A ROS-responsive micelle-gel complex for treating corneal neovascularization

Abstract

The present invention provides a ROS-responsive micelle-gel complex for treating corneal neovascularization and a preparation method, providing a new ocular drug delivery strategy. The excellent neovascular endothelial cell targeting and corneal adhesion of this hypotonic micelle-gel complex enable efficient and specific drug delivery to the diseased area and sustained release, thereby maintaining the therapeutic concentration required by the eye. It can significantly extend the drug release cycle, reduce patient compliance, and improve the clinical treatment effect of corneal neovascularization. This non-invasive drug administration method can also avoid the many ocular complications and toxic side effects brought to patients by invasive injection methods. The strong adhesion properties of the hypotonic gel itself also ensure that the drug remains in the diseased area for a long time, greatly improving the absorption and utilization of the therapeutic drug by the eye.

Description

Technical Field

[0001] The present invention provides a ROS-responsive micelle-gel complex for treating corneal neovascularization and a preparation method thereof. Specifically, the ROS-responsive micelle-gel complex encapsulating dexamethasone is prepared into a sustained-release gel using a hypotonic gel, belonging to the field of biopharmaceutical technology. Background Art

[0002] Corneal neovascularization is a pathological change caused by the invasion of limbal capillaries into the transparent zone of the cornea. Currently, most corneal diseases are accompanied by corneal neovascularization, and in severe cases, irreversible loss of vision can occur. Many pathological factors of the eye, such as infection, inflammation, hypoxia, trauma, corneal degeneration and corneal transplantation, can disrupt the "avascular" steady-state environment of the transparent zone of the cornea, thereby causing irreversible damage to eye vision. Currently, conventional treatments for corneal neovascularization include topical glucocorticoids and non-steroidal anti-inflammatory drugs, intraocular injection of anti-vascular endothelial growth factor drugs, and surgical treatments such as electrocoagulation and photodynamic therapy. However, the clinical effects of these treatments are not ideal.

[0003] Furthermore, treatments such as intraocular injections and surgery can present serious complications and side effects, including retinal detachment, vitreous hemorrhage, and a sharp rise in intraocular pressure. However, traditional instillation methods can also lead to issues such as short drug half-life, poor absorption, and low bioavailability due to eye clearance through blinking and nasolacrimal drainage. Furthermore, these nonspecific methods of administration can also cause other systemic side effects, including conjunctival hemorrhage, recurrent corneal epithelial defects, and corneal deposits. Summary of the Invention

[0004] The present invention provides a ROS-responsive micelle-gel complex for treating corneal neovascularization and a preparation method. The ROS-responsive micelle-gel complex encapsulating dexamethasone is prepared into a sustained-release gel using a hypotonic gel. The introduction of the cRGD peptide imparts targeting specificity to the complex, enabling it to improve the ocular adhesion of the preparation and slowly release the drug under high ocular oxidative stress (ROS) levels.

[0005] The present invention provides a method for preparing a ROS-responsive micelle-gel complex for treating corneal neovascularization, which is simple to operate and suitable for industrial production.

[0006] The method for preparing a dexamethasone-loaded ROS-responsive micelle-gel complex of the present invention comprises the following steps:

[0007] 1) Mix the synthesized PEG-PPS, Dex, and cRGD-PEG-PLGA at a mass ratio of 20:(1-6):(0-3). Dissolve 21-29 mg of the mixture in 1 mL of dichloromethane. In an ultrasonic bath over ice, inject the organic phase into 4-10 mL of deionized water at a constant rate over 1-5 minutes. After micelle self-assembly, stir at room temperature and evaporate the solvent for 1-8 hours to obtain a stable micelle solution.

[0008] 2) The micellar solution was magnetically stirred at room temperature for 8 h to evaporate the organic solvent in the solution. The solution was then centrifuged three times at 3500 rpm and 4°C for 20 min using a 3500 Da ultrafiltration tube and then stored at 4°C.

[0009] 3) Weigh 240-400 mg of poloxamer F127 powder according to the corresponding ratio and dissolve it in 2 mL of the prepared micelle solution. Stir until completely dissolved to obtain a hypotonic micelle-gel complex with a concentration of 12-20% (w / v).

[0010] The composite drug delivery system of the present invention presents a three-dimensional network structure, in which spherical micelles are embedded, with an average particle size between 120-150 nm and a potential of about -15-20 mV; it first releases a burst followed by a sustained release and can continue to release for 72 hours, achieving oxidative stress-responsive release behavior.

