A soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles and a preparation method and application thereof

Abstract

The application belongs to microneedle, and particularly relates to soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles and a preparation method and application thereof. 2+ The coordination center ion of the nanoparticles is selected from Zn 2+ , the ligand is selected from a mixture of 9-fluorenylmethyl oxycarbonyl modified histidine (Fmoc-H) and macrophage targeting peptide (Fmoc-HTKPR) modified by 9-fluorenylmethyl oxycarbonyl modified histidine derivatives, and methotrexate (MTX), a rheumatoid arthritis treatment drug containing a carboxyl group, and the microneedle carrier material is selected from a soluble polymer PVP. The application provides a scheme for realizing metal-drug coordination at normal temperature, and the nanoparticles are released by the microneedle in vivo, the nanoparticles can be actively targeted to inflammatory sites through the targeting peptide, and high-load MTX and active oxygen scavenging drugs are responsively released, so that high-efficiency and safe rheumatoid arthritis treatment is realized based on the synergistic effect of eliminating inflammatory macrophages and converting the same into an anti-inflammatory phenotype.

Description

Technical Field

[0001] This invention pertains to microneedles, specifically relating to a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is a common chronic, infectious, and autoimmune disease, often referred to as "the cancer that doesn't kill." Its clinical manifestations include joint swelling, synovitis, and significant cartilage damage and bone erosion. As the disease progresses, it can also cause extra-articular complications affecting multiple organs. In my country, RA is characterized by a large number of patients, delayed diagnosis and treatment, a high incidence of severe cases, and numerous complications, with a trend towards younger onset. Due to the complex pathogenesis of RA, the lack of a complete cure, and its long course and high disability rate, it has become a major public health problem urgently needing to be addressed. Currently, the widely accepted treatment options in clinical practice mainly include joint replacement surgery and treatment with chemical and biological drugs.

[0003] CN114939178A discloses an amino acid / peptide coordination polymer, its preparation method, and its application. It also discloses a metal-amino acid / peptide coordination polymer that can treat rheumatoid arthritis (RA), wherein the coordination center ion is Zn. 2+ Fe 3+ / 2+ Ca 2+ or Cu 2+ One or more of the following; the ligand is selected from histidine protected by 9-fluorenylmethoxycarbonyl, macrophage-targeting peptides modified with derivatives of histidine protected by 9-fluorenylmethoxycarbonyl, or mixtures of histidine protected by 9-fluorenylmethoxycarbonyl and macrophage-targeting peptides protected by 9-fluorenylmethoxycarbonyl, wherein the peptide contains a Tuftsin sequence. Based on a metal-amino acid / peptide framework, the inner layer encapsulates the drug methotrexate, and the outer layer encapsulates the drug Pt. However, due to the limitations of the framework itself, the drug loading capacity of this nanoparticle and similar metal-organic ligand structures is always limited (<10%). The treatment method proposed in this patent is intravenous administration, which, in addition to causing pain from long-term injection, will greatly reduce patient tolerance and medication adherence.

[0004] Microneedles (MN) are a novel physical permeation technology consisting of an array of micron-sized needle tips connected to a base. The needles are typically 10-2000 micrometers high and 10-50 micrometers wide. The length, size, and shape of the microneedles can be individually designed to meet specific treatment needs. Microneedles can penetrate the stratum corneum, creating micron-sized mechanical channels that deliver drugs directly to the epidermis or upper dermis, allowing them to participate in microcirculation and exert pharmacological responses without passing through the stratum corneum. As an emerging transdermal drug delivery method, the biggest advantage of microneedles compared to traditional patches is their ability to penetrate the stratum corneum and break down the skin barrier. However, due to size limitations, all types of microneedles suffer from low drug loading capacity.

[0005] Therefore, in order to achieve better therapeutic effects and overcome the technical defects of existing technologies such as amino acid / peptide coordination polymers or microneedles with low drug loading capacity, the present invention provides a novel microneedle. Summary of the Invention

[0006] A soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles, characterized in that the coordination center ion of the nanoparticles is selected from Zn. 2+ The first ligand is selected from a mixture of macrophage-targeting peptide Fmoc-H modified with 9-fluorenylmethyloxycarbonyl histidine Fmoc-H and a derivative of 9-fluorenylmethyloxycarbonyl histidine; the second ligand is selected from methotrexate; the microneedle carrier material is selected from PVP; the microneedles are prepared by the following steps:

[0007] The macrophage-targeting peptide Fmoc-H, modified with 9-fluorenylmethyloxycarbonyl histidine, was mixed with Fmoc-HTKPR, a derivative of 9-fluorenylmethyloxycarbonyl histidine, and methotrexate, and then Zn was added. 2+ The pH of the reaction system was adjusted to a slightly alkaline state under stirring, and metal-amino acid / peptide / drug coordination nanoparticles were self-assembled. These coordination nanoparticles were then mixed with a PVP solution and filled into a microneedle mold. After centrifugation and overnight drying, microneedle patches were obtained.

