Oxygen-enriched eye drops based on oxygen-containing liposome and application thereof

Abstract

The invention provides oxygen-enriched eye drops based on oxygen-containing lipidosome and application of the oxygen-enriched eye drops. The preparation method comprises the following steps: firstly, preparing blank liposome powder through a film dispersion-freeze drying technology, carrying out oxygenation treatment on the blank liposome powder to prepare oxygen-containing liposome, and then mixing the oxygen-containing liposome with sodium hyaluronate eye drops to obtain the oxygen-enriched eye drops. According to the oxygen-enriched eye drops, the permeability of oxygen in corneal tissues is improved, proliferation of corneal epithelial cells is remarkably promoted, and the apoptosis process of the corneal epithelial cells is delayed. The oxygen-enriched eye drops can promote repair after corneal epithelium injury.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to an oxygen-enriched eye drop based on oxygen-containing liposomes and its application. Background Technology

[0002] The cornea is a crucial refractive medium, and its transparency depends on the integrity of the corneal epithelium. When the corneal epithelium is damaged, visual function is often severely negatively affected. Persistent loss of epithelial integrity can lead not only to eye infections and corneal stromal edema, but also to corneal ulcers, and imperfect healing of corneal wounds can further cause damage to the ocular surface. Corneal epithelial cell repair after injury has always been a hot topic in ocular surface disease research. The corneal epithelial repair process is mainly accomplished by promoting the proliferation, division, and migration of peripheral corneal epithelial cells. Current research has confirmed that early oxygen therapy is of great significance in promoting corneal epithelial recovery. Li et al., by comparing the effects of two different oxygen delivery methods on corneal epithelial repair, confirmed that increasing the oxygen concentration in the periocular environment can significantly enhance the proliferative capacity of corneal epithelial cells and shorten the repair time after corneal epithelial injury, providing a theoretical basis for the treatment of corneal epithelial injury (Li S, Tian Q, Ding G, et al. The Impact of Different Oxygen Delivery Methods on Corneal Epithelial Repair after Injury [J]. Journal of ophthalmology, 2022, 2022:3260087.). Although early oxygen therapy has shown its potential in corneal epithelial repair, existing oxygen delivery methods still have some limitations, such as operational complexity and lack of convenience.

[0003] Liposomes have good biocompatibility and biodegradability, and can be taken up and metabolized by human cells. They are widely used in drug delivery, gene therapy, vaccine preparation and other fields. Among them, liposomes have shown significant application value in gene therapy and skin drug delivery, improving the efficiency and safety of treatment. There are also some studies on the preparation of eye drops using liposomes, but the application development of liposomes is still limited.

[0004] CN1170364A discloses a liposome eye drop composed of taurine, glucose and inorganic salts, used to relieve and treat symptoms caused by dry eyes.

[0005] CN109381707A discloses an azithromycin ion-paired liposome eye drop and its preparation method. Each 100 ml of the azithromycin ion-paired liposome eye drop contains: 0.5-2 g azithromycin, 5-10 g phospholipids, 1-5 g cholesterol derivatives, 5-10 g oil phase, 50-200 mg DSPE-PEG 2000, 1-5 mg preservative, and 30-80 mg antioxidant. The use of ion-pairing technology for drug loading improves the solubility and stability of the drug in the formulation, significantly increases the drug loading capacity, and makes it better suited for treating dry eye syndrome, with high bioavailability.

[0006] CN112804990A discloses a liposomal eye drop and its application in treating dry eye syndrome. The eye drop is composed of liposomes, which are made of non-hydrogenated phospholipids containing linseed oil, vitamin A palmitate, vitamin E polyethylene glycol succinate, aqueous vitamin B12, and pycnogenol. The liposomal eye drop contains a specific system composed of 2-amino-2-(hydroxymethyl)propane-1,3-diol, which acts as a salting agent for the water-soluble pycnogenol; and a borate buffer. The liposomal eye drop can play a role in protecting against UVA / UVB rays and reducing the negative effects of dry eye syndrome.

