阅读说明：本技术 一种用于修复损伤组织的细胞外囊泡生物光敏凝胶及其制备方法和应用 (Extracellular vesicle bio-photosensitive gel for repairing damaged tissues and preparation method and application thereof ) 是由 唐俊楠 张金盈 沈德良 崔小林 张增磊 徐彦彦 郭嘉城 刘刚琼 张力 路永政 秦 于 2021-07-14 设计创作，主要内容包括：一种用于修复损伤组织的细胞外囊泡生物光敏凝胶及其制备方法和应用。所述细胞外囊泡生物光敏凝胶包括细胞外囊泡、甲基丙烯酸酐化明胶和光引发剂。所述细胞外囊泡生物光敏凝胶的制备方法包括甲基丙烯酸酐化明胶的制备、细胞外囊泡的制备以及细胞外囊泡生物光敏凝胶的制备。所述细胞外囊泡生物光敏凝胶是通过喷洒和原位交联的方式来释放细胞外囊泡,以提高细胞外囊泡在损伤组织中的滞留率。将细胞外囊泡生物光敏凝胶喷洒到损伤组织表面,再用可见光照射30秒,可以实现作用效果增强的局部靶向治疗,这种策略将开启细胞外囊泡生物光敏凝胶在组织修复治疗的潜力。(An extracellular vesicle bio-photosensitive gel for repairing damaged tissues and a preparation method and application thereof. The extracellular vesicle biological photosensitive gel comprises extracellular vesicles, methacrylic acid anhydridized gelatin and a photoinitiator. The preparation method of the extracellular vesicle biological photosensitive gel comprises the steps of preparing methacrylic anhydridized gelatin, preparing extracellular vesicles and preparing the extracellular vesicle biological photosensitive gel. The extracellular vesicle bio-photosensitive gel releases extracellular vesicles in a spraying and in-situ crosslinking mode so as to improve the retention rate of the extracellular vesicles in damaged tissues. The extracellular vesicle biological photosensitive gel is sprayed on the surface of the damaged tissue and then irradiated by visible light for 30 seconds, so that local targeted therapy with enhanced effect can be realized, and the strategy can open the potential of the extracellular vesicle biological photosensitive gel in tissue repair therapy.)

1. An extracellular vesicle bio-photoactive gel for repairing damaged tissue, comprising extracellular vesicles, methacrylic anhydrified gelatin, and a photoinitiator.

2. The extracellular vesicle bio-photosensitive gel for repairing damaged tissues according to claim 1, wherein the extracellular vesicles are extracted from rodent mesenchymal stromal cells.

3. The extracellular vesicular biosensitive gel for repairing damaged tissue according to claim 1, wherein the photoinitiator comprises 0.5mM ruthenium and 5mM sodium persulfate.

4. A method of preparing an extracellular vesicular bio-photosensitive gel for repairing damaged tissue according to any one of claims 1 to 3, the method comprising:

step one, preparation of methacrylic anhydrified gelatin

Dissolving gelatin into phosphate buffer solution with pH of 7.3 according to volume ratio, stirring thoroughly, and heating to 50 deg.C; after complete dissolution, adding methacrylic anhydride, stirring, and reacting for 1 hour at 50 ℃; after the reaction is finished, dialyzing and purifying by using MilliQ water for 5 days, replacing once a day, and finally obtaining a solution which is purified methacrylic acid anhydridized gelatin, filtering and sterilizing the purified methacrylic acid anhydridized gelatin by using a 0.22 mu m filter, freeze-drying and purifying to obtain methacrylic acid anhydridized gelatin freeze-dried powder;

step two, preparation of extracellular vesicles

1) Culturing the bone marrow mesenchymal stromal cells of the mouse in a basal medium containing 10 percent fetal calf serum, flushing the cells for 3 times by using phosphate buffer solution with the pH value of 7.3 when the cell coverage rate reaches 80 percent, replacing a culture medium without fetal calf serum, and after continuously culturing the cells in the culture medium for 24 hours, replacing a new fresh culture medium without fetal calf serum to continuously culture the cells for 48 hours;