[0008]

[0009] Zn 2+ Structure after coordination with MTX

[0010] The soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles is characterized in that the molar ratio of the coordination center ion of the nanoparticle to the first ligand is 1:2.2, and the mass ratio of the first ligand to the second ligand is 1:1 to 9.

[0011] The soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles is characterized in that histidine Fmoc-H modified with 9-fluorenylmethyloxycarbonyl and macrophage-targeting peptide Fmoc-HTKPR modified with a derivative of histidine modified with 9-fluorenylmethyloxycarbonyl are mixed at a mass ratio of 1:0 to 0.3.

[0012] The application of a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles in the preparation of a drug delivery carrier.

[0013] The application is characterized in that the drug delivery carrier carries drugs and probes.

[0014] The application is characterized in that the drug delivery system comprises one or more of the following: small molecule compounds, polypeptides, proteins, polysaccharides, and nucleic acids.

[0015] The application is characterized in that the drug delivery system is 5-aminosalicylic acid.

[0016] The application of the aforementioned soluble microneedles based on metal-amino acid / peptide / drug coordination nanoparticles in anti-rheumatoid arthritis drugs.

[0017] Specifically:

[0018] The present invention discloses a method for preparing soluble microneedles based on metal-amino acid / peptide / drug coordination nanoparticles, which involves reacting Fmoc-H, Fmoc-HTKPR, and MTX with Zn. 2+ After mixing, adjust the pH of the reaction system to a slightly alkaline state (preferably pH 7-8), and stir until the metal-amino acid / peptide / drug coordination polymer self-assembles to form nanoparticles. Mix the coordination nanoparticles with the PVP solution, fill the mixture into a microneedle mold, centrifuge, and dry overnight to obtain the microneedle patch.

[0019] Preferably, the mass ratio of Fmoc-H to Fmoc-HTKPR in the first ligand is 1:0 to 0.3 (the percentage of Fmoc-HTKPR is 0% to 10%), more preferably 1:0.15 (the percentage of Fmoc-HTKPR is 5%); the mass ratio of the first ligand to the second ligand is 1:1 to 9, more preferably 3:7.

[0020] As a preferred method

[0021] (1) Mix Fmoc-H and Fmoc-HTKPR uniformly at a mass ratio of 1:0.15 (Fmoc-HTKPR feed percentage is 5%), then add a second ligand (MTX) solution to the system at a mass ratio of Fmoc-H and MTX of 3:7, and then add the first ligand and Zn2+ A zinc nitrate hexahydrate aqueous solution was added to the system at a molar ratio of 2.2:1;

[0022] (2) Add different types of substrates (such as 5-ASA) to the system according to the required function, and adjust the pH of the solution to a slightly alkaline state (preferably pH 7-8) under vigorous stirring to induce the formation of nanoparticles until particle growth is complete. The substrate-loaded metal-amino acid / peptide / drug coordination nanoparticles are obtained by dialysis purification. The nanoparticle solution is then uniformly mixed with PVP solution, placed in a microneedle mold, centrifuged, and dried overnight to obtain soluble microneedles of substrate-loaded metal-amino acid / peptide / drug coordination nanoparticles.

[0023] Another objective of this invention is the application of soluble microneedles based on metal-amino acid / peptide / drug coordination nanoparticles as carriers for delivering drugs or probes.

[0024] Specifically, the drug is one or more of small molecule compounds, peptides, proteins, polysaccharides, and nucleic acids. Further, the drug is 5-aminosalicylic acid (5-ASA). Co-incubating the coordination nanoparticles of this invention with an appropriate amount of 5-ASA yields coordination nanoparticles loaded with different masses of 5-ASA.

[0025] The mass of 5-ASA in the coordination nanoparticles is 0.6 mg to 3.0 mg, preferably 1.2 mg.

[0026] This invention primarily considers the following four aspects: ① Designing a nano-drug-carrying framework with inherent targeting and therapeutic capabilities, thereby enhancing drug accumulation at the lesion site through both active and passive targeting. ② Improving the unstable nature of metal-drug coordination bonds by doping the metal-drug coordination structure with strong metal-amino acid interactions, thereby increasing the stability of the metal-amino acid / drug coordination structure and the drug loading capacity. ③ Indirectly addressing the inherent limitation of low drug loading capacity in microneedles by increasing the drug loading capacity in nanoparticles. ④ Reducing pain and improving patient compliance through microneedle drug delivery.

