Novel gel-type microneedle patch for treating melanoma

Abstract

Provided is a novel gel-type microneedle patch for treating melanoma, which is loaded with dendritic cells (DC) and paclitaxel (PTX). The chemotherapeutic drug paclitaxel utilizes its own toxicity to

Description

CROSS REFERENCE TO THE RELATED APPLICATIONS

[0001] The present application is a national stage application of International Patent Application No. PCT / CN2024 / 082645, filed on March 07, 2024, which claims the benefit and priority of Chinese Patent Application No. CN202410261838.X entitled "NOVEL GEL-TYPE MICRONEEDLE PATCH FOR TREATING MELANOMA", all of which are incorporated herein by reference in their entities. TECHNICAL FIELD

[0002] The disclosure belongs to the field of medicine, and particularly relates to a novel gel-type microneedle patch for treating melanoma. BACKGROUND

[0003] Melanoma is a kind of skin malignant tumor, and the existing methods for treating melanoma comprise chemotherapy, radiotherapy and surgery. Among them, the drug accumulation in the tumor site is affected by the circulatory system when chemical drugs are used, and the toxicity of the chemical drugs used will cause great toxic and side effects to patients. This single treatment method will also produce immune tolerance, which is not conducive to the treatment of patients. Radiotherapy has strong radioactivity, which kills the tumor tissue and causes damage to its local normal tissue. Long-term radiotherapy will also cause some adverse reactions to patients, which will bring double pain to patients mentally and physically. Surgery will cause great trauma to patients, and unclean surgical resection or improper surgical care will also cause postoperative tumor recurrence. It can be seen that these treatments have many problems, such as large side effects, poor therapeutic effect, poor specificity, easy recurrence and easy drug resistance. At the same time, these treatments will bring pain, trauma and scar formation to patients to a certain extent. Therefore, it is necessary to find a milder, less invasive and effective treatment for melanoma.

[0004] Micro-needle is a new drug delivery system, which can effectively deliver drugs through the skin, increase the distribution of drugs in tumor sites and reduce the penetration of drugs into adjacent tissues. In the prior art, there has been a strategy of using microneedle technology to treat melanoma. For example, in the article "Discovering microneedles-based programmed delivery system for enhanced chemo-immunotherapy of melanoma.", Yu Tian et al., they developed a drug programmed delivery system based on microneedles. The system combines pH-sensitive liposomes containing chemotherapy drugs doxorubicin (DOX) and PD-L1 siRNA(siPD-Ll), which can target melanoma and release drugs in response to the pH of the tumor site, thus realizing chemotherapy immunotherapy of melanoma. The addition of adriamycin can not only fight against tumor itself, but also improve the immunotherapy of melanoma, trigger the immunogenic cell death effect and activate anti-tumor immunity. The physical auxiliary effect of soluble microneedles further enhances the effect of chemotherapy immunotherapy. Their in vitro and in vivo experimental results show that the microneedle can be quickly dissolved into the skin and has enough mechanical strength to penetrate the skin, showing better anti-tumor effect. However, it still has the defects of short anti-tumor immunity maintenance time and high tumor recurrence rate.

[0005] In order to solve these defects, a new type of microneedle patch with high efficiency, low recurrence rate, good targeting, high safety, non-invasive and painless is urgently needed in this field for treating melanoma. SUMMARY

[0006] The objective of the disclosure is to provide a novel gel-type microneedle patch for treating melanoma. The microneedle patch provided by the disclosure has good targeting and high safety, and can be used for efficient, non-invasive and painless treatment, and can reduce the recurrence rate.

[0007] In order to achieve the above objective, the present disclosure provides the following technical solution:

[0008] The disclosure provides a novel gel-type microneedle patch for treating melanoma, wherein the microneedle patch is loaded with dendritic cells (DC) and paclitaxel (PTX).

[0009] The disclosure adopts the microneedle technology to prepare the medicine into a patch, which can effectively transport the medicine to the lesion site and realize painless, noninvasive and accurate treatment of melanoma. Among them, the chemotherapy effect of paclitaxel can prevent the proliferation of tumor cells and make them apoptosis. At the same time, the combination of paclitaxel and dendritic cells can induce an anti-tumor immune response with high efficiency and low toxicity, strengthen the killing effect on tumors, help generate immune memory, and enable the body to obtain lasting anti-tumor immunity to reduce tumor recurrence. The disclosure first applies the living cell gel in the treatment of melanoma, and the combination of chemotherapy drugs and living cell gel shows the synergistic treatment of chemotherapy and immunity.

