Micro-needle patch capable of locally removing potassium ions and application of micro-needle patch

Abstract

The invention provides a microneedle patch capable of locally removing potassium ions and application thereof.The microneedle patch is characterized in that firstly, nano-sized particles are obtained through ball milling and screening of potassium-reducing medicine sodium zirconium cyclosilicate ZS-9, then the ZS-9 nano-particles are dispersed in 1-vinyl-2-pyrrolidone (NVP) according to a certain concentration, 1% (w / v) of a photoinitiator 2959 and a cross-linking agent N, N-dimethylformamide (NVP) are added, and the microneedle patch is prepared. The potassium ion removal microneedle patch (PR-MN) with PVP (polyvinylpyrrolidone) as a matrix is obtained through 365nm ultraviolet crosslinking of N, N '-methylene bisacrylamide (MBA) and N, N'-methylene bisacrylamide (MBA). The potassium-reducing microneedle patch has relatively high mechanical strength, and can effectively penetrate through the skin and penetrate into the interior of a tumor. Due to the array characteristic of the microneedle, multi-site Na < + >-K < + > exchange of the microneedle in a tumor is realized, and potassium ions in the tumor are effectively absorbed. The removal of potassium ions not only enhances the secretion of cell factors such as IFN gamma and the like by the CAR-T cells, but also promotes the formation of immunological synapses of the CAR-T cells, enhances the killing efficiency of the CAR-T cells and inhibits tumor growth.

Description

Technical Field

[0001] The present invention belongs to the field of drug delivery, and particularly relates to a microneedle patch capable of locally scavenging potassium ions and its application. Background Art

[0002] Although the efficacy of chimeric antigen receptor T cell (CAR-T) therapy for B-cell lymphoma is impressive, its success in treating solid tumors is still limited. Challenges such as tumor antigen heterogeneity, dense extracellular matrix barriers, and immunosuppressive microenvironments have hindered the efficacy of CAR-T cell therapy for solid tumors. In addition, the accumulation of metabolites in tumor interstitial fluid (TIF) can also impair the activity of immune cells, thus promoting tumor immune escape. Therefore, various strategies have been developed to enhance CAR-T infiltration, promote CAR-T cell proliferation, inhibit T cell exhaustion, and reduce side effects. Although synthetic biology modification of T cells and complex biological / chemical modifications have been used to enhance the function of T cells, alternative technologies that bypass direct cell modification provide a more simplified preparation process and protect T cells from potential damage. In addition, the continuously released toxic metabolites from necrotic cells in the tumor microenvironment (TME) emphasize the urgent need for a permanent and manageable solution to scavenge harmful by-products.

[0003] Potassium ion (K + ) is essential for maintaining the osmotic pressure of cells in the body and is also a major by-product of metabolic imbalance in the tumor microenvironment. In addition, K + homeostasis disorders may lead to significant physiological dysfunctions. Therefore, designing a locally controllable K + scavenging strategy while reducing the risk of systemic complications has become a key focus. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention prepares a K + removing microneedle patch by loading nano-scale sodium zirconium cyclosilicate particles. The patch can be locally inserted into the lesion site (such as a tumor), and through Na + -K + exchange to achieve the scavenging of K + ions in the lesion, maintain K + homeostasis, and enhance the function of CAR-T cells.

[0005] On the one hand, the present invention provides a microneedle patch, comprising any one or more potassium-lowering drugs such as sodium zirconium cyclosilicate, sodium polystyrene sulfonate, calcium polystyrene sulfonate, and sodium polystyrene sulfonate.

[0006] Microneedles generally consist of a three-dimensional array structure with a tip length of 25 - 2000 μm and a tip in the shape of an inclined plane or a symmetric cone. Microneedles can penetrate the skin stratum corneum barrier and form temporary microchannels on the skin surface. Using these microchannels, drugs can be directly delivered into the dermis, thereby improving the transdermal efficiency of the drugs. Microneedle drug delivery combines the dual advantages of local drug delivery and systemic injection, can bypass the first-pass effect of the liver, reduce the degradation of drugs by the gastrointestinal tract, and improve the bioavailability of drugs. At the same time, microneedles do not touch the dermis layer where pain nerves and blood vessels are distributed, and will not cause pain and bleeding, thus improving patient compliance and reducing the risk of infection.

