Alginate sulfate patch, preparation method and application thereof

Abstract

The application relates to a fucoidan sulfate patch as well as a preparation method and application thereof, and belongs to the technical field of medicines. Fucoidan sulfate is used as a main raw material of microneedles for the first time, the needle tip is completely composed of fucoidan sulfate, fucoidan sulfate plays a role of medicine and adjuvant in one, on one hand, fucoidan sulfate provides sufficient mechanical strength as a needle tip matrix to pierce a wart body and surrounding skin, and is more suitable for relevant warts with obvious skin thickening, on the other hand, fucoidan sulfate is diffused and released at a virus location, and plays a skin wart treatment effect by preventing the virus from entering and enhancing skin immunity, compared with a previously authorized patent scheme, the problem of insufficient entry into skin and absorption caused by fucoidan sulfate as a macromolecular polysaccharide is solved in the form of microneedles, fucoidan sulfate can be directly delivered to basal layer cells where HPV is located, and site-specific drug delivery is realized.

Description

Technical Field

[0001] This application relates to an alginate sulfate patch, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Human papillomavirus (HPV) is a non-enveloped circular double-stranded DNA virus with a genome containing 8kb of DNA sequence. It exhibits considerable tropism in human skin and mucous membrane epithelium. To date, more than 200 HPV genotypes have been identified, which can be classified into high-risk and low-risk HPV based on their carcinogenicity. HPV infection begins with minor skin or mucous membrane lesions. The infection process involves three key steps: First, the L1 protein binds to HSPG via its nonpolar K278-K361 sites. After viral internalization, the virus is transported through the endosome system. The viral capsid is disassembled or degraded, and the L2 protein, carrying the viral genome, escapes from the endosome into the cytoplasm and is transported to the nucleus via microtubules mediated by dyskinin. Next, the E6 and E7 proteins bind to p53 and Rb proteins, respectively, leading to the inactivation of these two tumor suppressor genes and causing unlimited cell growth. Finally, this unlimited growth results in two outcomes: high-risk HPV types such as 16 and 18 infecting tissues like the cervix and causing malignant transformation, while low-risk HPV types such as 1, 3, and 10 infecting skin tissues and causing benign verrucous hyperplasia.

[0003] HPV-induced skin warts are extremely common, with an incidence rate of 7-12%. Children and young adults are more susceptible, with the highest incidence in children aged 12-16. Clinically, they are classified according to their location, including common warts, filiform warts, flat warts, and plantar warts. Studies have also shown that different HPV types cause different types of skin warts. Common types of common warts include HPV1, 2, 4, 7, and 27; flat warts include HPV3, 10, 28, and 41; and plantar warts include HPV1, 2, and 4. What they have in common is that the lesions are painful to the touch, or may be asymptomatic. Under a skin microscope, pale yellow papules, thickened keratin rings, and round papillary hyperkeratosis are visible, interspersed with small black dots formed by ruptured capillaries. The corresponding pathological changes are epidermal hyperkeratosis, acanthosis, and elongation of rete ridges. The reason for this is that the virus exists in the cells of the stratum spinosum and basal layer, infecting and promoting the proliferation of keratinocytes, forming wart-like protrusions.

[0004] Generally, skin warts are self-limiting, not life-threatening, and in most cases eventually cleared by a cell-mediated immune response. However, HPV-related skin warts are highly refractory and can persist for many years, depending on the number of lesions and the location of infection. Treatment is necessary when there is pain, impact on appearance and social life, recurrence, or a large and persistent number of lesions.

[0005] Currently, the main treatments for skin warts are ablation of the warts and prevention of their formation, including methods such as medication, physical therapy, and surgical excision. Physical therapies such as cryotherapy, laser therapy, microwave therapy, and radiation therapy all achieve their therapeutic goals by inducing denaturation, coagulation, and necrosis of the wart tissue. Salicylic acid, a keratolytic agent, destroys virus-infected cells without affecting keratinocyte production, and is a commonly used drug for treating facial flat warts, plantar warts, and common hand warts. Antiviral and antimitotic drugs are also frequently used to treat refractory and recurrent skin warts. Ayman Elsayed et al. attempted intralesional injection of acyclovir to treat skin warts, and the results showed that 52.6% of patients in the acyclovir group experienced complete wart clearance, and 36.8% experienced partial remission, significantly higher than the saline control group, making it an effective and well-tolerated treatment for skin warts. Manal T. Barkat MD et al. injected bleomycin (1 mg / mL) intralesionally into the ablated plantar warts, once every two weeks, with a maximum of four injections. Dermoscopy and clinical assessments showed that complete clinical clearance of plantar warts was observed in 88.5% of patients (23 / 26), with 7.7% (2 / 26) showing clinical improvement and only 3.8% (1 / 26) experiencing clinical failure, which was significantly different from the placebo group.

[0006] Furthermore, due to the significant relationship between the course of HPV-induced warts and autoimmunity, systemic or local immunotherapy has received increasing attention. Imiquimod is a novel local immunomodulator that can induce various cytokines by stimulating peripheral immune cells such as Toll-like receptor 7, Langerhans cells, and keratinocytes at the application site, thereby producing immunomodulatory and indirect antiviral effects. Hengge et al. treated common warts with 5% imiquimod, and 30% of patients experienced complete wart regression, while 26% of patients saw their warts shrink by half. Autologous wart implantation is a traditional artificial immunotherapy. Its mechanism of action involves surgically embedding HPV antigens in the subcutaneous fat layer, stimulating the body to produce antibodies and triggering an autoimmune response.

[0007] Transdermal drug delivery (TDD) refers to the administration of drugs through the skin for local or systemic therapeutic purposes. Compared to other routes of administration (oral, injection, etc.), TDD avoids gastrointestinal digestive enzyme metabolism and the first-pass effect, continuously delivers drugs to maintain therapeutic concentrations, minimizes pain and infection risks, and improves patient compliance. However, only smaller molecules with moderate lipophilicity can freely penetrate the skin, while large molecule drugs are usually hindered by the stratum corneum (SC) barrier, resulting in very poor bioavailability. As a new generation of local drug delivery systems, microneedles (MN) exhibit superior properties. Microneedles possess strong mechanical strength, can penetrate the stratum corneum barrier, and create a series of temporary microchannels in the skin. This significantly increases transdermal drug penetration while avoiding contact with blood vessels and nerve fibers mainly located in the deep dermis, providing a minimally invasive and painless method to prevent bleeding at the application site.

