CN115400095A

CN115400095A - Intraocular injection based on micro-capsules and preparation method thereof

Info

Publication number: CN115400095A
Application number: CN202210990296.0A
Authority: CN
Inventors: 陶勇; 魏炜; 马光辉; 田颖; 包晗
Original assignee: Institute of Process Engineering of CAS; Beijing Chaoyang Hospital
Current assignee: Institute of Process Engineering of CAS; Beijing Chaoyang Hospital
Priority date: 2022-08-18
Filing date: 2022-08-18
Publication date: 2022-11-29
Anticipated expiration: 2042-08-18
Also published as: CN115400095B

Abstract

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

Description

Intraocular injection based on micro-capsules and preparation method thereof

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to an intraocular injection which comprises exosomes and a biodegradable polymer blend matrix.

Background

Retinal Ischemia Reperfusion Injury (RIRI) is a common pathological injury process in clinical multiple ophthalmic diseases, is related to diabetic retinopathy, glaucoma, central retinal artery occlusion and other ischemic retinopathy, and finally can cause irreversible vision damage and vision loss. At present, the clinical treatment is mainly conservative treatment, and no effective measure is available.

In recent years, researchers have explored the possibility of applying cell therapy in ophthalmology. It has been reported in the literature that intravitreal injection of mesenchymal stem cells reduces retinal cell death in the RIRI model in mice and has been used to treat refractory macular holes in clinical patients. Despite some positive therapeutic effects, cell-based therapies have encountered a range of problems in ophthalmic applications. From a medical point of view, the pathological microenvironment has a severe negative impact on the function of the therapeutic cells. Hypoxia, high oxidative stress and nutrient deprivation can result in low survival of therapeutic cells, limiting therapeutic efficacy. It has also been reported that stem cells are uncontrollably transformed into undesirable cells, such as myofibroblasts, which may also produce undesirable therapeutic effects and even side effects. In terms of clinical transformation, therapeutic cells require a long time to isolate and grow, and cannot be potentially applied as a ready-to-use product, which can be used in acute settings, limiting the clinical feasibility of these cell-based technologies.

At present, the current situation of treatment of retinal ischemia-reperfusion diseases is lack of a medicine which can overcome the defects of cell therapy and has the advantages of cell therapy. The invention can just make up the above disadvantages.

Disclosure of Invention

The invention aims to provide a microcapsule-based intraocular injection and a preparation method thereof.

In order to realize the purpose of the invention, the following technical scheme is provided:

an intraocular injection based on microcapsule is a sealed microcapsule made of exosome and macroporous microsphere; the exosome is loaded inside the macroporous microsphere, the macroporous microsphere is prepared from a hydrophobic polymer and an amphiphilic block copolymer, the surface of the macroporous microsphere is provided with openings, and the inside of the macroporous microsphere is provided with through channels; the average particle diameter of the microcapsule is 10-100 μm.

Preferably, the exosome is a mesenchymal stem cell-derived exosome or a regulatory T cell (Treg) -derived exosome.

Furthermore, the mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, such as mouse bone marrow mesenchymal stem cells. Regulatory T cells (tregs) are specifically exemplified by mouse spleen regulatory T cells.

According to a specific embodiment of the present invention, exosomes (MExo) derived from bone marrow mesenchymal stem cells are in the shape of cup-holder, have a particle size of about 120nm, and are positive for MExo's marker proteins ALIX, TSG101 and CD 63.

Specifically, the extraction method of the primary mesenchymal stem cells and the enrichment method of MExo are as follows: bone marrow Mesenchymal Stem Cells (MSCs) were extracted and purified from mouse bone marrow-derived nucleated cells, and culture supernatants of the MSCs were collected and enriched for MExo by ultracentrifugation.

Preferably, the average particle size of the microcapsules is 10 to 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 PELA is 40kDa.

