Composition for treating meibomian gland dysfunction based on chlorella and application

Abstract

The invention provides a meibomian gland dysfunction treatment composition based on chlorella, the composition comprises sodium hyaluronate gel and drug-loaded chlorella powder, and the drug-loaded chlorella powder comprises chlorella and loaded drug components. The invention also provides a preparation method of the meibomian gland dysfunction treatment composition based on chlorella, which comprises the following steps: (1) dispersing a chlorella suspension in a sodium hydroxyethyl cellulose solution, and adding the medicinal components in the stirring process; (2) carrying out ultrasonic treatment and freeze drying to obtain drug-loaded chlorella powder; and (3) resuspending the drug-loaded chlorella powder, and adding sodium hyaluronate to form the gelatinous composition. The invention also provides application of the composition in preparation of medicines for treating meibomian glands. The composition not only can deliver active ingredients of the medicine in a targeted manner, but also can supplement lipid components lacking in meibomian glands, so that meibomian gland dysfunction is effectively treated.

Description

Technical Field

[0001] The present invention specifically relates to the application of bioactive materials in the field of ophthalmic technology, and in particular to a Chlorella-based composition for treating meibomian gland dysfunction, and a preparation method and use thereof. Background Art

[0002] Meibomian Gland Dysfunction (MGD) is a chronic, diffuse meibomian gland disease characterized by obstruction of the terminal ducts of the meibomian glands and / or abnormalities in the quality or quantity of meibum. MGD can cause tear film abnormalities and ocular surface inflammation, leading to eye irritation symptoms. In severe cases, it may damage the cornea and affect visual function. MGD is the most common type of dry eye and requires long-term treatment and management. It seriously affects patients' work efficiency and quality of life, and has therefore become an important public health issue.

[0003] Traditional local drug treatments for MGD have some limitations, including rapid clearance and low bioavailability of topical medications, and the need for frequent dosing, which leads to decreased patient compliance. In order to overcome these problems, it is crucial to develop new, efficient and safe MGD treatment methods. For example, the Chinese patent with publication number CN1437947A discloses a drug active ingredient - ofloxacin - which is first made into a gel-type eye ointment (Dicloro eye ointment) for the treatment of ophthalmic bacterial infections, aiming to improve the bioavailability of the drug active ingredient, increase efficacy and medication safety; this invention is the first to make ofloxacin into a gel-type eye ointment, which not only solves the problem that eye drops are easily diluted by tears and have a short residence time in the eye; it also solves the problem that the treatment effect of systemic administration is not ideal. And Chinese patent publication number CN112618485A discloses a sustained-release levofloxacin eye drop and its preparation method and application. By using thickeners such as hydroxypropyl methylcellulose and polyvinyl pyrrolidone as adhesive polymers, the residence time of the drug in the eye is prolonged, the bioavailability of the drug in the eye is improved, the number of dosing times is reduced, and patient compliance is improved.

[0004] Chlorella (Chlo.) originates from the genus Chlorella, a single-cell green algae rich in a variety of bioactive ingredients. It contains abundant nutrients such as protein, fat, carbohydrates, vitamins, minerals, and bioactive substances such as chlorophyll and carotenoids. It can not only be used as a drug carrier, but also has multiple therapeutic effects such as anti-inflammatory, antioxidant, and immunomodulatory, forming a synergistic effect with drug ingredients. Therefore, how to use these characteristics of Chlorella to develop a comprehensive therapeutic composition targeting the symptoms and pathogenesis of MGD is expected to become a new and effective treatment for MGD. Summary of the invention

[0005] The purpose of the present invention is to provide a meibomian gland dysfunction treatment composition based on Chlorella, which can not only deliver active pharmaceutical ingredients in a targeted manner, but also supplement the lipid components lacking in the meibomian glands, thereby effectively treating meibomian gland dysfunction.

[0006] A chlorella-based composition for treating meibomian gland dysfunction comprises sodium hyaluronate gel and drug-loaded chlorella powder, wherein the drug-loaded chlorella powder comprises chlorella and loaded drug components.

[0007] Among them, the active pharmaceutical ingredients in the therapeutic composition include the loaded drugs and the biologically active substances of Chlorella itself; the loaded drugs and the biologically active substances of Chlorella itself include lipids and fat-soluble compounds, polysaccharides, peptides and phenolic compounds and other ingredients; in the therapeutic composition, Chlorella, as the main ingredient, can be used as a sustained-release carrier of the drug to prolong the residence time and bioavailability on the ocular surface, and also exert a synergistic therapeutic effect through its own biologically active ingredients.

