CN115400095B - Intraocular injection based on microcapsule and preparation method thereof - Google Patents

Intraocular injection based on microcapsule and preparation method thereof Download PDF

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CN115400095B
CN115400095B CN202210990296.0A CN202210990296A CN115400095B CN 115400095 B CN115400095 B CN 115400095B CN 202210990296 A CN202210990296 A CN 202210990296A CN 115400095 B CN115400095 B CN 115400095B
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macroporous
microcapsule
exosomes
microspheres
intraocular injection
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CN115400095A (en
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陶勇
魏炜
马光辉
田颖
包晗
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Institute of Process Engineering of CAS
Beijing Chaoyang Hospital
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Institute of Process Engineering of CAS
Beijing Chaoyang Hospital
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Abstract

The invention discloses an intraocular injection based on microcapsules and a preparation method thereof. The intraocular injection comprises an exosome and a biodegradable polymer blend matrix. The intraocular injection is in the form of microcapsule, the microcapsule contains multi-cavity structure, the average particle diameter of the microcapsule is 10-100 μm, preferably 10-30 μm, and the microcapsule is prepared by the following method: the method comprises the steps of preparing open-cell microspheres from a polymer blend, mixing the open-cell microspheres with a solution containing exosomes, and sealing the open-cell microspheres loaded with the exosome solution to form sealed microcapsules loaded with exosomes. The sealing condition of the microcapsule is 39-42 ℃,2-6h, and the optimal healing condition is 39 ℃ and 4h.

Description

Intraocular injection based on microcapsule and preparation method thereof
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an intraocular injection which comprises an exosome and a biodegradable polymer blend matrix.
Background
Retinal ischemia reperfusion injury (retinal ischemia reperfusion injury, ri) is a pathological injury process common to a variety of clinical ophthalmic diseases, and is associated with diabetic retinopathy, glaucoma, central retinal artery occlusion, etc. that cause ischemia, ultimately leading to irreversible vision impairment and vision loss. At present, the clinical treatment is still mainly conservative treatment, and no effective measures exist yet.
In recent years, researchers have explored the possibility of applying cell therapies in ophthalmology. It has been reported that intravitreal injection of bone marrow mesenchymal stem cells reduces retinal cell death in the mouse ri model and has been used to treat refractory macular holes in clinical patients. Despite the positive therapeutic effects achieved, cell-based therapies have encountered a number of problems in ophthalmic applications. From a medical point of view, the pathological microenvironment has a serious negative impact on the function of the therapeutic cells. Hypoxia stress, high oxidative stress and nutrient deprivation can lead to low therapeutic cell survival rates, limiting therapeutic efficacy. It has also been reported that stem cells can be uncontrollably transformed into undesirable cells, such as myofibroblasts, which can also produce undesirable therapeutic effects and even side effects. In terms of clinical transformation, therapeutic cells take a long time to isolate and grow, and cannot be potentially used as off-the-shelf products, in acute environments, which limits the clinical viability of these cell-based techniques.
At present, the current state of treatment of retinal ischemia reperfusion diseases is lack of a medicament which can overcome the defect of cell therapy and also can take the advantage of cell therapy into account. The present invention can exactly remedy the above-mentioned shortcomings.
Disclosure of Invention
The invention aims to provide an intraocular injection based on microcapsules and a preparation method thereof.
In order to achieve the purpose of the invention, the following technical scheme is provided:
an intraocular injection based on microcapsule is a sealed microcapsule made from exosomes and macroporous microspheres; the exosomes are loaded in the macroporous microspheres, the macroporous microspheres are prepared from hydrophobic polymers and amphiphilic block copolymers, and the surfaces of the macroporous microspheres are provided with open pores, and the inside of the macroporous microspheres is provided with a through pore channel; the average particle diameter of the microcapsules is 10-100 μm.
Preferably, the exosomes are mesenchymal stem cell-derived exosomes or regulatory T cell (Treg) -derived exosomes.
