A hydrogel of *Callicarpa nudiflora* loaded with aprestil, its preparation method and application

Abstract

This invention provides an apremilast-loaded hydrogel of *Callicarpa nudiflora* vesicles, its preparation method, and its application, relating to the field of pharmaceutical formulation technology. This invention utilizes *Callicarpa nudiflora* vesicles to encapsulate apremilast and then reconstitute it into a hydrogel, preparing a drug that can be directly applied to the affected skin of psoriasis patients via local transdermal administration. The hydrogel achieves synergistic treatment of psoriasis through apremilast and *Callicarpa nudiflora* vesicles, improving efficacy.

Description

Technical Field

[0001] This application relates to the field of pharmaceutical formulation technology, specifically to an aprestiger-loaded hydrogel of Callicarpa nudiflora, its preparation method, and its application. Background Technology

[0002] Psoriasis is an autoimmune inflammatory disease of the epidermis and dermis, characterized by epidermal hyperplasia accompanied by dermal telangiectasia. Clinical symptoms include itching, swelling, discomfort, and erythematous plaques composed of silvery scales. Unlike normal skin, due to abnormal excessive epidermal proliferation, parakeratosis, and preservation of keratinocyte nuclei, affected skin presents with typical scaly plaques. From a histopathological perspective, psoriatic skin exhibits epidermal hyperplasia, abnormal keratinocyte differentiation, excessive angiogenesis, and inflammatory infiltration. In recent years, psoriasis has been considered a systemic disease, often accompanied by psychological, metabolic, and cardiovascular complications. Plaque psoriasis is the most common phenotype, accounting for approximately 85%–90% of psoriasis patients.

[0003] Apremilast is a phosphodiesterase-4 (PDE4) specific inhibitor, approved by the FDA in 2014 and in China in 2021 for the treatment of moderate to severe plaque psoriasis. Its mechanism of action involves inhibiting PDE4, which increases intracellular cyclic adenosine monophosphate (cAMP) concentration, thereby activating protein kinase A (PKA) and transcription factors, ultimately reducing the levels of pro-inflammatory mediators such as interleukin-23, tumor necrosis factor, and interferon, thus alleviating skin inflammation symptoms.

[0004] Callicarpa nudiflora, a plant of the Verbenaceae family, has traditional medicinal properties including anti-inflammatory, detoxifying, dampness-resolving, and hemostatic effects. Current research indicates that Callicarpa nudiflora extract can upregulate apoptosis-related proteins (such as Caspase 3 and Bax) and downregulate anti-apoptotic proteins (such as BCL-2), inducing cell apoptosis by affecting the expression of related genes, thus showing potential pharmacological activity in the treatment of psoriasis.

[0005] Although apremilast has a proven efficacy in treating psoriasis and its current oral tablet form is convenient, its clinical application still faces significant challenges. Oral apremilast distributes throughout the body, easily causing systemic adverse reactions, primarily including headache, nausea, diarrhea, nasopharyngitis, and upper respiratory tract infections, which severely impact patient adherence and quality of life. To reduce systemic toxicity, topical administration has emerged as a potential alternative strategy. However, the stratum corneum at psoriatic lesions is often abnormally thickened and dense, forming a robust physical barrier that makes it difficult for conventional drug formulations to penetrate the skin and reach deeper lesions, resulting in low local bioavailability and limited efficacy.

[0006] In summary, existing single-drug administration methods are insufficient to balance efficacy and safety. Therefore, developing a novel drug delivery strategy or formulation system that can effectively avoid the systemic adverse reactions associated with oral apremilast, overcome the skin's stratum corneum barrier to achieve efficient transdermal drug delivery, and fully utilize the anti-inflammatory activity of traditional Chinese medicine for synergistic effects is of significant clinical importance and application value for improving the clinical treatment of psoriasis and enhancing patient prognosis. Summary of the Invention

[0007] To address the aforementioned issues, this application provides an apremilast-loaded hydrogel of Callicarpa nudiflora, its preparation method, and its application. This method constructs a quadruple synergistic mechanism of "nano-transdermal, amorphous solubilization, synergistic drug efficacy, and sustained gel release," resulting in significant reduction of toxicity and enhanced efficacy.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention aims to provide an apremilast-loaded Callicarpa nudiflora vesicle hydrogel, its preparation method, and its application. The hydrogel achieves deep penetration and long-lasting release through local transdermal drug delivery, and synergistically enhances the therapeutic effect of apremilast, Callicarpa nudiflora vesicles, and hydrogel matrix.

