Soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles as well as preparation method and application of soluble microneedle

Abstract

The invention belongs to microneedles, and particularly relates to a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles as well as a preparation method and application of the soluble microneedle. The coordination center ion of the nanoparticle is selected from Zn < 2 + >, and the ligand is selected from a mixture formed by 9-fluorenyl methyloxycarbonyl modified histidine (Fmoc-H) and macrophage targeting peptide (Fmoc-HTKPR) modified by a derivative of the 9-fluorenyl methyloxycarbonyl modified histidine and methotrexate (MTX) which contains a carboxyl group and is used for treating rheumatoid arthritis. The microneedle carrier material is selected from a soluble polymer PVP. The invention provides a scheme capable of realizing metal-drug coordination at normal temperature, nano-particles are dissolved and released in vivo through a microneedle, and the nano-particles can actively target an inflammation site through a targeting peptide and responsively release high-load MTX and an active oxygen scavenging drug, so that the aim of eliminating inflammation is fulfilled. Based on the synergistic effect of eliminating inflammatory macrophages and converting the inflammatory macrophages into anti-inflammatory phenotypes, efficient and safe treatment of rheumatoid arthritis is realized.

Description

Technical Field

[0001] The present invention belongs to microneedles, and specifically relates to a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles and a preparation method and application thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a common chronic, infectious and autoimmune disease, known as the "immortal cancer". Its clinical manifestations include joint swelling, synovial inflammation, and obvious cartilage damage and bone erosion. As the disease worsens, it can also cause multi-organ complications outside the joints. RA in my country is characterized by a large number of patients, many delays in diagnosis and treatment, many severe cases, and many complications, and the incidence rate is trending towards younger age. Due to the complex pathogenesis of RA, there is no complete cure, and its long course of disease and high disability rate have become a major public health issue that needs to be addressed urgently. Currently, the widely recognized 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 and its preparation method and application, discloses a metal-amino acid / peptide coordination polymer that can treat 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 9-Fmoc-protected histidine, a macrophage targeting peptide modified by a derivative of 9-Fmoc-protected histidine, or a mixture of 9-Fmoc-protected histidine and 9-Fmoc-protected macrophage targeting peptide, and the peptide contains a Tuftsin sequence. Based on the 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 nanoparticles with similar metal-organic ligand structures is always limited (<10%). The treatment method proposed in this patent is intravenous administration. Long-term injection will not only cause pain, but also greatly reduce the patient's tolerance and medication compliance.

[0004] Microneedles (MN) are a new type of physical penetration-enhancing technology, consisting of multiple micron-sized tiny needle tips connected in an array on a base. The needle body is generally 10-2000 microns high and 10-50 microns wide. The length, size and shape of the microneedles can be individually designed according to the needs of treatment. Microneedles can pass through the stratum corneum in a directional manner, produce micron-sized mechanical channels, and place drugs directly in the epidermis or upper dermis. They can participate in microcirculation and exert pharmacological reactions without passing through the stratum corneum. As an emerging transdermal drug delivery method, the biggest advantage of microneedles compared to traditional patches is that they can penetrate the stratum corneum and break the skin barrier. However, due to volume limitations, all types of microneedles have the problem of low drug loading.

[0005] Therefore, in order to obtain better therapeutic effects and overcome the technical defects of the prior art such as low drug loading of amino acid / polypeptide coordination polymers or microneedles, the present invention provides a new type of microneedle. Summary of the invention

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

[0007] After mixing 9-fluorenylmethyloxycarbonyl-modified histidine Fmoc-H, 9-fluorenylmethyloxycarbonyl-modified histidine derivative-modified macrophage targeting peptide Fmoc-HTKPR and methotrexate, Zn was added. 2+ , adjust the pH value of the reaction system to a weak alkaline state under stirring, and self-assemble to form metal-amino acid / peptide / drug coordination nanoparticles. The coordination nanoparticles are mixed with the PVP solution and filled into the microneedle mold, centrifuged, and dried overnight to obtain a microneedle patch.