[0007] A search revealed no current research on the use of liposomes for oxygen delivery in the treatment of ocular surface diseases. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides an oxygen-enriched eye drop based on oxygen-containing liposomes and its application. First, blank liposome powder is prepared using a thin-film dispersion-freeze-drying technique. This powder is then oxygenated to prepare oxygen-containing liposomes, which are subsequently mixed with sodium hyaluronate eye drops to obtain the oxygen-enriched eye drop. This oxygen-enriched eye drop increases oxygen permeability in corneal tissue, significantly promotes the proliferation of corneal epithelial cells (HCEC cells), and delays the apoptosis process of corneal epithelial cells. The oxygen-enriched eye drop can promote the repair of corneal epithelial damage. The oxygen-enriched eye drop of this invention is convenient and quick to use, providing a practical and effective means for the clinical treatment and repair of corneal epithelial damage.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: an oxygen-enriched eye drop based on oxygen-containing liposomes, characterized in that it is composed of oxygen-containing liposomes and sodium hyaluronate eye drops with a concentration of 0.1%~0.3%.

[0010] The sodium hyaluronate eye drops are composed of: sodium hyaluronate 0.1-0.3%, sodium chloride 0.7-0.9%, disodium hydrogen phosphate / sodium dihydrogen phosphate in appropriate amount (to adjust pH 7.2-7.4), and water for injection to make up to 100%.

[0011] Preferably, the mass ratio of the oxygenated liposomes to sodium hyaluronate eye drops is 1:0.5~3, more preferably 1:1.

[0012] The preparation method of the above-mentioned oxygenated liposomes specifically includes the following steps: (1) Dissolve liposomes in an organic solvent (such as chloroform), and then remove the solvent by rotary evaporation to form a uniform liposome film; (2) The liposome membrane obtained in step (1) is subjected to hydration and sonication to reduce the liposome particle size (particle size range after sonication: 100~250nm), then trehalose and mannitol are added and shaken well, and then freeze-dried to obtain dry blank liposomes; (3) The dry blank liposomes obtained in step (2) are sealed and oxygenated, and then placed to obtain oxygenated liposomes with an oxygen content of 65-75%.

[0013] Furthermore, the mass ratio of the liposomes, trehalose, and mannitol is 70:0.1~1:0.1~1.

[0014] Furthermore, the oxygenation time in step (3) is 3~10 min; the placement time is 40~60 h.

[0015] The present invention also provides the application of the above-mentioned oxygen-enriched eye drops in the preparation of a drug that promotes the regeneration and repair of damaged corneal epithelial cells.

[0016] The beneficial effects of this invention are: (1) This invention develops a novel oxygen-containing liposome. Blank liposome powder is prepared by thin-film dispersion-freeze-drying technology, and oxygen-containing liposomes are obtained by oxygenation treatment. After reconstitution treatment, the oxygen-containing liposomes can continuously release oxygen for a certain period of time, forming oxygen-carrying lipid nanobubbles. This novel oxygen delivery system not only improves the oxygen delivery efficiency, but also promotes cell proliferation and inhibits cell apoptosis.

[0017] (2) The oxygen-enriched eye drops prepared by the present invention using oxygen-containing liposomes and sodium hyaluronate eye drops improve the oxygen permeability in corneal tissue by optimizing the oxygen encapsulation and release mechanism, reducing the irritation to the eye, significantly promoting the proliferation of corneal epithelial cells, delaying the apoptosis process of corneal epithelial cells, and significantly reducing their apoptosis rate, thus helping to improve the survival status of cells. The oxygen-enriched eye drops can also promote the repair of corneal epithelial damage.