2) collecting culture medium supernatant, centrifuging for 15min with 5810R centrifuge at 3000g and 4 deg.C, placing the supernatant in a sterile container, adding 0.5 volume of ExoQuick-TC, mixing, and refrigerating at 4 deg.C for 12 hr to obtain ExoQuick-TC/supernatant mixture; centrifuging the ExoQuick-TC/supernatant mixture at 4 ℃ for 30min at 1500g, enriching to obtain extracellular vesicles, and re-suspending the extracellular vesicles with a phosphate buffer solution with the pH of 7.3 to obtain a phosphate buffer solution containing the extracellular vesicles;

step three, preparation of extracellular vesicle bio-photosensitive gel

Dissolving 100mg of purified methacrylic anhydrized gelatin freeze-dried powder in 1000 mu L of ultrapure water, filtering by using a 0.22 mu m filter, uniformly mixing the filtered methacrylic anhydrized gelatin with ruthenium, sodium persulfate and phosphate buffer solution containing extracellular vesicles according to the volume ratio of 50:1:1:48 to obtain extracellular vesicle biological photosensitive gel precursor, and adding the extracellular vesicle biological photosensitive gel precursor into the mixture at the concentration of 30mW cm^-2The visible light is excited to form the extracellular vesicle biological photosensitive gel.

5. The method for preparing an extracellular vesicle bio-photosensitive gel for repairing damaged tissues according to claim 4, wherein in the first step, the volume ratio of gelatin to phosphate buffer is 1: 10.

6. the method for preparing an extracellular vesicle bio-photosensitive gel for repairing damaged tissues according to claim 4, wherein in the first step, the amount of methacrylic anhydride added is 0.6g per 1g of gelatin.

7. The method for preparing extracellular vesicle bio-photosensitive gel for repairing damaged tissue according to claim 4, wherein the culture conditions of the culture are 5% CO₂And an incubator at 37 ℃.

8. The method according to claim 4, wherein in the second step, the total concentration of the protein in the enriched extracellular vesicles is 18 mg/mL.

9. Use of an extracellular vesicular bio-photosensitive gel according to any one of claims 1 to 3 for repairing damaged tissue for the manufacture of a medicament for repairing damaged tissue.

10. Use of an extracellular vesicular bio-photosensitive gel for repairing damaged tissue according to claim 9, wherein the extracellular vesicular bio-photosensitive gel is used in a method of 30mW cm^-2The precursor solution is sprayed on the surface of the tissue under the excitation of the visible light, and the extracellular vesicle biological photosensitive gel paved on the surface of the tissue organ can be formed after crosslinking for 30 seconds, thereby playing a role.

Technical Field

The invention relates to the technical field of biomedicine, in particular to an extracellular vesicle biological photosensitive gel for repairing damaged tissues and a preparation method and application thereof.

Background

Cardiovascular disease is a leading cause of death in the world. Myocardial infarction (MI, also known as heart attack) is one of the most common types of cardiovascular disease, which causes damage to the heart muscle, leading to Heart Failure (HF) and affecting the life span of the patient. Stem cell therapy has its inherent limitations, such as tumor risk, immune intolerance, low retention and transplant rates, and targeted drug delivery, and does not achieve satisfactory clinical results. Scientists have found that one of the major mechanisms by which stem cells repair the heart is the paracrine effect. The extracellular vesicles, as derivatives of stem cells, contain biologically active substances such as RNA and protein, and play important roles in inhibiting myocardial apoptosis after infarction, promoting cell proliferation, promoting angiogenesis and the like. In fact, extracellular vesicles from different cell sources have been reported to have a heart-protecting effect and show good therapeutic effects in a large number of in vitro studies and animal experiments. The use of extracellular vesicles in cardiac repair is becoming increasingly popular, but due to the drawbacks of delivery technology, extracellular vesicles do not perform their full potential. Currently, the problem of local low retention of the myocardium is the biggest problem faced in extracellular vesicle applications. Although hydrogels or other biomaterials are used as carriers to achieve local administration while increasing retention rates, the method of administration typically involves intramyocardial multiple injections. Myocardial injection is difficult to operate and easy to cause myocardial damage after the operation of opening the chest inevitably. Alternative delivery methods such as intravenous injection require surface modification of extracellular vesicles, increasing production costs, and the targeting efficiency of intravenous injection is not clear. As extracellular vesicles circulate throughout the body, they may accumulate in non-target tissues, resulting in an insufficient number of extracellular vesicles remaining in the infarcted tissue.

Disclosure of Invention

Therefore, the invention provides an extracellular vesicle bio-photosensitive gel for repairing damaged tissues and a preparation method and application thereof, and the retention rate of extracellular vesicles in damaged heart tissues is improved in a non-injection mode so as to solve the problem that the existing extracellular vesicles cannot fully play the role in heart repair.

