Algin sulfate (PMGS) patch as well as preparation method and application thereof

Abstract

The invention relates to an algin sulfate (PMGS) patch as well as a preparation method and application thereof, and belongs to the technical field of medicines. According to the application, the fucoidan polysaccharide sulfate (PMGS) is used as a main raw material of the microneedle for the first time, the needle tip is completely composed of PMGS, the PMGS plays a role in medicine and adjuvant integration, on one hand, the PMGS is used as a needle tip matrix to provide enough mechanical strength to pierce warts and surrounding skin and is more suitable for related warts with obvious skin thickening, and on the other hand, the PMGS is diffused and released at the position where viruses are located, so that the effect of preventing and treating warts is achieved. The skin wart treatment effect is achieved by preventing virus from entering and enhancing skin immunity, compared with a previous authorized patent scheme, the problems that PMGS as macromolecular polysaccharide enters the skin and is insufficient in absorption are solved in a microneedle mode, PMGS can be directly delivered to basal layer cells where HPV is located, and fixed-point drug delivery is achieved.

Description

Technical Field

[0001] The present application relates to a polymannuronate sulfate (PMGS) patch and its preparation method and application, belonging to the field of medical technology. Background Art

[0002] Human papillomavirus (HPV) is an enveloped circular double-stranded DNA virus. Its genome contains 8 kb of DNA sequence and shows considerable tropism for human skin and mucosal epithelium. So far, more than 200 HPV genotypes have been identified, which can be divided into high-risk HPV and low-risk HPV according to their carcinogenicity. HPV infection starts from minor skin and mucosal injuries, and there are three important steps in the infection process: First, the L1 protein binds to HSPG through its non-polar sites of K278-K361. After viral internalization, the virus is transported through the endosomal system, the disassembly or degradation of the viral capsid, the L2 protein carries the viral genome to escape from the endosome into the cytoplasm, and is transported along microtubules mediated by dynein to the nucleus; Next, the E6 and E7 proteins bind to the p53 and Rb proteins respectively, resulting in the inactivation of these two tumor suppressor genes and enabling the cells to grow infinitely; Finally, this infinite growth causes two results. The infection of high-risk HPV 16, 18, etc. in tissues such as the cervix leads to malignant carcinogenesis, and the infection of low-risk HPV 1, 3, 10, etc. in the skin tissue produces benign verrucous hyperplasia.

[0003] Skin warts caused by HPV are extremely common, with an incidence rate of 7-12%. Children and young people are more susceptible to the disease, and the incidence rate is the highest in children aged 12-16. Clinically, according to the different locations of occurrence, they are divided into verruca vulgaris, filiform warts, flat warts, plantar warts, etc. Another study shows that the HPV types causing different types of skin wart infections are also different. The common types of verruca vulgaris are HPV1, 2, 4, 7, 27, flat warts are HPV3, 10, 28, 41, and plantar warts are HPV1, 2, 4. The same is that the lesion sites of skin warts are painful when pressed, or there may be no symptoms; under a skin microscope, light yellow papules, thickened keratin rings and round papillary keratin hyperplasia can be seen, accompanied by small black dots formed by the rupture and bleeding of capillaries. The corresponding pathological changes are epidermal hyperkeratosis, acanthosis and papillary process elongation. The reason is that the virus exists in spinous layer and basal layer cells, infects and promotes the proliferation of keratinocytes, forming verrucous protrusions.

[0004] Generally speaking, skin wart lesions are self-limiting, do not endanger life, and will eventually be cleared by the cell-mediated immune response in most cases. However, HPV-related skin warts are highly refractory and last for many years, depending on the number of lesions and the infection site. When there are situations such as pain, affecting beauty and social interaction, repeated attacks, and a large number and continuous development, patients need treatment.

[0005] Currently, the main methods for treating skin warts are to ablate the warts and block the formation of warts, including methods such as drugs, physical therapy, and surgical resection. Physical therapies such as cryotherapy, laser, microwave, and radiation all achieve the treatment purpose by inducing degeneration, coagulation, and necrosis of the wart tissue; the keratolytic agent salicylic acid acts by destroying virus-infected cells without affecting the generation of keratinocytes, and it is a commonly used drug for treating flat warts on the face, plantar warts, and common warts on the hands; antiviral drugs and anti-mitotic drugs are also commonly used in the treatment of refractory recurrent skin warts. Ayman Elsayed et al. tried intralesional injection of acyclovir to treat skin warts. The results showed that complete clearance of warts was observed in 52.6% of the patients in the acyclovir group, and partial remission was observed in 36.8% of the patients, which was significantly higher than that in the normal saline control group. It can be an effective and well-tolerated treatment method for skin warts. Manal T. Barkat MD et al. injected bleomycin (1 mg / mL) intralesionally into the reduced plantar warts once every two weeks for a maximum of 4 times. The results of dermoscopic evaluation and clinical evaluation showed that clinical complete clearance of plantar warts was observed in 88.5% of the patients (23 / 26 patients), clinical improvement was observed in 7.7% of the patients (2 / 26 patients), and only 3.8% of the patients (1 / 26 patients) had clinical failure, showing a significant difference compared with the placebo group.

[0006] In addition, since the course of skin warts caused by HPV infection has an important relationship with autoimmunity, systemic or local immunotherapy has received increasing attention. Imiquimod is a new type of local immunomodulator that can induce various cytokines by stimulating peripheral immune cells such as Toll-like receptor 7, Langerhans cells, and keratinocytes at the local application site, thereby producing immunomodulatory and indirect antiviral effects. Hengge et al. treated common warts with 5% imiquimod, and complete regression of warts was observed in 30% of the patients, and the warts decreased by half in 26% of the patients. Autologous wart implantation is a traditional artificial immunotherapy, and its principle of action is to embed HPV antigen in the subcutaneous fat layer through surgery to stimulate the body to produce antibodies and trigger the body's cellular immune response.

