Application of polydopamine nanoparticles loaded with pharmaceutically active substances in preparation of drugs for treating posterior segment diseases of eyes

Abstract

The invention relates to application of polydopamine nanoparticles loaded with pharmaceutical active substances in preparation of drugs for treating posterior segment diseases of eyes, and belongs to the technical field of pharmaceutical preparations. According to the application of the polydopamine nanoparticles loaded with the pharmaceutical active substance in preparation of the medicine for treating the posterior ocular diseases, the polydopamine nanoparticles are solid polydopamine nanoparticles or mesoporous polydopamine nanoparticles, and the pharmaceutical active substance is the medicine for treating the posterior ocular diseases. According to the invention, the drug is loaded in the polydopamine nanoparticles, and the binding characteristic of polydopamine and melanin widely distributed in eye parts, especially fundus, is utilized, so that the transshipment capability of the drug to posterior eye segment tissues is enhanced in a non-invasive local administration manner, and the treatment effect on posterior eye segment diseases is improved; and the preparation method has a wide application prospect in local administration posterior segment drug delivery.

Description

TECHNICAL FIELD

[0001] The application relates to application of polydopamine nanoparticles loaded with a pharmacologically active substance in preparation of a medicine for treating ocular posterior segment diseases and belongs to the technical field of pharmaceutical preparations. BACKGROUND

[0002] With the aggravation of population aging, the wide use of electronic products and improper use of eyes, the incidence of various eye diseases is on the rise. Among them, the ocular posterior segment diseases at the fundus have become the main cause of blindness. At present, the way of vitreous injection of drugs to reach the ocular posterior segment disease site is generally adopted in clinical treatment of fundus diseases. Although this administration method can achieve a high therapeutic concentration in the ocular posterior segment, since the ocular posterior segment diseases are mostly chronic diseases that cannot be cured, patients often need to be injected for life, with a frequency of once every 1-3 months. This not only causes great adverse reactions, but also has high treatment cost and poor patient compliance. Therefore, seeking a non-invasive ocular posterior segment drug delivery strategy is the ultimate goal for the treatment of ocular posterior segment diseases.

[0003] Local administration is an ideal non-invasive administration method in the strategy of ocular administration, which can realize independent administration of patients. However, the traditional local administration method has the problem of fast drug clearance, which leads to low drug content in the eye and extremely low bioavailability of the ocular posterior segment tissue. In view of the problem that local administration is difficult to reach the ocular posterior segment tissue, currently, various strategies for increasing drug retention at the administration site and increasing transport to the ocular posterior segment lesion site are proposed, such as in-situ gel technology, nanotechnology, mucosal adhesion strategy and penetration promotion strategy. In addition to the above strategies for increasing passive transport, by analyzing the physiological structure of the eye, it is found that melanin, as an endogenous substance, has a wide distribution in the human body, among which the eyeball is the organ with the densest distribution of melanin, and more than 60% of which is distributed in the retinal pigment epithelial layer-choroid of the ocular posterior segment. The rich distribution of melanin makes many drugs accumulate in the retinal pigment epithelial layer (RPE)-choroid due to the specific binding ability with melanin, thereby increasing the retention of the drugs in the fundus and affecting the pharmacokinetics and pharmacodynamics behavior. However, everything has two sides. On the one hand, the combination of drugs and melanin can cause potential ocular toxicity, but on the other hand, this combination feature can be applied in the delivery system of the treatment drugs for fundus diseases to increase the targeting of the drugs and the driving force of active transport of the drugs. Based on this, some studies have proposed that the combination ability of some drugs with melanin in the RPE layer can be enhanced by modifying the compounds, so as to enhance the active transport ability of the drugs to the melanin-rich site and improve the targeting of the treatment drugs for fundus diseases.

[0004] Therefore, the ability to specifically bind to melanin is applied to carrier design, enabling the carrier to deliver a variety of drugs and thus becoming more versatile. Consequently, there is a need to develop delivery systems capable of binding to melanin, enhancing the ability of drug-carrying delivery systems to actively transport drugs to the posterior segment of the eye, ultimately improving the ability of local drug delivery to treat posterior segment eye diseases. Summary of the Invention

[0005] To address the challenges of delivering drugs locally to the posterior segment of the eye and the difficulty in selecting carrier materials with strong affinity for ocular melanin (i.e., different from modifying ordinary carrier surfaces with ligands that have active targeting effects) in existing technologies, this invention provides the application of pharmaceutically active polydopamine nanoparticles in the preparation of drugs for treating posterior segment eye diseases. This invention enhances drug delivery to the posterior segment of the eye through a non-invasive local administration method by loading drugs onto polydopamine nanoparticles and utilizing the binding properties of polydopamine to melanin, which is widely distributed in the eye, especially in the fundus. This improves the therapeutic effect of posterior segment eye diseases and has broad application prospects in local drug delivery to the posterior segment of the eye.

[0006] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the use of polydopamine nanoparticles loaded with a pharmaceutically active substance in the preparation of a drug for treating posterior segment ocular diseases, wherein the polydopamine nanoparticles are solid polydopamine nanoparticles or mesoporous polydopamine nanoparticles, and the pharmaceutically active substance is a drug for treating posterior segment ocular diseases.

[0007] In the above technical solution, the posterior segment eye disease is age-related macular degeneration, posterior uveitis, diabetic macular edema, diabetic retinopathy, choroidal neovascularization, retinitis pigmentosa, or fungal endophthalmitis.