[0027] This invention utilizes the strong coordination ability of metal ions with imidazole groups and Zn 2+ The ability of methotrexate to coordinate with carboxyl groups was observed in experiments to allow it to competitively bind to Zn when the content of methotrexate in the system was increased to a certain proportion while the content of histidine modified with 9-fluorenylmethyloxycarbonyl was decreased. 2+ The active sites of Zn are then optimized, transforming the metal-amino acid structure into a metal-amino acid / drug structure, ultimately increasing the proportion of therapeutically capable structures within the nanoframework. Furthermore, under relatively mild conditions, Zn... 2+It can form coordination compounds with the imidazole group in histidine modified with 9-fluorenylmethyloxycarbonyl and the dicarboxyl group in methotrexate, and can further self-assemble to form nanoparticles. This invention utilizes this property to modify the histidine of the targeting peptide with 9-fluorenylmethyloxycarbonyl, enabling the formed nanoparticles to actively target inflammatory macrophages in the joints of RA. Furthermore, 5-aminosalicylic acid (5-ASA), which has reactive oxygen species scavenging capabilities, can be further encapsulated within this nano-drug delivery framework. By scavenging reactive oxygen species in the inflammatory microenvironment, the macrophage phenotype is repolarized from a pro-inflammatory to an anti-inflammatory phenotype. Drug delivery is administered via a microneedle patch, where the needle tip dissolves and releases the nanoparticles. These nanoparticles accumulate in the inflamed joint due to both active and passive targeting, achieving a safe and efficient treatment of RA based on the dual effects of scavenging inflammatory macrophages and reversing their phenotype.

[0028] Beneficial effects

[0029] CN114939178A only uses methotrexate as the encapsulated drug, resulting in a low drug loading (less than 10%); while microneedles, due to inherent defects, also have a drug loading (less than 10%), neither of which can meet clinical needs. This invention unexpectedly discovered that methotrexate (MTX) can be used both as a therapeutic drug and as one of the ligands, namely Zn. 2+ It can form coordination compounds with the imidazole group in histidine modified with 9-fluorenylmethyloxycarbonyl and the dicarboxyl group in methotrexate to form nanoparticles. These nanoparticles can then encapsulate other drugs to form microneedles, which can solve the technical defect of low drug loading in microneedles.

[0030] Specifically:

[0031] In CN114939178A, the organic ligands Fmoc-H and Fmoc-H-Tuftsin are mixed, followed by the addition of MTX, and then Zn is added to the system. 2+ After the solution reacts completely, an amino acid / peptide coordination polymer is formed; due to Zn 2+ First, it reacts with ligands Fmoc-H and Fmoc-H-Tuftsin, Zn 2+ It is an appropriate amount, not an excessive amount. At this time, MTX cannot participate in the formation of the skeleton, so MTX is just a drug encapsulation.

[0032] The preparation method of this invention differs from CN114939178A, specifically by reducing the amount of Fmoc-H and increasing the amount of MTX, thus eliminating excess Zn. 2+ MTX can simultaneously form coordination compounds with the imidazole group in histidine modified with 9-fluorenylmethyloxycarbonyl and the dicarboxyl group in methotrexate, and then self-assemble into nanoparticles. In this case, MTX is both a drug and one of the ligands.

[0033] Furthermore, by adjusting the ratio of the two ligands, this invention optimizes the metal-amino acid framework into a metal-amino acid / drug framework, which greatly improves the solubility, drug loading (>30%), and stability of the poorly soluble drug MTX, while also endowing the nanoframework with certain disease treatment potential.

[0034] The soluble microneedles based on metal-amino acid / peptide / drug coordination nanoparticles described in this invention indirectly improve the inherent defect of low drug loading capacity in microneedles by increasing the drug loading capacity within the nanoparticles. These soluble microneedles are biodegradable, capable of self-degradation in vivo, and exhibit good biocompatibility and safety. They also possess a certain degree of mechanical strength, enabling them to penetrate the stratum corneum and release drugs into the dermis, thus breaking down the skin barrier and reducing pain during medication administration.

[0035] The coordination polymer described in this invention can form nanoparticles with a particle size of 50.0-60.0 nm under neutral or weakly alkaline conditions, and accelerates drug release after cleavage under acidic conditions. The water solubility, stability, and biocompatibility of the coordination nanoparticles meet the requirements of drug delivery carriers, making them particularly suitable for the treatment of rheumatoid arthritis (RA). This invention demonstrates that metal-amino acid / peptide / drug coordination nanoparticles loaded with MTX and 5-ASA can be released from microneedle patches and further accumulate in inflamed joints through active and passive targeting. Based on the dual effects of clearing inflammatory macrophages and reversing the inflammatory macrophage phenotype through reactive oxygen species scavenging, safe and efficient treatment of RA can be achieved. Attached Figure Description

[0036] Figure 1 Figure 1 shows the hydrodynamic diameter (DLS) of metal-amino acid / drug coordination nanoparticles (ZFM) with different Fmoc-H and MTX feeding ratios prepared in Example 1 of this invention (Figure A), and the drug loading (DLC) (Figure B) and encapsulation efficiency (DLE) (Figure C) of MTX.