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

[0008] Based on more than two decades of research, it is generally believed that MNs are divided into five categories: solid MNs, coated MNs, hollow MNs, dissolving MNs, and hydrogel MNs. The needles of solid microneedles, coated microneedles, and hollow microneedles are mostly made of materials such as silicon, titanium, and stainless steel, which have sufficient mechanical strength, but there are also problems such as poor biocompatibility, limited drug loading capacity, and difficult control of drug release. Dissolving microneedles and hydrogel microneedles are the current research hotspots, mostly using natural or synthetic polymeric materials with good biocompatibility. After insertion into the skin, the drugs loaded on the dissolving microneedles are released as the matrix dissolves, and the hydrogel MNs swell in the skin by absorbing tissue fluid to form porous aqueous microducts, through which the drugs contained in the reservoir can diffuse into the skin microcirculation.

[0009] Currently, microneedles have been widely used to deliver proteins, hydrophobic drugs, etc. for the treatment of diabetes, skin diseases, superficial skin tumors, etc. For example, Qu Xiaoying developed a dissolving microneedle based on HA / BSP for mediating 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 skin warts, some researchers have considered delivering drugs such as imiquimod and bleomycin directly to the lesion site through the microneedle formulation, increasing the local drug load while avoiding injection pain and improving patient compliance. Li Hansong et al. coated bleomycin on the tip of poly(lactic acid) (L-PLA) microneedles, and more than 80% of the bleomycin dissolved into the skin in vitro within 15 minutes. Compared with intralesional injection, the tip-coated microneedles more effectively distributed the drug to the subepidermal skin layer. Tsu-Man Chiu et al. prepared imiquimod-loaded dissolving microneedles using gelatin as the formulation, and the results showed that the delivery of imiquimod to the lower epidermal layer was achieved, playing a role in activating antigen-presenting cells and T cells.

[0010] Dissolving microneedles are usually made of materials with good biocompatibility and water solubility. They can completely dissolve in the skin and leave no sharp needles that can easily scratch the human body after use. At the same time, dissolving microneedles usually soften and dissolve in biological tissues when penetrating, thus preventing damage caused by mechanical forces related to application. Compared with synthetic polymer materials such as polyvinylpyrrolidone (PVP) and polyvinyl alcohol (PVA), polysaccharides have been widely used in the preparation of dissolving microneedles due to their good biocompatibility, solubility, and activities such as antibacterial, anti-inflammatory, and antioxidant. Tang Zhongming et al. used sulfated heteropolysaccharide - ulvan extracted from green algae as raw material and prepared ulvan dissolving microneedles with an average height of 655 μm and an aspect ratio of 2.63 by a two-step centrifugation method. It has sufficient mechanical strength to bypass the stratum corneum barrier of pig skin to the dermis layer, dissolves rapidly 2 minutes after insertion, and the needle height decreases by 90.3%. The loaded model drug can be released in situ in the skin layer, with advantages such as convenient use, good patient compliance, and good drug release performance.

[0011] Van der Maaden and his colleagues developed a chitosan pH-sensitive MNs array for delivering inactivated poliovirus vaccine particles, and in vivo tests showed an induced specific antibody response. Liu Wei et al. used hyaluronic acid and carboxymethyl chitosan as the back layer and the needle tip layer materials respectively to prepare multifunctional double-layer dual-drug-loaded microneedles, which sequentially released tetracycline hydrochloride (TH) and recombinant human epidermal growth factor (rh-EGF) locally. In an in vivo diabetic wound model, it showed effects such as inhibiting inflammation, promoting angiogenesis, collagen deposition, and tissue regeneration, and promoting the repair of diabetic wounds.

[0012] The applicant found in the prior Chinese authorized patent CN111481502B that the alginate sulfate (PMGS, such as Figure 1As shown, it has anti-HPV activity, has good inhibitory effects on both low-risk and high-risk viruses, and has low toxicity and side effects. Through Pull-down experiments, it was 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 the virus adsorbs to cells and inhibit virus infection. In addition, fucoidan sulfate also significantly inhibited HPV infection on the skin of BALB / c nude mice. PMGS activates the local immunity of mouse cervical tissues, recruits immune cells such as monocytes, dendritic cells, and macrophages, and activates the innate immune response of the host. By activating monocytes and macrophages, PMGS enhances their phagocytic ability, promotes the expression and secretion of cytokines by T cells and NK cells, enhances the immune function of lymphocytes, improves the killing effect of lymphocytes on target cells, and inhibits the expression of oncoproteins E6 / E7. It can be seen that PMGS can play an anti-HPV role by inhibiting virus binding and activating the body's immunity, and it is an effective anti-HPV active substance.

[0013] However, due to the large molecular weight of PMGS, the transdermal absorption effect is not good. Microneedles, as a currently highly concerned drug delivery method, in view of the fact that there are still few studies on the microneedle drug delivery method for PMGS in the current existing technologies, the feasibility of using PMGS as a dissolving microneedle matrix material to form needles alone or preparing microneedles loaded with PMGS by means of material compounding is still unknown. Therefore, in order to further solve the problems existing in the prior patent solutions, it is necessary for the applicant to provide a product that can more effectively prevent / treat HPV / skin warts. Summary of the Invention

[0014] In order to solve the above problems, a fucoidan sulfate (PMGS) patch, its preparation method and application are provided. In this application, fucoidan sulfate is used as the main raw material of microneedles for the first time, and the needle tips are completely composed of fucoidan sulfate, which can not only solve the problem of insufficient skin absorption caused by PMGS as a macromolecular polysaccharide, but also directly deliver PMGS to the basal cells where HPV is located to achieve targeted drug delivery.