[0010] A hydrogel containing aprescribing agent, comprising aprescribing agent-loaded *Callicarpa variegata* vesicles (APST-EV) and a hydrogel matrix; wherein the aprescribing agent-loaded *Callicarpa variegata* vesicles are dispersed in a three-dimensional network structure of the hydrogel matrix; wherein the mass ratio of the aprescribing agent-loaded *Callicarpa variegata* vesicles to the hydrogel matrix is ​​1:(5-10); the aprescribing agent loading in the aprescribing agent-loaded *Callicarpa variegata* vesicles is 1.49%-17.14%, and the encapsulation efficiency is 6.03%-41.49%.

[0011] Furthermore, the aprestin-loaded *Callicarpa nudiflora* vesicles are nanocomposites formed by the recombination of aprestin raw material and extracellular vesicles (EVs) derived from *Callicarpa nudiflora* through ultrasonic treatment and incubation; the aprestin exists in an amorphous form within the lipid bilayer structure of the extracellular vesicles derived from *Callicarpa nudiflora*; the average particle size of the aprestin-loaded *Callicarpa nudiflora* vesicles is 180-200 nm, and the polydispersity index (PDI) is less than 0.3.

[0012] Furthermore, the hydrogel matrix is ​​formed by neutralization and crosslinking of a 2%–7% (w / v) carbomer 940 solution; the hydrogel has a porous network structure and a pH value of 6–7.

[0013] The present invention also provides a method for preparing the above-mentioned aprestin-loaded *Callicarpa nudiflora* vesicle hydrogel, comprising the following steps: S1, extraction of carrier: extracellular vesicles (EVs) derived from *Callicarpa nudiflora* leaves are isolated and purified; S2, drug loading: aprestin is loaded into the EVs using ultrasound-assisted incubation technology to obtain aprestin-loaded *Callicarpa nudiflora* vesicles; S3, molding: a blank hydrogel matrix is ​​prepared, and the aprestin-loaded *Callicarpa nudiflora* vesicles are mixed evenly with the blank hydrogel matrix to obtain the final product.

[0014] Furthermore, in step S1, the separation and purification are performed using a combination of differential centrifugation and sucrose density gradient centrifugation. The differential centrifugation step includes: sequentially centrifuging at 1,000×g for 15 min, 5,000×g for 30 min, and 10,000×g for 60 min, and then taking the supernatant for centrifugation at 150,000×g for 2 h. The sucrose density gradient centrifugation collects bands in the 30%–45% sucrose density range.

[0015] Further, in step S2, the specific operation is as follows: Aprestin is dissolved in an organic solvent and mixed with EVs at a mass ratio of 1:(0.5-20); the ultrasonic treatment power is 10-40W and the time is 2-8min, using the ultrasonic effect to temporarily change the permeability of the vesicle membrane; then it is incubated at 37℃ for 1-3h to restore the integrity of the vesicle membrane; finally, the organic solvent and unencapsulated free aprestin are removed.

[0016] Furthermore, in step S3, the preparation of the blank hydrogel matrix includes dispersing carbomer powder in water to swell; after mixing evenly, the step further includes adding triethanolamine to adjust the pH value to 6-7 to induce the formation of a semi-solid structure in the gel matrix.

[0017] The present invention also provides the use of the above-mentioned Apster-loaded Callicarpa nudiflora vesicle hydrogel in the preparation of a drug for treating psoriasis.

[0018] Furthermore, the drug is a topical transdermal drug delivery formulation that utilizes the transdermal properties of Callicarpa nudiflora vesicles and the sustained-release characteristics of hydrogel to synergistically exert anti-inflammatory and immunomodulatory effects with apremilast.

[0019] In a first aspect, the present invention provides a hydrogel of *Callicarpa nudiflora* vesicles loaded with aprestil, the hydrogel comprising *Callicarpa nudiflora* vesicles loaded with aprestil and a blank gel matrix.

[0020] Secondly, the present invention provides a method for preparing Apstar-loaded vesicles of *Callicarpa nudiflora*, comprising the following steps: Aprestin was dissolved in anhydrous ethanol and mixed with vesicles of Callicarpa nudiflora at a certain feeding ratio. The mixture was then sonicated to allow the aprestin to enter the vesicles. After incubation at 37°C to restore the integrity of the vesicles, the mixture was stirred at 45°C for 30 min to evaporate the anhydrous ethanol and precipitate the free drug. The precipitated drug was removed by filtration to obtain vesicles of Callicarpa nudiflora loaded with aprestin.

[0021] Carbomer 940 powder was evenly sprinkled on the surface of ultrapure water and allowed to swell to obtain a blank gel matrix.

[0022] Aprester-loaded *Callicarpa nudiflora* vesicles were mixed with a blank gel and the pH was adjusted to 6-7 to prepare aprester-loaded *Callicarpa nudiflora* vesicle hydrogel.

