 2min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 2min, placing the mould in a sleeve 4500rpm for centrifugation for 8 min, scraping colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, and vacuumizing and centrifuging repeatedly until pinholes are filled;

s6, pouring the solution 230 mg of the micro-needle backing layer after bubble removal into a micro-needle mould, centrifuging at 2200rpm for 2min to enable the bottom of the micro-needle mould to be smooth and bubble-free, drying at 37 ℃ for 70h, and demoulding the micro-needle to obtain the paeonol soluble transdermal micro-needle for treating psoriasis.

The micro-scale administration channel is formed by using the micro-needle administration technology, the skin barrier is used for penetrating the hyperplastic lesion of the psoriasis horny layer, paeonol is directly delivered to the epidermis layer or the upper dermis layer, the stability and the transdermal absorption efficiency of the medicine are improved, the toxicity and the side effects are reduced, and the effect is very good, and by taking example 2, the related experimental data are as follows:

1. preparation of paeonol hydrophilic micelle

10 mg paeonol is weighed and dissolved in 10 mL methanol to be taken as paeonol methanol solution, 20 mg pluronic F-68 is weighed and dissolved in 10 mL methanol to be taken as pluronic methanol solution, and the two solutions are mixed into a eggplant-shaped bottle to be stirred at room temperature for 4 h. After the reaction is finished, the solution in the eggplant-shaped bottle is distilled at room temperature to remove redundant methanol, and a uniform and transparent film is obtained. Then adding 5mL deionized water into the centrifuge tube and preheating the centrifuge tube and the steamed eggplant-shaped bottle in a water bath kettle at 60 ℃ for 5 min. Adding preheated deionized water into an eggplant-shaped bottle, performing ultrasonic treatment for 5min, centrifuging the obtained solution at 3000rpm for 5min, and removing the excessive medicine with poor solubility to obtain paeonol-loaded micelle solution with the mass concentration of 3 mg/mL. Taking out part of paeonol micelle solution, putting into-80 ℃ for freezing, and then freeze-drying the frozen paeonol hydrophilic micelle by a freeze dryer to prepare paeonol hydrophilic micelle freeze-dried powder.

2. Particle size of paeonol hydrophilic micelle

The particle size distribution histogram is detected by a Nano-ZS dynamic laser particle size analyzer, and the result is shown in figure 1, so that the average particle size is 289nm, the polydispersity coefficient is 0.387, and the dispersibility is good.

3. Paeonol hydrophilic micelle observed by transmission electron microscope

And (3) dripping the paeonol hydrophilic micelle solution on a special copper mesh for a transmission electron microscope to take a picture, wherein the result is shown in figure 2, the pluronic successfully wraps the paeonol, and the particle size of the nanoparticle is 100 nm-200 nm.

4. Infrared spectrum of paeonol hydrophilic micelle

Taking paeonol hydrophilic micelle freeze-dried powder of 1 mg and KBr, fully grinding and uniformly mixing in a mortar, drying for 5min under an infrared baking lamp, adding into a die, repeatedly compacting by a tablet press to prepare tablets with uniform transparent distribution, and detecting in an infrared spectrometer. Paeonol and pluronic F68 tablets were prepared in the same manner. And (5) placing the sample into an infrared spectrometer for detection. Comparing infrared spectra of paeonol, pluronic F68 and paeonol hydrophilic micelle tablet, the results are shown in figure 3, wherein paeonol, pluronic F68 and paeonol hydrophilic micelle are respectively shown from top to bottom, and the paeonol hydrophilic micelle shows that the phenolic hydroxyl group of paeonol is 3400 and 3400 cm ^-1 The stretching vibration peak at this point also shows that the C-O-C bond in pluronic F68 is at 1610 and 1610 cm ^-1 The stretching vibration peak at the position, and after the pluronic F68 wraps paeonol, some characteristic peaks are providedIt became no longer apparent, indicating that pluronic F68 achieved successful encapsulation of paeonol.

