A microneedle patch loaded with dacarbazine and manganese salt, a preparation method thereof, and application thereof in the treatment of melanoma

Abstract

The present invention discloses a microneedle patch loaded with dacarbazine and manganese salt, a preparation method thereof, and an application in the treatment of melanoma, wherein the microneedle patch comprises a back-symmetric layer and a needle array arranged on the back-symmetric layer, wherein the back-symmetric layer and the needle array are both formed by an aqueous solution of a degradable polymer, and dacarbazine and manganese salt are loaded in the aqueous solution of the degradable polymer used to form the needle array. The drug-loaded microneedles obtained by the present invention can effectively inhibit the growth and metastasis of tumors, effectively prevent the recurrence of tumors, and achieve efficient killing of melanoma.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical device preparation, and specifically relates to a microneedle patch loaded with dacarbazine and manganese salt, a preparation method thereof, and an application thereof in the treatment of melanoma. Background Art

[0002] Melanoma is a malignant tumor that is caused by the excessive proliferation of abnormal melanocytes and is often found in the skin. According to statistics, there are about 200,000 new cases of melanoma each year in the world, and nearly 70,000 people die from melanoma and related diseases. The annual incidence rate is 3% to 5%, which is one of the fastest growing malignant tumors in the world and the second most deadly cancer after blood cancer. At present, early melanoma is often treated with surgical resection or radiotherapy of the primary lesion in clinical practice. However, melanoma often develops rapidly and has a very high recurrence and metastasis rate. Malignant melanoma cells will have skin transitional lesions, metastasize to lymph nodes, or metastasize to organs such as the lungs, liver, spleen, and brain with blood. Melanoma has high immunogenicity, and there are abundant immune cells around tumor cells. Immunotherapy is not affected by tumor mutations, and its therapeutic effect mainly depends on the patient's own immune status. Therefore, immunotherapy may be the most promising treatment for malignant melanoma. Recent cancer immunotherapy strategies have been developed based on regulating adaptive immune responses. Although enhancing adaptive immune responses has certain anti-cancer effects, it still cannot meet the clinical needs for cancer treatment and requires some auxiliary immune regulation methods. Considering that innate immunity is the first barrier of host cell defense and a bridge to adaptive immunity, activating innate immunity has become an emerging strategy to accelerate tumor immunotherapy.

[0003] In tumor response, the innate immune system cGAS-STING signaling pathway can activate dendritic cells (DCs), tumor-specific CD8 + T cells and natural killer cells (NK), thereby activating systemic anti-tumor immunity and killing cancer cells. 2+ As an agonist, it can effectively promote the activation of cGAS and STING. Immunity (2018, Vol. 48, pp. 675-687) reported that Mn 2+ Plays a key role in host defense against DNA viruses. 2+After viral infection, it is released from the membrane-enclosed organelles, accumulates in the cytoplasm, and directly binds to cGAS. It enhances the sensitivity of cGAS to double-stranded DNA (dsDNA) and its enzymatic activity, enabling cGAS to produce cGAMP in the presence of low concentrations of dsDNA, thereby activating STING, allowing the body to produce type I IFN and host antiviral capabilities. As the research deepened, Cell Research (2020, Vol. 30, pp. 966-979) reported that Mn 2+ It can also directly activate cGAS (completely independent of dsDNA), and Mn 2+ Catalysis and Mn 2+ -dsDNA catalyzes the synthesis of cGAMP from cGAS in a significantly different manner. 2+ -cGAS binding causes cGAS conformational changes similar to DNA-cGAS binding, but the structure of the catalytic center is significantly different, resulting in significant changes in the synthesis of cGAMP in the catalytic center of the cGAS enzyme. This study further proves that Mn 2+ It is the second cGAS activator besides DNA. 2+ It has great potential in activating the cGAS-STING signaling pathway to produce anti-tumor immune responses. In addition, dacarbazine, as an alkylating agent, can damage the DNA of tumor cells, which is similar to Mn 2+ The mediated cGAS-STING pathway has a synergistic effect and can enhance its effect. At present, dacarbazine is mainly used in clinical practice as dacarbazine citrate for injection. This type of dacarbazine needs to be prepared and used immediately. If it is left for a long time, the solution will turn light red. Summary of the invention

[0004] The present invention provides a microneedle patch containing dacarbazine and manganese salt, a preparation method thereof and an application thereof in the treatment of melanoma. The technical problem to be solved is to construct a transdermal drug delivery system to achieve Mn 2+ It can precisely release dacarbazine, reduce toxic side effects, improve the tumor microenvironment and activate the cGAS-STING signaling pathway to achieve tumor immunotherapy.