[0008] Furthermore, the lipid components in Chlorella can directly supplement tear film lipids.

[0009] Furthermore, the drug ingredients may include but are not limited to steroids, nonsteroidal anti-inflammatory drugs (NSAIDs), antibiotics, etc. These ingredients can play a therapeutic role on the symptoms and pathogenesis of MGD, such as inhibiting inflammatory response, improving meibomian gland function, etc.

[0010] More preferably, the drug ingredient is dexamethasone.

[0011] Furthermore, the size of the Chlorella is 1-10 μm.

[0012] Furthermore, the mass ratio of the chlorella to the medicinal component is 1:0.5-1:25.

[0013] The present invention also provides a method for preparing a composition for treating meibomian gland dysfunction based on Chlorella, the preparation method comprising:

[0014] (1) dispersing the chlorella suspension in the sodium hydroxyethylcellulose solution and adding the drug component during stirring;

[0015] (2) After ultrasonic treatment and freeze drying, drug-loaded Chlorella powder is obtained;

[0016] (3) After the drug-loaded Chlorella powder is resuspended, sodium hyaluronate is added to form a gel-like composition.

[0017] Furthermore, the drug component is dexamethasone, and the preparation method is: after centrifugal washing, the chlorella suspension is dispersed in a sodium hydroxyethyl cellulose solution, and dexamethasone is added during the stirring process.

[0018] (Dexamethasone, Dex). After ultrasonic treatment and freeze drying, drug-loaded Chlorella powder (Chlo.@Dex) was obtained. After the powder was resuspended, sodium hyaluronate was added to form a gel-like Chlo.@Dex gel.

[0019] Furthermore, in step (2), the ultrasonic treatment is carried out at 10-50W for 10-30min.

[0020] The present invention also provides an application of the above composition in preparing a medicine for treating meibomian glands.

[0021] The advantages of the present invention are: Chlorella as a carrier can slowly release the loaded drug ingredients (such as Dex) to provide a sustained drug effect; the lipid components rich in Chlorella itself can quickly replenish the missing lipids in the tear film to achieve immediate symptom improvement; the drug ingredients (such as Dex) loaded by Chlorella and the biologically active substances of Chlorella itself can synergistically play the therapeutic effects of inhibiting cell apoptosis, anti-inflammation, and promoting lipid synthesis; Chlorella has good dispersibility and a tiny size (1-10μm), which is conducive to the administration and absorption of the preparation. Based on the above advantages, the gel provided by the present invention (such as Chlo.@Dex gel) shows good safety, biocompatibility and therapeutic effects in both in vivo and in vitro experiments, providing a safe, effective candidate solution with clinical transformation prospects for the treatment of MGD. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are scanning electron microscopy and transmission electron microscopy images of the therapeutic composition prepared in the examples.

[0023] Figure 2 Hydrodynamic diameter and zeta potential plots for therapeutic compositions prepared for the Examples.

[0024] Figure 3 This is a drug sustained release curve of the therapeutic composition prepared in Example (the ordinate is the percentage of the cumulative released drug to the total amount of loaded drug).

[0025] Figure 4 These are fluorescent images of the distribution of the therapeutic composition prepared in Example on the ocular surface at different time points after eye instillation.

[0026] Figure 5 This is a graph showing the lipid content of the therapeutic composition prepared in the examples.

[0027] Figure 6 This is a diagram analyzing the therapeutic effect of the therapeutic composition prepared in Example in treating MGD.

[0028] Figure 7This is a staining diagram of meibomian gland sections showing the therapeutic effect of the therapeutic composition prepared in Examples on MGD.

[0029] Figure 8 This is a diagram showing the effect of the therapeutic composition prepared in the example on alleviating the inflammatory response of MGD.

[0030] Fig. 9 This is a diagram showing the effect of the therapeutic composition prepared in the example on promoting lipid synthesis in the meibomian gland. DETAILED DESCRIPTION

[0031] Example 1 Synthesis of Chlo.@Dex Gel

[0032] 5 mL of Chlorella (Chlo.) suspension was centrifuged (3200g, 10 min), thoroughly washed with phosphate buffered saline (PBS) to remove the culture medium, centrifuged, and the Chlorella was dispersed in 10 mL of sodium hydroxyethylcellulose solution, and 25 mg of dexamethasone (Dex) was added during gentle stirring (2 h). Subsequently, ultrasonic vibration (30W, 10 min) was performed in a dark environment at 4°C, and then freeze-dried to obtain a lyophilized powder of Chlorella (Chlo.@Dex) loaded with dexamethasone. The obtained lyophilized powder was washed with PBS and resuspended, and sodium hyaluronate (50 Da) was added to the above suspension to increase the viscosity and form a gel-like substance. After gentle stirring overnight, a therapeutic composition-drug-loaded gel (Chlo.@Dex Gel) was obtained. The therapeutic composition was collected and electron microscope pictures were taken. It can be seen that the synthesized therapeutic composition maintains the original spherical appearance of Chlorella, with an average size of about 3 μm ( Figure 1 and Figure 2 A).