[0007] Since microneedle drug delivery has more advantages than general injection or oral drug administration methods, the present invention combines a novel potassium-lowering drug with microneedles and designs a potassium ion scavenging microneedle patch, which improves the potassium ion scavenging efficiency. Considering that different resin-based potassium-lowering drug components and their effects are different, for example, sodium-type resin, compared with calcium-type resin, does not affect the concentrations of blood calcium, blood phosphorus, and parathyroid hormone in the body. Therefore, the present invention compared the differences in mechanical properties and potassium ion scavenging effects of microneedle patches prepared from several different resin-based potassium-lowering drugs. The results showed that zirconium silicate sodium ZS-9 has higher selectivity and affinity for potassium ions compared with other resin-based potassium-lowering drugs. Its cations preferentially capture potassium ions and prevent the binding of smaller ions such as magnesium, calcium, and sodium, and the potassium-lowering effect is more significant. And in terms of mechanical properties, the microneedle patch prepared from ZS-9 has stronger mechanical strength. Therefore, the potassium-lowering drug of ZS-9 is preferably used.

[0008] Furthermore, the microneedle patch further comprises any one or more of matrix solvents such as polyvinyl alcohol, polyvinylpyrrolidone, and hyaluronic acid.

[0009] Generally speaking, according to the structure of the microneedle and the drug delivery strategy, the microneedle can be divided into solid microneedles, coated microneedles and soluble microneedles, etc. In terms of drug delivery efficiency, the soluble microneedle can completely release the drug into the skin in a short time by virtue of its unique dissolution characteristics, ensuring the effective use of the drug. This efficient delivery method makes the soluble microneedle have broad application prospects in the field of transdermal drug delivery. In contrast, solid microneedles need to form channels on the skin surface by physical means, and the drug delivery speed is relatively slow. Therefore, the present invention considers the use of soluble microneedle patches. The matrix materials for preparing soluble microneedle patches can be divided into tough materials and brittle materials. Tough materials have better flexibility and strong plasticity. They are not easy to break when preparing microneedles, but the mechanical strength of the microneedles is insufficient and cannot penetrate the skin for drug delivery; brittle materials have good mechanical strength when preparing microneedles, but the microneedles are prone to being too brittle and breaking when administering drugs. The present invention compares several different matrix materials and finds that polyvinyl pyrrolidone, as a non-ionic polymer compound, has stable chemical properties and will not affect the activity of the drug; it also has good physiological inertness and excellent biocompatibility and is non-irritating to biological systems. Therefore, polyvinyl pyrrolidone is preferred.

[0010] Furthermore, the microneedle patch also includes a photocrosslinker and a photoinitiator, the photocrosslinker includes one or more of N,N'-methylenebisacrylamide (MBA) and ethylene glycol dimethacrylate, and the photoinitiator includes one or more of Irgacure 2959 and VA-086.

[0011] In another aspect, the present invention provides a method for preparing a microneedle patch, comprising the following steps:

[0012] S1. Preparation of nano-scale potassium-lowering drugs;

[0013] S2. potassium-lowering drug nanoparticles are dispersed in a matrix monomer solvent in proportion;

[0014] S3. Add a photocrosslinker and a photoinitiator to the solution obtained in step S2 to obtain a mixed solution;

[0015] S4. adding the mixed solution to the microneedle mold for cross-linking and curing;

[0016] S5. Add the substrate solution to the surface of the cured microneedles and further cross-link to obtain a microneedle patch.

[0017] Furthermore, the nano-scale potassium-lowering drug in step S1 is nano-scale drug particles obtained by physical ball milling and screening of the clinical potassium-lowering drug sodium zirconium cyclosilicate ZS-9. Nano-scale ZS-9 has a larger specific surface area than micron-sized ZS-9, which can not only enhance the potassium absorption capacity, but also facilitate the loading of microneedles.

[0018] Further, the addition ratio of the nanoparticles described in step S2 is 1.0 to 2.5 w / v%.

[0019] Preferably, the addition ratio of the nanoparticles described in step S2 is 2.0 to 2.5 w / v%.

[0020] In some embodiments, the matrix monomer solvent is 1-vinyl-2-pyrrolidone (NVP), which is an upstream raw material for synthesizing the matrix polyvinylpyrrolidone. The NVP monomers polymerize under the action of ultraviolet cross-linking to form polyvinylpyrrolidone, and the drug is finally dispersed in the polyvinylpyrrolidone.

[0021] Further, the addition ratio of the photo-crosslinking agent described in step S3 is 1.0 to 5 w / v%, and the addition ratio of the photoinitiator is 1.0 to 5 w / v%.

