Hydrogel loaded with targeted modified exosome as well as preparation method and application of hydrogel

Abstract

The invention discloses targeted modified exosome loaded hydrogel as well as a preparation method and application thereof, and belongs to the technical field of targeted modification and delivery systems of exosomes. The hydrogel loaded with the targeted modified exosome comprises the following components: carboxymethyl chitosan, oxidized dextran, a tannic acid-iron complex, the targeted modified exosome, PBS (Phosphate Buffer Solution) and water; the targeted modified exosome is obtained by mixing and incubating DSPE-PEG-AEAA and an exosome PBS (Phosphate Buffer Solution) derived from umbilical cord mesenchymal stem cells. The hydrogel disclosed by the invention has a targeting effect on activated hepatic stellate cells in hepatic fibrosis tissues, generates good anti-fibrosis capability, also has a remarkable effect of relieving hepatic fibrosis, and is expected to become a new way for treating hepatic fibrosis.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of targeted modification and delivery system of exosomes, and particularly relates to a hydrogel loaded with targeted modified exosomes and a preparation method and application thereof. BACKGROUND

[0002] Liver fibrosis is a chronic liver injury disease, and its occurrence is related to free radicals (ROS) generated after liver injury, which further activates hepatic stellate cells to secrete matrix proteins. Research reports show that more than 90% of hepatocellular carcinoma (HCC) cases occur in the background of chronic liver disease, and cirrhosis caused by liver fibrosis is the strongest factor for the occurrence of hepatocellular carcinoma. Therefore, the treatment of chronic liver fibrosis is a problem that needs to be solved for human beings.

[0003] Studies have shown that mesenchymal stem cells (MSCs) are a kind of multipotent cells, which not only have sustained self-renewal proliferation, differentiation and immunoregulatory activity, but also have significant anti-inflammatory effect. Human umbilical cord-derived mesenchymal stem cell exosomes (hucMSCs-Exo) not only have the common properties of mesenchymal stem cells, but also can play a role in anti-liver fibrosis. However, after systemic administration of exosomes by intravenous route, they will be rapidly eliminated by the reticuloendothelial system (RES). Moreover, although the homing ability of exosomes allows them to migrate to injured tissues, this process can be time-consuming and inefficient in vivo.

[0004] Hydrogel is a kind of three-dimensional polymer network gel, which can be cross-linked by covalent bond, cross-linked by non-covalent interaction, or cross-linked by the combination of the two, and can retain a large amount of water in the swollen state. At present, hydrogel has been applied in the fields of skin regeneration, wound dressing, diabetes treatment, liver regeneration, joint filling, drug delivery, etc. due to its variable physicochemical, structural characteristics (three-dimensional network and porosity) and biological properties, especially the characteristics of simulating in vivo tissue matrix, which is conducive to the encapsulation and expansion of cells in vitro and in vivo, can reproduce and maintain functions at the same time, and creates conditions for encapsulating cells and delivering drugs.

[0005] At present, there are still problems such as poor biocompatibility, poor activity and stability of exosomes, and poor tissue adaptability in loading exosomes by using hydrogel. SUMMARY

[0006] The purpose of the present application is to provide a hydrogel loaded with targeted modification, which has excellent performance in biocompatibility, exosome activity and stability, and tissue adaptability.

[0007] The technical scheme of the present application is described in detail as follows:

[0008] In a first aspect, the present invention provides a hydrogel loaded with targeted modified exosomes, the components of which include: carboxymethyl chitosan, oxidized dextran, tannic acid-iron complex, targeted modified exosomes, PBS and water;

[0009] The targeted modified exosomes were obtained by mixing and incubating DSPE-PEG-AEAA with PBS solution of exosomes derived from umbilical cord mesenchymal stem cells.

[0010] AEAA, or aminoacetyl anisamide, is chemically known as N-acetyl-p-methoxyaniline, with the molecular formula C9H. 11 NO2, with a molecular weight of 165.19, is a DSPE-PEG modified AEAA (DSPE-PEG-AEAA), composed of three parts: phospholipid (DSPE), polyethylene glycol (PEG), and aminoacetyl anisamide (AEAA). DSPE-PEG-AEAA binds to exosomes through electrostatic interactions or intermolecular forces during co-incubation, enabling the exosomes to target activated hepatic stellate cells. Tannic acid-iron complexes enhance the structural stability of the hydrogel, while carboxymethyl chitosan and oxidized dextran contribute to the hydrogel's good biocompatibility and degradability.

[0011] Optionally or preferably, the mass ratio of DSPE-PEG-AEAA to exosomes derived from umbilical cord mesenchymal stem cells in the above-mentioned hydrogel is 2–8:1, the incubation temperature is 25–40°C, and the incubation time is 0.5–2 h. The preferred mass ratio of DSPE-PEG-AEAA to exosomes derived from umbilical cord mesenchymal stem cells is 3–6:1, more preferably 5:1; the preferred incubation temperature is 30–40°C, more preferably 37°C; and the preferred incubation time is 0.5–1.5 h, more preferably 1 h.

[0012] Optionally or preferably, the molecular weight range of the DSPE-PEG-AEAA in the above-mentioned hydrogel is 3000±200 Da.

[0013] Secondly, the present invention provides a method for preparing any of the above-described hydrogels, comprising:

[0014] Oxidized dextran, targeted modified exosomes, and PBS were mixed to obtain an oxidized dextran exosome suspension;

[0015] A hydrogel loaded with targeted modified exosomes was obtained by mixing oxidized dextran exosome suspension, carboxymethyl chitosan PBS solution, and tannic acid-iron complex aqueous solution.

[0016] Optionally or preferably, in the above preparation method, the mass percentage concentration of the oxidized dextran exosome suspension is 0.5-1.5%; the mass percentage concentration of the carboxymethyl chitosan PBS solution is 2.0-4.0%; the mass percentage concentration of the tannic acid-iron complex aqueous solution is 20-25%; and the volume ratio of the oxidized dextran exosome suspension, the carboxymethyl chitosan PBS solution, and the tannic acid-iron complex aqueous solution is 6-12:6-12:1.

