Preparation method of diclofenac sodium eye drops

Abstract

The present invention belongs to the field of eye drops. In view of the problem that the existing diclofenac sodium ophthalmic preparations have poor efficacy and strong irritation on corneal neovascularization, the present invention provides a method for preparing diclofenac sodium eye drops. First, under ice bath conditions, a diclofenac solution is added to a ferrous sulfate solution. Subsequently, proanthocyanidins are coordinated with ferrous sulfate and diclofenac sodium to obtain an iron-based proanthocyanidin-diclofenac sodium nanozyme. The present invention obtains an iron-based proanthocyanidin-diclofenac sodium nanozyme by coordinating proanthocyanidins, ferrous sulfate and diclofenac sodium. The nanozyme is used for eye drops, which improves the inhibitory effect of diclofenac eye drops on corneal neovascularization, and can improve corneal sensitivity and maintain normal intraocular pressure.

Description

Technical Field

[0001] The invention belongs to the field of eye drops, and in particular relates to a method for preparing diclofenac sodium eye drops. Background Art

[0002] Corneal neovascularization (CNV) is a clinical disease that impairs vision and even causes corneal blindness. The imbalance of pro-angiogenic and anti-angiogenic factors caused by chemical burns, infectious keratitis, corneal transplant rejection, etc. is the main cause of corneal neovascularization. Corneal epithelial damage, corneal inflammation and oxidative stress can continuously affect and change the corneal microenvironment, leading to corneal neovascularization, corneal opacity, edema and ulcers, and ultimately lead to decreased vision. Therefore, timely and effective treatment of corneal neovascularization is very urgent and necessary. Corticosteroid eye drops, such as dexamethasone (DEX) eye drops, can effectively inhibit corneal inflammation and neovascularization, but can cause complications such as glaucoma, cataracts and repeated corneal infections. Anti-vascular endothelial growth factor (anti-VEGF) drugs have a certain effect on corneal neovascularization, but have poor effects on mature corneal blood vessels. Some plant extracts, such as curcumin, have strong antioxidant and anti-inflammatory capabilities, but their poor water solubility limits their clinical application in ophthalmology. Literature reports that the third-generation potent anthranilic acid nonsteroidal anti-inflammatory drug, diclofenac sodium, can inhibit the expression of VEGF and COX in the process of corneal neovascularization. -2The expression of diclofenac sodium ophthalmic preparations has a certain applicable prospect in the treatment of corneal neovascularization. However, both clinical and experimental studies have shown that the existing diclofenac sodium ophthalmic preparations are far less effective than dexamethasone preparations in controlling corneal neovascularization. In the animal model of corneal neovascularization in rats with alkali burns, the length of new blood vessels was used to calculate the area of ​​new blood vessels on the fourth and seventh days after injury. It was found that the occurrence and development of CNV in the experimental group was significantly inhibited, and there was a significant difference between the CNV in the control group (P < 0.05). The area of ​​new blood vessels in the experimental group was 52.11% (fourth day after burn) and 55.41% (seventh day after burn) of the control group, respectively. The area of ​​new blood vessels (X±s, mm 2) was 12.46±2.23, 19.01±3.45 and 23.91±3.59, 36.11±4.02, respectively. On the fourth day, P < 0.05, and on the seventh day, P < 0.01 (Study on the effect of diclofenac sodium on the regulation of VEGF on corneal neovascularization, Strait Pharmacy, Vol. 18, No. 5, 2006). In addition, diclofenac sodium ophthalmic preparations also have strong irritation, which can cause local irritation during use, such as symptoms such as redness and pain in the eyes. At present, the research on diclofenac sodium ophthalmic preparations mainly focuses on how to reduce irritation. For example, patent documents CN101352409A and CN104127377A both use osmotic pressure regulators to reduce the irritation of diclofenac sodium eye drops and improve drug efficacy. CN106806340A uses a bacteriostatic agent, sodium diacetate, to reduce irritation, and maintain the stability of the preparation to improve the patient's medication effectiveness and safety. There are few reports on how to improve the therapeutic effect of diclofenac sodium ophthalmic preparations in inhibiting corneal neovascularization. Patent document CN112043820A adopts transthyretin and diclofenac to cooperate synergistically to improve the therapeutic effect on eye diseases related to ocular angiogenesis and / or retinal leakage. The preparation mainly targets the neovascularization of the fundus, and the therapeutic effect on corneal neovascularization is unknown.

[0003] Nanozymes are nanomaterials with simple preparation, controllable size, adjustable functions, stable performance and enzyme catalysis. Nanozymes have unique potential and advantages in eye diseases. Nanozymes have similar effects to peroxidase (POD), catalase (CAT) and superoxide dismutase (SOD), and can effectively eliminate reactive oxygen species (ROS). As drug delivery carriers, nanozymes can prolong retention time and control drug release through strong mucosal adhesion. Summary of the invention

[0004] In view of the above-mentioned problems that the existing diclofenac sodium ophthalmic preparations have poor efficacy on corneal neovascularization and strong irritation, the present invention provides a method for preparing diclofenac sodium eye drops, and obtains iron-based proanthocyanidin-diclofenac sodium nanozyme through a metal coordination reaction of proanthocyanidins, diclofenac sodium and ferrous sulfate. The nanozyme is used in eye drops, which improves the inhibitory effect of diclofenac eye drops on corneal neovascularization, and can also improve corneal sensitivity and maintain normal intraocular pressure.