[0011] This provides a new strategy for ocular drug delivery. The good targeting of neovascular endothelial cells and corneal adhesion of this hypotonic micelle-gel complex can efficiently and specifically deliver drugs to the diseased area and continuously release them to maintain the therapeutic concentration required by the eye. In addition, it can greatly prolong the drug release cycle, reduce patient compliance and improve the clinical treatment effect of corneal neovascularization. It is worth noting that this non-invasive drug delivery method can also avoid the many eye complications and toxic side effects brought to patients by invasive injection methods. The strong adhesion properties of the hypotonic gel itself also ensure that the drug remains in the diseased area for a long time, greatly improving the absorption and utilization of therapeutic drugs by the eye. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Schematic diagram of the synthesis steps of PEG-PPS amphiphilic block copolymer;

[0013] Figure 2 It is an oxidative stress responsiveness assay of ROS-responsive micelles;

[0014] Figure 3 is the particle size distribution diagram of ROS-responsive micelles;

[0015] Figure 4 is the scanning electron microscopy image of ROS-responsive micelles;

[0016] Figure 5 To study the drug release behavior of ROS-responsive micelles under different oxidative stress levels;

[0017] Figure 6 Transmission electron microscopy image of the ROS-responsive micelle-gel complex;

[0018] Figure 7 This study investigates the drug release behavior of ROS-responsive micelle-gel complexes under different oxidative stress levels. Implementation Method

[0019] The present invention is further described by way of examples below, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or alteration of the present invention that can be easily implemented by a person skilled in the art will fall within the scope of the claims of the present invention.

[0020] Abbreviations given in the following examples: Dex represents dexamethasone; Hypo represents hypotonicity; Iso represents isotonicity; cRGD-DPPMs is the name of the micelle; cRGD-DPPMs / Gel is the name of the prepared micelle-gel complex; the schematic diagram of the synthesis steps of the PEG-PPS amphiphilic block copolymer is shown in Figure 1 . Example 1

[0021] PEG-PPS, Dex, and cRGD-PEG-PLGA were weighed and mixed in a mass ratio of 20:4:0.5. 24.5 mg of the mixture was dissolved in 1 mL of dichloromethane. The organic phase was uniformly injected into 6 mL of deionized water under ultrasonic ice bath conditions within 3 min. After micelle self-assembly, the solvent was evaporated by stirring at room temperature for 8 h. The mixture was then centrifuged at 3500 rpm and 4°C for 20 min using a 3500 Da ultrafiltration tube and washed three times with 1 mL of deionized water to obtain a stable micelle solution. Example 2

[0022] PEG-PPS, Dex, and cRGD-PEG-PLGA were weighed and mixed in a mass ratio of 20:4:1. 25 mg of the mixture was dissolved in 1 mL of dichloromethane. The organic phase was uniformly injected into 6 mL of deionized water under ultrasonic ice bath conditions within 3 min. After micelle self-assembly, the solvent was evaporated by stirring at room temperature for 8 h. The mixture was then centrifuged at 3500 rpm and 4°C for 20 min using a 3500 Da ultrafiltration tube and washed three times with 1 mL of deionized water to obtain a stable micelle solution. Example 3

[0023] PEG-PPS, Dex, and cRGD-PEG-PLGA were weighed and mixed in a mass ratio of 20:4:2. 26 mg of the mixture was dissolved in 1 mL of dichloromethane. The organic phase was uniformly injected into 6 mL of deionized water under ultrasonic ice bath conditions within 3 min. After micelle self-assembly, the solvent was evaporated by stirring at room temperature for 8 h, and then centrifuged at 3500 rpm and 4°C for 20 min using a 3500 Da ultrafiltration tube. The solution was then washed three times with 1 mL of deionized water to obtain a stable micelle solution. Example 4

[0024] PEG-PPS, Dex, and cRGD-PEG-PLGA were weighed and mixed in a mass ratio of 20:4:3. 27 mg of the mixture was dissolved in 1 mL of dichloromethane. The organic phase was uniformly injected into 6 mL of deionized water under ultrasonic ice bath conditions within 3 minutes. After micelle self-assembly, the solvent was evaporated by stirring at room temperature for 8 hours, and then centrifuged at 3500 rpm and 4°C for 20 minutes using a 3500 Da ultrafiltration tube. The solution was then washed three times with 1 mL of deionized water to obtain a stable micelle solution. Example 5

[0025] 2 mL of the micelle solution prepared in Example 3 was taken, and 240 mg of poloxamer F127 powder was weighed according to the corresponding proportion and stirred in the micelle solution until completely dissolved to obtain cRGD-DPPMs / Gel with a concentration of 12% (w / v). Example 6