[0008]

[0009] Zn 2+ The structural formula after coordination with MTX

[0010] The soluble microneedle based on metal-amino acid / peptide / drug coordinated nanoparticles is characterized in that the molar ratio of the coordinated central 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 1:1-9.

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

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

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

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

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

[0016] The application of the soluble microneedle preparation 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, wherein Fmoc-H, Fmoc-HTKPR and MTX are reacted with Zn 2+ After mixing, adjust the pH value of the reaction system to neutral to weakly alkaline (preferably pH range 7-8), and stir until the metal-amino acid / peptide / drug coordination polymer self-assembles to form nanoparticles. After the coordinated nanoparticles are mixed with the PVP solution, they are filled into the microneedle mold, centrifuged, and dried overnight to obtain a microneedle patch.

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

[0020] As a preferred method

[0021] (1) Fmoc-H and Fmoc-HTKPR were uniformly mixed at a mass ratio of 1:0.15 (the feed percentage of Fmoc-HTKPR was 5%), and then the second ligand (MTX) solution was added to the system at a mass ratio of Fmoc-H and MTX of 3:7.2+ Add zinc nitrate hexahydrate aqueous solution 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 functions, adjust the pH value of the solution to neutral to weak alkaline (preferably pH range of 7-8) under vigorous stirring, and induce the formation of nanoparticles until the particle growth is completed. The metal-amino acid / peptide / drug coordination nanoparticles loaded with the substrate are obtained by dialysis purification. After further uniformly mixing the nanoparticle solution with the PVP solution, it is placed in a microneedle mold, centrifuged, and dried overnight to obtain soluble microneedles loaded with metal-amino acid / peptide / drug coordination nanoparticles.

[0023] Another object of the present invention is to use a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles as a carrier for carrying drugs or probes.

[0024] Specifically, the drug is one or more of a small molecule compound, a polypeptide, a protein, a polysaccharide, and a nucleic acid. Further, the drug is 5-aminosalicylic acid (5-ASA). The coordinated nanoparticles of the present invention are co-incubated with an appropriate amount of 5-ASA to obtain coordinated nanoparticles loaded with different masses of 5-ASA.

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

[0026] The present invention mainly considers the following four aspects: ① Designing a nano drug loading framework with its own targeting and therapeutic capabilities, and improving the drug accumulation in the lesion site through the dual effects of active and passive targeting. ② Improving the unstable nature of the metal-drug coordination bond, by doping the metal-amino acid strong interaction coordination structure into the metal-drug coordination structure to improve the stability of the metal-amino acid / drug coordination structure and the drug loading capacity. ③ By increasing the drug loading capacity of the nanoparticles, indirectly improving the inherent defect of low drug loading capacity of microneedles. ④ Through the microneedle drug delivery method, pain is reduced and the patient's medication compliance is improved.

[0027] The present invention utilizes the strong coordination ability of metal ions and imidazole groups and Zn 2+ The ability to coordinate with carboxyl groups. In experiments, it was found that when the content of methotrexate in the system was increased to a certain proportion and the content of 9-fluorenylmethyloxycarbonyl-modified histidine was reduced, methotrexate could compete with 9-fluorenylmethyloxycarbonyl-modified histidine to bind Zn 2+ active sites, the metal-amino acid structure is optimized to the metal-amino acid / drug structure, which ultimately increases the proportion of structures with therapeutic ability in the nanoframe. 2+It can form coordination compounds with the imidazole group in the histidine modified by 9-fluorenylmethyloxycarbonyl and the dicarboxyl group in methotrexate, and can further undergo self-assembly to form nanoparticles. The present invention utilizes this characteristic, and modifies the histidine modified by 9-fluorenylmethyloxycarbonyl to the end of the targeting peptide, so that the formed nanoparticles have the ability to actively target inflammatory macrophages in RA diseased joints. On the other hand, 5-aminosalicylic acid (5-ASA) with active oxygen scavenging ability can be further encapsulated in the nano drug delivery framework, and the macrophage phenotype is repolarized from a pro-inflammatory phenotype to an anti-inflammatory phenotype by clearing the active oxygen in the inflammatory microenvironment. Through microneedle patch administration, the needle tip dissolves and releases the nanoparticles, which accumulate in the inflamed joints depending on the active and passive targeting effects of the nanoparticles, and based on the dual effects of clearing inflammatory macrophages and reversing their phenotypes, a safe and efficient treatment of RA is achieved.

