Preparation and application of drug-loaded micellar solution for treating corneal neovascularization

Abstract

The application provides a preparation and application of a drug-loaded micelle solution capable of treating corneal neovascularization. The preparation method comprises the following steps: 1) preparation of blank micelles: dissolving a high-molecular polymer in a good solvent, adding the good solvent into deionized water through a syringe pump under high-speed stirring, then removing the solvent to a thin film by using a rotary evaporator, and obtaining a blank micelle solution with uniform particle size by using a gradient hydration-ultrasonic crushing combined technology; and 2) preparation of drug-loaded micelles: dissolving a poorly soluble drug in a low-boiling good solvent, adding the low-boiling good solvent into the micelle solution in proportion, stirring in the dark for a certain time, and filtering to remove the unsolved drug, so as to obtain the drug-loaded micelle solution. The obtained drug-loaded system has high drug loading efficiency and excellent stability, can greatly improve the treatment effect of the drug on the corneal neovascularization, and provides an innovative delivery system with high safety for the eye for the clinical treatment of the corneal neovascularization.

Description

Technical Field

[0001] This invention relates to the field of ocular drug delivery, specifically to the preparation and application of a drug-loaded micelle solution for treating corneal neovascularization, which has good biocompatibility, can improve the bioavailability of poorly soluble drugs, and inhibit the formation of corneal neovascularization. Technical Background

[0002] Corneal injury is one of the most common causes of blindness worldwide, primarily stemming from mechanical trauma (such as abrasions, scratches, and punctures), chemical burns (accidental contact with acids or alkalis), microbial infections (fungi, bacteria, and viruses), epithelial loss induced by dry eye syndrome, and complications from refractive surgery. Corneal injury often leads to the abnormal growth of new blood vessels in the normally avascular corneal tissue, known as corneal neovascularization. These vessels typically invade the corneal center from the limbal vascular network, disrupting the cornea's immune clearance and optical transparency, resulting in visual impairment or even blindness. Besides corneal injury, corneal hypoxia caused by prolonged contact lens wear can also trigger corneal neovascularization. Current treatments include corticosteroid eye drops and intravitreal injections of anti-VEGF drugs, but these methods suffer from low local bioavailability, high invasiveness risks, limited drug penetration, and significant side effects. Therefore, the development of non-invasive, highly efficient novel drug delivery systems is urgently needed in clinical practice.

[0003] Currently, eye drops are the most common method of drug administration for treating eye diseases due to their advantages of ease of use, low cost, and high patient compliance, accounting for approximately 90% of the global ophthalmic drug market share. However, the main challenge in using eye drops is their low bioavailability. Due to the blink reflex and nasolacrimal duct drainage mechanisms, as well as the presence of a negatively charged mucin layer on the corneal surface that protects the ocular surface from external non-specific stimuli, drug absorption is also affected, ultimately resulting in less than 5% of the active drug ingredient being utilized. Therefore, developing a nanomedicine delivery system that combines high corneal permeability with long-lasting pre-corneal retention is of significant clinical value in improving the bioavailability of eye drops and thus enhancing therapeutic efficacy.

[0004] The development and application of polymer micelles is an ideal strategy for improving corneal penetration, maintaining drug levels, and enhancing the solubility and utilization of poorly soluble drugs. Their unique core-shell structure can encapsulate poorly soluble drugs to improve solubility and prevent drug precipitation and degradation, thereby improving drug stability in solution. Due to the interaction between the hydrophobic core and the poorly soluble drug, drug release can be delayed, achieving a sustained-release effect. Furthermore, the small size (<200 nm) and high stability of polymer micelles allow drugs to better penetrate the cornea and reach the lesion site to exert their efficacy. Currently, researchers have conducted relatively in-depth studies on cationic polymer micelles formed by incorporating natural polymers such as chitosan and dextran. However, compared to polymer micelles, they lack stability, which is highly detrimental to drug delivery.