In order to achieve the above purpose, the invention provides the following technical scheme:

according to a first aspect of the present invention there is provided an extracellular vesicular biosensitive gel for repairing damaged tissue, the extracellular vesicular biosensitive gel comprising extracellular vesicles, an anhydrified gelatin, and a photoinitiator.

Further, the extracellular vesicles are extracted from rodent mesenchymal stromal cells.

Further, the photoinitiator comprises 0.5mM of ruthenium and 5mM of sodium persulfate.

According to a second aspect of the present invention, there is provided a method for preparing the above extracellular vesicle bio-photosensitive gel for repairing damaged tissues, the method comprising:

step one, preparation of methacrylic anhydrified gelatin

Dissolving gelatin into phosphate buffer solution with pH of 7.3 according to volume ratio, stirring thoroughly, and heating to 50 deg.C; after complete dissolution, adding methacrylic anhydride, stirring, and reacting for 1 hour at 50 ℃; after the reaction is finished, dialyzing and purifying by using MilliQ water for 5 days, replacing the MilliQ water once a day to obtain a final solution which is purified methacrylic anhydridized gelatin, filtering and sterilizing the purified methacrylic anhydridized gelatin by using a 0.22 mu m filter, and freeze-drying to obtain purified methacrylic anhydridized gelatin freeze-dried powder;

step two, preparation of extracellular vesicles

1) Culturing the bone marrow mesenchymal stromal cells of the mouse in a basal medium containing 10 percent fetal calf serum, flushing the cells for 3 times by using phosphate buffer solution with the pH value of 7.3 when the cell coverage rate reaches 80 percent, replacing a culture medium without fetal calf serum, and after continuously culturing the cells in an incubator for 24 hours, replacing a new fresh culture medium without fetal calf serum to continuously culture the cells for 48 hours;

2) collecting culture medium supernatant, centrifuging for 15min at 3000g and 4 ℃ by a 5810R centrifuge, taking the centrifuged supernatant in a sterile container, adding ExoQuick-TC in a ratio of 5:1, mixing uniformly, refrigerating for 12h at 4 ℃ to obtain an ExoQuick-TC/supernatant mixture; centrifuging the ExoQuick-TC/supernatant mixture at 4 ℃ for 30min at 1500g, enriching to obtain extracellular vesicles, and re-suspending the extracellular vesicles with a phosphate buffer solution with the pH of 7.3 to obtain a phosphate buffer solution containing the extracellular vesicles;

step three, preparation of extracellular vesicle bio-photosensitive gel

Dissolving 100mg purified methacrylic anhydrified gelatin in 1000 μ L ultrapure water, filtering with 0.22 μm filter, mixing the filtered methacrylic anhydrified gelatin with ruthenium, sodium persulfate and phosphate buffer solution containing extracellular vesicles at a volume ratio of 50:1:1:48 to obtain extracellular vesicle bio-photosensitive gel precursor, adding into the mixture at 30mW cm^-2The visible light is excited to form extracellular vesicle biological photosensitive gel.

Further, in the first step, the volume ratio refers to a volume ratio of gelatin to phosphate buffer solution of 1: 10.

further, in the first step, the amount of the added methacrylic anhydride is 0.6g of methacrylic anhydride per 1g of gelatin.

Further, the culture conditions of the culture are all 5% CO₂And an incubator at 37 ℃.

Further, in the second step, the total concentration of the protein in the extracellular vesicles obtained by the enrichment is 18 mg/mL.

According to the third aspect of the invention, the extracellular vesicle biological photosensitive gel for repairing damaged tissues can be used for preparing medicines for repairing damaged tissues.

Further, the application method of the extracellular vesicle biological photosensitive gel is 30mW cm^-2The precursor solution is sprayed on the surface of the tissue under the excitation of the visible light, and the extracellular vesicle biological photosensitive gel paved on the surface of the tissue organ can be formed after crosslinking for 30 seconds, thereby playing a role.