[0005] The present invention is achieved through the following technical solutions:

[0006] The preparation method of diclofenac sodium eye drops provided by the present invention comprises the following steps:

[0007] (1) dissolving ferrous sulfate in deionized water and cooling the ferrous sulfate solution to -1°C to 2°C;

[0008] (2) dissolving diclofenac sodium in deionized water, and then adding the diclofenac sodium solution dropwise to the ferrous sulfate solution obtained in step (1), controlling the temperature of the system to be -1°C to 2°C, stirring while adding dropwise, so that the carboxyl group of diclofenac sodium and the iron ion undergo metal coordination, and the color of the system gradually changes from light green to white;

[0009] (3) dissolving proanthocyanidin in deionized water, and then adding the proanthocyanidin solution dropwise into the white system obtained in step (3), controlling the temperature of the system to -1°C to 2°C, stirring while adding dropwise, the phenolic hydroxyl group of the proanthocyanidin coordinates with the iron ion metal, and the color of the system gradually turns black to obtain a suspension;

[0010] (4) centrifuging the suspension at 8,000-15,000 rpm for 3-8 min, taking the supernatant, and dialysis using an 8000-14000 Da dialysis bag in distilled water to remove byproducts;

[0011] (5) vacuum freeze-drying the obtained dialysate to obtain iron-based proanthocyanidin-diclofenac sodium nanozyme;

[0012] (6) Dissolving the iron-based proanthocyanidin-diclofenac sodium nanozyme in water to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme eye drops.

[0013] Furthermore, the concentration of the ferrous sulfate solution in step (1) is 8.0-16.0 mg / mL, preferably 12.0 mg / mL.

[0014] Furthermore, the concentration of the diclofenac sodium solution in step (2) is 44.0-52.0 mg / mL, preferably 48.0 mg / mL.

[0015] Furthermore, the concentration of the proanthocyanidin solution in step (3) is 8.0-16.0 mg / mL, preferably 12.0 mg / mL.

[0016] Furthermore, in the iron-based proanthocyanidin-diclofenac sodium nanozyme in step (5), the mass ratio of ferrous sulfate, diclofenac sodium and proanthocyanidin is 1:2:(1-2).

[0017] Furthermore, the concentration of the iron-based proanthocyanidin-diclofenac sodium nanozyme eye drops in step (6) is 1.0-5.0 mg / mL, preferably 2-4 mg / mL.

[0018] Furthermore, in step (1), the ferrous sulfate solution is cooled to 0°C.

[0019] Furthermore, the reaction temperature of steps (2) and (3) is both 0°C.

[0020] Furthermore, before the vacuum freeze-drying in step (5), the dialysate is pre-frozen at -80 to -60°C.

[0021] Furthermore, in steps (1) to (3), ferrous sulfate, diclofenac sodium and proanthocyanidins are respectively dissolved by ultrasonic dissolution method.

[0022] Furthermore, in steps (2) and (3), the stirring speed is 300-600 rpm, and the stirring time is 8-12 min.

[0023] Furthermore, in step (4), the suspension is centrifuged at a speed of 10,000 rpm for 5 minutes.

[0024] The present invention has the following beneficial effects:

[0025] Nanozymes that use metal coordination bonds to connect proanthocyanidins and diclofenac sodium can release metal ions and bioactive drug molecules, show good water solubility, and have the advantages of inhibiting corneal neovascularization, anti-inflammation, anti-oxidation and promoting corneal epithelial repair; they can improve corneal sensitivity and maintain normal intraocular pressure; the preparation method is simple to operate and the preparation time is short; the obtained eye drops have good biocompatibility, stable properties, and can be stored at room temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Characterization of the iron-based proanthocyanidin-diclofenac sodium nanozyme (PC-DS NE) prepared in the example. (A) Transmission electron microscopy photo. (B) PC-DS NE particle size analysis. (C) Zeta potential of PC-DS NE. (DF) Transmission electron microscopy element distribution map (TEM mapping) of PC-DS NE. (G) X-ray photoelectron spectroscopy (XPS). (H) Energy dispersive spectrometer (EDS). (I) Photos of four solutions at room temperature, from left to right: physiological saline, dexamethasone (DEX), proanthocyanidin (PC), diclofenac sodium (DS) and PC-DS NE solution. (J) Fourier transform infrared spectroscopy (FTIR). (K) Thermogravimetric analysis. (L) In vivo drug release experiment of PC-DS NE in New Zealand white rabbits. (M) In vitro drug release experiment of PC-DS NE. (N) Appearance and color of PC-DS NE solutions with different concentrations, from left to right: 5 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL and 0.1 mg / mL. (O) Absorbance of PC-DS NE solutions with different concentrations.

