Oral or intraperitoneal injectable exosome hydrogel and preparation method thereof

Abstract

The invention discloses exosome hydrogel capable of being orally taken or intraperitoneally injected and a preparation method of the exosome hydrogel, and belongs to the technical field of exosome delivery. The exosome hydrogel is prepared from carboxymethyl chitosan, oxidized dextran, a tannic acid-iron complex, an umbilical cord mesenchymal stem cell-derived exosome, PBS (Phosphate Buffer Solution) and water. The preparation method comprises the following steps: mixing an oxidized dextran PBS solution and an umbilical cord mesenchymal stem cell-derived exosome PBS solution to obtain an oxidized dextran exosome suspension; the oxidized dextran exosome suspension, a carboxymethyl chitosan PBS solution and a tannic acid-iron complex aqueous solution are mixed, oscillated and left to stand, and the exosome hydrogel is obtained. The exosome hydrogel disclosed by the invention can be used by oral administration or intraperitoneal injection, has the advantages of good biocompatibility, good degradability, good structural stability, sustained release and the like, and lays a foundation for safe and accurate treatment of diseases related to exosome delivery.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of exosome delivery, in particular to an orally or intraperitoneally injectable exosome hydrogel and a preparation method thereof. BACKGROUND

[0002] Stem cells are a group of cell lines that can self-renew, continuously reproduce, and have the potential to differentiate into various functional cells. Mesenchymal stem cells (MSCs), as a kind of multipotent stem cells, play an important role in regenerative medicine. Related research reports suggest that the tissue remodeling and regeneration therapy of stem cells is related to their own paracrine function, and exosomes derived from stem cells are important paracrine substances. Mesenchymal stem cell-derived exosomes have been studied for the treatment of multiple sclerosis, cartilage tissue repair, ischemic stroke, renal toxicity, and anti-fibrosis diseases.

[0003] Although exosomes have great advantages in the treatment of various diseases, systemic administration of exosomes may be partially removed by the endothelial reticular system in the blood, which cannot meet the long-term preservation and sustainable release treatment requirements in the body. Therefore, it is necessary to provide an effective delivery platform for the slow release of exosomes. The medical application of hydrogels carrying exosomes for minimally invasive implantation shows great superiority due to their slow release effect. However, the current exosome-loaded hydrogel system still has problems such as unsatisfactory biocompatibility, poor degradability, and poor structural stability.

[0004] Analyzing the in vivo distribution of exosomes is a prerequisite for developing exosome-based therapeutic methods and drug delivery carriers, which can accurately predict the therapeutic dose and potential side effects. Therefore, determining the distribution of exosomes in various organs is crucial for precise treatment of diseases. There have been reports on the biodistribution of mesenchymal stem cells (MSCs) in animal models and the role of exosomes following systemic delivery of MSCs (Biodistribution of mesenchymal stem cells (MSCs) in animal models and implied role of exosomes following systemic delivery of MSCs: a systematic review. Am J Transl Res. 2022; 14(4): 2147-2161. ISSN: 1943-8141.). However, the distribution of exosomes encapsulated by hydrogels after oral or intraperitoneal injection for implantation into the body and slow release into important organs such as the heart, liver, spleen, lungs, kidneys, and brain has not been reported in the literature. SUMMARY

[0005] The present application aims to solve the problems of poor biocompatibility, degradability and structural stability of the exosome hydrogel delivery sustained-release system in the prior art, and the single delivery route, and provides an oral or intraperitoneal injection exosome hydrogel.

[0006] In a first aspect, the present application provides an oral or intraperitoneal injection exosome hydrogel made of carboxymethyl chitosan, oxidized dextran, tannic acid-iron complex, umbilical cord mesenchymal stem cell-derived exosomes, PBS and water.

[0007] In a second aspect, the present application provides a preparation method of the above-mentioned exosome hydrogel, comprising:

[0008] Mixing the oxidized dextran PBS solution and the umbilical cord mesenchymal stem cell-derived exosome PBS solution to obtain an oxidized dextran exosome suspension;

[0009] Mixing, oscillating and standing the oxidized dextran exosome suspension, the carboxymethyl chitosan PBS solution and the tannic acid-iron complex aqueous solution to obtain the exosome hydrogel.