[0005] NSC95397 is a selective Cdc25 bispecific phosphatase inhibitor with the ability to inhibit cell growth and induce cell cycle arrest. It also inhibits the interaction between Zeb1 and Ctbp, significantly reducing Zeb1 levels and thus inhibiting corneal neovascularization. Similarly, ethyl caffeate can also inhibit Zeb1 and has anti-inflammatory and antioxidant properties, showing good inhibitory effects on corneal neovascularization caused by ocular injury. However, due to the poor water solubility and stability of these drugs, they are difficult to penetrate the eye to the lesion site to exert their inhibitory effect on CNV formation. Summary of the Invention

[0006] To address the problems of existing technologies, this invention provides a method for preparing and applying a drug-loaded micelle solution for treating corneal neovascularization. It utilizes polymer micelle technology to prepare polymer micelles loaded with poorly soluble drugs, improving drug solubility and bioavailability in solution. This inhibits corneal neovascularization and maintains the integrity of corneal structure and function. The drug-loaded micelles provided by this invention are formed through self-assembly, and their particle size, potential, PDI, and osmotic pressure are all within the normal range suitable for the human eye. They exhibit excellent stability and good corneal permeability, thus effectively inhibiting corneal neovascularization.

[0007] To achieve the above objectives, the present invention will utilize the following technical solution:

[0008] A drug-loaded micelle solution for treating corneal neovascularization is prepared by the self-assembly of polymer micelles and a poorly soluble drug in solution; comprising the following steps:

[0009] (1) Preparation of blank micelles: The polymer was dissolved in a good solvent and added to deionized water by injection pump under high speed stirring. Then the solvent was removed by rotary evaporator until it became a thin film. After adding an appropriate amount of deionized water to dissolve it, the blank micelle solution with uniform size was obtained by ultrasonic disruption.

[0010] (2) Preparation of drug-loaded micelles: The poorly soluble drug is dissolved in a small amount of good solvent with a low boiling point, and then transferred to a blank micelle solution and stirred in the dark for a certain period of time until the organic solvent evaporates. After that, the undissolved drug is removed by filtration to obtain the drug-loaded micelle solution.

[0011] In the preferred embodiment of the above-described technical solution, the polymer is a cationic polymer with an amphiphilic polymer structure (PPICB-ODA). The main chain is a positively charged linear polypropyleneimide, the hydrophilic end is a propionate group, and the hydrophobic end is an octadecyl propionate ester. It is obtained by grafting tert-butyl acrylate and octadecyl acrylate onto the polypropyleneimide main chain. The preparation method includes the following steps: 1) synthesizing the monomer 2-methyloxazine using acetonitrile and 3-amino-1-propanol in the presence of a catalyst (zinc acetate, zinc chloride, or zinc acetate dihydrate); 2) subjecting the 2-methyloxazine monomer to cationic ring-opening polymerization to obtain polymethyloxazine; 3) hydrolyzing the polymethyloxazine under acidic conditions to obtain linear polypropyleneimine; 4) grafting tert-butyl acrylate and octadecyl acrylate onto the polypropyleneimine backbone to obtain an amphiphilic polymer; for details, please refer to the patent application number CN202410027381.6 entitled "A method for synthesizing an amphiphilic polymer and preparing blank micelles".

[0012] In the preferred embodiment of the above-described technical solution, in step (1), the good solvent is tetrahydrofuran or acetonitrile; in step (2), the good solvent with a lower boiling point is tetrahydrofuran, methanol or acetonitrile.

[0013] In the above-described technical solution, under preferred conditions, the poorly soluble drug is NSC95397 or ethyl caffeate; the mass ratio of the polymer to the poorly soluble drug is 100:1-5:1, preferably 20:1-5:1.

[0014] Based on the above-described technical solution, in the preferred case, in step (1), the ratio of the total amount of polymer, good solvent and deionized water is 5-20 mg: 0.5-2 mL: 6-24 mL, preferably 10-20 mg: 1-2 mL: 12-24 mL; in step (1), the volume ratio of the first addition of deionized water to the second addition of deionized water is 1-4: 5-20, preferably 2-4: 10-20; in step (2), the ratio of the poorly soluble drug and the good solvent with a low boiling point is 10-2000 μg: 20-500 μL, preferably 100-1000 μg: 50-250 μL.

[0015] In the preferred case, based on the above-described technical solution, in step (1), the stirring speed is 600-1200 rpm and the stirring time is 1-4 h; the injection pump rate is 500-1000 μL / min.

[0016] In the preferred case, based on the technical solution described above, in step (2), the stirring speed is 800-1500 rpm and the time is 12-24h.

