Iridium complex nanocrystalline microneedle as well as preparation method and application thereof

Abstract

The invention provides an iridium complex nanocrystalline microneedle as well as a preparation method and application thereof, and belongs to the technical field of medicines. The PIQ nanocrystal with small particle size and PDI is firstly prepared, the solubility is improved, and compared with PIQ, the PIQ nanocrystal has the advantages that the toxicity to A375 cells is remarkably improved, the migration inhibition capacity to the A375 cells is higher, and A375 cell apoptosis can be more remarkably promoted; chitosan and PVA are adopted as matrix materials and cooperate with the PIQ nanocrystals to prepare the iridium complex nanocrystal microneedle, the microneedle directly penetrates through the stratum corneum to act on the melanoma part, convenience is improved, meanwhile, the drug effect is improved, and toxic and side effects on normal tissue are reduced; the nanocrystalline microneedle enhances the solubility of PIQ and improves the percutaneous penetration capacity, has enough high mechanical strength, penetrates through the cuticle and directly acts on target tissue, and overcomes the defects that PIQ is poor in solubility and difficult to break through the cuticle.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to an iridium complex nanocrystal microneedle and a preparation method and application thereof. BACKGROUND

[0002] Melanoma originates from the malignant transformation of melanocytes, is a cancer that develops due to uncontrolled proliferation of melanocytes, and can invade mucosa everywhere in the body in addition to the skin. Due to its high invasiveness and fatality, it is called the "king of skin cancer", and the 5-year survival rate of patients in the late stage is less than 20%. At present, the conventional treatment method for melanoma is surgery, radiotherapy and chemotherapy. For patients with early malignant melanoma, surgery can cure and obtain a long survival period; but for high-risk patients with middle and late malignant melanoma, there may be factors such as lymph node metastasis, and the risk of recurrence after surgery is high, so surgery needs to be combined with non-surgical methods such as radiotherapy and chemotherapy to prevent further spread of the tumor.

[0003] Cisplatin, as the first generation of platinum-based antitumor drugs, is clinically used for the treatment of melanoma, which induces apoptosis of cancer cells by damaging DNA and interfering with repair, but has serious side effects such as nephrotoxicity, neurotoxicity and bone marrow suppression, and cancer cells can repair damaged DNA to affect drug efficacy. In order to overcome the limitations of platinum drugs, researchers are developing new metal complexes to improve efficacy and reduce toxicity. Transition metal drugs such as ruthenium, iridium, rhodium, osmium and palladium are becoming a new direction of research on antitumor drugs due to their potential advantages such as reducing adverse reactions and overcoming drug resistance. PIQ is an iridium metal complex designed and synthesized with 2-(4-isopropylphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline as a ligand, and its molecular formula is C 52 H 38 IrN6, which is a wine red powder, has lipophilicity, is difficult to dissolve in water, and is soluble in organic reagents such as acetone. It is found through in vitro cell studies that it has strong antitumor activity and good application development prospects. However, PIQ is a poorly soluble drug, which limits its development and application due to its low bioavailability. SUMMARY

[0004] The purpose of the present application is to provide an iridium complex nanocrystal microneedle and a preparation method and application thereof. The iridium complex nanocrystal microneedle prepared by the method provided by the present application promotes the transdermal delivery of the poorly water-soluble drug PIQ, inhibits the proliferation and migration ability of A375 cells, significantly inhibits the growth of melanoma and improves the damage to internal organs in vivo, improves the low bioavailability of PIQ, and expands its further development and application.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The application provides a preparation method of PIQ nanocrystal microneedles, comprising the following steps:

[0007] (1) mixing PIQ, an organic solvent, pure water and a stabilizer, and sequentially performing first stirring and homogenization to obtain a PIQ nanocrystal suspension;

[0008] mixing the PIQ nanocrystal suspension and a freeze-drying protective agent, and then performing freeze-drying to obtain PIQ nanocrystals;

[0009] the chemical formula of the PIQ is [Ir(piq)2(ZZIP)]PF6;

[0010] (2) dissolving chitosan in an acetic acid aqueous solution, and then adding polyvinyl alcohol PVA, and sequentially performing second stirring, heating and cooling to obtain a matrix liquid;

[0011] mixing the matrix liquid and the PIQ nanocrystals obtained in the step (1), and then performing third stirring, injection into a mold, drying and demolding to obtain iridium complex nanocrystal microneedles.

[0012] Preferably, the stabilizer in step (1) is selected from at least one of the following: polysorbate (including but not limited to Tween 20, Tween 40, Tween 60, Tween 80), cellulose (including but not limited to hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), methylcellulose (MC), sodium carboxymethylcellulose (CMC-Na), ethyl cellulose (EC)), poloxamer (including but not limited to poloxamer P188, poloxamer P407), polyvinylpyrrolidone (PVP, including but not limited to PVP K17, PVP K30, PVP K90), vitamin E derivative (including but not limited to vitamin E polyethylene glycol succinate), phospholipid (including but not limited to soybean phospholipid, lecithin, egg yolk phospholipid), anionic surfactant (including but not limited to sodium dodecyl sulfate (SDS), sodium dodecylbenzenesulfonate (SDBS), sodium stearate, sodium cholate, sodium deoxycholate), polyoxyethylene castor oil (including but not limited to polyoxyethylene castor oil (Cremophor EL), polyoxyethylene hydrogenated castor oil (Cremophor RH40)), sorbitan fatty acid ester (span, including but not limited to span 20, span 40, span 60, span 80), high molecular polymer (including but not limited to polyacrylic acid resin (Eudragit L100, Eudragit S100), chitosan and its derivatives (carboxymethyl chitosan, hydroxypropyl chitosan), gelatin, amino acid surfactant (including but not limited to sodium lauroyl glutamate, potassium cocoyl glycinate), polyethylene glycol derivative (including but not limited to mPEG-cholesterol, mPEG-stearic acid, mPEG-vitamin E succinate), more preferably at least one of Tween 80, hydroxypropyl methylcellulose HPMC, poloxamer P188 and polyvinylpyrrolidone PVP K30.

[0013] The concentration of the stabilizer used in the present application is preferably 0.1% to 5% (m / V, based on the total mass of the PIQ-NCs suspension), more preferably 0.5% to 2%; the stabilizer can be a single stabilizer or a composite stabilizer of two or more, and the mixing ratio of the composite stabilizer is 1:4 to 4:1 (mass ratio).

[0014] Preferably, the rotation speed of the first stirring in step (1) is 2800 to 4000 r / min; the number of homogenization is 20 to 40, and the pressure of homogenization is 1500 to 1800 bar.

[0015] Preferably, the freeze-drying protectant in step (1) is at least one of mannitol, sucrose, lactose, maltose, and trehalose.

[0016] Preferably, the average particle size of the PIQ nanocrystals in step (1) is 10-350 nm, and the polydispersity coefficient PDI is 0.01-0.3.

[0017] Preferably, the temperature of the heating in step (2) is 70-95 ℃, and the time of the heating is 20-30 min.

[0018] Preferably, the mass percentage of chitosan in the substrate solution in step (2) is 1%-5%, and the mass percentage of PVA in the substrate solution is 9%-20%.

[0019] Preferably, the mass of the PIQ nanocrystals in step (2) accounts for 1.5%-6% of the total mass of the iridium complex nanocrystal microneedle.

[0020] The application also provides an iridium complex nanocrystal microneedle prepared by the preparation method.

[0021] The application also provides an iridium complex nanocrystal microneedle prepared by the preparation method or the application of the iridium complex nanocrystal microneedle in the preparation of a microneedle patch for treating malignant melanoma.

[0022] The application provides a preparation method of a PIQ nanocrystal microneedle. First, PIQ is used as an active ingredient, an organic solvent and pure water are used as a solvent system, and a stabilizer is used as a dispersion aid. An initial product is prepared by a combination of anti-solvent precipitation and high-pressure homogenization technology, and then the initial product is freeze-dried to obtain PIQ nanocrystals (PIQ-NCs) with small particle size and PDI. The nanocrystals can significantly improve the solubility and bioavailability of PIQ and improve the biological activity of PIQ. Cell experiments prove that the toxicity, migration inhibition capacity and pro-apoptotic effect of PIQ-NCs on A375 cells are significantly enhanced compared with free PIQ. Then, chitosan and polyvinyl alcohol (PVA) are used as a composite matrix material to load PIQ-NCs and prepare an iridium complex PIQ nanocrystal microneedle (PIQ-NCs microneedle). The microneedle has excellent mechanical strength, can directly penetrate the stratum corneum and target melanoma sites, improves the drug compliance, reduces the toxic and side effects on normal tissues, and increases the solubility and transdermal penetration of PIQ, thereby systematically solving the key technical problems of poor solubility of PIQ and difficulty in breaking through the stratum corneum. The in-vitro anti-tumor and in-vivo preliminary pharmacodynamic results prove that the PIQ nanocrystal microneedle has excellent therapeutic performance, and provides a new technical idea and research direction for the use of a poorly soluble anti-tumor drug in the treatment of melanoma. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The appearance and transmission electron microscope images of the PIQ nanocrystals prepared in Example 1 in different states in the application, Figure 1A shows the suspension before freeze-drying (left) and the Tyndall effect of the suspension before freeze-drying (right); B shows the PIQ-NC lyophilized powder; C shows the reconstituted solution of the PIQ-NC lyophilized powder (left) and its Tyndall effect (right); D shows the transmission electron microscope image of PIQ-NCs; E shows the particle size distribution of PIQ-NCs; F shows the infrared spectra of PIQ prepared in this invention and PIQ-NCs prepared in Example 1; G shows the DSC diagram of PIQ prepared in this invention and PIQ-NCs prepared in Example 1.