[0017] In the oxidized dextran exosome suspension, the mass ratio of oxidized dextran to exosomes derived from umbilical cord mesenchymal stem cells is 2–8:1–3, preferably 3–6:1–2, and more preferably 3:1. The mixing speed is 150–250 rpm, preferably 180–220 rpm, and more preferably 200 rpm, for 3–7 min, preferably 4–6 min, and more preferably 5 min.

[0018] The concentration of the carboxymethyl chitosan PBS solution is 2.0–4.0%, preferably 2.5–3.5%, and more preferably 3%. The aqueous solution of tannic acid-iron complex is 0.05–0.3%, preferably 0.08–0.15%, and more preferably 0.12%.

[0019] Within the above concentration range, the volume ratio of the oxidized dextran exosome suspension, the carboxymethyl chitosan PBS solution, and the tannic acid-iron complex aqueous solution is 6–12:6–12:1, preferably 7–9:7–10:1, and more preferably 9:10:1. The mixing method is shaking, with a shaking time of 5–20 s, preferably 10–18 s, and more preferably 15 s.

[0020] Optionally or preferably, in the above preparation method, the oxidized dextran preparation method is as follows: a dextran aqueous solution is mixed and reacted with sodium periodate; after the reaction is completed, a quencher is added to terminate the reaction; dialysis is performed; the dialysate is collected and then dried after being cold-extracted by liquid nitrogen; the molecular weight of the dextran is 1×10⁻⁶. 4 ~5×10 5 Da, preferably 5×10 4 ~3×10 5 Da, further preferably 8×10 4 ~2×10 5 Da, more preferably 1×10 5 The concentration of Da, the dextran aqueous solution is 40-80 mg / mL, preferably 50-70 mg / mL, and more preferably 60 mg / mL.

[0021] When dextran aqueous solution is mixed with sodium periodate for reaction, the mass ratio of sodium periodate to dextran is 0.5–3:0.2–3, preferably 0.8–1.6:1, and more preferably 1.3:1. The reaction is carried out under light-protected conditions for 3–7 hours, preferably 4–6 hours, and more preferably 5 hours. The stirring speed during the reaction is 400–600 rpm, preferably 450–550 rpm, and more preferably 500 rpm. A quencher is added after the reaction to terminate it; the quencher is preferably ethylene glycol.

[0022] Optionally or preferably, in the above preparation method, the dialysis is performed using a dialysis bag with a molecular weight cutoff of 3000–4000 Da, the drying temperature is -15 to -25°C, and the time is 28–36 hours. The molecular weight cutoff of the dialysis bag is preferably 3200–3800 Da, more preferably 3400–3600 Da, and even more preferably 3500 Da. The drying temperature is preferably -18 to -22°C, more preferably -20°C, and the drying time is 28–36 hours, preferably 30–34 hours, and even more preferably 32 hours.

[0023] Thirdly, the present invention provides the application of hydrogels loaded with targeted modified exosomes as described above in the preparation of products that improve liver fibrosis.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The hydrogel loaded with targeted modified exosomes of the present invention exhibits good injectability, high self-healing and adhesion. It demonstrates excellent biocompatibility, with cell viability exceeding 91% after five days of culturing cells in the hydrogel extract. It exhibits good sustained-release effect; the exosome release curve shows rapid release of exosomes in the first four days, gradually slowing down after four days, reaching 79.8% on day 28. It demonstrates significant targeting performance; in vitro and in vivo experiments show that the uptake of exosomes by cells in the hydrogel of the present invention is very high, statistically significantly different from that of unmodified exosomes. It also exhibits strong anti-ROS properties, significantly superior to the unmodified exosome control group.

[0026] 2. The preparation method of the hydrogel of the present invention is simple, mild and environmentally friendly.

[0027] 3. Both in vitro and in vivo experiments have shown that the hydrogel of the present invention has a targeting effect on activated hepatic stellate cells in liver fibrotic tissue, producing good anti-fibrotic ability, and also has a significant effect on reducing liver fibrosis, which is expected to become a new way of treating liver fibrosis. Attached Figure Description

[0028] Figure 1 The particle size results of the exosomes prepared in Example 1 were detected by a nanoparticle tracking analyzer.

[0029] Figure 2 The results are TEM observations of the microstructure of exosomes in Example 1.

[0030] Figure 3 The results of detection of exosome surface membrane proteins by imaging with ELC luminescent solution in Example 1 are shown; control represents the control group, the experimental subject is the supernatant, and Exosomes represents the prepared exosome experimental group.

[0031] Figure 4 This is a confocal microscope image from Example 2 showing the distribution of exosomes in the hydrogel at different times; Gel / PKH-26-Exos represents exosomes stained with the fluorescent agent PKH-26 (CMC-OD / TA-Fe-AEAA-Exo), Brightfield represents brightfield, Merge represents the fused image of PKH-26-Exos and Brightfield, 3D Gel / PKH-26-Exos represents the stereoscopic image of Gel / PKH-26-Exos detected by confocal microscopy, Control represents the control group, and the experimental subject is the CMC-OD / TA-Fe-AEAA-Exo composite system without PKH-26 labeling.

[0032] Figure 5 The images show the SEM microstructure and elemental energy dispersive spectroscopy (EDS) results of the exosome-loaded hydrogel CMC-OD / TA-Fe-AEAA-Exos in Example 2. In the images, A shows the morphology of the exosomes adhering to the surface of the hydrogel under high and low magnification, and B shows the elemental energy dispersive spectroscopy (EDS) image of the hydrogel.

[0033] Figure 6 The Fourier transform infrared spectrum is the result of the hydrogel synthesis in Example 2.

[0034] Figure 7 The results of the comparative analysis of gelation time of CMC-OD-AEAA-Exos (represented by CMC-OD in the figure) and CMC-OD / TA-Fe-AEAA-Exos (represented by CMC-OD / TA-Fe in the figure) hydrogels in Example 2 are presented as mean ± SD, n = 3.

[0035] Figure 8 The results are the injectability performance test results of the hydrogel in Example 2.