[0028] Beneficial Effects

[0029] CN114939178A only uses methotrexate as a drug carrier, and its drug loading is low (less than 10%); and the drug loading of microneedles is also (less than 10%) due to inherent defects, and both cannot meet clinical needs. The present invention unexpectedly discovered that methotrexate (MTX) can be used as a therapeutic drug and as one of the ligands, namely Zn 2+ It can form coordination compounds with the imidazole group in 9-fluorenylmethyloxycarbonyl-modified histidine and the dicarboxyl group in methotrexate to self-assemble into nanoparticles. The nanoparticles can be loaded with other drugs to make microneedles, which can solve the technical defect of low drug loading capacity of microneedles.

[0030] Specifically:

[0031] In CN114939178A, the organic ligands Fmoc-H and Fmoc-H-Tuftsin are mixed, MTX is added, and then Zn is added to the system. 2+ solution, and after the reaction is complete, an amino acid / polypeptide coordination polymer is formed; due to Zn 2+ First react with the ligand Fmoc-H, 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 only encapsulates the drug.

[0032] The preparation method of the present invention is different from that of CN114939178A, that is, the feed amount of Fmoc-H is reduced and the feed amount of MTX is increased. 2+ It can simultaneously form coordination compounds with the imidazole group in histidine modified with 9-fluorenylmethyloxycarbonyl and the dicarboxyl group in methotrexate to self-assemble into nanoparticles. At this time, MTX is both a drug and one of the ligands.

[0033] Furthermore, the present invention optimizes the metal-amino acid framework to a metal-amino acid / drug framework by adjusting the ratio of the two ligands, thereby greatly improving the solubility, drug loading (>30%) and stability of the poorly soluble drug MTX, while giving the nanoframework a certain disease treatment potential.

[0034] The soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles of the present invention indirectly improves the inherent defect of low drug loading of microneedles by increasing the drug loading of the nanoparticles. The soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles of the present invention is a biodegradable microneedle that can self-degrade in the body and has good biocompatibility and safety. It also has a certain mechanical strength, can penetrate the stratum corneum, and release drugs in the dermis, breaking the skin barrier and reducing the pain of medication.

[0035] The coordination polymer of the present invention can form nanoparticles under neutral or weakly alkaline conditions, with a particle size of 50.0-60.0nm, and accelerates the release of drugs after cleavage under acidic conditions. The water solubility, stability and biosafety of the coordination nanoparticles meet the requirements of drug delivery carriers and are particularly suitable for the treatment of RA. The present invention has shown that the metal-amino acid / peptide / drug coordination nanoparticles loaded with MTX and 5-ASA can be released from the microneedle patch, and can further accumulate in the inflamed joints through active and passive targeting, based on the dual effects of clearing inflammatory macrophages and reversing the phenotype of inflammatory macrophages through active oxygen scavenging, to achieve safe and efficient treatment of RA. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0038] Figure 3 , DLS measurement results (Figure A) and cellular uptake statistical results (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 , are the DLS (Figure A) and Zeta potential (Figure B) measurement results of ZFM, ZFMA and pZFMA prepared in Examples 1, 2 and 4 of the present invention.

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

[0041] Figure 6 , is a transmission electron micrograph of pZFMA prepared in Example 4 of the present invention.

[0042] Figure 7 , is the X-ray diffraction pattern of pZFMA prepared in Example 4 of the present invention.

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

[0044] Fig. 9 , the cytotoxicity evaluation of ZFM, ZFMA and pZFMA prepared in Examples 1, 2 and 4 of the present invention.