[0010] Optionally or preferably, in the above preparation method, the preparation method of the oxidized dextran is: mixing and reacting the dextran aqueous solution with sodium periodate, adding a quenching agent to terminate the reaction after the reaction is completed, dialysis, collecting the dialysate and then drying after being cooled by liquid nitrogen;

[0011] The molecular weight of the dextran is 1x10 4 ~ 5x10 5 Da, and the concentration of the dextran aqueous solution is 40~80mg / mL.

[0012] The reaction is preferably carried out in the dark for 3~8h, and the stirring speed during the reaction is preferably 400~700rpm, and the quenching agent is preferably ethylene glycol.

[0013] Optionally or preferably, in the above preparation method, the dialysis is carried out using a dialysis bag with a molecular weight cut-off of 3000~4000Da, the drying temperature is-15~ -25℃, and the time is 28~36h.

[0014] Optionally or preferably, in the above preparation method, the mass ratio of sodium periodate to dextran is 0.5~3.0:0.2~3.0.

[0015] Optionally or preferably, in the above preparation method, the mass ratio of the oxidized dextran and the umbilical cord mesenchymal stem cell-derived exosomes is 2~8:1~3. The mixing speed is preferably 150~250rpm, and the mixing time is preferably 3~9min.

[0016] Optionally or preferably, in the preparation method, the concentration of carboxymethyl chitosan in the carboxymethyl chitosan PBS solution is 2.0-4.0%, and the concentration of tannic acid-iron complex in the tannic acid-iron complex aqueous solution is 20-25%.

[0017] Optionally or preferably, in the preparation method, 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.

[0018] Compared with the prior art, the present application has the following beneficial effects:

[0019] In the present application, carboxymethyl chitosan and oxidized dextran have good biocompatibility and degradability, and are used to form the hydrogel; the metal polyphenol substance tannic acid-iron complex (TA-Fe) can enhance the structural stability of the hydrogel. In the present application, carboxymethyl chitosan, oxidized dextran, and tannic acid-iron complex are used to form a composite hydrogel material delivery system to encapsulate umbilical cord stem cell-derived exosomes. The hydrogel is implanted locally into the stomach of a mouse through the oral route (oral administration in animal models is replaced by gavage), and the biodistribution of the released exosomes in the important organs of C57BL / 6J mice is preliminarily explored through small animal imaging technology and tissue immunofluorescence experiments. The present application lays a foundation for the safe and precise treatment of hydrogel / exosome delivery systems in diseases related to the liver, spleen, kidney, heart, lung, and brain.

[0020] Compared with intravenous injection of a systemic drug, the exosome hydrogel of the present application has high safety and efficiency, has a certain adhesion to tissues, and can slowly release mesenchymal stem cell-derived exosomes, thereby serving as a carrier for reaching important organs such as the liver, spleen, kidney, heart, lung, and brain to improve related diseases, and providing a reference for the safe and efficient treatment of liver, heart, spleen, lung, kidney, and brain diseases using hydrogel to carry stem cell-derived exosomes. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The particle size of the exosomes in Test Example 1 is shown in Table 1.

[0022] Figure 2 The microstructure of the exosomes observed by TEM in Test Example 1 is shown in Table 2.

[0023] Figure 3 The detection results of the surface membrane proteins of the exosomes in Test Example 1 are shown in Table 3.

[0024] Figure 4Microscopic morphology of the exosome hydrogel in Test Example 2

[0025] Figure 5 Fourier transform infrared spectrum of the synthesis of the hydrogel in Test Example 2

[0026] Figure 6 Gelation of the hydrogel in Test Example 2

[0027] Figure 7 Sustained-release properties of the hydrogel in Test Example 2

[0028] Figure 8 Results of the live-dead staining of the hydrogel in Test Example 2

[0029] Figure 9 Cell relative survival rate measured after treatment with the hydrogel extract in Test Example 2

[0030] Figure 10 Pathological changes of the hydrogel in Test Example 2 in important organs

[0031] Figure 11 Macroscopic state evaluation of the exosome hydrogel in Test Example 3 in mice

[0032] Figure 12 Evaluation of the state of the exosome hydrogel in vivo by small animal imaging technology in Test Example 3.

[0033] Figure 13 Evaluation of the distribution of the exosome hydrogel secreted by the hydrogel in the liver, spleen, kidney, heart, lung, and brain by small animal imaging technology in Test Example 3.