[0027] Figure 2 The multi-enzyme activity of PC-DS NE prepared in the examples. (A) Effect of pH value on TMB reaction; (B) Effect of tetramethylbenzidine (TMB) concentration on TMB reaction; (C) Effect of H2O2 concentration on TMB reaction; (D) Effect of PC-DS NE solubility on TMB reaction; (E) Effect of temperature on TMB reaction. (F) Effect of o-phenylenediamine (POD) at different pH and reaction time on TMB reaction. (G) Superoxide dismutase activity experiment. (H) Electron spin resonance (ESR) analysis.

[0028] Figure 3 Biocompatibility graphs of PC-DS NE prepared in the examples. (A) Live and dead cell staining. (BC) CCK-8 test of human corneal epithelial cells (HCEC) and human corneal stromal cells (HCSC). (D) Eye irritation test and corneal hematoxylin-eosin (HE) staining. (E) HE staining of mouse heart, liver, spleen, lung and kidney.

[0029] Figure 4 ROS scavenging ability of PC-DS NE prepared in the examples. (A) HCEC and HCSC ROS scavenging experiments. (B) Hydroxyl radical inhibition experiment (EP tubes from left to right are blank tube, standard tube, control tube, 12 mg / mL, 9 mg / mL and 6 mg / mL PC-DS NE solution). (C) ROS staining flow cytometric analysis of normal group, control group, DEX group and PC-DS NE group 7 days after alkali burn.

[0030] Figure 5Analysis of the anti-inflammatory activity of PC-DS NE prepared in Example. (AD) Real-time fluorescence quantitative PCR (RT-qPCR) of IL-1β, IL-6, IL-10 and IL-17α in the normal group, control group, DEX group and PC-DS NE group 32h after bone marrow-derived macrophages were treated with lipopolysaccharide (LPS). (E) Immunofluorescence of IL-1β in the normal group, control group and PC-DS NE group (scale bar = 100 μm). (F) Immunoblotting experiment of mouse corneal proteins on the 7th day after alkali burn (WB).

[0031] Figure 6 Corneal epithelial injury repair experiment of PC-DS NE prepared in Example. (A) Scratch test (scale: 60μm) and (B) injury repair rate (%). (C) Scrape corneal epithelium and take photos of fluorescein sodium staining at 0h, 12h, 24h and 48h (scale: 1mm) and (D) staining area analysis. (E) Tublin nerve staining spread (scale: 1mm) and (F) corneal nerve area analysis on the 7th day after eye drops of corneal epithelial injury in mice.

[0032] Figure 7 Alkali burn corneal neovascularization experiment of mice with PC-DS NE prepared in Example. (A) Animal treatment pattern diagram. (B) Slit lamp illumination and sodium fluorescein staining on the first day after alkali burn. (C) Slit lamp corneal limbal blood vessel photo after alkali burn (scale: 1 mm). (DE) Analysis of blood vessel length and area after alkali burn. (F) OCTA photo on the 7th day of alkali burn. (G) Alkali burn CD31 corneal staining spread (scale: 1 mm). (H) Alkali burn corneal HE staining (red arrow: blood vessel, scale: 100 μm). (I) Mouse corneal WB on the 7th day of alkali burn.

[0033] Figure 8 The umbilical vein endothelial cell (HUVEC) tube formation experiment of PC-DS NE prepared in the example and the treatment of corneal mature blood vessels 7 days after alkali burn. (A) HUVEC tube formation experiment. (B) Treatment of corneal mature blood vessels 7 days after alkali burn.

[0034] Fig. 9 Analysis of the efficacy of the PC-DS NE prepared in the example in the alkali burn experiment. (A) Optical coherence tomography (AS-OCT) of the anterior corneal segment after alkali burn and (B) corneal thickness analysis. (C) Comparison of corneal sensitivity after alkali burn. (D) Comparison of corneal transparency after alkali burn. (E) Intraocular pressure values ​​of each group after alkali burn. (F) Comparison of corneal mechanical strength after alkali burn. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below with reference to specific embodiments and accompanying drawings.

[0036] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0037] Example 1

[0038] (1) Add ferrous sulfate to deionized water and dissolve it by ultrasonic until a uniform solution is obtained. The concentration of the solution is 12.0 mg / mL. Add the prepared ferrous sulfate solution to a flask, place it in an ice box and use a magnetic stirrer at 400 rpm for 5 minutes to cool the temperature of the ferrous sulfate solution to 0°C.

[0039] (2) Diclofenac sodium was added to deionized water, and ultrasonically dissolved and mixed to obtain a diclofenac sodium solution with a concentration of 48.0 mg / mL. 5 mL of the diclofenac sodium solution was dropwise added to 10 mL of 0° C. ferrous sulfate solution. The system temperature was controlled to be 0° C., and the mixture was stirred at a speed of 400 rpm for 10 min while the addition was continued. The color of the system gradually changed from light green to white.