[0018] (3) The oxygenated liposomes of the present invention are portable, providing more possibilities for clinical application. They can be used as drug preparations for the regeneration and repair of damaged cells, providing a practical and effective means for the clinical treatment of corneal epithelial damage and repair. Attached Figure Description

[0019] Figure 1 This is a trend graph showing the cumulative oxygen release concentration (mg / L water) of oxygenated liposomes after different treatments in water. Figure 2 This is a graph showing the trend of oxygen content in oxygenated liposomes after different treatments following release into water; among them, Figure 1 and Figure 2 The oxygenated liposomes that underwent different treatments were: oxygenated liposomes that were oxygenated for 3 min, 6 min, and 10 min, and oxygenated liposomes that were oxygenated for 6 min and 10 min and then placed for 48 h. Figure 3 Corneal staining analysis was performed at 0h, 12h, 24h, 36h, 48h, 60h, and 72h postoperatively. Group A (experimental group): 0h (A1), 12h (A2), 24h (A3), 36h (A4), 48h (A5); Group B (control group): 0h (B1), 12h (B2), 24h (B3), 36h (B4), 48h (B5), 60h (B6); Group C (blank group): 0h (C1), 12h (C2), 24h (C3), 36h (C4), 48h (C5), 60h (C6), 72h (C7). Corneal fluorescence staining at 12h, 24h, 36h, 48h, 60h, and 72h postoperatively: The staining area in Group A at each time point was significantly smaller than that in Group B, and the staining area in Group B was smaller than that in Group C. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1: Preparation of oxygenated liposomes (1) Add the test sample liposomes (phospholipid 50mg + cholesterol 20mg + sucrose 310mg) to a round bottom flask and dissolve them in 380ml of chloroform (or other organic solvents that can dissolve liposomes can be used instead). Then use a rotary evaporator to remove the chloroform under reduced pressure at 40℃ to form a uniform liposome film. (2) 70 mg of liposome membrane was hydrated with 20 ml of water and sonicated at 30 kHz for 10 min to reduce the liposome particle size (particle size range after sonication: 100-250 nm) to achieve sample homogenization; then 0.3 mg of trehalose and 0.3 mg of mannitol were added and shaken thoroughly until dissolved; the liposome solution was dispensed into 2 ml vials and freeze-dried at -80℃ and 12 Pa for 10 h to obtain dry blank liposomes; (3) Seal the vial of the dry blank liposome and use an oxygen generator to oxygenate it. Replace the air in the vial with oxygen at a concentration of 80% and an oxygen flow rate of 2L / min. Oxygenate for 3 min, 6 min, and 10 min respectively to obtain oxygenated liposomes with different oxygenation times. The oxygen content of the oxygenated liposomes obtained with different oxygenation times is 68%, 69%, and 71% respectively.

[0022] Experimental Example 1: Oxygen-carrying capacity and oxygen release rate of liposomes The oxygenated liposomes prepared in Example 1, after being oxygenated for 6 min and 10 min, were placed for 48 h. Then, they were reconstituted with oxygenated liposomes after being oxygenated for 3 min, 6 min, and 10 min (2 ml for each treatment) using 2 mL of purified water. After thorough shaking and dissolution, the cumulative oxygen concentration and oxygen release rate of the oxygenated liposomes in water were measured using a DO-166MT-1SXS micro dissolved oxygen system at 5, 10, 20, 30, 40, 50, and 60 h, and the results were recorded. The trend of cumulative oxygen release concentration of the oxygenated liposomes in water after different treatments is shown in the figure below. Figure 1 As shown in the figure; the oxygen content trend of oxygenated liposomes after different treatments after release in water is shown in the figure. Figure 2 As shown.

[0023] like Figure 1 As shown, the cumulative oxygen concentration of oxygen-containing liposomes in water increases with the extension of oxygenation time during the measurement period; and after being placed for a period of time, the cumulative oxygen concentration of oxygen-containing liposomes obtained under the same oxygenation time increases, proving that oxygen-containing liposomes have oxygen-carrying capacity and that the oxygen-carrying capacity can increase with the extension of oxygenation time. Figure 2 As shown, the oxygen content of oxygenated liposomes after release into water and subsequent treatments gradually decreased, reaching its lowest point at 50 min (close to the oxygen content in the air). For comparison, oxygenated liposomes that had been oxygenated for 10 min and then placed for 48 h were selected as the material for subsequent experiments.