[0022] Preferably, the addition ratio of the photo-crosslinking agent described in step S3 is 1.0 to 2.5 w / v%, and the addition ratio of the photoinitiator is 1.0 to 2.5 w / v%.

[0023] Further, the base solution described in step S5 includes one or more ultraviolet curable agents such as NOA86, NOA86H, and NOA86TLH.

[0024] In some cases, the ultraviolet-cured potassium ion scavenging microneedle patch has strong mechanical properties, is easy to pierce the skin and tumor tissues, has high anti-solubility and biosecurity, can effectively prevent the leakage of ZS-9 nanoparticles loaded therein in the tissue, and avoid potential tissue fibrosis caused by particle leakage or the re-release of potassium ions after saturated absorption.

[0025] On the other hand, the present invention provides a use of a microneedle patch for preparing a device for reducing potassium ion levels. The microneedle patch includes any one or more potassium-lowering drugs such as sodium zirconium cyclosilicate, sodium poly(sulfophenyl)ethylene, calcium polystyrene sulfonate, and sodium polystyrene sulfonate.

[0026] In vivo potassium ion analysis shows that the potassium ion scavenging microneedle patch can effectively reduce the potassium ion content in tumors, while not affecting the potassium ion level in the serum, and has high safety.

[0027] On yet another aspect, the present invention provides a use of a microneedle patch for preparing a device for enhancing CAR-T cell function, characterized in that the microneedle patch includes any one or more potassium-lowering drugs such as sodium zirconium cyclosilicate, sodium poly(sulfophenyl)ethylene, calcium polystyrene sulfonate, and sodium polystyrene sulfonate.

[0028] Tumor K + level reduction not only enhances the secretion of cytokines such as IFNγ by CAR-T cells, but also inhibits K+ The induced depolarization of the cell membrane also enhances the formation of the CAR-T cell immune synapse, promoting the interaction between tumor cells and CAR-T cells. In a human tumor xenograft model, whether the CAR-T cells are injected intratumorally or intravenously, the potassium ion scavenging microneedle patch can effectively promote the killing of CAR-T cells, significantly inhibit tumor growth and prolong the survival of mice. In addition, in a large tumor model, the potassium ion removal microneedle patch can still strongly inhibit tumor growth, highly enhance CAR-T killing, drive a strong anti-tumor response in various mouse models and prolong the survival period.

[0029] Furthermore, the microneedle patch includes sodium zirconium cyclosilicate for potassium reduction.

[0030] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0031] 1. The constructed potassium ion scavenging microneedle patch realizes the physical removal of K + , providing a solution for specifically reducing the K + content in the lesion without affecting the systemic K + level;

[0032] 2. The array characteristics of the microneedles enable multi-point penetration and efficiently exert the potassium reduction effect;

[0033] 3. The microneedle patch provided by the present invention has good controllability and can achieve on-demand drug delivery to control the potassium ion level in the lesion;

[0034] 4. The novel potassium removal strategy provided by the present invention avoids side effects such as tissue fibrosis caused by tissue residue and reduces the potassium level;

[0035] 5. The potassium ion removal strategy provided by the present invention significantly improves the production of cytokines and the formation of immune synapses, thereby enhancing the function of CAR-T and inhibiting tumor growth;

[0036] 6. The present invention provides a novel mechanism, that is, reducing the potassium ions inside the tumor and inhibiting the depolarization of CAR-T cells, thereby improving the formation of immune synapses, while avoiding the increase in cost, the complexity of procedures and the damage to T cells caused by biological or chemical engineering transformation of T cells. + cost, the complexity of the process and the damage to T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0038] Figure 1 Schematic diagram for the preparation of potassium ion scavenging microneedle patch in Example 1;

[0039] Figure 2 ZS-9 nanoparticles after ball milling in Example 1;

[0040] Figure 3 Photograph of the potassium ion scavenging microneedle patch in Example 1 (a) and morphological characterization of the microneedles before and after loading ZS-9 (b);

[0041] Figure 4 For the potassium ion scavenging microneedle patch in Example 1 to absorb K + before (a) and after absorbing K + elemental analysis (b);

[0042] Figure 5 Mechanical properties of drug-free PVP-MN and potassium ion scavenging microneedle patch (PR-MN) in Example 1.