[0040] (3) Add proanthocyanidins into deionized water, dissolve and mix by ultrasonication to obtain a proanthocyanidin solution with a concentration of 12.0 mg / mL, add 10 mL of the proanthocyanidin solution dropwise into the system of step (3), control the system temperature to 0° C., stir at 400 rpm for 10 min, and the color of the system gradually changes from white to black to obtain a suspension;

[0041] (4) The obtained suspension was centrifuged at 10,000 rpm for 5 min, and the supernatant was taken and dialyzed in distilled water using a 10,000 Da dialysis bag. After dialysis for 48 h, the dialysate was placed in a centrifuge tube and placed in a -80 °C refrigerator overnight;

[0042] (5) After precooling in a vacuum freeze dryer for 20 minutes, the frozen centrifuge tube was quickly opened, sealed with a sealing film and pierced, and freeze-dried for 48 hours to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme;

[0043] (6) Dissolving the iron-based proanthocyanidin-diclofenac sodium nanozyme in water to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme eye drops.

[0044] Example 2

[0045] (1) Add ferrous sulfate to deionized water and dissolve it by ultrasonic until a uniform solution is obtained. The concentration of the solution is 8.0 mg / mL. Add the prepared ferrous sulfate solution to a flask, place it in an ice box and use a magnetic stirrer at 400 rpm for 5 minutes to cool the temperature of the ferrous sulfate solution to 0°C.

[0046] (2) Diclofenac sodium was added to deionized water, and ultrasonically dissolved and mixed to obtain a diclofenac sodium solution with a concentration of 44.0 mg / mL. 5 mL of the diclofenac sodium solution was dropwise added to 10 mL of 0° C. ferrous sulfate solution. The system temperature was controlled to be 0° C., and the mixture was stirred at a speed of 400 rpm for 10 min while the addition was continued. The color of the system gradually changed from light green to white.

[0047] (3) Add proanthocyanidins into deionized water, dissolve and mix by ultrasonication to obtain a proanthocyanidin solution with a concentration of 8.0 mg / mL, add 10 mL of the proanthocyanidin solution dropwise into the system of step (3), control the system temperature to 0° C., stir at a speed of 400 rpm for 10 min, and the color of the system gradually changes from white to black to obtain a suspension;

[0048] (4) The obtained suspension was centrifuged at 10,000 rpm for 5 min, and the supernatant was taken and dialyzed in distilled water using an 8000 Da dialysis bag. After dialysis for 48 h, the dialysate was placed in a centrifuge tube and placed in a -80 °C refrigerator overnight;

[0049] (5) After precooling in a vacuum freeze dryer for 20 minutes, the frozen centrifuge tube was quickly opened, sealed with a sealing film and pierced, and freeze-dried for 48 hours to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme;

[0050] (6) Dissolving the iron-based proanthocyanidin-diclofenac sodium nanozyme in water to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme eye drops.

[0051] Example 3

[0052] (1) Add ferrous sulfate to deionized water and dissolve it by ultrasonic until a uniform solution is obtained. The concentration of the solution is 16.0 mg / mL. Add the prepared ferrous sulfate solution to a flask, place it in an ice box and use a magnetic stirrer at 400 rpm for 5 minutes to cool the temperature of the ferrous sulfate solution to 0°C.

[0053] (2) Diclofenac sodium was added to deionized water, and ultrasonically dissolved and mixed to obtain a diclofenac sodium solution with a concentration of 52.0 mg / mL. 5 mL of the diclofenac sodium solution was dropwise added to 10 mL of 0° C. ferrous sulfate solution. The system temperature was controlled to be 0° C., and the mixture was stirred at 400 rpm for 10 min while the addition was continued. The color of the system gradually changed from light green to white.

[0054] (3) Add proanthocyanidins into deionized water, dissolve and mix by ultrasonication to obtain a proanthocyanidin solution with a concentration of 16.0 mg / mL, add 10 mL of the proanthocyanidin solution dropwise into the system of step (3), control the system temperature to 0° C., stir at 400 rpm for 10 min, and the color of the system gradually changes from white to black to obtain a suspension;

[0055] (4) The obtained suspension was centrifuged at 10,000 rpm for 5 min, and the supernatant was taken and dialyzed in distilled water using a 14000 Da dialysis bag. After dialysis for 48 h, the dialysate was placed in a centrifuge tube and placed in a -80 °C refrigerator overnight;

[0056] (5) After precooling in a vacuum freeze dryer for 20 minutes, the frozen centrifuge tube was quickly opened, sealed with a sealing film and pierced, and freeze-dried for 48 hours to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme;

[0057] (6) Dissolving the iron-based proanthocyanidin-diclofenac sodium nanozyme in water to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme eye drops.