[0008] In the above technical solution, the pharmaceutically active substance is a corticosteroid, a nonsteroidal anti-inflammatory drug, an immunosuppressant, a tyrosine kinase inhibitor, a carbonic anhydrase inhibitor, an antibiotic, an antifungal drug, an antiviral drug, an anticholinesterase drug, a β-receptor antagonist, a quinolone, a mydriatic, an antiallergen, an antiproliferator, or a decongestant.

[0009] Preferably, the corticosteroid is selected from triamcinolone acetonide, dexamethasone, dexamethasone acetate, betamethasone, fluocinolone acetonide, cortisone, betamethasone phosphate, budesonide, anecoxetine acetate, prednisolone acetate, or methylprednisolone sodium succinate.

[0010] Preferably, the nonsteroidal anti-inflammatory drug is selected from salicylates, celecoxib, indomethacin, ibuprofen, pranoprofen, diclofenac, flurbiprofen, piroxicam, or nabumetone.

[0011] Preferably, the immunosuppressant is selected from cyclosporine, azathioprine, or methotrexate.

[0012] Preferably, the tyrosine kinase inhibitor is selected from pazopanib.

[0013] Preferably, the carbonic anhydrase inhibitor is selected from brinzolamide.

[0014] Preferably, the antibiotic is selected from tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, brevicin, cephalexin, oxytetracycline, chloramphenicol, rifampin, ciprofloxacin, tobramycin, gentamicin, erythromycin, penicillin, sulfadiazine, sulfadiazine, sulfaacetamide, sulfadiazine, or sodium propionate.

[0015] Preferably, the antifungal drug is selected from amphotericin B or miconazole.

[0016] Preferably, the antiviral drug is selected from escitalopram, trifluorothymidine, acyclovir, or gapexicovir.

[0017] Preferably, the anticholinesterase drug is selected from pilocarpine, salicylates, carbachol, acetylcholine chloride, physostigmine, escythene, diisopropyl fluorophosphate, iodine phosphate, or cadmium bromide.

[0018] Preferably, the β-receptor antagonist is selected from timolol or betalol.

[0019] Preferably, the quinolone drug is selected from norfloxacin or levofloxacin.

[0020] Preferably, the mydriatic drug is selected from atropine or tropicamide.

[0021] Preferably, the anti-allergen is selected from sodium chromogenic acid, amprolium, methylpyridine, chlorpheniramine, cetirizine, pyramine, or acetanilide.

[0022] Preferably, the antiproliferative agent is selected from 1,3-cis-retinoic acid, 5-fluorouracil, paclitaxel, rapamycin, mitomycin C, or cisplatin.

[0023] Preferably, the decongestant is selected from phenylephrine, naphazoline, or tetrahydropyrazine.

[0024] In the above technical solution, the drug loading of the solid polydopamine nanoparticles is 2.0%~35%.

[0025] In the above technical solution, the drug loading of the mesoporous polydopamine nanoparticles is 15%~70%.

[0026] In the above technical solution, the particle size of the polydopamine nanoparticles is 5~250 nm.

[0027] In the above technical solution, the mesoporous polydopamine nanoparticles have a mesoporous pore size of 2~50 nm and a pore specific surface area of ​​50~100 m². 2 / g.

[0028] In the above technical solution, the polydopamine nanoparticles are prepared by one of the following methods: Solid polydopamine nanoparticles: The pH of the dopamine hydrochloride aqueous solution was adjusted to 8.0~11.0; then the pH-adjusted dopamine hydrochloride solution was added dropwise to the pharmaceutically active substance solution and stirred to obtain solid polydopamine nanoparticles; Mesoporous polydopamine nanoparticles: Add dopamine hydrochloride aqueous solution and pharmaceutically active substance solution dropwise to template agent solution, adjust pH to 8.0~11.0, stir, centrifuge, collect precipitate and redisperse with deionized water, then centrifuge again, repeat three times to obtain mesoporous polydopamine nanoparticles, wherein the template agent is selected from one or more of poloxamer F127 (F127), poloxamer 123 (P123), 1,3,5-trimethylbenzene (TMB), cyclohexane or decane.

[0029] Furthermore, the pharmaceutically active substance accounts for 2.0% to 35% of the mass percentage of the solid polydopamine nanoparticle formulation, and dopamine hydrochloride accounts for 65% to 98% of the mass percentage of the solid polydopamine nanoparticle formulation.

[0030] Furthermore, the pharmaceutically active substance accounts for 15% to 70% of the mass percentage of the mesoporous polydopamine nanoparticle formulation, dopamine hydrochloride accounts for 30% to 80% of the mass percentage of the mesoporous polydopamine nanoparticle formulation, and the template agent accounts for 0.8% to 3.5% of the mass percentage of the mesoporous polydopamine nanoparticle formulation.

[0031] Furthermore, the centrifugation conditions are centrifugation at a speed of 10,000~16,000 rpm / min for 10~30 min.

[0032] Furthermore, the stirring time is 2 to 24 hours.

[0033] In a second aspect, the present invention provides an eye drop for treating posterior segment eye diseases, the eye drop comprising polydopamine nanoparticles loaded with a pharmaceutically active substance, a liquid medium, and other pharmaceutically acceptable excipients.

[0034] Furthermore, the concentration of polydopamine in the eye drops of the polydopamine nanoparticles loaded with pharmaceutically active substances is 0.25~20.0 mg / mL.