[0023] Optionally, the ratio of Apstar to Callicarpa nudiflora vesicles is 1:(0.5-20), preferably 1:2.

[0024] Optionally, the ultrasonic treatment is performed by a probe ultrasonic instrument at 10-40W for 2-8 minutes, with a 5-second pause after every 5 seconds of operation, preferably 4 minutes at 20W.

[0025] Optionally, the incubation time at 37°C to restore the integrity of the vesicles of the naked flower *Callicarpa* is 1-3 hours, preferably 1 hour.

[0026] Optionally, the drug loading of the aprescribing vesicles of *Callicarpa nudiflora* is 1.49%–17.14%, and the encapsulation rate is 6.03%–41.49%, preferably 17.14% and 41.49%.

[0027] Optionally, the filter membrane used in the filtration process has a pore size of 450 nm.

[0028] Optionally, the concentration of the blank gel matrix is ​​2%–7%, preferably 5%.

[0029] Optionally, the mass ratio of the blank gel matrix to the Apstar-loaded vesicles of *Callicarpa nudiflora* is 1:(5-10), preferably 1:5.

[0030] Optionally, the reagent used to adjust the pH is triethanolamine, which can neutralize the acidity of the blank gel matrix and thicken it.

[0031] Ultrasound is a common method for loading drugs into vesicles, and it has a good loading effect on a variety of drugs. The effect of ultrasound temporarily changes the structure of the lipid bilayer of the vesicle, increasing the permeability of the membrane. Drug molecules enter the interior of the vesicle through passive diffusion. This change in membrane structure is usually reversible, and the integrity of the vesicle membrane can be restored after ultrasound treatment.

[0032] Thirdly, the present invention provides the application of apastin-loaded nude artichoke vesicle hydrogel in the preparation of a drug for treating psoriasis.

[0033] The Aprestigmine-loaded Callicarpa nudiflora vesicle hydrogel provided by this invention can reduce drug dosage, improve efficacy, and reduce side effects when used topically to treat psoriasis. In vivo pharmacodynamic studies have shown that the hydrogel of this invention significantly improves psoriatic skin lesions in mice (significantly reduced PASI score) while effectively reducing spleen coefficient, avoiding systemic immune responses and organ toxicity caused by oral administration, and has a safety profile superior to oral administration and simple drug gels.

[0034] Compared to ordinary drug gels, this invention utilizes nanovesicles to achieve highly efficient transdermal drug delivery and uses a gel matrix to achieve long-lasting sustained release for 24 hours (cumulative release rate of approximately 50%), solving the problems of high dosing frequency and low bioavailability.

[0035] The core principle behind this application in solving the problem lies in constructing a synergistic mechanism of "nanotransdermal - amorphous solubilization - drug efficacy synergy - gel sustained release": Nanocarrier transdermal mechanism (penetration): Utilizing the natural lipid bilayer structure and nanoscale size (approximately 180-200 nm) of extracellular vesicles (EVs) derived from Callicarpa nudiflora, it can mimic the properties of biological membranes and efficiently penetrate the dense stratum corneum barrier like a "Trojan horse" to deliver drugs to the dermis.

[0036] Amorphous solubilization mechanism (absorption): Using ultrasonic drug delivery technology, the poorly soluble drug apremilast is encapsulated in the hydrophilic core of vesicles or lipid bilayer, transforming it from a crystalline state to an amorphous state (confirmed by DSC and XRD), which significantly improves the drug's solubility and dissolution rate.

[0037] Synergistic mechanism of action (enhanced effect): Apremilast, as a PDE4 inhibitor, blocks the inflammatory pathway; the vesicles of Callicarpa nudiflora inherit the biological activity of the parent drug and can regulate apoptosis proteins (upregulate Caspase3 and Bax, downregulate BCL-2) to induce apoptosis in psoriatic cells. The combination of the two exerts a synergistic anti-inflammatory effect of "chemical drug + biological carrier".

[0038] Gel-based sustained-release mechanism (long-lasting): The three-dimensional network structure of carbomer hydrogel provides a physical barrier to delay drug release; at the same time, its excellent bioadhesion ensures that the drug remains on the surface of the lesion for a long time, reducing the frequency of administration.

[0039] The ultrasonic drug delivery process determined in this invention is simple and efficient, and the resulting formulation has a uniform particle size distribution. It can form a stable gel formulation at a wide range of matrix concentrations (2%-7%), and has good prospects for clinical translation. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 The structural formula for Apsted; Figure 2 An image showing the appearance of the Apstar-loaded Callicarpa nudiflora vesicle hydrogel prepared in Example 1; Figure 3 A scanning electron microscope image of the Apstar-loaded hydrogel of *Callicarpa nudiflora* vesicles prepared in Example 1; Figure 4 This is a differential scanning calorimetry (DSC) graph of the sample during performance testing in a specific implementation method. Figure 5 The image shows an X-ray diffraction pattern of the sample during performance testing in a specific implementation method. Figure 6 The cumulative release percentage of APST-EV and APST-EV-Gel prepared in Example 1 is used in the performance test of the specific implementation method.