[0043] Figure 6 Characterization of subcutaneous tissue fibrosis 21 days after ZS-9 injection in Example 4;

[0044] Figure 7 For the K in the pancreatic cancer KPC tumor of mice after treatment with PR-MN in Example 4 + level (a) and serum K + level (b);

[0045] Figure 8 Ratio of remaining elements (Si, Zr) in the tumor to the PR-MN payload (a) and leakage concentrations of Si and Zr in the tumor (b) in Example 4;

[0046] Figure 9 Ratio of important ion content in the tumor to serum in Example 5;

[0047] Figure 10 Gene expression of IFNG, GZMB, and PRF1 in CAR-T cells (a) and secretion of IFNγ (b) and CD4 + and CD8 + IFNγ in CAR-T cells + cell ratio and statistics (c) in different culture medium conditions in Example 5;

[0048] Figure 11 For CAR-T cells cultured in high K + medium, intracellular K + level after administration of ZS-9 particles (black dotted line indicates no high K +Intracellular K in treated CAR-T cells + level);

[0049] Figure 12 For the high K in Example 5 + Gene expression of IFNG, GZMB, and PRF1 in cultured CAR-T cells after ZS-9 administration (a); After ZS-9 treatment, with high K + Protein expression of GZMB and PRF in CAR T cells cultured in medium (b); High K + IFNγ secreted by cultured CAR-T cells after ZS-9 administration (c);

[0050] Figure 13 For Example 5, confocal images show the binding of CAR-T cells (dashed line) to target Capan2 cells, the relative distance between MTOC and synapses, and the superimposed area of F-actin in the immunological synapse (IS) (scale bar = 10 μm) (a); After Gram treatment, in high K + Membrane potential of CAR T cells cultured in medium (n = 4 culture replicates) (b); Representative confocal images of the interaction between Gram-treated CAR-T cells and Capan2 cells, the relative distance between MTOC and synapses, and the superimposed area of F-actin in the immunological synapse (IS) (scale bar = 10 μm) (c);

[0051] Figure 14 For Example 7, immunohistochemistry shows positive expression of mesothelin in PDX tumors;

[0052] Figure 15 For Example 7, after receiving 4×10 5 Tumor growth kinetics of different groups after intratumoral injection of CAR-T cells and treatment with PR-MN patches (a) and survival rate of mice receiving CAR-T and PR-MN patch treatment (b);

[0053] Figure 16 For Example 7, after receiving 1×10 6 Intratumoral injection of CAR-T cells and treatment with PR-MN patches, tumor growth curves (a), weights (b), and ex vivo tumor photos (c) of different groups of large-sized human pancreatic cancer tissue xenografted (PDX) tumors (≥400 mm 3 );

[0054] Figure 17 For Example 7, intravenous injection of CAR-T (4×10 6 ) and treatment of PDX tumors with PR-MN patches, tumor growth kinetics (a) and mouse survival rate (b). Detailed implementation mode

[0055] The present invention will be specifically described below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly thereby. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, rather than limiting the present invention.

[0056] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood to have the meanings as commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of any contradiction, this specification shall prevail.

[0057] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0058] Example 1: Preparation of Potassium Ion Scavenging Microneedle Patch (PR-MN)

[0059] The potassium ion scavenging microneedle patch provided by the present invention is constructed as Figure 1 shown. After thoroughly mixing ZS-9 particles with a polyvinylpyrrolidone solution, a photoinitiator and a photo-crosslinking agent are added, and then added to a mold to obtain a potassium ion removal microneedle patch by photo-crosslinking.

[0060] The specific steps are as follows:

[0061] (1) Preparation of nanoscale ZS-9 nanoparticles

[0062] Using a high-energy ball mill (SPEX-8000D), sodium zirconium cyclosilicate drug (purchased from AstraZeneca, Lixiana) is ball milled for 10 h, and nanoscale ZS-9 particles are screened by differential centrifugation. As Figure 2 shown, the average size of the obtained nanoscale ZS-9 particles is about 120 nm, and the uniformity is good.

[0063] (2) Preparation of PR-MN

[0064] The ZS-9 nanoparticles obtained by ball milling and screening were dispersed in 100 μL of 1-vinyl-2-pyrrolidone (purchased from Aladdin, V106155) solution at a ratio of 2.5 w / v%, and then 1.0 w / v% of the photo-crosslinking agent N,N'-methylenebisacrylamide (MBA) (purchased from Aladdin, M128783) and 1.0 w / v% of the photoinitiator 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure 2959, purchased from Sigma, 410896) were added in sequence. This mixed solution was added to the microneedle mold, the air bubbles were discharged by vacuum filtration, and it was crosslinked under 365 nm ultraviolet irradiation for 5 min to obtain potassium-lowering microneedles based on polyvinylpyrrolidone (PVP). Norland Optical Adhesive photocuring glue (purchased from Norland, NOA86) was used as the substrate and added to the surface of the crosslinked microneedles, and further crosslinked by ultraviolet for 5 min. After the prepared microneedle patch was removed, it was placed in a vacuum drying oven for drying and storage.