The invention provides a preparation method of an intraocular injection based on microcapsules, which comprises the following steps: preparing macroporous microspheres from a blend of a hydrophobic polymer and an amphiphilic block copolymer; the macroporous microspheres are then mixed with an exosome-containing solution, followed by sealing the macroporous microspheres loaded with exosome solution, resulting in sealed exosome-loaded microcapsules, i.e. the microcapsule-based intraocular injections.

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 condition of sealing the macroporous microspheres is incubation for 2-6h at 39-42 ℃, and the optimal condition is incubation for 4h at 39 ℃. The conditions are sealed under the conditions which are as mild as possible, and the denaturation of the protein components of the exosomes caused by high temperature is avoided to the maximum extent.

The preparation method of the microcapsule-based intraocular injection specifically 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 a solvent is an organic solvent; preparing an inner water phase solution and an outer water phase solution, and adding a surfactant into the outer water phase;

(2) Dispersing the inner 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 an aqueous-in-oil-in-aqueous double emulsion;

(3) Solidifying the oil phase by using a solvent removal method to obtain the macroporous microspheres with through-penetrating pores;

(4) Mixing the open-pore microspheres with a solution containing exosomes, and allowing the exosomes to enter an internal cavity from the surface of the macroporous microspheres to obtain macroporous microspheres loaded with exosomes;

(5) And (3) sealing the macroporous microspheres loaded with the exosomes to obtain the sealed microcapsule 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.

Preferably, the organic solvent may be ethyl acetate.

The solvent removal method in the step (3) is solvent extraction.

And (4) in the curing process in the step (3), fusing the inner aqueous phase and the outer aqueous phase to form a through hole. The macroporous microspheres with inner and outer through-holes in the step (3) are of a porous structure. In a preferred embodiment of the present invention, the macroporous microspheres of the present invention are surface porous microspheres.

According to a specific embodiment of the present invention, the preparation method of the macroporous microspheres comprises the following steps: 0.5mL of 0.5% sodium chloride was mixed with 2mL of ethyl acetate containing 150mg of the compound (PLGA and PELA, mass ratio 9:1) in an ice bath for 12s by sonication (120W); after homogenizing at 9000rpm for 120s, the mixture was added to 15mL of a 1.5% aqueous polyvinyl alcohol (PVA 217) solution, and the mixture was homogenized for 2 to 3min to obtain an emulsion. And vertically suspending the prepared emulsion by using a vertical suspension instrument at 45rpm for pre-curing for 25min, then adding the pre-cured emulsion into 500mL of deionized water, and magnetically stirring and curing at 100rpm for 10min to obtain the open-pore microspheres with through-pores. The microspheres with the particle size 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 the application of the microcapsule-based intraocular injection.

The application of the microcapsule-based intraocular injection provided by the invention is the application thereof in preparing products with the function of treating retinal ischemia-reperfusion injury.

The invention also protects a product with the function of treating the retinal ischemia-reperfusion injury.

The product comprises the microcapsule-based intraocular injection of the present invention.

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 a microcapsule-based intraocular injection 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 on the lower part of the vitreous body after being injected into the vitreous body cavity, and can continuously release therapeutic exosomes in the vitreous body, thereby achieving the treatment effect, and the curative effect is superior to that of clinical common medicines. Aims to make up the deficiency of the current clinical vitreoretinal disease treatment.

Drawings

FIG. 1 is a Transmission Electron Microscope (TEM) image of MExo in example 1.

FIG. 2 is a statistical chart of the MExo particle size and potential measured by the nanoparticle tracking analyzer of example 1.

FIG. 3 shows the Western blot analysis of marker proteins of MExo in example 1.

FIG. 4 is a Scanning Electron Microscope (SEM) image of microspheres from example 2 before healing.

FIG. 5 is an SEM image of the internal structure of the microsphere of example 2.

FIG. 6 is a confocal laser mapping of exosome-loaded encapsulated microspheres prepared in example 2.

FIG. 7 shows the loading ratio of exosome-loaded capped microspheres prepared in example 2.

FIG. 8 is an SEM image of encapsulated microspheres from example 2.

FIG. 9 is an SEM image of the internal structure of the encapsulated microspheres of example 2.

