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 kill tumor cells, while the dendritic cells activate an anti-tumor immune response, so that the killing effect on tumors is enhanced, and additionally, the problem that melanoma is prone to recurrence is solved. The provided microneedle patch has good targeting ability and high safety, and can effectively reduce the recurrence rate, thereby achieving painless, non-invasive and precise treatment of melanoma.

Description

A new gel-based microneedle patch for treating melanoma Technical Field

[0001] The present invention belongs to the field of medicine, and in particular relates to a novel gel-type microneedle patch for treating melanoma. Background Art

[0002] Melanoma, a malignant skin tumor, is currently treated with chemotherapy, radiotherapy, and surgery. Chemotherapy, however, can lead to drug accumulation at the tumor site, which is affected by the circulatory system. Furthermore, the toxicity of the chemotherapy drugs can cause significant side effects in patients. This single-dose treatment approach can also lead to immune tolerance, making it difficult for patients to recover. Radiotherapy, with its high radioactivity, kills tumor tissue while also damaging nearby normal tissue. Long-term radiotherapy can also cause adverse reactions, resulting in both mental and physical distress. Surgery can be highly traumatic, and incomplete resection or improper surgical care can lead to postoperative tumor recurrence. These treatments present significant side effects, poor efficacy, low specificity, high recurrence rates, and the development of drug resistance. Furthermore, these treatments can cause pain, trauma, and scarring to patients. Therefore, there is a need for more gentle, less invasive, and effective treatments for melanoma.

[0003] Microneedles are an emerging drug delivery system that can effectively deliver drugs through the skin, increasing drug distribution at the tumor site and reducing drug penetration into adjacent tissues. Prior art strategies using microneedle technology have been developed for the treatment of melanoma. For example, in the article "Dissolving microneedles-based programmed delivery system for enhanced chemo-immunotherapy of melanoma," Yu Tian et al. developed a microneedle-based programmed drug delivery system that combines pH-sensitive liposomes containing the chemotherapy drug doxorubicin (DOX) and PD-L1 siRNA (siPD-L1). This system targets melanoma and releases the drug in response to the pH at the tumor site, enabling chemoimmunotherapy of melanoma. The addition of doxorubicin not only protects against tumors on its own but also improves melanoma immunotherapy, triggering immunogenic cell death and activating anti-tumor immunity. The physical adjuvant effect of the soluble microneedles further enhances the efficacy of chemoimmunotherapy. Their in vitro and in vivo experimental results demonstrated that the microneedles rapidly dissolve into the skin and have sufficient mechanical strength to penetrate the skin, resulting in enhanced anti-tumor efficacy. However, it still has problems such as short maintenance time of anti-tumor immunity and high tumor recurrence rate.

[0004] To address these issues, this field urgently needs a new type of microneedle patch as a melanoma treatment method with high efficiency, low recurrence rate, good targeting, high safety, and non-invasive and painless.

[0005] Summary of the Invention

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

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

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

[0009] The microneedle technology employed in this invention prepares the drug into a patch, effectively delivering it to the lesion site, enabling painless, non-invasive, and precise treatment of melanoma. The chemotherapeutic effect of paclitaxel can inhibit tumor cell proliferation and induce apoptosis. Simultaneously, combining paclitaxel with dendritic cells can induce a highly effective, low-toxic anti-tumor immune response, enhancing tumor killing and helping to generate immune memory, enabling the body to acquire long-lasting anti-tumor immunity and reduce tumor recurrence. This invention represents the first application of live cell gel in the treatment of melanoma, demonstrating synergistic chemotherapy and immunotherapy through the combination of chemotherapy drugs and live cell gel.

[0010] Preferably, the paclitaxel is albumin-bound paclitaxel.

[0011] Albumin paclitaxel has outstanding efficacy, good biocompatibility, non-immunogenicity and biodegradability. Albumin particles serve as a carrier, allowing albumin paclitaxel to quickly enter tumor cells, making the drug act in tumor cells longer and increasing its efficacy.

[0012] The preparation method of the albumin-bound paclitaxel comprises the following steps: adding an albumin solution to a paclitaxel solution, stirring and ultrasonically treating the solution, and then volatilizing the solvent to allow the albumin to wrap the paclitaxel into nanoparticles.

[0013] Preferably, the solvent of the paclitaxel solution is ethanol.

[0014] Preferably, the albumin-bound paclitaxel in the microneedles is coupled to the surface of dendritic cells.

[0015] Preferably, the nab-paclitaxel-coupled dendritic cells in the microneedles are in 2% gelatin-culture medium.

[0016] Preferably, immature dendritic cells account for 100% of the dendritic cells.