[0015] This application provides a fucoidan sulfate (PMGS) patch, which includes: 1) A backing layer; 2) A needle tip layer, and the needle tip layer is one or more of λ / ι / κ-type carrageenan, polymannuronic acid sulfate, polyglucuronic acid sulfate, fucoidan sulfate, fucoidan, sulfated dextran, chondroitin sulfate, keratin, heparin and its analogues.

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

[0017] It should be noted that, in addition to the above materials, those skilled in the art can also select other polysaccharides with antiviral effects to be used alone as the tip layer, including but not limited to λ / ι / κ-carrageenan, polymannuronic acid sulfate, poly guluronic acid sulfate, alginate sulfate, fucoidan, sulfated dextran, chondroitin sulfate, cutin, heparin and its analogues, etc.

[0018] Optionally, the tip height in the tip layer is 200-2000 μm. This tip height can more easily make the tip layer reach the basal layer where HPV is located in the skin, reduce the generation of skin warts by acting on HPV, and fundamentally treat skin warts. Existing skin warts will gradually fall off, thus achieving the prevention / treatment of HPV / skin warts. Since the inhibition and binding of alginate sulfate to HPV virus have been verified, and in addition, the present application also reveals that alginate sulfate can activate the non-specific immunity of organisms and improve the immune level, so its preventive effect can be reasonably expected.

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

[0020] Optionally, the molecular weight range of the alginate sulfate is 10-300 kDa.

[0021] The inventors tried PMGS with molecular weights of 5 kDa, 10 kDa, 100 kDa, 270 kDa, and 300 kDa, and found that the PMGS with a smaller molecular weight (10 kDa) has slightly poor mechanical strength, but the tips with the required mechanical strength can also be prepared by adjusting the concentration. However, the tips of high-concentration PMGS with a low molecular weight are brittle after drying. The above PMGS molecular weight has both good antiviral effects and meets the requirements of mechanical strength, and the tips are less brittle after drying.

[0022] Optionally, the content of polymannuronic acid in the alginic acid sulfate is 5-95%, the content of poly guluronic acid is 5-95%, and the degree of substitution of sulfate ester groups is 5-15%.

[0023] Optionally, one or more of polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), chondroitin sulfate, chitosan, alginate sulfate, sodium carboxymethylcellulose (CMC-Na), sodium carboxymethyl starch (CMS-Na), and hyaluronic acid (HA).

[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 carboxymethylcellulose, and sodium carboxymethyl starch.

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

[0026] It can be understood that the main function of the backing layer in the solution of the present application is to load the needle tip layer. Those skilled in the art can select a suitable material as the backing layer material according to needs, and can also add other feasible medical components to the backing layer or modify the selected material of the backing layer.

[0027] For example, by quaternizing and chitosanizing polyvinyl alcohol, the backing layer comprises a composite material of quaternized chitosan and polyvinyl alcohol. The microneedle backing layer composed of the two cross-linked has good mechanical properties, solubility and biocompatibility. After the microneedle tip dissolves, the backing layer can be dissolved with a small amount of sterile water, and the backing layer becomes a gel and is applied to the wart. It not only plays a bactericidal and sedative role, relieves itching and swelling at the affected area, but also avoids the spread of skin warts caused by friction and scratching of patients. Active polysaccharides such as chitosan can also be added alone to the backing layer to provide antibacterial and anti-inflammatory effects, and can also relieve the redness and itching symptoms at the wart.

[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, is easy to prepare, and the prepared backing layer has a certain flexibility, is suitable for uneven wart-like protrusions, and has good skin adhesion.

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

[0031] Optionally, the drug loading amount of a single microneedle of the fucoidan sulfate (PMGS) patch is 100 - 500 μg.

[0032] Optionally, the needle tip in the needle tip layer is a conical needle or a pyramid shape. Those skilled in the art can also select other available shapes. For example, in order to have better mechanical strength, the pyramid shape can be preferentially selected.

[0033] The present application provides a preparation method of the above-mentioned fucoidan sulfate (PMGS) patch, which is characterized in that the preparation method comprises the following steps: 1) Prepare the backing layer solution for standby and prepare the needle tip layer solution for standby; 2) Aspirate the solution of the tip layer into the microneedle mold, perform the first centrifugation treatment, then remove the excess solution of the tip layer and add the solution of the backing layer, and perform the second centrifugation treatment; 3) After drying treatment, demold to obtain the alginate sulfate (PMGS) patch.

[0034] Optionally, 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, in the drying treatment, dry at 20 - 30 °C for 6 - 18 h.

[0035] After the solution of the tip layer is added, the above centrifugation conditions can make the solution of the tip layer fully centrifuged into the mold; after the solution of the backing layer is added, the above centrifugation conditions can make the solution of the backing layer fully contact with the solution of the tip layer, and the drying temperature is preferably maintained at 20 - 30 °C. If dried at a higher temperature, it is easy to cause the backing layer to be easily bent and uneven, affecting the subsequent use effect.

[0036] The present 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 verruca vulgaris, flat wart, filiform wart, plantar wart, etc. caused by HPV.

