Engineered exosome loaded with apatinib as well as preparation method and application of engineered exosome

Abstract

The invention provides an apatinib-loaded engineered exosome as well as a preparation method and application thereof, belongs to the technical field of ophthalmic preparations, and aims to solve the problem of low bioavailability of current eye drops. The preparation method comprises the following steps: step 1, preparing APA-loaded PLGA nanoparticles; step 2, separating the HCE-EXs; step 3, preparing the Apa nano particles wrapped by the HCE-EXs; step 4, preparing a phospholipid polymer modified by arginine; and 5, mixing the HCE-EXs coated APa nanoparticles prepared in the step 3 with the arginine modified phospholipid polymer prepared in the step 4, incubating the mixture at 37 DEG C for 30 minutes, and removing free impurities through volume exclusion chromatography to obtain the engineered exosome. The engineered exosome can release nitric oxide (NO) to effectively solve oxidative stress after corneal alkali burn. Secondly, compared with traditional medicine eye drops, the engineered exosome can be retained in cornea for a long time to continuously release apatinib for treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of ophthalmic preparations, and specifically relates to an engineered exosome loaded with apatinib, a preparation method and an application thereof. Background Art

[0002] There are many types of corneal injuries, among which chemical injuries are more difficult to treat clinically. Corneal alkali burns are more serious chemical injuries, which can lead to corneal epithelial necrosis and detachment, corneal conjunctival melting and perforation, and symblepharon, accompanied by the formation of a large number of new capillaries, which can cause corneal blindness in severe cases. At present, topical corticosteroids (including dexamethasone, prednisolone and triamcinolone acetonide) are the standard clinical treatment for CNV, and the medication time is usually up to 1 to 2 weeks. Because eye drops are non-invasive and the administration method is simple, eye drops are still the most commonly used method of administration for the treatment of ophthalmic diseases, accounting for 90% of commercialized ophthalmic drugs. Due to tear dilution, blinking and nasolacrimal duct drainage, 95% of the drugs are discharged into the body through the nasolacrimal duct, and the bioavailability of ordinary eye drops is extremely low (1-5%). In order to maintain effective therapeutic concentrations, frequent administration is always required, which increases the incidence of adverse reactions and greatly reduces patient compliance. However, long-term and frequent use of corticosteroid eye drops can lead to excessive drug concentration and excessive fluctuations, which may cause increased intraocular pressure and glaucoma.

[0003] In addition, chemical burns stimulate the production of reactive oxygen species (ROS) in the cornea. High levels of oxidative stress induce perturbations in the mitochondrial permeability transition pore, disrupt electron transfer, and ultimately lead to cell apoptosis or necrosis. As a second messenger, ROS triggers inflammation by activating nuclear factor-κB (NF-κB), which in turn stimulates the release of inflammatory cytokines and the expression of matrix metalloproteinases (MMPs). More seriously, the oxidative stress and persistent inflammatory response of the ocular surface caused by pathological damage will break the balance between angiogenic factors and anti-angiogenic factors, induce the formation of capillaries in the limbal vascular network, and then induce CNV, improve pharmacological bioavailability), which is of great benefit to the treatment of chemical corneal damage. Exosomes have unique advantages in drug delivery. They can not only continuously release drugs at the pathological tissue site, but also have negligible systemic toxicity. Compared with liquid eye drops, exosomes can stay on the ocular surface for a longer time to increase the absorption of ocular surface drugs. Summary of the invention

[0004] In view of the above problems existing in the prior art, the purpose of the present invention is to provide an engineered exosome loaded with apatinib, a preparation method and application thereof, wherein the engineered exosome can release nitric oxide (NO) to effectively solve the oxidative stress after corneal alkali burn. Secondly, compared with traditional drug eye drops, engineered exosomes can stay in the cornea for a long time and continuously release apatinib to provide treatment for it, solving the problem of low bioavailability.

[0005] The technical solution adopted by the present invention is: First, the present invention protects a method for preparing an injectable antioxidant hydrogel, which comprises the following steps: Step 1: Preparation of Apa-loaded PLGA nanoparticles; Step 2: Separation of HCE-EXs; Step 3: Preparation of HCE-EXs-encapsulated Apa nanoparticles; Step 4: Arginine-modified phospholipid polymer; Step 5: The Apa nanoparticles encapsulated by HCE-EXs prepared in step 3 were mixed with the arginine-modified phospholipid polymer prepared in step 4, the mixture was incubated at 37°C for 30 min, and free impurities were removed by size exclusion chromatography to obtain engineered exosomes; Among them, Apa stands for apatinib, HCE stands for human corneal epithelial cells, and EX stands for exosomes.