[0035] Furthermore, the present invention can regulate the concentration of polydopamine nanoparticles per unit volume by adjusting the concentration of dopamine hydrochloride, thereby adjusting its binding ability with melanin and changing its ability to be delivered to the posterior segment of the eye.

[0036] The beneficial effects of this invention are as follows: This invention uses polydopamine, an endogenous melanin structural analog, as a carrier. Leveraging the widespread distribution of melanin in the retina and the binding affinity between melanin and polydopamine, it provides a simple and readily available delivery system with posterior ocular transport capabilities. By utilizing the affinity between polydopamine and melanin, drug-loaded polydopamine nanoparticles can actively aggregate towards the melanin distribution sites in the posterior ocular region, improving posterior ocular transport efficiency. Furthermore, this invention can regulate the posterior ocular transport efficiency by adjusting the concentration of polydopamine nanoparticles per unit volume, thereby modulating its binding affinity to melanin. Attached Figure Description

[0037] Figure 1 The figure shows the effect of the triamcinolone acetonide-loaded polydopamine nanoparticle eye drops obtained in Example 1 and the triamcinolone acetonide-loaded polydopamine nanoparticle eye drops containing different concentrations of dopamine hydrochloride on the distribution of ocular tissue (n=4).

[0038] Figure 2 The figure shows the therapeutic effects of the triamcinolone acetonide-loaded polydopamine nanoparticle eye drops obtained in Example 1 and the triamcinolone acetonide-loaded polydopamine nanoparticle eye drops containing different concentrations of dopamine hydrochloride on posterior segment ocular diseases. Detailed Implementation

[0039] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0040] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0041] The particle size and zeta potential of the present invention are determined by the following method: the particle size, PDI and zeta potential are measured using a Malvern laser particle size analyzer, wherein the instrument scattering angle is 90° and the test temperature is 25 ± 0.5°C.

[0042] The pore size and specific surface area of ​​the mesoporous nanoparticles described in this invention are measured using a specific surface area analyzer.

[0043] The drug loading and encapsulation efficiency described in this invention are determined as follows: 0.1 mL of a polydopamine solution loaded with the pharmaceutically active ingredient is accurately measured and placed in a 10 mL brown volumetric flask. The solution is diluted with acetone and brought to the mark with acetonitrile. After high-speed centrifugation, the supernatant is collected, and the drug content is measured by high-performance liquid chromatography. The formulas for calculating the drug loading and encapsulation efficiency are as follows:

[0044] The high-performance liquid chromatography (HPLC) conditions are as follows: Chromatographic column: ZORBAX column (150 mm × 4.6 mm, 5 µm); flow rate: 1 mL / min; injection volume: 10 µL; column temperature: 30℃; mobile phase for triamcinolone acetonide determination: acetonitrile:water (v:v = 40:60), detection wavelength: 240 nm; mobile phase for celecoxib determination: acetonitrile:water (v:v = 30:70), detection wavelength: 254 nm; mobile phase for betalol determination: acetonitrile:10 mM KH2PO4 (v:v = 30:70), detection wavelength: 220 nm; mobile phase for pazopanib determination: acetonitrile:0.02 M ammonium acetate (v:v = 65:35), detection wavelength: 268 nm; mobile phase for dexamethasone determination: acetonitrile:water (v:v = 40:60), detection wavelength: 254 nm.

[0045] Example 1 A method for preparing an eye drop for treating posterior segment eye diseases, comprising the following steps: The prescription composition, by weight percentage, is: triamcinolone acetonide 2.4%; dopamine hydrochloride 97.6%.

[0046] Preparation process: Accurately weigh 6.0 mg of dopamine hydrochloride and dissolve it in 2 mL of distilled water, adjusting the pH of the solution to 8.0~11.0; then accurately weigh 10.0 mg of triamcinolone acetonide and dissolve it in 1 mL of methanol, and add 15 μL of the triamcinolone acetonide methanol solution dropwise to the above dopamine hydrochloride solution and continue stirring for 4 h to evaporate the organic solvent, to obtain a triamcinolone acetonide-loaded polydopamine nanoparticle solution with a polydopamine concentration of 3 mg / mL.

[0047] Example 2 The prescription composition, by weight percentage, is: celecoxib 20.6%; dopamine hydrochloride 79.4%.

[0048] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water, and adjust the pH of the solution to 8.0~11.0; 130 μL of 10 mg / mL celecoxib methanol solution is added dropwise to the above pH-adjusted dopamine hydrochloride solution and stirred for 4 h to obtain a celecoxib-loaded polydopamine nanoparticle solution.

[0049] Example 3 The prescription composition, by weight percentage, is: betalol 33.3%; dopamine hydrochloride 66.7%.

[0050] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water, and adjust the pH of the solution to 8.0~11.0; Measure 250 μL of 10 mg / mL betalol methanol solution and add it dropwise to the above pH-adjusted dopamine hydrochloride solution and continue stirring for 4 h to obtain betalol-loaded polydopamine nanoparticle solution.

[0051] Example 4 The prescription composition by weight percentage is: F127 0.8%; pazopanib 19.8%; dopamine hydrochloride 79.4%.

[0052] Preparation process: Accurately weigh 0.1 mg of the prescribed amount of F127 and dissolve it in 2.5 mL of distilled water. Then, add 5.0 mg of dopamine hydrochloride and 130 μL of pazopanib methanol solution (10 mg / mL) dropwise. Adjust the pH of the solution to 8.0~11.0 and continue stirring for 4 h. Centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water, and centrifuge again. Repeat this process three times. Finally, collect the lower precipitate and redisperse it in deionized water to obtain a pazopanib-loaded mesoporous polydopamine nanoparticle solution.