[0024] Experimental Example 2: Effects on corneal epithelial cells 1. Corneal epithelial cell culture Under aseptic conditions, cryopreserved corneal epithelial cells were preheated in a 37°C water bath and shaken until completely thawed. The cryopreservation solution was transferred to a centrifuge tube, 4 ml of complete human corneal epithelial cell culture medium was added, and the cells were mixed thoroughly by pipetting. The tube was then centrifuged at 1000 rpm for 5 min, and the supernatant was discarded. Another 5 ml of complete human corneal epithelial cell culture medium was added, and the cells were mixed thoroughly by pipetting. The cells were then seeded into 10 ml culture flasks and placed in a humidified CO2 incubator. The culture medium was changed every 2-3 days. When the cell density reached 80%, the old culture medium was aspirated, and the cells were washed 2-3 times with PBS buffer. Trypsin (enzyme activity 1.0 U / mg) was added to cover the bottom of the culture flask, and the flask was placed in an incubator (temperature: 37±0.5°C, CO2 concentration: 5%, humidity: ≥90%) for 1 min. When cell clumps were observed to float, 3 ml of complete human corneal epithelial cell culture medium was added to stop the digestion process, and the cells were quickly pipetted to form a suspension. The cell suspension was transferred to a centrifuge tube and centrifuged at 1000 rpm for 5 min. After 1 minute, aspirate the supernatant, add 1 ml of complete human corneal epithelial cell culture medium, mix well, and obtain corneal epithelial cell culture medium (1 × 10⁶ cells / mL). 5 ~2×10 5 (each / ml), for later use.

[0025] 2. Experimental Group Setup The experiment was divided into 4 groups.

[0026] Group A: This is the control group, which received no additional treatment in blank culture medium (complete human corneal epithelial cell culture medium); Group B: 5 ml blank culture medium + (100 µl) sodium hyaluronate eye drops; Group C: 5 ml blank culture medium + (100 µl) sodium hyaluronate eye drops + blank liposomes (100 µl); Group D: 5 ml blank culture medium + (100 µl) sodium hyaluronate eye drops + oxygenated liposomes (100 µl).

[0027] Sodium hyaluronate eye drops: Sodium hyaluronate content 0.2%, sodium chloride content 0.8%, appropriate amount of disodium hydrogen phosphate / sodium dihydrogen phosphate (to adjust pH 7.2-7.4), and water for injection to make up to 100%.

[0028] 3. Detection of the periodic distribution of corneal epithelial cells (1) Detection method Take 5 ml of the corneal epithelial cell culture medium obtained above, divide it into 4 groups, and then add 5 ml of the treatment medium of groups A, B, C, and D respectively. Incubate the above treatment groups in a 37℃, 5% CO2 cell culture incubator for 24 hours. Flow cytometry is used to detect the cell cycle of each group. After trypsin digestion and collection of cells from each group, adjust the cell number to 1×10⁻⁶ cells using 400 μL of complete human corneal epithelial cell culture medium. 6 Four groups of cells were collected and washed three times with PBS phosphate buffer. Then, pre-cooled 75% ethanol was added and the cells were fixed at 4°C for 24 h. The cell cycle distribution of each group was detected by flow cytometry according to the instructions of the cell cycle kit (Meilune). Each experiment was repeated three times.

[0029] (2) Results Analysis Generally, a higher proportion of cells in the S phase and G2 / M phase indicates stronger cell proliferation capacity. The proportion of cells in the S phase and G2 / M phase to the total number of cells is called the proliferation index, which is calculated using the following formula.