[0037] Figure 2 Figure 1 shows the DLS measurement results (Figure A) of metal-amino acid / drug coordination nanoparticles (ZFMA) with different 5-ASA feeding amounts prepared in Example 2 of the present invention, and the DLC (Figure B) and DLE (Figure C) measurement results of MTX and the DLC (Figure D) and DLE (Figure E) measurement results of 5-ASA.

[0038] Figure 3 Figure 1 shows the DLS measurement results (Figure A) and cellular uptake statistics (Figure B) of metal-amino acid / peptide / drug coordination nanoparticles (pZFM) with different Fmoc-H and Fmoc-HTKPR feed ratios prepared in Example 3 of the present invention.

[0039] Figure 4 Figure 1 shows the DLS (Figure A) and Zeta potential (Figure B) measurements of ZFM, ZFMA, and pZFMA prepared in Examples 1, 2, and 4 of this invention.

[0040] Figure 5 The images shown are Fourier transform infrared spectra of ZFM, ZFMA, and pZFMA prepared in Examples 1, 2, and 4 of this invention.

[0041] Figure 6 The image shown is a transmission electron microscope (TEM) image of the pZFMA prepared in Example 4 of this invention.

[0042] Figure 7 The image shows the X-ray diffraction pattern of pZFMA prepared in Example 4 of this invention.

[0043] Figure 8 The following is an evaluation of the cellular uptake of pZFMA prepared in Example 4 of the present invention, where A is the result of flow cytometry detection and B is the statistical result.

[0044] Figure 9 The cytotoxicity evaluation of ZFM, ZFMA and pZFMA prepared in Examples 1, 2 and 4 of this invention.

[0045] Figure 10 The following is an evaluation of the in vitro reactive oxygen species scavenging capacity of pZFMA prepared in Example 4 of the present invention, where A is the result of flow cytometry detection and B is the statistical result. Detailed Implementation

[0046] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. It should be understood that these embodiments are merely illustrative and not intended to limit the scope of the invention in any way. Various processes and methods not described in detail in the following embodiments are conventional methods known in the art. Unless otherwise specified, the materials, reagents, apparatuses, instruments, equipment, etc., used in the following examples are commercially available.

[0047] 9-fluorenylmethyloxycarbonyl modified histidine (Fmoc-H) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0048] The macrophage-targeting peptide (Fmoc-HTKPR) modified with a 9-fluorenylmethyloxycarbonyl histidine derivative was purchased from Shanghai Xishi Biotechnology Co., Ltd.

[0049] Methotrexate (MTX) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0050] Zinc nitrate hexahydrate was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0051] 5-Aminosalicylic acid (5-ASA) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0052] This invention constructs a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles. The coordination nanoparticles exhibit high water solubility, stability, and biocompatibility, and demonstrate a universal loading capacity for various drugs. The soluble microneedles loaded with MTX and 5-ASA based on metal-amino acid / peptide / drug coordination nanoparticles are suitable for in vivo anti-RA therapy. This invention demonstrates the anti-RA therapeutic potential of this soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles through in vitro characterization, drug loading capacity determination, cellular uptake studies, and in vitro efficacy studies.

[0053] Example 1: Construction of metal-amino acid / drug coordination nanoparticles (ZFM) with different Fmoc-H and MTX feed ratios.

[0054] First, accurately weigh the required amount of Fmoc-H and dissolve it in 0.07M hydrochloric acid to prepare a 0.026M Fmoc-H solution; then prepare 0.01M and 1M aqueous solutions of tris(hydroxymethyl)aminomethane (Tris) and 0.01M aqueous solution of zinc nitrate hexahydrate; finally, accurately weigh the required amount of MTX and dissolve it in 0.01M Tris buffer to prepare a 1 mg / mL MTX solution.

[0055] Under magnetic stirring, appropriate amounts of water, Fmoc-H solution, and MTX solution were mixed in a reaction flask and stirred thoroughly. Then, zinc nitrate hexahydrate aqueous solution was slowly added to maintain a mass ratio of Fmoc-H to MTX of 1:1–9 in the reaction system, while keeping the first ligand and Zn... 2+ The molar ratio was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to bring the pH to 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, purified by dialyzing, and stored at 4°C for subsequent experiments.

[0056] Characterization of the hydrodynamic diameter (DLS):

[0057] Take 1 mL of freshly prepared ZFM solutions with different Fmoc-H and MTX feeding ratios from Example 1 and perform DLS measurements on them using a particle size potentiometer.

[0058] DLS measurement results ( Figure 1 A) indicates that when the ratio of Fmoc-H to MTX is 1:9 to 4:6, the particle size of ZFM is less than 200 nm, which meets the size requirements for in vivo uptake of nanoparticles; among them, when the ratio of Fmoc-H to MTX is 3:7, the particle size of ZFM is the smallest.