Further, the mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, specifically, mouse bone marrow mesenchymal stem cells. Regulatory T cells (tregs) are specifically exemplified by mouse spleen regulatory T cells.
According to one embodiment of the invention, exosomes (MExo) derived from bone marrow mesenchymal stem cells are cup-shaped, have a particle size of about 120nm, and marker proteins ALIX, TSG101 and CD63 of MExo are positive.
Specifically, the extraction and MExo enrichment method of the primary mesenchymal stem cells is as follows: bone marrow Mesenchymal Stem Cells (MSCs) are extracted and purified from nucleated cells derived from mouse bone marrow, and culture supernatant of the MSCs is collected and enriched for MExo by ultracentrifugation.
Preferably, the average particle size of the microcapsules is 10-30 μm.
Preferably, the macroporous microspheres are made of PLGA and PELA; wherein the mass ratio of PLGA to PELA is 9:1.
The molecular weight of the PLGA is 21kDa; the molecular weight of the PELA was 40kDa.
The preparation method of the microcapsule-based intraocular injection provided by the invention comprises the following steps: firstly, preparing macroporous microspheres from a blend of a hydrophobic polymer and an amphiphilic block copolymer; and mixing the macroporous microspheres with a solution containing exosomes, and sealing the macroporous microspheres loaded with the exosome solution to obtain sealed microcapsules loaded with exosomes, namely the microcapsule-based intraocular injection.
Furthermore, the macroporous microspheres are prepared by adopting a double emulsion and solvent extraction method.
Further, the macroporous microspheres are made of PLGA and PELA.
Through exploration, the conditions of the macroporous microsphere sealing are 39-42 ℃ for 2-6h of incubation, and the optimal conditions are 39 ℃ for 4h of incubation. The conditions are sealed under as mild conditions as possible, and the denaturation of exosome protein components caused by high temperature is avoided to the greatest extent.
The preparation method of the microcapsule-based intraocular injection comprises the following steps:
(1) Preparing an oil phase, wherein the oil phase is a solution of a blend of a hydrophobic polymer and an amphiphilic block copolymer, and the solvent is an organic solvent; preparing an inner aqueous phase solution and an outer aqueous phase solution, and adding a surfactant into the outer aqueous phase;
(2) Dispersing an internal water phase into the oil phase to form a water-in-oil primary emulsion; dispersing the primary emulsion into an external water phase to form a water-in-oil-in-water double emulsion;
(3) Solidifying the oil phase by a solvent removal method to obtain macroporous microspheres with through channels;
(4) Mixing the open-pore microspheres with a solution containing exosomes, and allowing the exosomes to enter into the internal cavity from the surface of the macroporous microspheres to obtain macroporous microspheres loaded with the exosomes;
(5) And sealing the macroporous microspheres loaded with the exosomes to obtain the sealed microcapsules loaded with the exosomes.
Preferably in step (1), the oil phase is a solution of a blend of PLGA and PELA; wherein the mass ratio of PLGA to PELA is 9:1.
The molecular weight of the PLGA is 21kDa; the molecular weight of the PELA was 40kDa.
Preferably, the organic solvent may be ethyl acetate.
The solvent removal process in step (3) is solvent extraction.
In the curing process in the step (3), the inner water phase and the outer water phase are fused to form a through hole. And (3) the macroporous microspheres with inner and outer through channels are of porous structures. In a preferred embodiment of the invention, the macroporous microspheres of the invention are surface porous microspheres.
According to a specific embodiment of the invention, the preparation method of the macroporous microspheres comprises the following steps: mixing 0.5mL of 0.5% sodium chloride with 2mL of ethyl acetate containing 150mg of the compound (PLGA and PELA in a mass ratio of 9:1) in an ice bath for 12s by an ultrasonic method (120W); after homogenizing at 9000rpm for 120s, the mixture was added to 15mL of a 1.5% aqueous solution of polyvinyl alcohol (PVA 217), and homogenized for 2 to 3 minutes to obtain an emulsion. And (3) vertically suspending and pre-curing the prepared emulsion for 25 minutes by using a vertical suspension instrument at 45rpm, then adding the pre-cured emulsion into 500mL of deionized water, and curing for 10 minutes by using 100rpm magnetic stirring to obtain the open-cell microsphere with the through-channels. The microspheres with the particle size distribution of 10-30 mu m are screened by stainless steel screens with different sizes, which is more beneficial to intraocular injection.