[0008] Based on research over the past two decades, microneedles (MNs) are generally classified into five categories: solid MNs, coated MNs, hollow MNs, dissolved MNs, and hydrogel MNs. Solid microneedles, coated microneedles, and hollow microneedles are mostly made of materials such as silicon, titanium, and stainless steel, possessing sufficient mechanical strength, but also suffering from poor biocompatibility, limited drug loading capacity, and difficulty in controlling drug release. Dissolved microneedles and hydrogel microneedles are currently a research hotspot. They often utilize biocompatible natural or synthetic polymer materials. After insertion into the skin, the drug loaded in dissolved microneedles is released as the matrix dissolves. Hydrogel MNs, on the other hand, absorb tissue fluid and swell in the skin, forming porous aqueous microcatheters through which the drug contained in the reservoir can diffuse into the skin's microcirculation.

[0009] Currently, microneedles are widely used for delivering proteins and hydrophobic drugs in the treatment of diabetes, skin diseases, and superficial skin tumors. For example, Qu Xiaoying developed a HA / BSP-based dissolving microneedle to mediate the delivery of mesoporous polydopamine nanoparticles (MPDA) loaded with triamcinolone acetonide (TA) for the treatment of oral mucositis (OM). In recent years, for the treatment of persistent and refractory warts, some researchers have considered using microneedles as a formulation to directly deliver drugs such as imiquimod and bleomycin to the lesion site, increasing the local drug load while avoiding injection pain and improving patient compliance. Li Hansong et al. coated bleomycin onto the tip of polylactic acid (L-PLA) microneedles, and more than 80% of the bleomycin dissolved in vitro into the skin within 15 minutes. Compared with intralesional injection, the tip-coated microneedles more effectively distributed the drug to the subepidermal layer. Tsu-Man Chiu et al. prepared imiquimod-loaded dissolving microneedles using gelatin as a formulation. The results showed that imiquimod was delivered to the subepidermal layer, activating antigen-presenting cells and T cells.

[0010] Dissolving microneedles are typically made from biocompatible and water-soluble materials that dissolve completely in the skin, leaving no sharp needles that could scratch the body after use. Furthermore, dissolving microneedles usually soften and dissolve within biological tissue upon penetration, preventing damage from application-related mechanical forces. Compared to synthetic polymers such as polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA), polysaccharides have been widely used in the preparation of dissolving microneedles due to their excellent biocompatibility, solubility, and antibacterial, anti-inflammatory, and antioxidant activities. Tang Zhongming et al. used sulfated heteropolysaccharide-ulva polysaccharide extracted from green algae as raw material and prepared ulva polysaccharide dissolving microneedles with an average height of 655 μm and an aspect ratio of 2.63 using a two-step centrifugation method. These microneedles have sufficient mechanical strength to bypass the stratum corneum barrier of pig skin and reach the dermis. They dissolve rapidly within 2 minutes after insertion, with a 90.3% reduction in needle height. The loaded model drug can be released in situ in the skin layer. They have advantages such as convenient use, good patient compliance, and good drug release.

[0011] Van der Maaden and colleagues developed a chitosan pH-sensitive MN array for delivering inactivated poliovirus vaccine particles, which induced a specific antibody response in in vivo tests. Liu Wei et al. prepared multifunctional bilayer drug-loaded microneedles using hyaluronic acid and carboxymethyl chitosan as the backing layer and tip layer, respectively. These microneedles sequentially released tetracycline hydrochloride (TH) and recombinant human epidermal growth factor (rh-EGF) locally. In an in vivo diabetic wound model, they exhibited anti-inflammatory effects, promoted angiogenesis, collagen deposition, and tissue regeneration, thus promoting diabetic wound repair.

[0012] The applicant discovered in a prior Chinese patent CN111481502B that alginate sulfate (PMGS) is obtained by sulfonation of alginate. Figure 1As shown in the figure, it possesses anti-HPV activity, exhibiting good inhibitory effects against both low-risk and high-risk HPV types, with minimal toxic side effects. Pull-down experiments confirmed that biotinylated PMGS can bind to the capsid proteins of HPV16 and HPV45 pseudoviruses, as well as the L1 protein of HPV45, indicating that PMGS may bind to the virus before it adsorbs onto cells, thus inhibiting viral infection. Furthermore, alginate sulfate significantly inhibited HPV infection on the skin of BALB / c nude mice. PMGS activated local immunity in mouse cervical tissue, recruiting immune cells such as monocytes, dendritic cells, and macrophages, thereby activating the host's innate immune response. PMGS enhances the phagocytic capacity of monocytes and macrophages, promotes the expression and secretion of cytokines by T cells and NK cells, enhances the immune function of lymphocytes, increases the killing effect of lymphocytes on target cells, and inhibits the expression of oncogenes E6 / E7. Therefore, PMGS can exert its anti-HPV effect through both inhibiting viral binding and activating the body's immunity, making it an effective anti-HPV active substance.

[0013] However, due to the large molecular weight of PMGS, its transdermal absorption effect is poor. Microneedles are currently a much-discussed drug delivery method. However, given that there is still little research on microneedle drug delivery methods targeting PMGS in the existing technology, the feasibility of using PMGS as a dissolving microneedle matrix material to form needles alone or to prepare PMGS-loaded microneedles through material composite methods is still unknown. Therefore, in order to further solve the problems existing in the prior patent solution, it is necessary for the applicant to provide a product that can more effectively prevent / treat HPV / skin warts. Summary of the Invention

[0014] To address the aforementioned issues, this application provides a brown alginic acid sulfate (PMGS) patch, its preparation method, and its application. This application is the first to use brown alginic acid sulfate as the main raw material for microneedles, with the needle tip composed entirely of brown alginic acid sulfate. This not only solves the problem of insufficient skin absorption caused by PMGS being a large-molecule polysaccharide, but also allows direct delivery of PMGS to the basal cells where HPV is located, achieving targeted drug delivery.