[0036] Figure 9 The results show the self-healing properties of the hydrogel in Example 2.

[0037] Figure 10 The results show the adhesion energy of the hydrogel in Example 2. The top row represents CMC-OD-AEAA-Exos, and the bottom row represents CMC-OD / TA-Fe-AEAA-Exos.

[0038] Figure 11 The left figure shows the degradation and swelling characteristics of the hydrogel in Example 2, and the right figure shows the statistical results of the degradation performance.

[0039] Figure 12 Images showing the live and dead staining of L929 cells after different groups of hydrogel extracts were applied in Example 2, n=3; CMC-OD group: CMC-OD-AEAA-Exos hydrogel, Gel group: CMC-OD / TA-Fe hydrogel, Gel / AEAA-Exos group: CMC-OD / TA-Fe-AEAA-Exos hydrogel, Control group: normal culture medium.

[0040] Figure 13 The relative cell survival rate of L929 cells after treatment with different concentrations of hydrogel extract in Example 2 is shown in Figure 2. Different colors represent different concentrations.

[0041] Figure 14 The release rate of exosomes in the CMC-OD / TA-Fe-AEAA-Exos hydrogel in Example 2 is shown.

[0042] Figure 15 The results of the targeting evaluation of activated hepatic stellate cells by different experimental groups of hydrogel / exosome systems in Example 2 are shown. Gel / Exos represents the CMC-OD / TA-Fe-Exos hydrogel group, Gel / AEAA-Exos represents the CMC-OD / TA-Fe-AEAA-Exos hydrogel group, and PBS represents the PBS control group.

[0043] Figure 16 The results of the in vitro anti-ROS experiment of the hydrogel / exosome system in Example 2 are shown. Gel / Exos represents the CMC-OD / TA-Fe-Exos hydrogel group, and Gel / AEAA-Exos represents the CMC-OD / TA-Fe-AEAA-Exos hydrogel group.

[0044] Figure 17 The results of the in vitro antifibrotic experiment of the hydrogel / exosome system in Example 2 are shown. A represents the antifibrotic effect of the gel / AEAA-Exos system evaluated by the α-SMA index, and B represents the antifibrotic effect of the gel / AEAA-Exos system evaluated by the Colla I index (n=3). Gel / Exos represents the CMC-OD / TA-Fe-Exos hydrogel group, Gel / AEAA-Exos represents the CMC-OD / TA-Fe-AEAA-Exos hydrogel group, and PBS represents the PBS control group.

[0045] Figure 18The results of the experiment evaluating the targeting performance of exosomes on activated hepatic stellate cells by tissue immunofluorescence assay were obtained after the hydrogel / exosome systems of different treatment groups in Example 3 were implanted into SD rats for 7 days. Gel / Exos represents the CMC-OD / TA-Fe-Exos hydrogel group, and Gel / AEAA-Exos represents the CMC-OD / TA-Fe-AEAA-Exos hydrogel group.

[0046] Figure 19 The images show HE-stained liver, spleen, kidney, heart, and lungs of mice in the normal and experimental groups 7 days after the hydrogel was implanted in mice in Example 3. n=5. Gel / AEAA-Exos represents the CMC-OD / TA-Fe-AEAA-Exos hydrogel experimental group.

[0047] Figure 20 Example 3 evaluated the anti-liver fibrosis effects of different hydrogel systems using HE, Masson's stain, and Sirue red stain. Data are expressed as mean ± standard deviation (n=5), ***P<0.001, ****P<0.0001. Detailed Implementation

[0048] To enable those skilled in the art to better understand the present application, the present application will be clearly and completely described below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application. Unless otherwise specified, the instruments and reagents used in the embodiments are all from commercial channels.

[0049] Example 1: Preparation of exosomes derived from umbilical cord mesenchymal stem cells

[0050] The umbilical cord samples were obtained from full-term newborns with no obvious infectious diseases, family history of genetic disorders, or other illnesses. The sample acquisition was ethically sound, in accordance with national laws and regulations, and informed consent was obtained from the donor.

[0051] Rinse the fresh umbilical cord with 75% alcohol for 30 seconds, then rinse twice with physiological saline. Cut the umbilical cord into 1cm long segments and place them in a 10cm petri dish. Next, cut the umbilical cord along the umbilical vein, remove one umbilical vein and two umbilical arteries, rinse again with physiological saline, and then cut into approximately 1mm pieces with scissors. 3Small tissue pieces were carefully placed at the bottom of a T75 culture flask using tissue forceps. After incubating at 37°C for 3 hours, 15 mL of complete culture medium was added. Cell growth was observed on day 6, and the original culture medium was replaced on day 7. Cells were passaged when confluence reached 60–70%. For passage, cells were washed twice with PBS buffer, digested with 3 mL of trypsin for 2 min, centrifuged at 1000 rpm for 5 min after digestion, the supernatant was discarded, and the cells were resuspended and seeded in exosome-free serum-free medium. Cells were cultured until confluence reached 80–90%, the supernatant was collected, and the cells were passaged again.

[0052] Exosomes were extracted according to the kit instructions. First, cells were centrifuged at 3000g for 10 min to remove cells and some cell debris. The supernatant was collected and transferred to a new centrifuge tube, where it was centrifuged at 10000g for 15 min to further remove small debris and impurities. Then, the newly obtained supernatant was transferred in 15 mL increments to ultrafiltration centrifuge tubes (MWCO = 100 kDa), centrifuged at 5000 rpm for 15 min, and then concentrated to 20 mL. Next, 20 mL of the concentrated supernatant was weighed and gently mixed with 5 mL of ExoQuick-TC solution, and incubated at 4°C for 24 h. Subsequently, centrifugation at 10000g for 60 min was performed, and exosomes were observed precipitating on the centrifuge tube wall. The supernatant was gently aspirated. The obtained exosomes were resuspended in 200 μL of PBS and centrifuged again at 12000g for 2 min to obtain the exosome stock solution, which was then stored at -80°C for later use.