[0024] Figure 2 This is a graph showing the uptake results of PIQ and PIQ-NCs by A375 cells in this invention. Figure 2 (A is a microscopic image; B is a fluorescence intensity image; C is a quantitative fluorescence intensity analysis image) (n = 3, compared with the control group, ***p < 0.001)

[0025] Figure 3 The figures show the results of the cell scratch assay in this invention, where A is a microscopic image; B is the cell migration rate at 24 h; and C is the cell migration rate at 48 h. (n = 3, **p < 0.01, ***p < 0.001)

[0026] Figure 4 A) Appearance diagrams of soluble microneedles prepared with different matrix materials in this invention; B) Appearance diagrams of soluble microneedles prepared with different concentrations of PVA in this invention; C) Mechanical strength diagrams of soluble microneedles prepared with different concentrations of PVA in this invention.

[0027] Figure 5 The images show the appearance and mechanical strength of soluble microneedles prepared with different concentrations of PIQ-NCs in this invention. A is the appearance image, and B is the mechanical strength image.

[0028] Figure 6 The images show the overall appearance and partial needle tip views of the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 of this invention. Figure 6 A represents a blank microneedle. Figure 6 B in the middle is PIQ-NCs microneedles. Figure 6 The image in center C is a bright-field photograph of PIQ-NCs microneedles. Figure 6 The image in section D shows the PIQ-NCs microneedles captured using the GFP channel. Figure 6 E in the middle is a scanning electron microscope image. Figure 6 Image F in the middle is a partial needle tip image taken with a scanning electron microscope;

[0029] Figure 7 The graphs show the appearance changes and puncture rates of the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 when puncturing Parafilm® M membranes, respectively.Figure 7 A in the diagram represents the trend of appearance changes. Figure 7 Chart B shows the puncture rate statistics;

[0030] Figure 8 These are images showing the skin puncture results of the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 in this invention. Figure 8 A represents a blank microneedle. Figure 8 B in the middle is PIQ-NCs microneedles;

[0031] Figure 9 The graph shows the solubility test results of the blank microneedles and the PIQ-NCs microneedles (hereinafter referred to as drug-loaded microneedles) prepared in Example 1 in this invention;

[0032] Figure 10 This is a statistical chart showing the cumulative release of transdermal drug per unit in this invention;

[0033] Figure 11 These are images showing the healing process of blank microneedles and PIQ-NCs microneedles prepared in Example 1 after puncturing the skin of nude mice. Figure 11 Image A shows actual photos of the skin healing process in each group of nude mice. Figure 11 Image B shows the results of tissue sections of nude mouse skin after microneedle puncture, as well as the results of microneedle puncture in the blank microneedle and PIQ-NCs microneedle puncture groups prepared in Example 1.

[0034] Figure 12 The figures shown are the tumor inhibition results of each group of nude mice in this invention and the weight change of each group of nude mice. Figure 12 A shows the tumor in vitro, B shows the tumor volume change, C shows the tumor weight, D shows the tumor inhibition rate by volume, E shows the tumor inhibition rate by weight, and F shows the body weight change; (n = 4, compared with the negative control group, *p < 0.05; **p < 0.01, ***p < 0.001)

[0035] Figure 13 The figures show the results of TUNEL, H&E, and immunohistochemical analyses of tumor tissues from various groups of nude mice in this invention; among them, Figure 13 Image A shows TUNEL fluorescence staining images of tumor tissues from nude mice in each group, where blue represents DAPI-stained cell nuclei and red represents TUNEL-positive apoptotic cells. Figure 13 Figure B is a bar chart showing the statistical analysis results of the TUNEL fluorescence area in tumor tissues of nude mice in each group. Figure 13 C in the middle is stained with H&E. Figure 13In the middle section, D represents the immunohistochemical staining images of tumor tissues from nude mice in each group, and E represents the bar chart of the statistical analysis results of Ki67 positive expression rate in tumor tissues from nude mice in each group (n = 3, compared with the negative control group, **p < 0.01, ***p < 0.001; compared with the positive control group, ...). ### p < 0.001);

[0036] Figure 14 The image shows the H&E analysis results of the organs of each group of nude mice in this invention. Detailed Implementation

[0037] This invention provides a method for preparing iridium complex nanocrystalline microneedles, comprising the following steps:

[0038] (1) Mix iridium complex PIQ, organic solvent, pure water and stabilizer, and perform first stirring and homogenization in sequence to obtain PIQ nanocrystal suspension;

[0039] The PIQ nanocrystal suspension was mixed with a freeze-drying protectant and then freeze-dried to obtain PIQ nanocrystals.

[0040] The chemical formula of the iridium complex PIQ is [Ir(piq)2(ZZIP)]PF6;

[0041] (2) Dissolve chitosan in an aqueous acetic acid solution, then add polyvinyl alcohol (PVA), and perform a second stirring, heating and cooling in sequence to obtain a matrix solution;

[0042] After mixing the matrix liquid and the PIQ nanocrystals obtained in step (1), the mixture is stirred for the third time, injected into the mold, dried and demolded to obtain iridium complex nanocrystal microneedles.

[0043] Unless otherwise specified, all raw materials used in this invention are commercially available products in the art.

[0044] The present invention involves mixing PIQ, an organic solvent, pure water and a stabilizer, and then performing a first stirring and homogenization to obtain a PIQ nanocrystal suspension.

[0045] In this invention, the molecular formula of the PIQ is C 52 H 38 IrN6; The structural formula of the PIQ is:

[0046] .

[0047] In this invention, the organic solvent is preferably at least one selected from acetone, ethanol, and methanol, and more preferably acetone. PIQ has the highest solubility in acetone, and using acetone as a solvent facilitates the dissolution of PIQ. In this invention, the stabilizer is selected from at least one of the following: polysorbates (including but not limited to Tween 20, Tween 40, Tween 60, Tween 80), cellulose derivatives (including but not limited to hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), methylcellulose (MC), sodium carboxymethyl cellulose (CMC-Na), ethyl cellulose (EC)), poloxamer derivatives (including but not limited to poloxamer P188, poloxamer P407), polyvinylpyrrolidone derivatives (PVP, including but not limited to PVP K17, PVP K30, PVPK90), vitamin E derivatives (including but not limited to vitamin E polyethylene glycol succinate), phospholipids (including but not limited to soybean phospholipids, lecithin, egg yolk phospholipids), anionic surfactants (including but not limited to sodium dodecyl sulfate (SDS), sodium dodecylbenzene sulfonate (SDBS), sodium stearate, sodium cholate, sodium deoxycholate). The following are considered surfactants: deoxycholate, polyoxyethylene castor oil (including but not limited to polyoxyethylene castor oil (Cremophor EL), polyoxyethylene hydrogenated castor oil (Cremophor RH40)), fatty acid sorbitans (Span derivatives, including but not limited to Span 20, Span 40, Span 60, Span 80), high molecular weight polymers (including but not limited to polyacrylic acid resins (Eudragit L100, Eudragit S100), chitosan and its derivatives (carboxymethyl chitosan, hydroxypropyl chitosan), gelatin), amino acid surfactants (including but not limited to sodium lauroyl glutamate, potassium cocoyl glycinate), and polyethylene glycol derivatives (including but not limited to mPEG-cholesterol, mPEG-stearic acid, mPEG-vitamin E succinate). More preferably, at least one of Tween 80, hydroxypropyl methylcellulose HPMC, poloxamer P188, and polyvinylpyrrolidone PVP K30 is preferred, and more preferably, poloxamer P188 and PVP K30 / poloxamer P188 and HPMC are preferred. In this invention, the concentration of the stabilizer is preferably 0.1% to 5% (m / V, based on the total mass of the PIQ-NCs suspension), more preferably 0.5% to 2%; the stabilizer can be a single stabilizer or a combination of two or more stabilizers, and the mixing ratio of the combination stabilizers is 1:4 to 4:1 (mass ratio). In this invention, when the stabilizer is poloxamer P188 and PVP K30, the mass ratio of poloxamer P188 to PVP K30 is preferably 1:(1 to 3), more preferably 1:2.In this invention, due to the high surface energy of PIQ nanocrystals, they are prone to aggregation and Ostwald maturation, requiring a suitable stabilizer to adsorb onto the surface to maintain uniform dispersion. Poloxamer, as a nonionic surfactant, can reduce surface tension, prevent nanocrystal aggregation, and does not electrostatically adsorb with PIQ. PVP has strong surface activity and can quickly adsorb to form a stable protective layer. The steric hindrance effect of the combination of the two is beneficial for preparing nanocrystals with small particle size and PDI, and good stability. This invention controls the amount of stabilizer within the above-mentioned range to avoid insufficient dosage leading to incomplete surface coverage of nanocrystals and affecting stability, and also to prevent excessive dosage from causing polymer chain entanglement, reducing the stabilizing effect, or even promoting particle aggregation.