[0017] Based on the above-described technical solution, in the preferred case, in step (1), when using a rotary evaporator, the rotation speed is 40-100 rpm, the water bath temperature is 20-45℃, and the vacuum degree is 0-40 hPa; a blank micelle solution with uniform particle size is obtained by using gradient hydration-ultrasonic disruption combined technology. When using ultrasonic disruption, the external temperature is controlled at 0-4℃, the power is 100-300 watts, the ratio of intermittent time to ultrasonic time is 2:1-1:2, and the total ultrasonic disruption time is 10-20 min.

[0018] In the preferred embodiment of the above-described technical solution, in step (2), a filter head of 220-480 nm is used to filter the drug-loaded micelle solution to obtain a polymer micelle solution loaded with a poorly soluble drug.

[0019] In vitro release experiments were conducted using the prepared drug-loaded micelle solution via dialysis bag method: the release medium was simulated tear fluid (STF) solution (pH = 7.4). Release conditions were as follows: the release system oscillated at 100 rpm, the temperature was set at 34℃, and sample collection times were 0.5, 1, 2, 4, and 8 hours. All samples were measured three times.

[0020] Using prepared drug-loaded micelle solutions, in vitro corneal permeation experiments were conducted using porcine corneas via a Franz vertical diffusion cell. Different fluorescently labeled drug-loaded micelle solutions were added to the donor chamber, and STF solution was added to the recipient chamber. The temperature was maintained at 34°C, and the stirring speed was 100 rpm. After 10, 30, and 240 minutes, the porcine corneas were removed and observed under a confocal microscope.

[0021] The prepared drug-loaded micelle solution was used to perform a cell compatibility experiment on corneal epithelial cells. The CCK-8 method was used to evaluate the compatibility after 24 hours. There were three samples in each group.

[0022] Cell migration experiments of human umbilical vein endothelial cells were conducted using the prepared drug-loaded micelle solution. After 8 hours, the cells were observed and measured using an inverted fluorescence microscope. Each group had three samples.

[0023] The principle of this invention: Polymer micelles, composed of a hydrophilic outer end and a hydrophobic inner core, can self-assemble into an ordered structure in solution. The hydrophobic inner core can encapsulate poorly soluble drugs, forming drug-loaded micelles, thereby significantly improving the solubility and stability of poorly soluble drugs in solution. Furthermore, the interaction between the two cores allows for sustained drug release, thus improving drug bioavailability in the eye. Additionally, cationic polymer micelles can interact with the mucin layer of the ocular surface through positive and negative charge interactions, increasing the corneal retention time. Combined with their small size, ease of preparation, high encapsulation efficiency, and strong corneal penetration, polymer micelles have excellent potential for ocular applications.

[0024] This invention selects cationic polymer micelles as drug delivery carriers and NSC95397 or ethyl caffeate as model drugs, providing a method for preparing and applying a drug-loaded micelle solution for treating corneal neovascularization. Both drugs have extremely limited application in the eye. This invention offers a new approach to treating corneal neovascularization, achieving high drug loading, high release, and high therapeutic efficacy for poorly soluble drugs in eye drops, showing promising potential for clinical application.

[0025] This invention significantly reduces residual organic solvents through optimized formulation processes, resulting in a high level of ocular safety. The resulting drug delivery system combines high drug loading efficiency with excellent stability, and can also greatly improve the therapeutic effect of drugs on corneal neovascularization, providing an innovative delivery system with high ocular safety for the clinical treatment of corneal neovascularization.

[0026] Beneficial effects: Compared with existing technologies for treating corneal neovascularization, this invention has the following significant advantages: as an eye drop for treating corneal neovascularization, it avoids invasive drug administration and improves patient compliance; using cationic polymer micelles to load poorly soluble drugs can improve drug bioavailability, thereby achieving ideal treatment effects; the drugs selected in this invention have few precedents for ocular administration and are expected to become innovative drugs; the operation process is simple, easy to repeat, has a short cycle, and good effects, and has good clinical application value. Attached Figure Description

[0027] Figure 1 Particle size distribution (left) and data (right) of blank micelle solution and drug-loaded micelle solutions of different concentrations;

[0028] Figure 2 Zeta potential plots for blank micelle solution and drug-loaded micelle solutions of different concentrations;

[0029] Figure 3 The images show the FT-IR spectra before and after polymer micelle encapsulation of the drug.

[0030] Figure 4 To determine the drug loading and encapsulation efficiency after adding different concentrations of drug to micelles;

[0031] Figure 5 The stability curves of polymer micelles in artificial tears, including particle size and zeta potential.