[0037] The beneficial effects of the present application include but are not limited to: 1. According to the alginate sulfate (PMGS) patch, its preparation method and application of the present application, for the first time, fucoidan sulfate (PMGS) is used as the main raw material of the microneedle, and the tip is completely composed of PMGS. PMGS plays the role of both medicine and excipient. On the one hand, PMGS provides sufficient mechanical strength as the tip matrix to pierce the skin around the wart, and is more suitable for related wart diseases with obvious skin thickening. On the other hand, PMGS diffuses and releases at the site where the virus is located, and plays a therapeutic effect on skin warts by preventing virus adsorption and enhancing skin immunity.

[0038] 2. According to the alginate sulfate (PMGS) patch, its preparation method and application of the present application, based on the problems existing in the prior authorized patents, the feasibility of PMGS forming the tip alone is further studied. Finally, the problem of insufficient skin absorption caused by PMGS as a macromolecular polysaccharide is solved by dissolving the microneedle. And the design of the tip layer can directly deliver PMGS to the basal cells where HPV is located, so as to achieve targeted drug delivery, with good drug delivery effect, higher safety and better therapeutic effect.

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

[0040] The drawings described herein are used to provide a further understanding of the present application and constitute a part of the present application. The schematic embodiments and descriptions thereof of the present application are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings: Figure 1 It is a schematic structural diagram of the alginate sulfate (PGMS) related to the present application; Figure 2 It is a preparation flow chart of the alginate sulfate (PMGS) patch related to Example 1 of the present application; Figure 3 It is a SEM observation result diagram of the microneedle morphology related to Test Example 1 of the present application; Figure 4 It is a determination result diagram of the mechanical strength of the PMGS-PVP / PVA microneedle related to Test Example 2 of the present application; Figure 5 It is a confocal scanning 3D result diagram in the in vitro insertion depth determination of the PMGS-PVP / PVA microneedle related to Test Example 3 of the present application; Figure 6 It is a standard curve result diagram of the PMGS post-column derivation related to Test Example 4 of the present application; Figure 7 It is a liquid phase result diagram of the drug loading amount at the tip of the microneedle related to Test Example 4 of the present application; Figure 8 It is a result diagram of the needle hole conditions of the pig skin and mouse skin after trypan blue staining related to Test Example 5 of the present application; Figure 9 It is a result diagram of the mouse skin state at different times after the microneedle insertion related to Test Example 5 of the present application; Figure 10 It is a result diagram of the pig skin state at different times after the microneedle insertion related to Test Example 5 of the present application; Figure 11 It is a result diagram of the inhibitory effect of PMGS on HPV6 in HaCat cells related to Test Example 6 of the present application; Figure 12 It is a result diagram of the cytotoxicity detection of PMGS on HaCat cells related to Test Example 7 of the present application; Figure 13 It is a result diagram of the cytotoxicity detection of the microneedle leaching solution on HaCat cells related to Test Example 7 of the present application; Figure 14This is the result graph of the effect of PMGS involved in Test Example 8 of this application on the viability of mononuclear macrophage Raw264.7 cells; Figure 15 This is the result graph of the effect of PMGS involved in Test Example 8 of this application on the gene expression of cytokines in mononuclear macrophage Raw264.7 cells; Figure 16 This is the result graph of the effect of PMGS involved in Test Example 8 of this application on the phagocytic ability of mononuclear macrophage Raw264.7 cells; Figure 17 This is the result graph of the effect of PMGS microneedles involved in Test Example 9 of this application on the infiltration of partial immune cells in mouse skin; Figure 18 This is the result graph of the PMGS concentration screening involved in Experimental Example 2 of this application; Figure 19 This is the effect graph of PVA, PVP, and HA as backings involved in Experimental Example 3 of this application; Figure 20 This is the effect graph of PVP and PVA mixed as a backing involved in Experimental Example 3 of this application; Figure 21 This is the result graph of the in vitro simulated penetration of the needle tip layer involved in Comparative Example 2 of this application; Figure 22 This is the result graph of the morphology of the microneedles before and after treatment at 100 °C for 24 h involved in Test Example 10 of this application; Figure 23 This is the infrared spectrum of the microneedles before and after treatment at 100 °C for 24 h involved in Test Example 10 of this application. Detailed implementation manners

[0041] The following describes this application in detail with reference to the embodiments. However, this application is not limited to these embodiments. Unless otherwise specified, the raw materials and reagents in the embodiments of this application are purchased through commercial channels.

[0042] The following illustrates the solution of this application through specific embodiments.

[0043] Embodiment 1 1) Preparation of the backing layer: Accurately weigh 1 g of PVA0588, add 5 mL of pure water, fully dissolve it, and let it stand at room temperature to defoam until the solution is clear and transparent to prepare a 200 mg / mL PVA solution; Use a pipette to transfer 1 mL of the 200 mg / mL PVA solution, add 100 mg of PVP-K30, vortex and mix well, and after fully dissolving, ultrasonically defoam to obtain a 200 mg / mL PVA and 100 mg / mL PVP-K30 mixed solution for standby; 2) The solution of the tip layer uses a 150 mg / mL PMGS solution. In this example, the molecular weight of PMGS is selected as 270 kDa for preparation, and PMGS with other molecular weights can be selected. For example, an optional range is 10 - 300 kDa; 3) Product preparation: Micro - needles are prepared by a two - step centrifugation method. The steps are as Figure 2 shown. First, use a pipette to aspirate about 200 μL of the upper tip - layer solution into the micro - needle mold, and centrifuge at 3000 rpm / min for 20 min to fully fill the mold voids with the solution and eliminate air bubbles at the same time; then carefully remove the excess tip - layer solution and add about 200 μL of the backing - layer solution, and centrifuge at 3000 rpm / min for 10 min. After centrifugation, transfer it to a 25 °C oven and dry for 12 h. After demolding, store it in a desiccator to obtain the alginate sulfate (PMGS) patch.