[0059] Characterization of MTX drug loading (DLC) and encapsulation efficiency (DLE):

[0060] Take appropriate amounts of freshly prepared ZFM solutions with different Fmoc-H and MTX feed ratios from Example 1, freeze-dry them to prepare powders, dissolve them in anhydrous dimethyl sulfoxide by ultrasonication, filter the powders, and determine the DLC and DLE of MTX in a high-performance liquid chromatograph.

[0061] MTX DLC and DLE measurement results ( Figure 1 B and 1C) indicate that when the ratio of Fmoc-H to MTX is 3:7, the DLC (58.1%) and DLE (76.0%) of MTX are both at their highest.

[0062] Based on the above characterization results, the optimal feed ratio of Fmoc-H to MTX is 3:7.

[0063] Example 2: Construction of metal-amino acid / drug coordination nanoparticles (ZFMA) with different amounts of 5-ASA.

[0064] First, accurately weigh the required amount of Fmoc-H and dissolve it in 0.07M hydrochloric acid to prepare a 0.026M Fmoc-H solution; weigh the required amount of 5-ASA and dissolve it in 0.01M hydrochloric acid to prepare a 2 mg / mL 5-ASA solution; then prepare 0.01M and 1M aqueous solutions of tris(hydroxymethyl)aminomethane (Tris) and 0.01M aqueous solution of zinc nitrate hexahydrate; finally, accurately weigh the required amount of MTX and dissolve it in 0.01M Tris buffer to prepare a 1 mg / mL MTX solution.

[0065] Under magnetic stirring, appropriate amounts of water, Fmoc-H solution, MTX solution, and 5-ASA solution were mixed in a reaction flask and stirred thoroughly. Then, zinc nitrate hexahydrate aqueous solution was slowly added to maintain a Fmoc-H to MTX mass ratio of 3:7 and a 5-ASA feed mass of 0.6 mg to 3.0 mg, while keeping the first ligand and Zn... 2+ The molar ratio was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to bring the pH to 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, purified by dialyzing, and stored at 4°C for subsequent experiments.

[0066] DLS characterization:

[0067] Take 1 mL of freshly prepared ZFMA solutions with different 5-ASA feed amounts from Example 2 and perform DLS measurements on them using a particle size potentiometer.

[0068] DLS measurement results ( Figure 2A) indicates that as the amount of 5-ASA added to the system gradually increases, the particle size of ZFMA gradually increases. When the amount of 5-ASA added is 1.8 mg or less, the particle size of ZFMA is less than 200 nm, which meets the size requirements for in vivo uptake of nanoparticles. Therefore, based on the DLS results, the amount of 5-ASA added is preferably 1.8 mg or less.

[0069] DLC and DLE characterization of MTX and 5-ASA:

[0070] Take appropriate amounts of ZFMA solutions with different 5-ASA feed amounts prepared in Example 2, freeze-dry them to make powders, dissolve them in anhydrous dimethyl sulfoxide and potassium dihydrogen phosphate solution (pH=3) by ultrasonication, filter the membranes, and determine the DLC and DLE of MTX and 5-ASA by high performance liquid chromatography.

[0071] Based on DLS results, the optimal dosage of 5-ASA is 1.8 mg or less. Specifically, when the dosage of 5-ASA is 1.2 mg, the DLC of both MTX and 5-ASA reaches its highest values ​​(67.1% and 5.4%, respectively). Figure 2 B and 2D), while the DLE of MTX and 5-ASA showed no significant difference among the three groups ( Figure 2 (C and 2E).

[0072] Based on the above characterization results, the preferred dosage of 5-ASA is 1.2 mg.

[0073] Example 3: Construction of metal-amino acid / peptide / drug coordination nanoparticles (pZFM) with different Fmoc-H and Fmoc-HTKPR feed ratios and FITC-loaded pZFM-FITC.

[0074] First, accurately weigh the required amount of Fmoc-H and dissolve it in 0.07M hydrochloric acid to prepare a 0.026M Fmoc-H solution; then prepare a 10 mg / mL Fmoc-HTKPR aqueous solution, an 80 μg / mL FITC aqueous solution, 0.01M and 1M tris(hydroxymethyl)aminomethane (Tris) aqueous solutions, and a 0.01M zinc nitrate hexahydrate aqueous solution; finally, accurately weigh the required amount of MTX and dissolve it in 0.01M Tris buffer to prepare a 1 mg / mL MTX solution.

[0075] Under magnetic stirring, appropriate amounts of water, Fmoc-H solution, Fmoc-HTKPR solution, and MTX solution were mixed in a reaction flask and stirred thoroughly. Then, zinc nitrate hexahydrate aqueous solution was slowly added to maintain a Fmoc-H to Fmoc-HTKPR mass ratio of 1:0–0.3 (Fmoc-HTKPR feed percentage of 0%–10%) and a Fmoc-H to MTX mass ratio of 3:7, while maintaining the first ligand and Zn... 2+ The molar ratio was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to bring the pH to 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, purified by dialyzing, and stored at 4°C for subsequent experiments.