[0030] Proliferation index = (Number of cells in S phase + Number of cells in G2 / M phase) / Total number of cells × 100% As shown in Table 1, the cell proliferation indices in the S phase and G2 / M phase of group D were significantly higher than those in the other three groups, indicating that oxygenated liposomes have the potential to promote cell proliferation.

[0031] Table 1. Statistical table of corneal epithelial cell percentage at different cell cycles in each treatment group.

[0032] 4. Detection of corneal epithelial cell proliferation rate (1) Detection method Cell proliferation rate was determined using the CCK-8 assay. Human corneal epithelial cell culture medium prepared above was cultured for 24 h (culture conditions: 37℃, 5% CO2, ≥90% humidity). After most cells adhered to the culture vessel, the cells were shaken to obtain a corneal epithelial cell suspension. 24 wells were randomly selected from 96-well plates and divided into 4 groups of 6 wells each. 100 μL of the prepared corneal epithelial cell suspension was added to each well, followed by 10 μL of the treatment medium from groups A, B, C, and D, respectively. After cell treatment, 10 μL of CCK-8 solution (using the original concentration, no dilution required, typically 5-10 mM WST-8 containing the electron coupling agent 1-Methoxy PMS) was added to each well. Wells with blank cell culture and CCK-8 solution served as a blank control group. The 96-well plates were incubated in a cell culture incubator. At 24 h, 48 h, 72 h, and 96 h, the 96-well plates were incubated at 450 °C using a microplate reader. The absorbance was measured at nm and calculated.

[0033] Proliferation rate = (Average absorbance value of experimental group / Absorbance value of normal control group) × 100% (2) Results Analysis The CCK-8 assay is a commonly used method for detecting cell proliferation and cytotoxicity, reflecting cell proliferation by measuring intracellular ATP levels. As shown in Table 2, the proliferation rate of corneal epithelial cells under the influence of oxygenated liposomes was significantly higher than that of other experimental groups, confirming that oxygenated liposomes significantly promote the proliferation of corneal epithelial cells under in vitro culture conditions.

[0034] Table 2. Statistical table of corneal epithelial cell proliferation rate.

[0035] 5. Detection of corneal epithelial cell apoptosis rate (1) Detection method The corneal epithelial cell culture medium obtained above was divided into four groups, 2 ml per group. Then, 200 μL of the treatment medium from groups A, B, C, and D were added to each group. The treated groups were incubated at 37℃ in a 5% CO2 cell culture incubator for 24 hours. The apoptosis rate of each group was detected using the Annexin V-FITC method. After washing the cells with phosphate buffer, the cell concentration was adjusted to 1×10⁻⁶ cells / mL using 1×Binding Buffer. 4 Cell suspensions of 1 cell / mL were prepared. 400 µL of the cell suspension was taken and the apoptosis rate of each group was detected according to the Annexin V-PE / 7-AAD Apoptosis Detection Kit (BD Biosciences, Cat. No. 559763) instructions. The experiment was repeated 3 times.

[0036] Apoptosis rate = (early apoptotic cells + late apoptotic cells) / total number of cells × 100% (2) Results Analysis Apoptosis is a natural process of programmed cell death that plays an important physiological role in organisms, especially in the removal of aging or dysfunctional cells. Under normal physiological conditions, apoptosis is a mechanism for the body to self-regulate and maintain homeostasis.

[0037] As shown in Table 3, the test results indicate that oxygenated liposomes can significantly delay the apoptosis process of corneal epithelial cells (HCEC cells) and significantly reduce their apoptosis rate, which is statistically significant. p <0.001). Oxygenated liposomes have a positive regulatory effect on apoptosis in HCEC cells, thereby helping to improve cell survival.

[0038] Table 3. Statistical table of cell apoptosis rate

[0039] Experimental Example 3: Repair Effect on Corneal Epithelium 1. Materials and Methods Materials: 75 healthy New Zealand white rabbits, weighing 2-2.5 kg (no statistically significant difference in weight).

[0040] Test reagent: Oxygen-enriched eye drops.