[0048] In this invention, the preferred method for mixing the iridium complex PIQ, organic solvent, pure water, and stabilizer is to dissolve PIQ in an organic solvent to obtain a PIQ solution; and then add the PIQ solution to a mixture composed of pure water and a stabilizer.

[0049] In this invention, the stirring speed of the first stirring is preferably 2900~4500 r / min, more preferably 3000~4000 r / min. The stirring time of the first stirring is preferably 6~15 min, more preferably 8~13 min. By controlling the stirring speed and time within the above ranges, this invention adjusts the nucleation and growth rate of nanocrystals. A higher dispersion speed increases the probability of collisions between particles, avoiding the dominance of growth rate and the formation of larger nanocrystals, thus facilitating the obtaining of smaller PIQ nanocrystals. In this invention, the number of homogenization cycles is preferably 20~40, more preferably 25~35, and even more preferably 30; the homogenization pressure is preferably 1500~1800 bar, more preferably 1600~1790 bar, and even more preferably 1750 bar. This invention controls the number of homogenization cycles and pressure within the aforementioned range, using homogenized mechanical forces (shearing, impact, cavitation) to break up PIQ particles and reduce particle size; simultaneously, by reducing the particle size difference between particles, the PDI is reduced (more uniform distribution). By controlling the homogenization intensity (pressure, speed) and number of cycles within the aforementioned reasonable range to avoid the adverse effects caused by excessive agglomeration, the particle size and PDI of the prepared PIQ nanocrystals are adjusted, resulting in PIQ nanocrystals with smaller particle size and PDI.

[0050] After homogenization, the present invention preferably performs rotary evaporation on the homogenized product to obtain a PIQ nanocrystal suspension.

[0051] This invention does not impose any special restrictions on the rotary evaporation method; it is sufficient to remove residual organic solvents.

[0052] After obtaining the PIQ nanocrystal suspension, the present invention mixes the PIQ nanocrystal suspension with a freeze-drying protectant and then freeze-dries it to obtain PIQ nanocrystals.

[0053] In this invention, the freeze-drying protectant is preferably at least one selected from mannitol, sucrose, lactose, maltose, and trehalose, more preferably mannitol. In this invention, the mass ratio of the freeze-drying protectant to PIQ in the PIQ nanocrystal suspension is preferably (15~70):1, more preferably (40~50):1. In this invention, the freeze-drying time is preferably 24~96 h, more preferably 48 h. By controlling the freeze-drying temperature and time within the above ranges, this invention prepares PIQ-NCs with stable performance and uniform appearance. These PIQ-NCs retain the Tyndall effect after reconstitution, and the freeze-dried powder preparation further facilitates their storage.

[0054] In this invention, the average particle size of the PIQ nanocrystals is preferably 10-350 nm, more preferably 10-150 nm, and even more preferably 12-110 nm; the polydispersity index (PDI) of the PIQ nanocrystals is preferably 0.01-0.3, more preferably 0.11-0.22. In this invention, a smaller average particle size of the PIQ nanocrystals is more conducive to dissolution, and a smaller PDI and smaller particle size distribution range are beneficial to the stability of the nanoparticle solution, thereby improving the bioavailability of the nanocrystals. In this invention, the zeta potential of the PIQ nanocrystals is preferably -38 to -26 mV.

[0055] This invention, by adjusting factors including high-speed dispersion speed, homogenization times and pressure, type of stabilizer, type of composite stabilizer, ratio of composite stabilizer and amount added, ultimately prepares PIQ nanocrystals with small particle size and PDI and a certain ZETA potential. The PIQ-NCs prepared by selecting mannitol as the freeze-drying protectant have a uniform appearance and still exhibit the Tyndall effect after reconstitution. The freeze-dried powder preparation is more conducive to the storage of PIQ-NCs.

[0056] In this invention, chitosan is dissolved in an aqueous acetic acid solution, and then polyvinyl alcohol (PVA) is added. The mixture is then subjected to a second stirring, heating, and cooling process to obtain a matrix solution.

[0057] In this invention, the volume concentration of the acetic acid aqueous solution is preferably 1%. This invention does not impose any particular limitation on the method of the second stirring, as long as the components are fully swollen. In this invention, the heating temperature is preferably 80-95°C, more preferably 90°C. In this invention, the heating time is preferably 20-40 min, more preferably 30 min. This invention promotes complete dissolution of the components by controlling the heating temperature and time within the above ranges. In this invention, the cooling is preferably cooling to room temperature.

[0058] After obtaining the matrix liquid and PIQ nanocrystals, the present invention mixes the matrix liquid and the PIQ nanocrystals, performs a third stirring, injects into a mold, dries and demolds to obtain iridium complex nanocrystal microneedles.

[0059] In this invention, the mass percentage of chitosan in the matrix solution is preferably 1% to 5%, more preferably 2%. The mass percentage of PVA in the matrix solution is preferably 9% to 20%, more preferably 10% to 18%, and even more preferably 13%. This invention uses biocompatible and degradable brittle chitosan and tough PVA as the matrix. By controlling the amounts of both, problems such as high microneedle hardness leading to demolding breakage, fragmentation during drug administration, air bubbles, needle tip bending affecting use, insufficient mechanical strength preventing penetration of the stratum corneum, and easy breakage of the backing can be avoided. This results in microneedles with excellent overall performance, high mechanical strength, and good appearance.

[0060] This invention does not impose any special restrictions on the third stirring method, as long as the solution is clear, transparent, and free of bubbles. In this invention, the preferred method of injecting the solution into the mold is as follows: the mixed solution obtained by mixing the matrix liquid and the PIQ nanocrystals is filled into the mold, placed in a vacuum drying oven, evacuated to -0.08 MPa, held at pressure for 30 min, and then the bubbles are removed. This process of filling the mold with the remaining mixed solution and removing bubbles is repeated until all the material is used and the microneedles are prepared. In this invention, the drying temperature is preferably 30~50 ℃, more preferably 40 ℃; the drying time is preferably 8~18 h. This invention removes residual solvent through drying. In this invention, the mass of the PIQ nanocrystals preferably accounts for 1.5%~6% of the total mass of the iridium complex nanocrystal microneedles, more preferably 2%~5%.

[0061] This invention successfully prepared PIQ-NCs microneedles with a stable and reproducible preparation process. The microneedles promote the transdermal delivery of the poorly water-soluble drug PIQ, inhibit the proliferation and migration of A375 cells, significantly inhibit melanoma growth, and improve organ damage in vivo. The microneedles provide a new approach for the development of transdermal drug delivery for drugs with poor solubility and have the potential to treat melanoma through transdermal therapy.

[0062] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0063] Unless otherwise specified, all experiments were repeated three times, and the results are expressed as averages. In this invention, n represents the number of repetitions.

[0064] Example 1

[0065] A method for preparing iridium complex nanocrystalline microneedles, comprising the following steps:

[0066] (1) Dissolve PIQ in acetone to prepare a PIQ solution with a concentration of 2 mg / mL. Slowly add the PIQ solution to a mixture of pure water and stabilizer at a ratio of 1:10 (v / v). Stir at 3000 r / min for 10 min, and then homogenize at 1750 bar for 30 times to obtain a PIQ nanocrystal suspension. The stabilizer is poloxamer P188 and PVP K30 in a mass ratio of 1:2, and the mass of the stabilizer accounts for 1% of the total mass of the PIQ nanocrystal suspension.

[0067] The PIQ nanocrystal suspension and the freeze-drying protectant mannitol were mixed at a mass ratio of (40~50):1 and then freeze-dried for 48 hours to obtain PIQ nanocrystals.

[0068] The iridium complex PIQ has the chemical formula [Ir(piq)2(ZZIP)]PF6; the mass ratio of PIQ in the lyophilization protectant and the PIQ nanocrystal suspension is 50:1; the average particle size of the PIQ nanocrystals is 94.30 ± 1.38 nm, the PDI is 0.185, and the zeta potential is -31.56 ± 0.73 mV;

[0069] (2) Chitosan was weighed and dissolved in a 1% acetic acid aqueous solution. PVA was then slowly added and stirred for a second time to induce swelling. After full swelling, the solution was heated at 90 °C for 30 min to completely dissolve all components. After cooling to room temperature, a matrix solution was obtained. The matrix solution contained 2% chitosan by mass and 13% PVA by mass.

[0070] Add the PIQ nanocrystals prepared in step (1) to the matrix solution, and stir for the third time until the solution is clear, transparent, and free of bubbles to obtain a mixed solution. Fill the mold with the solution, place it in a vacuum drying oven, evacuate to -0.08 MPa, and maintain the pressure for 30 min to remove bubbles. Repeat the filling and bubble removal operations twice, and then place it in a drying oven at 40 ℃ for 12 h until completely dry. Demold to obtain iridium complex nanocrystal microneedles, in which the mass of PIQ-NCs accounts for 3% of the total mass of PIQ-NCs microneedles (246 μg PIQ / 90mg).