5. Paeonol hydrophilic micelle in-vitro hemolysis experiment

Fresh mouse blood (500. Mu.L) was taken, 5. 5mL physiological saline was added thereto, the supernatant was removed by repeated centrifugation, and the pellet was added to 10. 10 mL physiological saline to obtain a red blood cell suspension. 400 mu L of red blood cell suspension and 600 mu L of distilled water are taken as positive control, 400 mu L of red blood cell suspension and 600 mu L of physiological saline are taken as negative control, paeonol hydrophilic micelle solution is respectively diluted to 500 mu g/mL, 400 mu g/mL, 300 mu g/mL, 200 mu g/mL and 100 mu g/mL by physiological saline, each concentration volume is 600 mu L, 400 mu L of red blood cell suspension is added in each concentration, all groups are incubated at the constant temperature of 37 ℃ for 4 h, 10000g are centrifuged for 5min, the supernatant is taken and added into a 96-well plate, and the absorbance of each group at 577 nm is detected by an enzyme-labeling instrument, and the hemolysis rate is calculated.

Wherein, the hemolysis ratio= (Abs experimental group-Abs negative group)/(Abs positive group-Abs negative group) ×100%.

As shown in figure 4, the haemolysis rates of paeonol hydrophilic micelles in different concentration groups are all lower than 10%, which shows that the paeonol hydrophilic micelles have good in-vitro biosafety, and provide a basis for the subsequent use of the paeonol hydrophilic micelles in a mouse psoriasis model.

6. Paeonol hydrophilic micelle cytotoxicity experiment on HUVEC

The cultured HUVEC cells were collected and prepared into 5X 10 cells after cell counting ⁴ The cell concentration per mL was inoculated in a 96-well plate at 100. Mu.L/well, and 5% CO was added ₂ Incubate 24 h at 37 ℃. Discarding original culture medium, adding fresh culture medium containing paeonol hydrophilic micelle with concentration of 50 μg/mL, 100 μg/mL, 200 μg/mL, 300 μg/mL, 400 μg/mL and 500 μg/mL, respectively, and adding 5% CO ₂ The incubator at 37℃continued to incubate 24 h. The culture medium containing paeonol hydrophilic micelle is discarded, and the volume ratio of the MTT PBS solution with the volume ratio of 5 mg/mL to the culture medium is 1: 9, after fully mixing, adding 100 mu L/hole into a pore plate, continuously incubating 2.5 h of the waste liquid, adding 150 mu L of DMSO into each hole, vibrating for 5min, detecting absorbance at 490 nm by using an enzyme-labeling instrument, taking absorbance of cells which are not treated as a control group, and calculating the pair of the paeonol hydrophilic micelles with different concentrations to HUVEInfluence of C-cell Activity.

As shown in FIG. 5, the HUVEC cells were treated with paeonol hydrophilic micelles of different concentrations 24 to h, and the cell viability was not significantly reduced, indicating that the paeonol hydrophilic micelles were less toxic.

7. Preparation and morphology of paeonol-soluble transdermal microneedle

600 mg of sodium hyaluronate with 5 ten thousand molecular weight is added to 3 mg/mL paeonol hydrophilic micelle EP tube in portions, each addition is vigorously stirred by a 1 mL gun head, and the mixture is completely dissolved by ultrasound for 10 min to serve as a matrix. 400 mg of pullulan EP tube with 20 ten thousand molecular weight is added to 2 mL deionized water in portions, each of which is vigorously stirred with a 1 mL gun head, and the mixture is completely dissolved as a backing layer by ultrasonic treatment for 10 min. The matrix solution and the backing layer solution were placed in a centrifuge and centrifuged at 3000rpm for 3min, and subjected to degassing and bubbling treatment. Then adding the matrix material into a microneedle mould, vacuumizing, centrifuging at 4200rpm for 10 min, scraping off the redundant matrix material with bubbles, supplementing new matrix material, and centrifuging under repeated vacuumizing until the pinholes are filled. Filling 230 mg backing layer material into a microneedle mould, centrifuging at 2000rpm for 3min to make the bottom of the microneedle smooth and bubble-free, drying in a 37 ℃ oven for 72h, and demoulding to obtain paeonol hydrophilic micelle microneedle.

The paeonol-soluble transdermal microneedles obtained through experiments are shown in fig. 6, and the transdermal microneedles are arranged in a needle array.