[0015] This application provides an alginate sulfate (PMGS) patch, comprising: 1) Backing layer; 2) The needle tip layer is one or more of the following: λ / ι / κ type carrageenan, polymannuron sulfate, polyguluron sulfate, alginate sulfate, fucoidan, sulfated dextran, chondroitin sulfate, keratin, heparin and its analogues.

[0016] Optionally, the needle tip layer is alginate sulfate.

[0017] It should be noted that, in addition to the materials mentioned above, those skilled in the art may also select other polysaccharides with antiviral effects as the tip layer alone, including but not limited to λ / ι / κ type carrageenan, polymannuron sulfate, polyguluron sulfate, alginate sulfate, fucoidan, sulfated dextran, chondroitin sulfate, keratin, heparin and its analogues.

[0018] Optionally, the needle tip height in the needle tip layer is 200~2000μm. This needle tip height makes it easier for the needle tip layer to reach the basal layer where HPV is located in the skin. By acting on HPV, it reduces the production of warts, fundamentally treating warts. Existing warts will also gradually fall off, thus achieving prevention / treatment of HPV / warts. Since the HPV virus inhibition binding of alginate sulfate has been verified, and this application also reveals that alginate sulfate can activate the non-specific immunity of organisms and improve the immune level, its preventive efficacy can be reasonably expected.

[0019] Optionally, the needle tip height in the needle tip layer is 500~1000μm.

[0020] Optionally, the molecular weight of the alginate sulfate ranges from 10 to 300 kDa.

[0021] The inventors experimented with PMGS with molecular weights of 5kDa, 10kDa, 100kDa, 270kDa, and 300kDa. They found that PMGS with a lower molecular weight (10kDa) had slightly lower mechanical strength, but needle tips with the required mechanical strength could be prepared by increasing the concentration. However, high-concentration PMGS needle tips with low molecular weight were brittle after drying. The PMGS with the above molecular weights have the advantages of good antiviral effect, satisfactory mechanical strength, and low brittleness after drying.

[0022] Optionally, the content of polymannuronic acid in the alginate sulfate is 5-95%, the content of polyguluronic acid is 5-95%, and the degree of substitution of sulfate ester group is 5-15%.

[0023] Optionally, the polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), chondroitin sulfate, chitosan, alginate sulfate, sodium carboxymethyl cellulose (CMC-Na), sodium carboxymethyl starch (CMS-Na), and hyaluronic acid (HA) may be present.

[0024] Optionally, the backing layer includes a first material and a second material; the first material is selected from one or more of chitosan and its derivatives, polyvinylpyrrolidone and its derivatives, alginate sulfate, and chondroitin sulfate. The second material is selected from one or more of polyvinyl alcohol and its derivatives, hyaluronic acid, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, and sodium carboxymethyl starch.

[0025] Optionally, the backing layer comprises polyvinylpyrrolidone and polyvinyl alcohol.

[0026] It is understood that the main function of the backing layer in this application is to support the needle tip layer. Those skilled in the art can select appropriate materials as the backing layer material as needed, and can also add other feasible medical components to the backing layer or modify the selected materials of the backing layer.

[0027] For example, polyvinyl alcohol undergoes quaternization and chitosanization treatment. The backing layer comprises a composite material of quaternized chitosan and polyvinyl alcohol. The microneedle backing layer, composed of cross-linked components, exhibits excellent mechanical properties, solubility, and biocompatibility. After the microneedle tips dissolve, the backing layer can be dissolved with a small amount of sterile water, transforming into a gel-like substance that, when applied to the wart, not only exerts bactericidal and soothing effects, relieving itching and redness, but also prevents the spread of warts caused by friction and scratching. Alternatively, active polysaccharides such as chitosan can be added separately to the backing layer to provide antibacterial and anti-inflammatory effects, further alleviating redness and itching at the wart site.

[0028] Optionally, the backing layer comprises polyvinyl alcohol and polyvinylpyrrolidone. Optionally, the mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is (0.5~4):1.

[0029] Here, a backing layer is prepared by combining polyvinyl alcohol and polyvinylpyrrolidone in a certain proportion. The backing layer solution has good fluidity and is easy to prepare. The prepared backing layer has a certain degree of flexibility and is suitable for uneven wart-like protrusions with good skin adhesion.

[0030] Optionally, the mass ratio of polyvinyl alcohol to polyvinylpyrrolidone is (1~3):1.

[0031] Optionally, the drug loading of a single microneedle in the alginate sulfate (PMGS) patch is 100~500 μg.

[0032] Optionally, the needle tip in the needle tip layer can be conical or pyramidal. Those skilled in the art can also choose other available shapes. For example, a pyramidal shape may be preferred for better mechanical strength.

[0033] This application provides a method for preparing the above-mentioned alginate sulfate (PMGS) patch, characterized in that the preparation method includes the following steps: 1) Prepare the backing layer solution and the needle tip layer solution for later use; 2) Pipette the tip layer solution into the microneedle mold, centrifuge for the first time, then remove the excess tip layer solution and add the backing layer solution, and centrifuge for the second time; 3) After drying, the alginate sulfate (PMGS) patch is obtained.

[0034] Optionally, the first centrifugation treatment may involve centrifuging at 2500–3500 rpm for 10–30 min; and / or, In the second centrifugation treatment, centrifuge at 2500~3500 rpm for 5~15 min; and / or, Dry at 20-30℃ for 6-18 hours during the drying process.

[0035] After the needle tip layer solution is added, the above centrifugation conditions can ensure that the needle tip layer solution is fully centrifuged into the mold; after the backing layer solution is added, the above centrifugation conditions can ensure that the backing layer solution is fully in contact with the needle tip layer solution. The drying temperature is best kept at 20~30℃. If the drying temperature is higher, the backing layer is prone to bending and unevenness, which will affect the subsequent use effect.