[0071] Comparative Example

[0072] Blank microneedles were prepared according to the method of Example 1, except that no PIQ nanocrystals were added, and only the matrix solution prepared in Example 1 was used as the raw material.

[0073] Investigating the effects of some preparation parameters on PIQ nanocrystals

[0074] (1) The speed of the first stirring, the number of homogenization cycles and the pressure

[0075] PIQ nanocrystals were prepared according to the method of Example 1, except that the speed of the first stirring, the number of homogenizations and the pressure were adjusted, as shown in Table 1.

[0076] The particle size and PDI of PIQ nanocrystals prepared under different conditions after the above adjustments were tested using particle size and PDI detection methods. The results are shown in Table 1: The particle size increases with the increase of dispersion speed, because the high speed accelerates the nucleation and growth rate, and the increased probability of particle collision makes growth dominant. The particle size is smaller at 3000 r / min. The particle size and PDI decrease after 30 homogenization cycles compared to 20 cycles, but the change is not significant compared to 40 cycles. Considering the energy consumption and efficiency of large-scale production, 30 cycles are more suitable. The particle size and PDI decrease with the increase of homogenization pressure, and the effect is better at 1750 bar.

[0077] Table 1. Particle size and PDI (n = 3) of PIQ-NCs prepared under different conditions

[0078] High speed dispersion rotation speed (r / min) Particle size (nm) PDI 3000 98.31 ± 1.01 0.132 ± 0.007 5000 117.81 ± 0.79 0.142 ± 0.027 7000 128.53 ± 0.98 0.141 ± 0.005 High pressure homogenization times (times) Particle size (nm) PDI 20 104.50 ± 1.11 0.217 ± 0.001 30 103.06 ± 0.41 0.194 ± 0.008 40 102.22 ± 1.68 0.192 ± 0.015 High pressure homogenization pressure (bar) Particle size (nm) PDI 1250 105.25 ± 2.51 0.214 ± 0.005 1500 103.23 ± 0.46 0.202 ± 0.011 1750 102.22 ± 1.68 0.192 ± 0.015

[0079] (2) Effect of stabilizer ratio on PIQ nanocrystals

[0080] PIQ nanocrystals were prepared according to the method of Example 1, except that the proportion and amount of stabilizer and the type of lyophilization protectant were adjusted. The particle size and PDI of the prepared PIQ-NCs were detected using the same method as above, as shown in Table 2.

[0081] As shown in Table 2, when the stabilizer ratio is poloxamer P188:PVP K30 = 1:2, the particle size and PDI are relatively small.

[0082] Table 2. Particle size and PDI (n = 3) of PIQ-NCs prepared with different stabilizer ratios

[0083] Stabilizer ratio (poloxamer P188: PVP K30) Particle size (nm) PDI 4：1 123.41 ± 4.12 0.219 ± 0.023 2：1 116.97 ± 1.77 0.215 ± 0.012 1：1 112.38 ± 0.56 0.213 ± 0.015 1：2 105.21 ± 0.70 0.211 ± 0.005 1：4 128.78 ± 1.55 0.248 ± 0.026

[0084] The appearance of the PIQ nanocrystals prepared in Example 1 under different states was observed, and their transmission electron microscopy (TEM) images, particle size distribution, infrared spectra, and DSC images were detected. The results are as follows: Figure 1 , Figure 1Image A shows the suspension before freeze-drying (left) and the Tyndall effect of the suspension before freeze-drying (right); Image B shows the PIQ-NC lyophilized powder; Image C shows the reconstituted solution of the PIQ-NC lyophilized powder (left) and its Tyndall effect (right); Image D shows the transmission electron microscope image of PIQ-NCs; Image E shows the particle size distribution of PIQ-NCs; Image F shows the infrared spectra of PIQ prepared in this invention and PIQ-NCs prepared in Example 1; Image G shows the DSC diagram of PIQ prepared in this invention and PIQ-NCs prepared in Example 1. Figure 1 It can be seen that both the suspension before and after freeze-drying and the reconstituted solution are clear, without flocculation or precipitation, and both exhibit the Tyndall effect. Figure 1 (A, B); The surface of the freeze-dried PIQ-NCs is smooth, without obvious cracks, collapses or delamination. Figure 1 (C); PIQ-NCs exhibit an irregular shape and a size of approximately 250 nm ( Figure 1 (E).

[0085] The particle size, PDI, and potential of the particles prepared in Example 1 were measured using a NanoBrook Omni. The results showed that the average particle size of the particles prepared in Example 1 was 94.30 ± 1.38 nm, the PDI was 0.185, and the zeta potential was -31.56 ± 0.73 mV.

[0086] The equilibrium solubility of PIQ and PIQ-NCs prepared in Example 1 was detected by the shake flask method. The solubility of PIQ-NCs in water was measured to be 0.894 μg / mL, while PIQ was almost insoluble in water. The results show that preparing PIQ into nanocrystals in Example 1 can effectively improve its solubility.

[0087] PIQ and PIQ-NCs prepared in Example 1 were detected using an infrared spectrometer. The results are shown in the figure. Figure 1 The main characteristic absorption peak of PIQ active pharmaceutical ingredient is 3030 cm⁻¹. -1 (Aromatic ring CH stretching vibration), 1650-1550 cm -1 (C=N stretching vibration), 1600-1450 cm -1 (Aromatic ring C=C skeletal vibration), 1370-1450 cm -1 (-CH3 and -CH2 tert-butyl bending vibrations), 650-850 cm -1 (Aromatic ring CH out-of-plane bending vibration), 554 cm -1 (Metal-nitrogen coordination). The above characteristic peaks are present but weak in the physical mixture (due to the small amount of drug added), indicating that the excipients did not undergo significant chemical reaction with the drug; 554 cm⁻¹ in PIQ-NCs -1 (Metal-nitrogen coordination peak) redshifted to 548 cm⁻¹ -1It is speculated that the excipients interact with PIQ through hydrogen bonding and other forces, and play a stabilizing role, possibly due to the enhanced hydrogen bonding.

[0088] Differential scanning calorimetry (DSC) was used to detect the DSC patterns of PIQ and PIQ-NCs prepared in Example 1, respectively. The results are as follows: Figure 1 As shown in Figure G, the endothermic peak of PIQ raw material is around 332 °C, and the exothermic peak of mannitol is around 167 °C. The physical mixture retains the above peaks. However, in PIQ-NCs, a significant exothermic peak can be observed at around 155 °C. This peak may be due to recrystallization of some amorphous or metastable regions in the sample during the heating process. It may also be due to the increased specific surface area after preparation into nanocrystals, thus resulting in the appearance of this exothermic peak.

[0089] This invention employs various cell lines for experiments. All cells are cultured in a basal medium containing 10% fetal bovine serum and 1% penicillin-streptomycin, with A375 and HaCaT cells cultured in DMEM high-glucose medium. Cells are seeded in culture plates of different sizes and cultured at 37°C in a 5% CO2 incubator. The medium is changed every 2-3 days, and cells are passaged when they reach approximately 80% confluence to maintain stable growth and reproduction.

[0090] Investigating the in vitro antitumor activity of PIQ-NCs

[0091] (1) Cytotoxicity

[0092] The cytotoxicity of PIQ and PIQ-NCs to A375 and HaCaT cells was detected using the CCK-8 assay.

[0093] First, the toxicity of blank nanocrystalline excipients (poloxamer P188 and PVP K30) to A375 and HaCaT cells was investigated. The results showed that the cell survival rate exceeded 80%, indicating that the nanocrystalline excipients had no significant toxicity. Inhibition experiments on A375 cells showed that the toxicity of both free PIQ and PIQ-NCs increased with increasing drug concentration in a concentration-dependent manner; moreover, PIQ-NCs were significantly more toxic than PIQ (***p<0.001), indicating that the efficacy was enhanced after nanocrystalline preparation.

[0094] (2) Cellular uptake

[0095] A375 cells in the logarithmic phase were used at a rate of 5 × 10⁻⁶ 5 / wells were inoculated into 6-well plates and incubated at 37 ℃ and 5% CO2 for 24 h. Then, the inoculum was replaced with an IC50 solution at a final concentration of 100 mg / L. 50After culturing in drug-containing medium for 24 h, cells were collected, washed with PBS, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and then stained with DAPI staining solution for 5 min. The staining solution was discarded, and the cells were washed three times with PBS before observation and photography under a fluorescence microscope. The quantitative analysis procedure was as follows: after staining the cell nuclei with DAPI staining solution for 5 min and discarding the staining solution, DAPI staining solution was added again for 5 min and discarded. The cells were then digested with trypsin without EDTA, washed three times, and the fluorescence intensity was measured using flow cytometry. The experiment was repeated three times. The results of A375 cell uptake of PIQ and PIQ-NCs are as follows: Figure 2 As shown, where, Figure 2 Image A is a microscopic image; image B is a fluorescence intensity graph; image C is a quantitative fluorescence intensity analysis graph (n = 3, ***p < 0.001 compared to the Control group). Figure 2 It was found that both the PIQ group and PIQ-NCs exhibited green fluorescence, but the fluorescence intensity of the PIQ-NCs group was stronger than that of the PIQ group. Preliminary inference suggests that preparing PIQ into nanocrystals promoted the uptake of PIQ by A375 cells. Quantitative uptake results are as follows... Figure 2 As shown in Figure C, compared with the control group, PIQ and PIQ-NCs exhibited stronger fluorescence intensity and showed a significant difference (***p < 0.001), indicating that preparing PIQ into nanocrystals promotes PIQ entry into A375 cells.

