CN108743620B - Bioactive material for promoting stem cell-derived exosome to treat corneal injury - Google Patents

Abstract

The invention relates to a biomaterial hydrogel eye drop containing exosomes secreted by stem cells, which has good cell compatibility and bioactivity. The active hydrogel can be used as eye drops to slowly release exosomes to damaged tissues; the hydrogel can enhance the retention rate and local concentration of exosome at an injured part, promote the injury repair of eye tissues, promote the survival and proliferation of corneal cells, reduce apoptosis, promote the functional recovery of an injured area, inhibit pathological angiogenesis and inhibit the inflammatory reaction of eyes. Meanwhile, after being combined with the bioactive material, the medicine can prolong the residence time of exosome, reduce the administration times and facilitate more accurate control of the medicine dosage.

Description

Bioactive material for promoting stem cell-derived exosome to treat corneal injury

Technical Field

The invention belongs to the technical field of tissue engineering and new medicines. In particular to preparation of a bioactive material hydrogel eye drop combined with exosomes derived from stem cells, and also relates to a treatment effect of the material as the eye drop applied to corneal tissue injury repair.

Background

The stem cell can be widely applied to various diseases due to totipotency and multidirectional differentiation potential, and can be divided into adult stem cells and embryonic stem cells, wherein the mesenchymal stem cells are adult stem cells with wide sources, only express low-level HLA/MHC class I molecules, and have low immunogenicity and certain immunosuppressive capacity. It exhibits pluripotency and a phenotype that enables differentiation into many connective tissue cell types such as osteoblasts, chondrocytes and adipocytes in vitro and in vivo. Become an important tool in stem cell therapy. In current research, mesenchymal stem cells have shown potential in the treatment of a variety of diseases, including autoimmune diseases, solid organ transplant survival, liver cirrhosis, kidney diseases, neurological and muscular degenerative diseases, and diseases and injuries caused by myocardial infarction or spinal cord. The placenta-derived mesenchymal stem cell may have a stronger proliferation and differentiation ability as a cell derived from a young donor.

It is considered that the paracrine action of mesenchymal stem cells is an important mechanism that plays a role in the treatment of diseases, in addition to tissue cells that undergo transdifferentiation instead of injury after cell transplantation. Typical mesenchymal stem cell secretion profiles include growth factors, cytokines, extracellular matrix proteases, hormones, lipid mediators, and the like. In the early 80 s of the 20 th century, researchers first described small endothelial vesicles (30 nm-100 nm) secreted by reticulocytes and named using exosomes (exosomes). The exosomes are vesicle-like bodies composed of lipids, proteins, cytoskeletal elements, molecular chaperones and signaling molecules, and are secreted extracellularly by cell motility and are uniform in size. Several studies have shown that communication between mesenchymal stem cells and other cells can be via exosomes (exosomes). It has been shown that exosomes also contain genetic material, including messenger rna (mrna) and microrna (microrna). Various types of cells, including cardiomyocytes, vascular endothelial cells and mesenchymal stem cells, secrete exosomes containing mirnas and cytokines, and miRNA-mediated communication between endothelial cells and cardiovascular cells has also been demonstrated.

Hydrogels, a crosslinked form of a hydrophilic polymer, have been shown to be a biomaterial with great potential in the biological and medical fields. These hydrophilic polymer networks generally have a high affinity for water but do not dissolve in water because they form a crosslinked network through physical or chemical action, while water can penetrate between the polymer networks of the polymer, subsequently causing swelling to form a hydrogel pattern. As an implant, the implant can be simply mixed to load a plurality of therapeutic drugs without presetting the shape of a bracket, and is an ideal drug carrier, an embolism material and a tissue engineering bracket material. In addition, the hydrogel serving as a natural macromolecular compound has certain physical parameters such as mechanical strength, degradability and the like, has biocompatibility and biological characteristics of providing a biological related microenvironment, and has wide application in the medical field. In addition, the temperature response type hydrogel has good injectability, and can avoid damage caused by placing the formed cell scaffold, so the temperature response type hydrogel can be used as the cell scaffold for tissue engineering to transplant cells. The natural hydrogel has a loose and porous reticular structure, can simulate a natural extracellular matrix and provides support for cell adhesion, migration and proliferation; in addition, natural hydrogels can be degraded by lysosomes, have good biodegradability in the organism, and can be completely biodegradable so as to be completely replaced by biological components after tissue repair.