The invention also provides application of the microcapsule-based intraocular injection.
The application of the microcapsule-based intraocular injection provided by the invention is the application of the microcapsule-based intraocular injection in preparing a product with the effect of treating retinal ischemia reperfusion injury.
The invention also provides a product with the function of treating retinal ischemia reperfusion injury.
The product comprises the microcapsule-based intraocular injection.
The product described in the present invention may be a medicament or a pharmaceutical formulation.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides an intraocular injection based on microcapsules, which is formed by mixing MSCs exosomes and macroporous microspheres and has the function of slowly and continuously releasing MExo in eyes. The system can be settled at the lower part of the vitreous body after being injected through the vitreous cavity, and continuously releases therapeutic exosomes in the vitreous body, so that the therapeutic effect is achieved, and the curative effect is better than that of the clinically common medicines. Aims at overcoming the defect of the current clinical treatment of vitreoretinal diseases.
Drawings
Fig. 1 is a Transmission Electron Microscope (TEM) image of MExo in example 1.
FIG. 2 is a statistical plot of MExo particle size and potential measured by a nanoparticle tracking analyzer in example 1.
FIG. 3 shows the marker protein of MExo analyzed by Western blotting in example 1.
Fig. 4 is a Scanning Electron Microscope (SEM) image of the microspheres of example 2 before healing.
FIG. 5 is an SEM image of the internal structure of the microspheres in example 2.
Fig. 6 is a laser confocal plot of the exosome-loaded capped microspheres prepared in example 2.
Figure 7 shows the loading rate of the exosome-loaded capped microspheres prepared in example 2.
FIG. 8 is an SEM image of the capped microspheres of example 2.
FIG. 9 is an SEM image of the internal structure of the seal microsphere in example 2.
Figure 10 images of small animals from the ri model mice in example 3.
FIG. 11 is a fundus phase of RIRI model mice in example 3.
FIG. 12 is a photograph of an isolated eyeball of a RIRI model mouse of example 3.
Fig. 13 is the total retinal and ganglion cell layer thickness of the ri model mice observed by Optical Coherence Tomography (OCT) in example 4.
FIG. 14 is an Electroretinogram (ERG) observation of visual function in RIRI model mice in example 4.
FIG. 15 is a section HE staining of heart, liver, spleen, lung and kidney of RIRI model mice in example 4.
FIG. 16 shows the measurement of intraocular pressure in RIRI model mice using the contact tonometer of example 4.
FIG. 17 is a blood routine of mice in the RIRI model for intravenous blood collection test in example 4.
Fig. 18 is a TEM image of trero in example 5.
Fig. 19 is a statistical chart of the nanoparticle tracking analyzer measuring the diameter of the tremo particle and the potential in example 5.
FIG. 20 shows the Western blot analysis of marker proteins of TrExo in example 5.
Fig. 21 is a laser confocal plot of the trero-loaded capped microspheres prepared in example 5.
FIG. 22 shows the amount of inflammatory cytokines in the ocular fluid of the PMU model mice of example 5.
FIG. 23 is H & E sections of retina after treatment in a PMU model mouse of example 5.
FIG. 24 is H & E staining of sections of heart, liver, spleen, lung and kidney of PMU model mice in example 5.
FIG. 25 shows the measurement of the ocular tension of mice in the PMU model by the contact tonometer of example 5.
FIG. 26 is a blood routine of a PMU model mouse for intravenous blood sampling test in example 5.