[0036] This application provides the use of the above-mentioned alginate sulfate (PMGS) patch in the prevention and / or treatment of HPV and / or skin warts, including but not limited to common warts, flat warts, filiform warts, plantar warts, etc. caused by HPV.

[0037] The beneficial effects of this application include, but are not limited to: 1. Based on the alginate sulfate (PMGS) patch, its preparation method, and its application, this application is the first to use alginate sulfate (PMGS) as the main raw material for microneedles. The needle tip is entirely composed of PMGS, which plays a combined role of drug and adjuvant. On the one hand, PMGS, as the needle tip matrix, provides sufficient mechanical strength to pierce the skin around the wart, making it more suitable for wart diseases with significant skin thickening. On the other hand, PMGS diffuses and releases at the site of the virus, achieving the therapeutic effect of skin warts by both preventing virus adsorption and enhancing skin immunity.

[0038] 2. Based on the alginate sulfate (PMGS) patch, its preparation method, and its application, and considering the problems existing in the prior authorized patent, the feasibility of PMGS being used as a needle tip was further studied. Ultimately, the problem of insufficient skin absorption of PMGS as a large molecule polysaccharide was solved by dissolving microneedles. Furthermore, the design targeting the needle tip layer can directly deliver PMGS to the basal cells where HPV is located, thereby achieving targeted drug delivery with better drug delivery effect, higher safety, and better treatment effect.

[0039] 3. Based on the alginate sulfate (PMGS) patch, its preparation method, and its application, this application provides a polysaccharide microneedle with excellent properties and good activity for anti-HPV and treatment of skin warts. This not only provides a research and material basis for the clinical application of skin warts, but also has important market and economic value. Attached Figure Description

[0040] The accompanying drawings, which are included to provide a further understanding of this application and form part of this application, illustrate exemplary embodiments and are used to explain this application, but do not constitute an undue limitation of this application. In the drawings: Figure 1 This is a schematic diagram of the structural formula of the brown alginate sulfate (PGMS) involved in this application; Figure 2 This is a flowchart illustrating the preparation process of the alginate sulfate (PMGS) patch involved in Example 1 of this application; Figure 3 This is a SEM image showing the microneedle morphology observed in Test Example 1 of this application. Figure 4 The figure shows the results of the PMGS-PVP / PVA microneedle mechanical strength test involved in Test Example 2 of this application; Figure 5 This is a 3D image of the confocal scanning results for the in vitro insertion depth measurement of PMGS-PVP / PVA microneedles involved in Test Example 3 of this application; Figure 6 This is a graph showing the results of the PMGS post-column derived standard curve involved in Test Example 4 of this application; Figure 7 This is a liquid chromatography result of the drug loading at the microneedle tip involved in Test Example 4 of this application; Figure 8 This is a result image of the pinhole situation on pig skin and rat skin after trypan blue staining, as shown in Test Example 5 of this application. Figure 9 The images show the results of mouse skin condition at different times after microneedle insertion in Test Example 5 of this application. Figure 10 The images show the results of pigskin condition at different times after microneedle insertion in Test Example 5 of this application. Figure 11 This is a graph showing the inhibitory effect of PMGS on HPV6 in HaCat cells in Test Example 6 of this application. Figure 12 This is a graph showing the PMGS cytotoxicity test results for HaCat cells involved in Test Example 7 of this application; Figure 13 This is a graph showing the cytotoxicity test results of the microneedle extract on HaCat cells involved in Test Example 7 of this application; Figure 14The figure shows the effect of PMGS on the viability of Raw264.7 mononuclear macrophages, as described in Test Example 8 of this application. Figure 15 The figure shows the effect of PMGS on the expression of cytokine genes in Raw264.7 monocytes and macrophages involved in Test Example 8 of this application. Figure 16 The figure shows the effect of PMGS on the phagocytic capacity of Raw264.7 mononuclear macrophages involved in Test Example 8 of this application. Figure 17 This is a diagram showing the effect of PMGS microparticles on partial immune cell infiltration in mouse skin, as described in Test Example 9 of this application. Figure 18 This is a graph showing the PMGS concentration screening results involved in Experiment Example 2 of this application; Figure 19 This is a diagram showing the effect of using PVA, PVP, and HA as backing materials in Experimental Example 3 of this application. Figure 20 This is an illustration of the effect of using a mixture of PVP and PVA as a backing in Experiment Example 3 of this application. Figure 21 This is a diagram showing the results of an in vitro simulation of needle tip insertion in Comparative Example 2 of this application; Figure 22 The image shows the morphological results of the microneedles before and after treatment at 100°C for 24 hours in Test Example 10 of this application. Figure 23 The infrared spectra of the microneedles before and after treatment at 100°C for 24 hours are shown in Test Example 10 of this application. Detailed Implementation

[0041] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of the present application are all purchased through commercial channels.

[0042] The present application solution will be described below through specific embodiments.

[0043] Example 1 1) Preparation of backing layer: Accurately weigh 1g PVA0588, add 5mL of pure water, dissolve completely, let stand at room temperature to defoam until the solution is clear and transparent, and prepare a 200mg / mL PVA solution; pipette 1mL of 200mg / mL PVA solution, add 100mg PVP-K30, vortex mix, dissolve completely, and then sonicate to defoam, to obtain a mixed solution of 200mg / mL PVA and 100mg / mL PVP-K30 for later use; 2) The needle tip layer solution is prepared using 150 mg / mL PMGS solution. In this example, the molecular weight of PMGS is selected to be 270 kDa. Other molecular weights of PMGS can be selected, such as 10~300 kDa. 3) Product preparation: Microneedles are prepared using a two-step centrifugation method, as follows: Figure 2 As shown. First, approximately 200 μL of the tip layer solution was pipetted into the microneedle mold and centrifuged at 3000 rpm for 20 min to ensure the solution fully filled the mold gaps and eliminate air bubbles. Then, after carefully removing the excess tip layer solution, approximately 200 μL of the backing layer solution was added and centrifuged at 3000 rpm for 10 min. After centrifugation, the mold was transferred to a 25°C oven for drying for 12 h. After demolding, the mold was stored in a desiccator to obtain the alginate sulfate (PMGS) patch.