Scientists have confirmed the presence of exosomes in culture media of human-derived mesenchymal stem cells, and injection of these exosomes into ischemia-reperfusion model rats showed excellent myocardial protection. The above results indicate that exosomes secreted by MSCs may have the potential to promote tissue repair. In view of the cleaning effect of the eye, there has been a problem with ophthalmic administration that the drug stays on the corneal surface for a short time. Therefore, the current method of ocular administration is multiple administrations, but repeated drug stimulation may generate inflammatory reaction, and the drug usage amount is increased, and the patient compliance is poor. From the point, the exosome is combined with the bioactive material to prepare the hydrogel eye drops, so that the exosome retention can be promoted, the treatment effect is improved, and a new visual angle is provided for tissue injury repair.

Disclosure of Invention

The invention relates to a technical means for wrapping and delivering exosome by using biomaterial hydrogel and enhancing the treatment effect of the exosome in corneal injury.

The invention uses exosome secreted by stem cells to be combined with hydrogel of bioactive material through physical mixing, so as to release the exosome into surrounding tissues and regulate the release speed and release mode of the exosome.

The hydrogel is bioactive material hydrogel eye drops, and the hydrogel has the physical and chemical properties of the biomaterial and also has the biological activity of exosomes secreted by stem cells. The biological material can be chitosan, but is not limited to chitosan, and also comprises hyaluronic acid, sodium carboxymethyl cellulose and other materials which can be applied to ocular administration.

The exosome is derived from stem cells, and the stem cells comprise adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, placenta mesenchymal stem cells, urine-derived stem cells, endothelial progenitor cells, cardiac stem cells and the like.

The invention can effectively enhance the retention rate of the exosome at the damaged part, simultaneously improve the stability of effective components such as protein, microRNA and the like in the exosome, slowly release the exosome to the damaged part, further improve the treatment effect of the exosome and promote the recovery of the damaged tissue structure and function.

The tissue damage includes corneal damage, skin damage, and the like.

Drawings

Fig. 1 is an identification of exosomes characteristic of secreted by mesenchymal stem cells.

FIG. 2 shows the chemical formula of natural chitosan and the good biocompatibility of the present invention.

FIG. 3 shows that exosomes can be endocytosed by cells to function.

Figure 4 shows that the hydrogel wraps the slow-release exosome to better promote the recovery after corneal injury. H & E staining showed best corneal structure repair in the hydrogel-encapsulated exosome-treated group, with more regular stroma arrangement.

Detailed Description

In the following examples, unless otherwise specified, all methods used are conventional and all reagents used are commercially available.

Example 1, the present invention provides a method for the extraction and identification of exosomes derived from stem cells.

Cell culture media, double antibody, pancreatin and other reagents are purchased from Gibco company; cell culture consumables were purchased from Corning.

Serum required for cell culture is fetal bovine serum without exosomes, purchased from BI company, and processed by the following steps: fetal bovine serum was placed in an ultracentrifuge tube, centrifuged at 120,000 g for 2 h at 4 ℃, the supernatant was taken out on a clean bench, filtered through a 0.22 μm needle filter, and stored in a refrigerator at-80 ℃ for further use.

The procedures of cell biology experiment operations such as cell subculture, cryopreservation and recovery are described in animal cell culture (sixth edition).