Detailed Description
The invention will be further illustrated with reference to the following specific examples, but the invention is not limited to the following examples. The methods are conventional methods unless otherwise specified. The starting materials are available from published commercial sources unless otherwise specified.
The molecular weight of the PLGA described in the examples below was 21kDa; the molecular weight of the PELA is 40kDa; the mass ratio of PLGA to PELA was 9:1.
Example 1 in vitro expansion of Primary bone marrow mesenchymal Stem cell cells and enrichment of MExo
(1) Mesenchymal Stem Cells (MSCs) were extracted and purified from whole nuclear cells derived from mouse bone marrow. After 72h, non-adherent cells were removed and adherent cells were cultured in MEM-a medium (containing 10% exosome-free fetal bovine serum, 1% penicillin) supplemented with 2mM L-glutamine and 55mM 2-mercaptoethanol. Cell cultures were grown in a 5% carbon dioxide incubator.
(2) Collecting cell culture supernatant of 7-14 days, centrifuging at 300g for 15min to remove impurities; then centrifuged at 10,000g for 15min to remove cell debris; centrifuge at 100,000g for 120min, discard supernatant, collect pellet, and then re-suspend in PBS to give MExo solution. The morphology is observed by using a Transmission Electron Microscope (TEM), the particle size and the potential distribution of the nanoparticle are measured by Nanoparticle Tracking Analysis (NTA), and the expression of the exosome characteristic protein is measured by using a western blotting method.
The results are shown in FIGS. 1-3. As can be seen, MExo is cup-holder shaped, has a particle size of about 120nm, and is positive for marker proteins ALIX, TSG101 and CD63 of MExo.
EXAMPLE 2 preparation of sealed microcapsules loaded with exosomes (denoted MExoCap)
(1) The microcapsule is prepared by adopting a double emulsion and solvent extraction method. 0.5mL of 0.5% sodium chloride was mixed with 2mL of ethyl acetate containing 150mg of the compound (PLGA and PELA) in an ice bath by sonication (120W) for 12s. After homogenizing at 9000rpm for 120s, the mixture was added to 15mL of an aqueous solution of polyvinyl alcohol (PVA 217) having a mass volume concentration of 1.5%, and homogenized for 120s to obtain an emulsion. And (3) vertically suspending and pre-curing the prepared emulsion for 25 minutes by using a vertical suspension instrument at 45rpm, then adding the pre-cured emulsion into 500mL of deionized water, and curing for 10 minutes by using 100rpm magnetic stirring to obtain the open-cell microsphere with the through-channels. The microspheres with the particle size distribution of 10-30 mu m are screened by stainless steel screens with different sizes, which is more beneficial to intraocular injection.
(2) The microsphere suspension was pipetted in 50. Mu.L (containing about 50. Mu.g of microspheres) and dropped onto tinfoil and left to dry at room temperature. And (3) sticking the tinfoil paper containing the sample on a sample preparation table by using conductive adhesive, spraying gold, and observing the surface morphology of the microsphere by using SEM.
As shown in FIG. 4, the surface pore diameter was 3-4. Mu.m, the structure of the surface opening was obtained, and the average particle diameter of the microspheres was 25. Mu.m. Meanwhile, in order to observe the internal structure of the microsphere, the dried microsphere is chopped by an ultrathin blade. The samples were then attached to a conductive gel and sprayed with gold and observed with SEM. As shown in FIG. 5, the interior of the microsphere has a porous and interpenetrating structure, and the internal pore diameter is about 4-5. Mu.m.