[0044] Example 2 1) Backing - layer preparation: Accurately weigh 1 g of PVA0588, add 5 mL of pure water, fully dissolve it, and let it stand at room temperature to defoam until the solution is clear and transparent to prepare a 200 mg / mL PVA solution; use a pipette to transfer 1 mL of the 200 mg / mL PVA solution, add 100 mg of PVP - K30, vortex and mix well, and after fully dissolving, ultrasonically defoam to obtain a 200 mg / mL PVA and 100 mg / mL PVP - K30 mixed solution for standby; 2) The solution of the tip layer uses a 150 mg / mL PMGS solution; 3) Product preparation: Micro - needles are prepared by a two - step centrifugation method. First, use a pipette to aspirate about 200 μL of the upper tip - layer solution into the micro - needle mold, and centrifuge at 2500 rpm / min for 30 min to fully fill the mold voids with the solution and eliminate air bubbles at the same time; then carefully remove the excess tip - layer solution and add about 200 μL of the backing - layer solution, and centrifuge at 2500 rpm / min for 15 min. After centrifugation, transfer it to a 30 °C oven and dry for 6 h. After demolding, store it in a desiccator to obtain the alginate sulfate (PMGS) patch.

[0045] Example 3 1) Backing - layer preparation: Accurately weigh 1 g of PVA0588, add 5 mL of pure water, fully dissolve it, and let it stand at room temperature to defoam until the solution is clear and transparent to prepare a 200 mg / mL PVA solution; use a pipette to transfer 1 mL of the 200 mg / mL PVA solution, add 100 mg of PVP - K30, vortex and mix well, and after fully dissolving, ultrasonically defoam to obtain a 200 mg / mL PVA and 100 mg / mL PVP - K30 mixed solution for standby; 2) The solution of the tip layer uses a 150 mg / mL PMGS solution; 3) Product preparation: Microneedles were prepared by a two-step centrifugation method. First, approximately 200 μL of the upper tip layer solution was aspirated with a pipette into a microneedle mold and centrifuged at 3500 rpm / min for 10 min to fully fill the mold gaps with the solution and eliminate air bubbles simultaneously. Then, after carefully removing the excess tip layer solution, approximately 200 μL of the backing layer solution was added and centrifuged at 3500 rpm / min for 5 min. After centrifugation, it was transferred to a 20 °C oven and dried for 18 h. After demolding, it was placed in a desiccator for storage to obtain the fucoidan sulfate (PMGS) patch.

[0046] Test Example 1 SEM Observation of Microneedle Morphology For the SEM morphological observation of the microneedle patch product, before observation, the HA / PVP-MN sample was taken out and fixed on a sample plate with conductive tape. After vacuum sputtering with gold, the overall image of the 10×10 microneedle array and the surface morphology image of a single needle were observed under a scanning electron microscope. The results are as Figure 3 shown. The left figure is the overall image of the prepared 10×10 microneedle array at 80 times magnification, and the right figure is the single needle height measurement image at 200 times magnification. Specifically, the tip-to-tip distance is 515 μm, the bottom size of the microneedle is 310 μm×310 μm, the size of the backing layer is 9 mm×9 mm, and the number of microneedle arrays is 10×10. It can be seen from the left figure that the conical morphological structure of the prepared microneedles is clear, the array is neat, there is no obvious needle breakage or missing phenomenon within the visual field, the backing layer is flat, without shrinkage or bubbles; the height measurement result of the right figure shows that the height of a single needle is about 720 μm.

[0047] Test Example 2 Determination of the Mechanical Strength of PMGS-PVP / PVA Microneedles The mechanical strength of PMGS-PVP / PVA microneedles was determined. A mobile sensor of appropriate size was selected, the microneedle array was placed in the chassis, and the backing layer was in contact with the chassis. The program was set for single extrusion. The mobile sensor contacted the tip part from top to bottom. When the bottom of the sensor touched the topmost part of the microneedle, the travel of the sensor and the force applied to the microneedle were recorded. The results are as Figure 4 shown.

[0048] As can be seen from the picture, when 150 mg / mL PMGS was used as the tip, at a displacement of 640 μm, the force borne by each single microneedle in each group was higher than the force theoretically required to penetrate the stratum corneum (0.15 N). Therefore, it has the ability to penetrate the stratum corneum.

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

[0050] After trimming the edge of the backing of RhB-MN, the tip was forcefully inserted into ex vivo porcine skin for 15 min. The skin sample after insertion was taken out and placed upside down in a confocal dish, and the fluorescence intensity was detected at 561 nm using a confocal laser microscope for visualization to observe the vertical distribution of Rhodamine in the skin. The plane parallel to the surface of porcine skin was defined as the XY plane, and the plane perpendicular to the porcine skin downward was the Z plane. The initial scanning plane of the skin was when an obvious microneedle array pattern appeared in the stratum corneum scan, and at this time Z = 0 μm. Starting from the initial scanning surface, an optical image was captured every 20-μm interval along the Z plane downward until no red fluorescence was observed, and finally the diffusion depth of Rhodamine in ex vivo porcine skin was obtained. The results are as Figure 5 shown.

[0051] As can be seen from the pictures, after the microneedles loaded with Rhodamine B were inserted into the porcine skin, neat tip holes were formed, and Rhodamine B diffused and released in the porcine skin. Judging from the depth where the fluorescence existed, the depth of microneedle insertion into the porcine skin was about 280 μm, which was much greater than the thickness of the stratum corneum.