[0017] Immature dendritic cells take up melanoma-specific antigens such as MART1, Melan-A, and gp 100 at the tumor site, and after processing, they develop into mature dendritic cells. Mature dendritic cells secrete and express cytokines such as IL-6, TNF-α, and interferon. These cytokines can enable dendritic cells to smoothly chemotaxis 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, ultimately killing tumor cells, and at the same time helping the body to produce immune memory and obtain lasting anti-tumor immunity.

[0018] Preferably, the shell material of the microneedle mold is a hydrophobic copolymer VP-CO-MMA.

[0019] The preparation method of the microneedle patch is to load albumin-bound paclitaxel-coupled dendritic cells into a microneedle mold in a 2% gelatin-culture medium through vacuum. Beneficial effects:

[0020] (1) The present invention uses microneedle technology to load living cell gel and chemotherapy drugs, directly targeting the lesion site, ensuring accurate drug delivery and efficient treatment.

[0021] (2) Based on paclitaxel and dendritic cells, the present invention adopts a combined chemo-immunotherapy method, using paclitaxel to prevent the proliferation of tumor cells and promote their apoptosis, and activate the anti-tumor immune response after combining with dendritic cells, thereby enhancing the killing effect on tumors, helping the human body to produce immune memory, and enabling the body to obtain long-lasting anti-tumor immunity to reduce tumor recurrence.

[0022] (3) The present invention combines chemotherapy drugs with living cells to work synergistically, accelerating the elimination and recovery process of melanoma.

[0023] (4) The paclitaxel in the present invention is albumin-modified, safe and non-toxic, with good biocompatibility, and high safety during the treatment process.

[0024] (3) The present invention uses microneedle therapy, which is painless, noninvasive, and easy to operate, significantly reducing the pain associated with treatment. The microneedles are biodegradable and, after being applied as a patch to the lesion, can be degraded by esterases in the body, thus achieving painless and noninvasive treatment of melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 is a schematic diagram of the preparation principle of the microneedle patch.

[0027] Figure 2 is a morphology image of the microneedle patch analyzed under a transmission electron microscope.

[0028] FIG3 shows the cell survival rate under the action of different concentrations of paclitaxel.

[0029] FIG4 shows the proliferation of untreated / treated dendritic cells after culturing for 1, 2, 4, and 6 days. DETAILED DESCRIPTION

[0030] This invention provides a novel gel-type microneedle patch for treating melanoma. This patch, created by infusing albumin-paclitaxel and dendritic cell gel into perforated microneedles, effectively delivers the drug to the lesion site and precisely releases it, enabling noninvasive and painless treatment of melanoma. The microneedle patch of this invention provides efficient, long-lasting, and painless treatment for melanoma.

[0031] Albumin is biodegradable, safe and non-toxic. It can be used as a carrier to encapsulate paclitaxel and deliver it to the tumor site. Compared with paclitaxel, it has better solubility and can prevent paclitaxel from being cleared from the blood in the body, thereby increasing the targeting of the tumor.

[0032] The chemotherapy drug paclitaxel uses its own toxicity to kill tumor cells, while significantly increasing local tumor oxygen levels and inhibiting HIF-1α expression, alleviating tumor hypoxia and regulating the tumor microenvironment. Dendritic cells activate anti-tumor immune responses, enhancing tumor-killing effects and addressing the high recurrence rate of melanoma.

[0033] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the art, and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0034] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art.

[0035] Example 1

[0036] Preparation process of a new gel-based microneedle patch for treating melanoma

[0037] (1) Preparation of albumin-bound paclitaxel

[0038] Albumin was dissolved in a buffer solution and thoroughly dissolved by stirring and sonication, after which impurities and bubbles were removed. Paclitaxel was dissolved in ethanol to form a paclitaxel solution. 10 ml of the albumin solution (2 mg / ml) was added to 150 μl of a paclitaxel solution (20 mg / ml) pre-dissolved in ethanol. After stirring overnight at room temperature, the solution was centrifuged at 10,000 rpm for 10 minutes to remove excess paclitaxel, resulting in an albumin-bound paclitaxel solution. The solvent then evaporated, allowing the albumin to encapsulate the paclitaxel into nanoparticles. Ultrafiltration and other techniques were used to obtain the albumin-bound paclitaxel, with a paclitaxel content of 10%. The molar ratio of albumin to paclitaxel was approximately 1:5.

[0039] (2) Albumin-bound paclitaxel coupled to dendritic cells

[0040] Albumin-bound paclitaxel was coupled to the surface of dendritic cells through sugar metabolism and click chemistry. Thermoreversible gelatin was selected as the cell loading matrix, and the dendritic cells coupled with albumin-bound paclitaxel were transferred into 2% gelatin-AIM-V culture medium.

[0041] The sugar metabolism and click chemistry method involves rapidly synthesizing various molecules by piecing together small building blocks. In biological applications, glycan structures are modified and functional groups are introduced. When non-native monosaccharides come into contact with cells, the cells produce their own glycans through intrinsic metabolic pathways. The specific steps are: Immature dendritic cells are cultured in a culture medium containing the azidosugar Ac4ManNAz for three days to attach an azide tag. Following azide tagging, albumin-bound paclitaxel is covalently attached to the azide-labeled DCs.