8. Preparation and morphology of doxorubicin micropins

600 mg of 5-ten thousand molecular weight sodium hyaluronate was added in portions to an EP tube of 1 mg/mL doxorubicin solution, each with vigorous stirring at a 1 mL tip, and sonicated for 10 min to completely dissolve as matrix. 400 mg of pullulan EP tube with 20 ten thousand molecular weight is added to 2 mL of ultrapure water in portions, each of which is vigorously stirred by a 1 mL gun head, and the mixture is completely dissolved by ultrasonic treatment for 10 minutes to serve as a backing layer. The EP tube was placed in a centrifuge and centrifuged at 3000rpm for 3min for de-bubbling treatment. Then adding the matrix material into a microneedle mould, vacuumizing, centrifuging at 4200rpm for 10 min, scraping off the redundant matrix material with bubbles, supplementing new matrix material, and centrifuging under repeated vacuumizing until the pinholes are filled. Filling the backing layer material into a microneedle mould, centrifuging at 2000rpm for 3min to make the bottom of the microneedle smooth and bubble-free, drying in a 37 ℃ oven for 72h, and demoulding to obtain the doxorubicin microneedle.

The experiment shows that doxorubicin in a needle array arrangement instead of paeonol-soluble transdermal microneedles are shown in fig. 7, and the whole microneedle exhibits vivid red color. As shown in FIG. 8, under the excitation of ultraviolet light, the red-violet fluorescence is presented, and the uniform distribution of the medicine can be more intuitively observed through a fluorescence picture, so that the appearance of the needle body is complete.

9. Preparation and morphology of trypan blue microneedle

600 mg of sodium hyaluronate with a molecular weight of 5 ten thousand was added in portions to an EP tube of an aqueous solution of trypan blue of 5 mg/mL, each of which was vigorously stirred with a 1 mL gun head, and sonicated for 10 min to completely dissolve as a matrix. 400 mg of pullulan EP tube with 20 ten thousand molecular weight is added to 2 mL of ultrapure water in portions, each of which is vigorously stirred by a 1 mL gun head, and the mixture is completely dissolved by ultrasonic treatment for 10 minutes to serve as a backing layer. The EP tube was placed in a centrifuge and centrifuged at 3000rpm for 3min for de-bubbling treatment. Then adding the matrix material into a microneedle mould, vacuumizing, centrifuging at 4200rpm for 10 min, scraping off the redundant matrix material with bubbles, supplementing new matrix material, and centrifuging under repeated vacuumizing until the pinholes are filled. Filling 230 mg backing layer material into a microneedle mould, centrifuging at 2000rpm for 3min to make the bottom of the microneedle smooth and bubble-free, drying in a 37 ℃ oven for 72h, and demoulding to obtain the trypan blue soluble transdermal microneedle.

The trypan blue soluble transdermal microneedles obtained by the experiment are shown in fig. 9A, and the transdermal microneedles are arranged in a needle array.

10. Trypan blue microneedle solubility test

To better show the solubility of the microneedles in subcutaneous tissues, the microneedles were prepared using trypan blue instead of nano drug-loaded micelle solution, and dissolved in the ears of mice, after one Balb/c female mouse was anesthetized, the ears of mice were wetted with a cotton ball containing PBS for 10 min, then the ear surface liquid was wiped with absorbent paper, and a trypan blue microneedle patch cut 1/4 was fixed on the microneedle syringe, and after pricking into the ears of mice, the substrate was dissolved by fixing for 10 min, and the dissolution of the microneedles was observed.

The results are shown in fig. 9B, and the mouse ears show blue microneedle arrays, which indicate that the microneedles prepared by trypan blue instead of the nano drug-loaded micelle solution can be dissolved transdermally, and provide a basis for the subsequent release of drugs from paeonol microneedles through the stratum corneum of the skin to treat psoriasis.