[0096] (3) Cell migration ability

[0097] The effect of PIQ and PIQ-NCs prepared in Example 1 on the migration rate of A375 cells was investigated: The effect of the drugs on cell migration ability was evaluated using a cell scratch assay. Logarithmic-phase A375 cells were sputtered at 5 × 10⁻⁶ cells / year. 5 The cells were seeded at a density of / well in 6-well plates and incubated at 37°C and 5% CO2 for 24 h. The inoculum was then replaced with a final concentration of IC50. 50 Cells were cultured in drug-containing medium for 24 and 48 hours; cell migration was recorded under a microscope, and the scratch area was analyzed using ImageJ software. Figure 3 The results of the cell scratch assay are shown in Figure A, where A is a microscopic image; B is the cell migration rate at 24 h; and C is the cell migration rate at 48 h. (n = 3, **p < 0.01, ***p < 0.001). Figure 3Experimental results showed that cell growth in the scratched area was less in the drug-treated group than in the control group, with the PIQ-NCs group showing the best inhibitory effect. Image J quantitative analysis showed that the cell migration rate in the PIQ-NCs group was significantly lower than that in the PIQ group and the control group at 24 h and 48 h after drug administration (***p<0.001), and the PIQ group was also significantly lower than the control group (**p<0.01).

[0098] (4) Apoptosis

[0099] To investigate the apoptotic effects of PIQ and PIQ-NCs prepared in Example 1 on A375 melanoma cells, Annexin V-FITC / PI double staining was used for analysis.

[0100] A375 cells in the logarithmic phase were used at a rate of 5 × 10⁻⁶ 5 / wells were inoculated into 6-well plates and incubated at 37 ℃ and 5% CO2 for 24 h. Then, the inoculum was replaced with an IC50 solution at a final concentration of 100 mg / L. 50 After culturing in the drug-containing medium for 24 h, cells were collected, washed with PBS, digested with trypsin without EDTA, centrifuged, and the supernatant was discarded. The cells were then washed twice with PBS. Binding Buffer was added to resuspend the cells, and Annexin V-FITC and PI staining solution were added. After incubation at room temperature in the dark for 15 min, the cells were observed and photographed under a fluorescence microscope. Alternatively, after processing according to the method, the fluorescence intensity was measured using an ultra-high-speed flow cytometer (Attune NxT, Thermo Fisher Scientific, USA). The experiment was repeated 3 times.

[0101] The results of the detection of apoptosis in A375 melanoma cells by PIQ and PIQ-NCs prepared in Example 1 showed that: the Control group had only weak green fluorescence, while the PIQ group had some red fluorescence that overlapped with the green fluorescence (green fluorescence indicates cell localization, and red fluorescence indicates that the PI dye enters the cell nucleus after the cell membrane is destroyed after apoptosis). The PIQ-NCs group showed strong red fluorescence, which preliminarily indicates that PIQ-NCs can promote apoptosis.

[0102] Flow cytometry analysis of apoptosis showed that almost no apoptosis occurred in the Control group, while apoptosis occurred in the PIQ and PIQ-NCs groups. The apoptosis effect in the PIQ-NCs group was significantly stronger than that in the PIQ group (**p < 0.01), indicating that PIQ-NCs can promote apoptosis.

[0103] (5) Cell cycle detection

[0104] To further investigate the anti-tumor mechanism, flow cytometry was used to explore the cell cycle arrest effect of PIQ and PIQ-NCs prepared in Example 1 on A375 cells. Cells treated with the drugs were collected, washed with PBS, and fixed in pre-chilled 75% ethanol overnight at -20°C. After centrifugation to remove the ethanol, cells were resuspended in RNase A, incubated at 37°C for 30 min, and then stained with 50 μg / mL PI staining solution at 4°C in the dark for 30 min. Fluorescence intensity was detected using the PI channel of an ultra-high-speed flow cytometer, and the percentage of cells in G1, S, and G2 / M phases (n=3) was analyzed using FlowJo software.

[0105] The results of the detection of the inhibitory effect of PIQ and PIQ-NCs prepared in Example 1 on A375 cells showed that the number of cells in the G1 phase increased significantly after drug administration (***p < 0.001), and the number of cells in the G1 phase in the PIQ-NCs group was significantly higher than that in the PIQ group (**p < 0.01). The G1 phase is mainly the stage of cell growth and preparation for DNA replication. The increase in G1 phase cells and the decrease in S phase cells after drug administration indicate that PIQ mainly inhibits cell proliferation by preventing DNA replication and synthesis.

[0106] The present invention uses the following methods to detect the needle content and mechanical strength of microneedles, respectively.

[0107] Needle content: The microneedles were placed on a glass slide and observed column by column under a polarizing microscope (4 ×). The needle content (N) of the microneedle tip was calculated according to Equation 3. eff ).

[0108] (Equation 3)

[0109] Among them, T n This refers to the total number of needles in the microneedle array; D n The number of defective needle tips.

[0110] Mechanical strength: Measured using a Texture Analyzer (TA-XT plus, Stable Micro Systems, UK). The microneedle was placed on a platform with the tip facing upwards. The microneedle was pressed down at a speed of 0.01 mm / s using a P / 2 probe (2 mm diameter), with a trigger force of 0.05 N. After reaching 90% deformation, the pressure was maintained for 5 s, and the force-displacement curve was recorded. The force corresponding to a displacement of 650 μm was taken as the tip mechanical strength.

[0111] Investigating the process optimization and characterization of 3PIQ-NCs microneedles

[0112] (1) Different matrix materials

[0113] Chitosan and PVP were selected as brittle materials, while PVA and HA were selected as toughening materials to prepare microneedles. The solution compatibility among PVPK30, PVA, chitosan, and HA (10-20 w, 20-40 w, and 80-150 w) was investigated. If a homogeneous phase could be formed, further investigation was conducted, and a better blank matrix formulation was screened. Observation revealed that a phase inversion phenomenon occurred when chitosan was mixed with HA of different molecular weights, failing to form a homogeneous solution. Analysis of their molecular structures showed that because chitosan carries a positive charge and hyaluronic acid carries a negative charge, electrostatic adsorption occurred when they were mixed, forming a polyelectrolyte complex. This interaction led to tight intermolecular bonding, resulting in phase separation. Furthermore, the microneedles prepared by mixing chitosan and PVP, both brittle materials, were prone to breakage during demolding due to their high hardness. The appearance of soluble microneedles prepared with different matrix materials is shown in the following figures. Figure 4 As shown in Figure A, cracks appeared in the backing layer and the needle tip broke, which could easily cause the microneedles to break and lose the drug during administration. When preparing microneedles using a composite of PVP and HA, it was found that due to the high viscosity of HA, phenomena such as air bubbles and needle tip bending occurred, which could easily prevent the needle tip from effectively penetrating the skin to release the drug during administration. When chitosan and PVA were combined, the needle content was better than that of the PVP and PVA combination. Therefore, chitosan and PVA were chosen as matrix materials for further research.

[0114] (2) PVA at different concentrations

[0115] The effects of different concentrations of PVA (9%, 11%, 13%, 15%, 17%) on microneedle performance were investigated. Since the addition of both materials affects the appearance of the microneedles, the effects of different PVA concentrations on the appearance and mechanical strength of the microneedles were also examined. The appearance of soluble microneedles prepared with different PVA concentrations was obtained as follows: Figure 4 As shown in Figure B, the mechanical strength of soluble microneedles prepared with different concentrations of PVA is as follows: Figure 4 As stated in C, when the PVA concentration is 13%, its mechanical strength is the greatest and its appearance is also better. Therefore, 2% chitosan and 13% PVA were selected as blank matrix materials.

[0116] (1) PIQ-NCs concentration

[0117] The effect of different PIQ-NCs concentrations (3%, 5%, 7%) on microneedle performance. Since PIQ-NCs use poloxamer P188 and PVP K30 as stabilizers and mannitol as a lyophilization protectant, the addition of PIQ-NCs to microneedles may affect their properties due to the presence of polymeric materials. Therefore, the effects of 3%, 5%, and 7% addition amounts on microneedles were investigated. The appearance of soluble microneedles prepared with different concentrations of PIQ-NCs is shown in the figures below. Figure 5As shown in Figure 5A, when 5% and 7% are added, the increased amount of polymer material causes needle tip bending, affecting the needle content, and the mechanical strength also decreases with increasing addition (see Figure 5A). Figure 5 B), and the microneedles have better appearance and higher mechanical strength when 3% is added. Therefore, the addition amount is selected as 1.5% to 6% (m / V), and 3% is preferred, as the appearance and mechanical strength of the microneedles are better.