[0053] Example 2: Preparation of hydrogels loaded with targeted modified exosomes

[0054] (1) Add 1.0g of dextran (Shanghai Aladdin Biochemical Technology Co., Ltd., molecular weight 1×10⁻⁶) to the dextran. 5 Da) was dissolved in 16.7 mL of double-distilled water to obtain a dextran aqueous solution with a concentration of 60 mg / mL.

[0055] (2) Add 0.80 g of sodium periodate (NaIO4) to the dextran aqueous solution obtained in step (1), wherein the molar ratio of NaIO4 to dextran is 1.3:1. Mix and react for 5 h at room temperature (25℃) in the dark, with a stirring speed of 500 rpm. After the reaction, add 2 mL of ethylene glycol to terminate the reaction for 1 h. Dialyze using a dialysis bag with a cutoff of 3500 Da for 3 days, changing the solution every 4 hours until there is no periodate in the dialysate (the method for detecting periodate is: add 0.5 mL of dialysate to 0.5 mL of 1% silver nitrate solution; if there is no precipitate, it indicates that there is no periodate). After dialysis, quench the dialysate with liquid nitrogen and then quickly place it in a freeze dryer and freeze dry at -20℃ for 32 h to obtain oxidized dextran (OD).

[0056] (3) DSPE-PEG-AEAA was mixed with exosomes derived from umbilical cord mesenchymal stem cells and incubated to obtain targeted modified exosomes DSPE-PEG-AEAA-Exo; the incubation temperature was 37℃; and the incubation time was 1 h. The specific steps are as follows:

[0057] Weigh 0.05 g of the oxidized dextran prepared in step (2) and dissolve it in 5 mL of 1×PBS buffer solution. Add DSPE-PEG-AEAA-Exo containing 2 mg of exosomes and stir for 5 min in a magnetic stirrer at 200 rpm to obtain an oxidized dextran-exosome suspension with a mass fraction of 1%. The mass ratio of oxidized dextran to exosomes in the suspension is 3:1.

[0058] (4) Prepare a ferric chloride solution with a concentration of 9 mM by dissolving ferric chloride hexahydrate in distilled water; prepare a tannic acid solution with a concentration of 27 mM by dissolving tannic acid in distilled water; mix the ferric chloride solution and the tannic acid solution to obtain an aqueous solution of tannic acid-iron complex with a mass percentage concentration of 23.25%.

[0059] (5) Dissolve carboxymethyl chitosan powder (90% deacetylation, 80% carboxymethyl) in PBS buffer to obtain a 3% carboxymethyl chitosan PBS solution.

[0060] 400 μL of 3% carboxymethyl chitosan PBS solution, 40 μL of 23.25% tannic acid iron complex aqueous solution, and 360 μL of 1% oxidized dextran exosome suspension obtained in step (3) were shaken and mixed for 10 s, and then allowed to stand to form a gel, thus obtaining a hydrogel loaded with AEAAA-targeted modified exosomes, abbreviated as CMC-OD / TA-Fe-AEAA-Exos.

[0061] Simultaneously, hydrogels without added exosomes (CMC-OD / TA-Fe) and hydrogels containing exosomes without AEAA modification (CMC-OD / TA-Fe-Exos) were prepared according to step (5).

[0062] CMC-OD / TA-Fe: Mix 360 μL of 3% carboxymethyl chitosan PBS solution and 50 μL of 23.25% tannic acid iron complex aqueous solution by shaking and allowing to stand to form a gel.

[0063] CMC-OD / TA-Fe-Exos: Step (3) Mix 400 μL of 1 g / ml oxidized dextran PBS solution, 400 μL of 3% carboxymethyl chitosan PBS solution and 50 μL of 23.25% tannic acid iron complex aqueous solution by shaking and allowing to stand to form a gel.

[0064] Example 1: Characterization and function of exosomes

[0065] 1. Exosome diameter

[0066] Take 40 μL of the exosome stock solution prepared in Example 1, dilute it to 1 mL, and then perform particle size analysis using a nanoparticle tracking analyzer (NTA). The NTA results are as follows: Figure 1 As shown, the diameter of the exosomes is approximately 130 nm.

[0067] 2. Exosome structure

[0068] After fixing the exosome stock solution with 2.5% glutaraldehyde solution overnight at 4°C, 10 μL was dropped onto a copper grid and allowed to stand at room temperature for 5 min. The liquid was then absorbed by filter paper. Staining solution (saturated uranium acetate-acetic acid solution) was added to the copper grid and stained for 1 min, followed by absorption of the liquid by filter paper. ddH₂O was added to the copper grid and allowed to stand at room temperature for 5 min, then the liquid was absorbed by filter paper; this process was repeated once. The mixture was dried at room temperature and observed and photographed using TEM. The TEM results are shown below. Figure 2 As shown, exosomes have a clear membrane structure resembling a saucer or cup, with clear edges, and are evenly distributed without aggregation.

[0069] 3. Detection of exosome markers

[0070] After exosome concentration detection, 20 μg of exosomes (with an equal volume of culture supernatant as a control) were added to 5×SDS-PAGE Loading Buffer and mixed thoroughly. The mixture was then heated at 95℃ for 5 min. SDS-PAGE electrophoresis: Acrylamide separating gel concentration was 12%. The supernatant from the above heating treatment was added to each sample well, and electrophoresis was performed (70V / 30 min, 120V / 60 min). Transfer: Wet transfer at 250 mA constant current for 90 min. Blocking: Block with 5% skim milk powder at room temperature for 1 h. Incubation with primary antibody: The antibody was diluted with 5% skim milk powder (antibody diluted 1000 times) and incubated overnight at 4℃. Incubation with secondary antibody: The primary antibody was aspirated, and the membrane was washed 3 times with TBST (5 min / wash). HRP-labeled secondary antibody (1:5000) was added, and the membrane was incubated at room temperature for 1 h. Imaging: After secondary antibody incubation, the membrane was washed 3 times with TBST (5 min / wash) and then imaged using ELC chemiluminescence buffer. Results of exosome surface membrane protein detection are as follows Figure 3 As shown, exosome markers TSG101, CD9, and CD63 are positively expressed.

