Eye drops as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses eye drops and a preparation method and application thereof.The eye drops per liter contain the following growth factors with the final concentration: 4.29 [mu] g / L-8. 06 [mu] g / L of platelet-derived growth factor AA, 28.36 [mu] g / L-38.30 [mu] g / L of platelet-derived growth factor AB, 0.08 [mu] g / L-0. 11 [mu] g / L of platelet-derived growth factor BB, 41.39 [mu] g / L-64.13 [mu] g / L of transforming growth factor-beta, 0.14 [mu] g / L-0. 23 [mu] g / L of epidermal growth factor and the balance of water. The fibroblast growth factor is 0.01-0.03 [mu] g / L, and the balance is a NaCl solution with the mass fraction of 0.9%. The eye drops provided by the invention contain rich growth factors, can promote ocular surface regeneration, and can be used for treating xerophthalmia.

Description

Technical Field

[0001] This invention relates to the field of technology, specifically to an eye drop solution, its preparation method, and its application. Background Technology

[0002] Dry eye syndrome, also known as keratoconjunctivitis sicca, is a common eye condition characterized by an imbalance in the homeostasis of tears and the ocular surface, leading to eye discomfort, visual disturbances, and tear film instability. Dry eye syndrome affects a large number of people, and its prevalence increases with age and poor eye habits. Common symptoms include eye fatigue, a foreign body sensation, and dryness. In severe cases, burning, soreness, redness, pain, and photophobia may occur, and it can potentially lead to corneal damage. If left untreated, dry eye syndrome can develop into corneal ulcers, perforation, or secondary infections, seriously threatening vision.

[0003] Existing products are primarily artificial tears, whose main components are water and lubricants such as polyvinyl alcohol, sodium carboxymethyl cellulose, and sodium hyaluronate. They aim to mimic the lubricating and moisturizing functions of natural tears. They mainly relieve dry eyes by replenishing the moisture in tears, providing lubrication and hydration, but they cannot directly promote the repair of ocular surface tissues. Some products contain preservatives, which may cause irritation or damage to the ocular surface with long-term use. Therefore, it is necessary to provide an eye drop specifically for treating dry eye syndrome. Summary of the Invention

[0004] To develop a drug for treating dry eye syndrome, this invention provides an eye drop, its preparation method, and its application. The eye drop provided by this invention contains abundant growth factors, which can promote the regeneration of the ocular surface and can be used to treat dry eye syndrome.

[0005] This invention provides an eye drop solution, each liter of which contains the following final concentration of growth factor: Platelet-derived growth factor AA 4.29ug / L~8.06ug / L, platelet-derived growth factor AB 28.36ug / L~38.30ug / L, platelet-derived growth factor BB 0.08ug / L~0.11ug / L, transforming growth factor-β 41.39ug / L~64.13ug / L, epidermal growth factor 0.14ug / L~0.23ug / L, fibroblast growth factor 0.01ug / L~0.03ug / L, with the balance being a 0.9% NaCl solution.

[0006] The eye drops provided by this invention contain abundant growth factors, which can promote the regeneration of the ocular surface and can be used to treat dry eye syndrome.

[0007] Furthermore, each liter of the eye drops contains the following final concentration of growth factors: Platelet-derived growth factor AA 6.175 ug / L, platelet-derived growth factor AB 33.33 ug / L, platelet-derived growth factor BB 0.095 ug / L, transforming growth factor-β 52.76 ug / L, epidermal growth factor 0.185 ug / L, fibroblast growth factor 0.02 ug / L, with the balance being a 0.9% NaCl solution.

[0008] The present invention also provides a method for preparing the aforementioned eye drops, comprising the following steps: Preparation of white membrane from whole blood: After centrifugation of whole blood, red blood cells, white membrane, and plasma are separated. Platelet-rich plasma was obtained by centrifugation of the white membrane. Platelet-rich plasma was naturally depolymerized at 23℃~25℃ to obtain concentrated platelet suspension; Concentrated platelet suspension was subjected to intermittent ultrasonic lysis to obtain platelet lysates; Platelet lysate was centrifuged, the supernatant was collected, and filtered to obtain the platelet lysate product. The platelet lysate was diluted with 0.9% NaCl solution to obtain eye drops.