[0045] Fig.10 , is the in vitro reactive oxygen scavenging ability evaluation of pZFMA prepared in Example 4 of the present invention, wherein A is the flow cytometry detection result and B is the statistical result. DETAILED DESCRIPTION

[0046] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that this embodiment is only for the purpose of illustrating the present invention, and does not limit the scope of the present invention in any way. In the following examples, the various processes and methods not described in detail are conventional methods well known in the art. The materials, reagents, devices, instruments, equipment, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

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

[0048] The macrophage targeting peptide modified with a derivative of histidine modified with 9-fluorenylmethyloxycarbonyl (Fmoc-HTKPR) 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] The present invention constructs a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles, wherein the coordinated nanoparticles have high water solubility, stability and biocompatibility, and show universal loading capacity for different types of drugs. The soluble microneedles of metal-amino acid / peptide / drug coordination nanoparticles loaded with MTX and 5-ASA are suitable for in vivo anti-RA treatment. The present invention demonstrates the anti-RA therapeutic potential of the soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles by in vitro characterization, drug loading capacity determination, cell uptake research and in vitro efficacy research.

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

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

[0055] Under magnetic stirring, mix appropriate amount of water, Fmoc-H solution and MTX solution in a reaction bottle and stir thoroughly. Then slowly add zinc nitrate hexahydrate aqueous solution to make the mass ratio of Fmoc-H and MTX in the reaction system 1:1-9, and keep the first ligand and Zn 2+ The molar ratio of 2.2:1 was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to make the solution pH reach 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, dialyzed for purification, and stored in a refrigerator at 4°C for subsequent experiments.

[0056] Hydrodynamic diameter (DLS) characterization:

[0057] 1 mL of each of the freshly prepared ZFM solutions of Example 1 with different Fmoc-H and MTX feed ratios was taken and subjected to DLS measurement on a particle size potential analyzer.

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

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

[0060] Appropriate amounts of the freshly prepared ZFM solutions of Example 1 with different Fmoc-H and MTX feed ratios were freeze-dried to prepare powders, dissolved by ultrasonication in anhydrous dimethyl sulfoxide, and filtered through a membrane. The DLC and DLE of MTX were determined by a high performance liquid chromatograph.

[0061] The DLC and DLE results of MTX ( Figure 1 B and 1C) show that when the feed ratio of Fmoc-H and MTX is 3:7, the DLC (58.1%) and DLE (76.0%) of MTX reach the highest.

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

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

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

[0065] Under magnetic stirring, mix appropriate amounts of water, Fmoc-H solution, MTX solution and 5-ASA solution in a reaction bottle and stir thoroughly. Then slowly add zinc nitrate hexahydrate aqueous solution to make the mass ratio of Fmoc-H and MTX in the reaction system 3:7, the feed mass of 5-ASA 0.6mg~3.0mg, and keep the first ligand and Zn 2+ The molar ratio of 2.2:1 was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to make the solution pH reach 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, dialyzed for purification, and stored in a refrigerator at 4°C for subsequent experiments.

[0066] DLS Characterization:

[0067] Take 1 mL of the freshly prepared ZFMA solutions with different 5-ASA dosages in Example 2 and perform DLS measurement on them on a particle size potentiometer.

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

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

[0070] Appropriate amounts of freshly prepared ZFMA solutions with different 5-ASA dosages in Example 2 were freeze-dried to prepare powders, which were ultrasonically dissolved with anhydrous dimethyl sulfoxide and potassium dihydrogen phosphate solution (pH=3), respectively. The membranes were filtered, and the DLC and DLE of MTX and 5-ASA were determined in a high performance liquid chromatograph, respectively.