[0118] The integrity, wrinkling, and bubble formation of the microneedle backing layer were observed using a stereomicroscope, the needle tip was observed using a polarizing and fluorescence microscope, and the needle body structure and morphology were observed using a scanning electron microscope (after gold sputtering, low-vacuum scanning at 10 kV). The observation results are as follows: Figure 6 As shown: Both blank and drug-loaded microneedles (containing 3% PIQ-NCs) were molded after drying and curing. After demolding, the backing layer was intact and there were no visible air bubbles. The needle content was 97.33±0.32% and 96.81±0.13%, respectively, with a difference of <1%. Drug loading has little impact on appearance and high drug delivery efficiency. The blank microneedles were pale yellow and transparent, while the drug-loaded microneedles were yellow and semi-transparent. Figure 6 (A, B); Because PIQ exhibits autofluorescence in the green fluorescence channel, the drug-eluting microneedles appear green under a fluorescence microscope ( Figure 6 In the middle D region, blank microneedles were not visible. Scanning electron microscopy showed (…). Figure 6 (E, F) The microneedle body is tightly connected to the backing layer, without hollows or bubbles, and the needle tip has a multi-layered four-sided pyramid structure, which is sharp and without bending or breakage.

[0119] (2) Mechanical strength of microneedles

[0120] The mechanical strength of the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 were tested using the above method. The results were as follows: the mechanical strength of the blank microneedles was 87.57 ± 2.24 N, and the mechanical strength of the PIQ-NCs microneedles prepared in Example 1 was 634.32 ± 8.34 N. Since the mold used to prepare the microneedles was a 225-needle array (each microneedle contained 225 needle tips), the breaking force of each needle tip was 0.39 ± 0.01 N and 2.82 ± 0.04 N, respectively, which is greater than the 0.2 N required to overcome the skin barrier. Furthermore, the mechanical strength of the PIQ-NCs microneedles prepared in Example 1 was significantly higher than that of the blank microneedles (***p < 0.001), indicating that the PIQ-NCs microneedles prepared in Example 1 can successfully penetrate the skin to release the drug and exert its effect.

[0121] (3) The puncture depth and puncture efficiency of microneedles

[0122] For the PIQ-NCs microneedles prepared in Example 1, skin penetration was simulated by pressing the Parafilm® M membrane with the microneedles. The penetration depth of the microneedles was examined by analyzing the changes in the membrane puncture rate. Eight layers of Parafilm® M membrane (each layer approximately 127 μm thick) were flattened and stacked on a 5 mm thick polystyrene foam (simulating elastic skin). After pressing the microneedles for 30 seconds, the number of punctures in each layer was counted, and the puncture rate (Peff) was calculated according to Equation 4. A graph was plotted with Peff as the ordinate and the number of puncture layers as the abscissa. The penetration depth of the microneedles and whether they effectively reached different depths of the skin were examined by analyzing the changes in the puncture rate of each layer.

[0123] (Equation 4)

[0124] Where, N m This refers to the number of micropores on the membrane; T n This refers to the total number of needles in the microneedle array.

[0125] The blank microneedles and the PIQ-NCs microneedles prepared in Example 1 were detected to puncture Parafilm. ® The appearance change trend of the M membrane is shown in the figure below. Figure 7 As shown in A, Figure 7 Chart B shows the puncture rate statistics, from... Figure 7 It can be seen that obvious puncture marks can be observed in the first two membranes, with a puncture efficiency of 100%. The first membrane also shows that the bottom of the PIQ-NCs microneedle prepared in Example 1 is quadrilateral. The third and fourth membranes show marks, but the puncture efficiency drops significantly. The fifth and sixth membranes show imprints, but the puncture efficiency is low. The seventh and eighth membranes have a puncture efficiency of 0%. The thickness of each membrane is about 127 μm, indicating that the PIQ-NCs microneedles prepared in Example 1 can penetrate to about 500 μm, which is sufficient to penetrate the stratum corneum of the skin and reach a certain depth.

[0126] Fresh, isolated rat skin tissue was used to investigate the ex vivo skin puncture efficiency of the PIQ-NCs microneedles prepared in Example 1 using trypan blue staining. Ex vivo rat skin was placed on a texture analyzer platform, and 0.4% (m / V) trypan blue microneedles were prepared. These were attached to a P / 50 probe (50 mm in diameter) and pressed down at a speed of 2 mm / s. After a trigger force of 0.05 N, the speed was reduced to 0.5 mm / s, and then maintained at 30 N for 1 min. The number of skin pores was counted, and the puncture efficiency was calculated according to Equation 4.

[0127] The results of skin puncture using blank microneedles and PIQ-NCs microneedles prepared in Example 1 are shown in the figure below. Figure 8As shown, the effective rates of skin puncture by the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 were 91.56 ± 0.93% and 92.44 ± 1.18%, respectively, indicating that the PIQ-NCs microneedles prepared in Example 1 can effectively penetrate the stratum corneum barrier to exert their therapeutic effects.

[0128] (4) Solubility of microneedles

[0129] To investigate whether the dissolution rate of soluble microneedles met the requirements for drug release, isolated rat skin was washed and dried, and laid flat on the texture analyzer platform. The microneedles were attached to the P / 50 probe (50 mm in diameter) and lowered at a rate of 2 mm / s. When the trigger force reached 0.05 N, the rate was lowered to 0.5 mm / s. After the pressure reached 30 N, the probe was held for 5 s, 30 s, 1 min, 2 min, 5 min, and 10 min, respectively. Then the probe was moved up, the microneedles were removed, and the swelling and dissolution were observed and recorded under a polarizing microscope.

[0130] The solubility test results of the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 are as follows: Figure 9 As shown, the tip of the PIQ-NCs microneedle dissolves in 10 seconds, swells in 1 minute, and almost completely dissolves within 10 minutes; there is no significant difference in the dissolution rate between the two, indicating that the PIQ-NCs microneedle can meet the requirements for drug release and is an ideal transdermal delivery carrier.

[0131] (5) PIQ content in microneedles

[0132] The drug content of the microneedles was determined by ultraviolet spectrophotometry. The mass of each microneedle tablet was 90 ± 0.21 mg. Three PIQ-NC@DMNs tablets were cut into small pieces, and 90 mg was accurately weighed and completely dissolved in 50% methanol. The PIQ concentration was determined using a validated ultraviolet spectrophotometric method (λ = 283 nm), and the drug loading was calculated. Each microneedle tablet was found to contain approximately 246 μg of PIQ.

[0133] (6) External safety performance

[0134] The cytotoxicity of the blank microneedles to A375 and HaCat cells was investigated using the CCK-8 assay described above. The results showed that the blank microneedles did not exhibit significant cytotoxicity in either cell type, and the cell viability was higher than 80%, indicating that the blank microneedles have good biocompatibility.

[0135] (7) Transdermal permeability

[0136] (S1) Model skin treatment: Scrape off the hair on the abdomen of the rat, apply hair removal cream and let it stand for 5 minutes, then wash off the fine hair with warm water; euthanize the rat 24 hours after hair removal, quickly remove the abdominal skin, peel off the subcutaneous and connective tissue, soak and wash three times with physiological saline to remove residue, and trim it with a scalpel to the appropriate size of the Franz diffusion cell.

[0137] (S2) Diffusion apparatus; a Franz vertical diffusion cell apparatus is used, with a diffusion cell diameter of 2 cm and an effective diffusion area of ​​3.14 cm². 2 The receiving pool has a volume of 8 mL. Place the skin between the supply pool and the receiving pool, with the stratum corneum facing the supply side and the dermis facing the receiving pool side, and secure the diffusion cup with a horseshoe clamp.

[0138] (S3) Transdermal test: The in vitro transdermal diffusion behavior of PIQ solution, PIQ-NCs solution and PIQ-NCs microneedles was investigated. A solution containing 1% SDS PBS (pH 7.4) was selected as the receiving solution.

[0139] The fixed diffusion cup was placed in a transdermal diffusion apparatus at a rotation speed of 300 r / min and a water bath temperature of 32 ℃. At 0.5, 1, 2, 4, 8, 12, 24, and 48 h after the start of the experiment, 4 mL of receiving solution was taken from the receiving cell and immediately replenished with an equal volume of receiving solution. The sample solution was processed as described above, with 1% SDS-PBS (pH 7.4) as a blank control. The UV absorbance at 283 nm was measured, and the cumulative permeation per unit area (Qn) was calculated according to Equation 5.

[0140] (Equation 5)

[0141] In the formula C n Let C be the concentration of PIQ in the subcutaneous receiving fluid at the nth time point. i V represents the PIQ concentration in the subcutaneous receiving fluid at the i-th time point. s Let V be the volume of the receiving pool, V be the sampling volume of the receiving liquid, and S be the effective permeation area of ​​the diffusion pool. Plot time t on the x-axis, and the cumulative permeation per unit area Q. n Plot Q with the vertical axis as the ordinate. n -t curves. Q-t curves of PIQ, PIQ-NCs, and PIQ-NCs microneedles were analyzed using Origin software. n -t curve equation is fitted.