[0044] Example 2 1) Preparation of backing layer: Accurately weigh 1g PVA0588, add 5mL of pure water, dissolve completely, let stand at room temperature to defoam until the solution is clear and transparent, and prepare a 200mg / mL PVA solution; pipette 1mL of 200mg / mL PVA solution, add 100mg PVP-K30, vortex mix, dissolve completely, and then sonicate to defoam, to obtain a mixed solution of 200mg / mL PVA and 100mg / mL PVP-K30 for later use; 2) The needle tip layer solution used was 150 mg / mL PMGS solution; 3) Product preparation: Microneedles were prepared using a two-step centrifugation method. First, approximately 200 μL of the tip layer solution was pipetted into the microneedle mold and centrifuged at 2500 rpm / min for 30 min to ensure the solution fully filled the mold gaps and eliminate air bubbles. Then, after carefully removing excess tip layer solution, approximately 200 μL of backing layer solution was added and centrifuged at 2500 rpm / min for 15 min. After centrifugation, the microneedles were transferred to a 30°C oven for drying for 6 h. After demolding, the microneedles were stored in a desiccator to obtain the alginate sulfate (PMGS) patch.

[0045] Example 3 1) Preparation of backing layer: Accurately weigh 1g PVA0588, add 5mL of pure water, dissolve completely, let stand at room temperature to defoam until the solution is clear and transparent, and prepare a 200mg / mL PVA solution; pipette 1mL of 200mg / mL PVA solution, add 100mg PVP-K30, vortex mix, dissolve completely, and then sonicate to defoam, to obtain a mixed solution of 200mg / mL PVA and 100mg / mL PVP-K30 for later use; 2) The needle tip layer solution used was 150 mg / mL PMGS solution; 3) Product preparation: Microneedles were prepared using a two-step centrifugation method. First, approximately 200 μL of the tip layer solution was pipetted into the microneedle mold and centrifuged at 3500 rpm / min for 10 min to ensure the solution fully filled the mold gaps and eliminate air bubbles. Then, after carefully removing excess tip layer solution, approximately 200 μL of backing layer solution was added and centrifuged at 3500 rpm / min for 5 min. After centrifugation, the microneedles were transferred to a 20°C oven for drying for 18 h. After demolding, the microneedles were stored in a desiccator to obtain the alginate sulfate (PMGS) patch.

[0046] Test Example 1: SEM Microneedle Morphology Observation SEM morphology observation was performed on the microneedle patch product. Before observation, the HA / PVP-MN sample was removed and fixed to the sample tray with conductive tape. After vacuum sputtering with gold, the overall image of the 10×10 microneedle array and the surface morphology of individual needles were observed under a scanning electron microscope. The results are as follows: Figure 3 As shown, the left image is an overall view of the prepared 10×10 microneedle array at 80x magnification, and the right image is a single-needle height measurement at 200x magnification. Specifically, the needle tip spacing is 515μm, the microneedle base size is 310μm×310μm, the backing layer size is 9mm×9mm, and the number of microneedles in the array is 10×10. The left image shows that the prepared microneedles have a clear conical morphology, a neat array, and no obvious needle breakage or missing parts within the field of view. The backing layer is flat, without wrinkles or bubbles. The height measurement results in the right image show that the single-needle height is approximately 720μm.

[0047] Test Example 2: Determination of the Mechanical Strength of PMGS-PVP / PVA Microneedles The mechanical strength of PMGS-PVP / PVA microneedles was measured. A suitable-sized motion sensor was selected, and the microneedle array was placed in a chassis with the backing layer in contact with the chassis. The program was set to a single compression. The motion sensor contacted the needle tip from top to bottom. When the bottom of the sensor contacted the very tip of the microneedle, the sensor's travel and the force applied to the microneedle were recorded. The results are as follows: Figure 4 As shown.

[0048] As shown in the image, when 150 mg / mL PMGS is used as the needle tip, the force that each microneedle can withstand when the displacement is 640 μm is higher than the theoretical force (0.15 N) required to penetrate the stratum corneum. Therefore, it has the ability to penetrate the stratum corneum.

[0049] Test Example 3: In Vitro Insertion Depth Measurement of PMGS-PVP / PVA Microneedles The in vitro insertion depth of PMGS-PVP / PVA microneedles was determined. First, rhodamine B-loaded microneedles were prepared. Rhodamine B (RhB), as a model drug, exhibits strong fluorescence at an excitation wavelength of approximately 550 nm. To directly observe the depth of microneedle insertion into the skin, rhodamine-loaded microneedles (RhB-MN) were prepared by loading the needle tip with 6 mg / mL rhodamine for subsequent observation.

[0050] After trimming the backing edge of the RhB-MN needle, the needle tip was forcefully inserted into the ex vivo pig skin for 15 minutes. The inserted skin sample was then removed and placed upside down in a confocal microscopy dish. Fluorescence intensity was detected at 561 nm using a confocal laser microscope for visualization, observing the vertical distribution of rhodamine in the skin. The plane parallel to the surface of the pig skin was defined as the XY plane, and the plane perpendicular to the downward direction of the pig skin was defined as the Z plane. The initial scanning plane for the skin was defined when a clear microneedle array pattern appeared in the stratum corneum scan image; at this point, Z = 0 μm. Starting from the initial scanning surface, an optical image was captured every 20 μm along the Z plane downwards until no red fluorescence was observed. The diffusion depth of rhodamine in the ex vivo pig skin was then determined, and the results are as follows: Figure 5 As shown.

[0051] As shown in the image, after the microneedles loaded with Rhodamine B were inserted into the pigskin, they formed a neat array of needle tips. Rhodamine B diffused and was released within the pigskin. Judging from the depth of fluorescence, the depth of insertion of the microneedles into the pigskin was approximately 280 μm, which is much greater than the thickness of the stratum corneum.