[0053] Example 5 The prescription composition by weight percentage is: P123 0.8%; triamcinolone acetonide 52.5%; dopamine hydrochloride 46.7%.

[0054] Preparation process: Accurately weigh 0.2 mg P123 and dissolve it in 2.5 mL of distilled water. Then, add 5.0 mg of dopamine hydrochloride aqueous solution and 570 μL of triamcinolone acetonide methanol solution (10 mg / mL) dropwise. Adjust the pH of the solution to 8.0~11.0 and continue stirring for 4 h. Centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water, and centrifuge again. Repeat three times. Finally, collect the lower precipitate and redisperse it in deionized water to obtain a triamcinolone acetonide-loaded mesoporous polydopamine nanoparticle solution.

[0055] Example 6 The prescription composition by weight percentage is: TMB 0.8%; dexamethasone 66.1%; dopamine hydrochloride 33.1%.

[0056] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water; accurately weigh 0.3 mg of TMB reagent and 1.0 mL of dexamethasone methanol solution (10 mg / mL) and add them dropwise to the dopamine hydrochloride solution. Adjust the pH of the solution to 8.0~11.0, continue stirring for 4 h, centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water and centrifuge again, repeat three times, and finally collect the lower precipitate and redisperse it in deionized water to obtain a dexamethasone-loaded mesoporous polydopamine nanoparticle solution.

[0057] Example 7 The formulation consists of the following components by weight percentage: cyclohexane 2.2%; prednisolone 19.6%; dopamine hydrochloride 78.2%.

[0058] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water; accurately weigh 0.3 mg of cyclohexane reagent and 130 μL of prednisolone methanol solution (10 mg / mL) and add them dropwise to the dopamine hydrochloride solution. Adjust the pH of the solution to 8.0~11.0, continue stirring for 4 h, centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water and centrifuge again. Repeat three times. Finally, collect the lower precipitate and redisperse it in deionized water to obtain a solution of prednisolone-loaded mesoporous polydopamine nanoparticles.

[0059] Example 8 The formulation consists of the following components by weight percentage: F127 1.0%; TMB 1.2%; triamcinolone acetonide 51.8%; and dopamine hydrochloride 46.0%.

[0060] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water; accurately weigh 0.2 mg of F127 and 0.3 mg of TMB, and add 130 μL of triamcinolone acetonide methanol solution (10 mg / mL) dropwise to the dopamine hydrochloride solution, adjust the pH of the solution to 8.0~11.0, continue stirring for 4 h, centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water and centrifuge again, repeat three times, finally collect the lower precipitate and redisperse it in deionized water to obtain a triamcinolone acetonide-loaded mesoporous polydopamine nanoparticle solution.

[0061] Example 9 The formulation consists of the following components by weight percentage: decane 2.2%; celecoxib 65.2%; dopamine hydrochloride 32.6%.

[0062] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water; accurately weigh 0.4 mg of decane reagent and 1.0 mL of celecoxib methanol solution (10 mg / mL) and add them dropwise to the dopamine hydrochloride solution. Adjust the pH of the solution to 8.0~11.0, continue stirring for 4 h, centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water and centrifuge again. Repeat three times. Finally, collect the lower precipitate and redisperse it in deionized water to obtain a celecoxib-loaded mesoporous polydopamine nanoparticle solution.

[0063] Example 10 The formulation, by weight percentage, consists of: P123 2.0%; TMB 1.5%; Pazopanib 19.3%; Dopamine Hydrochloride 77.2%. Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water; accurately weigh 0.2 mg of P123 and 0.1 mg of TMB, and add 125 μL of pazopanib methanol solution (10 mg / mL) dropwise to the dopamine hydrochloride solution, adjust the pH of the solution to 8.0~11.0, continue stirring for 4 h, centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water and centrifuge again, repeat three times, finally collect the lower precipitate and redisperse it in deionized water to obtain a pazopanib-loaded mesoporous polydopamine nanoparticle solution.

[0064] Example 11 The prescription composition by weight percentage is: TMB 3.5%; triamcinolone acetonide 51.1%; dopamine hydrochloride 45.4%.

[0065] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water; accurately weigh 0.4 mg of TMB reagent and 560 μL of triamcinolone acetonide methanol solution (10 mg / mL) and add them dropwise to the dopamine hydrochloride solution. Adjust the pH of the solution to 8.0~11.0, continue stirring for 4 h, centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water and centrifuge again, repeat three times, and finally collect the lower precipitate and redisperse it in deionized water to obtain a triamcinolone acetonide-loaded mesoporous polydopamine nanoparticle solution.

[0066] Example 12 The prescription composition by weight percentage is: F127 3.5%; triamcinolone acetonide 64.3%; dopamine hydrochloride 32.2%.

[0067] Preparation process: Accurately weigh 5.0 mg of dopamine hydrochloride and dissolve it in 2.5 mL of distilled water; accurately weigh 0.6 mg of TMB reagent and 1.0 mL of triamcinolone acetonide methanol solution (10 mg / mL) and add them dropwise to the dopamine hydrochloride solution. Adjust the pH of the solution to 8.0~11.0, continue stirring for 4 h, centrifuge at 10,000 rpm / min for 10 min, discard the supernatant, take the lower precipitate, redisperse it with deionized water and centrifuge again. Repeat three times. Finally, collect the lower precipitate and redisperse it in deionized water to obtain a triamcinolone acetonide-loaded mesoporous polydopamine nanoparticle solution.