[0006] In one implementation, the specific operations of step 1 are as follows: Apa and PLGA were dissolved in dimethyl sulfoxide at a mass ratio of 1:5, and the mixture was slowly added dropwise into a certain amount of deionized water with stirring. After the mixture was dialyzed in deionized water for 3-5 days, the Apa-loaded PLGA NPs solution was collected.

[0007] In one implementation, the specific operations of step 2 are as follows: Step 201: incubating HCE in serum-free medium for 48 hours; Step 202: collecting the supernatant and performing gradient centrifugation; Step 203: Filter the remaining supernatant with a 0.22 μm filter and ultracentrifuge at 120,000 g for 90 min to precipitate HCE-EXs; Step 204: The HCE-EXs were resuspended in PBS and ultracentrifuged again to remove residual proteins. The purified HCE-EXs were stored at -80°C.

[0008] In one implementation, the specific operation of gradient centrifugation of the supernatant in step 202 is: collecting the supernatant, and centrifuging the supernatant in sequence according to the gradient setting of 200g centrifugation for 5 min, 3000g centrifugation for 10 min, and 16000g centrifugation for 30 min.

[0009] In one implementation, the specific operations of step 3 are as follows: The Apa-loaded PLGA nanoparticles prepared in step 1 were mixed with the HCE-EXs suspension prepared in step 2, and the mixture was treated with ultrasound for 10 min to obtain exosome-encapsulated apatinib nanoparticles, which were designated as Apa-EXs.

[0010] In some implementations, the concentration of the apatinib-loaded PLGA nanoparticles in step 3 ranges from 1 mg / m to 10 mg / ml, and the concentration of the HCE-EXs suspension ranges from 2 mg / m to 15 mg / ml.

[0011] In some implementations, the concentration of the Apa-loaded PLGA nanoparticles in step 3 ranges from 1 mg / ml to 10 mg / ml, and the concentration of the HCE-EXs suspension ranges from 2 mg / ml to 15 mg / ml.

[0012] In one implementation, the specific operations of step 4 are as follows: The DMSO solution of DSPE-PEG-NHS and the DMSO solution of Arg were mixed in a molar ratio of 1:1, stirred in the dark for 24 hours, dialyzed in an aqueous solution to remove DMSO, and freeze-dried to obtain DSPE-PEG-Arg, that is, DSPE-PEG-Arg is an arginine-modified phospholipid polymer.

[0013] In some implementations, in step 5, Apa-EXs and DSPE-PEG-Arg are mixed at a volume ratio of 1:2, and the concentration of Apa-EXs is 5 μg / ml, and the concentration of DSPE-PEG-Arg is 10 μg / ml.

[0014] Secondly, the present invention also protects an engineered exosome loaded with apatinib, and the engineered exosome is prepared by the above-mentioned method for preparing an engineered exosome loaded with apatinib.

[0015] Finally, the present invention also protects the use of engineered exosomes loaded with apatinib in the preparation of drugs for treating corneal alkali burns.

[0016] Beneficial effects of the present invention: 1. The arginine on the surface of the engineered exosomes of the present invention can respond to the oxidative stress environment of the cornea after alkali burns and inhibit oxidative stress and inflammatory response by releasing NO; 2. The engineered exosomes of the present invention can slowly release anti-angiogenic drugs to inhibit limbal neovascularization; 3. The engineered exosomes of the present invention are modified by the exosomes of corneal epithelial cells, have excellent biocompatibility and tissue compatibility, and have no adverse reactions in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 is a transmission electron microscopy image of the engineered exosomes in the experimental example of the present invention; Figure 2 is the release curve of engineered exosome NO in the experimental example of the present invention; Figure 3 is the release curve of apatinib from engineered exosomes in the experimental example of the present invention; Figure 4 These are pictures of the healing of corneal alkali burns at different time points during treatment in the experimental examples of the present invention. DETAILED DESCRIPTION

[0019] To make the purpose, technical solution and advantages of the embodiments of the present invention more clear, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0020] The test materials used in the following examples are chemically pure unless otherwise specified. Example 1

[0021] This example mainly introduces a method for preparing engineered exosomes loaded with apatinib, and the preparation steps are as follows: (1) Preparation of apatinib-loaded PLGA nanoparticles (NPs) 2 mg of apatinib and 10 mg of PLGA were dissolved in 1 ml of dimethyl sulfoxide. Subsequently, the solution was added dropwise to a certain amount of deionized water under slow stirring. Finally, the mixture was dialyzed in deionized water for 3 days. Then, the apatinib-loaded PLGA NPs solution was collected.