Harvesting hP-MSCs conditioned media containing exosomes: when cultured at 75 cm²The hP-MSCs in the cell culture bottle are in logarithmic growth phase, when the cell confluency reaches 80%, the culture medium is completely sucked up, PBS is used for washing twice, 10 ml of prepared complete culture medium containing 10% of FBS without exosome is added into each bottle, after the culture is continued for 24 h, the culture medium is collected into a centrifuge tube, and the culture medium is a conditioned culture medium rich in exosome.

Separating and extracting exosome by an ultracentrifugation method: firstly, centrifuging the condition culture obtained in the step for 10 min at the temperature of 4 ℃ under the condition of 300 g, and removing cell debris; ② centrifuging the obtained supernatant for 20 min at the temperature of 4 ℃ at 12,000 g to remove large cell debris such as apoptotic bodies; thirdly, filtering the obtained supernatant by using a needle type filter with the diameter of 0.22 mu m to remove micro-vesicles with the diameter of more than 200 nm; placing the filtered supernatant into an ultracentrifuge tube, centrifuging for 70 min at the temperature of 4 ℃ by 100,000 g, removing the supernatant, adding a proper amount of PBS (phosphate buffer solution) to resuspend the sediment at the bottom of the tube, and storing at the temperature of-80 ℃.

Identifying the form of the exosome by using a transmission electron microscope, dripping the exosome extracted in the previous step on a 200-mesh sample net, standing at room temperature for 2 min, and sucking redundant liquid by using filter paper; dripping 20 mg/mL uranium acetate solution on the sample net, standing at room temperature for 1 min, carrying out negative dyeing on the sample, sucking excess liquid by using filter paper, and airing the sample net; and (3) observing the prepared sample under a transmission electron microscope, and collecting a photo. As shown in FIG. 1, exosomes are of a cup-vesicle-like structure, with a diameter of about 70-100 nm.

The exosome marker proteins CD9 and CD63 were detected by Western blot (fig. 1).

1) Protein sample preparation: adding RIPA lysis solution into the exosome precipitate obtained after ultracentrifugation to lyse exosomes, repeatedly blowing and beating, transferring into a clean 1.5 mLEP tube, lysing on ice for 30 min, carrying out vortex oscillation once every 10 min, centrifuging at 4 ℃, 12,000 rpm for 15 min, and transferring the supernatant into a new EP tube; measuring the protein concentration of the exosome by using a BCA method, adding 5 Xloading buffer solution into the rest protein solution, boiling for 10 min in boiling water, and storing in a refrigerator at-80 ℃ for later use.

2) Polyacrylamide gel electrophoresis: and (3) mounting the clean and dried glass plate on a glue making frame, enabling the bottom edges to be tightly fitted, and checking leakage by using distilled water. Preparing 10% separating gel solution according to the separating gel formula, mixing well, adding 4.5 ml separating gel solution into the gap of the glass plate by using a liquid transfer device, immediately and gently adding distilled water to flatten the liquid surface of the separating gel, and solidifying the separating gel after about 20 min. Preparing 5% concentrated glue solution according to the formula, pouring 1.5 ml of concentrated glue solution above the separation glue, immediately inserting a comb, and using after the concentrated glue is solidified. Placing the prepared rubber plate into an electrophoresis tank, paying attention to the inward side of the short glass plate, adding electrophoresis liquid between the two glass plates, pulling out a comb, unifying the sample loading amount according to the measured protein concentration, adding a protein sample into a sample loading hole, adding the electrophoresis liquid to the mark position of the electrophoresis tank, covering an electrophoresis tank cover, paying attention to connection of a positive electrode and a negative electrode, starting electrophoresis at 90V, adjusting the voltage to 120V when bromophenol blue runs to separation gel, and stopping electrophoresis until the bromophenol blue is close to the bottom of the glass plates.