[0068] The basic physicochemical properties of the drug-loaded polydopamine nanoparticles obtained in Examples 1-12 are shown in Table 1.

[0069] Table 1. Physicochemical properties of polydopamine nanoparticles with different formulations (n=3)

[0070] Example 13 To demonstrate the advantages of the polydopamine nanoparticles provided by this invention in the delivery of posterior ocular tissue, a solution of polydopamine nanoparticles loaded with triamcinolone acetonide was used as the drug delivery formulation. In addition, a triamcinolone acetonide solution, a polydopamine gel containing triamcinolone acetonide, and a PLGA-PEG NPs solution containing triamcinolone acetonide were used as control groups to evaluate the delivery effect of the polydopamine nanoparticles provided by this invention in posterior ocular tissue.

[0071] (1) The preparation process of the control group is as follows: Triamcinolone-loaded polydopamine nanoparticle solutions: 1.0 mg, 2.0 mg, 4.0 mg, and 6.0 mg of dopamine hydrochloride were accurately weighed and dissolved in 2 mL of distilled water, and the pH of the solution was adjusted to 8.0–11.0. Then, 10.0 mg of triamcinolone was accurately weighed and dissolved in 1 mL of methanol. 20 μL, 40 μL, 80 μL, and 120 μL of the triamcinolone methanol solution were added dropwise to the above dopamine hydrochloride solution, and the mixture was stirred for 4 h to evaporate the organic solvent. The mixture was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the lower precipitate was redispersed with deionized water and centrifuged again. This process was repeated three times. Finally, the lower precipitate was collected and redispersed in deionized water to obtain triamcinolone-loaded polydopamine nanoparticle solutions with polydopamine concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 3 mg / mL, respectively.

[0072] Triamcinolone (TA) solution: Accurately weigh 6.0 mg of triamcinolone and 120 mg of PEG400 and dissolve them in 1 mL of distilled water. Stir well to obtain a triamcinolone solution.

[0073] Triamcinolone acetonide-containing polydopamine gel (Gel): Accurately weigh 150 mg poloxamer 407 (P407) and 40 mg hydroxypropyl methylcellulose (HPMC 606W) and dissolve them in 1 mL of distilled water, stirring to obtain a gel solution. Then, accurately weigh 2.0 mg dopamine hydrochloride and 3.0 mg triamcinolone acetonide and slowly add them to the above gel solution, stirring until homogeneous to obtain triamcinolone acetonide polydopamine gel.

[0074] PLGA-PEG NPs solution containing triamcinolone acetonide: Accurately weigh 10.0 mg PLGA 30000 -PEG 2000 1.0 mg of triamcinolone acetonide was dissolved in 1 mL of ethyl acetate, and 0.1% (v / v) Tween 80 and 0.1% (v / v) anhydrous ethanol were added to obtain the oil phase. The aqueous phase was a 0.1% (w / v) PVA solution. Under high-speed shearing conditions of 9000 rpm, 1 mL of the oil phase was injected into 2 mL of the aqueous phase, and the emulsification process lasted for 40 s to obtain an O / W type primary emulsion. The primary emulsion was sonicated using a probe at 200 W power for 2 min (3 s on, 3 s off), and then the solution was stirred at 25 °C for 24 h to evaporate the solvent, thus obtaining a nanoparticle solution containing 16.7% PEG.

[0075] (2) Experimental methods The distribution of eye tissue was studied using male Japanese white rabbits (albino rabbits) and Dutch rabbits (pigmented rabbits) weighing 1.8–2.0 kg, provided by the Animal Center of Shenyang Pharmaceutical University.

[0076] The experiment consisted of seven groups: triamcinolone acetonide solution (12% PEG400 with a drug loading of 50 μg TA); triamcinolone acetonide polydopamine gel (with a drug loading of 50 μg TA); PLGA-PEG NPs solution containing 16.7% PEG and loaded with triamcinolone acetonide (with a drug loading of 50 μg TA); polydopamine nanoparticle solution with a dopamine hydrochloride concentration of 0.5 mg / mL (with a drug loading of 50 μg TA); polydopamine nanoparticle solution with a dopamine hydrochloride concentration of 1 mg / mL (with a drug loading of 50 μg TA); polydopamine nanoparticle solution with a dopamine hydrochloride concentration of 2 mg / mL (with a drug loading of 50 μg TA); and polydopamine nanoparticle solution with a dopamine hydrochloride concentration of 3 mg / mL (with a drug loading of 50 μg TA).

[0077] Rabbits were uniformly administered the drug intraconjunctival sac via the lower eyelid. 50 μL was slowly injected over 2 minutes to prevent leakage. The upper and lower eyelids were closed for 10 seconds after administration, and timing was started at this point. Rabbits were euthanized by injecting air into the marginal ear vein at 0.25 h, 0.5 h, 2 h, and 6 h, respectively. The eyeballs were rapidly enucleated, rinsed with physiological saline, and aqueous humor and vitreous humor were aspirated. The cornea, conjunctiva, iris, sclera, and choroid-retina were harvested, accurately weighed, and homogenized in buffered saline solution.