[0071] Example 2: Performance of hydrogels loaded with targeted modified exosomes

[0072] 1. Distribution of exosomes in hydrogels

[0073] The state of exosomes carried in the hydrogel was evaluated using PKH-26 red fluorescent dye. PKH-26 was added to the CMC-OD / TA-Fe-AEAA-Exos prepared in Example 2, and the exosomes were incubated according to the manufacturer's instructions; this mixture was referred to as Gel / PKH-26-Exos. An unlabeled CMC-OD / TA-Fe-AEAA-Exos mixture was used as a control. Excess dye was removed by centrifugation, and the mixture was observed under an inverted fluorescence microscope. It was then stored at 4°C until the exosomes were encapsulated in the hydrogel for evaluation using confocal microscopy.

[0074] The results are as follows Figure 4 As shown, the results indicate that there was no significant difference in the detection results at 3h and 72h after exosomes were encapsulated in hydrogel, suggesting that the exosomes were relatively stable in the hydrogel, which is beneficial for the sustained-release experiment.

[0075] 2. Microstructure of the exosome-loaded hydrogel

[0076] After freeze-drying the CMC-OD / TA-Fe-AEAA-Exos hydrogel prepared in Example 2, the microscopic morphological characteristics of the hydrogel were measured using scanning electron microscopy (SEM); the elemental contents of C, O, N, Fe and P in the hydrogel were analyzed using energy dispersive spectroscopy (EDS) to characterize the composition of the hydrogel.

[0077] The results are as follows Figure 5 Under high magnification, numerous exosomes can be observed adhering to the surface of the hydrogel (see...). Figure 5 A) Elemental energy dispersive spectroscopy (EDS) analysis of hydrogels carrying exosomes, such as... Figure 5 As shown in Figure B, the CMC-OD / TA-Fe-AEAA-Exos gel is composed of C, N, O, P, and Fe elements. Since iron tannate contains Fe, this preliminarily explains the synthesis of the CMC-OD / TA-Fe hydrogel. The presence of N may be related to the presence of N in exosomes and AEAAA, while the presence of P may be related to the exosomes attached to the hydrogel. The exosome membrane is composed of a phospholipid bilayer, and its surface is embedded with transmembrane proteins.

[0078] 3. Fourier transform infrared spectroscopy evaluation of hydrogels

[0079] The gelation mechanism of carboxymethyl chitosan, oxidized dextran, and TA-Fe was evaluated using Fourier transform infrared spectroscopy (FTIR), confirming the successful synthesis of the hydrogel material.

[0080] like Figure 6 Mid-Fourier transform spectroscopy showed that carboxymethyl chitosan (CMC) was at 1409.7 cm⁻¹. -1 and 1594.3cm -1 The site exhibits typical characteristic peaks. The oxidized dextran (OD) has a peak at 1731.8 cm⁻¹. -1 A new absorption peak appears at 1608.9 cm⁻¹ in the spectrum of the CMC-OD / TA-Fe-AEAA-Exos hydrogel. -1 The presence of a characteristic peak for an imine bond indicates a Schiff base reaction between the -NH₂ group of CMC and the -CH=O group of OD. The TA (tannic acid) absorption band is at 3446.7 cm⁻¹. -1 The absorption band at 3451.5 cm⁻¹ in CMC-OD -1 All correspond to the stretching vibration of -OH, and migrate to 3399.5 cm⁻¹ in CMC-OD / TA-Fe(III). -1 This indicates that the CMC-OD / TA-Fe(III) hydrogel was successfully synthesized.

[0081] 4. Hydrogel formation

[0082] We prepared CMC-OD-AEAA-Exos and CMC-OD / TA-Fe-AEAA-Exos hydrogels in transparent ampoules to verify the role of tannic acid iron complexes in the hydrogels.

[0083] CMC-OD-AEAA-Exos hydrogel: 400 μL of 3% carboxymethyl chitosan PBS solution + 400 μL of 1% oxidized dextran exosome suspension, shake and mix for 10 seconds, then let stand to form a gel.

[0084] CMC-OD / TA-Fe-AEAA-Exos hydrogel: 400 μL of 3% carboxymethyl chitosan PBS solution + 50 μL of 0.12% tannic acid iron complex aqueous solution + 400 μL of 1% oxidized dextran exosome suspension, shake and mix for 10 s, then let stand to form a gel.

[0085] like Figure 7 As shown, the CMC-OD-AEAA-Exos hydrogel is colorless and transparent (CMC-OD is represented in the figure). After the addition of TA-Fe (i.e., CMC-OD / TA-Fe-AEAA-Exos, CMC-OD / TA-Fe is represented in the figure), the color turns purplish-brown. The gelation time of the two different hydrogels was determined by the inverted test tube method. It was found that the gelation time of both hydrogels was less than 1 min, and the gelation time of CMC-OD / TA-Fe-AEAA-Exos was shorter than that of CMC-OD-AEAA-Exos. This indicates that the addition of TA-Fe promoted the cross-linking of the hydrogel, thus shortening the gelation time.

[0086] 5. Injectability, self-healing properties, and adhesive properties of hydrogels

[0087] To test the injectability of the hydrogel, such as Figure 8 As shown, we used a 5ml syringe to inject CMC-OD / TA-Fe-AEAA-Exos hydrogel and wrote "Li lab" in a 10cm diameter culture dish. After standing the dish upright, we observed that "Li lab" never detached from the dish wall. The injection of CMC-OD / TA-Fe-AEAA-Exos hydrogel was relatively smooth during this procedure, indicating that CMC-OD / TA-Fe-AEAA-Exos hydrogel has good injectability.

[0088] Figure 9 The self-healing properties of the hydrogel were demonstrated. First, the prepared heart-shaped CMC-OD / TA-Fe-AEAA-Exos hydrogel was cut in half with a blade and then put back together. After 30 minutes, the hydrogel was stretched to both sides with appropriate force using tweezers. It was found that the hydrogel did not separate from the break, indicating that the hydrogel has good self-healing properties.