3) Film transfer: soaking a membrane transferring clamp, sponge and filter paper in a precooled membrane transferring buffer solution, placing polyacrylamide gel on one side of a black clamp plate, shearing a PVDF membrane with a proper size, placing the PVDF membrane in methanol for activating for 60 s, placing the PVDF membrane on the gel, removing bubbles, covering the filter paper and the sponge, sequentially clamping the membrane transferring clamp according to the sequence of a negative electrode (black) of the membrane transferring clamp, the sponge-filter paper-the gel-the membrane-the filter paper-the sponge-a positive electrode (white), placing the membrane transferring clamp in a membrane transferring groove, adding a membrane transferring solution, and transferring the membrane for 2 h at a constant pressure of ice bath of 120V.

4) And (3) sealing: the PVDF membrane after the membrane transfer is taken out, the gel residue is washed out by TBST solution, and the gel residue is placed in 5 percent of skimmed milk (sealing solution) and sealed for 1 hour at room temperature by a horizontal shaking table at 100 rpm.

5) Antibody hybridization: first-antibody incubation: diluting the primary antibody with a blocking solution according to the instruction (Gapdh, 1:5000 dilution, CD9, 1:1000 dilution, CD63, 1:1000 dilution), sucking 2 ml of the primary antibody and placing the primary antibody in an antibody incubation box, cutting a target strip by a control protein Marker, soaking the cut strip in the corresponding primary antibody, and incubating the cut strip at 4 ℃ overnight; and secondly, secondary antibody incubation, namely washing the strips with TBST solution for 3 times, each time for 5 min, then adding corresponding secondary antibody, incubating for 2 h at room temperature by a horizontal shaking table at 100 rpm, and washing for 3 times, each time for 5 min, with TBST after incubation is finished.

6) Luminescence detection: mixing the luminous liquid A/B in the ratio of 1 to prepare working liquid, dripping the luminous liquid on a film in a dark room, and exposing, developing and fixing by using a film when a target strip emits green fluorescence.

Example 2, the present invention provides a method for preparing a chitosan hydrogel.

The preparation method of the temperature response chitosan hydrogel comprises the following steps: chitosan (Mn = 50,000) powder was dissolved in 0.1M acetic acid, filter-sterilized at 0.22 μ M filter, and prepared as a 2% chitosan solution in ice bath.

Example 3, the present invention provides a method for delivery of exosomes using hydrogel encapsulation.

Dissolving 0.2% hydrogel solution and exosome derived from stem cells in equal volume, preparing into 1 × solution containing exosome, keeping the solution in 4 deg.C environment, and pre-cooling with injector at 4 deg.C when using.

Embodiment 4, the present invention provides a method for treating corneal alkali injury by using exosome-conjugated biomaterial chitosan hydrogel eye drops secreted by mesenchymal stem cells.

Constructing a mouse corneal alkali damage model: a piece of filter paper (11 μm) was cut into a plurality of 2mm circles for use. A1M NaOH solution was prepared by dissolving 2g of NaOH in 50ml of distilled water. Stored at room temperature. 4% chloral hydrate is injected into the abdominal cavity for anesthesia, and the anesthesia takes 1-2 minutes approximately. The depth of anesthesia can be determined by gently pinching the mouse's toes or tail and should not respond if the amount of anesthesia is sufficient. Note that: care should be taken to ensure that mice do not experience hypothermia due to anesthesia. Mice can be transferred to a heating pad post-operatively. Surgical instruments were cleaned in advance and autoclaved, and a piece of filter paper soaked with NaOH was picked up using sterile forceps. Note that if excess NaOH is observed to stick to or drip from the filter paper, the excess NaOH should be removed and the filter paper is tapped gently onto the dried filter paper to absorb the excess NaOH. Animals were randomized into 4 groups: PBS group, Chitosan (CS) group, exosome (Exo) group, CS encapsulated exosome (CS-Exo) group. A piece of NaOH soaked filter paper was placed over the central cornea. Standing for 30 seconds produces an acute alkali burn of about 2 x 2mm square. The filter paper was removed. Immediately thereafter, the eye was gently rinsed twice with 10 ml PBS using a 10 ml syringe, and the residual NaOH was washed away.