[0058] (A) Characterization of iron-based proanthocyanidin-diclofenac sodium nanozyme.

[0059] The iron-based proanthocyanidin-diclofenac sodium nanozyme (PC-DS NE) prepared in Example 1 was characterized. Figure 1 (A) Transmission electron microscopy photo shows that PC-DS NE is a spherical structure. (B) PC-DS NE particle size analysis shows that the diameter of the nanozyme is about 40nm. (C) The Zeta potential of PC-DS NE shows that the nanozyme has good dispersibility. (D~F) Transmission electron microscopy element distribution map (TEM mapping) of PC-DS NE shows that PC-DS NE contains O and Fe elements. (G) X-ray photoelectron spectroscopy technology (XPS) shows that PC-DS NE contains C, N, O, and Fe elements. (H) Energy dispersive spectrometer (EDS) shows that PC-DS NE contains C, N, O, and Fe elements and their content. Figure 1(I) pH values ​​of four solutions at room temperature, from left to right: physiological saline, dexamethasone (DEX), proanthocyanidin (PC), diclofenac sodium (DS) and PC-DS NE solution. The pH value of PC-DS NE solution is 5.85±0.02. (J) Fourier transform infrared spectroscopy (FTIR), showing that proanthocyanidin has absorption peaks at 3385cm-1, 1610cm-1 and 1110cm-1. Diclofenac sodium has absorption peaks at 3385cm-1, 1574cm-1 and 746cm-1, and the intensity of these characteristic peaks is significantly reduced in the FTIR of PC-DS NE. This shows that PC-DS NE was successfully prepared by the reaction. (K) Thermogravimetric analysis shows that PC-DS NE has good thermal stability. (L) PC-DS NE in vivo drug release experiment in New Zealand white rabbits. From 15min to 120min, the concentration of proanthocyanidins in the aqueous humor of New Zealand white rabbits showed a gradual upward trend, while the concentration of diclofenac sodium showed a trend of first increasing and then decreasing, indicating that diclofenac sodium can also be released rapidly in vivo. (M) PC-DS NE in vitro drug release experiment showed that as time increased, the proanthocyanidins in PC-DS NE were first released rapidly within 48h and then released steadily after 48h, while diclofenac sodium in PC-DS NE was released rapidly within 48h and then released slowly after 48h. (N) The appearance and color of PC-DS NE solutions of different concentrations, from left to right: 5mg / mL, 2mg / mL, 1mg / mL, 0.5mg / mL and 0.1mg / mL, indicating that at low concentrations, the nanozyme solution had no obvious color. When the concentration exceeded 2mg / mL, the higher the concentration, the darker the color. (O) The absorbance of PC-DS NE solutions with different concentrations showed that there was no obvious absorbance when the wavelength of PC-DS NE solution was >300 nm and the concentration was ≤2 mg / mL.

[0060] (II) Multienzyme activity of PC-DS NE

[0061] The PC-DS NE prepared in Example 1 was subjected to a multi-enzyme activity test. Figure 1(A) pH value experiment shows that as time increases, OD 652 at pH = 2 shows a downward trend, while OD 652 at pH = 3 shows an upward trend and eventually becomes the highest point. Therefore, the present application uses PBS at pH = 3 as the subsequent reaction condition. (B) Tetramethylbenzidine (TMB) concentration shows that OD 652 shows an upward trend as TMB concentration increases, and this trend begins to slow down after TMB = 4. (C) Within the H2O2 concentration range of 50 to 500 mmol / L, the OD 652 value does not change significantly when the H2O2 concentration (50-500 mM) is changed. ; (D) It shows that OD652 increases with the increase of PC-DS NE concentration. (E) The effect of temperature on TMB reaction, OD 652 is the highest at 37°C, the lowest at 4°C, and higher than room temperature at 60°C. (F) O-phenylenediamine (POD) experiment at different pH and time. In the OPD experiment of PC-DS NE, the OD 450 value was the highest at pH = 1, and OD 450 gradually decreased as the pH increased. (G) Superoxide dismutase activity experiment. Compared with the control group, the PC-DS NE solution can significantly reduce the absorption peak of the product at 320nm, indicating that PC-DS NE has superoxide dismutase activity. (H) Electron spin resonance (ESR) analysis shows that there are obvious signals when PC-DS NE interacts with H2O2, while these signals are significantly weakened when PC-DS NE or H2O2 is detected alone. This shows that in the presence of H2O2, PC-DS NE has the function of catalyzing H2O2 to produce hydroxyl free radicals.