[0052] Those skilled in the art know that the skin is the largest organ of the human body and is divided into the epidermis, dermis, and subcutaneous tissue. The epidermis is the outermost layer of the skin, generally with a thickness of 0.035 - 0.15 mm. According to the differentiation and characteristics of keratinocytes, the epidermis is divided into 5 layers from the outside to the inside, namely the stratum corneum, stratum lucidum (only present in the palms and soles), stratum granulosum, stratum spinosum, and stratum basale. The stratum basale is connected to the dermis by means of a basement membrane, and the stratum basale is located at the bottom layer of the epidermis and is only a layer of columnar or cubic basal cells, about 10 - 14 μm in size. From the in vitro insertion depth experiment of microneedles, the depth of microneedle insertion into the porcine skin was about 280 μm, which was sufficient to reach the basal cells (150 μm), and the HPV virus infects the basal cells, and the excessive proliferation of basal cells leads to skin warts. Therefore, it can be known that PMGS in the microneedles of this application can be successfully delivered to the basal cells to prevent virus adsorption and play an antiviral role.

[0053] Test Example 4 Determination of the drug loading capacity of PMGS-PVP / PVA microneedles Weigh 150 mg of PMGS, dissolve it in 1 mL of pure water, and let it stand and swell at room temperature until the solution becomes uniform and transparent, obtaining a PMGS solution with a concentration of 150 mg / mL. Use a pipette to aspirate 200 μL of the above PMGS solution into a microneedle mold, centrifuge at 3000 rpm for 20 min. After centrifugation, scrape off the excess solution at the tip of the needle, transfer it to an oven at 25 °C and dry for 12 h. Use tape to stick out the dried needle tip, dissolve it with 0.3 mL and 0.5 mL of mobile phase respectively, filter through a 0.22 μm filter membrane, and then detect it by post-column derivation method in liquid chromatography. The results of the PMGS post-column derivation standard curve are as Figure 6 shown, and the results of the drug loading amount in the microneedle tip by liquid chromatography are as Figure 7 shown.

[0054] As can be seen from the pictures, substituting the peak area into the standard curve, the calculated PMGS contents are 0.1987 mg and 0.1919 mg respectively. It can be known that the drug loading amount of a single microneedle tip is about 195 μg.

[0055] Test Example 5 Insertion ability of PMGS into pig skin and skin recovery after insertion Test on the insertion ability of microneedles: Take out the pre-treated pig skin from the refrigerator, thaw it, and incubate it in PBS (pH 7.4) solution for 1 h. Then dry the solution on the stratum corneum side of the pig skin for later use. Place the ex vivo pig skin on a tissue paper soaked with PBS (pH 7.4) solution, and place the ex vivo pig skin at 37 °C to simulate the in vivo environment. Insert the microneedles into the ex vivo pig skin, and pull them out after inserting for a certain time (60 s). Use a Best magnifying microscope to record the morphological changes of the microneedles after pulling them out. And expose the skin to trypan blue solution for 5 minutes. Wash the tissue with normal saline to remove the dye and observe the skin.

[0056] Test on the skin recovery ability after insertion: Insert PMGS-MN into the skin of hairless mice / ex vivo pig skin. Press the tip of the microneedle forcefully into the skin for 60 s, then carefully remove the backing layer with forceps, take a photo to record the initial skin state, and then record it every 10 / 30 min to observe the number of micropores left on the skin and the skin recovery at different time points.

[0057] The pinhole conditions of pig skin (left picture) and mouse skin (right picture) after trypan blue staining are as Figure 8 shown, and the skin states of mice at different times (left picture 0 min, right Figure 10 min) after microneedle insertion are as Figure 9 shown, and the skin states of pig skin at different times (left picture 0 min, middle Figure 10 min, right Figure 20 min) after microneedle insertion are as Figure 10 shown.

[0058] As can be seen from the pictures, obvious blue pinholes can be observed on both pigskin and mouse skin, proving that PMGS microneedles can be well inserted into the skin for drug delivery. The pinholes on the mouse skin disappeared within 10 minutes, and after 20 minutes, the punctured area of the pigskin was hardly distinguishable, and the skin basically fully recovered, indicating that PMGSP-MN has good biocompatibility, causes no obvious damage to the skin, and has good safety.

[0059] Test Example 6 Anti-HPV6 pseudovirus effect of PMGS HaCat cells were seeded in a 96-well cell culture plate at a density of 8×10 4 cells / mL. After overnight culture, 5 μL of HPV6 pseudovirus particle mixture was added to the HaCat cells. A total of 5 concentrations of fucoidan sulfate were set at 0.2, 0.1, 0.05, 0.025, and 0.0125 μg / mL, with 3 replicates for each concentration. A blank well and a virus control well were established, and the cells were cultured for another 24 h; the culture medium was discarded, and the cells were washed twice with PBS. 100 μL of lysis buffer was added to each well, and after shaking in an ice bath, the lysis buffer was transferred to a 96-well white colorimetric plate using a multi-channel pipette. Subsequently, the colorimetric solution was added, and the relative fluorescence intensity in each well was measured using a fluorescence microplate reader to compare the fluorescence values of the drug-treated group and the virus control group. The inhibition rate of the sample on the virus = (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 and Figure 11 as follows.

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

[0061] As can be seen from the pictures and the table, the IC 50 value of PMGS against HPV6 pseudovirus in the HaCat cell line was 0.097 μg / mL, indicating that PMGS has a significant in vitro anti-low-risk HPV virus effect.