[0041] 2. Methods: Rabbits were randomly divided into an experimental group, a control group, and a blank group, with 25 New Zealand white rabbits in each group. One eye was used as the experimental eye. The cornea was marked with a 5 mm trephine and then scraped with a corneal epithelial scraper for 2 hours each time. All three groups were given levofloxacin eye drops 4 times / day to prevent infection. The experimental group received oxygen-enriched eye drops (prepared in Example 1) 6 times daily at 8, 10, 12, 14, 16, and 18 o'clock. The control group received betamethasone eye drops 6 times daily at 8, 10, 12, 14, 16, and 18 o'clock. The blank group received no treatment and waited for natural healing. Corneal fluorescein staining was performed at 12h, 24h, 36h, 48h, 60h, and 72h after injury. The corneal epithelial repair status of the three groups was observed under a slit lamp, and the corneal epithelial cell damage grading was recorded.

[0042] 3. Statistical Analysis: Statistical analysis. The Kruskal-Wallis H rank-sum test was used to compare the number of corneal epithelial cell layers among different groups. All data are expressed as mean ± standard deviation. For overall comparisons, P < 0.05 was considered statistically significant; to avoid Error I due to multiple comparisons among multiple sampling rates, it is recommended to change the level to P = 0.05 / 3 = 0.017. For paired comparisons, P < 0.017 was considered statistically significant.

[0043] 4. Results like Figure 3 As shown, the stained area of ​​the three groups at each time point follows the order of experimental group < control group < blank group, indicating that corneal epithelial healing is fastest in the experimental group, followed by the control group, and slowest in the blank group.

[0044] Conclusion: Oxygen-enriched eye drops can promote the repair of corneal epithelial damage.

Claims

1. An oxygen-enriched eye drop based on oxygen-containing liposomes, characterized in that, It consists of oxygen-containing liposomes and sodium hyaluronate eye drops with a concentration of 0.1%~0.3%; The method for preparing the oxygenated liposomes specifically includes the following steps: (1) Dissolve liposomes in an organic solvent, and then remove the organic solvent by rotary evaporation to form a uniform liposome film; (2) The liposome membrane obtained in step (1) was subjected to hydration and sonication to reduce the liposome particle size, and then trehalose and mannitol were added and shaken well. The membrane was then freeze-dried to obtain dry blank liposomes. (3) The dry blank liposomes obtained in step (2) are sealed and oxygenated, and then placed to obtain oxygenated liposomes with an oxygen content of 65-75%.

2. The oxygen-enriched eye drops based on oxygen-containing liposomes as described in claim 1, characterized in that, The sodium hyaluronate eye drops are composed of the following components by mass ratio: sodium hyaluronate 0.1-0.3%, sodium chloride 0.7-0.9%, disodium hydrogen phosphate / sodium dihydrogen phosphate to adjust the pH to 7.2-7.4, and water for injection to make up to 100%.

3. The oxygen-enriched eye drops based on oxygen-containing liposomes as described in claim 1, wherein the mass ratio of the oxygen-containing liposomes to sodium hyaluronate eye drops is 1:0.5~3.

4. The oxygen-enriched eye drops based on oxygen-containing liposomes as described in claim 1, characterized in that, The mass ratio of the liposomes, trehalose, and mannitol is 70:0.1~1:0.1~1.

5. The oxygen-enriched eye drops based on oxygen-containing liposomes as described in claim 1, characterized in that, The oxygenation time in step (3) is 3~10 min; the placement time is 40~60 h.

6. The oxygen-enriched eye drops based on oxygen-containing liposomes as described in claim 1, characterized in that, Particle size range after ultrasound: 100~250nm.

7. The oxygen-enriched eye drops based on oxygen-containing liposomes as described in claim 1, characterized in that, The organic solvent is chloroform.

8. The use of the oxygen-enriched eye drops based on oxygen-containing liposomes according to any one of claims 1-7 in the preparation of a medicament for promoting the regeneration and repair of damaged corneal epithelial cells.

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