[0010] In some embodiments, the paclitaxel is albumin-bound paclitaxel.

[0011] Albumin paclitaxel has outstanding curative effect, good biocompatibility, no immunogenicity and biodegradability. Albumin particles as a carrier can make albumin paclitaxel enter tumor cells quickly, which makes the drug act in tumor cells for a longer time and increase the curative effect.

[0012] The method for preparing albumin-bound paclitaxel comprises: adding an albumin solution into a paclitaxel solution for stirring and ultrasonic treatment, and then volatilizing a solvent to make the albumin encapsulate the paclitaxel into nanoparticles.

[0013] In some embodiments, the solvent of the paclitaxel solution is ethanol.

[0014] In some embodiments, the albumin-bound paclitaxel in microneedle is coupled to the surface of the dendritic cells.

[0015] In some embodiments, the albumin-bound taxol-coupled dendritic cells in microneedle are in 2% gelatin-medium.

[0016] In some embodiments, immature dendritic cells account for 100% of the dendritic cells.

[0017] Immature dendritic cells ingest specific antigen of melanoma, such as MARTI, Melan-A and gp 100, etc., and after processing, they develop into mature dendritic cells, which secrete and express cytokines such as IL-6, TNF- a and interferon, and the cytokines enable dendritic cells to successfully migrate to peripheral lymphoid organs to interact with T cells, thereby presenting antigens to T cells, prompting T cells to differentiate into cytotoxic T cells to initiate antigen-specific immune responses, and ultimately kill tumor cells, while helping the body to generate immune memory and obtain long-lasting anti-tumor immunity.

[0018] In some embodiments, a shell material of a microneedle mold is hydrophobic copolymer VP-CO-MMA.

[0019] A preparation method of the microneedle patch comprises: loading albumin-bound taxol-coupled dendritic cells in 2% gelatin-medium into a microneedle mold by vacuum.

[0020] Beneficial effects:

[0021] (1) The disclosure adopts the microneedle to load the living cell gel and chemotherapy drugs, which directly hits the lesion site, thus ensuring the accurate delivery and efficient treatment of drugs.

[0022] (2) Based on paclitaxel and dendritic cells, the disclosure adopts a chemical-immune combined therapy method, which uses paclitaxel to prevent the proliferation of tumor cells and promote their apoptosis, and activates anti-tumor immune response after combining with dendritic cells, so as to strengthen the killing effect on tumors, help the human body to generate immune memory, and enable the body to obtain lasting anti-tumor immunity to reduce tumor recurrence.

[0023] (3) The disclosure combines chemotherapy drugs with living cells for synergistic effect, and accelerates the elimination and rehabilitation of melanoma.

[0024] (4) The paclitaxel in the disclosure is modified by albumin, which is safe and nontoxic, has good biocompatibility and high safety in the treatment process.

[0025] (5) The disclosure adopts microneedle therapy, which is painless, non-invasive and simple to operate, and can greatly alleviate the pain caused by treatment. Microneedles are degradable, and can be degraded by esterase in vivo after they play a therapeutic role in the lesion in the form of patches, thus achieving painless and noninvasive treatment of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to explain the embodiments of the present disclosure or the technical scheme in the prior art more clearly, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present disclosure. For ordinary skills in the art, other drawings can be obtained according to these drawings without creative labor work.

[0027] FIG. 1 is a schematic diagram of the preparation of microneedle patche.

[0028] FIG. 2 is a morphological diagram of the microneedle patch under a transmission electron microscope.

[0029] FIG. 3 shows the cell survival rate under the action of different concentrations of paclitaxel.

[0030] FIG. 4 shows the proliferation of untreated / untreated dendritic cells cultured for 1, 2, 4 and 6 days. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0031] The disclosure provides a novel gel-type microneedle patch for treating melanoma. Albumin paclitaxel combined with dendritic cell gel is poured into a perforated microneedle to make a patch, which effectively delivers and accurately releases drugs to the lesion site, thus realizing the non-invasive and painless treatment of melanoma. The microneedle patch of the disclosure can treat melanoma efficiently, persistently and painlessly.

[0032] Albumin is biodegradable, safe and non-toxic. As a carrier, the albumin wraps paclitaxel and enters into the tumor site, it has better solubility than paclitaxel, and can prevent paclitaxel from being cleared by blood in vivo and increase the targeting of tumor.