[0005] In order to solve the technical problem, the present invention adopts the following technical solution:

[0006] The present invention firstly discloses a microneedle patch loaded with dacarbazine and manganese salt, which is characterized in that: the microneedle patch comprises a back-symmetric layer and a needle array arranged on the back-symmetric layer; the back-symmetric layer and the needle array are both formed by an aqueous solution of a degradable polymer, and dacarbazine and manganese salt are loaded in the aqueous solution of the degradable polymer used to form the needle array.

[0007] Furthermore, the manganese salt is at least one of manganese sulfate, manganese chloride, manganese nitrate and manganese acetate.

[0008] Furthermore, in the aqueous solution of the degradable polymer used to form the backing layer, the concentration of the degradable polymer is 1-2 g / mL.

[0009] Furthermore, in the aqueous solution of the degradable polymer used to form the needle array, the concentration of the degradable polymer is 0.1-0.2 g / mL, the mass ratio of the manganese salt to the degradable polymer is 1:2-10, and the mass ratio of dacarbazine to the manganese salt is 0.5-1:1.

[0010] Furthermore, the degradable polymers used in the back surface layer and the needle array are independently selected from at least one of hyaluronic acid, gelatin, polyvinyl pyrrolidone and polyvinyl alcohol.

[0011] Furthermore, each needle in the needle array is in a cone shape with a height of 400 to 1000 μm and a bottom diameter of 150 to 400 μm.

[0012] The present invention also discloses a method for preparing the microneedle patch loaded with dacarbazine and manganese salt, comprising the following steps:

[0013] Dissolving the degradable polymer in deionized water to obtain a back-layer solution;

[0014] Dissolve the degradable polymer in deionized water, add dacarbazine and manganese salt, and stir at room temperature in the dark for 12-24 hours to obtain a needle body mixture;

[0015] Through the micro-transfer molding method, the needle body mixture is filled into the needle body micropores of the microneedle mold, the excess part outside the micropores is removed, and then the backing layer solution is added above the micropores, dried and demolded to obtain a microneedle patch loaded with dacarbazine and manganese salt.

[0016] The microneedle patch loaded with dacarbazine and manganese salt obtained by the present invention can be used for transdermal administration to treat melanoma and inhibit metastasis. The present invention constructs a bioresponsive polymer microneedle drug release system loaded with manganese salt and chemotherapy drugs (dacarbazine) for activating innate immunity and adaptive immunity to treat melanoma. The released Mn 2+ and the chemotherapy drug dacarbazine induce ICD to kill melanoma cells and initiate the exposure of tumor-specific antigen molecules including CRT, ATP, HMGB1, IFN-β, CXCL10, etc., thereby activating adaptive immunity; Mn 2+Accumulation in tumor cells can enhance the activation of cGAS-STING-I type interferon, activate innate immunity, increase the secretion of IFN-β, and ultimately achieve good tumor inhibition effects on primary and metastatic tumors, providing an experimental basis and data support for improving the effective response rate of current clinical tumor immunotherapy.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] 1. The present invention prepares soluble microneedles loaded with dacarbazine and manganese salt for the first time. The preparation method is simple, the reaction conditions are mild, the obtained product has uniform morphology, and the production cost is low, which is suitable for industrial scale-up production.

[0019] 2. The microneedles loaded with dacarbazine and manganese salt prepared by the present invention can be rapidly dissolved and delivered through transdermal administration above the tumor site to achieve more precise drug release, with a smaller dosage and better biocompatibility.