[0009] Furthermore, whole blood centrifugation includes a first centrifugation and a second centrifugation; The first centrifugation conditions were: 2250g, 8 min; The second centrifugation conditions were: 180g, 12 min.

[0010] Furthermore, the platelet lysate was centrifuged at 4°C and 4000g for 20 minutes.

[0011] Furthermore, filtration was performed at 0.22µm.

[0012] Furthermore, the conditions for the intermittent ultrasonic lysis are: ultrasonic treatment for 30 seconds, stop for 10 seconds, cycled 15 times, ultrasonic frequency 25KHz, and ultrasonic power 3600w.

[0013] The present invention also provides the use of the eye drops in the preparation of a drug for treating dry eye syndrome.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a platelet lysis solution eye drop, which, with its rich growth factors, ability to promote ocular surface regeneration, and good safety profile, demonstrates significant advantages in the treatment of ocular surface diseases.

[0015] Platelet lysate is rich in various growth factors that promote cell repair and regeneration, which is particularly important for the treatment of dry eye syndrome. Secondly, it has anti-inflammatory effects: the anti-inflammatory cytokines in platelet lysate help reduce ocular inflammation, which is an important therapeutic mechanism for dry eye patients. Thirdly, it promotes corneal epithelial repair: platelet lysate can repair corneal epithelial cells, which is crucial for improving dry eye symptoms. Fourthly, it enhances tear film stability: platelet lysate can increase tear film height and delay tear film breakup time, thereby enhancing tear film stability. Fifthly, it reduces tear film osmotic pressure: platelet lysate can reduce tear film osmotic pressure, improving discomfort in dry eye patients. In summary, platelet lysate has multiple advantages in treating dry eye syndrome, including mechanisms such as promoting corneal repair, anti-inflammation, and improving tear film stability, as well as therapeutic advantages such as definite efficacy, good safety, and improvement of tear parameters. This invention application provides a standardized eye drop rich in platelet lysate and its preparation method, which has important value for the biotherapy of dry eye syndrome and other ocular surface diseases. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 This is a flowchart illustrating the preparation process of the eye drops in Example 1 of the present invention.

[0018] Figure 2 Microscopic image of human corneal epithelial cells used in platelet lysis fluid.

[0019] Figure 3 Microscopic image of rabbit corneal epithelial cells after platelet lysis fluid application. Detailed Implementation

[0020] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0021] This invention provides a method for preparing eye drops, comprising: Screening of autologous and allogeneic blood donors: must meet GB 18467-2011, pass infectious disease testing, and pass blood sterility tests.

[0022] The white membrane layer in whole blood is separated using a blood component separator.

[0023] Platelet-rich plasma (PRP) preparation: Platelet-rich plasma (PRP) was obtained by two centrifugations (2250g, 8min and 180g, 12min).

[0024] PRP was allowed to stand at room temperature for 1 hour to depolymerize and then concentrated into a platelet suspension, which was stored at (22±2)℃ with shaking.

[0025] Platelet lysate preparation: ultrasonic lysis (4°C, 25kHz, 3600W, 30s on / 10s off x 15 times).

[0026] The platelet lysate was centrifuged at 4°C (400g, 20min) to collect the supernatant.

[0027] Purification was achieved by filtration through a 0.22μm membrane.

[0028] Preparation of eye drops: dilute with 0.9% NaCl → aseptically dispense → pass sterility test → store at 4℃.

[0029] Example 1: An eye drop solution, its preparation method and application.