[0042] Figure 7 This is an image showing the skin lesion status of mice in a pharmacodynamic study of a specific implementation method. Figure 8-11 A scoring chart of mouse skin lesion status in a pharmacodynamic study of a specific implementation method; Figure 12-13 The accompanying diagrams show the observation of mouse spleen status and the statistical chart of mouse spleen coefficients in the pharmacodynamic study, illustrating the specific implementation method. Figure 12 This is an image showing the condition of a mouse spleen. Figure 13 This is a statistical graph of the spleen coefficient in mice during a pharmacodynamic study. Detailed Implementation

[0043] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0044] This invention provides a hydrogel of *Callicarpa nudiflora* vesicles loaded with aprestin, the hydrogel comprising *Callicarpa nudiflora* vesicles loaded with aprestin and a blank gel matrix, wherein the mass ratio of the *Callicarpa nudiflora* vesicles loaded with aprestin to the blank gel matrix is ​​1:(5-10), the aprestin loading in the *Callicarpa nudiflora* vesicles is 1.49%-17.14%, and the encapsulation efficiency is 6.03%-41.49%.

[0045] In some embodiments, the mass ratio of the Apstar-loaded nude artichoke vesicles to the blank gel matrix is ​​preferably 1:5.

[0046] In some embodiments, the aprepitant loading in the vesicles of the naked flower *Callicarpa* is preferably 17.14%.

[0047] In some embodiments, the encapsulation rate of apres in the vesicles of the naked flower violet is preferably 41.49%.

[0048] This invention also provides a method for preparing a hydrogel of *Callicarpa nudiflora* vesicles loaded with aprestil, comprising the following steps: S1. Dissolve apremilast in anhydrous ethanol, mix with the vesicles of Callicarpa nudiflora, treat with an ultrasonic instrument, incubate at 37°C, and finally remove the ethanol by rotary evaporation to precipitate the free drug. Filter to remove the free drug to obtain vesicles of Callicarpa nudiflora loaded with apremilast.

[0049] S2. Carbomer 940 powder is evenly dispersed on the water surface and allowed to swell to obtain a blank gel matrix. The Apres-loaded *Callicarpa nudiflora* vesicles are mixed with the blank gel matrix, and triethanolamine is added to adjust the pH to 6-7 to obtain an Apres-loaded *Callicarpa nudiflora* vesicle hydrogel.

[0050] In some embodiments, when performing step S1, the ultrasonic treatment includes: processing with a probe ultrasonic instrument at 10w-40w for 2min-8min, stopping for 5s every 5s, preferably processing with 20w for 4min.

[0051] In some embodiments, when performing step S1, the incubation time at 37°C is 1-3 hours, preferably 2 hours.

[0052] In some embodiments, when performing step S1, the rotary evaporation temperature is 45°C and the time is 30 minutes.

[0053] In some embodiments, when performing step S1, the filtration device is a 450nm filter membrane.

[0054] In some embodiments, since apremilast is insoluble in water, the ethanol in the solution is removed by rotary evaporation at 45°C for 30 minutes to precipitate free apremilast, which is then removed by passing it through a 450nm filter membrane.

[0055] In some embodiments, when performing step S2, the mass ratio of the Apstar-loaded nude Callicarpa vesicles to the blank gel matrix is ​​1:(5-10), preferably 1:5.

[0056] The Apres used in this embodiment of the invention was purchased from Jiangxi Qingfeng Pharmaceutical Co., Ltd.; the Callicarpa nudiflora used in this embodiment of the invention was purchased from Jiangxi Puzheng Pharmaceutical Co., Ltd.; and the Carbomer 940 used in this embodiment of the invention was purchased from Shanghai Yi'en Chemical Reagent Co., Ltd.

[0057] Example 1 Example 1 of this invention provides a method for preparing Apstar-loaded Callicarpa nudiflora vesicular hydrogel (APST-EV-Gel), comprising the following steps: S1. Dissolve 2 mg of apremilast (APST) in 1 ml of anhydrous ethanol to prepare a 2 mg / ml APST solution. Mix 0.5 ml of the APST solution with 1 ml of 1 mg / ml Callicarpa nudiflora vesicles (EV). The mass ratio of APST to EV is 1:2. Treat the mixture with a probe sonicator at 20 W for 4 min, incubate at 37 °C for 1 h, and finally stir at 45 °C for 30 min to evaporate the ethanol, allowing the free drug to precipitate. Remove the free drug through a 0.45 μm filter membrane to obtain apremilast-loaded Callicarpa nudiflora vesicles (APST-EV).