And observing the molding condition one day after the operation, observing the eyes of the mouse on the first day after the operation after the construction of the corneal injury model is completed, and observing a white turbid area with the size of 2mm, wherein the eyelids are not damaged and adhered, and the eyes of the mouse can be opened normally, so that the molding is successful.

15 microliter of exosome hydrogel eye drops are added three times a day, and the administration concentration of exosome is 100 micrograms per day. The application is repeated for 3 times/day for 14 days.

Mice were examined daily under an operating microscope and were based on corneal haze, NV and blood vessel size. Representative eye images were taken using a digital camera for 5 days. Clinical evaluation was performed directly or mice were sacrificed and eyes were used for corneal conventional paraffin/cryohistological evaluation.

Example 5, the present invention provides a method for testing the effect of a hydrogel on the therapeutic function of a stem cell-derived exosome.

The effect of the treatment was observed by stereomicroscope: for 14 consecutive days. Mice were examined daily under an operating microscope and were based on corneal haze, NV and blood vessel size. Using a digital camera, a representative eye image is taken.

Corneal fluorescein staining: on day seven, fluorescein sodium staining of the cornea was performed. After the mice were anesthetized with 4% chloral hydrate in the abdominal cavity, the sodium fluorescein filter paper was wetted with sterilized PBS, lightly applied to the mouse cornea, and then rinsed with PBS to remove excess fluorescein. The damage and staining condition of the cornea is observed under ultraviolet light, and a digital camera is used for photographing.

Immunostaining of frozen sections of corneal tissue: taking out the slices from a refrigerator at the temperature of-20 ℃, and standing for 30 minutes at room temperature; placing the slices into acetone stock solution (precooled for 30 minutes at the temperature of minus 20 ℃) for fixing for 10 minutes; the slices were air dried for 30 minutes; sections were washed with PBS (5 min each, 2 times); membrane breaking: 0.1% Triton X-100, room temperature, 10 minutes; PBS wash, 5 min, 2 times; sealing serum, adding primary antibody at room temperature for one hour, and rewarming at 4 deg.C overnight in the morning for 1 hour; PBS wash, 5 min, 4 times; adding a secondary antibody (in the dark), and keeping the temperature at room temperature for 2 hours; PBS wash for 5 min each, 3 times; DAPI mounting, observing under a fluorescence microscope, and taking a picture.

Example 6, the present invention provides a method of constructing a model of skin injury under mice.

Constructing a mouse skin injury model: weighing the weight of the mouse, and injecting the mouse into the abdominal cavity for anesthesia according to the dosage of 330 mg/kg (10 mu L/g) of 4 percent chloral hydrate; cutting off the whole skin and the flesh membrane on the back of the mouse, and exposing the muscle layer to cause a circular skin defect with the diameter of 1 cm; covering the wound surface with IV3000 sterile impervious wound dressing, adhering the edge of the adhesive film to the back skin of the nude mouse with wound adhesive DIAN, and replacing the wound surface dressing every 2 days from day 3; post-operative animals were randomized into 4 groups: PBS group, Chitosan (CS) group, exosome (Exo) group, CS-encapsulated exosome (CS-Exo) group, and PBS, CS, Exo and CS-Exo were injected at the skin injury site, respectively.

Claims (4)

1. The eye drops are characterized by being prepared from biomaterial hydrogel, wherein the biomaterial hydrogel is prepared by wrapping human placenta-derived mesenchymal stem cell exosome with temperature-response chitosan hydrogel.

2. The method for preparing an ophthalmic solution according to claim 1, wherein the preparation method comprises combining human placenta-derived mesenchymal stem cell exosomes with temperature-responsive chitosan hydrogel by physical mixing.

3. An ophthalmic solution according to claim 1, wherein the ophthalmic solution can carry human placenta-derived mesenchymal stem cell exosomes at different concentrations.

4. Use of the ophthalmic solution of claim 1 for the preparation of a medicament for the treatment of corneal injury.

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