The invention has the following advantages:

1) the invention releases the extracellular vesicles in a spraying and in-situ crosslinking mode, so as to improve the retention rate of the extracellular vesicles in damaged tissues and avoid the damage caused by intramuscular injection. 2) According to the invention, after the visible light is irradiated for 30 seconds, the extracellular vesicle gel is sprayed on the surface of the myocardial infarction area, the extracellular vesicle can be physically captured in a methacrylic anhydridized gelatin network, the local targeted drug delivery with controllable concentration and enhanced action time is realized, and the strategy can open the potential of the extracellular vesicle biological photosensitive gel in tissue repair treatment.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

FIG. 1 is a characteristic analysis of extracellular vesicles, wherein A is a schematic shape under a transmission electron microscope of the extracellular vesicles delivered without injection provided by the invention; b is a morphological schematic diagram under an atomic force microscope of the extracellular vesicles; c is a diameter distribution detection schematic diagram of the extracellular vesicles; wherein the scale bar is 100 nm.

FIG. 2 is a scanning electron microscope image of the extracellular vesicle bio-photosensitive gel provided by the invention; wherein the scale bar is 20 μm.

FIG. 3 shows hydrogen nuclear magnetic resonance spectroscopy analysis of GelMA photosensitive gel provided by the present invention.

Fig. 4 is a schematic diagram of the cross-linking test of GelMA photosensitive gel on the 3D printed cardiac surface.

FIG. 5 is a comparison of mass loss (%) and swelling ratio (%) of extracellular vesicle bio-photosensitizing Gel (Gel-E) and GelMA photosensitizing Gel (Gel).

FIG. 6 is a photograph of the heart after the light-induced gel of extracellular vesicle bio-photosensitive gel on the surface of the mouse heart.

FIG. 7 is a graph showing the retained fluorescence of the mouse heart after 48 hours of surface-irradiation of extracellular vesicle bio-photosensitive gel. A: an extracellular vesicle bio-photosensitive gel treatment group; b: and (3) an extracellular vesicle processing group.

FIG. 8 is a representation of the observation under a fluorescence microscope of the entry of extracellular vesicles into cardiac tissue in a sprayed extracellular vesicle bio-photosensitive gel. Blue for nuclear staining DAPI, green for myocardial tissue staining α -SA, red for extracellular vesicle staining PKH26, scale bar, 50 μm.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

AFM Key Sight 7500 USA

SEM JFOL JSM-7000F USA

Fetal Bovine Serum (FBS) Biological industries Israel

Minimal basal Medium (MEM) gibco USA

5810R centrifuge eppendorf Germany

ExoQuick-TC 1mL/2mL SBI US

BCA protein assay kit PC0020 Solambio Beijing China

Phosphate PBS buffer Biological industries Israel

Stereolithography Formlabs form 2

PKH26 kit (Sigma, P9691)

Mouse bone marrow mesenchymal stromal cells are from cell bank of Chinese academy of sciences

Example 1 preparation of extracellular vesicle Biophotosensitive gel for repairing damaged tissues

(I) preparation and analysis of extracellular vesicles

Bone marrow mesenchymal stromal cells of mice were cultured in basal medium containing 10% fetal bovine serum in 5% CO₂Standing in 37 deg.C incubator, washing with phosphate PBS buffer solution for 3 times when cell coverage reaches 80%, and continuing to use culture medium without fetal calf serum and 5% CO₂After culturing at 37 ℃ for 24 hours in an incubator, fresh non-fetal bovine serum MEM was replaced and incubated for 48 hours, and the culture medium supernatant was collected. Collecting culture medium supernatant, centrifuging at 3000g and 4 deg.C for 15min with 5810R centrifuge, removing cells and cell debris, collecting supernatant, mixing with ExoQuick-TC at a ratio of 5:1, refrigerating at 4 deg.C overnight (at least 12 hr)) (ii) a The ExoQuick-TC/supernatant mixture was centrifuged at 1500 × g at 4 ℃ for 30 minutes to collect extracellular vesicles, and the extracellular vesicles were resuspended in phosphate buffer at pH 7.3 to obtain phosphate buffer containing extracellular vesicles.

The morphology of the "goblet" as shown in FIG. 1A and the particle size of the extracellular vesicles as shown in FIG. 1B were confirmed by Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) images, and the nanoparticle tracking system showed that more than 80% of the isolated extracellular vesicles had diameters between 110 and 140nm as shown in FIG. 1C.

Preparation of (di) methacrylic anhydridized gelatin (GelMA) and scanning electron microscope morphology detection

45g of gelatin was dissolved in 450mL of phosphate buffer solution (PBS solution) having pH 7.3, sufficiently stirred, and then heated to 50 ℃. After completely dissolving, methacrylic anhydride (0.6 g of methacrylic anhydride per 1g of gelatin) was added, stirred, and reacted at 50 ℃ for 1 hour. After the reaction was completed, 3 liters of MilliQ water was added for dialysis and purification for 5 days, and the water was changed every 1 day to ensure that the unreacted monomers were removed completely, and the final solution was purified methacrylic anhydrified gelatin (GelMA). The collected GelMA was sterilized by filtration and then lyophilized for storage.