[0142] The cumulative release of transdermal drug units was statistically analyzed, as shown in the following graph. Figure 10 As shown, by Figure 10It was found that after PIQ was processed into PIQ-NCs and PIQ-NCs microneedles in Example 1, the permeation per unit area increased. Origin software fitting of the permeation data revealed the transdermal mechanism: the cumulative permeation per unit area of ​​both PIQ-NCs and PIQ-NCs microneedles over 48 hours conformed to the Ritger-Peppas equation, with release indices of 0.31 and 0.39, respectively, indicating that drug release was Fick diffusion, mainly controlled by diffusion. The fitting effects of PIQ with the zero-order, first-order, Higuchi, and Ritger-Peppas equations were all poor, which may be due to the poor solubility of the PIQ raw material.

[0143] Investigating the safety of four PIQ-NCs microneedles

[0144] (1) Evaluation of healing at the puncture site

[0145] Both the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 were pressed perpendicularly to the dorsal skin surface of healthy nude mice for approximately 30 seconds. The microneedles were then removed, and the skin recovery was observed at different time points until the skin fully recovered to its pre-puncture state. The observations were recorded, and the healing status of the blank microneedles and the PIQ-NCs microneedles prepared in Example 1 after puncturing the skin of nude mice is shown in the figure below. Figure 11 As shown in A, by Figure 11 As shown in Figure A, obvious puncture marks can be observed immediately after the microneedles are removed, with a puncture rate of over 90%. Five minutes after the microneedles are removed, the channels begin to heal, and the diameter gradually decreases. At 10 minutes, some channels gradually heal, with less than 50% of the channels showing visible drug administration traces. At 15 minutes, the condition is basically the same as before drug administration, with no obvious drug administration marks and no erythema or wounds.

[0146] (2) H&E sections of tissue at the puncture site

[0147] The depth of microneedle insertion and the condition of the skin tissue after puncture were evaluated by performing hematoxylin and eosin (H&E) staining on skin sections after the above-mentioned punctures. The H&E-stained skin sections were observed and photographed under a microscope. The results of the tissue sections after puncture of nude mice in the normal group without microneedle puncture, the blank microneedle group, and the PIQ-NCs microneedle puncture group prepared in Example 1 are as follows: Figure 11 As shown in Figure B, both the blank microneedles and the drug-loaded microneedles were able to effectively penetrate the stratum corneum barrier of the skin after puncture, reaching a depth of approximately 300 μm, which is about 50% of the needle tip length. The fact that the prepared microneedles did not reach the actual length of insertion into the skin may be due to the elasticity of living skin. However, this depth is sufficient to penetrate the stratum corneum. Therefore, it can be concluded that this transdermal delivery system can break through the stratum corneum barrier to reach the dermis and release drugs, thus promoting transdermal drug delivery.

[0148] Investigating the in vivo tumor-suppressing effect of 5PIQ-NCs microneedles

[0149] Establishment of a melanoma model: A375 melanoma cell suspension was injected subcutaneously to construct the model: Log-phase A375 cells were collected and prepared into 5×10⁻⁶ cells. 7 A suspension of 100 μL was injected into the right forelimb axilla of Balb / c nude mice, leaving the needle in place for a few seconds before removal. Observations were made every two days. After six days, a movable, indurated mass without inflammation appeared at the inoculation site. The tumor was allowed to grow to 100 mm. 3 The model has been successfully established and can now be used for further research.

[0150] Experimental grouping: Sixteen tumor-bearing Balb / c nude mice were randomly divided into four experimental groups: ① negative control group (blank microneedle) (n = 4), ② positive control group (vemurafenib) (n = 4), ③ PIQ-NCs microneedle group (low dose) (n = 4), ④ PIQ-NCs microneedle group (high dose) (n = 4), and four non-tumor-bearing Balb / c nude mice were selected as the normal group.

[0151] Dosage Regimen: The positive control group used vemurafenib as the positive control drug. Vemurafenib is used to treat unresectable or metastatic melanoma with BRAF V600E mutation and is a first-line clinical treatment, approved by the FDA in 2011. Vemurafenib was administered in 100 μL via gavage using 5% DMSO, 5% PEG400, and 90% PBS as solvents. Specific dosing regimen is as follows:

[0152] ① Normal group; ② Negative control group: Blank microneedle transdermal drug delivery, administered every other day, for a total of 7 doses;

[0153] ③ Positive control group: administered by gavage at a dose of 30 mg / Kg every other day for a total of 7 times; ④ Low-dose PIQ-NCs microneedle group: administered by percutaneous microneedle puncture at a dose of 4 mg / Kg every other day for a total of 7 times; ⑤ High-dose PIQ-NCs microneedle group: administered by percutaneous microneedle puncture at a dose of 8 mg / Kg every other day for a total of 7 times.

[0154] Skin sectioning method: After euthanizing nude mice following the puncture experiment, skin tissue was collected and fixed with 4% paraformaldehyde for 24 h. The tissue was then processed using an automated dehydrator: 70% ethanol for 4 h → 85% ethanol for 2 h → 90% ethanol for 2 h → 95% ethanol for 1 h → anhydrous ethanol I, II, and III for 30 min each → benzene for 5-10 min → xylene I and II for 5-10 min each → paraffin I, II, and III at 65 ℃ for 1 h each. Paraffin embedding: After embedding in an embedding machine, the paraffin was cooled and solidified at -20 ℃, and the paraffin block was trimmed. Paraffin sectioning: 4 μm thick sections were cut using a microtome, spread out, and dried overnight in a 60 ℃ oven for later use. H&E staining: White slides were dewaxed by immersion in environmentally friendly dewaxing solutions I and II for 20 min each, followed by gradient ethanol dehydration (anhydrous ethanol I for 15 min → II for 5 min → 75% ethanol for 5 min), and washed with pure water; treated with high-definition constant staining pretreatment solution for 1 min, stained with hematoxylin for 5 min, rinsed with tap water for 2 min, differentiated with differentiation solution, washed with water and then stained with blue solution, rinsed with running water, dehydrated with 95% ethanol for 1 min, and stained with eosin for 15 s; dehydrated and mounted: anhydrous ethanol I, II, III for 2 min each → n-butanol I, II for 2 min each → xylene I, II for 2 min each for clearing, and mounted with neutral resin.

[0155] (1) Weight analysis

[0156] The weight of nude mice in each group was observed and recorded every other day. The differences in weight changes before and after drug administration were compared to preliminarily infer the safety of the drug. The weight changes of nude mice in each group during the drug administration process are shown in the graph below. Figure 12 As shown in Figure F, when measured at the same time point, there was no significant difference in the weight of tumor-bearing nude mice in each group compared with the normal group. After drug administration, the weight of nude mice in each group showed an upward trend. Although there were slight fluctuations in weight during the treatment period, this may be related to the fact that the nude mice themselves were relatively light. After the treatment, the weight was higher than before the treatment, indicating that the mice were in good condition and the drug was highly safe.

[0157] (2) Evaluation of tumor suppression effect

[0158] On the 15th day after administration, nude mice were subjected to enucleation to collect blood, followed by deep anesthesia, cervical dislocation, and euthanasia. The nude mice were then dissected to collect tumors and organs such as the heart, liver, spleen, lungs, and kidneys. The tumors and spleen were weighed, and the tumor weight inhibition rate, tumor volume inhibition rate, and spleen index were calculated using the following formulas.

[0159] (Formula 6);

[0160] (Equation 7);

[0161] (Equation 8);

[0162] (Equation 9);

[0163] Tumors and organs were fixed with 4% paraformaldehyde and H&E stained sections. Tumor sections were also stained with TUNEL and observed under a fluorescence microscope.

[0164] Immunohistochemical procedure: Paraffin sections were prepared according to the TUNEL staining method. After antigen retrieval and cooling, the sections were destained with PBS (pH 7.4) and washed three times (5 min each time) on a shaker. The sections were then incubated in 3% hydrogen peroxide solution at room temperature in the dark for 25 min, followed by three washes with PBS (5 min each time). 3% BSA was added to the histochemistry zone to cover the tissue, and the sections were blocked at room temperature for 30 min. After discarding the blocking solution, diluted primary antibody was added, and the sections were incubated overnight at 4 ℃ in a humidified chamber. The sections were washed three times with PBS (5 min each time), and HRP-labeled secondary antibody of the corresponding species was added to cover the tissue. The sections were incubated at room temperature for 50 min. The sections were washed three times with PBS (5 min each time), and fresh DAB chromogenic solution was added. The color development was controlled under a microscope (positive results were brownish-yellow), and the color development was stopped with tap water. Hematoxylin was counterstained for 3 min, washed with water, differentiated for a few seconds with differentiation solution, washed with water, and then blued with blue solution. The sections were rinsed with running water. The sections were then sequentially stained with 75% alcohol, 85% alcohol, anhydrous ethanol I and II, n-butanol, and xylene I for 5 minutes each. After dehydration and clearing, the slide is slightly dried, then sealed with mounting adhesive and observed under a microscope for imaging.

