Eye protection preparation based on exosome as well as preparation method and application of eye protection preparation

Abstract

The invention belongs to the field of biological medicine, and particularly discloses an exosome-based eye protection preparation and a preparation method and application thereof.The eye protection preparation comprises a retinal pigment epithelial cell-derived exosome and a carrier, the particle size of the exosome is 40-100 nm, and the exosome expresses CD63, CD81 and RPE65 proteins; the exosome derived from retinal pigment epithelial cells is taken as a core, superoxide dismutase and catalase contained in the exosome can remove active oxygen, miR-26a can block an inflammation cascade reaction, and the exosome can penetrate through a blood-eye barrier to realize targeted delivery; according to the present invention, by matching with the appropriate carrier, the stability and the cornea permeability can be enhanced, the deep pathological mechanism of eye diseases can be effectively intervened, the safety is good, no significant irritation or systemic toxicity exists, the retina function is significantly improved, the oxidative damage is inhibited, and the innovative treatment scheme is provided for xerophthalmia, AMD and other diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an eye protection preparation based on exosomes, and a preparation method and application thereof. Background Art

[0002] In modern life, prolonged use of electronic devices leads to eye fatigue, dryness and increased myopia, and age-related eye diseases such as AMD have become one of the main causes of blindness worldwide.

[0003] Traditional treatments such as artificial tears can only relieve symptoms but lack intervention in the underlying pathological mechanisms;

[0004] Oxidative stress and chronic inflammation are common causes of most eye diseases, but existing antioxidants (such as vitamin C) and anti-inflammatory drugs (such as glucocorticoids) have limitations such as short duration of action and obvious side effects.

[0005] Exosomes are nanoscale membrane vesicles (30-150 nm) secreted by cells, naturally carrying bioactive substances such as proteins, lipids, and nucleic acids, which can penetrate the blood-ocular barrier and deliver their contents to recipient cells in a targeted manner;

[0006] Compared with chemical drugs, exosomes have low immunogenicity, high biocompatibility and modifiability. For example, mesenchymal stem cell (MSC) exosomes have been shown to inhibit corneal inflammation through anti-inflammatory proteins (such as TSG-6) and miRNA (such as miR-146a).

[0007] However, existing exosome research is mostly focused on disease diagnosis or tumor treatment, and traditional eye drops have a drug absorption rate of less than 5% due to the corneal barrier. Summary of the Invention

[0008] The purpose of the present invention is to provide an exosome-based eye protection preparation and its preparation method and application, to prepare an eye protection preparation with cellular exosomes as the core active ingredient, and to solve the problems raised in the above-mentioned background technology by targeted delivery of antioxidant enzymes, anti-inflammatory miRNAs and neuroprotective factors.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] An exosome-based eye protection preparation comprises: exosomes derived from retinal pigment epithelial cells and a carrier, wherein the exosomes have a particle size of 40-100 nm and express CD63, CD81 and RPE65 proteins.

[0011] Preferably, the surface of the exosomes is modified with a retinal targeting peptide for precise delivery to photoreceptor cells.

[0012] Preferably, the exosomes contain superoxide dismutase, catalase and miR-26a.

[0013] Preferably, the retinal pigment epithelial cells are cells differentiated from induced pluripotent stem cells.

[0014] Preferably, the carrier is 0.5%-2% w / v sodium hyaluronate, and the exosome concentration is 1×10 8 -1×10 11 particles / mL.

[0015] Preferably, the carrier is 0.1-1 mM DSPE-PEG and exosomes, wherein the molar ratio of exosomes to DSPE-PEG is 1:2.

[0016] A method for preparing an exosome-based eye protection preparation, comprising:

[0017] S1. Cultivate retinal pigment epithelial cells in serum-free medium to 80% confluence to obtain conditioned medium.

[0018] S2, centrifuging the conditioned medium at 300×g for 10 minutes, and then filtering through a 0.22 μm filter membrane to obtain a primary clarified solution;

[0019] S3, filtering the primary clarified liquid through tangential flow filtration using a 100 kDa hollow fiber column at an operating pressure of 0.2 MPa to obtain a concentrated exosome solution;

[0020] S4. Purifying the concentrated solution through a Sepharose CL-4B size exclusion chromatography column, collecting the elution peak with a retention time of 15-20 minutes to obtain exosomes with a particle size of 40-100 nm;

[0021] S5. Compounding the exosomes with the carrier to form an eye protection preparation.

[0022] Preferably, the exosomes are coupled to the retinal targeting peptide via EDC / NHS reagent at a molar ratio of 30-70:1, and the reaction conditions are 25° C., pH 6.0, and shaking for 2 hours.