[0022] (2) Isolation of exosomes from human corneal epithelial cells (HCE) HCE with a density of 85% was incubated in serum-free medium for 48 h. Then, the supernatant was collected and centrifuged at 200 g for 5 min, 3000 g for 10 min, and 16000 g for 30 min. Subsequently, the remaining supernatant was filtered with a 0.22 μm filter and ultracentrifuged at 120000 g for 90 min to precipitate HCE-exosomes (EX). HCE-EX was resuspended in PBS and ultracentrifuged again to remove residual proteins, and the purified HCE-EXs were stored at -80 °C.

[0023] (3) Preparation of HCE-EXs-coated Apa nanoparticles The drug-loaded nanoparticles (5 mg / ml) prepared in step (1) were mixed with the HCE-EXs suspension (15 mg / ml) obtained in step (2), and exosome-encapsulated Apa nanoparticles (Apa-EXs) were obtained after ultrasonic treatment for 10 min.

[0024] (4) Arginine (Arg) modified phospholipid polymers The DMSO solution of dioleoylphosphatidylethanolamine (DSPE)-polyethylene glycol (PEG)-active ester (NHS) and the DMSO solution of Arg were mixed at a molar ratio of 1:1, stirred in the dark for 24 hours, dialyzed in an aqueous solution to remove DMSO, and freeze-dried to obtain DSPE-PEG-Arg.

[0025] (5) Preparation of engineered exosomes 10 μL Apa-EXs (5 μg / ml) was mixed with 20 μL DSPE-PEG-Arg (10 μg / ml). The mixture was incubated at 37 °C for 30 min, and free impurities were removed by size exclusion chromatography to obtain Arg-Apa-EXs.

[0026] Example 2 Product Arg-Apa-EXs Test and Application

[0027] 1. Characterization and determination of Arg-Apa-EXs The morphology of exosomes was observed by transmission electron microscopy. First, Arg-Apa-EXs (1 mg / ml) were fixed with 1% glutaraldehyde and 5-10 μl was added to the copper grid. Then, 2% phosphotungstic acid solution (pH 6.5) was used for staining at room temperature for 2 min. Finally, TEM was used to observe the image. Figure 1 ) It can be seen that the particle size distribution of the prepared engineered exosomes is between 70-100 nm.

[0028] The in vitro release of Apa and NO from Arg-Apa-EXs was studied. Briefly, Arg-Apa-EXs were prepared as described above, sealed in dialysis bags (MWCO = 3.5 kDa), placed in 50 mL centrifuge tubes with 10 mL of artificial tears containing 20% ​​hydrogen peroxide, and then incubated at 37°C in a constant temperature shaker (120 rpm) for 72 h. At the required intervals, artificial tears (0.5 mL) containing the release medium were collected and supplemented with an equal volume of fresh solution. The concentrations of NO and Apa in the release medium were examined by Griess reagent (Beyotime Biotechnology) (10 min) and high-performance liquid chromatography at room temperature, respectively. The release experiments were repeated three times. Figure 2 and Figure 3 It can be seen that Arg-Apa-EXs will release NO and encapsulated Apa explosively within 24 hours, with the release amounts reaching 20.35 uM and 52.34%, respectively, and then slowly release NO and Apa within the following 48 hours.

[0029] 2. Arg-Apa-Exs for the treatment of corneal alkali burns To investigate the effect of Arg-Apa-EXs on the healing of corneal neovascularization, healthy 8-week-old female SD rats were used, and a corneal alkali burn model was established. The rats were first anesthetized and oxybuprocaine hydrochloride eye drops were instilled, then a round filter paper (2 mm in diameter) pre-soaked in 1 N NaOH was placed in the center of the right cornea for 40 seconds, and then the wound was rinsed with PBS for 1 minute. The alkali-burned rats were randomly divided into four groups: a control group (treated with PBS), an Apa-EXs group, and an Arg-Apa-EXs group. On the first day before medication, the opacity of the corneas of the three groups was comparable, indicating that the modeling had good homogeneity. On the fifth day, the corneal clarity of the Apa-EXs and Arg-Apa-EXs groups began to recover, while that of the PBS group was unclear, and obvious limbal neovascularization and anterior chamber hemorrhage were observed. On the ninth day, the Arg-Apa-EXs group more effectively inhibited CNV than the Apa-EXs group (e.g. Figure 4 ).