FIG. 10 is a small animal image of RIRI model mouse in example 3.

FIG. 11 is the fundus phase of the RIRI model mouse of example 3.

Fig. 12 is an image of a small animal of ex vivo eyeball of RIRI model mouse in example 3.

FIG. 13 is the observation of the thickness of the retina hololayer and ganglion cell layer of RIRI model mice by Optical Coherence Tomography (OCT) in example 4.

FIG. 14 is an Electroretinogram (ERG) observation of the visual function of RIRI model mice in example 4.

FIG. 15 is HE staining of sections 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 by the contact tonometer in example 4.

FIG. 17 is a blood routine for the intravenous blood collection test of RIRI model mice in example 4.

Fig. 18 is a TEM image of TrExo in example 5.

FIG. 19 is a statistical graph of the TrExo particle size and potential measured by the nanoparticle tracking analyzer of example 5.

FIG. 20 shows the Western blot analysis of TrExo marker protein in example 5.

Fig. 21 is a confocal laser map of TrExo loaded capped microspheres prepared in example 5.

FIG. 22 shows the content of inflammatory cytokines in the eye fluid of the PMU model mouse in example 5.

FIG. 23 is an H & E section of retina after treatment of PMU model mouse in example 5.

FIG. 24 is H & E staining of heart, liver, spleen, lung and kidney sections of the PMU model mouse in example 5.

FIG. 25 shows the measurement of intraocular pressure of mouse model PMU by the contact tonometer in example 5.

FIG. 26 is a routine of blood sampling of PMU model mice in example 5.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The starting materials are commercially available from the open literature unless otherwise specified.

The molecular weight of the PLGA described in the examples below is 21kDa; the molecular weight of the PELA is 40kDa; the PLGA to PELA mass ratio was 9:1.

Example 1 in vitro expansion of Primary mesenchymal Stem cells and enrichment of MExo

(1) Mesenchymal Stem Cells (MSCs) were extracted and purified from mouse bone marrow-derived whole nuclear cells. After 72h, nonadherent cells were removed and adherent cells were cultured in MEM-a medium (containing 10% exosome-free fetal bovine serum, 1% streptomycin) supplemented with 2mM L-glutamine and 55mM 2-mercaptoethanol. The cell cultures were cultured in a 5% carbon dioxide incubator.

(2) Collecting cell culture medium supernatant of 7-14 days, and centrifuging at 300g for 15min to remove impurities; then centrifuged at 10,000g for 15min to remove cell debris; the mixture was centrifuged at 100,000g for 120min, the supernatant was discarded, and the pellet was collected and then resuspended in PBS to obtain MExo solution. The appearance of the protein is observed by using a Transmission Electron Microscope (TEM), the particle size and the potential distribution of the protein are measured by Nanoparticle Tracking Analysis (NTA), and the protein expression of the exosome characteristic is measured by a western blot method.

The results are shown in FIGS. 1-3. As can be seen, MExo is in the shape of a cup holder with a particle size of about 120nm, and MExo is positive for the marker proteins ALIX, TSG101 and CD 63.

Example 2 preparation of exosome-loaded encapsulated microcapsules (denoted as MExoCap)

(1) The microcapsule is prepared by double emulsion and solvent extraction. 0.5mL of 0.5% sodium chloride was mixed with 2mL of ethyl acetate containing 150mg of compound (PLGA and PELA) in an ice bath for 12s using sonication (120W). After homogenizing at 9000rpm for 120s, the mixture was added to 15mL of a 1.5% by mass/volume aqueous polyvinyl alcohol (PVA 217) solution to give an emulsion after homogenizing for 120 s. And vertically suspending the prepared emulsion by using a vertical suspension instrument at 45rpm for pre-curing for 25min, then adding the pre-cured emulsion into 500mL of deionized water, and magnetically stirring and curing at 100rpm for 10min to obtain the open-pore microspheres with through-pores. The microspheres with the particle size of 10-30 mu m are screened by stainless steel screens with different sizes, which is more beneficial to intraocular injection.