[0052] Those skilled in the art will understand that the skin is the largest organ in the human body, consisting of the epidermis, dermis, and subcutaneous tissue. The epidermis is the outermost layer of skin, generally 0.035–0.15 mm thick. Based on the differentiation and characteristics of keratinocytes, the epidermis is divided into five layers from the outside in: the stratum corneum, the stratum lucidum (found only in the palms and soles), the stratum granulosum, the stratum spinosum, and the stratum basale. The stratum basale is connected to the dermis via the basement membrane and is located at the bottom of the epidermis, consisting of only a single layer of columnar or cubic basal cells, approximately 10–14 μm in size. In vitro microneedle insertion depth experiments show that the microneedles penetrate pig skin to a depth of approximately 280 μm, sufficient to reach the basal cells (150 μm). HPV infection of basal cells leads to excessive proliferation of these cells, resulting in warts. Therefore, it is evident that the PMGS in the microneedles of this application can be successfully delivered to the basal cells, preventing viral adsorption and exerting an antiviral effect.

[0053] Test Example 4: Determination of Drug Loading Capacity of PMGS-PVP / PVA Microneedles Weigh 150 mg of PMGS and dissolve it in 1 mL of pure water. Allow it to swell at room temperature until the solution is homogeneous and transparent to prepare a PMGS solution with a concentration of 150 mg / mL. Pipette 200 μL of the above PMGS solution into a microneedle mold and centrifuge at 3000 rpm for 20 min. After centrifugation, scrape off any excess solution from the needle tip and transfer the microneedle to a 25°C oven to dry for 12 h. Remove the dried needle tip with tape and dissolve it in 0.3 mL and 0.5 mL of mobile phase, respectively. Filter the solution through a 0.22 μm filter and then inject it into the liquid phase for post-column derivatization analysis. The PMGS post-column derivatization standard curve results are shown below. Figure 6 As shown in the figure, the liquid chromatography results of the drug loading at the microneedle tip are as follows: Figure 7 As shown.

[0054] As shown in the image, when the peak area is substituted into the standard curve, the calculated PMGS content is 0.1987 mg and 0.1919 mg, respectively, indicating that the drug loading of a single microneedle tip is approximately 195 μg.

[0055] Test Example 5: PMGS pigskin insertion capability and skin recovery after insertion Microneedle insertion capability test: Pre-treated pigskin was removed from the freezer, thawed, and incubated in PBS (pH 7.4) solution for 1 hour. The solution on the keratinous side of the pigskin was then wiped dry. The isolated pigskin was placed on a paper towel soaked in PBS (pH 7.4) solution and kept at 37°C to simulate the in vivo environment. Microneedles were inserted into the isolated pigskin and withdrawn after a certain time (60 seconds). Morphological changes after microneedle withdrawal were recorded using a Bester magnifying microscope. The skin was then exposed to trypan blue solution for 5 minutes. The tissue-labeled dye was washed with physiological saline, and the skin was observed.

[0056] Skin recovery ability test after insertion: PMGS-MN was inserted into the skin of hairless mice / ex vivo pig skin. The microneedle tip was pressed into the skin for 60 seconds and the backing layer was carefully removed with tweezers. The initial skin condition was recorded by taking a picture. The record was then taken every 10 / 30 minutes to observe the number of micropores left on the skin and the skin recovery at different time points.

[0057] The pinhole condition of pig skin (left) and mouse skin (right) after trypan blue staining is as follows: Figure 8 As shown, at different times after microneedle insertion (left image 0 min, right image 0 min), Figure 10 (min) Mouse skin condition as Figure 9 As shown, at different times after microneedle insertion (left image 0 min, middle image 0 min), Figure 10 min, right Figure 20 min) Pigskin condition as follows Figure 10 As shown.

[0058] As shown in the images, obvious blue needle holes were observed on both pig and mouse skin, proving that PMGS microneedles can be effectively inserted into the skin for drug delivery. Within 10 minutes, the needle holes disappeared on the mouse skin, and after 20 minutes, the needle holes on the pig skin were barely visible, indicating that the skin had almost completely recovered. This demonstrates that PMGSP-MN has good biocompatibility, causes no significant damage to the skin, and has good safety.

[0059] Test Example 6: PMGS's effect against HPV6 pseudovirus HaCat cells at 8 × 10 4 Cells were seeded at a density of [number] cells / mL in 96-well cell culture plates and cultured overnight. Then, 5 μL of a mixture of HPV6 pseudovirus particles was added to HaCat cells. Five concentrations of alginate sulfate were prepared: 0.2, 0.1, 0.05, 0.025, and 0.0125 μg / mL, with three replicates for each concentration. Blank wells and virus control wells were also included. Cells were cultured for another 24 hours. The culture medium was discarded, and the cells were washed twice with PBS. 100 μL of lysis buffer was added to each well, and the cells were shaken on ice. The lysis buffer was then transferred to a 96-well white chromogenic plate using a pipette. Chromogenic buffer was then added, and the relative fluorescence intensity in each well was measured using a fluorescence microplate reader. The fluorescence intensity of the treated group and the virus control group was compared. The inhibition rate of the sample against the virus was calculated as: (Fluorescence intensity of the virus group - Fluorescence intensity of the experimental group) / (Fluorescence intensity of the virus group - Fluorescence intensity of the blank group). 100%, the results are shown in Table 1 below and Figure 11 As shown.

[0060] Table 1. Inhibition rate of HPV6 virus at different concentrations of PMGS

[0061] As shown in the images and tables, PMGS in the HaCat cell line achieved an IC50 response to HPV6 pseudovirus. 50 The value was 0.097 μg / mL, indicating that PMGS has a significant effect on low-risk HPV viruses in vitro.