[0071] Based on the DLS results, the optimal dosage of 5-ASA was 1.8 mg or less. When the dosage of 5-ASA was 1.2 mg, the DLC of MTX and 5-ASA reached the highest values ​​(67.1% and 5.4%) ( Figure 2 B and 2D), while the DLE of MTX and 5-ASA had 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, the required Fmoc-H was accurately weighed and dissolved in 0.07 M hydrochloric acid to prepare a 0.026 M Fmoc-H solution; then, a 10 mg / mL Fmoc-HTKPR aqueous solution, an 80 μg / mL FITC aqueous solution, 0.01 M and 1 M tris(hydroxymethyl)aminomethane (Tris) aqueous solutions, and a 0.01 M zinc nitrate hexahydrate aqueous solution were prepared; finally, the required MTX was accurately weighed and dissolved in 0.01 M Tris buffer to prepare a 1 mg / mL MTX solution.

[0075] Under magnetic stirring, mix appropriate amount of water, Fmoc-H solution, Fmoc-HTKPR solution and MTX solution in a reaction bottle and stir thoroughly. Then slowly add zinc nitrate hexahydrate aqueous solution to make the mass ratio of Fmoc-H and Fmoc-HTKPR in the reaction system 1:0-0.3 (the feed percentage of Fmoc-HTKPR is 0%-10%), the mass ratio of Fmoc-H and MTX is 3:7, and keep the first ligand and Zn 2+ The molar ratio of 2.2:1 was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to make the solution pH reach 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, dialyzed for purification, and stored in a refrigerator at 4°C for subsequent experiments.

[0076] The same preparation method as above was used to prepare FITC-loaded pZFM-FITC by adding 0.05 μM FITC to pZFM.

[0077] DLS Characterization:

[0078] 1 mL of pZFM solution with different Fmoc-H and Fmoc-HTKPR feed ratios freshly prepared in Example 3 was taken and subjected to DLS measurement on a particle size potentiometer.

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

[0080] Cellular uptake assay:

[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 maintained at 37°C and 5% CO 2 After subculturing the cells in the logarithmic growth phase, they were seeded in 6-well plates (5×10 5 / well). Inflammatory macrophages were obtained by stimulating and activating with 100 ng / mL LPS for 24 hours. The culture medium was removed, and the FITC dose was 0.05 μM, and pZFM-FITC with different Fmoc-H and Fmoc-HTKPR feed ratios were incubated for 4 hours. The culture medium was then removed, washed 3 times with PBS, and centrifuged at 350g for 5 minutes. The centrifuged cells were redispersed in PBS for flow cytometry detection. For each sample, a single FITC channel was used to compare the uptake differences of RAW264.7 cells between different preparations.

[0082] Cellular uptake results ( Figure 3 B) shows that when the feed ratio of Fmoc-H and Fmoc-HTKPR is 1:0-0.15 (i.e., the feed percentage of Fmoc-HTKPR is 5% and below), as the feed ratio of Fmoc-HTKPR increases, the cell uptake gradually increases. When the feed ratio of Fmoc-H and Fmoc-HTKPR is 1:0.3 (i.e., the feed percentage of Fmoc-HTKPR is 10%), the cell uptake is significantly reduced, which may be due to the fact that the particle size of pZFM is too large at this time, which is not conducive to cell uptake.

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

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

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

[0086] Under magnetic stirring, mix appropriate amounts of water, Fmoc-H solution, Fmoc-HTKPR solution, MTX solution and 5-ASA solution in a reaction bottle and stir thoroughly. Then slowly add zinc nitrate hexahydrate aqueous solution to make the mass ratio of Fmoc-H and Fmoc-HTKPR in the reaction system 1:0.15 (i.e., the Fmoc-HTKPR feed percentage is 5%), the mass ratio of Fmoc-H and MTX is 3:7, the feed mass of 5-ASA is 1.2 mg, and keep the first ligand and Zn2+ The molar ratio of 2.2:1 was 2.2:1. Finally, 1M Tris solution was added dropwise to the reaction system to make the solution pH reach 7.0. After the reaction reached equilibrium, it was allowed to stand for half an hour, dialyzed for purification, and stored in a refrigerator at 4°C for subsequent experiments.