[0034] Figure 14 Evaluation of the distribution of the exosome hydrogel secreted by the hydrogel in the liver, spleen, kidney, heart, lung, and brain by tissue immunofluorescence technology in Test Example 3.

[0035] Figure 15 Comparative analysis of the distribution of the exosome hydrogel secreted by the hydrogel in the liver, spleen, kidney, heart, lung, and brain in Test Example 3.

[0036] Figure 16 Macroscopic state evaluation of the exosome hydrogel in Test Example 4 in mice

[0037] Figure 17 Evaluation of the state of the exosome hydrogel in vivo by small animal imaging technology in Test Example 4.

[0038] Figure 18 Evaluation of the distribution of the exosome hydrogel secreted by the hydrogel in the liver, spleen, kidney, heart, lung, and brain by small animal imaging technology in Test Example 4.

[0039] Figure 19 In Experiment 4, the distribution of exosomes secreted by the hydrogel in the liver, spleen, kidney, heart, lungs, and brain was evaluated using tissue immunofluorescence.

[0040] Figure 20 In Experiment 4, the distribution of exosomes secreted by the hydrogel in the liver, spleen, kidney, heart, lungs, and brain was compared and analyzed. Detailed Implementation

[0041] 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.

[0042] Example 1: Preparation of exosomes derived from umbilical cord mesenchymal stem cells

[0043] 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.

[0044] 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. 3 Small 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.

[0045] Exosome extraction was performed according to the kit method. First, the cells were centrifuged at 3000 g for 10 min to remove the cells and part of the cell fragments; the supernatant was collected and transferred to a new centrifuge tube and centrifuged at 10000 g for 15 min to further remove fine fragments and impurities; then the newly obtained supernatant was transferred to an ultrafiltration centrifuge tube (MWCO 100 kd) at a time of 15 mL, and after centrifugation at 5000 rpm for 15 min, the supernatant was concentrated to 20 ml. Then 20 mL of the concentrated supernatant was weighed and mixed with 5 mL of ExoQuick-TC liquid by gently blowing, and was allowed to react at 4°C in the refrigerator for 24 h. Then centrifuged at 10000 g for 60 min, the exosomes were precipitated on the wall of the centrifuge tube, and the supernatant was gently aspirated. The obtained exosomes were resuspended with 200 μL PBS, and then resuspended by centrifugation at 12000 g for 2 min, to obtain the exosome stock solution, which was then placed in a -80°C refrigerator for standby.

[0046] Example 2 Preparation of exosome hydrogel

[0047] (1) 1.0 g of dextran (Dextran, Shanghai Aladdin Biochem Technology Co., Ltd., molecular weight 1 x 10 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.

[0048] (2) 0.80 g of sodium periodate (NaIO4) was added to the dextran aqueous solution obtained in step (1), and the molar ratio of NaIO4 to dextran was 1.3:1. The mixture was reacted at room temperature (25°C) in the dark for 5 h, and the stirring speed during the reaction was 500 rpm. After the reaction, 2 mL of ethylene glycol was added to terminate the reaction for 1 h. The dialysis bag with a cut-off component of 3500 Da was used for dialysis for 3 days, and the liquid was changed every 4 hours until there was no periodate in the dialysate (the detection method of periodate was: 0.5 mL of dialysate was added to 0.5 mL of 1% silver nitrate solution, and if there was no precipitate, it indicated that there was no periodate). After dialysis, the dialysate was quickly placed in a freeze dryer after being quenched by liquid nitrogen, and was freeze-dried at -20°C for 32 h to obtain oxidized dextran (OD).

[0049] (3) 0.05 g of oxidized dextran prepared in step (2) was weighed and dissolved in 5 mL of 1 x PBS buffer solution, and 2 mg of exosome stock solution of umbilical cord mesenchymal stem cell-derived exosomes prepared in Example 1 was added. The mixture was stirred in a magnetic stirrer at a speed of 200 rpm for 5 min to obtain a 1% oxidized dextran exosome suspension.