[0062] Test Example 7: Cytotoxic Effects of PMGS and Microneedle Extract on HaCat Cells To evaluate the biocompatibility of PMGS with PVP and PVA, the effect of microneedle matrix materials on the viability of human immortalized keratinocytes (HaCaT) was determined using the CCK-8 assay. Cells were seeded at a density of 5000 cells / well in 96-well plates (100 μL per well). After overnight culture, the original culture medium was aspirated, and 100 μL of culture medium containing the test sample was added. Four concentrations of PMGS were set: 1, 0.75, 0.5, and 0.25 mg / mL. Four concentrations of microneedle extract were set: 0.2, 0.1, 0.05, and 0.025 mg / mL. 24 h after administration, 10 μL of CCK-8 solution was added, and the cells were incubated for 1–2 h. Cell viability was measured using a microplate reader at 450 nm. The results are shown below. Figure 12 and Figure 13 As shown, Figure 12 The results of PMGS assay for HaCat cell cytotoxicity are as follows. Figure 13 The results show the cytotoxicity of microneedle extract on HaCat cells.

[0063] As shown in the images, after 24 hours of co-incubation, the viability of HaCaT cells remained above 80%, indicating that the microneedle matrix material has no obvious cytotoxicity and is biosafe.

[0064] Test Example 8: Immune activation effect of PMGS on Raw264.7 In the study of the effect of PMGS on the viability of Raw264.7 monocytes and macrophages, the cytotoxicity of PMGS was first detected using the same method as described above. The results are as follows: Figure 14 As shown.

[0065] As shown in the image, PMGS can promote the proliferation of Raw264.7 cells at concentrations ranging from 0 to 1000 μg / mL, and this effect is dose-dependent.

[0066] In the effect of PMGS on cytokine expression in Raw264.7 monocytes and macrophages, at a concentration of 1×10⁻⁶... 6 Cells were cultured in 6-well plates with 2 mL of complete culture medium per well. PMGS was administered at concentrations of 1, 5, 25, 50, and 100 µg / mL, while the positive control (LPS) was administered at 100 ng / mL. An equal volume of culture medium was added to the blank control group. After 6 h of culture, the culture medium was removed, and cells were washed twice with PBS. RNA was extracted for subsequent qRT-PCR detection. Raw264.7 monocytes / macrophages were stimulated with different concentrations of PMGS, and the transcriptional levels of highly expressed cytokines such as TNFα and IL-1β were detected. The results are as follows: Figure 15 As shown.

[0067] As shown in the image, PMGS can activate Raw264.7 cells and promote the expression of cytokines such as TNFα, IL-1β, and IFNα.

[0068] In the study on the effect of PMGS on the phagocytosis of Raw264.7 cells, Raw264.7 cells were collected and processed at a concentration of 1×10⁻⁶ cells per cell. 5 Cells were seeded at a density of 0.2 mL / well in 96-well plates. After 12 h of incubation, different concentrations of sample (5, 25, 50, 100, 500 µg / mL) or LPS (1 µg / mL) were added to each well. An equal volume of culture medium was added to the blank control group. After 24 h of incubation, the supernatant was discarded, and 100 µL of 0.075% neutral red solution was added to each well. Incubation was continued at 37 °C for 5 min. Cells were washed three times with PBS to remove free neutral red. Cells were lysed using lysis buffer at room temperature, and OD values ​​were measured at 570 nm. A neutral red phagocytosis assay was used to investigate the effect of PMGS on the phagocytic ability of Raw264.7 cells. The results are as follows: Figure 16 As shown.

[0069] As shown in the images, PMGS significantly enhances the phagocytic capacity of Raw264.7 cells in a dose-dependent manner, thereby increasing their antigen-presenting ability. Antigen presentation by Raw264.7 cells contributes to the activation of T lymphocytes and the response of the host's adaptive immune system, thus enhancing the host's ability to recognize and eliminate pathogens.

[0070] Test Example 9: Effects of PMGS microbes on the skin immune microenvironment of healthy mice Three days prior to microneedling, mice underwent hair removal with depilatory cream to eliminate the influence of cream irritation on the experiment. The skin on the back was held firmly at the application site for one minute with the thumb and forefinger, and then secured with medical tape. The BMN was inserted into the mouse skin as completely as possible; when the MN was removed after 10 minutes, the needle dissolved in the skin. Six hours after administration, mice were euthanized by cervical insemination. Immunohistochemical staining was performed on the skin sections from the application site and inguinal lymph nodes. The effects of PMGS microneedling on partial immune cell infiltration in mouse skin were analyzed. Figure 17 As shown.

[0071] As shown in the images, immunohistochemical analysis revealed that microneedle administration can recruit immune cells such as monocytes, dendritic cells, macrophages, and NK cells in the innate immune system, but has no effect on T lymphocytes, which play an important role in acquired immunity. There was no infiltration of T lymphocytes in the skin tissue, which may also be related to the relatively weak immunogenicity of polysaccharides.

[0072] Test Example 10: Stability Test The morphological changes and infrared spectra of the prepared microneedle products before and after being placed at 100℃ for 24 hours are shown in the figure. Figure 22 and Figure 23 As shown. Figure 22 The two images on the left are of the product before treatment, and the two images on the right are of the product after treatment at 100℃ for 24 hours. Figure 23 The upper middle section shows the infrared spectrum of the product before treatment, while the lower section shows the infrared spectrum of the product after treatment at 100℃ for 24 hours.

[0073] The results show that it has good high-temperature stability and can withstand 100℃. Furthermore, previous test results indicate that it also has good puncture effect and fast dissolution rate.

[0074] Comparative Example 1 In this comparative example, the substrate material of the needle tip layer is a stainless steel needle tip, and PMGS is prepared as a coating solution and coated on the surface of the needle tip layer. The coating solution is a 200 mg / mL PMGS solution.

[0075] Comparative Example 2 This comparative example is basically the same as Example 1, except that the main matrix material of the needle tip layer is HA, while PMGS is prepared by adding it to HA as an additive, and the ratio of HA to PMGS is 1:1 (100 mg / mL HA + 100 mg / mL PMGS).

[0076] Comparative Example 3 This comparative example is the cream formulation in the applicant's prior authorized patent CN111481502B.