[0020] 3. The microneedles prepared in the present invention can release Mn 2+ Thus, it consumes the overexpressed GSH and Mn in the tumor. 2+ It can also react with endogenous H 2 O 2 Generate ·OH, change the redox homeostasis of the tumor microenvironment, and kill tumor cells.

[0021] 4. The microneedles prepared in the present invention can release Mn 2+ Activating the cGAS-STING signaling pathway is expected to promote immune cells to produce type I IFN and related anti-tumor cytokines, thereby increasing the infiltration of cytotoxic T cells and NK cells in the tumor site, achieving effective immunotherapy for melanoma.

[0022] 5. The present invention combines dacarbazine into microneedles to form a solid, which can achieve long-term storage at room temperature, and local transdermal release of dacarbazine through microneedles, which not only significantly reduces the dosage and toxic side effects of dacarbazine, but also can induce a stronger immune effect and show better therapeutic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is an optical picture of the microneedle prepared in Example 1;

[0024] Figure 2 This is an inverted fluorescence microscope image of the microneedle prepared in Example 1 in bright field;

[0025] Figure 3 This is a scanning electron microscope (SEM) photograph of the microneedle prepared in Example 1;

[0026] Figure 4The ultraviolet absorption of the microneedles prepared in Example 2 dissolved in phosphate buffered saline solution;

[0027] Figure 5 The curves of tumor volume changes after dissection of each treatment group in Example 6;

[0028] Figure 6 HE staining images of tumor tissues dissected from each treatment group in Example 6;

[0029] Figure 7 This is a TUNEL staining image of the tumor tissues dissected from each treatment group in Example 6; DETAILED DESCRIPTION

[0030] The following is a detailed description of an embodiment of the present invention. This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiment.

[0031] Example 1

[0032] This example prepares microneedles without dacarbazine and manganese salt:

[0033] Weigh 1300 mg of hyaluronic acid and add it to 10 mL of deionized water and stir for 24 hours in the dark to form a transparent and uniform gel mixture. Transfer it to a 10 mL beaker and add the microneedle mold. Then put the beaker into a centrifuge tube and centrifuge it at 4000 rpm for 5 minutes.

[0034] After centrifugation, take out the mold from the beaker, remove the parts outside the micropores, add 1.5 g / mL PVP solution, and then put the mold into a 40°C oven to dry overnight away from light. Finally, gently peel off the formed microneedles from the mold.

[0035] Figure 1 This is an optical picture of the microneedle prepared in this example. It can be seen from the picture that the back surface layer of the microneedle patch is a square with a side length of 9 mm.

[0036] Figure 2 This is a bright field side view of the microneedle prepared in this example under an inverted fluorescence microscope (Olympus CKX41 biological inverted microscope). The sharp tip of the microneedle can be seen from the figure.

[0037] Figure 3 The scanning electron microscope image (ZEISS GeminiSEM 300 field emission scanning electron microscope) of the microneedle prepared in this example clearly shows the overall appearance of the microneedle.

[0038] Example 2

[0039] In this example, microneedles containing only dacarbazine were prepared:

[0040] 325 mg of dacarbazine (dacarbazine accounts for 25% of the mass of hyaluronic acid) and 1300 mg of hyaluronic acid were weighed and added to 10 mL of deionized water and stirred for 24 hours in the dark to form a milky white uniform colloidal mixture. The mixture was transferred to a 10 mL beaker, and the microneedle mold (the same as in Example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5 min.

[0041] After centrifugation, take out the mold from the beaker, remove the parts outside the micropores, add 1.5 g / mL PVP solution, and then put the mold into a 40°C oven to dry overnight away from light. Finally, gently peel off the formed microneedles from the mold.

[0042] The microneedles were dissolved in 8 mL of phosphate buffered saline, and the UV absorption of dacarbazine was measured by UV spectrophotometer to facilitate quantification. Figure 4 The maximum absorption peak is shown at 328 nm.

[0043] Example 3

[0044] This example prepares microneedles containing only manganese salt:

[0045] 650 mg of manganese chloride and 1300 mg of hyaluronic acid were weighed and added to 10 mL of deionized water and stirred for 24 h in the dark to form a brown uniform colloidal mixture. The mixture was transferred to a 10 mL beaker, and the microneedle mold (same as in Example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5 min.