[0087] DLS and Zeta Potential Characterization:

[0088] Take 1 mL of each of the freshly prepared ZFM, ZFMA and pZFMA in Examples 1, 2 and 4, and measure the Zeta potential of the ZFM, ZFMA and pZFMA using a particle size potential meter.

[0089] DLS test results ( Figure 4 A) shows that the hydrodynamic diameter of ZFM is 105.7 nm, the hydrodynamic diameter of ZFMA is 168.5 nm, and the hydrodynamic diameter of pZFMA is 177.8 nm. The increase in the hydrodynamic diameters 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 of each of the raw materials Fmoc-H, MTX, 5-ASA, freeze-dried powders of freshly prepared ZFM, ZFMA, pZFMA in Examples 1, 2, and 4, and physical mixed powders of each raw material according to the feed ratio, grind them with an appropriate amount of KBr, and perform Fourier transform infrared absorption measurement by the tablet method.

[0093] FT-IR results ( Figure 5 ) showed that the 1697 cm -1 Fmoc-H and Zn 2+ Stretching vibration of C=O after coordination, 1396cm -1 MTX and Zn 2+ The -OH peak on the coordinated carboxyl group. In addition, the peak at 1652 cm -1 The stretching vibration of C=O on the carboxyl group of 5-ASA is at 1406 cm. -1 The characteristic peak at 2+ Coordination occurs.

[0094] Transmission electron microscopy (TEM) characterization:

[0095] The pZFMA freshly prepared in Example 4 was diluted to a certain multiple and then dropped onto a copper grid. After being naturally air-dried, the morphology and size of the material were characterized using a transmission electron microscope.

[0096] TEM results ( Figure 6 ) showed that pZFMA had a good spherical morphology with a particle size of 50.0-60.0 nm.

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

[0098] The pZFMA solution freshly prepared in Example 4 was lyophilized to powder, and then ground until there was no granularity. The crystal form was analyzed by XRD, with a scanning angle range of 5-90°, a scanning step length of 0.02° / step, a scanning speed of 0.03° / s, and a scanning time of 10 min.

[0099] XRD results ( Figure 7 ) showed that pZFMA had a characteristic amorphous diffraction peak near the 2theta angle of 20°, indicating that it was in an amorphous form.

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

[0101] Referring to the synthesis steps of FITC-loaded pZFM in Example 3, 5-ASA was replaced with 0.05 μM FITC in Example 4 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 maintained at 37°C and 5% CO 2 After subculturing the cells in the logarithmic growth phase, they were seeded in 6-well plates (5×10 5 / well), and inflammatory macrophages were stimulated and activated with 100 ng / mL LPS for 24 hours. The culture medium was removed, and the FITC dose was 0.05 μM, and the nanoparticles were incubated for 0, 2, 4, 8, and 12 hours, respectively. Then the culture medium was removed, washed 3 times with PBS, and centrifuged at 350g for 5 minutes. The centrifuged cells were redispersed in PBS for flow cytometry detection. For each sample, a single FITC channel was used to compare the uptake differences of RAW264.7 cells between the same preparations at different times.

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

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

[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 maintained at 37°C and 5% CO 2 After subculturing the cells in the logarithmic growth phase, they were seeded in 96-well plates (5×10 3 / well) and cultured for 12 hours until the cells adhered to the wall. Inflammatory macrophages were obtained by stimulating and activating with 100 ng / mL LPS for 24 hours. The culture medium was removed and cultured in DMEM culture medium containing ZFM, ZFMA and pZFMA with different MTX concentrations for 24 hours. The culture medium was removed and washed twice with PBS. Then, the cells were 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 DMSO was added to each well to dissolve the purple crystals. The absorbance of the purple crystal product at 490 nm was detected by an enzyme marker, and 3 samples were set up in each group. The formula for calculating cell viability is as follows: Cell viability (%) = (sample-A 0 ) / (Control Group-A 0 )×100%. 0 is the absorbance of the blank well at 490 nm.