(3) Sucking 1mL of the open microsphere suspension prepared in the step (1) with the dry weight of 30mg into a 1.5mL centrifuge tube, centrifuging to remove the supernatant, adding 500 mu L of an exosome solution with the dry weight of 20mg/mL into the centrifuge tube, blending with the microspheres, and placing the mixture on a vertical suspension instrument for suspension for 4h (300 rpm), so that exosome fully enters the cavities of the microspheres by using the through channels of the microspheres. Incubating for 2-6h at 39-42 deg.C in an incubator, respectively, and exploring the healing mode with the lowest temperature and the shortest time. The microsphere can be sealed without affecting the morphology of the microsphere and the function of the exosome. And then placing the vertical suspension instrument and the microspheres in a 39 ℃ incubator for heating and sealing, wherein the suspension speed is 100rpm in the process, ensuring that the microspheres are heated uniformly in the whole heating process and do not settle at the same time, sealing after 4 hours of reaction, centrifuging (500 g,5 min) to remove the supernatant, and obtaining the sealed microcapsule loaded with the exosomes. The loading of the microcapsules on the exosomes was observed using a confocal microscope and the results are shown in fig. 6. As can be seen from fig. 6, in the confocal laser image, sky blue represents the microsphere scaffold and yellow represents the exosomes. In the 2D scan mode, a sky blue microsphere backbone can be seen, which contains many circular cavities, while the yellow exosomes fill the cavities of the microspheres. The loading rate is shown in fig. 7.
(4) Sucking 50 mu L of sealed microcapsule suspension containing exosomes, dripping on tinfoil paper, and air drying at room temperature. And (3) sticking the tinfoil paper containing the sample on a sample preparation table by using conductive adhesive, spraying gold, and observing the surface morphology of the microcapsule by using SEM. As shown in fig. 8, the surface pores are completely closed, forming a capped microcapsule. And simultaneously, in order to observe the internal structure of the sealed microcapsule, the aired microcapsule is chopped by an ultrathin blade. The samples were then attached to a conductive gel and sprayed with gold and observed with SEM. As shown in fig. 9, the microcapsule still has an internally porous structure, and the pore diameter is about 4 to 5 μm, but the internally penetrating porous structure becomes a closed and independent porous structure.
EXAMPLE 3 retention and distribution of microspheres in mice eyes
(1) C57BL/6 mice were anesthetized with 0.5% pentobarbital (0.1 mL/10 g.bw) by intraperitoneal injection, and 1% compound Topirocamine eye drops were pupil-expanded. Subsequently, the drug was administered by the intravitreal injection route and administered only once throughout the course, MExo, MSCs and MExoCap were injected into the vitreous cavity with Hamilton's microinjector (single intravitreal injection of 5 μg MExo/eye, 4×10 6 MSCs per eye, 5 μg MExo per eye loaded in 50 μg microsphere, 6 eyes of 6 mice per group.
(2) The 0, 3, 7, 14, 21, 28 and 35 Gastrodia elata drunk mice after intravitreal injection were imaged using a small animal imaging system, and as a result, the intraocular residence time was significantly prolonged after encapsulation of the exosomes in microspheres as shown in fig. 10. On the third day after intravitreal injection, fundus phases were photographed using a fundus camera after general anesthesia mydriasis. The results are shown in FIG. 11. Afterwards, the mice are sacrificed by cervical dislocation, the optic nerve is clamped by forceps, the eyeballs are lifted up and separated, and the eyeballs are completely taken out. The eyeballs were immediately placed in a petri dish and the distribution of MExo, MSCs and MExoCap in the eyes was observed in a small animal imaging system. As shown in fig. 12, a small amount of residual MExo is uniformly distributed in the preretinal region and MSCs are distributed on the posterior lens surface, possibly due to the adherent nature of MSCs. While MExoCap is distributed primarily in the lower vitreous cavity and its released MExo is distributed in the preretinal area. The results prove that: MExoCap settles in the lower part of the vitreous cavity after intravitreal injection and does not affect the optical path.