[0078] a. Pretreatment of aqueous humor and vitreous fluid: Accurately pipette 100 μL of aqueous humor and 200 μL of vitreous fluid, and add 200 μL and 300 μL of internal standard solution (prednisolone methanol solution 5 μg / mL), respectively. Then add 100 μL and 200 μL of protein precipitant (0.1% formic acid methanol solution), respectively. Vortex for 5 min, centrifuge at 12,000 rpm for 10 min, and evaporate to dryness with nitrogen. Redissolve in 100 μL of mobile phase, and centrifuge at 12,000 rpm for 10 min. Inject 10 μL of the supernatant.

[0079] b. Pretreatment of the cornea, conjunctiva, iris, sclera, and choroid-retina Corneal, conjunctival, iris, sclera, and choroid-retinal tissues were blotted dry with filter paper and accurately weighed. Each tissue was thoroughly minced and homogenized using a tissue homogenizer with 700 μL of buffered saline. 200 μL of homogenate was accurately pipetted into a vortex mixer, and 200 μL of internal standard solution and 1 mL of protein precipitant were added. The mixture was vortexed for 5 min, centrifuged at 12,000 rpm for 10 min, and allowed to evaporate to dryness under nitrogen. The mixture was reconstituted with 100 μL of mobile phase and centrifuged at 12,000 rpm for 10 min. 10 μL of the supernatant was injected as a sample.

[0080] Because the common Japanese white rabbits, which are commonly used in ocular animal experiments, lack melanin in their eyes and are often referred to as albino rabbits, this invention, in order to investigate the interaction with intraocular melanin, selected Dutch rabbits with melanin distribution in their eyes for comparison with common Japanese white rabbits. The aim was to explore the differences in the distribution of melanin-specific polydopamine nanoparticles (PDA NPs) and ordinary PLGA-PEG NPs in the eyes of pigmented and albino rabbits, and whether the concentration of polydopamine PDA (dopamine hydrochloride can be almost completely converted into polydopamine) affected the distribution in the eyes of pigmented rabbits. The results are as follows: Figure 1 As shown in Table 2, the calculated pharmacokinetic parameters are shown in Table 2.

[0081] First, the distribution of the same formulation in the ocular tissues of albino and pigmented rabbits was compared. In all ocular tissues, there were no significant differences in the AUC (Average Values) between the TA solution group and the PLGA-PEG NPs group in both albino and pigmented rabbit tissues. This indicates that the distribution of ordinary solutions and conventional NPs that do not bind to melanin in the eye is unrelated to the distribution of melanin. Although the gel group contained polydopamine, due to the gel structure, the polydopamine could not be completely released into the eye; therefore, only in the iris of pigmented rabbits was the AUC 1.2 times higher than that of the iris of albino rabbits. p <0.05). There were no statistically significant differences between albino and pigmented rabbits in other tissues.

[0082] However, the presence of melanin significantly affected the distribution of PDA NPs in ocular tissues. In the conjunctiva, the drug concentrations of different concentrations of PDA NPs in pigmented rabbits were significantly higher than those in albino rabbits, and the drug concentration in the conjunctiva of pigmented rabbits continued to increase with increasing PDA concentration. The AUC values ​​of 2 and 3 mg / mL PDA NPs in the conjunctiva of pigmented rabbits were 1.3 and 1.6 times higher than those in albino rabbits, respectively. p <0.05). Results for the sclera and conjunctiva were similar. Pharmacokinetic calculations showed that as the PDA concentration increased from 0.5 mg / mL to 3 mg / mL, the AUC values ​​of the sclera in pigmented rabbits were 1.3, 1.4, 1.6, and 1.7 times that of albino rabbits, respectively. p <0.05). In the posterior segment choroid-retina, as the PDA concentration increased from 0.5 mg / mL to 3 mg / mL, the AUC values ​​in the choroid-retina of pigmented rabbits were 1.3, 1.4, 2.0, and 2.2 times that of albino rabbits, respectively. p <0.05). Furthermore, differences in melanin distribution were also observed in the cornea due to its presence. The AUC value in the cornea of ​​pigmented rabbits was lower than that of albino rabbits; the AUC values ​​in the 0.5, 1, 2, and 3 mg / mL PDA NPs groups were reduced by 17.1%, 21.4%, 28.9%, and 23.5% respectively compared to albino rabbits. p <0.05). The above results indicate that PDA NPs can serve as nanoparticles with a specific binding ability to melanin. Specifically, PDA NPs exhibit a significant melanin-oriented distribution, which significantly increases the distribution of PDA NPs in melanin-rich rabbit tissues. Furthermore, this melanin-oriented distribution ability becomes stronger with increasing PDA concentration, further promoting the drug transport efficiency in the posterior segment of the eye.

[0083] Table 2. Major pharmacokinetic parameters of various tissues in the rabbit eye after administration of eye drops (n=4)

[0084] & p <0.05: Comparison with albino rabbits of the same prescription Example 14 To demonstrate the therapeutic effect of the polydopamine nanoparticles provided by this invention on posterior ocular diseases, choroidal neovascularization was used as a disease model. A solution of polydopamine nanoparticles loaded with triamcinolone acetonide was used as the drug delivery formulation, and a triamcinolone acetonide solution and a PLGA-PEG NPs solution containing triamcinolone acetonide were used as control groups to evaluate the therapeutic effect of the polydopamine nanoparticles provided by this invention on posterior ocular diseases.

[0085] (1) The preparation process of the control group is the same as that of the control group in Example 13. The polydopamine nanoparticle solution loaded with triamcinolone was selected with polydopamine concentrations of 1 mg / mL and 3 mg / mL.