[0089] like Figure 10As shown, we simulated and explored the elasticity and tissue adhesion of CMC-OD / TA-Fe-AEAA-Exos hydrogel through a porcine skin torsion experiment, evaluating the adaptive changes of the hydrogel in complex environments such as intestinal peristalsis within the abdominal cavity. A control group was set up with CMC-OD-AEAA-Exos hydrogel without tannic acid iron complex. Porcine skin with the attached gel was soaked in 0.9% physiological saline for 48 hours, then removed and torsioned in different directions, finally inverted. It was found that the CMC-OD-AEAA-Exos gel fractured, with only a small portion adhering to the porcine skin (indicated by the red arrow), while the CMC-OD / TA-Fe-AEAA-Exos gel remained structurally intact and did not detach from the porcine skin (indicated by the yellow arrow). These results indicate that the CMC-OD / TA-Fe-AEAA-Exos gel possesses strong viscoelasticity.

[0090] 6. Degradation and swelling properties of hydrogels

[0091] 400 μl of CMC-OD-AEAA-Exos and CMC-OD / TA-Fe-AEAA-Exos hydrogels (m0) were immersed in 5 mL centrifuge tubes containing 4 mL of phosphate-buffered saline (PBS, pH 7.4) and placed in a 37°C incubator. The tubes were removed from the PBS at time points (T = 1 day, 3 days, 5 days, 7 days, 9 days, 12 days, 15 days, 18 days, 21 days, and 24 days), lyophilized, and weighed. The degradation rate was calculated using the formula: from Figure 11 As we can see, in terms of overall degradation rate, the degradation rate of CMC-OD / TA-Fe-AEAA-Exos hydrogel is slower than that of CMC-OD-AEAA-Exos gel. This may be because the addition of TA-Fe enhances the structural strength of the gel, resulting in a slower degradation rate of CMC-OD / TA-Fe-AEAA-Exos hydrogel.

[0092] The freeze-dried CMC-OD-AEAA-Exos and CMC-OD / TA-Fe-AEAA-Exos hydrogels (m0) were immersed in 5 mL centrifuge tubes containing 3 mL of phosphate-buffered saline (PBS, pH 7.4) and placed in a 37°C incubator. The tubes were removed from the PBS at time points (T = 0.5 h, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, and 8 h), excess PBS was wiped off, and the weight of each sample (m1) was recorded until the sample weight no longer increased. The swelling rate was calculated using the following formula: Swelling rate (%) = (m1 - m0) / m0 × 100%. Experimental results are as follows: Figure 11As shown, the CMC-OD / TA-Fe-AEAA-Exos hydrogel exhibits good swelling behavior, and swelling equilibrium can be achieved within 300 min.

[0093] 7. Biocompatibility assessment

[0094] This study evaluated the biosafety performance of the hydrogel / exosome delivery system using cell viability / staining and CCK-8 cytotoxicity assays.

[0095] (1) Analysis of cell viability and mortality by hydrogel staining

[0096] We used different groups of hydrogels as experimental groups: CMC-OD group: CMC-OD-AEAA-Exos hydrogel, Gel group: CMC-OD / TA-Fe hydrogel, Gel / AEAA-Exos group: CMC-OD / TA-Fe-AEAA-Exos hydrogel, and normal culture medium as control group.

[0097] L929 cells were cultured in each of the above groups, and the effect of hydrogels on cell viability was detected using a live / dead staining kit. The results are shown in [Figure number missing]. Figure 12 .from Figure 12 It was observed that as time went on, the number of cells in different groups gradually increased, the cell morphology did not change significantly, and the number of dead cells in different groups was relatively small.

[0098] (2) Cell relative survival rate analysis

[0099] We evaluated the cytotoxicity of the experimental group Gel / AEAA-Exos hydrogel, namely CMC-OD / TA-Fe-AEAA-Exos hydrogel, using the CCK-8 cytotoxicity assay.

[0100] like Figure 13 As shown, when cells were cultured using Gel / AEAA-Exos hydrogel extract, the cell viability was found to be above 91.7% after culturing L929 cells for 1, 3, and 5 days with different concentrations of extract (10%, 30%, 50%, and 100%). The experimental results indicate that the Gel / AEAA-Exos hydrogel exhibits good biosafety properties.

[0101] 8. Evaluation of sustained-release exosome characteristics

[0102] Using CMC-OD / TA-Fe-AEAA-Exos hydrogel as the experimental subject, a PKH-26-labeled hydrogel system was first prepared in a 5 ml EP tube with a volume of 800 μl, and allowed to stand for 30 min. Then, an appropriate volume of PBS buffer was added to the EP tube, and the tube was incubated at 37℃ with gentle shaking at a medium speed. The total exosome release over time was quantitatively analyzed using a microplate reader, and the cumulative release over 28 consecutive days was measured (n=3 per group).

[0103] See results Figure 14 The exosome release curves show that the hydrogel-released exosomes are released at a relatively fast rate in the first 4 days. After 4 days, the release rate of exosomes gradually slows down, and then the release of exosomes gradually flattens out, reaching 79.8% on day 28.

[0104] 9. In vitro targeting assessment

[0105] This study used DSPE-PEG-AEAA to engineer exosomes derived from umbilical cord mesenchymal stem cells, enabling the exosomes to target and bind to Sigma receptors on the surface of activated hepatic stellate cells, thereby improving the therapeutic efficacy of exosomes.

[0106] The targeting performance of exosomes modified with AEAAA was evaluated using an experiment involving uptake of exosomes by activated hepatic stellate cells. The experiment was conducted in three groups:

[0107] Non-vectorized exosomes (Gel / Exos): CMC-OD / TA-Fe-Exos hydrogel;

[0108] Vectorized exosomes (Gel / AEAA-Exos): CMC-OD / TA-Fe-AEAA-Exos hydrogel;

[0109] Control group: PBS.