[0062] Test Example 7 Cytotoxic effect of PMGS and microneedle extract on HaCat cells To evaluate the biocompatibility of PMGS with PVP and PVA, the CCK-8 method was used to determine the effect of the microneedle matrix material on the viability of human immortalized keratinocytes (HaCaT). Cells were seeded in 96-well plates at a density of 5000 cells / well, 100 μL per well. After overnight culture of the seeded cells, the original culture medium was aspirated, and 100 μL of the culture medium containing the test sample was added. Four concentrations of PMGS were set: 1, 0.75, 0.5, and 0.25 mg / mL, and four concentrations of the microneedle extract were set: 0.2, 0.1, 0.05, and 0.025 mg / mL. After 24 h of drug administration, 10 μL of CCK-8 solution was added, and the cells were cultured in an incubator for 1 - 2 h. The cell viability was measured with a microplate reader at a wavelength of 450 nm. The results are as Figure 12 and Figure 13 shown, Figure 12 which is the cytotoxicity test result of PMGS on HaCaT cells, Figure 13 and this is the cytotoxicity test result of the microneedle extract on HaCaT cells.

[0063] As can be seen from the pictures, after co-incubation for 24 h, the viability of HaCaT cells remained above 80%, indicating that the microneedle matrix material had no obvious cytotoxicity and had biosafety.

[0064] Test Example 8 Immune Activation Effect of PMGS on Raw264.7 In the study of the effect of PMGS on the viability of mononuclear macrophage Raw264.7 cells, the cytotoxicity of PMGS was first detected by the same method as above. The results are as Figure 14 shown.

[0065] As can be seen from the pictures, at concentrations of 0 - 1000 μg / mL, PMGS could promote the proliferation of Raw264.7 cells in a dose-dependent manner.

[0066] In the study of the effect of PMGS on the cytokine expression of Raw264.7 mononuclear macrophages, cells were cultured in 6-well plates at a density of 1×10 6 / well, 2 mL of complete culture medium per well. The administration concentrations of PMGS were 1, 5, 25, 50, and 100 μg / mL, the administration concentration of the positive control lipopolysaccharide LPS was 100 ng / mL, and the blank control group was added with an equal volume of culture medium. After 6 h of culture, the culture medium was removed, and the cells were washed twice with PBS solution. Then RNA was extracted for subsequent qRT-PCR detection. After stimulating Raw264.7 mononuclear macrophages with different concentrations of PMGS, the transcriptional levels of cytokines such as TNFα and IL-1β with high expression after activation were detected. The results are as Figure 15 shown.

[0067] As can be seen from the picture, PMGS can activate Raw264.7 cells and promote the expression of cytokines such as TNFα, IL-1β, and IFNα.

[0068] In the effect of PMGS on the phagocytosis of Raw264.7 cells, Raw264.7 cells were collected and cultured at 1×10 5 / mL of cells were seeded in a 96-well plate, 0.2mL / well. After 12h of culture, different concentrations of samples (5, 25, 50, 100, 500µg / mL) or LPS (1µg / mL) were added to each well, and an equal volume of culture medium was added to the blank control group. After incubation for 24h, the supernatant was discarded, and 100µL of 0.075% neutral red solution was added to each well. After further incubation at 37℃ for 5min, the cells were washed 3 times with PBS to remove free neutral red. The cells were lysed at room temperature with lysis buffer, and the OD value was measured at a wavelength of 570nm. The effect of PMGS on the phagocytic ability of Raw264.7 cells was investigated using a neutral red phagocytosis experiment. The results are shown in the figure. Figure 16 shown.

[0069] As can be seen from the picture, PMGS can significantly enhance the phagocytic ability of Raw264.7 cells in a dose-dependent manner and improve the antigen presentation ability of Raw264.7 cells. The antigen presentation of Raw264.7 cells helps activate T lymphocytes and the response of the host's adaptive immune system, thereby enhancing the host's ability to recognize and eliminate pathogens.

[0070] Test Example 9 Effect of PMGS Microneedles on the Skin Immune Microenvironment of Healthy Mice Depilatory cream was used to remove hair in the mice 3 days before the microneedle application to eliminate the effect of depilatory cream stimulation on the experiment. The back skin was pinched with the thumb and index finger for 1 minute to firmly fix it at the application site and fixed with medical tape. The intact BMN was inserted into the mouse skin as much as possible. When the MN was removed after 10 min, the needle dissolved in the skin. The mice were killed by cervical distension 6 hours after administration, and the skin and inguinal lymph nodes at the administration site were taken for immunohistochemical section staining. The effect of PMGS microneedles on the infiltration of immune cells in the mouse skin is shown in the following figure. Figure 17 shown.

[0071] As can be seen from the picture, immunohistochemistry testing found that after microneedle administration, immune cells such as monocytes, dendritic cells, macrophages and NK cells in the innate immune system can be recruited, but it has no effect on T lymphocytes that play an important role in acquired immunity. There is no infiltration of T lymphocytes in 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°C for 24 h are shown inFigure 22 and Figure 23 as shown Figure 22 Among them, the two on the left are product diagrams before treatment, and the two on the right are product diagrams after treatment at 100 °C for 24 h. Figure 23 In the upper part, it is the infrared spectrum diagram of the product before treatment, and in the lower part, it is the infrared spectrum diagram of the product after treatment at 100 °C for 24 h.

[0073] According to the results, it has good high-temperature stability and can withstand 100 °C. And according to the previous test results, it also has good puncture effect and fast dissolution rate.

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

[0075] Comparative Example 2 This comparative example is basically the same as Example 1. The difference is that the main matrix material of the needle tip layer is HA, and PMGS is added to HA in the form of an additive. 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 agent in the applicant's previously authorized patent CN111481502B.