[0165] The tumor inhibition results of each group of nude mice are shown in the figure below. Figure 12 As shown in Figures A through E, by Figure 12 As shown in Figures A through E, after the administration was completed, the tumor volume and weight of the tumor-bearing nude mice in each treatment group were significantly different from those in the negative control group (***p < 0.001). The tumor-inhibiting effect of each treatment group was evaluated by the tumor inhibition rate based on volume and weight. The tumor inhibition rates, from highest to lowest, were: high-dose PIQ-NCs microneedle group, positive control group, and low-dose PIQ-NCs microneedle group. Compared with the positive control group, the tumor inhibition rates of the high-dose PIQ-NCs microneedle group were 82.27 ± 1.18% and 73.83 ± 3.08% respectively, showing a significant difference (**p < 0.01). This indicates that the transdermal drug delivery system has a strong anti-tumor effect and shows good therapeutic potential in the treatment of melanoma.

[0166] (3) Observation of tumor sections

[0167] To gain a deeper understanding of tumor factor expression, H&E analysis was performed on tumor tissues from each group of nude mice. The results are as follows: Figure 13As shown in Figure C. In the negative control group, tumor cells were arranged in a disordered and tightly packed manner, with a disordered tissue structure, frequent nuclear division, and abundant new blood vessels. Compared with the negative control group, the ratio of cell nucleus to cytoplasm was lower in each treatment group, and there were varying degrees of cell necrosis. Among them, the high-dose PIQ-NCs microneedle group showed significant reduction in tumor cell size, nuclear shrinkage, and the appearance of immune cells such as neutrophils, with vacuoles between cells, indicating the best effect.

[0168] (4) Hematologic and epithelial analysis of tumor tissues from nude mice in each group

[0169] TUNEL signaling indicates apoptosis or necrosis. The TUNEL analysis results of tumor tissues from each group of nude mice are as follows: Figure 13 As shown in Figure A. (As per...) Figure 13 As shown in Figure A, apoptotic positive cells exhibit red fluorescence. Analysis was performed using ImageJ software, as shown below. Figure 13 As shown in Figure B, all treatment groups showed significant differences compared to the negative control group (***p < 0.001). Among them, the high-dose PIQ-NCs microneedle group showed the strongest apoptosis-inducing effect. In vitro and in vivo experiments demonstrate that PIQ-NCs can promote melanoma apoptosis.

[0170] Ki67 is a nuclear protein associated with cell proliferation; higher Ki67 expression levels are associated with increased tumor cell proliferation activity and malignancy. The immunohistochemical analysis results of tumor tissues from various groups of nude mice are shown in the figure below. Figure 13 As shown in D, by Figure 13 The results showed that there were significant differences in positive expression between the drug-treated groups and the negative control group (**p<0.01), indicating that the drug could significantly inhibit Ki67 expression and tumor cell proliferation; the high-dose PIQ-NCs microneedle group was significantly different from the positive control group (###p<0.001), indicating that its effect on inhibiting tumor cell proliferation in vivo was better than that of the positive drug vemurafenib.

[0171] (5) Observation of tissue sections

[0172] Based on the blood biochemistry study, H&E staining analysis of nude mouse organs was performed using the above method to further investigate the drug's effects: Nude mice were dissected 24 hours after the last drug administration, and sections of the heart, liver, spleen, lung, and kidney were prepared and stained with H&E for microscopic observation. The H&E analysis results of the organs from each group of nude mice are shown in the figure below. Figure 14 As shown, by Figure 14The results showed that the cardiac cardiomyocytes in all groups of nude mice exhibited clear striations and no vacuoles, indicating no difference from the normal group, suggesting that the drug had no significant cardiotoxicity. In the liver tissue, the negative control group showed necrosis with disappearance of hepatic cords, vacuoles, and unclear lobular structure, while the other treated groups were no different from the normal group, indicating that the drug was non-toxic to the liver and could alleviate the lesions. In the spleen, the negative control group showed some vacuoles and unclear boundaries between the white and red pulp (possibly due to tumor-induced splenic hyperimmune response), while the treated groups were no different from the normal group, suggesting that the drug could reduce excessive immune response. There were no significant differences in lung and kidney tissue among the groups (normal alveolar structure and clear glomeruli), indicating that the drug had no significant toxic side effects on the lungs and kidneys.

[0173] The spleen is an immune organ of the body. The spleen index was used as an indicator to evaluate the effect of different drug administration methods on the spleen of nude mice. The spleen index results of each group of nude mice were obtained. The results showed that the spleen index of the negative control group was significantly increased (***p <0.001), and the spleen index of the drug decreased to varying degrees after drug administration. It can be inferred that the drug can alleviate spleen damage while exerting a therapeutic effect.

[0174] In summary, since melanoma is a type of skin cancer, this invention, through the preparation of PIQ-NCs microneedles, increases convenience while allowing the drug to directly penetrate the stratum corneum to reach the dermis and exert its effect, reducing toxic side effects on normal tissues. The PIQ-NCs microneedles have sharp tips with no breakage, a pyramidal structure, a mechanical strength of 634.32 ± 8.34 N, and can penetrate to a depth of 380 μm to reach the dermis, with a puncture efficiency exceeding 90%. The needles dissolve almost completely within 10 minutes of skin penetration. Compared with other treatment groups, the high-dose PIQ-NCs microneedle group showed less hepatotoxicity and could also slow down tumor-induced liver damage. Compared with the negative control group, the tumor-suppressing effects of each treatment group were significantly different. Further evaluation of the tumor-suppressing effects of each treatment group was conducted using volumetric and weight-based tumor inhibition rates. Compared with the positive control group using the first-line treatment drug vemurafenib, the high-dose PIQ-NCs microneedle group showed a more significant therapeutic effect and better safety, possibly due to the in-situ treatment at the tumor site. Observation of H&E-stained sections of tumor sites showed that all groups had therapeutic effects. The positive control group and the high-dose PIQ-NCs microneedling treatment group had comparable therapeutic effects, exhibiting phenomena such as extensive nuclear shrinkage and cell necrosis. Further TUNEL staining and immunohistochemical analysis of Ki67 protein expression in tumor tissue sections revealed that the high-dose PIQ-NCs microneedling group significantly promoted cell apoptosis compared to other treatment groups, demonstrating a significant therapeutic effect. Observation of H&E-stained sections of nude mouse organs in each treatment group showed that each treatment group caused less substantial damage to the organs and could also alleviate tumor-induced immune organ damage. Spleen index analysis showed that each treatment group could improve splenomegaly to varying degrees.

[0175] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing iridium complex nanocrystalline microneedles, characterized in that, Includes the following steps: (1) Mix iridium complex PIQ, organic solvent, pure water and stabilizer, and perform first stirring and homogenization in sequence to obtain PIQ nanocrystal suspension; The PIQ nanocrystal suspension was mixed with a freeze-drying protectant and then freeze-dried to obtain PIQ nanocrystals. The chemical formula of the iridium complex PIQ is [Ir(piq)2(ZZIP)]PF6; (2) Dissolve chitosan in an aqueous acetic acid solution, then add polyvinyl alcohol (PVA), and perform a second stirring, heating and cooling in sequence to obtain a matrix solution; After mixing the matrix liquid and the PIQ nanocrystals obtained in step (1), the mixture is stirred for the third time, injected into the mold, dried and demolded to obtain iridium complex nanocrystal microneedles.

2. The preparation method according to claim 1, characterized in that, The stabilizer in step (1) is selected from at least one of the following: polysorbate, cellulose, poloxamer, polyvinylpyrrolidone, vitamin E derivatives, phospholipids, anionic surfactants, polyoxyethylene castor oil, fatty acid sorbitan, high molecular weight polymers, chitosan and its derivatives, gelatin, amino acid surfactants, and polyethylene glycol derivatives.

3. The preparation method according to claim 1, characterized in that, In step (1), the first stirring speed is 2800~4000 r / min; the number of homogenizations is 20~40; and the homogenization pressure is 1500~1800 bar.

4. The preparation method according to claim 1, characterized in that, In step (1), the freeze-drying protectant is at least one of mannitol, sucrose, lactose, maltose, and trehalose.

5. The preparation method according to claim 1, characterized in that, In step (1), the average particle size of PIQ nanocrystals is 10~350 nm and the polydispersity index (PDI) is 0.01~0.

3.

6. The preparation method according to claim 1, characterized in that, The heating temperature in step (2) is 70~95℃, and the heating time is 20~40 min.

7. The preparation method according to claim 1, characterized in that, In step (2), the mass percentage of chitosan in the matrix solution is 1% to 5%, and the mass percentage of PVA in the matrix solution is 9% to 20%.

8. The preparation method according to claim 1, characterized in that, In step (2), the mass of PIQ nanocrystals accounts for 1.5% to 6% of the total mass of the iridium complex nanocrystal microneedles.

9. An iridium complex nanocrystal microneedle prepared by the preparation method according to any one of claims 1 to 8.

10. The use of an iridium complex nanocrystal microneedle prepared by the preparation method according to any one of claims 1 to 8, or the iridium complex nanocrystal microneedle according to claim 9, in the preparation of a microneedle patch for treating malignant melanoma.

Images