[0050] (4) Dissolve ferric chloride hexahydrate in distilled water to prepare a ferric chloride solution with a concentration of 9 mM; dissolve tannic acid in distilled water to prepare a tannic acid solution with a concentration of 27 mM; mix the ferric chloride solution with the tannic acid solution to obtain a tannic acid-iron complex aqueous solution with a mass percentage concentration of 23.25%;

[0051] (5) Dissolve carboxymethyl chitosan powder (degree of deacetylation 90%, carboxymethyl 80%) in PBS buffer to obtain a carboxymethyl chitosan PBS solution with a mass percentage concentration of 3%; mix 400 μL of the 3% carboxymethyl chitosan PBS solution, 50 μL of the 23.25% tannic acid-iron complex aqueous solution, and 400 μL of the 1% oxidized dextran exosome suspension obtained in step (3) by oscillation for 10 s, and then obtain an exosome hydrogel (CMC-OD / TA-Fe-Exos) after the gelation.

[0052] Characterization and function of exosomes in test example 1

[0053] 1. Diameter of exosomes

[0054] Take 40 μL of the exosome stock solution prepared in Example 1, dilute to 1 mL, and then perform particle size detection using a nanoparticle tracking analyzer (NTA). The NTA results are shown in Figure 1 , which show that the diameter of the exosomes is about 90 nm.

[0055] 2. Structure of exosomes

[0056] After fixing the exosome stock solution with 2.5% glutaraldehyde solution at 4°C overnight, take 10 μL and drop it onto a copper mesh, and then stand at room temperature for 5 min, and then absorb the liquid with filter paper. Add staining solution (saturated uranyl acetate solution) to the copper mesh and stain for 1 min, and then absorb the liquid with filter paper. Add ddH2O to the copper mesh and stand at room temperature for 5 min, and then absorb the liquid with filter paper; repeat once. Dry at room temperature, and then observe and take pictures under a TEM. The TEM results are shown in Figure 2 , which show that the exosomes have clear membrane structures in the form of tea tray or cup, with clear edges and uniform distribution without aggregation.

[0057] 3. Detection of exosome markers

[0058] After the exosome concentration was detected, 20 μg of exosomes (an equal amount of culture supernatant was taken as a control) were mixed with 5x SDS-PAGE Loading Buffer and heated at 95°C for 5 min. SDS-PAGE electrophoresis: the acrylamide separation gel concentration was 12%, and each sample well was added with the above-mentioned heated supernatant for electrophoresis (70V / 30min, 120V / 60min). Membrane transfer: wet transfer at 250mA for 90min. Blocking: blocking at room temperature for 1h with 5% skimmed milk. Incubation of primary antibody: the antibody was diluted with 5% skimmed milk (antibody dilution 1000 times), and incubated at 4°C overnight. Incubation of secondary antibody: the primary antibody was aspirated, the membrane was washed with TBST for 3 times (5min / time), HRP-labeled secondary antibody (1:5000) was added, and incubated at room temperature for 1h. Development and imaging: after the incubation of secondary antibody, the membrane was washed with TBST for 3 times (5min / time), and developed and imaged with ELC luminescent solution. The exosome surface membrane protein detection results are shown in Figure 3 It can be seen that the exosome markers TSG101, CD63 and CD9 are positively expressed.

[0059] Test Example 2: Exosome hydrogel performance

[0060] 1. Micro-morphology of exosome hydrogel

[0061] After the exosome hydrogel prepared in Example 2 was freeze-dried, the micro-morphological characteristics of the hydrogel were measured using a scanning electron microscope (SEM), and the elemental content of C, O, N, Fe and P in the hydrogel was analyzed using an energy dispersive spectrometer (EDS) to characterize the composition of the hydrogel. The successful synthesis of oxidized dextran was evaluated using a Fourier transform infrared spectrometer (FTIR). A large number of exosomes can be seen attached to the surface of the hydrogel under a high-power lens (see Figure 4 A) of Figure 4 B). In addition to C, N and O elements, P and Fe elements were also found, as shown in Figure 4 B, which may be related to the encapsulation of exosomes and TA-Fe in the hydrogel, because the exosome membrane contains P elements, and the tannic acid-iron complex contains Fe elements.

[0062] 2. Fourier infrared spectrum evaluation of exosome hydrogel

[0063] As shown in the Fourier spectrum in Figure 5 , the peak intensity of oxidized dextran (OD) as a whole decreased compared with dextran (Dex), and a new absorption peak appeared at 1734.1 cm -1 , which may be the result of the mutual influence of the aldehyde group after the oxidation of dextran and the inherent groups of dextran, confirming that dextran has been successfully oxidized and a large number of aldehyde groups have been formed.