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

[0078] As Figure 21 can be seen, from the results of in vitro simulated penetration data, the single PMGS group has higher mechanical strength. In addition, the single PMGS group also has a higher drug loading capacity, which has better advantages compared with Comparative Example 2. And the single PMGS group only contains the PMGS drug component and can achieve targeted drug delivery. Polymannuronate sulfate (PMGS) can play a good role in treating skin warts through two ways: activating skin immunity and inhibiting virus adsorption. At the same time, as a kind of polysaccharide material derived from the ocean, PMGS has good biocompatibility and can also avoid the addition of other polymer materials, ensuring the safety of the microneedles.

[0079] In addition, the PMGS microneedles prepared in this application have good mechanical properties, dissolution rate, and transdermal efficiency. The PMGS microneedles solve the problem of insufficient mechanical strength of most soluble microneedles and do not require the addition of extra reinforcing materials. As a water-soluble polysaccharide material, PMGS dissolves rapidly after piercing the skin, which is conducive to drug delivery. The solution of this application solves the problem of poor transdermal effect of macromolecular polysaccharides, directly delivers macromolecular PMGS to the basal cells where the virus is located, realizes local targeted drug delivery, and achieves a better therapeutic effect on skin warts. In addition, the tip of the needle is completely composed of PMGS, with a large drug loading capacity per single needle, simple preparation, natural safety. Of course, according to needs, PMGS microneedles can also be loaded with a variety of drugs in the future, acting as a drug carrier, and simultaneously playing roles such as immune activation and antioxidant to assist in treatment.

[0080] Experimental Example 1 The inventors respectively tried PMGS with molecular weights of 5 kDa, 10 kDa, 100 kDa, 270 kDa, and 300 kDa, and found that smaller molecular weight PMGS (10 kDa) can also prepare tips with certain mechanical strength through the adjustment of increasing concentration. However, the tips of high-concentration PMGS with low molecular weight are brittle after drying. In actual operation, the molecular weight range of PMGS can be selected as 10 - 300 kDa, at which time it has both good antiviral effect and mechanical strength.

[0081] Experimental Example 2 Using a PM membrane 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 the PMGS concentration, the results are as Figure 18 shown. It was found that the puncture rate increased with the increase of concentration. At high concentration (200 mg / mL), there were also problems such as poor solution fluidity and more difficult preparation process. In practice, a concentration of 150 mg / mL can be selected for preparation.

[0083] Experimental Example 3 After testing by the experimenters, it was found that choosing PVA (such as PVA0588, PVA1788, etc.), PVP series (PVP-K30, PVP-K90), and HA (10 - 400 kDa) are all feasible. The results are as Figure 19 shown. The effect diagrams with PVA, PVP, and HA as the backing from left to right in the figure only slightly have problems such as air bubbles, bending, and brittleness, and can still function normally as the backing layer.

[0084] As Figure 20As shown, it is the back lining morphology of the microneedles obtained by mixing PVP and PVA. The back lining of the microneedles is flat without bubbles and has good toughness. It should be noted that for the selection and preparation of the back lining layer, those skilled in the art can make adjustments and selections. For those skilled in the art, a basic requirement for the back lining layer is to carry the microneedle layer. Those skilled in the art can adjust and change the back lining layer according to the situation, which is the conventional skill of those skilled in the art.

[0085] As mentioned above, it is only the embodiment of the present application. The protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the technical idea and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A phycocolloid sulfate (PMGS) patch, characterized in that, It includes: 1) A backing layer; 2) A tip layer, which is one or more of lambda / iota / kappa-carrageenan, polymannuronic acid sulfate, poly guluronic acid sulfate, alginate sulfate, fucoidan, sulfated dextran, chondroitin sulfate, keratan, heparin and its analogues.

2. The alginate sulfate (PMGS) patch according to claim 1, characterized in that, The tip layer is alginate sulfate, and the molecular weight range of the alginate sulfate is 10 - 300 kDa.

3. The alginate sulfate (PMGS) patch according to claim 1, characterized in that, In the alginic acid sulfate, the content of polymannuronic acid is 5 - 95%, the content of poly guluronic acid is 5 - 95%, and the degree of substitution of sulfate ester groups is 5 - 15%.

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

5. The alginate sulfate (PMGS) patch according to claim 1, characterized in that, 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, chondroitin sulfate; The second material is selected from one or more of polyvinyl alcohol and its derivatives, hyaluronic acid, hydroxypropyl methylcellulose, sodium carboxymethyl cellulose, sodium carboxymethyl starch.

6. The alginate sulfate (PMGS) patch according to claim 1, characterized in that, The backing layer includes polyvinyl alcohol and polyvinylpyrrolidone.

7. The alginate sulfate (PMGS) patch according to claim 1, characterized in that, The tip height in the tip layer is 200 - 2000 μm.

8. A preparation method of the alginate sulfate (PMGS) patch according to any one of claims 1 to 7, characterized in that, The preparation method includes the following steps: 1) Prepare a backing layer solution for standby and a tip layer solution for standby; 2) Aspirate the tip layer solution into a microneedle mold, perform the first centrifugation treatment, then remove the excess tip layer solution and add the backing layer solution, and perform the second centrifugation treatment; 3) After drying treatment, demold to obtain the alginate sulfate (PMGS) patch.

9. The preparation method of the fucoidan sulfate (PMGS) patch according to claim 8, 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, In the drying treatment, dry at 20 - 30 °C for 6 - 18 h.

10. Use of the alginate sulfate (PMGS) patch according to any one of claims 1 - 7 in the prevention and / or treatment of HPV and / or skin warts.

Images