Preparation of (tri) GelMA photosensitive gel

Dissolving 100mg of methacrylic anhydrified gelatin (GelMA) in 1000. mu.L of ultrapure water, and filtering the solution through a 0.22 μm filter to obtain a GelMA solution; uniformly mixing GelMA solution with ruthenium (Ru), Sodium Persulfate (SPS) and phosphate buffer solution (PBS solution) at a volume ratio of 50:1:1:48 to obtain GelMA photosensitive gel precursor (liquid) at 30mW cm^-2Can be cross-linked into a lattice-like GelMA photosensitive gel within 30 seconds under the excitation of visible light. As shown in fig. 2, SEM observations of the structure of GelMA photoactivated gel, which allows release of extracellular vesicles to the injured tissue.

(IV) preparation of extracellular vesicle bio-photosensitive gel

GelMA (100 mg) was dissolved in 1000. mu.L of ultrapure water and filtered through a 0.22 μm filter to obtain a GelMA solution; resuspending the extracellular vesicles in PBS solution; GelMA solution is mixed with Ru, SPS, and extracellular vesiclesThe PBS solution is uniformly mixed according to the volume ratio of 50:1:1:48, and the extracellular vesicle biological photosensitive gel precursor (liquid state) can be formed, wherein the volume ratio is 30mW cm^-2The visible light is excited to form extracellular vesicle biological photosensitive gel.

Experimental example 1 NMR Hydrogen Spectroscopy analysis of GelMA Photoactivatable gel in extracellular vesicle BioPhotoactivatable gel for repairing damaged tissue

GelMA gelatin and GelMA photosensitive gels were analyzed using NMR hydrogen spectroscopy. As shown in fig. 3, the peak at δ of 2.9ppm in the GelMA photosensitizing gel correlates with the signal of the lysine proton. Peaks at δ 5.33ppm and δ 5.77ppm were observed in the GelMA photosensitive gel, indicating the presence of conjugated vinyl groups.

Experimental example 2GelMA Photosensitive gel Cross-linking test on 3D-printed Heart surface

A 3D printed heart model (Formlabs, form 2) was used to evaluate the feasibility of spraying GelMA photosensitive gel on the heart surface and cross-linking analysis. As shown in fig. 4, the GelMA photosensitive gel was completely crosslinked and remained in the inkjet area within 30 seconds of irradiation under visible light excitation.

EXAMPLE 3 comparison of mass loss (%) and swelling ratio (%)% of extracellular vesicle Biophotosensitive Gel (Gel-E) and GelMA Bio-Gel (Gel)

Preparation of extracellular vesicle bio-photosensitive gel:

mass loss and swelling ratio measurements were performed to determine that extracellular vesicles did not affect the crosslinking of GelMA photogel precursors. According to the invention, the quality loss and swelling ratio detection shows that no matter whether extracellular vesicles are added into the GelMA photosensitive gel or not, the quality loss and swelling ratio difference of the two groups has no statistical significance (p is more than 0.05). Indicating that the presence of extracellular vesicles had no effect on the cross-linking of GelMA photosensitive gels, as shown in figure 5.

Experimental example 4 mouse heart surface was exposed to extracellular vesicle-type bio-photosensitive gel light to gel

Selecting C57/BL6 male mouse, performing intraperitoneal injection anesthesia (dosage is 40mg/kg) with 1% sodium pentobarbital, shaving the hair between the armpit to the level of xiphoid process, the anterior midline to the left posterior axillary line, and supinely fixing at 37 deg.C constant temperature water bath cushionThe above. The trachea cannula is directly inserted into a small animal respirator for ventilation, the tidal volume is 0.5mL, the exhale ratio is 1:2, and the respiratory frequency is 80 times/minute. Adjusting the body position of the mouse to the right side lying position of 45 degrees, disinfecting the operation area, horizontally and sequentially cutting the skin, pectoralis major, serratus anterior and intercostal muscles along the 4 th intercostal space of the left chest, placing the mouse special thoracic cage spreader to expose the heart, and stripping the pericardium. Adding the extracellular vesicle biological photosensitive gel precursor (liquid) into a lightproof spraying container, spraying 100 μ L of solution, and simultaneously administering 30mW cm^-2May direct light toward the heart. As shown in FIG. 6, the left side is the heart without gel ejection, the right side is the heart with gel ejection, and the gel is visible in the red dotted circle and distributed on the surface of the heart tissue.