11. Preparation and morphology of near infrared dye IR820 soluble transdermal microneedle

600 mg of sodium hyaluronate with a molecular weight of 5 ten thousand was added in portions to 50. Mu.g/mL of IR820 solution EP tube under light-shielding conditions, each addition was vigorously stirred with a 1 mL gun head, and sonicated for 10 min to completely dissolve as matrix. 400 mg of pullulan EP tube with 20 ten thousand molecular weight is added to 2 mL deionized water in portions, each of which is vigorously stirred with a 1 mL gun head, and the mixture is completely dissolved as a backing layer by ultrasonic treatment for 10 min. The EP tube was placed in a centrifuge and centrifuged at 3000rpm for 3min for de-bubbling treatment. Then adding the matrix material into a microneedle mould, vacuumizing, centrifuging at 4200rpm for 10 min, scraping off the redundant matrix material with bubbles, supplementing new matrix material, and centrifuging under repeated vacuumizing until the pinholes are filled. Filling 230 mg backing layer material into a microneedle mould, centrifuging at 2000rpm for 3min to make the bottom of the microneedle smooth and bubble-free, drying in a 37 ℃ oven for 72h, and demoulding to obtain the near infrared dye IR820 soluble transdermal microneedle.

The fluorescence image of the near infrared dye IR820 soluble transdermal microneedle is shown in fig. 10, indicating that IR820 was successfully prepared as a microneedle patch. The comparison of the near infrared dye IR820 soluble transdermal microneedle and paeonol soluble transdermal microneedle is shown in FIG. 11, which shows that the near infrared dye IR820 soluble transdermal microneedle has better fluorescence stability.

12. Near infrared dye IR820 soluble transdermal microneedle solubility performance experiment

The solubility of the microneedle in subcutaneous tissue is displayed by utilizing the living body imaging of a small animal, the near infrared dye IR820 is used for replacing nano drug-loaded micelle solution to prepare the microneedle, the microneedle is dissolved in the ear of the mouse, the change of the ear of the mouse and the dissolution condition of the IR820 microneedle are observed, after one Balb/c female mouse is anesthetized, the ear of the mouse is moistened for 10 min by utilizing a cotton ball containing PBS, then the surface liquid of the ear is wiped by using absorbent paper, the IR820 microneedle patch cut by 1/4 is fixed on a microneedle injector, and the substrate is dissolved by fixing for 10 min after the microneedle patch is pricked into the ear of the mouse, and the dissolution condition of the microneedle is observed by the living body imaging of the small animal.

As shown in figure 12, the mouse ear shows fluorescence, which shows that the near infrared dye IR820 soluble transdermal microneedle is effectively absorbed by skin tissue, and shows that the microneedle prepared by the matrix of 5 ten thousand molecular weight sodium hyaluronate and the backing layer of 20 ten thousand molecular weight pullulan can be dissolved and released into the skin to release medicine, thus laying foundation for treating psoriasis by paeonol micro-needle.

13. Paeonol-soluble transdermal microneedle in vivo treatment effect experiment

16 coholic female Balb/c mice were prepared, 4 of which were normal healthy mice, as a control group. The remaining 12 mice were randomly divided into 3 groups of 4, each group being a model group, a paeonol hydrophilic micelle treatment group and a paeonol soluble transdermal microneedle treatment group, and the right ear of the anesthetized mice was smeared with imiquimod ointment, and the psoriasis model was constructed while being treated.

The mice were anesthetized on days 2, 4, and 6 after the first application of imiquimod, the ears of the mice were moistened with cotton balls containing PBS for 10 min, then the ear surface liquids were rubbed with absorbent paper, and paeonol hydrophilic micelle application and paeonol microneedle administration were performed on the lesions of the mice in the paeonol hydrophilic micelle treatment group and the paeonol soluble transdermal microneedle treatment group, respectively. Ear thickness, ear inflammation and body weight were recorded daily for each group of mice.