[0046] After centrifugation, take out the mold from the beaker, remove the parts outside the micropores, add 1.5 g / mL PVP solution, and then put the mold into a 40°C oven to dry overnight away from light. Finally, gently peel off the formed microneedles from the mold.

[0047] Placing the microneedle in this embodiment in a potassium permanganate solution will cause a color change. This is because the divalent manganese ions in the microneedle react with the heptavalent manganese ions in the potassium permanganate to form manganese ions with an intermediate valence, thereby causing a color change.

[0048] Example 4

[0049] In this example, the microneedles were prepared according to the mass ratio of dacarbazine to manganese salt of 0.5:1:

[0050] 325 mg of dacarbazine, 650 mg of manganese chloride and 1300 mg of hyaluronic acid were weighed and added to 10 mL of deionized water and stirred for 24 h in the dark to form a milky uniform colloidal mixture. The mixture was transferred to a 10 mL beaker, and the microneedle mold (same as in Example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5 min.

[0051] After centrifugation, take out the mold from the beaker, remove the parts outside the micropores, add 1.5 g / mL PVP solution, and then put the mold into a 40°C oven to dry overnight away from light. Finally, gently peel off the formed microneedles from the mold.

[0052] Example 5

[0053] In this example, microneedles were prepared according to a mass ratio of dacarbazine to manganese salt of 1:1:

[0054] 650 mg of dacarbazine, 650 mg of manganese chloride and 1300 mg of hyaluronic acid were weighed and added to 10 mL of deionized water and stirred for 24 h in the dark to form a milky uniform colloidal mixture. The mixture was transferred to a 10 mL beaker, and the microneedle mold (same as in Example 1) was added thereto. The beaker was then placed in a centrifuge tube and centrifuged at 4000 rpm for 5 min.

[0055] After centrifugation, take out the mold from the beaker, remove the parts outside the micropores, add 1.5 g / mL PVP solution, and then put the mold into a 40°C oven to dry overnight away from light. Finally, gently peel off the formed microneedles from the mold.

[0056] Example 6

[0057] This example tests the therapeutic effect of the microneedles prepared in the above example on melanoma in the following steps:

[0058] The mice were randomly divided into the following groups: control microneedle group, dacarbazine microneedle group, manganese salt microneedle group, dacarbazine and manganese salt 0.5:1 group and dacarbazine and manganese salt 1:1 group, with 5 mice in each group. The modeling method was to inject mouse melanoma cells (B16F10) subcutaneously in the back to construct a B16F10 subcutaneous tumor model. The treatment method was to treat the tumor when the volume was about 100mm 3 , the microneedle patch was applied to the tumor site of the mice and treated according to the groups, once every three days.

[0059] Control microneedle group: pure hyaluronic acid microneedles of Example 1 were administered to model mice;

[0060] Dacarbazine microneedle group: The microneedles containing only dacarbazine in Example 2 were administered to the modeled mice;

[0061] Manganese salt microneedle group: The microneedles containing only manganese salt in Example 3 were administered to the modeled mice;

[0062] 0.5:1 group: microneedles containing dacarbazine and manganese salt in a mass ratio of 0.5:1 of Example 4 were administered to model mice;

[0063] 1:1 group: microneedles containing dacarbazine and manganese salt in a mass ratio of 1:1 as described in Example 5 were administered to model mice.

[0064] The volume of the mouse tumor was measured every day, and the volume was calculated and recorded according to the formula of volume = a*b*b / 2 (a is the maximum length of the mouse tumor, b is the minimum length of the mouse tumor). At the end of 14 days, the mice were killed and the tumors were removed. Immunohistochemistry and immunofluorescence pathological analysis were performed on the tumor tissues to detect the corresponding necrosis and apoptosis factors, verifying the killing effect of the microneedles loaded with dacarbazine and manganese salt on tumors in animals.