[0033] The drug loading amount of the drug-loaded gel prepared in this example is 50.67%.

[0034] Test Example 1: Drug loading performance verification

[0035] The Zeta potential of the therapeutic composition synthesized in Example 1 was detected by a Zeta potential meter, showing that the zeta potential of Chlo. was -16.97±0.78 mV, and the zeta potential of Chlo.@Dex was -19.15±0.75 mV. The Zeta potential changed, proving that Dex was successfully loaded on Chlo. ( Figure 2 B).

[0036] Test Example 2 In vitro drug release performance test

[0037] Take 1 mL of the Chlo.@Dex gel prepared in the example and dissolve it in 50 mL of PBS buffer, place it in a shaker at 37°C, and take samples at the following time points: 5 min, 10 min, 20 min, 30 min, 40 min, 1 h, 2 h, 4 h, and 6 h. Take 500 μL of sample each time and add the same volume of PBS buffer. Measure the sample absorbance at 242 nm using PBS buffer as a reference solution. The results show that with PBS as the release medium, Dex is slowly released from the Chlo.@Dex gel, and no obvious burst release occurs during the 6-hour observation period. The drug is basically completely released in 2 hours, indicating that the Chlo.@Dex gel can control the release of Dex, help maintain a stable concentration of the drug within a given time, and improve the therapeutic effect ( Figure 3 ).

[0038] Test Example 3: Detection of distribution and retention on the ocular surface at different time points after eye application

[0039] Freeze-dried Chlorella was dispersed in PBS to prepare Chlo.Eye Drop. Chlo.Eye Drop was used as the control group, and 60 μL Chlo.@Dex gel and Chlo. eye drops were dripped into each eye respectively. At specific time points of 0min, 5min, 15min, 30min, 1h, and 2h, 2% sodium pentobarbital 1mL / kg was intravenously injected to anesthetize New Zealand white rabbits. Subsequently, the changes in the fluorescence intensity of the rabbit eyes were recorded using an in vivo imaging system. The results showed that the fluorescence intensity of the drug-loaded gel group was significantly higher than that of the eye drops group over time. This shows that the drug-loaded gel has good adhesion and retention, and can stay on the ocular surface for a longer time, thereby increasing the drug's action time and effect ( Figure 4 ).

[0040] Test Example 4: Supplementation of tear film lipids by the therapeutic composition

[0041] Gently wipe the moisture and dirt around the eyes with a clean gauze. Then rinse the eyes with normal saline to ensure the cleanliness of the ocular surface. Next, take 60μL of Dex gel (10mL sodium hydroxyethylcellulose solution, and add Dex 25mg and sodium hyaluronate during gentle stirring to prepare dexamethasone gel (Dex Gel)) and Chlo.@Dex gel and drip them into the eyes topically. After dripping, blink manually 2-3 times, and then adjust the head position so that the outer canthus is at the bottom and the inner canthus is at the top. Take a clean EP tube and place it in the outer canthus position. Take 200μL of normal saline, rinse the ocular surface from the inner canthus, gently pull down the lower eyelid, let the liquid flow naturally into the EP tube, indicate the sample name, and proceed to the next step of lipid content test. Use the triglyceride (TG enzymatic method) test kit to set up the blank group, standard group, Dex Gel group and Chlo.@Dex Gel group according to the instructions of the reagent manufacturer. 10 μL of each sample was added to 1 mL of working solution and incubated at 37°C for 10 minutes. Then, the absorbance of the sample was measured at a wavelength of 500 nm using PBS buffer as a reference solution. The results showed that the lipid content of Chlo.@Dex gel was 0.359 mmol / L, and the lipid content of Dex gel was -0.017 mmol / L. This shows that Chlo.@Dex gel has a significant effect in replenishing tear film lipids, and its lipid content is significantly higher than that of Dex gel ( Figure 5 ).