(2) The above microsphere suspension was pipetted in 50. Mu.L (containing about 50. Mu.g of microspheres), dropped on a tin foil, and left to dry at room temperature. And (3) pasting the tin foil paper containing the sample on a sample preparation table by using a conductive adhesive, and observing the surface appearance of the microsphere by using an SEM (scanning Electron microscope) after gold spraying.

As shown in FIG. 4, the surface pore size is 3-4 μm, the microspheres have a structure with open surface, and the average particle size of the microspheres is 25 μm. Meanwhile, in order to observe the internal structure of the microspheres, the dried microspheres are cut up by an ultrathin blade. The sample was then adhered to a conductive gel and gold-sprayed and observed by SEM. As shown in FIG. 5, the microspheres also have a structure with internal pores penetrating through each other, and the internal pore diameter is about 4-5 μm.

(3) Sucking 1mL of the suspension of the open microspheres with the dry weight of 30mg prepared in the step (1) into a centrifugal tube with the volume of 1.5mL, centrifuging to remove the supernatant, adding 500 mu L of 20mg/mL of the solution of the exosomes to blend with the microspheres, placing the mixture on a vertical suspension instrument to suspend for 4 hours (300 rpm), and enabling the exosomes to fully enter the cavities of the microspheres by utilizing pores penetrated by the microspheres. Respectively incubating for 2h-6h in an incubator at 39-42 ℃, and exploring the healing mode with the lowest temperature and the shortest time. The microsphere can be sealed without affecting the shape of the microsphere and the function of exosome. And then placing the vertical suspension instrument and the microspheres in a 39 ℃ thermostat, heating and sealing, wherein the suspension speed is 100rpm in the process, ensuring that the microspheres are uniformly heated in the whole heating process, simultaneously, not settling, completing sealing after reacting for 4 hours, centrifuging (500g, 5 min) and removing supernatant to obtain the sealed microcapsules loaded with exosomes. The loading of the microcapsules on exosomes was observed using confocal microscopy, and the results are shown in fig. 6. As can be seen from fig. 6, in the confocal laser confocal image, sky blue represents the microsphere skeleton, and yellow represents the exosome. In 2D scanning mode, sky blue microsphere skeleton is seen, which contains many circular cavities, while yellow exosomes fill the cavities of the microspheres. The loading rate is shown in fig. 7.

(4) Sucking 50 μ L of the sealed microcapsule suspension containing exosome, dropping on tin foil paper, and airing at room temperature. And (3) pasting the tin foil paper containing the sample on a sample preparation table by using a conductive adhesive, and observing the surface appearance of the microcapsule by using SEM after gold spraying. As shown in fig. 8, the surface porosity was completely closed to form sealed microcapsules. Meanwhile, in order to observe the internal structure of the sealed microcapsule, the dried microcapsule is cut into pieces by an ultrathin blade. The sample was then adhered to a conductive gel and gold-sprayed and observed by SEM. As shown in fig. 9, the inside of the microcapsule still has an internal porous structure and a pore diameter of about 4 to 5 μm, but the internal porous structure is a closed and independent porous structure.

Example 3 retention and distribution of microspheres in the eyes of mice

(1) C57BL/6 mice were anesthetized by intraperitoneal injection with 0.5% pentobarbital (0.1 mL/10g · bw), and mydriasis was dilated with 1% compound topicamide eye drops. Subsequently, the administration was by the intravitreal injection route and was done only once all the time, and MExo, MSCs and MExoCap were injected into the vitreous cavity using Hamilton's microinjector (single intravitreal injection amount 5 μ g MExo/eye, 4X 10 ⁶ One MSCs per eye, 5 μ g MExo loaded in 50 μ g microspheres), 6 eyes of 6 mice were used per group.