[0064] 3. Exosome hydrogel gelation

[0065] As shown in Figure 6 , we prepared CMC-OD / TA-Fe-Exos exosome hydrogel in a transparent ampoule. It can be observed that CMC-OD / TA-Fe-Exos is purple-brown and becomes a solid jelly after gelation.

[0066] 4. Exosome hydrogel sustained-release characteristics

[0067] In order to detect the sustained-release characteristics of the exosome hydrogel, CMC-OD / TA-Fe-Exos was used as the experimental object. First, a PKH-26-labeled hydrogel system was prepared in a 5-ml EP tube, with a volume of 800 μl hydrogel, and left to stand for 30 min. Then, an appropriate volume of PBS buffer was added to the EP tube, which was placed in a 37°C incubator and shaken slowly at medium speed. The total amount of exosomes released over time was quantitatively analyzed using a microplate reader, and the cumulative release amount was detected for 10 consecutive days (n = 3 for each group).

[0068] As shown in Figure 7 , through the exosome release curve, it can be found that the release rate of the hydrogel-sustained exosomes is faster in the first 3 days, and the release rate of the exosomes gradually slows down after 3 days, and the release is relatively stable, indicating that the exosome hydrogel has good sustained-release characteristics.

[0069] 5. Biological safety evaluation

[0070] In this study, the biological safety performance of the hydrogel / exosome delivery system was evaluated by cell live / dead staining and CCK-8 cytotoxicity test.

[0071] (1) Live / dead staining analysis of cells by hydrogel

[0072] CMC-OD group: Mix equal volumes of 3% carboxymethyl chitosan PBS solution and 1% oxidized dextran PBS solution to obtain CMC-OD hydrogel.

[0073] CMC-OD / TA-Fe group: Prepared according to the method of Example 2, except that no exosomes were added, and the remaining steps were the same as those of Example 2.

[0074] We used different groups of hydrogels as experimental groups (CMC-OD group and CMC-OD / TA-Fe group), and normal culture medium as the control group (Control), and cultured L929 cells to detect the effect of hydrogel on cell activity by live / dead staining kit. From Figure 8 it can be observed that there is no significant difference in the number of cells in different groups, and the cell morphology has no significant change.

[0075] (2) Analysis of relative survival rate of cells

[0076] We evaluated the cytotoxicity of hydrogel by CCK-8 test. As shown in Figure 9 Figure 2, the L929 cells were cultured with the extract of CMC-OD / TA-Fe-Exos prepared in Example 2, and it was found that the survival rate of cells was above 80% after 1, 2 and 3 days of culture with different concentrations of extract (10%, 30%, 50% and 100%). The experimental results showed that the CMC-OD / TA-Fe-Exos exosome hydrogel had good biological safety performance.

[0077] 6. In vivo safety evaluation

[0078] We analyzed the histomorphology of liver, spleen, kidney, lung, heart and brain of normal mice and C57BL / 6J mice implanted with CMC-OD / TA-Fe-Exos for 1 day by HE staining to evaluate the biological safety of the gel.

[0079] As shown in Figure 10 Figure 6 are the pathological images of main organs (liver, spleen, kidney, heart, lung and brain) of mice implanted with OD-CMC / TA-Fe-Exos exosome hydrogel by gavage for 24 h. Through the HE tissue staining pictures, it was found that the morphology of parenchymal cells in liver, spleen, kidney, heart, lung and brain tissues was not obviously abnormal, and there was no obvious inflammatory cell infiltration in the tissues at different times. The above results showed that the OD-CMC / TA-Fe-Exos exosome hydrogel had good biological safety performance.

[0080] Example 3 In vivo study and evaluation of exosome hydrogel after implantation by gavage

[0081] 1. Evaluation of the macroscopic state of exosome hydrogel in mice in vivo

[0082] Mice were implanted with OD-CMC / TA-Fe-Exos exosome hydrogel by gavage, and the residual situation of exosome hydrogel was observed at different times after implantation.

[0083] After implantation of exosome hydrogel by gavage, the abdominal cavity and gastrointestinal tract of the mouse were opened, and it could be clearly observed that a large amount of exosome hydrogel remained in the stomach of the mouse 1 h after gavage. With the passage of time, the residual amount at 4 h, 8 h, 12 h and 24 h was significantly reduced compared with that at 1 h observation point. Especially at the 12 h observation point, the residual amount of exosome hydrogel in the stomach of the mouse was almost difficult to observe, but a certain amount of residual was found in the small intestine. As shown in Figure 11 .