[0032] Figure 6 The in vitro release curves of the encapsulated polymer micelles in simulated tear fluid (STF) are shown.

[0033] Figure 7 Images showing the permeation of polymer micelles encapsulating Nile Red in isolated porcine corneas;

[0034] Figure 8 Images showing the permeation of FITC-labeled polymer micelles in an isolated porcine cornea;

[0035] Figure 9 This is a diagram showing the cell compatibility of the blank micelle solution with corneal epithelial cells.

[0036] Figure 10 This is a diagram showing the cell compatibility of free drug and drug-loaded micelles with corneal epithelial cells;

[0037] Figure 11 The left image shows the experimental results (left) and quantitative data (right) of polymer micelles encapsulated with drugs on cell migration of human umbilical vein endothelial cells.

[0038] Figure 12 Images (left) and total vessel length data (right) of corneal neovascularization formed in mice with different alkali burn treatment solutions. Detailed Implementation

[0039] The following specific embodiments are for further explanation of the content of the present invention and should not be construed as limiting the present invention in any way.

[0040] In the following examples, the polymer is an amphiphilic polymer (PPICB-ODA), with a main chain of positively charged linear polypropyleneimide, a hydrophilic end with a propionate group, and a hydrophobic end with an octadecyl propionate ester. It is prepared by grafting tert-butyl acrylate and octadecyl acrylate onto the polypropyleneimide main chain. For specific preparation methods, please refer to step 5) of Example 1 in patent application number CN202410027381.6, entitled "A Method for Synthesizing an Amphiphilic Polymer and Preparing Blank Micelles," to obtain PPICB-ODA.

[0041] Example 1

[0042] 10 mg of the polymer PPICB-ODA was dissolved in 1 mL of tetrahydrofuran and added to 1 mL of deionized water at a rate of 500 μL / min using a syringe pump at 1000 rpm. The mixture was stirred for 1 h. The solvent was then removed using a rotary evaporator under the following conditions: rotation speed: 60 rpm; water bath temperature: 35 °C; vacuum: 20 hPa, until a thin film was formed. 10 mL of deionized water was then added, and the mixture was sonicated in a water bath until the film was completely dissolved. Next, the blank micelle solution was treated using an ultrasonic disruptor at 0 °C, with a power of 150 W, and an interval-to-ultrasonic time ratio of 1:1, for a total disruption time of 10 min. This yielded a uniformly sized blank micelle solution, denoted as a 1 mg / mL blank micelle solution.

[0043] The particle size, PDI, and Zeta potential of the blank micelle solution were measured, and its FT-IR spectrum was measured after lyophilization. The particle size results are as follows: Figure 1 The blank micelles were 165.48±2.72 nm, the PDI was 0.34±0.01, and the Zeta potential was as follows. Figure 2 The "blank micelles" shown in the image have a value of 52.54 ± 3.31 mV. The FT-IR spectrum is shown below. Figure 3 The blank micelles are shown in the image.

[0044] Example 2

[0045] Dissolve 1 mg of NSC95397 drug in 100 μL of tetrahydrofuran, and add it to 10 mL of the 1 mg / mL blank micelle solution prepared in Example 1 at 1000 rpm. Stir for 24 h in the dark to remove the organic solvent, and then filter the solution using a 220 nm filter to finally form a drug-loaded micelle solution, denoted as drug-loaded micelle-1 mg.

[0046] The particle size, PDI, Zeta potential, encapsulation efficiency, and drug loading of the drug-loaded micelle solution were measured. The FT-IR spectrum of the solution was then measured after lyophilization. The particle size results are as follows: Figure 1The "drug-loaded micelles - 1 mg" are shown to have a wavelength of 106.65 ± 1.02 nm, a PDI of 0.31 ± 0.01, and a zeta potential as shown. Figure 2 The value of "drug-loaded micelles - 1 mg" is 34.97 ± 0.55 mV, and the FT-IR spectrum is shown below. Figure 3 As shown in the image, “Drug-loaded micelles - 1 mg”. Changes in particle size and potential indicate an interaction between the poorly soluble drug NSC95397 and the polymer micelles. The masking of some characteristic peaks in the FT-IR spectrum confirms that the drug is encapsulated within the polymer micelle core. Figure 4 As shown in the "drug-loaded micelles - 1mg" sample, the encapsulation efficiency was 83.76% and the drug loading was 8.38%, indicating that polymer micelles can effectively encapsulate drugs and greatly improve drug solubility.