[0065] The potassium ion scavenging microneedle patch constructed was analyzed by scanning electron microscopy, and the results showed that the ZS-9 nanoparticles were evenly distributed inside the microneedles ( Figure 3 ). The microneedle patch was immersed in 50 mM KCl solution for 12 h, then dried and subjected to elemental analysis to evaluate its performance of absorbing K + .

[0066] As Figure 4 shown, after absorbing K + , the K + in the potassium ion scavenging microneedle patch increased significantly, while the Na + decreased significantly, indicating that good Na + -K + exchange was achieved. The mechanical properties of the microneedles were tested using a press, and the results showed that the microneedles loaded with ZS-9 nanoparticles had similar mechanical strength to those before loading ( Figure 5 ).

[0067] Example 2. Screening of potassium-lowering drugs

[0068] Since the components and functions of different potassium-lowering drugs are different, the prepared microneedle patches may be different in mechanical strength and potassium ion scavenging effect. In order to improve the K + scavenging efficiency of the microneedle patch, four resin-based potassium-lowering drugs shown in Table 1 were provided in this example. After being ground into nanoscale particles, potassium ion scavenging microneedle patches were prepared. The remaining materials and steps were the same as in Example 1. Then, the microneedle patches containing different potassium-lowering drugs were subjected to elemental analysis and mechanical property tests, and the results are shown in the following table.

[0069] Table 1 Test analysis results of microneedle patches containing different potassium-lowering drugs

[0070]

[0071] As shown in the data of Table 1, the microneedle patch prepared with the calcium-type polystyrene resin potassium-lowering drug has better selectivity for the absorption of K + than the sodium-type resin, and the mechanical strength of a single microneedle is also higher. The microneedle patch of sodium styrene sulfonate has a higher mechanical strength of a single microneedle than the former two, but the absorption performance for K + is lower; the microneedle patch of sodium zirconium cyclosilicate drug has a stronger Na + -K + exchange ability, stronger affinity for K + , and the absorption performance and mechanical strength for K + are significantly better than those of the other three microneedle patches. Therefore, in this example, sodium zirconium cyclosilicate potassium-lowering drug is preferably used.

[0072] Example 3. Screening of matrix materials

[0073] Microneedle patches prepared from different matrix materials have differences in many properties such as biocompatibility and mechanical strength. In order to further explore the differences in the mechanical properties, K + clearance, etc. of microneedle patches prepared from different matrix materials and screen out the most suitable matrix materials, in this example, several matrix materials shown in the following table are used to prepare microneedles, and the other materials and steps are the same as those in Example 1. Then, the mechanical properties of microneedle patches with different matrix materials are tested and analyzed, and the test results are shown in Table 2.

[0074] Table 2 Test analysis results of microneedle patches with different matrix materials

[0075]

[0076] The data in Table 2 show that the potassium-lowering abilities of the three microneedle patches are comparable. Among them, the potassium-lowering microneedle patch constructed with polyvinylpyrrolidone has a higher absorption performance for K + and stronger mechanical properties, and is easier to penetrate into tissues; after penetrating the skin and tumors, it has better anti-swelling properties. Therefore, it is preferable to use the potassium-lowering microneedle patch constructed with the matrix solvent of polyvinylpyrrolidone.

[0077] Example 4. In vivo safe potassium-lowering test based on potassium ion scavenging microneedle patch

[0078] (1) Safety of ZS-9

[0079] To evaluate the safety of ZS-9 treatment, ZS-9 nanoparticles were implanted subcutaneously in mice for 21 days. The skin tissue containing ZS-9 was removed, subjected to Masson staining and observed microscopically. The results are as Figure 6 shown that ZS-9 did not degrade in mice for 21 days. After 21 days of implantation, an obvious fibrous capsule was formed, leading to tissue fibrosis.