[0026] 2 mL of the micelle solution prepared in Example 3 was taken, and 320 mg of poloxamer F127 powder was weighed according to the corresponding proportion and stirred in the micelle solution until completely dissolved to obtain cRGD-DPPMs / Gel with a concentration of 16% (w / v). Example 7

[0027] 2 mL of the micelle solution prepared in Example 3 was taken, and 360 mg of poloxamer F127 powder was weighed according to the corresponding proportion and stirred in the micelle solution until completely dissolved to obtain cRGD-DPPMs / Gel with a concentration of 18% (w / v). Example 8

[0028] 2 mL of the micelle solution prepared in Example 3 was taken, and 400 mg of poloxamer F127 powder was weighed according to the corresponding proportion and stirred in the micelle solution until completely dissolved to obtain cRGD-DPPMs / Gel with a concentration of 20% (w / v).

[0029] Experimental Example 1

[0030] ROS-responsiveness assay of micelles

[0031] Place 3 mL of the micelle solution prepared in Example 3 in a vial. Add an appropriate amount of 30% H₂O₂ (w / w) solution to a final H₂O₂ concentration of 5% (w / w). Incubate on a shaker at 37°C. Observe and record changes in the macroscopic state of the micelles. Measure the particle size and macroscopic changes of the samples at 0, 4, and 8 hours.

[0032] The results showed that within 0-8 h, the micelle size first increased from ~142 nm to ~202 nm and then decreased to ~116 nm, indicating that the hydrophobic core of the micelle PPS first expanded and then was oxidized and disintegrated by reactive oxygen free radicals. In addition, after incubation, the solution changed from milky white to clear, further demonstrating that the responsive disintegration of the micelle core enhanced the water solubility of the micelles; for details, see Figure 2 a and Figure 2 b.

[0033] Experimental Example 2

[0034] Characterization experiment of properties of ROS-responsive micelles

[0035] 1 mL of the micelle solution prepared in Example 3 was diluted and added to the sample cell. The solution was equilibrated in a ZS90 particle size potential analyzer for 60 s at a detection temperature of 25 °C. Each sample was measured in parallel three times.

[0036] 1 mL of the micellar solution and methanol prepared in Example 3 were measured and mixed to break the emulsion. 2 mL of mobile phase was added and filtered through a 220 μm microporous membrane. The total drug concentration (C tot ). Another 1 mL of micelle solution was placed in a 3500Da ultrafiltration centrifuge tube and centrifuged at 3500 rpm for 30 min. The lower filtrate was taken and the mobile phase was added to 2 mL, mixed and filtered, and the free drug concentration (C free ).

[0037] The particle size, dispersity (PDI), potential (Zeta), drug loading and encapsulation efficiency of the micelles prepared in Examples 1-4 were investigated. The results are shown in Table 1 below:

[0038] Table 1.

[0039]

[0040] As can be seen from the table, there is no significant difference in the particle size, dispersion and potential of the micelles prepared in Examples 1-4, but the micelle solution obtained in Example 3 has the highest drug loading and encapsulation efficiency. Therefore, it can be seen that the optimal amount of the targeting peptide copolymer input into the micelle is 2 mg; the micelle particle size distribution diagram in Example 3 is shown in FIG. Figure 3 .

[0041] Experimental Example 3

[0042] ROS-responsive micelle morphology experiments

[0043] An appropriate amount of the micelle solution prepared in Example 3 was dripped onto a smooth silicon wafer, dried at room temperature, sprayed with gold, and tested on a scanning electron microscope (SEM) sample stage to examine the micelle morphology.

[0044] The results showed that the micelles were complete spherical structures with uniform particle size, high dispersion and no aggregation and adhesion. The size distribution was about 90-110 nm, slightly smaller than the measured hydrated size. The scanning electron microscopy images of the micelles are shown in Figure 4 .

[0045] Experimental Example 4

[0046] Oxidative stress-responsive release behavior experiment of ROS-responsive micelles

[0047] Accurately weigh 2 mL of the micellar solution from Example 3 and place it into a 3500Da MWCO dialysis bag. Place the solution in artificial tears and add the corresponding volumes of hydrogen peroxide solution to achieve concentrations of 0%, 1%, 2.5%, and 5%. Place the centrifuge tube in a 37°C incubator at 100 rpm. At specific time points, collect 2 mL of the supernatant and add 2 mL of the corresponding fresh release medium. Add 2 mL of a 1:1 methanol:acetonitrile solution to the sample solution. After ultrasonic demulsification, determine the Dex concentration at each time point by HPLC.