[0058] S2. Take 5g of carbomer 940 powder and sprinkle it evenly on the surface of 100ml of ultrapure water. Let it stand and swell to obtain a 5% blank gel matrix.

[0059] The 5% blank gel matrix was mixed with the aprestin-loaded *Callicarpa nudiflora* vesicles at a mass ratio of 1:5. The pH of the system was adjusted to 7 with triethanolamine, and the air bubbles were removed by vacuum treatment to obtain the aprestin-loaded *Callicarpa nudiflora* vesicle hydrogel (APST-EV-Gel). Figure 2 As shown.

[0060] Example 2 Example 2 of this invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the probe is used for ultrasonic treatment at 20W for 2 minutes in step S1.

[0061] Example 3 Example 3 of the present invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the probe is used for treatment at 20W for 6 minutes in step S1.

[0062] Example 4 Example 4 of this invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the probe is used for ultrasonic treatment at 20W for 8 minutes in step S1.

[0063] Example 5 Example 5 of the present invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the probe is used for ultrasonic treatment at 10W for 4 minutes in step S1.

[0064] Example 6 Example 6 of the present invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the probe is treated with an ultrasonic instrument at 30W for 4 minutes in step S1.

[0065] Example 7 Example 7 of the present invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the probe is used for treatment at 40W for 4 minutes in step S1.

[0066] Example 8 Example 8 of the present invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the mass ratio of APST to EV in step S1 is 1:0.5.

[0067] Example 9 Example 9 of the present invention provides a method for preparing Apst-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the mass ratio of APST to EV in step S1 is 1:1.

[0068] Example 10 Example 10 of the present invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the mass ratio of APST to EV in step S1 is 1:5.

[0069] Example 11 Example 11 of the present invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the mass ratio of APST to EV in step S1 is 1:10.

[0070] Example 12 Example 12 of the present invention provides a method for preparing Apst-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the mass ratio of APST to EV in step S1 is 1:20.

[0071] Example 13 Example 13 of this invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the incubation time at 37°C in step S1 is 2 hours.

[0072] Example 14 Example 14 of this invention provides a method for preparing Apstar-loaded nude flower purple vesicle hydrogel (APST-EV-Gel), which differs from Example 1 in that the incubation time at 37°C in step S1 is 3 hours.

[0073] Example 15 Example 15 of this invention provides a method for preparing Apster-loaded nude flower purple vesicle hydrogel, which differs from Example 1 in that, in step S2, 2g of carbomer 940 powder is weighed and dissolved in 100ml of ultrapure water to obtain a 2% blank gel matrix.

[0074] Example 16 Example 16 of this invention provides a method for preparing Apster-loaded nude flower vesicle hydrogel, which differs from Example 1 in that, in step S2, 7g of carbomer 940 powder is weighed and dissolved in 100ml of ultrapure water to obtain a 7% blank gel matrix.

[0075] Performance testing The performance of APST-EV and APST-EV-Gel prepared in Example 1 was tested, including... (1) The average particle size and polydispersity index of APST-EV were measured by dynamic laser scattering particle size analyzer for 7 consecutive days. The measurements were performed in parallel three times, and the results are shown in Table 1.

[0076] The results showed that the particle size of APST-EV did not change much over 7 consecutive days and the dispersion coefficient was less than 0.3, indicating that the particle size was uniform, the particle dispersion was uniform and stable.

[0077] Table 1. Particle size and polydispersity index of APST-EV

[0078] (2) Appearance and shape and observation by scanning electron microscopy The appearance and shape of APST-EV-Gel were observed using an experimental scanning electron microscope.

[0079] Appearance shape Figure 2 As shown, APST-EV-Gel is translucent, reddish-brown, semi-solid, with good adhesion, and is easy to apply to the skin.

[0080] 0.1 g of APST-EV-Gel lyophilized powder was spread evenly on an aluminum head coated with double-layer conductive tape, and gold was sputtered using an ion sputtering system. The coated sample was then randomly imaged using scanning electron microscopy to observe its morphological characteristics and take photographs. The results are as follows: Figure 3 As shown in the figure, the internal structure of Gel and APST-EV-Gel is a porous network cross-linked structure.