[0023] An eye protection preparation based on exosomes is used in the preparation of a drug for treating eye diseases, wherein the eye diseases include dry eye, age-related macular degeneration or diabetic retinopathy.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The present invention uses exosomes derived from retinal pigment epithelial cells as the core. The superoxide dismutase and catalase contained in them can scavenge reactive oxygen species, miR-26a can block the inflammatory cascade reaction, and the exosomes can penetrate the blood-ocular barrier to achieve targeted delivery. When combined with a suitable carrier, it can enhance stability and corneal permeability, effectively intervene in the deep pathological mechanisms of eye diseases, and has good safety, without significant irritation and systemic toxicity.

[0026] The present invention uses tangential flow filtration technology to extract high-purity exosomes and prepare them into eye protection preparations. This preparation can penetrate the blood-ocular barrier, targetedly deliver antioxidant enzymes and anti-inflammatory miRNAs, significantly improve retinal function and inhibit oxidative damage, providing innovative treatment options for diseases such as dry eye and AMD. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the specification scheme.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0030] Example 1: This example provides an exosome-based eye protection preparation, comprising: exosomes derived from retinal pigment epithelial cells and a carrier, the exosome particle size is 40-100 nm (verified by dynamic light scattering), and expresses surface markers CD63, CD81 and RPE65 protein (verified by Western Blot).

[0031] Specifically, the surface of exosomes is modified with retinal targeting peptides (such as RRR), which bind to retinal heparan sulfate proteoglycans through electrostatic adsorption, enhancing cellular uptake and photoreceptor cell targeting for precise delivery to photoreceptor cells.

[0032] Specifically, exosomes contain superoxide dismutase, catalase, and miR-26a.

[0033] Exosomes contain superoxide dismutase (SOD) and catalase (CAT), which directly remove reactive oxygen species (ROS) and inhibit the NF-κB pathway through miR-26a, thereby blocking the inflammatory cascade.

[0034] Specifically, retinal pigment epithelial cells are retinal pigment epithelial cells (RPE) obtained by directed differentiation of human induced pluripotent stem cells (iPSCs), and exosomes are exosomes secreted by retinal pigment epithelial cells (RPE).

[0035] Specifically, the carrier is 0.5%-2% w / v sodium hyaluronate, and the exosome concentration is 1×10 8 -1×10 11 particles / mL.

[0036] A method for preparing an exosome-based eye protection preparation, comprising:

[0037] S1. Culture retinal pigment epithelial cells in StemPro serum-free medium to 80% confluence to obtain conditioned medium at 37°C and 5% CO2.

[0038] Serum-free culture avoids contamination of animal-derived exosomes, ensuring the purity of subsequent extraction, and 80% confluence maximizes exosome secretion (electron microscopy shows high secretion activity).

[0039] S2. Centrifuge the conditioned medium at 300 × g for 10 minutes and filter through a 0.22 μm filter to obtain the primary clarified fluid (supernatant without cell debris);

[0040] S3. The primary clarified liquid was filtered through a 100 kDa hollow fiber column with tangential flow filtration at an operating pressure of 0.2 MPa to obtain a concentrated exosome solution with a recovery rate of 85%;

[0041] S4. The concentrated solution was purified by Sepharose CL-4B size exclusion chromatography column, and the elution peak with a retention time of 15-20 minutes was collected to obtain 1×10 8 -1×10 11 Particles / mL high-purity exosomes (CD63+ / CD81+);

[0042] Isolate highly pure, structurally intact exosomes from complex culture media.

[0043] Tangential flow concentration directly determines the amount of sample loaded on the chromatogram (insufficient concentration leads to failure of peak separation), and size exclusion chromatography precisely controls the particle size to ensure corneal permeability of subsequent preparations.

[0044] S5, 1×10 8 -1×10 11Particles / mL exosomes were complexed with sodium hyaluronate to form an ophthalmic gel, the pH was adjusted to 7.4, and the gel was sterilely packaged.

[0045] Specifically, exosomes were coupled with retinal targeting peptides using EDC / NHS reagents at a molar ratio of 30-70:1, and the reaction conditions were 25°C, pH 6.0, and shaking for 2 hours.

[0046] Preferably, the invention is used for preparing a drug for treating dry eye.

[0047] Exosomes are suspended in a hyaluronic acid matrix, prolonging their retention time in the eye and treating dry eye (as confirmed by the Schirmer test, which increased tear secretion by 40%).

[0048] From the above, we can see that exosomes are nano-sized vesicles of 30-150nm, which can penetrate the blood-ocular barrier and deliver active ingredients to intraocular cells through membrane fusion mechanism.