[0080] (2) Potassium reduction in vivo

[0081] KPC cells at a concentration of 1×10 6 were subcutaneously injected to construct KPC tumor-bearing mice. When the tumor grew to 200 mm 3 , the microneedle patch prepared in Example 1 was covered on the tumor area and inserted into the tumor at different times (0, 2, 4, 6, 8, 10, 12, 24 h). Tumors and sera were collected. The tumor tissue was wrapped with a 10-μm sieve and the tumor interstitial fluid was collected by centrifugation at 400 g for 10 min. The main ion contents in the tumor interstitial fluid were detected using a blood biochemical analyzer (purchased from Hitachi, model 7180). The results are as Figure 7 shown in a. After PR-MN treatment, the K + content in the tumor was significantly reduced. After 12 h of insertion, the K + in the tumor decreased by more than 3-fold compared with the 0 h point (control group). At the same time, the PR-MN treatment did not affect the K + level in the serum ( Figure 7 b).

[0082] (3) Drug residue in tissues

[0083] Human pancreatic cancer tissues were xenografted into mice to construct PDX tumors. PR-MN was inserted into the PDX tumors for 24 h. The tumor tissues were lysed sufficiently with nitric acid, and the contents of Zr and Si in the tumor tissues were analyzed using inductively coupled plasma-mass spectrometry (ICP-MS) (purchased from PerkinElme, USA, model NexION 300XX). As Figure 8 shown, in the tumor area, the residual rates of Si and Zr (the main elements of ZS-9 nanoparticles) relative to the PR-MN payload were approximately 0.39% and 0.06% respectively. The residual amounts of Si and Zr in every 100 mg of tissue were 10.13 μg and 1.38 μg respectively, confirming the lowest residual rate of ZS-9 in tumors and avoiding tissue fibrosis caused by ZS-9 residue in vivo.

[0084] Example 5. Potassium reduction and enhanced CAR-T cell function of ZS-9 in vitro

[0085] To further explore the effect of ZS-9 on clearing potassium ions from chimeric antigen receptor T cells (CAR-T cells), the present invention further subcutaneously established B16F10 and KPC tumors, and the analysis of major ions within the tumors showed ( Figure 9 ) that the K + in the tumors was significantly elevated, while the levels of other ions such as sodium (Na + ), chloride (Cl - ), and calcium (Ca 2+ ) were similar to those in the serum. To evaluate clinical relevance, in this example, samples of human hepatocellular carcinoma (HCC) after clinical surgery were collected to assess the concentration of major ions in tumor interstitial fluid (TIF). As Figure 9 shown, the K + concentration generated by TIF in HCC reached approximately 70 mM, which was significantly higher than the physiological concentration (3.5 - 5.5 mM).

[0086] On this basis, the present invention further studied the effect of a high K + microenvironment on the function of CAR-T cells specifically recognizing mesothelin (MSLN) constructed by lentiviral transfection. The CAR-T cells were cultured in a high Na + , high K + medium, and the expression of cell proteins related to tumor suppression was analyzed.

[0087] Real-time quantitative polymerase chain reaction (qPCR) analysis revealed ( Figure 10 a) that compared with the control group and the high Na + (50 mM) medium group, the expression of interferon γ (IFNG), granzyme B (GZMB), and perforin (PRF1) in CAR-T cells cultured in a high K + medium was significantly inhibited. In addition, enzyme-linked immunosorbent assay (ELISA) showed ( Figure 10 b) that compared with high Na + treatment, the secretion of the tumor-killing factor IFNγ was inhibited after high K + treatment. In addition, flow cytometry analysis showed ( Figure 10 c) that in a high K + environment, the proportion of IFNγ + in CD4 + and CD8 + CAR-T cells decreased significantly.

[0088] To explore the K + clearing effect of ZS-9 in a high K + environment, flow cytometry was used to further detect the K + inside CAR-T cells in a high K +Concentration, and it was found that compared with untreated cells, the K + inside CAR-T cells cultured in high-K + medium increased by about 1.2 times; after adding 200 μg / mL ZS-9 nanoparticles alone, the excessive K + in CAR-T cells was effectively cleared and showed a dose-dependent decrease ( Figure 11 ).

[0089] Furthermore, qPCR analysis was performed, and the results showed ( Figure 12 a) that the high-K + microenvironment reduced the expression of tumor-suppressing related cytokines, including IFNG, GZMB, and PRF1. However, after incubating with ZS-9 for 6 hours, the expression of these genes in CAR-T cells was significantly restored. In addition, the elevated K + significantly inhibited the protein expression of GZMB and PRF, and the expression of GZMB and PRF was effectively reversed after adding ZS-9 ( Figure 12 b). ELISA analysis showed ( Figure 12 c) that the treatment with ZS-9 restored the impaired IFNγ secretion of CAR-T cells.