(2) After the intravitreal injection, the

number

0, 3, 7, 14, 21, 28, and 35 gastrodia elata drunken mice were imaged by using a small animal imaging system, and as a result, as shown in fig. 10, the retention time in the eye was significantly prolonged after the exosomes were encapsulated in the microspheres. On the third day after intravitreal injection, fundus images were taken using a fundus camera after conventional anesthesia for mydriasis. The results are shown in FIG. 11. Then, the mouse is sacrificed by cervical dislocation, and the optic nerve is clamped by forceps to lift the eyeball and separate the optic nerve, thereby completely taking out the eyeball. The eyeballs were immediately placed in a petri dish and the distribution of MExo, MSCs and MExoCap within the eye was observed in a small animal imaging system. As shown in fig. 12, the small amount of residual MExo was evenly distributed in the anterior retinal area and the MSCs were distributed on the posterior surface of the lens, possibly due to the adherent nature of MSCs. Whereas MExoCap is distributed mainly in the lower part of the vitreous chamber, MExo released is distributed in the anterior zone of the retina. The results prove that: MExoCap settles in the lower part of the vitreous cavity after injection into the vitreous cavity and does not affect the optical path.

Example 4 in vivo efficacy and safety validation of RIRI model mice

(1) A Retinal Ischemia Reperfusion Injury (RIRI) mouse model was constructed. C57BL/6 mice were anesthetized by intraperitoneal injection with 0.5% pentobarbital (0.1 mL/10g · bw), and mydriasis was dilated with 1% compound topicamide eye drops. An infusion bottle filled with 100mL of sterile physiological saline is connected with a disposable sterile insulin syringe through an infusion apparatus, and air in the infusion apparatus is exhausted. After mydriasis, the needle is held by hand and inserted along the temporal limbus parallel to the longitudinal axis of the mouse body. After the needle head is fixed by the adhesive plaster, the water gate of the infusion apparatus is opened, the infusion bottle is slowly lifted, the final height from the mouse is 150cm, and the intraocular pressure formed at the height is 110mmHg. After the infusion bottle is lifted, the iris and the eyeground of the mouse can be observed to become pale, which indicates the formation of retinal ischemia. After 1h of ischemia, the height of the infusion bottle is reduced, the needle head is pulled out, and then the blood flow of the iris and the eyeground is recovered, namely the retinal reperfusion is formed.

(2) After 1 day of molding, the drug was administered by intravitreal injection and only once. After anesthetizing RIRI model mice with intraperitoneal injection of 0.5% pentobarbital (0.1 mL/10g · bw), the mice were separately injected with Hamilton's microinjectorPBS, 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 ⁶ One MSCs per eye, 5 μ g MExo/eye loaded in 50 μ g microspheres, 3 μ g NGF/eye), 6 eyes of 6 mice were used per administration group.

(3) 28 days after administration, RIRI model mice were anesthetized with 0.5% pentobarbital (0.1 mL/10g · bw) by intraperitoneal injection, and 1% compound topicamide eye drops were mydriasis, examined by OCT and ERG. See fig. 13, 14. The results show that: the reduction of the thickness of the whole retina layer and the ganglion cell layer in the MExoCap treatment group is the smallest, and the reduction of the amplitude of A wave and B wave of the electroretinogram is the smallest. After the mouse is killed after the cervical vertebra dislocation, the eyeball is removed, and after the eyeball is embedded and fixed, a paraffin section with the thickness of 3-4 mu m/slice is made, and the result shows that: the nucleus layer number of the inner nucleus layer and the outer nucleus layer of the MExoCap treatment group is reduced least, and the effect is better than that of the nerve growth factor which is commonly used in clinic.

(4) After 28 days of administration, RIRI model mice were anesthetized and their intraocular pressure was measured using a small animal tonometer, see FIG. 16. The results show that: MExoCap treatment did not have an effect on intraocular pressure. Venous blood from mice was taken to test the blood routine of RIRI model mice, and the results are shown in FIG. 17, showing no significant change in blood routine. RIRI model mice were then sacrificed and their internal organs (heart, liver, spleen, lung, kidney) were fixed and paraffin embedded and 3-4 μm/piece sections were made, stained with hematoxylin and eosin (H & E) and observed using an optical microscope, see FIG. 15. The results show that: no obvious abnormalities were seen in the gut after MExoCap treatment.