[0086] (2) Experimental methods The distribution of eye tissue was studied using male Japanese white rabbits (albino rabbits) and Dutch rabbits (pigmented rabbits) weighing 1.8–2.0 kg, provided by the Animal Center of Shenyang Pharmaceutical University.

[0087] A rabbit model of choroidal neovascularization was induced by injecting vascular endothelial growth factor (VEGF). The specific method is as follows: After anesthesia via the marginal ear vein, 10 μL of PBS solution containing 1 μg of recombinant human VEGF165 was injected into the vitreous body using a 30G needle. On the 7th day after injection, albino rabbits and pigmented rabbits were randomly divided into groups of 4 rabbits each: (1) saline model; (2) intravitreal injection of TA solution; (3) PLGA-PEG NPs eye drops; (4) 1 mg / mL PDA NPs eye drops; (5) 3 mg / mL PDA NPs eye drops for 14 consecutive days (total dose 0.28 g). Intravitreal injection was administered only once on the first day, and eye drops were administered twice daily at 8-hour intervals (10 μg TA each time, 20 μg TA daily).

[0088] Blood samples were collected from the marginal ear vein on days 1, 5, 10, and 14 after the start of treatment. The collected blood samples were centrifuged at high speed (12,000 rpm, 10 min), and plasma was collected and stored at -20°C. After 14 days of treatment, the rabbits were euthanized, and the eyeballs were removed. The choroid-retina was separated and processed according to the method described in Example 13. The levels of VEGF in plasma and TNF-α and MCP-1 in the choroid-retina were determined using enzyme-linked immunosorbent assay (ELISA). Eyeballs from each group were fixed with 4% paraformaldehyde for 24 h, and whole-eye paraffin sections were stained with hematoxylin and eosin. The specimens were observed under an optical microscope. Results are as follows: Figure 2 As shown.

[0089] First, the treatment efficacy was evaluated by measuring VEGF levels in plasma. Results were as follows: Figure 2 As shown in -B and 2-C, the VEGF level in the saline group (disease model group) was significantly higher than that in the healthy group on day 0. p A value <0.001 indicates that the disease model was successfully established. After 14 days of treatment, the VEGF level in the disease model group was still 2.5 times that of the healthy group. p <0.05%. The IVT group achieved the most significant reduction in VEGF levels on day 5 post-injection, at which point VEGF levels were comparable to those in the healthy group. However, starting from day 5, VEGF levels began to rise. By the final day 14, the VEGF levels in the IVT group were 33.8% lower than those in the model group (…). p <0.05%. Among all NP administration combinations, PLGA-PEG NPs and 1 mg / mL PDA NPs showed comparable therapeutic effects in albino and pigmented rabbits. However, 3 mg / mL PDA NPs showed superior VEGF downregulation in pigmented rabbits compared to albino rabbits, reducing VEGF levels by 37.1% and 30.7% respectively compared to the model group. p <0.05).

[0090] Besides VEGF levels, two other factors are also considered to be associated with angiogenesis and inflammatory responses: tumor necrosis factor-α (TNF-α), a cytokine that regulates cell-mediated angiogenesis and anti-inflammatory responses, and also promotes the production of inflammatory cytokines, including monocyte chemoattractant protein-1 (MCP-1), by retinal endothelial cells and retinal vasculature. Therefore, the levels of these two proteins in the choroid-retina were measured after 14 days of continuous administration. Results are as follows... Figure 2 - (DE) shows that the NPs group significantly downregulated TNF-α levels in both albino and pigmented rabbits. However, unlike PLGA-PEG NPs, which showed similar downregulation effects in both albino and pigmented rabbits, 3 mg / mL PDA NPs downregulated VEGF levels more effectively in pigmented rabbits than in albino rabbits. p <0.05). A similar trend was observed in MCP-1. PLGA-PEG NPs showed similar downregulation effects in albino and pigmented rabbits, downregulating by 53.6% and 51.1% respectively compared to the model group. Meanwhile, 3 mg / mL PDA NPs downregulated MCP-1 by 50.2% and 63.8% in albino and pigmented rabbits, respectively. p <0.05). Ultimately, the levels of TNF-α and MCP-1 in the pigmented rabbits administered 3 mg / mL PDA NPs were comparable to those in the intravitreal injection group.

[0091] In a normal retina, the layers are neatly arranged with clear boundaries. However, in the disease model group, neovascularization penetrated the entire retinal structure. Figure 2 -G). After treatment, the area of ​​neovascularization decreased, and the structures of different layers of the retina recovered to varying degrees. Specifically, the thickness of the neovascularization area was significantly reduced in the vitreous injection group, but still significantly higher than that in the healthy group. PLGA-PEG NPs reduced the thickness of the neovascularization area in albino and pigmented rabbits by 59.0% and 56.5%, respectively, but there was no statistically significant difference between the two types of rabbits ( p >0.05). However, the thickness of the neonatal region in pigmented rabbits was comparable to that in the intravitreal injection group with 3 mg / mL PDA NPs. Furthermore, the treatment effect was superior in pigmented rabbits compared to albino rabbits, as shown by quantitative results ( Figure 2 -F), the thickness of neonatal regions in albino and pigmented rabbits treated with 3 mg / mL PDA NPs was reduced by 61.2% and 69.7%, respectively, compared to the disease model group. p <0.05).