[0090] Since an increase in extracellular K + can increase the membrane potential and may lead to cell membrane depolarization, in this example, a membrane potential probe was used to monitor the change of cell membrane potential, and it was found that the elevated extracellular K + induced an increase in membrane potential, while the treatment with ZS-9 alleviated this increase. At the same time, because the change of cell membrane surface charge is closely related to cell activities (such as cell migration), the present invention speculates that the change of membrane potential may affect the formation of the immunological synapse (IS), which is a key index of T cell activity. In this example, confocal 3D imaging was further used to observe the formation of IS between CAR-T cells and human pancreatic cancer Capan2 cells. Confocal imaging showed ( Figure 13 a) that in CAR-T cells exposed to the high-K + environment, the accumulation of F-Actin in the synaptic region decreased, and at the same time, the distance between the microtubule organizing center (MTOC) and the IS increased. After treatment with ZS-9, the damaged synapses between CAR-T and tumor cells were restored. To further confirm whether K + directly disrupts the formation of IS in CAR-T cells, gramicidin (Gram) was used to reduce intracellular K + without reducing the membrane potential ( Figure 13 b). Confocal imaging showed ( Figure 13 c) that after Gram administration, there was no such difference in the IS region and the distance from the microtubule organizing center of CAR-T cells to the IS, indicating that K +It will not directly affect the formation of IS, but the formation of IS will be inhibited by the depolarization of the cell membrane.

[0091] Example 6. In vitro verification of the effect of PR-MN on the function of CAR-T cells

[0092] According to the results of Example 1 and Example 2, it can be seen that when using different potassium-lowering drugs or matrix materials, the prepared microneedle patches have different clearance efficiencies for K + . Therefore, there may also be differences in the synergistic effects of microneedle patches prepared with different components on the function of CAR-T cells. To further verify the effects of different microneedle patches on the function of CAR-T cells, in this example, two potassium-lowering drugs, calcium polystyrene sulfonate and sodium zirconium cyclosilicate, in Example 2, and two matrix materials, polyvinyl alcohol and polyvinylpyrrolidone, in Example 3, were combined in pairs to prepare four potassium ion-clearing microneedle patches as shown in Table 3, and the method steps were the same as those in Example 1. The four microneedle patches were used to treat the co-culture system of human pancreatic cancer Capan2 cells and CAR-T cells constructed in Example 5 for 6 h. The control was the microneedle patch without added drug. Then, the levels of related cytokines IFNγ and IL-2 secreted by CAR-T cells in the high K + medium were detected, and the results are shown in the following table.

[0093] Table 3 Analysis of the effects of microneedle patches with different components on the function of CAR-T cells

[0094]

[0095] According to the data in Table 3, it can be seen that compared with the control, the high K + microenvironment reduced the expression of tumor suppression-related cytokines and inhibited the secretion of IFNγ and IL-2 cytokines; after incubation with microneedle patches for a period of time, the expression of these genes in CAR-T cells was restored. However, the therapeutic effect of the microneedle patches using calcium polystyrene sulfonate as the potassium-lowering drug (Combination 3 and Combination 4) was worse than that of sodium zirconium cyclosilicate, and the restoration effect of the microneedle patch prepared with sodium zirconium cyclosilicate and polyvinyl alcohol (Combination 1) on the secretion of IFNγ and IL-2 cytokines was not as good as that of sodium zirconium cyclosilicate and polyvinylpyrrolidone (Combination 2). Therefore, the combination of sodium zirconium cyclosilicate and polyvinylpyrrolidone is preferred.

[0096] Example 7. Effect of PR-MN on CAR-T treatment in vivo

[0097] After studying the K + clearance ability of ZS-9 nanoparticles in tumors, this example further evaluated the therapeutic effect of potassium ion-clearing microneedle patches in vivo.

[0098] First, a patient-derived PDX tumor model was established. Subsequently, the PR-MN prepared in Example 1 was used to cover the PDX tumor to evaluate the anti-tumor effect of PR-MN patch-assisted CAR-T cells. Immunohistochemical staining showed that MSLN was significantly expressed in the PDX tumor ( Figure 14 ). The size of the PR-MN was further adjusted to cover the PDX tumor area, and the PR-MN was inserted into the tumor 4 h after injecting CAR-T cells into the tumor. When the tumor size reached 50 mm 3 , the mice were respectively treated with PBS, MNs, PR-MNs, CAR-T cells, and the combination of CAR-T lymphocytes with MNs and PR-MNs. The treatment with CAR-T cells combined with MNs (CAR-T+MNs) showed an anti-tumor effect similar to that of CAR-T injection, and the treatment with CAR-T cells combined with PR-MNs (CAR-T+PR-MNs) showed a more satisfactory therapeutic effect ( Figure 15 a). In addition, 60% of the mice survived 130 days after treatment with CAR T+PR-MN. In contrast, all the mice in the other groups died within 100 days ( Figure 15 b).