[0077] Compared with the product in Comparative Example 1, the proposed solution has many advantages. Taking penetration effect as an example, the PMGS content of the cream in Comparative Example 3 is 1-5%, with a cumulative penetration rate of 64%. A single 10×10 array microneedle patch theoretically contains 195 μg of PMGS, which can be completely dissolved in the skin in 10 minutes. To achieve the same subcutaneous drug penetration, based on the cream drug content and penetration rate, the required amount of cream is 6.09-30.47 mg, which is 31.25-156.25 times the mass of a single microneedle tip.

[0078] like Figure 21 As shown in the in vitro simulation data, the PMGS-only group exhibits higher mechanical strength and drug loading capacity, demonstrating superior advantages compared to Comparative Example 2. Furthermore, the PMGS-only group contains only PMGS drug components, enabling targeted drug delivery. Brown algae polysaccharide sulfate (PMGS) can effectively treat warts through both activating skin immunity and inhibiting viral adsorption. As a marine-derived polysaccharide material, PMGS possesses excellent biocompatibility and avoids the need for other polymer materials, ensuring the safety of microneedles.

[0079] Furthermore, the PMGS microneedles prepared in this application possess excellent mechanical properties, dissolution rate, and transdermal efficiency. PMGS microneedles solve the problem of insufficient mechanical strength in most soluble microneedles and require no additional reinforcing materials. As a water-soluble polysaccharide material, PMGS dissolves rapidly after insertion into the skin, facilitating drug delivery. The solution in this application addresses the issue of poor transdermal efficacy of macromolecular polysaccharides, directly delivering macromolecular PMGS to the basal cells where the virus resides, achieving localized drug delivery and resulting in better treatment of warts. In addition, the needle tip is entirely composed of PMGS, allowing for a large drug loading capacity per needle, and the preparation is simple, natural, and safe. Of course, depending on the needs, PMGS microneedles can also be loaded with various drugs in the future, serving as drug carriers and simultaneously exerting immune activation, antioxidant, and other effects to assist in treatment.

[0080] Experimental Example 1 The inventors experimented with PMGS with molecular weights of 5 kDa, 10 kDa, 100 kDa, 270 kDa, and 300 kDa, and found that even PMGS with a smaller molecular weight (10 kDa) could produce needle tips with a certain mechanical strength by increasing the concentration. However, high-concentration PMGS needle tips with low molecular weight are brittle after drying. In practice, the molecular weight range of PMGS can be selected from 10 to 300 kDa, which combines good antiviral effects with mechanical strength.

[0081] Experiment Example 2 Using PM membranes to simulate in vitro puncture experiments, it was found that at the same concentration, PMGS alone showed a higher puncture rate than HA, PVP, and the mixed group.

[0082] Further screening of PMGS concentrations yielded the following results: Figure 18 As shown, the puncture rate increased with increasing concentration. However, at high concentrations (200 mg / mL), there were problems such as poor solution fluidity and more difficult preparation processes. In practice, a concentration of 150 mg / mL can be selected for preparation.

[0083] Experimental Example 3 Experiments by researchers have shown that selecting PVA (such as PVA0588, PVA1788, etc.), PVP series (PVP-K30, PVP-K90), and HA (10~400kDa) are all feasible, as shown in the results. Figure 19 As shown in the figure, from left to right, the images show the effects of using PVA, PVP, and HA as backing materials. Although there are slight issues such as bubbles, bending, and brittleness, they can still function normally as backing layers.

[0084] like Figure 20The image shows the morphology of the microneedle backing obtained by mixing PVP and PVA. The microneedle backing is flat, bubble-free, and has good toughness. It should be noted that those skilled in the art can adjust and select the backing layer and its preparation. For those skilled in the art, a basic requirement for the backing layer is that it can support the microneedle layer. Those skilled in the art can adjust and change the backing layer as needed, which is a common skill of those skilled in the art.

[0085] The above description is merely an embodiment of this application, and the scope of protection of this application is not limited to these specific embodiments, but is determined by the claims of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principles of this application should be included within the scope of protection of this application.

Claims

1. A patch, characterized in that, It includes: 1) Backing layer; 2) The needle tip layer is alginate sulfate; The structure of the alginate sulfate is as follows: ； The alginate sulfate has a molecular weight range of 10-300 kDa, and the content of polymannuronic acid in the alginate sulfate is 5-95%, the content of polyduronic acid is 5-95%, and the degree of substitution of sulfate groups is 5-15%.

2. The patch according to claim 1, characterized in that, The backing layer comprises one or more of polyvinyl alcohol, polyvinylpyrrolidone, hydroxypropyl methylcellulose, chondroitin sulfate, chitosan, alginate sulfate, sodium carboxymethyl cellulose, sodium carboxymethyl starch, and hyaluronic acid.

3. The patch according to claim 1, characterized in that, The backing layer comprises a first material and a second material; The first material is selected from one or more of chitosan, polyvinylpyrrolidone, alginate sulfate, and chondroitin sulfate; The second material is selected from one or more of polyvinyl alcohol, hyaluronic acid, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, and sodium carboxymethyl starch.

4. The patch according to claim 1, characterized in that, The backing layer comprises polyvinyl alcohol and polyvinylpyrrolidone.

5. The patch according to claim 1, characterized in that, The needle tip height in the needle tip layer is 200~2000μm.

6. A method for preparing a patch as described in any one of claims 1 to 5, characterized in that, The preparation method includes the following steps: 1) Prepare the backing layer solution and the needle tip layer solution for later use; 2) Pipette the tip layer solution into the microneedle mold, centrifuge for the first time, then remove the excess tip layer solution and add the backing layer solution, and centrifuge for the second time; 3) After drying, the film is demolded to obtain the patch.

7. The method for preparing the patch according to claim 6, characterized in that, In the first centrifugation treatment, centrifuge at 2500~3500 rpm for 10~30 min; and / or, In the second centrifugation treatment, centrifuge at 2500~3500 rpm for 5~15 min; and / or, Dry at 20-30℃ for 6-18 hours during the drying process.

8. The use of the patch according to any one of claims 1 to 5 in the preparation of products having the effect of preventing and / or treating HPV and / or skin warts.

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