[0076] Similar to the preparation method described above, to prepare FITC-loaded pZFM-FITC, an additional 0.05 μM of FITC needs to be added to pZFM.

[0077] DLS characterization:

[0078] Take 1 mL of each of the freshly prepared pZFM solutions with different Fmoc-H and Fmoc-HTKPR feed ratios from Example 3, and perform DLS measurements on them using a particle size potentiometer.

[0079] DLS measurement results ( Figure 3 A) indicates that as the Fmoc-HTKPR feed ratio increases, the particle size of pZFM gradually increases. When the Fmoc-H and Fmoc-HTKPR feed ratio is between 1:0 and 0.15 (i.e., the Fmoc-HTKPR feed percentage is 5% or less), the particle size of pZFM is less than 200 nm, which meets the size requirements for in vivo uptake of nanoparticles. Therefore, based on the DLS results, the preferred Fmoc-H and Fmoc-HTKPR feed ratio is 1:0 to 0.15 (i.e., the Fmoc-HTKPR feed percentage is 5% or less).

[0080] Cellular uptake experiment:

[0081] RAW264.7 cells were routinely cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. RAW264.7 cells were incubated at 37°C and 5% CO2. After passage in the logarithmic growth phase, cells were seeded in 6-well plates (5 × 10⁻⁶ cells / well). 5Inflammatory macrophages were obtained by stimulating and activating them with 100 ng / mL LPS for 24 h. The culture medium was removed, and pZFM-FITC at a dose of 0.05 μM was incubated for 4 h with different Fmoc-H and Fmoc-HTKPR feed ratios. The culture medium was then removed, and the cells were washed three times with PBS and centrifuged at 350 g for 5 min. The centrifuged cells were redispersed in PBS for flow cytometry analysis. For each sample, the uptake differences of RAW264.7 cells between different formulations were compared using a single-channel FITC assay.

[0082] Cellular uptake results ( Figure 3 B) indicates that when the Fmoc-H to Fmoc-HTKPR feed ratio is between 1:0 and 0.15 (i.e., the Fmoc-HTKPR feed percentage is 5% or less), cellular uptake gradually increases with the increase of the Fmoc-HTKPR feed ratio. However, when the Fmoc-H to Fmoc-HTKPR feed ratio is 1:0.3 (i.e., the Fmoc-HTKPR feed percentage is 10%), cellular uptake decreases significantly. This may be because the pZFM particle size is too large at this point, which is not conducive to cellular uptake.

[0083] Based on the above characterization results, the preferred feed ratio of Fmoc-H and Fmoc-HTKPR is 1:0.15 (i.e., the feed percentage of Fmoc-HTKPR is 5%).

[0084] Example 4: Construction of metal-amino acid / peptide / drug coordination nanoparticles (pZFMA) loaded with 5-ASA.

[0085] First, accurately weigh the required amount of Fmoc-H and dissolve it in 0.07M hydrochloric acid to prepare a 0.026M Fmoc-H solution; weigh the required amount of 5-ASA and dissolve it in 0.01M hydrochloric acid to prepare a 2 mg / mL 5-ASA solution; then prepare a 10 mg / mL Fmoc-HTKPR aqueous solution, 0.01M and 1M tris(hydroxymethyl)aminomethane (Tris) aqueous solutions, and a 0.01M zinc nitrate hexahydrate aqueous solution; finally, accurately weigh the required amount of MTX and dissolve it in 0.01M Tris buffer to prepare a 1 mg / mL MTX solution.

[0086] Under magnetic stirring, appropriate amounts of water, Fmoc-H solution, Fmoc-HTKPR solution, MTX solution, and 5-ASA solution were mixed in a reaction flask and stirred thoroughly. Then, zinc nitrate hexahydrate aqueous solution was slowly added to maintain a Fmoc-H to Fmoc-HTKPR mass ratio of 1:0.15 (i.e., Fmoc-HTKPR feed percentage of 5%), a Fmoc-H to MTX mass ratio of 3:7, and a 5-ASA feed mass of 1.2 mg, while maintaining the first ligand and Zn...2+ The molar ratio was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to bring the pH to 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, purified by dialyzing, and stored at 4°C for subsequent experiments.

[0087] DLS and Zeta potential characterization:

[0088] Take 1 mL each of freshly prepared ZFM, ZFMA and pZFMA from Examples 1, 2 and 4, and measure the zeta potential of DLS on them using a particle size potentiometer.

[0089] DLS test results ( Figure 4 A) shows that the hydrodynamic diameter of ZFM is 105.7 nm, that of ZFMA is 168.5 nm, and that of pZFMA is 177.8 nm. The increase in the hydrodynamic diameter of ZFMA and pZFMA is due to the doping of 5-ASA and Fmoc-HTKPR in the system.

[0090] Zeta potential test results ( Figure 4 B) shows that the surface potential of ZFM is -29.2 mV; the surface potential of ZFMA is -33.0 mV; and the surface potential of pZFMA is -29.5 mV. The increase in potential is due to the presence of positively charged arginine in the Fmoc-HTKPR sequence.