[0030] I. Growth Factor Content in Eye Drops Each liter of eye drops contains the following final concentration of growth factors: Platelet-derived growth factor AA (PDGF-AA) 6.175 ug / L, platelet-derived growth factor AB (PDGF-AB) 33.33 ug / L, platelet-derived growth factor BB (PDGF-BB) 0.095 ug / L, transforming growth factor-β (TGF-β1) 52.76 ug / L, epidermal growth factor (EGF) 0.185 ug / L, fibroblast growth factor (bFGF) 0.02 ug / L, balance 0.9% NaCl solution.

[0031] II. Experimental Methods Preparation steps of eye drops: 1. Selection of blood donors (including autologous and allogeneic blood donors) (1) The selection criteria for blood donors shall be in accordance with the "Requirements for Health Examination of Blood Donors" (GB 18467-2011), especially that blood donors have not used platelet inhibitors (such as aspirin) or anti-inflammatory drugs within 7 days before blood collection.

[0032] (2) Blood donors who pass the tests for human immunodeficiency virus (HIV) infection markers, hepatitis B virus (HBV) infection markers, hepatitis C virus (HCV) infection markers, syphilis infection markers, alanine aminotransferase (ALT) and other indicators as stipulated by the national and provincial health administrative departments, as well as those who pass the tests for local and time-limited transfusion-related infectious disease markers.

[0033] (3) Blood from donors who meet the criteria of (1) and (2) can be used to prepare platelet lysate only after passing the sterility test according to the "Blood Station Technical Operation Procedures".

[0034] 2. Preparation of concentrated platelets ① Preparation of white membrane from whole blood separation: Whole blood (collected and stored at 22 ℃, prepared within 8 hours) was collected from a 400 mL disposable plastic blood bag (mother bag) with ACD-B anticoagulation (product of Sichuan Nangeer Biotechnology Co., Ltd.). The blood was centrifuged for the first time at a speed of 2250g for 8 min at a temperature of (22±2)℃. After centrifugation, the blood bag was removed and vibration was avoided. The blood bag and its tubing were checked to ensure there was no leakage. Red blood cells, white membrane and plasma were separated using a blood component separator. The white membrane was collected in a blood bag for subsequent experiments.

[0035] The 400 mL six-pack (mother bag) of ACD-B anticoagulant is simply referred to as the six-pack.

[0036] ② Preparation of platelet-rich plasma by separation of the white membrane layer: Place the blood bag containing the white membrane and its connecting tubes, transfer bag 1 and transfer bag 2, flat into a centrifuge cup and centrifuge a second time at a speed of 180g for 12 min at a temperature of (22±2)℃. After centrifugation, transfer bag 1 and transfer bag 2 of the aforementioned six-bag system are transferred.

[0037] After the second centrifugation, the blood bag and transfer bag were taken out and placed on the plasma separator. The blood components in transfer bag 1 and transfer bag 2 were separated into two layers from top to bottom. The upper layer was platelet-rich plasma and the lower layer was a sediment of red blood cells and white blood cells. All the upper platelet-rich plasma in transfer bag 1 was squeezed into transfer bag 2 to obtain platelet-rich plasma.

[0038] ③ Platelet deagglomeration: The prepared platelet-rich plasma was allowed to stand at 25°C for 1 hour to allow for natural deagglomeration. Afterward, the platelet suspension was gently shaken in transfer bag 2 to prepare a concentrated platelet suspension, which was then stored at (22±2)°C with shaking. The platelet count of the concentrated platelet suspension was 950 x 10⁻⁶. 9 / L.

[0039] 3. Preparation of platelet lysates ① Place transfer bag 2 in an ultrasonic lyser and fix it in place. Perform intermittent ultrasonic lysis of platelet-rich plasma under a water bath at 4℃±2℃ to obtain platelet lysate. The conditions for intermittent ultrasonic lysis are: ultrasonic treatment for 30s, stop for 10s, 15 cycles, ultrasonic frequency of 25KHz, and ultrasonic power of 3600w.

[0040] ② Centrifuge the platelet lysate at 4℃ and 4000g for 20 min, collect the supernatant and filter it through a filter membrane with a pore size ≤0.22um to obtain the platelet lysate product.

[0041] 4. Preparation of eye drops rich in platelet lysate.