[0047] Example 3

[0048] Stability testing of drug-loaded micelle solutions in simulated tear fluid (STF):

[0049] The drug-loaded micelles-1mg solution prepared in Example 2 was lyophilized and then reconstituted in artificial tears (STF, 6.78 mg / ml NaCl, 2.18 mg / ml NaHCO3, 1.38 mg / ml KCl, 0.084 mg / ml CaCl2·2H2O, solvent: deionized water, pH 7.4) to a concentration of 1 mg / mL. The solution was placed at 4°C, and the particle size and zeta potential of the solution were measured on days 1, 2, 3, 5, 7, 10, 14, 17, 21, and 28.

[0050] During the 28-day storage period, the particle size and zeta potential of the drug-loaded micelle solution remained at a relatively stable level, as shown in the following results. Figure 5 As shown, this confirms that the drug delivery system formed by loading drugs into polymer micelles has good stability and can also avoid drug precipitation and degradation.

[0051] Example 4

[0052] The difference between this embodiment and Embodiment 2 is that the amount of NSC95397 drug added is 2mg, and the final drug-loaded micelle solution is denoted as drug-loaded micelle-2mg.

[0053] The particle size, PDI, Zeta potential, encapsulation efficiency, and drug loading of the drug-loaded micelle solution were measured. The FT-IR spectrum of the solution was then measured after lyophilization. The particle size results are as follows: Figure 1 The "drug-loaded micelles - 2mg" are shown to be 115.00±0.60nm, with a PDI of 0.27±0.01, and a Zeta potential as shown. Figure 2 The value of "drug-loaded micelles - 2 mg" is shown as 30.70 ± 0.69 mV, and the FT-IR spectrum is as follows. Figure 3 As shown in the description "Drug-loaded micelles - 2mg". Figure 4 As shown in the "drug-loaded micelles - 2mg" sample, the encapsulation efficiency was 79.44% and the drug loading was 15.89%. These results also demonstrate that polymer micelles can effectively encapsulate poorly soluble drugs.

[0054] Example 5

[0055] The difference between this embodiment and Embodiment 2 is that the amount of NSC95397 drug added is 3mg, and the final drug-loaded micelle solution is denoted as drug-loaded micelle-3mg.

[0056] The particle size, PDI, Zeta potential, encapsulation efficiency, and drug loading of the drug-loaded micelle solution were measured. The particle size results are as follows: Figure 1 The wavelength of the drug-loaded micelles (3 mg) is 124.62 ± 3.41 nm, the PDI is 0.25 ± 0.03, and the zeta potential is as shown. Figure 2 The value of "drug-loaded micelles - 3mg" is shown as 21.47±2.53mV, and the FT-IR spectrum is as follows. Figure 3 As shown in the description "Drug-loaded micelles - 3mg". Figure 4 As shown in the "drug-loaded micelles - 3mg" sample, the encapsulation efficiency was 78.20% and the drug loading was 23.46%. These results also demonstrate that polymer micelles can effectively encapsulate poorly soluble drugs.

[0057] Example 6

[0058] In vitro release experiment of drug-loaded micelle solution:

[0059] 5 mL of 0.2 mg / mL free drug solution (1 mg NSC95397 dissolved in 500 μL dimethyl sulfoxide, then diluted to 5 mL with deionized water) and 5 mL of drug-loaded micelle solutions (drug-loaded micelles - 1 / 2 / 3 mg) prepared in Examples 2, 4, and 5, respectively, were transferred to dialysis bags (3400 Da) and completely immersed in 50 mL of artificial tears under continuous shaking at 34 °C and 100 rpm. 1 mL of permeate was aspirated at different time points to determine the cumulative drug release.

[0060] The results are as follows Figure 6 As shown, the release of the free drug solution exhibits a clear burst release pattern, with nearly 100% released within 1 hour. In contrast, the drug-loaded micelles show a more stable release curve, achieving sustained release over 8 hours. This indicates that polymer micelles can control drug release at a relatively slow and sustained rate, thereby ensuring continuous drug delivery to the lesion site.