[0030] The embodiments described above are only preferred specific implementation modes of the present invention, and the protection scope of the present invention is not limited thereto. Any simple changes or equivalent replacements of the technical solutions that can be obviously obtained by any technician familiar with the field within the technical scope disclosed in the present invention belong to the protection scope of the present invention.

Claims

1. A method for preparing engineered exosomes loaded with apatinib, characterized in that: The preparation method comprises the following steps: Step 1: Preparation of Apa-loaded PLGA nanoparticles; Step 2: Separation of HCE-EXs; Step 3: Preparation of HCE-EXs-encapsulated Apa nanoparticles; Step 4: Arginine-modified phospholipid polymer; Step 5: The Apa nanoparticles encapsulated by HCE-EXs prepared in step 3 were mixed with the arginine-modified phospholipid polymer prepared in step 4, the mixture was incubated at 37°C for 30 min, and free impurities were removed by size exclusion chromatography to obtain engineered exosomes; Among them, Apa stands for apatinib, HCE stands for human corneal epithelial cells, and EX stands for exosomes.

2. The method for preparing an engineered exosome loaded with apatinib according to claim 1, characterized in that: The specific operations of step 1 are as follows: Apa and PLGA were dissolved in dimethyl sulfoxide at a mass ratio of 1:5, and the mixture was slowly added dropwise into a certain amount of deionized water with stirring. After the mixture was dialyzed in deionized water for 3-5 days, the Apa-loaded PLGA NPs solution was collected.

3. The method for preparing an engineered exosome loaded with apatinib according to claim 1, characterized in that: The specific operations of step 2 are as follows: Step 201: incubating HCE in serum-free medium for 48 hours; Step 202: collecting the supernatant and performing gradient centrifugation; Step 203: Filter the remaining supernatant with a 0.22 μm filter and ultracentrifuge at 120,000 g for 90 min to precipitate HCE-EXs; Step 204: The HCE-EXs were resuspended in PBS and ultracentrifuged again to remove residual proteins. The purified HCE-EXs were stored at -80°C.

4. The method for preparing an engineered exosome loaded with apatinib according to claim 3, characterized in that: The specific operation of performing gradient centrifugation on the supernatant in step 202 is: collecting the supernatant, and centrifuging the supernatant in sequence according to the gradient setting of 200g centrifugation for 5 min, 3000g centrifugation for 10 min, and 16000g centrifugation for 30 min.

5. The method for preparing an engineered exosome loaded with apatinib according to claim 1, characterized in that: The specific operations of step 3 are as follows: The Apa-loaded PLGA nanoparticles prepared in step 1 were mixed with the HCE-EXs suspension prepared in step 2, and the mixture was treated with ultrasound for 10 min to obtain exosome-encapsulated apatinib nanoparticles, which were designated as Apa-EXs.

6. The method for preparing an engineered exosome loaded with apatinib according to claim 5, characterized in that: In step 3, the concentration range of the Apa-loaded PLGA nanoparticles is 1 mg / m to 10 mg / ml, and the concentration range of the HCE-EXs suspension is 2 mg / m to 15 mg / ml.

7. The method for preparing an engineered exosome loaded with apatinib according to claim 1, characterized in that: The specific operations of step 4 are as follows: The DMSO solution of DSPE-PEG-NHS and the DMSO solution of Arg were mixed in a molar ratio of 1:1, stirred in the dark for 24 hours, dialyzed in an aqueous solution to remove DMSO, and freeze-dried to obtain DSPE-PEG-Arg, that is, DSPE-PEG-Arg is an arginine-modified phospholipid polymer.

8. The method for preparing an engineered exosome loaded with apatinib according to claim 1, characterized in that: In step 5, Apa-EXs and DSPE-PEG-Arg were mixed at a volume ratio of 1:2, and the concentration of Apa-EXs was 5 μg / ml, and the concentration of DSPE-PEG-Arg was 10 μg / ml.

9. An engineered exosome loaded with apatinib, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the engineered exosomes loaded with apatinib according to claim 9 in the preparation of a drug for treating corneal alkali burns.

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