Example 4 in vivo efficacy and safety validation of RIRI model mice
(1) A mouse model of retinal ischemia reperfusion injury (ri) was constructed. C57BL/6 mice were anesthetized with 0.5% pentobarbital (0.1 mL/10 g.bw) by intraperitoneal injection, and 1% compound Topirocamine eye drops were pupil-expanded. The infusion bottle filled with 100mL of sterile physiological saline is connected with a disposable sterile insulin syringe through an infusion apparatus, and the air in the infusion apparatus is exhausted. After the pupil is dilated, the hand-held needle is inserted parallel to the longitudinal axis of the mouse body along the limbus of the temporal side. After the needle is fixed by the adhesive tape, the sluice of the transfusion system is opened, and the transfusion bottle is slowly lifted to make the final height from the mouse be 150cm, and the intraocular pressure formed by the height is 110mmHg. After the infusion bottle was raised, the iris and fundus of the mouse were observed to become pale, indicating retinal ischemia formation. After ischemia for 1h, the height of the infusion bottle is reduced, the needle is pulled out, and at the moment, the blood flow of the iris and the fundus is restored, namely, the retinal reperfusion is formed.
(2) After molding for 1 day, the drug was administered by intravitreal injection route and once only. After intraperitoneal injection of anesthesia RIRI model mice with 0.5% pentobarbital (0.1 mL/10 g.bw), PBS, MExo, MSCs, MExoCap and NGF (murine nerve growth factor) were injected into the vitreous cavity (single intravitreal injection of 2. Mu.L PBS/eye, 5. Mu.g MExo/eye, 4X 10) using Hamilton's microinjector 6 MSCs per eye, 5. Mu.g MExo per eye, 3. Mu.g NGF per eye loaded in 50. Mu.g microspheres, 6 eyes of 6 mice per administration group.
(3) 28 days after administration, RIRI model mice were anesthetized with 0.5% pentobarbital (0.1 mL/10 g.bw) intraperitoneal injection, 1% compound Topirocamine eye drops mydriasis, OCT and ERG examination. See fig. 13, 14. The results show that: the MExoCap treatment group had the least decrease in thickness of the retinal whole layer and ganglion cell layer, and the least decrease in amplitude of electroretinogram a-wave and B-wave. After the cervical dislocation of the mice is killed, eyeballs are removed, paraffin sections of 3-4 mu m/piece are made after embedding and fixing, and the results show that: the nuclear layer number of the inner nuclear layer and the outer nuclear layer of the MExoCap treatment group is reduced to the minimum, and the effect is superior to that of the nerve growth factor commonly used in clinic.
(4) 28 days after administration, the RIRI model mice were anesthetized and their ocular pressure was measured using a small animal tonometer, see FIG. 16. The results show that: MExoCap treatment did not affect eye pressure. The blood routine of the RIRI model mice was tested with venous blood from the mice, see FIG. 17, and the results showed no significant change in blood routine. The RIRI model mice were then sacrificed, their viscera (heart, liver, spleen, lung, kidney) were removed, fixed, paraffin-embedded, 3-4 μm/slice sections were made, hematoxylin and eosin (H & E) stained, and observed using an optical microscope, see FIG. 15. The results show that: no obvious abnormalities were seen in viscera following MExoCap treatment.
Example 5 extraction of Treg exosomes
After obtaining a cell suspension from mouse spleen milling solution, methoprene CD4 was used + CD25 + Regulatory T cell isolation kit, operating according to instructions, treg cells were obtained. Treg cells were cultured using X-Vivo complete medium formulated with exosome-free serum (containing 10% exosome-free serum and 1% diabody), cultured for 48h after passage and cell supernatants were collected. Centrifuging at 300g for 15min to remove impurities; then centrifuged at 10,000g for 15min to remove cell debris; centrifuge at 100,000g for 120min, discard supernatant, collect pellet, then re-suspend in PBS to give TrExo solution. The morphology is observed by using a TEM, the particle size and the potential distribution of the NTA are measured, and the expression of the exosome characteristic protein is measured by using a western blotting method.
The results are shown in FIGS. 18-20. As can be seen, trExo is cup-holder-shaped, has a particle size of about 120nm, and is positive for the marker proteins ALIX, TSG101, CD63, CTLA-4 and IL-10.
Example 6 production of sealed microcapsules loaded with Treg exosomes (denoted TrExoCap).