Claims

1. The application of polydopamine nanoparticles loaded with pharmaceutically active substances in the preparation of drugs for treating posterior segment ocular diseases, characterized in that: The polydopamine nanoparticles are solid polydopamine nanoparticles or mesoporous polydopamine nanoparticles, and the pharmaceutically active substance is a drug for treating diseases of the posterior segment of the eye.

2. The application according to claim 1, characterized in that: The posterior segment eye diseases mentioned are age-related macular degeneration, posterior uveitis, diabetic macular edema, diabetic retinopathy, choroidal neovascularization, retinitis pigmentosa, or fungal endophthalmitis.

3. The application according to claim 1, characterized in that: The medications used to treat diseases of the posterior segment of the eye include corticosteroids, nonsteroidal anti-inflammatory drugs, immunosuppressants, tyrosine kinase inhibitors, carbonic anhydrase inhibitors, antibiotics, antifungals, antivirals, anticholinesterase drugs, beta-receptor antagonists, quinolones, mydriatics, antiallergens, antiproliferative agents, or decongestants.

4. The application according to claim 3, characterized in that: The corticosteroids are selected from triamcinolone acetonide, dexamethasone, dexamethasone acetate, betamethasone, fluocinolone acetonide, cortisone, betamethasone phosphate, budesonide, anecoxetine acetate, prednisolone acetate, or methylprednisolone sodium succinate; the nonsteroidal anti-inflammatory drugs are selected from salicylates, celecoxib, indomethacin, ibuprofen, pranoprofen, diclofenac, flurbiprofen, piroxicam, or nabumetone; the immunosuppressants are selected from cyclosporine, azathioprine, or methotrexate; the tyrosine kinase inhibitors are selected from pazopanib; the carbonic anhydrase inhibitors are selected from brinzolamide; the antibiotics are selected from tetracycline, chlortetracycline, bacitracin, neomycin, polymyxin, brevicin, cephalexin, oxytetracycline, chloramphenicol, rifampin, ciprofloxacin, tobramycin, gentamicin, erythromycin, penicillin, sulfadiazine, sulfadiazine, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole, sulfamethoxazole The antifungal drug is selected from amphotericin B or miconazole; the antiviral drug is selected from escitalidine, trifluorothymidine, acyclovir, or gapexicocyclovir; the anticholinesterase drug is selected from pilocarpine, salicylates, carbachol, acetylcholine chloride, physostigmine, esethene, diisopropyl fluorophosphate, iodine phosphate, or cadmium bromide; the β-receptor antagonist is selected from timolol or betalol; the quinolone... The ketone drugs are selected from norfloxacin or levofloxacin; the mydriatic drugs are selected from atropine or tropicamide; the anti-allergen drugs are selected from sodium chromogenic acid, amprolium, methylpyridine, chlorpheniramine, cetirizine, pyramine, or acetanilide; the antiproliferative agents are selected from 1,3-cis-retinoic acid, 5-fluorouracil, paclitaxel, rapamycin, mitomycin C, or cisplatin; and the decongestants are selected from phenylephrine, naphazoline, or tetrahydropyrazine.

5. The application according to claim 1, characterized in that: The drug loading of the solid polydopamine nanoparticles is 2.0% to 35%; the drug loading of the mesoporous polydopamine nanoparticles is 15% to 70%.

6. The application according to claim 1, characterized in that: The polydopamine nanoparticles have a particle size of 5-250 nm; the mesoporous polydopamine nanoparticles have a mesoporous pore size of 2-50 nm and a pore specific surface area of ​​50-100 m². 2 / g.

7. The application according to claim 1, characterized in that: The polydopamine nanoparticles loaded with the pharmaceutically active substance were prepared by one of the following methods: Solid polydopamine nanoparticles loaded with pharmaceutically active substances: The pH of the dopamine hydrochloride aqueous solution was adjusted to 8.0~11.0; then the pH-adjusted dopamine hydrochloride solution was added dropwise to the pharmaceutically active substance solution, and the mixture was stirred to obtain solid polydopamine nanoparticles; Mesoporous polydopamine nanoparticles loaded with pharmaceutically active substances: Dopamine hydrochloride aqueous solution and pharmaceutically active substance solution are added dropwise to template agent solution, pH is adjusted to 8.0~11.0, stirred, centrifuged, precipitate is collected and redispersed with deionized water, and centrifuged again. This process is repeated three times to obtain mesoporous polydopamine nanoparticles. The template agent is selected from one or more of poloxamer F127, poloxamer 123, 1,3,5-trimethylbenzene, cyclohexane or decane.

8. The application according to claim 7, characterized in that: The pharmaceutically active substance accounts for 2.0% to 35% of the mass percentage of the solid polydopamine nanoparticle formulation, and dopamine hydrochloride accounts for 65% to 98% of the mass percentage of the solid polydopamine nanoparticle formulation. The pharmaceutically active substance accounts for 15% to 70% of the mass percentage of the mesoporous polydopamine nanoparticle formulation, dopamine hydrochloride accounts for 30% to 80% of the mass percentage of the mesoporous polydopamine nanoparticle formulation, and the template agent accounts for 0.5% to 3.5% of the mass percentage of the mesoporous polydopamine nanoparticle formulation.

9. An eye drop for treating posterior segment eye diseases, characterized in that: The eye drops contain polydopamine nanoparticles loaded with pharmaceutically active substances, a liquid medium, and other pharmaceutically acceptable excipients.

10. The eye drops according to claim 9, characterized in that: The concentration of polydopamine in the polydopamine nanoparticles loaded with pharmaceutically active substances in the eye drops is 0.25~20.0 mg / mL.

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