Example 5 extraction of Treg cell exosomes

After obtaining cell suspension from mouse spleen lapping liquid, use Meitian and whirlpool CD4 ⁺ CD25 ⁺ And (3) obtaining the Treg cells by using the regulatory T cell separation kit according to the operation of the instruction. Treg cells were cultured using X-Vivo complete medium (containing 10% of exosome-depleted serum and 1% of double antibody) formulated with exosome-depleted serum, 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; the mixture was centrifuged at 100,000g for 120min, the supernatant was discarded, and the pellet was collected and then resuspended in PBS to obtain TrExo solution. By usingTEM observes the appearance, NTA measures the particle size and potential distribution, and Western blotting determines the protein expression of its exosome characteristics.

The results are shown in FIGS. 18-20. As can be seen from the figure, trExo is in a cup shape, the particle size is about 120nm, and the TrExo marker proteins ALIX, TSG101, CD63, CTLA-4 and IL-10 are positive.

Example 6 preparation of sealed microcapsules loaded with Treg cell exosomes (referred to as TrExoCap).

Open microspheres were prepared as in step (1) of example 2.

Sucking 1mL of open microsphere suspension with the dry weight of 30mg into a centrifugal tube with the dry weight of 1.5mL, centrifuging to remove the supernatant, adding 500 mu L of 20mg/mL exosome solution to blend with the microspheres, placing on a vertical suspension instrument to suspend for 4h (300 rpm), and enabling exosome to fully enter the cavity of the microspheres by utilizing the pore canals penetrated by the microspheres. Respectively incubating for 2h-6h in an incubator at 39-42 ℃, and exploring the healing mode with the lowest temperature and the shortest time. The microsphere can be sealed without affecting the shape of the microsphere and the function of exosome. And then placing the vertical suspension instrument and the microspheres in a 39 ℃ thermostat, heating and sealing, wherein the suspension speed is 100rpm in the process, ensuring that the microspheres are uniformly heated in the whole heating process, simultaneously, not settling, completing sealing after reacting for 4 hours, centrifuging (500g, 5 min) and removing supernatant to obtain the sealed microcapsules loaded with exosomes. The loading of the exosomes by the microcapsules was observed by confocal microscopy, and the results are shown in fig. 21. As can be seen from fig. 21, in the confocal laser confocal image, sky blue represents the microsphere skeleton, and pink represents exosome. In 2D scan mode, a sky blue microsphere skeleton can be seen, containing many circular cavities, while pink exosomes fill the cavities of the microspheres.

Example 7: PMU model mouse in vivo effect and safety verification

(1) A PMU mouse model was constructed. C57BL/6 mice were anesthetized by intraperitoneal injection with 0.5% pentobarbital (0.1 mL/10g · bw), and mydriasis was dilated with 1% compound topicamide eye drops. On day-9, mice were injected subcutaneously with 100 μ g of inactivated Mycobacterium tuberculosis H37Ra antigen dissolved in 0.1mL of incomplete Freund's adjuvant. After 7 days, after general anesthetization of the mice, the right eye was instilled with surface anesthetic (oxybuprocaine hydrochloride eye drops) and mydriatic agent (compound tropicamide eye drops), followed by intravitreal injection of 3 μ g of mycobacterium tuberculosis antigen H37Ra dissolved in 1 μ L of PBS buffer per mouse right eye. The administration was performed 2 days later.

(2) After molding, the drug was administered by intravitreal injection and only once. After intraperitoneal injection of 0.5% pentobarbital (0.1 mL/10 g. Bw) to anesthetized PMU model mice, PBS, trExo, treg cells, trExoCap and TA (triamcinolone acetonide) were injected into the vitreous cavity with Hamilton's microsyringe (single intravitreal injection of 2 μ L PBS/eye, 5 μ g TrExo/eye, 4X 10 ⁶ Treg cells/eye, 5 μ g TrExo/eye loaded in 50 μ g microspheres, 80 μ g TA/eye), 6 eyes of 6 mice were used per administration group.