[0061] Example 7

[0062] In vitro porcine corneal permeation experiment using drug-loaded micelle solutions:

[0063] Isolated porcine corneas were placed between the recipient and donor regions of a vertical Franz diffusion cell. The donor region was provided with 1 mL of a blank micelle solution labeled with FITC (prepared by mixing PPICB-ODA (0.2 mg / mL) and FITC (in a 1:3 molar ratio) in deionized water for 2 h, then transferring to a dialysis bag (3400 Da) and dialyzing in deionized water for 24 h to obtain FITC-PPICB-ODA, which was then diluted with deionized water to 0.2 mg / mL), and 1 mL of a polymeric micelle solution loaded with Nile Red (prepared using the same method as the drug-loaded micelle solution in Example 2, except that NSC95397 was replaced with Nile Red). 5 mL of PBS buffer solution with a pH of 7.4 was added to the recipient region. The mixture was stirred at 100 rpm in a constant temperature water bath at 34°C. After 10, 30, and 240 min, stirring was stopped, and the corneas were observed under a confocal microscope.

[0064] like Figure 7 , 8 As shown, over time, the micelles penetrate deeper into the cornea. Furthermore, the micelles can carry Nile Red and penetrate evenly throughout the cornea, thus reaching the designated area to exert their effectiveness.

[0065] Example 8

[0066] Cell compatibility evaluation experiment of blank micelles:

[0067] With 1×10 4 Human corneal epithelial cells were seeded into 96-well plates at a density of 1 cell / well and cultured overnight at 37°C in a CO2 incubator. After 24 hours, the cell culture medium was discarded, and the cells were washed with 1×PBS to remove residual culture medium. Then, 100 μL of blank micelle solutions prepared in Example 1, diluted to different concentrations (0.1, 0.2, 0.3 mg / mL) with DMEM medium, and 100 μL of DMEM medium were added as controls, with 4 replicates per group. After co-incubating the plates at 37°C for 24 hours, 10 μL of LCK-8 solution (purchased from APE×BIO) was added under light-protected conditions, and the plates were incubated at 37°C for 2 hours. Cell viability was calculated by measuring the absorbance of each well at 450 nm using a microplate reader.

[0068] After co-incubating blank micelle solution with human corneal epithelial cells for 24 hours, the relative cell viability was as follows: Figure 9 As shown, when the micelle concentration is below 0.3 mg / mL, the relative cell viability is above 90%, indicating good cell compatibility (p>0.5), making it safe for ocular administration.

[0069] Example 9

[0070] Cell compatibility evaluation experiments of free drug and drug-loaded micelles:

[0071] The difference between this embodiment and Example 8 is that the added solutions are free drug (5, 10 μM, 15.5 and 31.0 μg of NSC95397 dissolved in 100 μL of dimethyl sulfoxide and diluted to 10 mL with deionized water) and drug-loaded micelle solutions (5, 10 μM, prepared in the same way as in Example 2, but the amount of drug added is changed to 15.5 and 31.0 μg).

[0072] The result is as follows Figure 10 As shown, drug-loaded micelles with a concentration less than 5 μM exhibit good cell compatibility. This suggests good cell compatibility (p>0.5).

[0073] Example 10

[0074] Cell migration assays using drug-loaded micelles:

[0075] The ability of drug-loaded micelles to inhibit angiogenesis was evaluated using a migration assay of human umbilical vein endothelial cells (HUVECs). The ibidi insert was placed in a culture dish, and 70 μL of cell suspension (2 × 10⁻⁶ cells / mL) was added to the insert. 5 Cells were cultured at 37°C and 5% CO2 for 24 hours. The insert was removed using sterile forceps, and the cell layer was gently rinsed three times with PBS solution to remove non-adherent cells. Then, 2 mL of vascular endothelial cell culture medium, blank micelles (0.1 mg / mL, diluted 10-fold with vascular endothelial cell culture medium as described in Example 1), free NSC95397 (1, 3, 5 μM, prepared using the same method as in Example 9, but with the drug amount changed to 3.1, 10, 15.5 μg), and drug-loaded micelles (1, 3, 5 μM, prepared using the same method as in Example 2, but with the NSC95397 amount changed to 3.1, 10, 15.5 μg) were added respectively. After incubation at 37°C for 8 hours, the cells were removed and their migration was observed using an inverted fluorescence microscope.