Open microspheres were prepared as in step (1) of example 2.
1mL of the open microsphere suspension with the dry weight of 30mg is sucked into a centrifuge tube with the dry weight of 1.5mL, the supernatant is removed by centrifugation, 500 mu L of exosome solution with the dry weight of 20mg/mL is added to be blended with the microspheres, and the mixture is placed on a vertical suspension instrument to be suspended for 4h (300 rpm), so that exosome fully enters the cavity of the microspheres by utilizing a pore canal penetrated by the microspheres. Incubating for 2-6h at 39-42 deg.C in an incubator, respectively, and exploring the healing mode with the lowest temperature and the shortest time. The microsphere can be sealed without affecting the morphology of the microsphere and the function of the exosome. And then placing the vertical suspension instrument and the microspheres in a 39 ℃ incubator for heating and sealing, wherein the suspension speed is 100rpm in the process, ensuring that the microspheres are heated uniformly in the whole heating process and do not settle at the same time, sealing after 4 hours of reaction, centrifuging (500 g,5 min) to remove the supernatant, and obtaining the sealed microcapsule loaded with the exosomes. The loading of the microcapsules on the exosomes was observed using a confocal microscope and the results are shown in fig. 21. As can be seen from fig. 21, in the confocal laser image, sky blue represents the microsphere scaffold and pink represents the exosomes. In the 2D scan mode, a sky blue microsphere backbone can be seen, which contains many circular cavities, while the pink exosomes fill the cavities of the microspheres.
Example 7: verification of in-vivo effects and safety of PMU model mice
(1) A PMU mouse model was constructed. C57BL/6 mice were anesthetized with 0.5% pentobarbital (0.1 mL/10 g.bw) by intraperitoneal injection, and 1% compound Topirocamine eye drops were pupil-expanded. On day-9, mice were subcutaneously injected with 100 μg of inactivated Mycobacterium tuberculosis H37Ra antigen dissolved in 0.1mL of incomplete Freund's adjuvant. After 7 days of normal anesthesia, the mice were given surface anesthesia (oxybuprocaine hydrochloride eye drops) and mydriatic (compound topicamine eye drops) by right eye drops, followed by intravitreal injection of 3 μg of mycobacterium tuberculosis antigen H37Ra dissolved in 1 μl PBS buffer per mouse right eye. The administration was after 2 days.
(2) After molding, the drug was administered via intravitreal injection route and only once. After intraperitoneal injection of anesthetized PMU model mice with 0.5% pentobarbital (0.1 mL/10 g.bw), PBS, trExo, treg cells, trExoCap and TA (triamcinolone acetonide) were injected into the vitreous cavity (single vitreous cavity) respectively using a Hamilton's microinjectorThe injection amount was 2. Mu.L PBS/eye, 5. Mu.g TrExo/eye, 4X 10 6 Each Treg cell/eye, 5 μg trex/eye loaded in 50 μg microsphere, 80 μg TA/eye) 6 eyes of 6 mice were used per dosing group.
(3) After 28 days of administration, PMU model mice were anesthetized by intraperitoneal injection of 0.5% pentobarbital (0.1 mL/10 g.bw), 1% compound tropamide eye drops were pupil expanded, and the anterior aqueous humor of the mice was collected, see FIG. 22, and the results showed that the TrExoCap group mice had the lowest inflammatory cytokines (IL-1 beta, IL-6, IL-8 and TNF) content in the intraocular fluid. After the cervical dislocation of the mice is killed, eyeballs are removed, paraffin sections of 3-4 mu m/piece are made after embedding and fixing, as shown in fig. 23, and the results show that: mice in the TrExoCap treatment group have the lowest infiltration degree of vitreous and retina inflammatory cells, and the effect is better than that of a hormone medicine triamcinolone acetonide commonly used in clinic.