[0042] Test Example 5 Therapeutic effect of the therapeutic composition on meibomian gland dysfunction

[0043] Two weeks after treatment, the treatment effect of MGD was evaluated under slit lamp, referring to the Meibum Quality Score (MQS) and Lid Margin Abnormality Score (LMAS) scoring standards. MQS ranges from 0 to 3, 0: clear fluid, 1: turbid fluid, 2: turbid granular fluid, 3: concentrated toothpaste-like fluid. LMAS ranges from 0 to 3, 0: irregular eyelid margin, 1: vascular congestion, 2: blockage of meibomian gland opening, 3: anterior and posterior displacement of mucosa-skin junction. After slit lamp examination, the experimental animals were killed by air embolism, and the upper eyelid tissue was obtained, fixed with 4% PFA for 24 hours, embedded in paraffin, sliced ​​at 4 μm thickness, stained with HE, and sealed with neutral resin. Placed in a high-speed slice scanning system for photography and pathological histological analysis. The results showed that each treatment group (Dex Gel, Chlo.Gel and Chlo.@Dex) showed an improvement trend in the state of the eyelid margin, the clarity of the meibomian gland opening and the quality of meibum compared with the control group ( Figure 6 and Figure 7 ).

[0044] The preparation method of the Chlo.Gel group is as follows: freeze-dried Chlorella is dispersed in PBS, and sodium hyaluronate is added during gentle stirring to prepare Chlo.Gel.

[0045] Test Example 6: Effect of the therapeutic composition on the alleviation of inflammatory response to meibomian gland dysfunction

[0046] After the animals were sacrificed, pathological sections were prepared for immunohistochemical staining. The results showed that the staining in the Dex Gel group was lighter than that in the Control group, indicating that the expression of TNF-α decreased, suggesting that the inflammatory response was alleviated. However, the expression of TNF-α in the Chlo.Gel group did not decrease significantly, while the staining in the Chlo.@Dex Gel group became significantly lighter, indicating that it had the strongest anti-inflammatory effect. On the other hand, compared with the Control group, the Tunel staining positivity rate in the treatment group decreased significantly, while the Chlo.@Dex Gel group had the lowest staining positivity rate ( Figure 8 ).

[0047] Test Example 7 Effect of the therapeutic composition on promoting lipid synthesis in the meibomian gland

[0048] After sampling, the meibomian gland tissue was stained with Oil Red O and Sudan IV. The results showed that compared with the other treatment groups, the meibomian glands in the Control group were sparsely arranged, and there was glandular atrophy. The staining area in the Dex Gel group, Chlo.Gel group, and Chlo.@Dex Gel group increased significantly, and the meibomian glands were arranged more closely, among which the Chlo.@Dex Gel group had the most significant effect. In order to further observe the subtle changes in the glands, frozen sections were prepared for magnified observation. Compared with the other three groups, the staining area in the Control group was smaller, the number of glands was smaller, and proliferating fibrous tissue components were visible between the glands. The comprehensive results showed that in the treatment group, the staining area of ​​the meibomian glands increased, the glands were arranged more closely, and the lipid synthesis function was improved ( Fig. 9 ).

Claims

1. A composition for treating meibomian gland dysfunction based on Chlorella, characterized in that: The composition comprises sodium hyaluronate gel and drug-loaded chlorella powder, wherein the drug-loaded chlorella powder comprises chlorella and loaded drug components.

2. The Chlorella-based composition for treating meibomian gland dysfunction according to claim 1, characterized in that: The pharmaceutical ingredient is selected from steroids, nonsteroidal anti-inflammatory drugs or antibiotics.

3. The composition for treating meibomian gland dysfunction based on Chlorella according to claim 1, characterized in that: The drug ingredient is dexamethasone.

4. The Chlorella-based composition for treating meibomian gland dysfunction according to claim 1, characterized in that: The size of the Chlorella is 1-10 μm.

5. The Chlorella-based composition for treating meibomian gland dysfunction according to claim 1, characterized in that: The mass ratio of the chlorella to the medicinal component is 1:0.5-1:

25.

6. A method for preparing a composition for treating meibomian gland dysfunction based on Chlorella according to any one of claims 1 to 5, characterized in that: The preparation method comprises: (1) dispersing the chlorella suspension in the sodium hydroxyethylcellulose solution and adding the drug component during stirring; (2) After ultrasonic treatment and freeze drying, drug-loaded Chlorella powder is obtained; (3) After the drug-loaded Chlorella powder is resuspended, sodium hyaluronate is added to form a gel-like composition.

7. The method for preparing the Chlorella-based composition for treating meibomian gland dysfunction according to claim 1, characterized in that: In step (2), the ultrasonic treatment conditions are 10-50W, 10-30min.

8. Use of the composition according to any one of claims 1 to 5 in the preparation of a medicament for treating meibomian glands.

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