[0099] Since the increase in tumor cell necrosis in large tumors can lead to more K + leakage, increasing K + in the tumor microenvironment (TME), it may lead to a more severe loss of T cell function. To further verify the therapeutic effect of PR-MN in large tumors, this example further constructed large tumors (≥400 mm 3 ), and treated the mice bearing large tumors with PBS, CAR-T cells, and CAR-T+PR-MN.

[0100] As Figure 16 shown, after intravenous treatment with CAR-T cells, limited anti-tumor efficacy could be observed. However, when the PR-MN was inserted to remove K + in the TIF, the CAR-T cells significantly inhibited tumor growth by 4.5-fold compared with free CAR-T injection. Subsequently, CAR-T cells were intravenously injected to further simulate clinical treatment. In the CAR-T+PR-MN group, 83% of the tumors regressed, indicating a significant improvement in anti-tumor efficacy. Notably, these cured mice from the CAR-T+PR-MN group survived tumor-free for more than 200 days ( Figure 17 ). The above results indicate that PR-MN can enhance the function of CAR-T and inhibit tumor growth.

[0101] Finally, it should also be noted that the terms "comprise", "comprising" or any other variant thereof are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also includes other elements not expressly listed, or elements inherent to such process, method, article or apparatus.

[0102] Although the preferred embodiments of the present invention have been described, additional changes and modifications can be made to these embodiments by those skilled in the art once they learn of the basic inventive concept. Therefore, the appended claims are intended to be construed to include the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0103] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention is also intended to include these modifications and variations.

Claims

1. A microneedle patch, characterized in that: The potassium-lowering drugs include any one or more of sodium zirconium cyclosilicate, sodium polystyrene sulfonate, calcium polystyrene sulfonate, and sodium polystyrene sulfonate.

2. The microneedle patch according to claim 1, characterized in that It also includes any one or more base solvents selected from polyvinyl alcohol, polyvinyl pyrrolidone, and hyaluronic acid.

3. The microneedle patch according to claim 1, characterized in that The invention also comprises a photocrosslinking agent and a photoinitiator. The photocrosslinking agent comprises one or more of N,N'-methylenebisacrylamide and ethylene glycol dimethacrylate, and the photoinitiator comprises one or more of Irgacure 2959 and VA-086.

4. A method for preparing a microneedle patch, characterized in that: The following steps are involved: S1. Preparation of nano-scale potassium-lowering drugs; S2. potassium-lowering drug nanoparticles are dispersed in a matrix monomer solvent in proportion; S3. Adding a photocrosslinker and a photoinitiator to the solution obtained in step S2 to obtain a mixed solution; S4. adding the mixed solution to the microneedle mold for cross-linking and curing; S5. Add the substrate solution to the surface of the cured microneedles and further cross-link to obtain a microneedle patch.

5. The method according to claim 4, characterized in that The addition ratio of the nanoparticles in step S2 is 1.0-2.5 w / v%.

6. The method according to claim 4, characterized in that In step S3, the addition ratio of the photocrosslinking agent is 1.0-5.0 w / v%, and the addition ratio of the photoinitiator is 1.0-5.0 w / v%.

7. The method according to claim 4, characterized in that The base solution described in step S5 includes any one or more photocurable materials selected from NOA86, NOA86H, and NOA86TLH.

8. A use of a microneedle patch for preparing a device for reducing potassium ion levels, characterized in that: The microneedle patch comprises any one or more potassium-lowering drugs selected from sodium zirconium cyclosilicate, sodium polystyrene sulfonate, calcium polystyrene sulfonate, and sodium polystyrene sulfonate.

9. Use of a microneedle patch for preparing a device for enhancing CAR-T cell function, characterized in that: The microneedle patch comprises any one or more potassium-lowering drugs selected from sodium zirconium cyclosilicate, sodium polystyrene sulfonate, calcium polystyrene sulfonate, and sodium polystyrene sulfonate.

10. The use according to claim 9, characterized in that The microneedle patch includes sodium zirconium cyclosilicate potassium-lowering drug.

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