[0084] 2. Small animal imaging to evaluate the state of exosome hydrogel in vivo

[0085] Figure 12The bio-distribution of hydrogel-released exosomes in mice was evaluated over time by small-animal live imaging. It was observed that the signal was stronger at 4h than at 1h, which might be due to the fact that the hydrogel in the 1h observation point was blocked by food in the stomach. The signal was relatively weak at 12h and 24h, which might be due to the fact that the hydrogel was eliminated from the body through the digestive tract. However, strong signals were still observed at 96h and 144h, and the signals were located in the upper abdomen, which was consistent with the location of the liver and spleen. It was considered that the signal was formed by the exosomes released by the hydrogel reaching and accumulating in the liver tissue and being excited by the excitation light.

[0086] 3. Small-animal imaging evaluation of the distribution of hydrogel-released exosomes in various organs in vivo

[0087] In addition to live imaging of mice, the bio-distribution of exosomes in the liver, spleen, kidney, heart, lung, brain and other organs was evaluated by small-animal imaging at the 24h and 144h observation points, and the fluorescence signal intensity of different groups of organs was quantitatively analyzed.

[0088] As shown in Figure 13 , at the 24h observation point, the liver tissue showed a stronger fluorescence signal than other organs. At the 144h observation point, the accumulation of exosomes in the liver was significantly reduced. This might be due to the consumption of exosomes in the body and the elimination of hydrogel from the body through the digestive tract. This result was consistent with the results shown in Figure 12 . In summary, the fluorescence intensity evaluation of mice in vivo and organs by small-animal imaging showed that the oral administration of exosome hydrogel (gel jelly) could release exosomes to the liver, and the accumulation of exosomes in the liver was significantly more than in other organs.

[0089] 4. Evaluation of the distribution characteristics of hydrogel-released exosomes in various organs in vivo by tissue immunofluorescence technology

[0090] After the exosome hydrogel was administered to mice by gavage, the bio-distribution of PKH-26-labeled exosomes in the liver, spleen, kidney, lung, heart and brain tissues at different time observation points (1h, 4h, 8h, 12h, 24h) was evaluated by inverted microscope, and the red area represented the exosome-rich area, with 3 mice in each group.

[0091] From Figure 14It can be clearly seen that the red area of each organ gradually increases with time, indicating that the accumulation of exosomes in the liver, spleen, kidney, lung, heart and brain tissue gradually increases. The quantitative analysis curve also shows the corresponding experimental results. It can also be found that the distribution of exosomes in the liver is first from the portal area to the central vein area, and finally spreads to the whole liver tissue. The reason for this distribution feature is that the portal vein is in the portal area, and the exosomes administered by the oral route first reach the liver through the portal vein.

[0092] 5. Comparative analysis of the distribution characteristics of hydrogel-secreted exosomes in various organs in vivo

[0093] After the exosome hydrogel was implanted into the mouse body by the oral administration route, the fluorescence intensity of PKH-26-labeled exosomes in the liver, spleen, kidney, lung, heart and brain tissue at different time observation points (4h, 12h, 24h) was quantitatively analyzed.

[0094] Reference Figure 15 From the column chart, it can be found that the most distributed exosome hydrogel sustained-release umbilical cord mesenchymal stem cell-derived exosomes are in the liver, followed by the spleen, and the least accumulated in the brain. It is worth noting that there is no report on the research of exosome hydrogel (gel jelly) sustained-release exosomes reaching various organs by oral implantation. In addition, according to the results of this study, we speculate that under the condition of multiple administrations, exosomes administered by the oral administration route may also have the advantage of accumulating in large quantities in various organs, and the treatment method is safer than the traditional invasive implantation method. In view of the above research results, we believe that this study provides a new choice for using hydrogel-wrapped exosomes to treat liver, lung, brain, heart, kidney and other diseases in the future.

[0095] Test Example 4 In vivo research evaluation of exosome hydrogel after implantation by intraperitoneal injection

[0096] 1. Macroscopic state evaluation of exosome hydrogel in mice in vivo

[0097] Mice were implanted with OD-CMC / TA-Fe-Exos exosome hydrogel by intraperitoneal injection, and the residual exosome hydrogel was observed at different times after implantation.