[0081] (3) Determination of encapsulation efficiency (EE%) and drug loading (DL%) Chromatographic conditions: The chromatography workstation was an Agilent 1260 Infinity liquid chromatography system, and the chromatographic column was a Supersil ODS2 (4.6 mm × 250 mm, 5 μm). The mobile phase was methanol:water = 64:36 (v / v); the detection wavelength was 230 nm, the flow rate was 0.5 mL / min, the injection volume was 10 μL, and the column temperature was 30 °C.

[0082] Take 1 ml of APST-EV, add 3 ml of acetonitrile, and treat with an ultrasonic probe at 200W for 30 min to completely release the drug from the vesicles of *Callicarpa nudiflora*. After mixing and filtering through a 0.22 μm filter membrane, perform HPLC to determine the drug concentration. Calculate the encapsulation efficiency and drug loading according to the following formulas. Perform three parallel determinations and take the average value. The drug loading was 17.14 ± 0.13%, and the encapsulation efficiency was 41.49 ± 0.48%. The determination results under different preparation processes are shown in Table 2. The results show that when the feed ratio is 1:0.5 (Example 8), the lowest drug loading is 1.49%, and the lowest encapsulation efficiency is 6.03%; under the optimal process (Example 1), the highest drug loading can reach 17.14%, and the highest encapsulation efficiency can reach 41.49%.

[0083] DL% = m1 / (m0+m1) × 100% EE% = m1 / m2 × 100% Where: m1 is the amount of aprestil in the vesicles of Callicarpa nudiflora; m0 is the amount of vesicles of Callicarpa nudiflora; m2 is the amount of aprestil added.

[0084] Table 2 shows the encapsulation efficiency and drug loading of apremilast-loaded vesicles under different preparation processes.

[0085]

[0086] As shown in Table 2, the ultrasonic process parameters and feed ratio have a significant impact on the physicochemical properties of drug-loaded vesicles. In the investigation of ultrasonic power and time (Examples 1-7), too low power or too short time (e.g., Examples 2 and 5) cannot effectively open the vesicle membrane gaps, resulting in low drug encapsulation efficiency; while too high power or too long time (e.g., Examples 4 and 7) may produce excessive cavitation effects, destroying the integrity of the vesicle structure, leading to a significant decrease in encapsulation efficiency and drug loading. The results indicate that 20W power for 4 minutes is the optimal ultrasonic drug loading condition.

[0087] In the investigation of feed ratios (Examples 8-12), as the amount of *Callicarpa nudiflora* vesicles added increased, the encapsulation efficiency and drug loading showed a trend of first increasing and then stabilizing. When the mass ratio of APST to EVs was 1:0.5 (Example 8), the lowest drug loading was measured to be 1.49% and the lowest encapsulation efficiency was 6.03% due to the limited number of carriers. This data provided experimental evidence for the lower limit of the scope of protection of this invention. When the ratio reached 1:2 (Example 1), the drug loading performance reached its optimal level (encapsulation efficiency 41.49%, drug loading 17.14%). Further increasing the amount of carrier (Examples 11-12) did not significantly improve the drug loading performance and resulted in carrier waste.

[0088] Furthermore, Examples 13-14 show that incubation time has a certain impact on membrane recovery, with 1 hour being the most suitable incubation time. Examples 15 and 16 further confirm that changing the concentration of Carbomer 940 in the hydrogel matrix (within the range of 2%–7%) does not affect the encapsulation efficiency and drug loading of the drug-loaded vesicles themselves, indicating that the drug delivery system has good formulation adaptability. In summary, this invention, through multi-dimensional process optimization, has determined the optimal preparation conditions and can obtain stable traditional Chinese medicine formulations within the numerical ranges protected by the claims.

[0089] (4) Differential scanning calorimetry (DSC) Differential scanning calorimetry (DSC) analysis was performed on apremilast raw material (APST), vesicles of Callicarpa nudiflora (EV), the physical mixture of APST and EV, and APST-EV. The results are as follows: Figure 4 As shown.

[0090] APST exhibits a characteristic endothermic peak at 160℃, confirming its crystallization characteristics. EV does not show a phase transition signal in the detection temperature range. The endothermic peak of APST is still retained in the physical mixture, while no endothermic peak is detected in APST-EV. This indicates that APST is incorporated into EV in an amorphous form.

[0091] (5) X-ray diffraction analysis (XRD) X-ray diffraction analysis was performed on the apremilast raw material (APST), vesicles of Callicarpa nudiflora (EV), the physical mixture of APST and EV, and APST-EV. The results are as follows: Figure 5 As shown APST active pharmaceutical ingredient exhibits characteristic diffraction peaks at diffraction angles of 10.2°, 12.5°, 13.6°, 20.9°, and 27.1°, indicating that it exists in crystalline form. The characteristic diffraction peaks of APST can still be detected in the physical mixture of APST and EV, while the characteristic diffraction peaks of APST disappear in APST-EV, indicating that APST changes from a crystalline state to an amorphous state in EV.