[0091] Fourier transform infrared spectroscopy (FT-IR) characterization:

[0092] Take 2 mg each of the raw materials Fmoc-H, MTX, 5-ASA, freshly prepared ZFM, ZFMA, and pZFMA freeze-dried powders from Examples 1, 2, and 4, as well as the physical mixture powders of each raw material according to the feeding ratio, grind them with an appropriate amount of KBr, and then perform Fourier transform infrared absorption measurement by tablet compression.

[0093] FT-IR results ( Figure 5 The results showed that 1697 cm was observed in ZFM, ZFMA, and pZFMA. -1 Fmoc-H and Zn 2+ Stretching vibration of C=O after coordination, 1396 cm -1 MTX and Zn 2+ The -OH peak on the coordinated carboxyl group. Additionally, a peak at 1652 cm⁻¹ was observed in both ZFMA and pZFMA. -1 The stretching vibration of the C=O group on the carboxyl group in 5-ASA was observed. In the physically mixed powder, the -OH group on the -COOH group in MTX showed a stretching vibration at 1406 cm⁻¹. -1 The characteristic peak at this location indicates that MTX is not associated with Zn. 2+ Coordination occurs.

[0094] Transmission electron microscopy (TEM) characterization:

[0095] The freshly prepared pZFMA from Example 4 was diluted by a certain factor and dropped onto a copper grid. After air drying, the morphology and size of the material were characterized by a transmission electron microscope.

[0096] TEM results ( Figure 6 The results show that pZFMA has a good spherical morphology with a particle size of 50.0-60.0 nm.

[0097] X-ray diffraction (XRD) characterization:

[0098] The freshly prepared pZFMA solution from Example 4 was cooled and dried to form a powder, which was then ground until it was free of particles. XRD was used to analyze its crystal form, with a scanning angle range of 5-90°, a scanning step size of 0.02° / step, a scanning speed of 0.03° / s, and a scanning time of 10 min.

[0099] XRD results ( Figure 7 The data shows that pZFMA has a characteristic amorphous diffraction peak near the 2theta angle of 20°, indicating that it has an amorphous morphology.

[0100] Example 5: Cellular uptake study of metal-amino acid / peptide / drug coordination nanoparticles (pZFMA) loaded with 5-ASA.

[0101] Following the synthesis steps of FITC-loaded pZFM in Example 3, in Example 4, 5-ASA was replaced with 0.05 μM FITC to obtain nanoparticles.

[0102] RAW264.7 cells were routinely cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. RAW264.7 cells were incubated at 37°C and 5% CO2. After passage in the logarithmic growth phase, cells were seeded in 6-well plates (5 × 10⁶ cells / well). 5 Inflammatory macrophages were obtained by stimulating and activating the cells with 100 ng / mL LPS for 24 h in ( / well). The culture medium was removed, and the cells were incubated with nanoparticles at a FITC dose of 0.05 μM for 0, 2, 4, 8, and 12 h, respectively. The culture medium was then removed, and the cells were washed three times with PBS and centrifuged at 350 g for 5 min. The centrifuged cells were redispersed in PBS for flow cytometry analysis. For each sample, the uptake differences of the same formulation in RAW264.7 cells at different time points were compared using a single-channel FITC assay.

[0103] Flow cytometry results ( Figure 8The results showed that RAW264.7 cells had the highest cellular uptake of nanoparticles after 8 hours of culture, indicating that metal-(amino acid / peptide / drug) coordination nanoparticles are highly efficient drug delivery carriers for inflammatory macrophages.

[0104] Example 6: Cytotoxicity evaluation of metal-amino acid / peptide / drug coordination nanoparticles (pZFMA) loaded with 5-ASA.

[0105] RAW264.7 cells were routinely cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. RAW264.7 cells were incubated at 37°C and 5% CO2. After passage in the logarithmic growth phase, cells were seeded in 96-well plates (5 × 10⁶ cells / well). 3 Cells were cultured in each well for 12 hours until adherence was observed. Inflammatory macrophages were obtained by stimulation with 100 ng / mL LPS for 24 hours. The culture medium was removed, and the cells were cultured in DMEM containing different concentrations of ZFM, ZFMA, and pZFMA (MTX) for 24 hours. The culture medium was then removed, and the cells were washed twice with PBS. The cells were then cultured in DMEM containing 0.5 mg / mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) for 4 hours. The supernatant was then removed, and 100 μL of DMSO was added to each well to dissolve the purple crystals. The absorbance of the purple crystal product at 490 nm was measured using a microplate reader. Three samples were used per group. Cell viability was calculated using the following formula: Cell viability (%) = (sample - A0) / (control group - A0) × 100%. A0 is the absorbance of the blank well at 490 nm.