[0049] Example 2: This example is basically the same as Example 1, except that the carrier is 0.1-1 mM DSPE-PEG and exosomes, wherein the molar ratio of exosomes to DSPE-PEG is 1:2.

[0050] Exosomes were complexed with DSPE-PEG to form nano-eye drops.

[0051] Preferably, the method is used for preparing a drug for treating age-related macular degeneration.

[0052] Exosomes complexed with PEGylated phospholipids improve corneal permeability and age-related macular degeneration (confirmed by a 60% increase in ERGa wave amplitude).

[0053] Example 3: Animal model verification of the eye protection preparation (exosome eye gel) prepared in Example 1;

[0054] Model details: C57BL / 6 mice were exposed to blue light (450 nm, 10,000 lux, 4 h / day × 7 days);

[0055] Dosage regimen:

[0056] Group deal with result Experimental group Exosome gel, 5 μL / eye / day ERGa wave amplitude ↑60%* control group PBS gel MDA↓45%, SOD↑230%

[0057] Results: Electroretinogram (ERG): The a-wave amplitude in the experimental group increased by 60% compared with that in the control group (p<0.01);

[0058] Oxidative stress level: Retinal MDA content decreased by 45% and SOD activity increased by 2.3 times.

[0059] Example 4: Safety verification of the eye protection preparation (exosome eye gel) prepared in Example 1;

[0060] Eye irritation: The Draize test (0-4) was performed on a New Zealand rabbit model and scored after 7 consecutive days of administration. The results showed that the scores for corneal opacity, conjunctival congestion and secretion edema were all grade 0.

[0061] Systemic toxicity:

[0062] index Before administration 7 days after administration Range of change ALT(U / L) 35±3 38±4 +8.6％(p>0.05) BUN (mg / dL) 18±2 20±3 +11.1％(p>0.05)

[0063] Results: There was no significant difference in blood biochemical indicators (ALT, BUN) compared with before administration, proving that exosomes had no systemic toxicity.

[0064] It will be appreciated that in the development of any actual embodiment, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will, for those of ordinary skill having the benefit of this disclosure, be a routine undertaking of design, fabrication, and production without undue experimentation.

[0065] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. An eye protection preparation based on exosomes, characterized in that include: Exosomes and vectors derived from retinal pigment epithelial cells, wherein the exosomes have a particle size of 40-100 nm and express CD63, CD81 and RPE65 proteins.

2. The exosome-based eye protection preparation according to claim 1, characterized in that: The surface of the exosomes is modified with retinal targeting peptides for precise delivery to photoreceptor cells.

3. The exosome-based eye protection preparation according to claim 2, characterized in that: The exosomes contain superoxide dismutase, catalase and miR-26a.

4. The exosome-based eye protection preparation according to claim 3, characterized in that The retinal pigment epithelial cells are cells differentiated from induced pluripotent stem cells.

5. The exosome-based eye protection preparation according to claim 4, characterized in that: The carrier is 0.5%-2% w / v sodium hyaluronate, and the exosome concentration is 1×10 8 -1×10 11 particles / mL.

6. The exosome-based eye protection preparation according to claim 4, characterized in that: The carrier is 0.1-1 mM DSPE-PEG and exosomes, wherein the molar ratio of exosomes to DSPE-PEG is 1:

2.

7. The method for preparing an exosome-based eye protection preparation according to any one of claims 1 to 6, characterized in that: include: S1. Cultivate retinal pigment epithelial cells in serum-free medium to 80% confluence to obtain conditioned medium. S2, centrifuging the conditioned medium at 300×g for 10 minutes, and then filtering through a 0.22 μm filter membrane to obtain a primary clarified solution; S3, filtering the primary clarified liquid through tangential flow filtration using a 100 kDa hollow fiber column at an operating pressure of 0.2 MPa to obtain a concentrated exosome solution; S4. Purifying the concentrated solution through a Sepharose CL-4B size exclusion chromatography column, collecting the elution peak with a retention time of 15-20 minutes to obtain exosomes with a particle size of 40-100 nm; S5. Compounding the exosomes with the carrier to form an eye protection preparation.

8. The method for preparing an exosome-based eye protection preparation according to claim 7, characterized in that: The exosomes were coupled to the retinal targeting peptide using EDC / NHS reagent at a molar ratio of 30-70:

1. The reaction conditions were 25° C., pH 6.0, and shaking for 2 hours.

9. Use of an exosome-based eye protection preparation according to claims 1-6 in the preparation of a medicament for treating eye diseases, wherein the eye diseases include dry eye, age-related macular degeneration or diabetic retinopathy.