[0033] Paclitaxel, a chemotherapeutic drug, can kills tumor cells by its own toxicity, at the same time, it significantly increases the local oxygen level of tumor and inhibits the expression of HIF-1 a , thus alleviating tumor hypoxia and regulating tumor microenvironment. Dendritic cells can activate anti-tumor immune response, strengthen the killing effect on tumors and solve the problem of easy recurrence of melanoma.

[0034] In order to further illustrate the present disclosure, the technical solution provided by the present disclosure will be described in detail with the attached drawings and examples, but they cannot be understood as limiting the protection scope of the present disclosure. Unless otherwise specified, the production process, experimental method or detection method involved in the embodiment of the present disclosure are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and definite in the relevant use fields. According to the names, skills in the art can understand the conventional process steps and apply corresponding equipment to implement them according to the conventional conditions or the conditions suggested by manufacturers.

[0035] Various instruments, equipments, raw materials or reagents used in the embodiment of the present disclosure are all conventional products that can be purchased through formal commercial channels, and can also be prepared according to conventional methods well known to those skilled in the art..

[0036] EXAMPLE 1

[0037] Preparation process of novel gel-type microneedle patch for treating melanoma

[0038] (1) Preparing albumin-bound paclitaxel

[0039] Albumin was dissolved in the buffer, which was fully dissolved by stirring and ultrasonic treatment, and then impurities and bubbles were removed. Paclitaxel was dissolved in ethanol to form paclitaxel solution, and 10 ml of albumin solution (2 mg / ml) was added to 150ul of paclitaxel solution pre-dissolved in ethanol (paclitaxel concentration was 20mg / ml). After stirring overnight at room temperature, the mixed solution was centrifuged at lOOOOrpm for lOmin, and then excess paclitaxel was removed, and finally albumin-bound paclitaxel solution was otained. Then, paclitaxel was encapsulated into nanoparticles by albumin through solvent volatilization, and albumin-bound paclitaxel was obtained by ultrafiltration, and the content of paclitaxel in the obtained albumin-bound paclitaxel was 10%, the molar ratio of albumin to paclitaxel is about 1:5.

[0040] (2) Coupling albumin-bound paclitaxel with dendritic cells

[0041] Albumin-bound paclitaxel was coupled to the surface of dendritic cells by the methods of glucose metabolism and click chemistry, thermoreversible gelatin was selected as the cell-loading matrix, and the albumin-bound taxol-coupled dendritic cells were transferred to 2% gelatin -AIM-V medium.

[0042] The methods of glucose metabolism and click chemistry are as follows: click chemistry is a rapid chemical synthesis of various molecules by splicing small units. Glucose metabolism is to change the structure of glycans and introduce functional groups in biological applications. When unnatural monosaccharides come into contact with cells, cells make their own glycans through internal metabolic pathways. The specific steps are as follows: immature dendritic cells were cultured in azido Ac4ManNAz medium for three days, and they were labeled with azide. After azide labeling, albumin-bound paclitaxel was covalently modified on azide labeled DCs.

[0043] The source and culture of immature dendritic cells: after the mouse cervical vertebra was dislocated and killed, the bone was moved to an ultra-clean table, soaked in 70% alcohol for 2-5 minutes, and then washed twice with sterile PBS; the bone was moved into PBS, both ends of the bone were cut off with scissors, PBS was extracted with a syringe, and the needle was inserted into the bone marrow cavity from both ends of the bone, and the bone marrow was repeatedly washed out into the culture dish until the bone became completely white. Bone marrow suspension was collected and filtered with 200 mesh nylon net to remove small fragments and muscle tissue; the filtrate was centrifuged at 1200 rpm for 5min minutes, and the supernatant was discarded. 2 ml ammonium chloride erythrocyte lysate (lx) was added to the remaining sediment, the cells were resuspended, and incubated at room temperature for 3-10min. The suspended cells were removed, and continued to culture with AIM-V containing GM-CSF(500-1000 U / mL) and IL-4 (500 U / mL), and half liquid change was performed every 2-3 days, and GM-CSF and IL-4 were supplemented, finally, immature dendritic cells were obtained.