The thickness results of the ears of the mice are shown in fig. 13A, after 8 days of treatment, the severity of psoriasis lesions in the model group is increased, the skin horny layer of the ears of the mice is proliferated, the skin of the ears is thickened, and the thickness of the ears of the mice treated by the paeonol hydrophilic micelle treatment group and the paeonol soluble transdermal microneedle treatment group is obviously lower than that of the model group, which indicates that the lesions are better recovered. Compared with a paeonol hydrophilic micelle treatment group, the paeonol soluble transdermal microneedle treatment group has better inhibition degree of mouse lesion, the treatment effect is more similar to that of a control group, and the paeonol soluble transdermal microneedle patch on the surface forms a micron-sized administration channel by penetrating a superficial epidermis barrier, so that paeonol is directly delivered to an epidermis layer or an upper dermis layer, the stability and the transdermal absorption efficiency of a medicine are improved, the transdermal administration rate is improved by 47.1%, and the aim of accurately treating psoriasis by paeonol is fulfilled.

The results of the ear inflammation of the mice are shown in fig. 13B, the body weight of the mice in the model group is gradually reduced, which indicates that the health condition is gradually reduced, and the health degree of the mice in the paeonol hydrophilic micelle treatment group and the paeonol soluble transdermal microneedle treatment group is good, which indicates that both administration modes have lower toxic and side effects and in vivo safety.

Meanwhile, the experiment is carried out on the embodiment 2, and the same experiment is carried out on the embodiments 1 and 3, so that the same and similar results as those of the embodiment 2 are obtained, and the results are not listed here, so that the invention has good effect and stable and reliable curative effect.

In summary, the paeonol-soluble transdermal microneedle for treating psoriasis can be prepared into paeonol microneedle patches by adjusting the size of a diseased area. Increase Rong Cailiao pluronic to improve paeonol water solubility, and prepare paeonol hydrophilic micelle by using a film hydration method, so as to reduce irritation caused by methanol. The paeonol hydrophilic micelle is prepared into the microneedle patch, and the microneedle patch is prevented from contacting important nerves and capillaries in epidermis while penetrating a shallow epidermis barrier to form a micron-sized administration channel, and penetrates a psoriasis horny layer hyperplasia lesion through the shallow epidermis barrier, so that the paeonol is directly delivered to an epidermis layer or an upper dermis layer, the stability and transdermal absorption efficiency of a medicine are improved, the transdermal administration rate is improved by 47.1%, the medicine compliance of a patient is improved, the aim of accurately treating psoriasis by the paeonol is fulfilled, the paeonol is a great innovation on a medicine formulation for treating psoriasis, the technical support is provided for the medicine formulation for treating psoriasis, and the social and economic benefits are remarkable.

It is noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and the present invention is not limited thereto, and that any person skilled in the art can make modifications or variations within the scope of the present invention without departing from the scope of the present invention.

Claims (5)

1. A method for preparing paeonol-soluble transdermal microneedles for treating psoriasis, which is characterized by comprising the following steps:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at a solute mass ratio of 1:2 at room temperature, steaming for 15-25min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ to preheat for 4-5 min, adding the preheated deionized water into the film for ultrasonic treatment for 4-6 min, centrifuging for 4-6 min at 2800-3200rpm, and removing excessive drugs with poor solubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with molecular weight of 5 ten thousand into paeonol hydrophilic micelle with concentration of 2 mL of 3 mg/mL, stirring uniformly, and performing ultrasonic treatment for 10-12 min to completely dissolve the paeonol hydrophilic micelle to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 10-12 min to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 2800-3200rpm for 2-5min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 1-2min, placing the mould in a sleeve at 4000-4500rpm for centrifugation for 8-12 min, scraping the colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, vacuumizing repeatedly, and centrifuging until pinholes are filled;

s6, pouring the solution 230 mg of the backing layer of the micro needle after bubble removal into a micro needle mould, centrifuging at 1800-2200rpm for 2-4min to enable the bottom of the micro needle mould to be flat and bubble-free, drying at 32-37 ℃ for 70-75h, and demoulding the micro needle to obtain the paeonol soluble transdermal micro needle for treating psoriasis.