[0076] The results are as follows Figure 11As shown, the migration area of ​​the control group was defined as 100%. After 8 hours, the polymeric micelles PPICB-ODA alone did not show a significant inhibitory effect (93.18±7.54%), while the relative migration rates of the free drug at the three concentrations were 96.90±4.25%, 50.80±4.21%, and 48.42±3.09%, respectively, which showed an inhibitory effect on HUVEC migration compared to the control group. Compared with the control group or the free drug group, the drug-loaded micelles at the same concentration showed a stronger inhibitory effect (p<0.5), namely 81.04±6.33%, 19.98±5.58%, and 11.04±5.63%, respectively.

[0077] Example 11

[0078] In vivo treatment of CNV in a mouse model of corneal neovascularization induced by alkali burn.

[0079] A sterile circular filter paper disc with a diameter of 2.5 mm was immersed in 1M NaOH solution for 60 seconds and placed in the center of the cornea of ​​the right eye of a mouse for 30 seconds. Then, the cornea was rinsed with 10 mL of physiological saline (0.95% NaCl solution) for 60 seconds to terminate the alkali burn, and 5 μL of commercially available levofloxacin eye drops were immediately administered to prevent ocular infection in the mice. The mice were randomly divided into four groups: ① Control group (PBS solution group with pH 7.4); ② Blank micelle solution group (PPICB-ODA, prepared in Example 1, diluted with PBS solution to 0.1 mg / mL); ③ Free NSC95397 group (Free Drug, concentration of 3 μM, prepared in the same way as in Example 10); ④ Drug-loaded micelle solution group (NSC@PPICB-ODA, NSC95397 drug concentration of 3 μM, prepared in the same way as in Example 10). To avoid affecting corneal epithelial repair in the early stages of alkali burns, mice in each group received a local instillation of 5 μL of the corresponding solution (corresponding to groups ①-④) into the right eye 24 hours after modeling. The medication was administered twice daily for 21 days. The dynamic evolution of corneal neovascularization was monitored using a slit lamp on days 1, 3, 5, 7, 10, 14, and 21, and the total length of corneal neovascularization was quantified using ImageJ software. At least three fields of view were taken from each cornea for these analyses.

[0080] The results are as follows Figure 12As shown, in the initial stage of damage repair (3-5 days), blood vessels broke through the limbus and grew upwards in the cornea of ​​each group of mice, forming corneal neovascularization. By day 7, a clear vascular network had appeared inside the cornea, including some well-formed and relatively thick vessels, which was most evident in the control group with PBS. In contrast, the corneas of mice with drug-loaded micelles remained smooth and transparent, with only a small amount of neovascularization. This indicates that drug-loaded micelles can inhibit the formation of corneal neovascularization and fibrosis in the early stage of corneal repair. In the middle stage of corneal damage repair (14 days), significant corneal neovascularization was observed in the mouse corneas. In the control group, blood vessels had invaded the normal area of ​​the cornea, and corneal fibrosis was becoming increasingly severe, with the pupil and iris barely visible, seriously affecting the normal vision of the mouse's right eye. Furthermore, the neovascularization in the blank micelle group was also severe, with symptoms of corneal fibrosis also appearing. The angiogenesis in the free drug group was not as severe as in the control group, due to the presence of NSC95397 drug. In contrast, the corneal neovascularization in the drug-loaded mice showed partial regression by day 7, and the pupil and iris were also observable, indicating an effective inhibition of corneal fibrosis. The total CNV length in the drug-loaded mice was then measured using ImageJ. Figure 12 (b) shows a length of 17.76±4.83 mm, which significantly inhibited corneal angiogenesis compared to the control group (43.99±8.86 mm), the blank micelle group (39.29±6.38 mm), and the free drug group (37.59±11.96 mm) (p<0.5). By day 21, neovascularization in mice in the control group (56.69±13.23 mm) and the blank micelle group (49.43±13.72 mm) almost completely filled the cornea, while the symptoms in the free drug group only showed slight relief (47.15±5.62 mm). At this time, the cornea of ​​mice in the drug-loaded micelle group remained clear and transparent, and the number and length of neovascularization (19.56±2.61 mm) did not increase significantly, and even showed a tendency to regress, demonstrating a very significant inhibitory effect on corneal angiogenesis (p<0.5).

[0081] Example 12

[0082] The difference between this embodiment and Embodiment 1 is that the solvent used to dissolve the polymer micelles is acetonitrile.