Experimental example 5 analysis of amount of retained fluorescence after 48 hours of spraying extracellular vesicle bio-photosensitive gel on the surface of mouse heart

Construction of extracellular vesicle bio-photosensitive gel with fluorescent agent:

extracellular vesicle staining with PKH 26: labeling was performed using PKH26 fluorescence kit. The specific method comprises the following steps: 50 μ L of extracellular vesicles were diluted with 100 μ L of solution C from PKH26 kit. Another 100. mu.L of solution C was dissolved with 0.5. mu.L of PKH26 stain. The two liquids were mixed well and incubated at room temperature for 5 min. Immediately after the incubation was complete, staining was stopped using 5% BSA in PBS. Then adding the mixture into a mixed sample for stopping dyeing by using an ExoQuick-TC exosome extraction kit according to the volume ratio of 1:5, uniformly blowing the mixture, and standing the mixture overnight at the temperature of 4 ℃. After standing, the mixture was centrifuged at 1500g for 30min, the supernatant was discarded, and the mixture was centrifuged at 1500g for 5min again, and the supernatant was discarded. Resuspend the pellet with 50 μ L PBS, load into a lightproof centrifuge tube, and store the stained extracellular vesicles at-80 ℃ for later use.

GelMA (100 mg) was dissolved in 1000. mu.L of ultrapure water and filtered through a 0.22 μm filter to obtain a GelMA solution; resuspending PKH26 stained extracellular vesicles in PBS solution; the GelMA solution, Ru, SPS and PBS solution containing the stained extracellular vesicles are uniformly mixed according to the volume ratio of 50:1:1:48, and the stained extracellular vesicle biological photosensitive gel precursor (liquid state) can be formed.

A C57/BL6 male mouse is selected, 1% sodium pentobarbital is used for intraperitoneal injection anesthesia (the dosage is 40mg/kg), hairs between the armpit to the level of the xiphoid process and the front median line to the left rear armpit line are shaved, and the male mouse is fixed on a constant-temperature water bath cushion at 37 ℃ in a supine position. The trachea cannula is directly inserted into a small animal respirator for ventilation, the tidal volume is 0.5mL, the exhale ratio is 1:2, and the respiratory frequency is 80 times/minute. Adjusting the body position of the mouse to the right side lying position of 45 degrees, disinfecting the operation area, horizontally and sequentially cutting the skin, pectoralis major, serratus anterior and intercostal muscles along the 4 th intercostal space of the left chest, placing the mouse special thoracic cage spreader to expose the heart, and stripping the pericardium. The extracellular vesicle bio-photosensitive gel precursor (liquid) was added to a dark spray container, and 100. mu.L of the solution was sprayed while 30mW cm-2 of light-directed heart irradiation was given. At the same time, 100. mu.L of extracellular vesicles were administered as a control group, and sprayed onto the surface of the mouse heart. Taking down the dilator, expanding the lung lobes under positive pressure, and closing the intercostal space, the chest wall muscles and the skin layer by 8-0 non-invasive suture. The mice were returned to their cages after surgery and were monitored until complete awakening.

The fluorescence amount distribution was observed under a small animal fluorescence tracking system, as shown in fig. 7A, and after 48 hours, the fluorescence was visible at the thoracic position, indicating that there was still extracellular vesicle bio-photosensitive gel remaining in the heart. Also, the amount of residual fluorescence was greater than that of the group treated with only extracellular vesicles (fig. 7B).

Experimental example 6 analysis of the entrance of extracellular vesicles into cardiac tissue in a sprayed extracellular vesicle bio-photosensitive gel by fluorescence microscopy

Extracellular vesicle bio-photosensitive gels were ejected on mouse hearts and gelled in the same procedure as in experimental example 5. After 24 hours, heart tissue was removed for section staining and observed under a fluorescent microscope. As shown in fig. 8, the penetration of a large number of PKH 26-stained extracellular vesicles (red fluorescence) in the myocardial tissue (green fluorescence staining) was seen, indicating that the photosensitive gel could support the entry of extracellular vesicles into the myocardial tissue.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.