[0044] (3) Loading into a microneedle mold

[0045] Albumin-bound taxol-coupled dendritic cells in 2% gelatin-medium were loaded into the microneedle mold by vacuum. The shell material of microneedle mould was hydrophobic copolymer VP-CO-MMA, which was a hydrophilic medium for maintaining cell vitality and could be degraded by esterase in vivo. The middle was hollow and perforated, which was beneficial to the release of drugs and living cells.

[0046] EXAMPLE 2

[0047] Verification of therapeutic effect of microneedle patch

[0048] (1) The morphology of microneedle patch under transmission electron microscope was analyzed. (FIG. 2)

[0049] (2) The cell survival rate under different concentrations of paclitaxel was analyzed. (FIG. 3)

[0050] In vitro cytotoxicity experiment: melanoma cells were inoculated into 96-well plates at the density of 1 X 104 cells per well overnight, then incubated with different concentrations of paclitaxel for 48 hours, and then the cells were counted, and the relative survival rate of the cells was determined by kit -8 (CCK-8).

[0051] (3) Proliferation of untreated / untreated dendritic cells after 1, 2, 4 and 6 days of culture. (FIG. 4)

[0052] Microneedles carrying albumin-bound taxol-coupled dendritic cells were placed in a 24-well plate and added with culture medium to release the cells and incubate them in the 24-well plate. At the same time, the same type of cells were inoculated at a density of 1 X 105 cells per well as positive control cells. On the 1st, 2nd, 4th and 6th day, alamarBlue was measured: alamarBlue reagent was added to each well, and the final concentration was 10% (vol / vol). After incubation at 37°C for 4 hours, the fluorescence intensity (excitation 540nm, emission 590nm) was measured. The fluorescence intensity of each designated culture time was divided by the fluorescence intensity after one day of culture to obtain the quantification of cell proliferation. The results are shown in FIG. 4, which shows that the dendritic cells in microneedles still have good activity after release and can play an immune role.

[0053] From the above examples, it can be seen that paclitaxel has the function of inhibiting tumor cells, and albumin, as a carrier of paclitaxel, is biodegradable and can encapsulate paclitaxel into tumor sites, prevent paclitaxel from being cleared by blood, and enhance the therapeutic effect of paclitaxel. Albumin-bound taxol-coupled dendritic cells still have good activity after being released in microneedles, and can play an immune role.

[0054] Although the above embodiment has described the disclosure in detail, it is only a part of the embodiment of the disclosure, not the whole embodiment. People can also obtain other embodiments according to this embodiment without inventing, and these embodiments all belong to the scope of protection of the disclosure.

Claims

1. A novel gel-type microneedle patch for treating melanoma, wherein the microneedle patch is loaded with dendritic cells and a paclitaxel.

2. The microneedle patch according to claim 1, wherein the paclitaxel is an albumin-bound paclitaxel.

3. The microneedle patch according to claim 2, wherein a method for preparing the albumin-bound paclitaxel comprises: adding an albumin solution into a paclitaxel solution for stirring and ultrasonic treatment, and then volatilizing a solvent to make the albumin encapsulate the paclitaxel into nanoparticles.

4. The microneedle patch according to claim 3, wherein the solvent of the paclitaxel solution is ethanol.

5. The microneedle patch according to claim 2, wherein the albumin-bound paclitaxel in microneedle is coupled to a surface of the dendritic cells.

6. The microneedle patch according to claim 5, wherein albumin-bound taxol-coupled dendritic cells in microneedle are in a 2% gelatin-medium.

7. The microneedle patch according to claim 1, wherein immature dendritic cells account for 100% of the dendritic cells.

8. The microneedle patch according to claim 1, wherein a shell material of a microneedle mold is a hydrophobic copolymer VP-CO-MMA.