[0083] The particle size, PDI, and Zeta potential of the blank micelles were measured. The particle size was 180.58 ± 5.28 nm, the PDI was 0.23 ± 0.08 nm, and the Zeta potential was 45.32 ± 5.69 mV.

[0084] Example 13

[0085] The difference between this embodiment and Embodiment 2 is that the poorly soluble drug used is ethyl caffeate.

[0086] The particle size, PDI, Zeta potential, encapsulation efficiency, and drug loading of the drug-loaded micelles were measured. The particle size was 130.58±4.25 nm, the PDI was 0.22±0.01, the Zeta potential was 25.23±1.69 mV, the encapsulation efficiency was 75.69%, and the drug loading was 7.25%.

[0087] Example 14

[0088] The difference between this embodiment and Embodiment 2 is that the solvent used to dissolve the poorly soluble drug is acetonitrile.

[0089] The particle size, PDI, and Zeta potential of the drug-loaded micelles were measured. The particle size was 148.55 nm ± 8.69 nm, the PDI was 0.34 ± 0.02, and the Zeta potential was 28.16 ± 5.26 mV.

[0090] Example 15

[0091] The difference between this embodiment and Embodiment 2 is that the solvent used to dissolve the poorly soluble drug is methanol.

[0092] The particle size, PDI, and Zeta potential of the drug-loaded micelles were measured. The particle size was 129.49 nm ± 5.26 nm, the PDI was 0.29 ± 0.06, and the Zeta potential was 24.69 ± 3.58 mV.

Claims

1. A method for preparing a drug-loaded micelle solution for treating corneal neovascularization, characterized in that, Includes the following steps: (1) Preparation of blank micelles: The polymer was dissolved in a good solvent and added to deionized water by injection pump while stirring. The solvent was then removed by rotary evaporator until it became a thin film. After dissolving in deionized water, the blank micelle solution was obtained by ultrasonic disruption. (2) Preparation of drug-loaded micelles: The poorly soluble drug is dissolved in a good solvent with a low boiling point, and then transferred to a blank micelle solution and stirred in the dark for a certain period of time until the organic solvent evaporates. After that, the undissolved drug is removed by filtration to obtain the drug-loaded micelle solution.

2. The preparation method according to claim 1, characterized in that: In step (1), the polymer is an amphiphilic polymer with a main chain rich in positively charged linear polypropyleneimide, a hydrophilic end with a propionate group, and a hydrophobic end with an octadecyl propionate ester. It is obtained by grafting tert-butyl acrylate and octadecyl acrylate onto the polypropyleneimide main chain.

3. The preparation method according to claim 1, characterized in that: In step (1), the good solvent is tetrahydrofuran or acetonitrile; In step (2), the good solvent with a low boiling point is tetrahydrofuran, methanol or acetonitrile.

4. The preparation method according to claim 1, characterized in that: The poorly soluble drug is NSC95397 or ethyl caffeate; the mass ratio of the polymer to the poorly soluble drug is 100:1-5:

1.

5. The preparation method according to claim 1, characterized in that: In step (1), the ratio of polymer, good solvent and deionized water is 5-20 mg: 0.5-2 mL: 6-24 mL; the volume ratio of the first addition of deionized water to the second addition of deionized water is 1-4: 5-20. In step (2), the amount of poorly soluble drug and good solvent is 10-2000 μg: 20-500 μL.

6. The preparation method according to claim 1, characterized in that: In step (1), the stirring speed is 600-1200 rpm and the stirring time is 1-4 h; the injection pump rate is 500-1000 μL / min. In step (2), the stirring speed is 800-1500 rpm and the time is 12-24h.

7. The preparation method according to claim 1, characterized in that: In step (1), when using a rotary evaporator, the rotation speed is 40-100 rpm, the water bath temperature is 20-45℃, and the vacuum degree is 0-40 hPa; when using ultrasonic disruption, the ambient temperature is controlled at 0-4℃, the power is 100-300 watts, the ratio of intermittent time to ultrasonic time is 2:1-1:2, and the total ultrasonic disruption time is 10-20 min.

8. The preparation method according to claim 1, characterized in that: In step (2), the drug-loaded micelle solution is filtered using a filter head with a diameter of 220-480 nm.

9. A drug-loaded micelle solution for treating corneal neovascularization prepared according to the preparation method of any one of claims 1-8.

10. The use of the drug-loaded micelle solution according to claim 9 in the preparation of ophthalmic formulations.

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