[0042] ① The blood bags containing platelet lysate were diluted with 0.9% NaCl solution using a seamless nozzle technique, thoroughly mixed, and then dispensed into sterile plastic bottles under aseptic conditions to prepare platelet lysate-rich eye drops, which were stored at 4°C.

[0043] ②The eye drops rich in platelet lysate have passed the sterility test.

[0044] ③The concentration range of related growth factors in eye drops rich in platelet lysate is shown in Table 1.

[0045] Table 1. Growth factor concentrations in platelet lysate eye drops Corneal epithelial cells were cultured in a medium containing platelet lysis buffer, resulting in cells with typical morphology and high purity. The effects of the platelet lysis buffer eye drops of this invention on the proliferative activity of human and rabbit corneal epithelial cells were detected using a CCK-8 assay kit (GLPbio product, batch number GK10001). The platelet lysis buffer showed superior proliferative effect and stability compared to platelet-rich plasma (PRP) and serum.

[0046] Microscopic images of human corneal epithelial cells prepared using the platelet lysis fluid of Example 1 of this invention and microscopic images of rabbit corneal epithelial cells prepared using the platelet lysis fluid of Example 1 are shown below. Figure 2 and Figure 3 As shown.

[0047] Although preferred embodiments of the invention have been described, those skilled in the art, once they have learned the basic inventive concept, can make other changes and modifications to these embodiments.

[0048] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. An eye drop, characterized in that, Each liter of the eye drops contains the following final concentration of growth factors: Platelet-derived growth factor AA 4.29ug / L~8.06ug / L, platelet-derived growth factor AB 28.36ug / L~38.30ug / L, platelet-derived growth factor BB 0.08ug / L~0.11ug / L, transforming growth factor-β 41.39ug / L~64.13ug / L, epidermal growth factor 0.14ug / L~0.23ug / L, fibroblast growth factor 0.01ug / L~0.03ug / L, with the balance being a 0.9% NaCl solution.

2. The eye drops according to claim 1, characterized in that, Each liter of the eye drops contains the following final concentration of growth factors: Platelet-derived growth factor AA 6.175 ug / L, platelet-derived growth factor AB 33.33 ug / L, platelet-derived growth factor BB 0.095 ug / L, transforming growth factor-β 52.76 ug / L, epidermal growth factor 0.185 ug / L, fibroblast growth factor 0.02 ug / L, with the balance being a 0.9% NaCl solution.

3. A method for preparing the eye drops according to claim 1 or 2, characterized in that, Includes the following steps: Preparation of white membrane from whole blood: After centrifugation of whole blood, red blood cells, white membrane, and plasma are separated. Platelet-rich plasma was obtained by centrifugation of the white membrane. Platelet-rich plasma was naturally depolymerized at 23℃~25℃ to obtain concentrated platelet suspension; Concentrated platelet suspension was subjected to intermittent ultrasonic lysis to obtain platelet lysates; Platelet lysate was centrifuged, the supernatant was collected, and filtered to obtain the platelet lysate product. The platelet lysate was diluted with 0.9% NaCl solution to obtain eye drops.

4. The method for preparing eye drops according to claim 3, characterized in that, Whole blood centrifugation includes a first centrifugation and a second centrifugation; The first centrifugation conditions were: 2250g, 8 min; The second centrifugation conditions were: 180g, 12 min.

5. The method for preparing eye drops according to claim 3, characterized in that, The centrifugation conditions for platelet lysate were: 4℃, 4000g for 20min.

6. The method for preparing eye drops according to claim 3, characterized in that, Filtering at 0.22µm.

7. The method for preparing eye drops according to claim 3, characterized in that, The conditions for the intermittent ultrasonic lysis are: ultrasonic treatment for 30 seconds, stop for 10 seconds, cycled 15 times, ultrasonic frequency 25KHz, and ultrasonic power 3600w.

8. The use of the eye drops according to claim 1 or 2 in the preparation of a medicament for treating dry eye syndrome.

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