[0048] The results showed that the cumulative release rate of the drug was positively correlated with the level of oxidative stress. Therefore, the micelles have good ROS responsiveness, which can not only release the therapeutic drug to the maximum extent in the oxidative stress environment, but also ensure the therapeutic effect of the drug in the eye through sustained release / controlled release. The ROS responsive release behavior curve of the micelles is shown in Figure 5 .

[0049] Experimental Example 5

[0050] Gelation experiment of ROS-responsive micelle-gel complex

[0051] 2 mL of the cRGD-DPPMs / Gel prepared in Examples 5-8 was placed in a water bath. The gelation temperature, gelation time, gelation state, pH, and osmotic pressure of the sample were investigated. The results are shown in Table 2: (Gelation time: the time from the start of the mixed solution at 25° to the 30 s after the test tube was tilted 90° when the liquid level did not move)

[0052] Table 2.

[0053]

[0054] It can be seen from the results that the gelation temperature, gelation time, gel state, pH and osmotic pressure of Example 5 are all optimal.

[0055] Experimental Example 6

[0056] Appearance experiment of ROS-responsive micelle-gel complex

[0057] The micelle-gel complex solution prepared in Example 5 was dropped onto a copper mesh, and then a drop of uranyl acetate solution was added for counterstaining. The sample was prepared by freeze-drying and the size and morphology of the sample were observed under a transmission electron microscope.

[0058] Transmission electron microscopy results showed that the micelles were still spherical in the hypotonic gel and embedded in the three-dimensional gel network structure, and the gel matrix itself had no significant effect on the particle size and morphology of the liposomes; the transmission electron microscopy image of the micelle-gel complex is shown in Figure 6 .

[0059] Experimental Example 7

[0060] Oxidative stress-responsive release behavior experiment of ROS-responsive micelle-gel complex

[0061] Accurately weigh 2 mL of the micelle-gel solution from Example 5 into a 3500Da MWCO dialysis bag, place it in artificial tears, and add the corresponding volumes of hydrogen peroxide solution to concentrations of 0%, 1%, 2.5%, and 5%. Place the centrifuge tube in a 37°C incubator at 100 rpm. At specific time points, 2 mL of supernatant were collected and 2 mL of fresh release medium were added. To the sample solution, 2 mL of a 1:1 methanol:acetonitrile solution was added. After ultrasonic demulsification, the Dex concentration at each time point was determined by HPLC, and the cumulative release rate was calculated.

[0062] There were significant differences in the in vitro release of PMs / Gel at different H2O2 concentrations. When the H2O2 concentration was 5%, cRGD-DPPMs were able to rapidly release approximately 37% of the drug within 12 to 24 hours, and to continuously and rapidly release 72.52% of the drug within 72 hours. In addition, compared with a single micelle solution, cRGD-DPPMs / Gel had stronger sustained / controlled release and ocular adhesion within the first 48 hours. The results showed that the encapsulation of micelles by gel did not affect the ROS responsiveness of the micelles themselves; the oxidative stress responsive release curve of the ROS-responsive micelle-gel complex is shown in Figure 7 .

Claims

1. A ROS-responsive micelle-gel complex for treating corneal neovascularization, comprising the following steps: 1) Mix the synthesized PEG-PPS, Dex, and cRGD-PEG-PLGA at a mass ratio of 20:(1-6):(0.5-3). Dissolve 21-29 mg of the mixture in 1 mL of dichloromethane. In an ultrasonic bath over ice, inject the organic phase into 4-10 mL of deionized water at a constant rate over 1-5 minutes. After micelle self-assembly, stir at room temperature and evaporate the solvent for 1-8 hours to obtain a stable micelle solution. 2) The micellar solution was magnetically stirred at room temperature for 8 h to evaporate the organic solvent in the solution. The solution was then centrifuged three times at 3500 rpm and 4°C for 20 min using a 3500 Da ultrafiltration tube and then stored at 4°C. 3) Weigh 240-400 mg of Poloxamer F127 powder according to the corresponding ratio and dissolve it in 2 mL of the prepared micelle solution. Stir until completely dissolved to obtain a hypotonic micelle-gel complex with a concentration of 12-20% (w / v).

2. The ROS-responsive micelle-gel complex for treating corneal neovascularization according to claim 1, wherein: The preferred mass ratio of PEG-PPS, Dex and cRGD-PEG-PLGA in step 1) is 20:4:0.5.

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