9. A preparation method of the microneedle patch according to claim 1, wherein the preparation method comprises: loading albumin-bound taxol-coupled dendritic cells in a 2% gelatin-medium into a microneedle mold by vacuum.INTERNATIONAL SEARCH REPORT International application No. PCT / CN2024 / 082645A. CLASSIFICATION OF SUBJECT MATTER A61K9 / 00(2006.01)i; A61K9 / 70(2006.01)i; A61K 31 / 337(2006.01)i; A61K 35 / 15(2015.01)i; A61K47 / 42(2017.01)i; A61P 35 / 00(2006.01)i According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols) IPC: A61K, A61P Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) CNABS. CNTXT, DWPI, WPABS, ENTXT, VCN, ISI Web of Science, PubMed, STNext, 1¾ BAIDU, CJFD, DUXIU ACADEMIC: W, IfeX W, Wx, BK, TO |XU §1, microneedle, paclitaxel, PTX, DC, dendritic cell, melanoma, conjugate, gel, patch, VP-CO-MMA C. DOCUMENTS CONSIDERED TO BE RELEVANT Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No. Y CN 116687833 A (SUN YAT-SEN UNIVERSITY) 05 September 2023 (2023-09-05) claims 9-10, and description, paragraphs [0047]-[0048] and [0054]-[0059] 1-9 Y Y US 2021060146 Al (SOTIO A.S.) 04 March 2021 (2021-03-04) claims 1 and 14, and description, paragraphs [0005] and [0063] US 2021153848 Al (IMCOMET B.V.) 27 May 2021 (2021-05-27) claims 1-12, and description, paragraph [0001] 1-9 1-9 A US 2015368612 Al (BAYLOR RESEARCH INSTITUTE) 24 December 2015 (2015-12-24) claims 48 and 71-72 1-9 A US 2022062606 Al (CITY UNIVERSITY OF HONG KONG) 03 March 2022 (2022-03-03) claims 1-23 1-9 A CN 117379459 A (GUANGZHOU UNIVERSITY OF CHINESE MEDICINE) 12 January 2024 (2024-01-12) claims 1-10 1-9 | Z | Further documents are listed in the continuation of Box C. | J | See patent family annex. * Special categories of cited documents: “p” later document published after the international filing date or priority “A" document defining the general state of the art which is not considered date and not in conflict with the application but cited to understand the to be of particular relevance principle or theory underlying the invention “D" document cited by the applicant in the international application “X” document of particular relevance; the claimed invention cannot be “E" earlier application orpatent but published on or after the international considered novel or cannot be considered to involve an inventive step filing date when the document is taken alone “L" document which may throw doubts on priority claim(s) or which is “Y” document of particular relevance; the claimed invention cannot be cited to establish the publication date of another citation or other considered to involve an inventive step when the document is special reason (as specified) combined with one or more other such documents, such combination “O” document referring to an oral disclosure, use, exhibition or other being obvious to a person skilled in tile art means “&” document member of the same patent family “P” document published prior to the international filing date but later than the priority date claimed Date of the actual completion of the international search 25 February 2025 Date of mailing of the international search report 25 February 2025 Name and mailing address of the ISA / CN China National Intellectual Property Administration (ISA / CN) China No. 6, Xitucheng Road, Jimenqiao, Haidian District, Beijing 100088 Authorized officer Telephone No.INTERNATIONAL SEARCH REPORT International application No. PCT / CN2024 / 082645C. DOCUMENTS CONSIDERED TO BE RELEVANTCategory*Citation of document, with indication, where appropriate, of the relevant passagesRelevant to claim No.FUKUDA K. et al. "Peptide-Pulsed Dendritic Cell Vaccine in Combination With Carboplatin and Paclitaxel Chemotherapy for Stage IV Melanoma"Melanoma Research. 31 December 2017 (2017-12-31),pages 1-9Paclitaxel on Biological Activity of Human Dendritic Cells)"BBBBBtR (China Medical Herald), Vol. 10, No. 33, 30 November 2013 (2013-11-30), pages 15-19JUNG J. B. et al. "Microneedle-Directed Drug Delivery to Tumor-Draining Lymph Node for Synergistic Combination Chemoimmunotherapy for Metastatic Cancer"Advanced Therapeutics, Vol. 5, 12 February 2022 (2022-02-12),pages 1-101-91-9INTERNATIONAL SEARCH REPORT International application No.Information on patent family members PCT / CN2024 / 082645Patent document cited in search report Publication date (day / month / year) Patent family member(s) Publication date (day / month / year) CN 116687833 A 05 September 2023 None US 2021060146 Al 04 March 2021 EA 202091743 Al 08 December 2020 KR 20200116113 A 08 October 2020 JP 2021511384 A 06 May 2021 CA 3087823 Al 01 August 2019 EP 3743099 Al 02 December 2020 WO 2019145471 Al 01 August 2019 US 2021153848 Al 27 May 2021 AU 2020273354 Al 10 June 2021 US 2015368612 Al 24 December 2015 US 10023841 B2 17 July 2018 US 2022062606 Al 03 March 2022 US 12005221 B2 11 June 2024 CN 117379459 A 12 January 2024 None

Images