[0110] See results Figure 15 ,Depend on Figure 15 Numerous red granules were observed around the nuclei of activated hepatic stellate cells in both the non-vectored exosome group (Exos) and the vectored exosome group (Gel / AEAA-Exos) (left image). These red granules are PKH-26-labeled exosomes, indicating that both Exos and Gel / AEAA-Exos can be taken up by activated hepatic stellate cells. Quantification results of exosomes (right image) show that the amount taken up by activated hepatic stellate cells in the Gel / AEAA-Exos group was significantly greater than that in the Exos group, with a statistically significant difference (P<0.01). This indicates that exosomes modified with AEAA have significant targeting properties.

[0111] 10. ROS resistance assessment

[0112] The anti-ROS effect of exosomes was detected using the DCFH-DA fluorescent probe method, a reactive oxygen species (ROS) detection method. The following three groups were set up for the experiment:

[0113] Non-vectorized exosomes (Gel / Exos): CMC-OD / TA-Fe-Exos hydrogel;

[0114] Vectorized exosomes (Gel / AEAA-Exos): CMC-OD / TA-Fe-AEAA-Exos hydrogel;

[0115] Positive control group (PC): Reactive oxygen species positive control reagent (Rosup).

[0116] See results Figure 16 ,from Figure 16 The left image shows that green fluorescence is distributed in activated hepatic stellate cells from the positive control group (PC), Gel / Exos group, and Gel / AEAA-Exos group. This is because a large amount of ROS in the cells oxidizes DCFH-DA to DCFH, resulting in green fluorescence. The quantification results of fluorescence intensity (right image) show that vectorized exosomes have stronger anti-ROS properties compared to non-vectored exosomes.

[0117] 11. Evaluation of in vitro antifibrotic effect

[0118] We further evaluated the therapeutic effect of vectored exosomes on fibrosis in vitro. The experiment was conducted in three groups:

[0119] Non-vectorized exosomes (Gel / Exos): CMC-OD / TA-Fe-Exos hydrogel;

[0120] Vectorized exosomes (Gel / AEAA-Exos): CMC-OD / TA-Fe-AEAA-Exos hydrogel;

[0121] Control group: PBS.

[0122] See results Figure 17 ,like Figure 17 As shown in Figure A, we observed α-SMA (red fluorescent signal) distributed around the blue cell nuclei in different groups. This experimental result indicates that both Gel / Exos and Gel / AEAA-Exos can clear α-SMA produced by activated hepatic stellate cells, and that vectorized exosomes exhibit a stronger ability to clear α-SMA compared to non-vectored exosomes. Figure 17As shown in Figure B, the green fluorescent signal around the cell nucleus represents CollaI produced by activated hepatic stellate cells. This experimental result indicates that both Gel / Exos and Gel / AEAA-Exos can scavenge CollaI produced by activated hepatic stellate cells, and that vectorized exosomes exhibit a stronger ability to scavenge CollaI compared to non-vectored exosomes. In conclusion, these experimental results suggest that the CMC-OD / TA-Fe-AEAA-Exos hydrogel delivery system possesses good anti-fibrotic ability in vitro.

[0123] 12. In vivo targeting assessment

[0124] Two experimental groups were set up: the non-vectored exosome group (Gel / Exos): CMC-OD / TA-Fe-Exos hydrogel; and the vectored exosome group (Gel / AEAA-Exos): CMC-OD / TA-Fe-AEAA-Exos hydrogel. SD rats were used as the research subjects (n=5). The targeting effect of this delivery system was evaluated using tissue immunofluorescence.

[0125] The experiment evaluating the targeting effect of exosomes using tissue immunofluorescence was divided into two groups: Group 1 and Group 2. The experimental group was treated with PKH-26-labeled vectorized exosomes (Gel / AEAA-Exos), while the control group was treated with PKH-26-labeled non-vectored exosomes (Gel / Exos). Liver tissue nuclei were stained with DAPI. Activated hepatic stellate cells were located using blue fluorescent labeling via α-SMA high-expression sites, and green fluorescent labeling was used. The purpose of this experiment was to observe and compare the targeting ability of AEAAA-modified exosomes. Rats were injected with 400 μg of the corresponding exosomes once.

[0126] This study used α-SMA as a marker for activated hepatic stellate cells. Liver tissues from two groups of SD rats were collected at the 7-day observation point for histoimmunofluorescence analysis. Figure 18 As shown, a large number of exosomes (red fluorescent signal) were found to accumulate around α-SMA (green fluorescent signal) in the Gel / AEAA-Exos group, while the red signal area around α-SMA was relatively small in the Gel / Exos group. These experimental results indicate that vectored exosomes have better targeting performance than non-vectored exosomes.

[0127] 13. In vivo safety assessment

[0128] We evaluated the biocompatibility of the hydrogel / exosome delivery system by analyzing the histomorphology of the liver, spleen, kidney, lung, and heart of C57BL / 6J mice 7 days after implantation with the Gel / AEAA-Exos delivery system, as well as normal mice. The experimental group (vectored exosome group, Gel / AEAA-Exos) consisted of mice injected with CMC-OD / TA-Fe-AEAA-Exos hydrogel; the control group consisted of normal mice. Each mouse was given 800 μg of the corresponding exosomes once.

[0129] The results are as follows Figure 19 As shown, the morphology of liver cells and the structure of liver lobules in the Gel / AEAA-Exos group were not significantly abnormal, and there was no obvious inflammatory cell infiltration in the tissues, showing no significant difference from the normal group. In other organs of the experimental group, such as the spleen, kidneys, lungs, heart, and brain, the morphology of parenchymal cells was not significantly abnormal, and there was no obvious inflammatory cell infiltration in the tissues, showing no significant difference from the normal group. These results indicate that Gel / AEAA-Exos has good biological safety.

[0130] 14. In vivo anti-fibrotic assessment

[0131] Animal experimental group for anti-liver fibrosis treatment: Mice were randomly divided into 5 groups (n=5): normal group, model group, gel group, gel / Exos group, and gel / AEAA-Exos group. Hydrogels were prepared according to the aforementioned proportions, with a volume of 300 μL per mouse. Treatments for each group were as follows:

[0132] Normal group: Mice were fed normal rat diet for 12 weeks.