[0062] (III) Biocompatibility of PC-DS NE

[0063] The biocompatibility of the PC-DS NE prepared in Example 1 was studied. Figure 1 (A) Live and dead cell staining showed that PC-DSNE had no toxicity to HCEC and HCSC, and had the activity of promoting HCEC cell proliferation. (BC) CCK-8 experiment of human corneal epithelial cells (HCEC) and human corneal stromal cells (HCSC), there was no significant difference in the activity of HCEC and HCSC cells between the PC-DS NE group and the control group, indicating that PC-DS NE had no cytotoxicity. (D) Eye irritation test and corneal hematoxylin-eosin (HE) staining, no obvious corneal epithelial defect, corneal edema and pigmentation were found in the PC-DS NE group by slit lamp and sodium fluorescein staining, and no edema and inflammation were found in the PBS group and PC-DS NE group by HE staining. (E) HE staining of the heart, liver, spleen, lung and kidney of mice showed no obvious degeneration, necrosis, inflammatory cell infiltration, cell regeneration and tissue fibrosis in the heart, liver, spleen, lung and kidney of the PBS group and PC-DS NE group.

[0064] (IV) ROS scavenging ability of PC-DS NE

[0065] The ROS scavenging ability of the PC-DS NE prepared in Example 1 was studied. Figure 4 (A) HCEC and HCSC ROS scavenging experiments showed that PC-DS NE can scavenge ROS produced by HCEC and HCSC cells under H2O2 stimulation. (B) Hydroxyl free radical inhibition experiment (EP tubes from left to right are blank tube, standard tube, control tube, 12mg / mL, 9mg / mL and 6mg / mL PC-DS NE solution), showing that the ability of PC-DS NE to scavenge hydroxyl free radicals gradually increases with the increase of concentration. (C) ROS staining flow cytometric analysis of the normal group, control group, DEX group and PC-DS NE group 7 days after alkali burn, showing that PC-DS NE can reduce ROS in the corneal tissue of alkali-burned mice.

[0066] (V) Analysis of anti-inflammatory activity of PC-DS NE

[0067] The anti-inflammatory activity of PC-DS NE prepared in Example 1 was analyzed. Figure 5 (AD) Real-time fluorescence quantitative PCR (RT-qPCR) of IL-1β, IL-6, IL-10 and IL-17α in the normal group, control group, DEX group and PC-DS NE group 32 hours after the addition of lipopolysaccharide (LPS) to bone marrow-derived macrophages showed that the mRNA expression levels of pro-inflammatory factors IL-1β, IL-6 and IL-17α in the PC-DS NE group were significantly reduced compared with the control group (P < 0.05 =), while the expression of anti-inflammatory factor IL-10 was significantly increased (P < 0.05 =). This indicates that PC-DS NE may achieve anti-inflammatory effects by transforming M1 macrophages into M2 macrophages. (E) Immunofluorescence of IL-1β in the normal group, control group and PC-DS NE group (scale bar = 100 μm), indicating that PC-DS NE can control the inflammatory response after corneal alkali burn by reducing the expression level of inflammatory factor IL-1β. (F) Western blotting experiment of mouse corneal proteins on the 7th day after alkali burn (WB), indicating that PC-DS NE can control the inflammatory response after corneal alkali burn by reducing the expression levels of various inflammatory factors.

[0068] (VI) Corneal epithelial damage repair experiment

[0069] The PC-DS NE prepared in Example 1 was applied to a corneal epithelial damage repair experiment.

[0070] Experimental procedure: 8-week-old male C57 mice were intraperitoneally injected with 0.6% sodium pentobarbital (300 mg sodium pentobarbital powder + 50 mL normal saline), with an injection volume of 14.5 μL / g, about 0.32 mL / mouse. 0.5% proparacaine was dripped into the eye for surface anesthesia. A 2.5 mm trephine was used to inscribe the center of the corneal epithelium, and the corneal epithelium was scraped off using an Alger Brush II corneal rust ring remover. The mice were divided into three groups: control group, DEX group, and PC-DS NE group. The control group was dripped with PBS, and the DEX group and PC-DS NE group were dripped with 1 mg / mL DEX solution and 1 mg / mL PC-DS NE solution, respectively. Twice a day, 5 μL each time. For a total of 7 days. Slit lamp sodium fluorescein staining was performed and photographed at 0 h, 12 h, 24 h, and 48 h. The staining area was analyzed by ImageJ software.

[0071] Figure 6 The results of the application of PC-DS NE in corneal epithelial damage repair. The results of the HCEC scratch test showed that the HCEC scratch damage repair rates of the control group, 0.1mg / mL PC-DS NE group and 0.01mg / mL PC-DS NE group at 12h were (33.87±3.89)%, (63.73±9.03)% and (75.37±10.64)%, respectively. The HCEC scratch damage repair rates of the 0.1mg / mL PC-DS NE group and 0.01mg / mL PC-DS NE group were significantly higher than those of the control group (P<0.05). This shows that PC-DS NE can significantly promote HCEC cell migration, proliferation and damage repair. Similarly, fluorescein sodium staining at 0h, 12h, 24h and 48h after mouse epithelial scraping showed that compared with the control group, there was no obvious fluorescein sodium staining in the PC-DS NE group at 48h, while there was still a large area of ​​fluorescein sodium staining in the DEX group at 48h. This indicates that PC-DS NE solution can significantly promote the repair of corneal epithelial damage, while DEX solution can delay corneal epithelial repair. On the 7th day, corneal nerve Tublin staining spreads showed that the corneal nerves in the PC-DS NE group could grow into the center of the cornea, and the small nerves were dense, while the corneal nerves in the control group and the DEX group could not grow into the center of the cornea, and the small nerves were sparse. Statistical analysis results showed that the area of ​​sodium fluorescein staining of PC-DS NE was significantly lower than that of the control group and the DEX group at 48h (P < 0.05). The area of ​​corneal nerves in the PC-DS NE group on the 7th day was significantly higher than that in the DEX group (P < 0.05). The above results indicate that DEX will cause delayed healing of corneal epithelium and nerves, while PC-DS NE has the effect of promoting the proliferation and repair of corneal epithelium and nerves.