[0098] As shown in Figure 16 , after implanting the exosome hydrogel by intraperitoneal injection, it can be clearly observed that the hydrogel at different time points is in good condition, with a purple color. Since the TA-Fe complex is contained in the hydrogel, the gel structure is more stable, and there is no obvious sign of fracture.

[0099] 2. Small animal imaging evaluation of the state of the gel in vivo

[0100] Figure 17 We showed the biodistribution of hydrogel-released exosomes in mice over time by small-animal live imaging. In the intraperitoneal injection group, a relatively strong signal was clearly observed in the mouse peritoneal cavity at different time points, which was due to the signal presented by the hydrogel carrying Dir fluorescein-labeled exosomes under the excitation of the corresponding waveband light of the small-animal imager. It was also found that the hydrogel was relatively stable at different observation points, and the area of the strong signal region in the mouse peritoneal cavity at individual observation points increased slightly over time, for example, at the 96 h and 144 h observation points, as shown in FIG. 6B, which may be due to the swelling of the hydrogel caused by the absorption of peritoneal fluid. Figure 17

[0101] 3. Small-animal imaging evaluation of the distribution of gel-secreted exosomes in various organs in vivo

[0102] In addition to live imaging of mice, we anesthetized mice at the 24 h observation point and the 144 h observation point, evaluated the biodistribution of exosomes in the liver, spleen, kidney, heart, lung, brain and other organs by small-animal imaging, and quantitatively analyzed the fluorescence signal intensity of different groups of organs. As shown in FIG. 6C, at the 24 h observation point, the liver tissue in the intraperitoneal injection administration route group showed a relatively strong fluorescence signal than other organs, indicating that the intraperitoneal administration route resulted in a larger amount of exosomes taken up by the liver than other organs. At the 144 h observation point, the accumulation of exosomes in the liver in the intraperitoneal injection administration group showed no significant difference from that at 24 h, which may be due to the combined effects of the slow release of the hydrogel in the peritoneal cavity and the consumption of exosomes by the liver under the current exosome load. In summary, the evaluation of the fluorescence intensity of mice in vivo and organs by small-animal imaging showed that the implantation of the hydrogel / exosome system in the peritoneal cavity could release exosomes to the liver, and the accumulation of exosomes in the liver was significantly more than that in other organs. Figure 18 4. Evaluation of the distribution characteristics of hydrogel-secreted exosomes in various organs in vivo by tissue immunofluorescence technology

[0103]

[0104] Figure 19 As shown in FIG. 7, after the intraperitoneal injection of exosome hydrogel into mice, the biodistribution of PKH-26-labeled exosomes in the liver, spleen, kidney, lung, heart and brain tissues at different time observation points (1 h, 4 h, 8 h, 12 h, 24 h) was evaluated by an inverted microscope, and the red area represented the exosome-rich region, with 3 mice in each group. As shown in FIG. 7, the exosomes were mainly distributed in the liver and spleen at 1 h, 4 h and 8 h, and the accumulation of exosomes in the liver was significantly more than that in other organs. At 12 h and 24 h, the exosomes were mainly distributed in the liver, and the accumulation of exosomes in the liver was significantly more than that in other organs. Figure 19 ​​It can be clearly seen that the red area of each organ gradually increases with time, indicating that the accumulation of exosomes in the liver, spleen, kidney, lung, heart and brain tissue gradually increases. The quantitative analysis curve also shows the corresponding experimental results. It can also be found that the distribution of exosomes in the liver starts from the portal tract and gradually extends to the central vein area, and finally spreads to the whole liver tissue. The accumulation of exosomes in the abdominal injection administration route is also related to the blood circulation route. Research reports that most of the anti-tumor drugs injected into the abdominal cavity are absorbed by the visceral peritoneum, enter the portal vein system and reach the liver. In theory, the abdominal sustained-release exosomes should reach the liver through this route, thus leading to the above results.