[0133] Model group: Mice were intraperitoneally injected with 2.5 μl / g of CCl4 / olive oil (4 / 1, V / V) twice a week for 8 weeks. Starting from week 9, rats were intraperitoneally injected with 300 μl of PBS and observed for 30 days.

[0134] Gel group: Mice were intraperitoneally injected with 2.5 μl / g CCl4 / olive oil twice a week for 8 consecutive weeks. Starting from week 9, mice were intraperitoneally injected with hydrogel (CMC-OD / TA-Fe hydrogel) and observed for 30 days.

[0135] Gel / Exos group: Mice were intraperitoneally injected with 2.5 μl / g of CCl4 / olive oil twice a week for 8 weeks. Starting from week 9, mice were intraperitoneally injected with the gel / Exos (CMC-OD / TA-Fe-Exos hydrogel) system and observed for 30 days.

[0136] Gel / AEAA-Exos group (Gel / AEAA-Exos): Mice were intraperitoneally injected with 2.5 μl / g of CCl4 / olive oil twice a week for 8 weeks. Starting from week 9, rats were intraperitoneally injected with the gel / AEAA-Exos (CMC-OD / TA-Fe-AEAA-Exos hydrogel) system and observed for 30 days. Both the gel / Exos group and the gel / AEAA-Exos group were given 800 μg / mouse of the corresponding exosomes.

[0137] Thirty days after the intervention, the mice were anesthetized by inhalation of the anesthetic isoflurane. Liver tissue was collected for further analysis at the end of the experiment.

[0138] like Figure 20 As shown, visual observation revealed significant fibrotic scarring in the livers of the Model and Gel groups, while there was no significant difference between the Gel / Exos and Gel / AEAA-Exos groups. The livers of the Normal group mice had relatively smooth surfaces with no obvious fibrotic scarring. Masson staining results showed that both the Model and Gel groups exhibited obvious pathological features of liver fibrosis, with collagen fibers forming ring-like strands that created pseudolobules (blue strands) connecting various portal areas and even the central venous area. However, the pseudolobule structures formed by liver fibrosis in the Gel / Exos and Gel / AEAA-Exos groups were less pronounced than those in the Model and Gel groups, and some were even absent. Quantitative analysis of the fibrotic tissue revealed a significant reduction in fibrosis in the Gel / Exos and Gel / AEAA-Exos groups compared to the Model and Gel groups. Moreover, the amount of fibrosis in the Gel / AEAA-Exos group was significantly less than that in the Gel / Exos group. Furthermore, Sirus staining results showed the same findings as Masson staining. These experimental results confirm that CMC-OD / TA-Fe has no significant anti-liver fibrosis effect, while hydrogel containing exosomes has a significant anti-fibrosis effect. However, compared with the non-vectored exosome group, the hydrogel / AEAA-Exos system has a significant effect in reducing liver fibrosis.

[0139] As can be seen from the above embodiments, the present invention provides a hydrogel loaded with targeted modified exosomes, its preparation method, and its application. The hydrogel loaded with exosomes prepared by the present invention has good biocompatibility, can stably and sustainably release AEAAA-targeted modified exosomes, has a targeting effect on activated hepatic stellate cells in liver fibrosis tissue, and can significantly alleviate liver fibrosis, showing promise as a new approach for the treatment of liver fibrosis.

[0140] This article uses specific examples to illustrate the inventive concept in detail. The description of the above embodiments is only for the purpose of helping to understand the core idea of ​​the present invention. It should be noted that any obvious modifications, equivalent substitutions or other improvements made by those skilled in the art without departing from the inventive concept should be included within the protection scope of the present invention.

Claims

1. A hydrogel loaded with targeted modified exosomes, characterized in that, The ingredients include: carboxymethyl chitosan, oxidized dextran, tannic acid-iron complex, targeted modified exosomes, PBS and water; The targeted modified exosomes were obtained by mixing and incubating DSPE-PEG-AEAA with PBS solution of exosomes derived from umbilical cord mesenchymal stem cells.

2. The hydrogel according to claim 1, characterized in that, The mass ratio of DSPE-PEG-AEAA to exosomes derived from umbilical cord mesenchymal stem cells was 2–8:1, the incubation temperature was 25–40℃, and the incubation time was 0.5–2 h.

3. The hydrogel according to claim 1, characterized in that, The molecular weight range of the DSPE-PEG-AEAA is 3000±200 Da.

4. The method for preparing the hydrogel according to any one of claims 1 to 3, characterized in that, include: Oxidized dextran, targeted modified exosomes, and PBS were mixed to obtain an oxidized dextran exosome suspension; A hydrogel loaded with targeted modified exosomes was obtained by mixing oxidized dextran exosome suspension, carboxymethyl chitosan PBS solution, and tannic acid-iron complex aqueous solution.

5. The method according to claim 4, characterized in that, The mass percentage concentration of the oxidized dextran exosome suspension is 0.5-1.5%; the mass percentage concentration of the carboxymethyl chitosan PBS solution is 2.0-4.0%; the mass percentage concentration of the tannic acid-iron complex aqueous solution is 20-25%; and the volume ratio of the oxidized dextran exosome suspension, the carboxymethyl chitosan PBS solution, and the tannic acid-iron complex aqueous solution is 6-12:6-12:

1.

6. The preparation method according to claim 4, characterized in that, The method for preparing oxidized dextran is as follows: a dextran aqueous solution is mixed with sodium periodate and reacted. After the reaction is completed, a quenching agent is added to terminate the reaction. Dialysis is performed, and the dialysate is collected and freeze-dried. The molecular weight of dextran is 1×10⁻⁶. 4 ~5×10 5 The concentration of Da, dextran aqueous solution is 40-80 mg / mL.

7. The preparation method according to claim 6, characterized in that, The dialysis was performed using a dialysis bag with a molecular weight cutoff of 3000–4000 Da, at a drying temperature of -15 to -25°C, for a time of 28–36 hours.

8. The use of the hydrogel loaded with targeted modified exosomes as described in any one of claims 1 to 4 in the preparation of products that improve liver fibrosis.

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