[0072] (VII) Experimental treatment of corneal alkali burn neovascularization

[0073] The PC-DS NE prepared in Example 1 was used to treat corneal alkali burn neovascularization.

[0074] Experimental procedure: 8-week-old male C57 mice were intraperitoneally injected with 0.6% sodium pentobarbital. 0.5% proparacaine was dripped into the eye for surface anesthesia. A 2.0 mm trephine was used to drill a filter paper disc and immersed in a 1 M NaOH solution. The filter paper was taken out and placed in the center of the cornea for 40 seconds, and then immediately rinsed with normal saline for 40 seconds. After wiping off the residual liquid with a cotton swab, ofloxacin eye ointment was applied once. The mice were divided into 6 groups: normal group (Normal), control group (Control), proanthocyanidin group (PC), diclofenac sodium group (DS), dexamethasone group (DEX), and proanthocyanidin-diclofenac sodium nanozyme group (PC-DS NE). The mice in the normal group were fed normally, the control group was dripped with PBS, and the PC group, DS group, DEX group, and PC-DS NE group were dripped with 5 mg / mL of the corresponding solution, 3 times / d, for a total of 7 days.

[0075] Figure 7 The results of the experiment on corneal alkali burn neovascularization using PC-DS NE. On the first day after the mouse alkali burn model was established, corneal slit lamp and sodium fluorescein photography showed that the degree of epithelial defect in the six groups of mouse corneal alkali burn models was balanced. On the third day after the mouse alkali burn, the blood vessels in the control group began to grow from the corneal limbus to the center of the cornea, and entered the corneal visual center on the seventh day. Pigmentation was visible in the proanthocyanidin group. The diclofenac sodium group showed corneal thinning and dissolution. No obvious neovascularization was observed in the DEX group and the PC-DS NE group. Compared with the control group, the reduction in vascular length and vascular area in the PC-DS NE group was statistically different (P < 0.05). Among the six groups of data, the vascular length (0.20 ± 0.09 mm) and area (0.23 ± 0.13 mm) of the PC-DS NE group on the 7th day were significantly reduced. 2) was the lowest. OCTA results showed that thick and dense blood vessels were visible in both the control group and the proanthocyanidin group, and the blood vessel area in the control group was large and close to the center of the cornea. The density and area of ​​corneal neovascularization in the DEX and PC-DS NE groups were both small. At the same time, CD31 corneal vascular staining spreads showed that the control group had fine and dense neovascularization growing into the center of the cornea, while the neovascularization area in the DEX and PC-DS NE groups was small, similar to the normal group, and existed in the limbus. HE staining results showed that the control group had inflammatory cell infiltration, anterior iris adhesion, and some vascular structures were visible, while the PC-DS NE group had no obvious inflammatory cell infiltration and vascular structure. In addition to VEGF, the formation of pathological neovascularization is also closely related to corneal stromal fibrosis. Therefore, myofibroblast marker (α-SMA) is also generally considered to be a tissue detection indicator of CNV. The results of mouse corneal tissue WB showed that on the 7th day after alkali burn, the expression levels of VEGFA and α-SMA proteins in the PC-DS NE group were lower than those in the control group. The above results indicate that PC-DS NE can inhibit the formation of CNV by inhibiting the expression of angiogenic factors such as CD31, VEGF and α-SMA.

[0076] (VIII) Experimental treatment of corneal mature blood vessels

[0077] The PC-DS NE prepared in Example 1 was used to treat mature blood vessels in corneal alkali burns.

[0078] Experimental process: After establishing the mouse alkali burn model according to (VII), the mice were fed normally for 7 days. Starting from the 8th day, they were divided into three groups: control group, DEX group and PC-DS NE group. PBS, 5mg / mL DEX solution and 5mg / mL PC-DS NE solution were dripped into both eyes, 5μL each time, 3 times a day, for 7 days. Slit lamp limbal vascular photography was performed on the 7th and 14th days.