[0065] Figure 5 The volume change curve of the tumor tissue dissected from each treatment group in this example. As can be seen from the figure, compared with the control group without intervention, the tumor size of the dacarbazine and manganese salt 1:1 group is much smaller than the tumor size of the other groups after treatment.

[0066] Figure 6 HE staining of tumor tissues dissected and removed from each treatment group in this embodiment. As can be seen from the figure, the number of blue cell nuclei in the control microneedle group and the manganese salt microneedle group is large and complete, indicating that tumor cell necrosis is less. The microneedle groups loaded with dacarbazine all have varying degrees of necrosis, indicating that dacarbazine and manganese salts alone have a certain killing effect on tumor cells. And the dacarbazine and manganese salt group has the strongest killing effect, with a small number of blue cell nuclei and broken, indicating that the prepared microneedles loaded with dacarbazine and manganese salts can effectively cause tumor cell necrosis, proving that it has a significant therapeutic effect on melanoma.

[0067] Figure 7 This is a TUNEL staining diagram of the tumor tissue dissected and removed from each treatment group in this embodiment. It can be seen from the figure that compared with the green fluorescence representing cell apoptosis in the control microneedle group and the manganese salt microneedle group, the dacarbazine microneedle group and the dacarbazine and manganese salt microneedle group have different degrees of cell apoptosis, indicating that both dacarbazine and manganese salt alone have a certain killing effect on tumor cells. And the dacarbazine and manganese salt group has the strongest killing effect, resulting in the most green fluorescence, indicating that the prepared microneedles loaded with dacarbazine and manganese salt can effectively cause tumor cell apoptosis, proving that it has a significant therapeutic effect on melanoma.

[0068] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A microneedle patch containing dacarbazine and manganese salt for treating melanoma, characterized in that: The microneedle patch includes a back-symmetric layer and a needle array arranged on the back-symmetric layer; the back-symmetric layer and the needle array are both formed by an aqueous solution of a degradable polymer, and dacarbazine and manganese salt are loaded in the aqueous solution of the degradable polymer used to form the needle array, and the mass ratio of dacarbazine to manganese salt is 0.5:1; the degradable polymer used in the needle array is hyaluronic acid.

2. The microneedle patch containing dacarbazine and manganese salt for treating melanoma according to claim 1, characterized in that: The manganese salt is at least one of manganese sulfate, manganese chloride, manganese nitrate and manganese acetate.

3. The microneedle patch containing dacarbazine and manganese salt for treating melanoma according to claim 1, characterized in that: In the aqueous solution of the degradable polymer used to form the back surface layer, the concentration of the degradable polymer is 1-2 g / mL.

4. The microneedle patch containing dacarbazine and manganese salt for treating melanoma according to claim 1, characterized in that: In the aqueous solution of the degradable polymer used to form the needle array, the concentration of the degradable polymer is 0.1-0.2 g / mL, and the mass ratio of the manganese salt to the degradable polymer is 1:2-10.

5. The microneedle patch containing dacarbazine and manganese salt for treating melanoma according to claim 1, characterized in that: The degradable polymer used in the back layer is at least one selected from hyaluronic acid, gelatin, polyvinyl pyrrolidone and polyvinyl alcohol.

6. The microneedle patch containing dacarbazine and manganese salt for treating melanoma according to claim 1, characterized in that: Each needle in the needle array is in a cone shape with a height of 400-1000 μm and a bottom diameter of 150-400 μm.

7. A method for preparing a microneedle patch containing dacarbazine and a manganese salt as claimed in any one of claims 1 to 6, characterized in that: The steps include: Dissolving the degradable polymer in deionized water to obtain a back-layer solution; Dissolve the degradable polymer in deionized water, add dacarbazine and manganese salt, and stir at room temperature in the dark for 12-24 hours to obtain a needle body mixture; Through the micro-transfer molding method, the needle body mixture is filled into the needle body micropores of the microneedle mold, the excess part outside the micropores is removed, and then the backing layer solution is added above the micropores, dried and demolded to obtain a microneedle patch loaded with dacarbazine and manganese salt.

8. Use of the microneedle patch containing dacarbazine and manganese salt according to any one of claims 1 to 6 in the preparation of a drug for treating melanoma.

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