[0106] Cytotoxicity results ( Figure 9 The results showed that ZFM (IC50 of 56.17 μg / mL) and ZFMA (IC50 of 58.00 μg / mL) exhibited similar cytotoxicity, which was due to the lower 5-ASA loading in ZFMA; while pZFMA (IC50 of approximately 44.19 μg / mL) showed the greatest cytotoxicity, which was due to the presence of the targeting peptide increasing the cellular uptake of pZFMA by inflammatory macrophages.

[0107] Example 7 Evaluation of the in vitro reactive oxygen species scavenging capacity of metal-amino acid / peptide / drug coordination nanoparticles (pZFMA) loaded with 5-ASA.

[0108] RAW264.7 cells were routinely cultured in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin. RAW264.7 cells were incubated at 37°C and 5% CO2. After passage in the logarithmic growth phase, cells were seeded in 6-well plates (5 × 10⁶ cells / well). 5Inflammatory macrophages were obtained by stimulating and activating them with 100 ng / mL LPS for 24 h in ( / well). The culture medium was removed, and the cells were cultured in DMEM medium containing the same concentrations of ZFM, ZFMA, and pZFMA for 24 h. The culture medium was then removed, and the cells were washed three times with PBS. DCFH-DA probe dilution buffer (probe stock solution to incomplete culture medium volume ratio of 1:1000) was added, and the cells were incubated in the dark for 30 min. The culture medium was removed, and the cells were washed three times with PBS. The cells were collected in PBS, centrifuged at 350 g for 5 min, and then redispersed in PBS for flow cytometry analysis. Cells without LPS stimulation and cells without drug administration were used as controls. The differences in intracellular reactive oxygen species (ROS) levels between different formulations were compared using a FITC single-channel assay.

[0109] Results of reactive oxygen species scavenging capacity ( Figure 10 The results showed that LPS stimulation significantly increased intracellular reactive oxygen species (ROS) levels. Both ZFM and ZFMA were able to kill inflammatory macrophages by loading MTX, thereby reducing intracellular ROS levels. However, the ZFMA group, due to the loading of 5-ASA, exhibited a further enhanced ability to scavenge intracellular ROS. pZFMA showed the strongest ability to scavenge intracellular ROS due to increased cellular uptake caused by the presence of the targeting peptide.

[0110] Example 8: Preparation of a soluble microneedle patch (pZFMA-MN) based on metal-amino acid / peptide / drug coordination nanoparticles (pZFMA) loaded with 5-ASA.

[0111] Take an appropriate amount of the freshly prepared pZFMA solution from Example 4, mix it thoroughly with the PVP solution, add it to the microneedle mold, centrifuge to distribute the nanoparticles on the needle tip layer, and dry it overnight to obtain a soluble microneedle patch based on metal-(amino acid / peptide / drug) coordination nanoparticles loaded with 5-ASA.

Claims

1. A soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles, characterized in that, The coordination center ion of the nanoparticles is selected from Zn. 2+ The first ligand is selected from a mixture of macrophage-targeting peptide Fmoc-H modified with 9-fluorenylmethyloxycarbonyl histidine Fmoc-H and a derivative of 9-fluorenylmethyloxycarbonyl histidine; the second ligand is selected from methotrexate; the microneedle carrier material is selected from PVP; the microneedles are prepared by the following steps: The macrophage-targeting peptide Fmoc-H, modified with 9-fluorenylmethyloxycarbonyl histidine, was mixed with Fmoc-HTKPR, a derivative of 9-fluorenylmethyloxycarbonyl histidine, and methotrexate, and then Zn was added. 2+ The pH of the reaction system was adjusted to a slightly alkaline state under stirring, and metal-amino acid / peptide / drug coordination nanoparticles were formed by self-assembly. The coordination nanoparticles were mixed with PVP solution and filled into a microneedle mold. After centrifugation and overnight drying, microneedle patches were obtained.

2. The soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles as described in claim 1, characterized in that, The molar ratio of the coordination center ion to the first ligand is 1:2.2, and the mass ratio of the first ligand to the second ligand is 1:1~9.

3. The soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles as described in claim 1, characterized in that, The macrophage-targeting peptide Fmoc-H, modified with 9-fluorenylmethyloxycarbonyl histidine, and the macrophage-targeting peptide Fmoc-HTKPR, modified with a derivative of 9-fluorenylmethyloxycarbonyl histidine, were mixed at a mass ratio of 1:0~0.

3.

4. The application of a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles as described in any one of claims 1-3 in the preparation of a drug delivery carrier; wherein the drug delivery carrier carries a drug or probe; and wherein the drug carried is 5-aminosalicylic acid.

5. The application as described in claim 4, characterized in that, The feed mass of 5-ASA is 0.6 mg to 3.0 mg.

6. The application of the soluble microneedles based on metal-amino acid / peptide / drug coordination nanoparticles as described in claim 4 in anti-rheumatoid arthritis drugs.

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