(4) After 28 days of administration, PMU model mice were anesthetized and their ocular pressure was measured using a small animal tonometer, see FIG. 24. The results show that: the TrExoCap treatment did not affect eye pressure. The blood routine of the mice in the PMU model was examined by taking venous blood from the mice, see FIG. 25, and the results showed no significant change in blood routine. The PMU model mice were then sacrificed, their viscera (heart, liver, spleen, lung, kidney) were removed, fixed, paraffin-embedded, 3-4 μm/slice sections were made, hematoxylin and Eosin (HE) stained, and observed using an optical microscope, see FIG. 26. The results show that: there were no obvious abnormalities in viscera after TrExoCap treatment.

Claims (9)

1. An intraocular injection based on microcapsule is a sealed microcapsule made from exosomes and macroporous microspheres; the exosome is loaded in the macroporous microsphere, the macroporous microsphere is prepared from PLGA and PELA, and the surface of the macroporous microsphere is provided with open pores and a through hole in the macroporous microsphere; the average particle diameter of the microcapsule is 10-30 mu m;
PLGA and PELA in the mass ratio of 9 to 1;
the preparation method of the microcapsule-based intraocular injection comprises the following steps: firstly, preparing macroporous microspheres from a blend of PLGA and PELA; then mixing the macroporous microspheres with a solution containing exosomes, and sealing the macroporous microspheres loaded with the exosome solution to obtain sealed microcapsules loaded with exosomes, namely the microcapsule-based intraocular injection;
the condition of the macroporous microsphere sealing is that the macroporous microsphere is incubated for 2-6h at 39-42 ℃.
2. The microcapsule-based intraocular injection according to claim 1, wherein: the exosomes are mesenchymal stem cell-derived exosomes or regulatory T cell-derived exosomes.
3. The microcapsule-based intraocular injection according to claim 2, wherein: the mesenchymal stem cells are derived from bone marrow mesenchymal stem cells.
4. A method of preparing a microcapsule-based intraocular injection according to any one of claims 1-3, comprising the steps of: firstly, preparing macroporous microspheres from a blend of PLGA and PELA; then mixing the macroporous microspheres with a solution containing exosomes, and sealing the macroporous microspheres loaded with the exosome solution to obtain sealed microcapsules loaded with exosomes, namely the microcapsule-based intraocular injection;
the macroporous microspheres are prepared from PLGA and PELA;
the condition of the macroporous microsphere sealing is that the macroporous microsphere is incubated for 2-6h at 39-42 ℃.
5. The method of manufacturing according to claim 4, wherein: the macroporous microspheres are prepared by adopting a double emulsion and solvent extraction method.
6. The method of manufacturing according to claim 4, wherein: the condition of the macroporous microsphere sealing is 39 ℃ incubation for 4 hours.
7. The production method according to any one of claims 4 to 6, characterized in that: the preparation method of the microcapsule-based intraocular injection comprises the following steps:
(1) Preparing an oil phase, wherein the oil phase is a solution of a blend of PLGA and PELA, and the solvent is an organic solvent; preparing an inner aqueous phase solution and an outer aqueous phase solution, and adding a surfactant into the outer aqueous phase;
(2) Dispersing an internal water phase into the oil phase to form a water-in-oil primary emulsion; dispersing the primary emulsion into an external water phase to form a water-in-oil-in-water double emulsion;
(3) Solidifying the oil phase by a solvent removal method to obtain macroporous microspheres with through channels;
(4) Mixing the macroporous microspheres with a solution containing exosomes, and allowing the exosomes to enter into an internal cavity from the surface of the macroporous microspheres to obtain macroporous microspheres loaded with the exosomes;
(5) And sealing the macroporous microspheres loaded with the exosomes to obtain the sealed microcapsules loaded with the exosomes.
8. Use of a microcapsule-based intraocular injection according to any one of claims 1-3 for the preparation of a product having therapeutic effect on retinal ischemia reperfusion injury.
9. A product having a therapeutic effect on retinal ischemia reperfusion injury comprising the microcapsule-based intraocular injection according to any one of claims 1-3.
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