[0106] Cytotoxicity results ( Fig. 9 ) 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 loading amount of 5-ASA in ZFMA; while pZFMA (IC50 of approximately 44.19 μg / mL) had the greatest cytotoxicity, which was due to the presence of the targeting peptide that increased the cellular uptake of pZFMA by inflammatory macrophages.

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

[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 maintained at 37°C and 5% CO2 After subculturing the cells in the logarithmic growth phase, they were seeded in 6-well plates (5×10 5 / well), stimulated and activated with 100 ng / mL of LPS for 24 hours to obtain inflammatory macrophages. Remove the culture medium, culture in DMEM culture medium containing the same concentration of ZFM, ZFMA and pZFMA for 24 hours, remove the culture medium, wash 3 times with PBS, add DCFH-DA probe diluent (the volume ratio of the probe stock solution to the incomplete culture medium is 1: 1000), and incubate in the dark in an incubator for 30 minutes. Remove the culture medium, wash 3 times with PBS, collect the cells in PBS, centrifuge at 350g for 5 minutes, and redisperse the cells after centrifugation in PBS for flow cytometry detection. At the same time, cells without LPS stimulation and cells without drug administration were used as controls. FITC single channel was used to compare the differences in the levels of reactive oxygen species in RAW264.7 cells between different preparations.

[0109] Active oxygen scavenging ability results ( Fig.10 ) showed that after LPS stimulation, the level of intracellular reactive oxygen species increased significantly. Both ZFM and ZFMA can kill inflammatory macrophages through the loaded MTX, thereby reducing the content of intracellular reactive oxygen species, and in the ZFMA group, due to the loading of 5-ASA, its ability to scavenge intracellular reactive oxygen species is further enhanced. Due to the presence of targeting peptides in pZFMA, cell uptake increases, so its ability to scavenge intracellular reactive oxygen species is the strongest.

[0110] Example 8 Preparation of 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 in Example 4, mix it thoroughly with the PVP solution, add it to the microneedle mold, centrifuge it to distribute the nanoparticles in the needle tip layer, and dry it overnight to obtain a soluble microneedle patch based on metal-(amino acid / peptide / drug) coordinated 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 nanoparticle is selected from Zn 2+ ; The first ligand is selected from a mixture of 9-fluorenylmethyloxycarbonyl-modified histidine Fmoc-H and a 9-fluorenylmethyloxycarbonyl-modified histidine derivative-modified macrophage targeting peptide Fmoc-HTKPR; the second ligand is selected from methotrexate; the microneedle carrier material is selected from PVP; the microneedle is prepared by the following steps: After mixing 9-fluorenylmethyloxycarbonyl-modified histidine Fmoc-H, 9-fluorenylmethyloxycarbonyl-modified histidine derivative-modified macrophage targeting peptide Fmoc-HTKPR and methotrexate, Zn was added. 2+ , adjust the pH value of the reaction system to a weak alkaline state under stirring, and self-assemble to form metal-amino acid / peptide / drug coordination nanoparticles. The coordination nanoparticles are mixed with the PVP solution and filled into the microneedle mold, centrifuged, and dried overnight to obtain a microneedle patch.

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

3.

4. Use of a soluble microneedle based on metal-amino acid / peptide / drug coordination nanoparticles as described in any one of claims 1 to 3 in the preparation of a drug delivery carrier.

5. The use according to claim 4, characterized in that Drug delivery vehicles carry drugs or probes.

6. The use according to claim 5, characterized in that The drug carrier is one or more of small molecule compounds, polypeptides, proteins, polysaccharides, and nucleic acids.

7. The use according to claim 6, characterized in that The drug carried is 5-aminosalicylic acid.

8. The use according to claim 7, characterized in that The feed mass of 5-ASA is 0.6 mg to 3.0 mg.

9. Use of the soluble microneedle preparation based on metal-amino acid / peptide / drug coordination nanoparticles as claimed in claim 4 or 8 in anti-rheumatoid arthritis drugs.

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