[0092] (6) In vitro drug release behavior study In vitro release behavior was studied using aprescribing hydrogel of *Callicarpa nudiflora* loaded with aprescribing agent (APST-EV-Gel). The specific procedure was as follows: APST methanol, APST-EV, APST-Gel, and APST-EV-Gel with the same drug concentration were placed into pretreated dialysis bags (molecular weight cutoff 3500 Da), tied at both ends, and then placed in a container containing 50 mL of release medium. The mixture was kept at a constant temperature of 37℃ and stirred at a constant speed of 100 rpm. At predetermined time intervals (h1, 2, 4, 8, 12, and 24), 1 mL of release medium was aspirated, and 1 mL of isothermal release medium was added simultaneously. The released medium was filtered through a 0.22 μm microporous membrane, and the concentration of aprescribing agent in the release medium was determined by HPLC. The cumulative release percentage (Q%) of aprescribing agent was calculated using the following formula. The results are as follows: Figure 6 As shown, APST-EV has a certain sustained-release effect compared to APST methanol solution, and can better control drug release. APST-EV-Gel prolongs the duration of action.

[0093] Q% = Wt / Wtotal × 100% Where: Wt is the cumulative amount of apremilast released in the release medium at time point t; Wtotal is the total amount of apremilast contained in the formulation before the release test.

[0094] (7) Pharmacodynamic studies Preparation method of the control preparation: APST-Gel: Accurately weigh APST raw material powder, grind it through a 200-mesh sieve, and then directly disperse it in the above-mentioned blank carbomer gel matrix. Stir evenly to prepare a suspension gel with the same drug concentration as in Example 1.

[0095] Nude Callicarpa vesicle gel (EV-Gel): Take a blank nude Callicarpa vesicle suspension without drugs, mix it evenly with a blank carbomer gel matrix according to the ratio of Example 1, and adjust the pH to 7 to obtain the gel.

[0096] Tacrolimus group (TAC): Use commercially available 5% tacrolimus ointment directly.

[0097] Newly purchased C57BL / 6J mice were acclimatized for one week. The hair on the backs (2cm × 3cm) of the mice was carefully removed using a razor and depilatory cream, taking care not to damage the skin. The mice were allowed 24 hours to recover after hair removal to allow their skin to return to normal. Mice were randomly divided into four groups: Control, Model, Tacrolimus (TAC), Apremilast Gel (APST-Gel), EV-Gel, and Apremilast-loaded EV-Gel, with six mice in each group.

[0098] 62.5 mg of 5% imiquimod cream (IMQ) was evenly applied to the backs of mice in the Model group, TAC group, APST-Gel group, EV-Gel group, and APST-EV-Gel group, once daily. Four hours later, 0.4 g of medical petroleum jelly, 5% tacrolimus ointment, APST-Gel, EV-Gel, and APST-EV-Gel were applied to the backs of the mice, respectively, for 7 days. The control group mice received only an equal amount of petroleum jelly applied to their backs once daily for 7 days. Four hours after the administration on day 7, all mice were euthanized by cervical dislocation.

[0099] The skin lesions on the backs of mice in each group were recorded daily and scored according to the internationally accepted Psoriasis Area and Severity Index (PASI) scoring standard. The specific PASI scoring criteria are shown in Table 3. The total score obtained by adding the three scores is the severity of the skin lesions (total score 12 points). The higher the score, the more severe the skin lesions.

[0100] Table 3 PASI Scoring Criteria

[0101] Continuous observation revealed that the control group mice did not exhibit erythema, scaling, or thickening of the skin on their backs during days 1-7. In the model group mice, the skin on the backs slightly thickened and reddened on day 3 after medication, with a small number of mice developing a few scales; on day 4, the erythema worsened further, the skin became moderately raised, and most mice developed a small amount of white scales; on day 5, the skin lesions continued to worsen; and on day 6, the lesions peaked, becoming deep red, with thickened skin and almost completely covered by white scales, resembling the skin lesions of human psoriasis, indicating successful model establishment. The erythema score, thickness score, scaling score, and total score of the other groups continued to increase from day 1-7, but the values ​​were all lower than those of the model group. The APST-EV-Gel group showed the most significant improvement in mouse skin, as shown in the results below. Figure 8-11 As shown.