[0079] Figure 8 The umbilical vein endothelial cell (HUVEC) tube formation experiment of PC-DS NE prepared in Example 1 and the treatment of corneal mature blood vessels 7 days after alkali burn. (A) In the HUVEC tube formation experiment, the normal group and the DEX group had obvious vascular lumen structures, while the cells in the PC-DSNE group did not form vascular lumen structures, indicating that PC-DS NE can effectively inhibit the formation of HUVEC cell vascular lumen. (B) Treatment of corneal mature blood vessels 7 days after alkali burn. Compared with the control group and the DEX group, the PC-DS NE group can effectively control the growth of corneal mature blood vessels.

[0080] (IX) Analysis of the efficacy of alkali burn experiment

[0081] Fig. 9Analysis of the efficacy of the PC-DS NE prepared in Example 1 in an alkali burn experiment. (A) Optical coherence tomography (AS-OCT) of the anterior corneal segment after alkali burn and (B) analysis of corneal thickness. The central corneal thickness (108.33±13.43μm) of the PC-DS NE group on the 7th day was not significantly different from that of the normal group (105±6.51μm) (P>0.05). The corneal morphology and thickness of the PC-DS NE group remained relatively normal. (C) Comparison of corneal sensitivity after alkali burn, indicating that PC-DS NE can improve the sensitivity of the cornea after alkali burn. (D) Comparison of corneal transparency after alkali burn. Compared with the control group, the corneal transparency of the PC-DS NE group was better and the corneal inflammation was lighter. (E) Intraocular pressure values ​​of each group after alkali burn. The control group, PC group, DS group and DEX group all had varying degrees of increased intraocular pressure, while the intraocular pressure of the PC-DS NE group could be maintained at a normal level. (F) Comparison of corneal mechanical strength after alkali burn, indicating that PC-DS NE group can improve the mechanical strength of mouse cornea after alkali burn.

[0082] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for preparing diclofenac sodium eye drops, characterized in that: The following steps are involved: (1) dissolving ferrous sulfate in deionized water and cooling the ferrous sulfate solution to -1°C to 2°C; (2) dissolving diclofenac sodium in deionized water, and then adding the diclofenac sodium solution dropwise to the ferrous sulfate solution obtained in step (1), controlling the temperature of the system to -1°C to 2°C, stirring while adding dropwise, so that the carboxyl group of diclofenac sodium reacts with the iron ion, and the color of the system gradually changes from light green to white; (3) dissolving proanthocyanidin in deionized water, and then adding the proanthocyanidin solution dropwise into the white system obtained in step (3), controlling the temperature of the system to -1°C to 2°C, stirring while adding dropwise, the phenolic hydroxyl group of the proanthocyanidin reacts with the iron ion, the color of the system gradually turns black, and a suspension is obtained; (4) Centrifuge the suspension at 8,000-15,000 rpm for 3-8 min, take the supernatant, and dialyze it in distilled water using a 8000-14000 Da dialysis bag to remove by-products; (5) vacuum freeze-drying the obtained dialysate to obtain iron-based proanthocyanidin-diclofenac sodium nanozyme; (6) Dissolving the iron-based proanthocyanidin-diclofenac sodium nanozyme in water to obtain the iron-based proanthocyanidin-diclofenac sodium nanozyme eye drops.

2. The preparation method according to claim 1, characterized in that: The concentration of the ferrous sulfate solution in step (1) is 8.0 to 16.0 mg / mL; the concentration of the diclofenac sodium solution in step (2) is 44.0 to 52.0 mg / mL; and the concentration of the proanthocyanidin solution in step (3) is 8.0 to 16.0 mg / mL.

3. The preparation method according to claim 1, characterized in that: The concentration of the ferrous sulfate solution in step (1) is 12.0 mg / mL; the concentration of the diclofenac sodium solution in step (2) is 48.0 mg / mL; and the concentration of the proanthocyanidin solution in step (3) is 12.0 mg / mL.

4. The preparation method according to claim 1, characterized in that: The mass ratio of ferrous sulfate, diclofenac sodium and proanthocyanidins in the iron-based proanthocyanidin-diclofenac sodium nanozyme in step (5) is 1:2:

1.

5. The preparation method according to claim 1, characterized in that: The concentration of the iron-based proanthocyanidin-diclofenac sodium nanozyme eye drops in step (6) is 1.0 to 5.0 mg / mL.

6. The preparation method according to claim 1, characterized in that: In step (1), the ferrous sulfate solution is cooled to 0°C; and the reaction temperatures in steps (2) and (3) are both 0°C.

7. The preparation method according to claim 1, characterized in that: In the step (5), the dialysate is pre-frozen at -80 to -60°C before vacuum freeze drying.

8. The preparation method according to claim 1, characterized in that: The steps (1) to (3) respectively use an ultrasonic dissolution method to dissolve ferrous sulfate, diclofenac sodium and proanthocyanidins.

9. The preparation method according to claim 1, characterized in that: The stirring speeds in steps (2) and (3) are both 300-600 rpm, and the stirring time is both 8-12 min.

10. The preparation method according to claim 1, characterized in that: The suspension is centrifuged at a speed of 10,000 rpm in step (4) for 5 min.

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