[0042] Source and culture of immature dendritic cells: Mice were sacrificed by cervical dislocation, and their bones were transferred to a clean bench and soaked in 70% alcohol for 2-5 minutes, then washed twice with sterile PBS. The bones were transferred to PBS, and the ends of the bones were cut with scissors. PBS was withdrawn with a syringe, and a needle was inserted into the bone marrow cavity at each end of the bone. The bone marrow was repeatedly flushed out into a culture dish until the bone turned completely white. The bone marrow suspension was collected and filtered through a 200-mesh nylon mesh to remove small debris and muscle tissue. The filtrate was centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. 2 ml of ammonium chloride red blood cell lysis buffer (1x) was added, and the cells were resuspended and incubated at room temperature for 3-10 minutes. The suspended cells were removed and the cells were cultured with AIM-V containing GM-CSF (500-1000 U / mL) and IL-4 (500 U / mL). Half of the medium was changed every 2-3 days, and GM-CSF and IL-4 were supplemented to obtain immature dendritic cells.

[0043] (3) Loading into microneedle mold

[0044] Dendritic cells coupled with albumin-bound paclitaxel in 2% gelatin-based culture medium were vacuum loaded into the microneedle mold. The outer shell of the microneedle mold is made of a hydrophobic copolymer VP-CO-MMA, which is a hydrophilic medium that helps maintain cell viability and can be degraded by esterases in the body; the center is hollow and perforated to facilitate the release of drugs and living cells.

[0045] Example 2

[0046] Verification of the therapeutic effect of microneedle patches

[0047] (1) Analysis of the morphology of the microneedle patch under a transmission electron microscope (Figure 2)

[0048] (2) The cell survival rate under the action of different concentrations of paclitaxel was analyzed. (Figure 3)

[0049] In vitro cytotoxicity assay, melanoma cells were first plated at 1×10 4 The cells were seeded in 96-well plates at a density of 100 cells / mL overnight and then incubated with different concentrations of paclitaxel for 48 hours. The cells were then counted and the relative cell viability was determined using CCK-8.

[0050] (3) Proliferation of untreated / treated dendritic cells after 1, 2, 4, and 6 days of culture (Figure 4)

[0051] The microneedles carrying albumin-bound paclitaxel-coupled dendritic cells were placed in a 24-well plate and then culture medium was added to release the cells and incubated in the 24-well plate. 5The cells were inoculated with the same type of cells at a density of 100 cells per well as a positive control. AlamarBlue assays were performed on days 1, 2, 4, and 6: alamarBlue reagent was added to each well at a final concentration of 10% (vol / vol). After incubation at 37°C for 4 hours, fluorescence intensity was measured (excitation 540 nm, emission 590 nm). The fluorescence intensity at each designated incubation time was divided by the fluorescence intensity after 1 day of incubation to quantify cell proliferation. The results are shown in Figure 4, which demonstrate that dendritic cells released from the microneedles retain good activity and are able to exert immune effects.

[0052] As demonstrated in the above examples, paclitaxel has a tumor cell-inhibiting effect. Albumin is biodegradable and, as a carrier for paclitaxel, can encapsulate paclitaxel and deliver it to the tumor site, preventing its clearance from the blood and enhancing its therapeutic efficacy. Dendritic cells coupled to albumin-bound paclitaxel retain significant activity after release from the microneedles, demonstrating their ability to exert immune responses.

[0053] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A novel gel-type microneedle patch for treating melanoma, characterized in that: The microneedle patch is loaded with dendritic cells and paclitaxel.

2. The microneedle patch according to claim 1, characterized in that The paclitaxel is albumin-bound paclitaxel.

3. The microneedle patch according to claim 2, characterized in that The preparation method of the albumin-bound paclitaxel comprises the following steps: adding an albumin solution to a paclitaxel solution, stirring and ultrasonically treating the solution, and then volatilizing the solvent to allow the albumin to wrap the paclitaxel into nanoparticles.

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

5. The microneedle patch according to claim 2, characterized in that Albumin-bound paclitaxel in the microneedles is coupled to the surface of dendritic cells.

6. The microneedle patch according to claim 5, characterized in that The nab-paclitaxel-coupled dendritic cells in the microneedles were in 2% gelatin-culture medium.

7. The microneedle patch according to claim 1, characterized in that Among the dendritic cells, immature dendritic cells account for 100%.

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

9. The method for preparing the microneedle patch according to claim 1, characterized in that: The preparation method is to load albumin-bound paclitaxel-coupled dendritic cells into a microneedle mold in a 2% gelatin-culture medium by vacuum loading.

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