[0102] After euthanizing the mice, their spleens were harvested, photographed, and weighed to calculate the organ coefficients for each group. The results are as follows: Figures 12-13As shown, compared with the spleen of the control group mice, the spleen of the model group mice was enlarged, dark red in color, and had a higher spleen index. This indicates that the mouse immune system was abnormally activated, stimulating abnormal activation and proliferation of immune cells, which in turn caused spleen cell proliferation, spleen enlargement, and a significant increase in the spleen index, accompanied by an increase in the level of inflammatory factors. This may lead to microcirculatory disturbances in the spleen, making the spleen appear dark red. Compared with the model group, the spleen index of each treatment group was reduced and the symptoms were relieved. Among them, the tacrolimus group showed the most significant relief of spleen symptoms.

[0103] Supplementary observation of skin irritation: Throughout the entire pharmacodynamic experiment, the drug-loaded area on the back of mice and the surrounding normal skin were observed daily. The results showed that no irritation reactions such as redness, swelling, ulceration, or exudation were observed in the skin of mice in the blank group, EV-Gel group, and APST-EV-Gel group, indicating that the drug-loaded vesicle hydrogel prepared in this invention has good biocompatibility and skin safety.

[0104] In-depth analysis of the synergistic mechanism: Combined with DSC and XRD results, it was found that in the APST-Gel control group, the drug mainly existed as micron-sized crystalline particles with low solubility and difficulty penetrating the stratum corneum. However, in the APST-EV-Gel of this invention, apremilast is transformed into a high-energy amorphous form and encapsulated in nanovesicles. This method enables the drug to overcome the stratum corneum barrier of psoriatic lesions. Furthermore, pharmacodynamic results showed that the efficacy of APST-EV-Gel was superior to the sum of APST-Gel and EV-Gel (based on the PASI score trend), confirming a significant synergistic anti-inflammatory effect between the chemical drug and the bioactive carrier. In summary, the Aprestil-loaded Callicarpa nudiflora hydrogel provided by this invention can improve the efficacy of psoriasis treatment, and has a sustained-release effect and good biocompatibility, avoiding the side effects of systemic administration.

Claims

1. A naked Callicarpa macrophylla vesicular hydrogel loaded with apremilast, characterized in that, The hydrogel comprises Aprestor-loaded *Callicarpa nudiflora* vesicles and a hydrogel matrix; the Aprestor-loaded *Callicarpa nudiflora* vesicles are dispersed in the three-dimensional network structure of the hydrogel matrix; wherein the Aprestor-loaded *Callicarpa nudiflora* vesicles are nanocomposites formed by encapsulating Aprestor inside extracellular vesicles derived from *Callicarpa nudiflora*.

2. The hydrogel of claim 1, wherein, The aprestinate exists in an amorphous form within the lipid bilayer structure of the extracellular vesicles derived from *Callicarpa nudiflora*; the *Callicarpa nudiflora* vesicles loaded with aprestinate have nanoscale particle sizes and are uniformly distributed.

3. The hydrogel of claim 1, wherein, The hydrogel matrix includes carbomer and a pH adjuster; the hydrogel matrix has a porous network structure.

4. The hydrogel of claim 1, wherein, The mass ratio of the Apstar-loaded nude Callicarpa vesicles to the hydrogel matrix ensures that the hydrogel remains in a semi-solid state and does not separate into layers.

5. A method for preparing the hydrogel according to any one of claims 1 to 4, characterized in that, Includes the following steps: S1. Extraction of the carrier: Extracellular vesicles derived from *Callicarpa nudiflora* leaves were isolated and purified; S2. Drug loading: Apremilast was loaded into the extracellular vesicles derived from *Callicarpa nudiflora* using ultrasound-assisted incubation technology to obtain *Callicarpa nudiflora* vesicles loaded with apremilast; S3. Molding: A blank hydrogel matrix was prepared, and the *Callicarpa nudiflora* vesicles loaded with apremilast were mixed evenly with the blank hydrogel matrix to obtain the final product.

6. The production method according to claim 5, characterized by, In step S1, differential centrifugation combined with density gradient centrifugation is used to remove impurities and obtain vesicles with a specific sedimentation coefficient.

7. The preparation method according to claim 5, characterized in that, Step S2 specifically includes: dissolving apremilast in an organic solvent and mixing it with extracellular vesicles derived from Callicarpa nudiflora; sonicating the mixture; incubating it under constant temperature conditions; and removing the organic solvent and unencapsulated free apremilast.

8. The preparation method according to claim 7, characterized in that, During the ultrasound treatment, the drug enters the vesicles without damaging the overall structure of the vesicles; the incubation temperature is the physiological temperature.

9. The preparation method according to claim 5, characterized in that, In step S3, the preparation of the blank hydrogel matrix includes dispersing carbomer in water to swell; mixing evenly and then adding an alkaline pH adjuster to adjust the pH value.

10. The use of a hydrogel as described in any one of claims 1 to 4 in the preparation of a medicament for treating psoriasis, characterized in that, The drug is a topical transdermal delivery formulation.

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