[0105] We can also find that exosomes are mainly distributed in the red pulp area of the spleen, while almost no exosomes are observed in the white pulp area. This is because the red pulp area of the spleen has a large number of splenic blood sinuses or capillary distribution, while the white pulp area is a lymphocyte accumulation area. Exosomes are transported by blood, so the above phenomenon occurs. Exosomes in the lung tissue are mainly distributed in the tissue around the bronchioles and terminal bronchioles. Since the blood vessels in the lung are accompanied by bronchioles, forming a capillary network to nourish the bronchioles, such anatomical structure and function promote the distribution of exosomes transported by blood circulation in the lung tissue as shown in the figure. The distribution of exosomes in the heart tissue is from the lateral myocardial tissue to the inner side. According to the anatomical characteristics of coronary circulation, the left and right coronary arteries of the heart are emitted from the root of the ascending aorta, and their main stems and large branches run on the surface of the heart. Small branches often penetrate the myocardium in a direction perpendicular to the surface of the heart, along the way emitting branches, and finally branching into a network under the endocardium. In view of this, we speculate that exosomes may first reach the lateral part of the heart along the blood flow of the coronary vascular branches, and then reach the medial part of the heart, so the accumulation of exosomes in the heart presents a phenomenon of extending from the lateral to the inner side. The accumulation of exosomes in the brain is in a uniform and diffuse distribution state.

[0106] 5、Comparison and analysis of the distribution characteristics of gel-secreted exosomes in various organs in vivo

[0107] Figure 20As shown, after the exosome hydrogel was implanted into the mouse body by intraperitoneal injection, the fluorescence intensity of the PKH-26 labeled exosomes in the liver, spleen, kidney, lung, heart and brain tissues at different time observation points (4h, 12h, 24h) was quantitatively analyzed. From the column chart, it can be found that the umbilical cord mesenchymal stem cell-derived exosomes released by the hydrogel jelly in 24h are mostly distributed in the liver, followed by the spleen, and the least in the brain. It is worth noting that the research on the exosomes released by the hydrogel / exosome system through intraperitoneal injection for reaching various organs has not been reported. In view of the above research results, we believe that the present study provides a new choice for using exosome hydrogel to treat liver, lung, brain, heart, kidney and other diseases in the future.

[0108] From the above examples, it can be seen that the exosome hydrogel prepared by the present application has good biocompatibility and can stably release exosomes into the liver, spleen, kidney, lung, heart and brain tissue, and is expected to become a new way for the treatment of diseases related to the regeneration or damage repair of important organs such as liver, spleen, kidney, heart, lung and brain. Especially, it opens up a precedent for the future to add suitable edible flavoring pigments in the oral exosome hydrogel, to develop easy-to-swallow medicinal liver-protecting jelly, and to protect the kidney, spleen, heart, lung and brain jelly.

[0109] The specific examples are applied in the present application to elaborate the inventive concept in detail, and the above examples are only used to help understand the core idea of the present application. It should be pointed out that any obvious modification, equivalent replacement or other improvement made by those skilled in the art without departing from the inventive concept should be included in the protection scope of the present application.

Claims

1. An exosome hydrogel that can be taken orally or injected intraperitoneally, characterized in that, It is made from carboxymethyl chitosan, oxidized dextran, tannic acid-iron complex, exosomes derived from umbilical cord mesenchymal stem cells, PBS and water.

2. The method for preparing the exosome hydrogel according to claim 1, characterized in that, include: Oxidized dextran PBS solution and exosome PBS solution derived from umbilical cord mesenchymal stem cells were mixed to obtain an oxidized dextran exosome suspension; The exosome hydrogel was obtained by mixing and shaking the oxidized dextran exosome suspension, carboxymethyl chitosan PBS solution, and tannic acid-iron complex aqueous solution, and allowing it to stand.

3. The preparation method according to claim 2, 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, the dialysate is collected and then dried after being cold-extracted by liquid nitrogen. 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.

4. The preparation method according to claim 3, 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 28–36 hours.

5. The preparation method according to claim 3, characterized in that, The mass ratio of sodium periodate to dextran is 0.5–3.0:0.2–3.

0.

6. The preparation method according to claim 2, characterized in that, The mass ratio of oxidized dextran to exosomes derived from umbilical cord mesenchymal stem cells was 2–8:1–3.

7. The preparation method according to claim 2, characterized in that, The concentration of carboxymethyl chitosan in the PBS solution was 2.0–4.0%, and the concentration of tannic acid-iron complex in the aqueous solution was 20–25%.

8. The preparation method according to claim 2, characterized in that, The volume ratio of oxidized dextran exosome suspension, carboxymethyl chitosan PBS solution, and tannic acid-iron complex aqueous solution was 6–10:6–10:1.

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