2. A method for preparing paeonol-soluble transdermal microneedles for treating psoriasis according to claim 1, comprising the steps of:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at the solute mass ratio of 1:2 at room temperature, steaming for 15min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ to preheat for 4min, adding the preheated deionized water into the film to carry out ultrasonic treatment for 4min, centrifuging at 2800rpm for 6 min, and removing the medicines with poor dissolubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with 5 ten thousand molecular weight into paeonol hydrophilic micelles in batches, uniformly stirring, and performing ultrasonic treatment for 10 min to completely dissolve the paeonol hydrophilic micelles to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 10 minutes to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 2800rpm for 5min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 1min, placing the mould in a sleeve at 4000rpm for centrifugation for 12 min, scraping the colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, and vacuumizing and centrifuging repeatedly until pinholes are filled;

s6, pouring the solution 230 mg of the micro-needle backing layer after bubble removal into a micro-needle mould, centrifuging at 1800rpm for 4min to enable the bottom of the micro-needle mould to be smooth and bubble-free, drying at 32 ℃ for 75h, and demoulding the micro-needle to obtain the paeonol soluble transdermal micro-needle for treating psoriasis.

3. A method for preparing paeonol-soluble transdermal microneedles for treating psoriasis according to claim 1, comprising the steps of:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at the solute mass ratio of 1:2 at room temperature, steaming for 20min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ for preheating for 5min, adding the preheated deionized water into the film for ultrasonic treatment for 5min, centrifuging at 3000rpm for 5min, and removing the drugs with poor dissolubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with 5 ten thousand molecular weight into paeonol hydrophilic micelles in batches, uniformly stirring, and performing ultrasonic treatment for 10 min to completely dissolve the paeonol hydrophilic micelles to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 10 minutes to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 3000rpm for 3min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 2min, placing the mould in a sleeve 4200rpm for centrifugation for 10 min, scraping the colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, and vacuumizing and centrifuging repeatedly until pinholes are filled;

s6, pouring the solution 230 mg of the micro-needle backing layer after bubble removal into a micro-needle mould, centrifuging at 2000rpm for 3min to enable the bottom of the micro-needle mould to be smooth and bubble-free, drying at 35 ℃ for 72h, and demoulding the micro-needle to obtain the paeonol soluble transdermal micro-needle for treating psoriasis.

4. A method for preparing paeonol-soluble transdermal microneedles for treating psoriasis according to claim 1, comprising the steps of:

s1, uniformly mixing paeonol methanol solution with the mass concentration of 1 mg/mL and pluronic F68 methanol solution with the mass concentration of 2mg/mL at the solute mass ratio of 1:2 at room temperature, steaming for 25min to remove methanol to obtain a uniform and transparent film, respectively placing deionized water and the film in a water bath kettle at 60 ℃ to preheat for 5min, adding the preheated deionized water into the film to carry out ultrasonic treatment for 6 min, centrifuging at 3200rpm for 4min, and removing the medicines with poor solubility to obtain paeonol hydrophilic micelles with the mass concentration of 3 mg/mL;

s2, mixing sodium hyaluronate 600 mg with 5 ten thousand molecular weight into paeonol hydrophilic micelles in batches, uniformly stirring, and performing ultrasonic treatment for 12 min to completely dissolve the paeonol hydrophilic micelles to obtain a microneedle matrix solution;

s3, adding deionized water into the pullulan with the molecular weight of 20 ten thousand, stirring and dissolving, and performing ultrasonic treatment for 12 minutes to completely dissolve the pullulan to obtain a microneedle backing layer solution with the mass concentration of 200 mg/mL;

s4, respectively centrifuging the microneedle substrate solution and the microneedle backing layer solution at 3200rpm for 2min to remove bubbles;

s5, pouring the micro-needle matrix solution with bubbles removed into a micro-needle mould to fill the surface, vacuumizing for 2min, placing the mould in a sleeve 4500rpm for centrifugation for 8 min, scraping colloid with bubbles, pouring the micro-needle matrix solution into the mould to fill the surface, and vacuumizing and centrifuging repeatedly until pinholes are filled;

s6, pouring the solution 230 mg of the micro-needle backing layer after bubble removal into a micro-needle mould, centrifuging at 2200rpm for 2min to enable the bottom of the micro-needle mould to be smooth and bubble-free, drying at 37 ℃ for 70h, and demoulding the micro-needle to obtain the paeonol soluble transdermal micro-needle for treating psoriasis.

5. Use of a paeonol-soluble transdermal microneedle according to any one of claims 1-4 for the manufacture of a medicament for the treatment of psoriasis.