(3) 28 days after administration, PMU model mice were anesthetized with 0.5% pentobarbital (0.1 mL/10 g. Bw) by intraperitoneal injection, the 1% compound tropicamide eye drops were used for dilation of the pupil, and the eye fluid was collected from the anterior chamber of the mice, as shown in FIG. 22, and the results showed that the content of inflammatory cytokines (IL-1. Beta., IL-6, IL-8 and TNF) in the eye fluid of the mice in the TrExoCap group was the lowest. After the mice are killed by dislocation of cervical vertebrae, the eyeballs are removed, and 3-4 μm/paraffin section is made after embedding and fixing, as shown in FIG. 23, the results show: the mice in the TrExoCap treatment group have the lowest infiltration degree of vitreous body and retina inflammatory cells, and the effect is better than that of the clinically common hormone drug triamcinolone acetonide.

(4) After administration for 28 days, the PMU model mice were anesthetized and their intraocular pressure was measured using a small animal tonometer, see fig. 24. The results show that: trExoCap treatment did not have an effect on intraocular pressure. Venous blood of the mice is taken for routine blood detection of the PMU model mice, and the result is shown in FIG. 25, and the routine blood is not obviously changed. PMU model mice were then sacrificed and their internal organs (heart, liver, spleen, lung, kidney) were fixed, paraffin-embedded and 3-4 μm/piece sections were made, stained with Hematoxylin and Eosin (HE), and observed using an optical microscope, see FIG. 26. The results show that: no obvious abnormalities were seen in the gut after TrExoCap treatment.

Claims

1. An intraocular injection based on microcapsule is a sealed microcapsule prepared from exosome and macroporous microsphere; the exosome is loaded inside the macroporous microsphere, the macroporous microsphere is prepared from a hydrophobic polymer and an amphiphilic block copolymer, the surface of the macroporous microsphere is provided with openings, and the inside of the macroporous microsphere is provided with through-holes; the average particle diameter of the microcapsule is 10-100 μm.

2. The microcapsule-based intraocular injection according to claim 1, characterized in that: the exosome is an exosome derived from mesenchymal stem cells or an exosome derived from regulatory T cells;

preferably, the mesenchymal stem cell is derived from a bone marrow mesenchymal stem cell.

3. The microcapsule-based intraocular injection according to claim 1 or 2, characterized in that: the average particle diameter of the microcapsule is 10-30 μm.

4. The microcapsule-based intraocular injection according to any one of claims 1 to 3, characterized in that: the macroporous microspheres are made of PLGA and PELA; wherein the mass ratio of PLGA to PELA is 9:1.

5. The method for preparing the microcapsule-based intraocular injection according to any one of claims 1 to 4, comprising the steps of: preparing macroporous microspheres from a blend of a hydrophobic polymer and an amphiphilic block copolymer; the macroporous microspheres are then mixed with an exosome-containing solution, followed by sealing the macroporous microspheres loaded with exosome solution, resulting in sealed exosome-loaded microcapsules, i.e. the microcapsule-based intraocular injections.

6. The method of claim 5, wherein: the macroporous microspheres are made of PLGA and PELA; the macroporous microspheres are prepared by adopting a double-emulsion and solvent extraction method.

7. The production method according to claim 5 or 6, characterized in that: the condition of sealing the macroporous microspheres is incubation for 2-6h at 39-42 ℃, and the optimal condition is incubation for 4h at 39 ℃.

8. The production method according to any one of claims 5 to 7, characterized in that: the preparation method of the microcapsule-based intraocular injection specifically comprises the following steps:

9. Use of the microcapsule-based intraocular injection according to any one of claims 1 to 4 for the preparation of a product having a therapeutic effect on retinal ischemia-reperfusion injury.

10. A product having a therapeutic effect on retinal ischemia reperfusion injury comprising the microcapsule-based intraocular injection according to any one of claims 1 to 4.