Preparation method of a biomimetic temperature-sensitive hydrogel loaded with adipose stem cell exosomes
By constructing microencapsulated exosomes combined with thermosensitive hydrogels, the problems of rapid clearance and controlled release of exosomes in vivo were solved, achieving long-term sustained release of exosomes and a suitable microenvironment to promote tissue repair.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIAXING UNIV
- Filing Date
- 2026-03-17
- Publication Date
- 2026-06-09
Smart Images
Figure CN122163531A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedical materials and regenerative medicine, and in particular to a method for preparing a biomimetic thermosensitive hydrogel loaded with adipose-derived stem cell exosomes. Background Technology
[0002] Exosomes, as nanoscale extracellular vesicles, are rich in bioactive molecules such as proteins, mRNA, and miRNA, playing a crucial role in intercellular communication, immune regulation, and tissue regeneration. In particular, adipose-derived stem cell-derived exosomes (ADSC-Exos) show great potential in tissue engineering and regenerative medicine due to their ability to promote angiogenesis, regulate inflammatory responses, and accelerate cell proliferation and migration.
[0003] However, the application of exosomes in vivo faces two major challenges: first, they are easily cleared by the immune system after direct injection, and their short half-life results in a low local retention rate; second, there is a lack of effective controlled-release carriers, making it difficult to maintain a long-term and stable therapeutic concentration at the site of injury.
[0004] Thermosensitive hydrogels, due to their excellent properties of being liquid at low temperatures (e.g., 4°C) and transforming into a gel state in situ at physiological temperatures (37°C), have become ideal carriers for delivering exosomes. Existing technologies, such as CN108743619A, disclose a technique for encapsulating and delivering exosomes using temperature-responsive hydrogels to enhance their therapeutic effects and improve retention rates in damaged tissues. Furthermore, current research has also explored the use of materials such as chitosan, poloxamer, or hyaluronic acid to construct exosome carriers.
[0005] Despite some progress in existing technologies, the following shortcomings remain: 1) Most hydrogels have a simple network structure and lack biomimetic simulation of the nanotopology of the natural extracellular matrix (ECM), which is detrimental to cell adhesion and function; 2) Exosomes are prone to burst release or loss of activity during gel preparation or in vivo release; 3) The mechanical properties and degradation rate of hydrogels are difficult to perfectly match with the growth rate of new tissue. Therefore, developing a biomimetic thermosensitive hydrogel that can simulate the natural ECM microenvironment, achieve precise controlled release of exosomes, and possess excellent bioactivity remains a pressing technical challenge in this field. Summary of the Invention
[0006] In order to overcome the above-mentioned defects of the prior art, the present invention provides a method for preparing a biomimetic thermosensitive hydrogel loaded with adipose stem cell exosomes, so as to solve the problems existing in the background art.
[0007] This invention provides the following technical solution: a method for preparing a biomimetic thermosensitive hydrogel loaded with adipose-derived stem cell exosomes, comprising the following steps: Step 1: Prepare amination-modified temperature-responsive polymer A; Step 2: Prepare oxidatively modified natural polysaccharide B; Step 3: Preparation of adipose stem cell exosome microcapsules: Using layer-by-layer self-assembly technology, polyelectrolyte multilayer membranes are alternately coated on the surface of adipose stem cell exosomes to form microencapsulated exosomes; Step 4: Preparation of biomimetic temperature-sensitive composite hydrogel: The amination-modified temperature-responsive polymer A obtained in Step 1 and the oxidized modified natural polysaccharide B obtained in Step 2 are dissolved and then mixed with the microencapsulated exosomes obtained in Step 3. Through temperature-induced physical cross-linking and the synergistic effect of dynamic covalent bonds formed between amine and aldehyde groups, the mixture is solidified into a gel.
[0008] Furthermore, the amination-modified temperature-responsive polymer A in step 1 is a poly(N-isopropylacrylamide) copolymer with amine groups introduced into the end groups or side chains, preferably an amination-modified poly(N-isopropylacrylamide-co-acrylamide).
[0009] Furthermore, in the poly(N-isopropylacrylamide-co-acrylamide), the molar ratio of N-isopropylacrylamide to acrylamide is (85-95):(15-5).
[0010] Furthermore, the oxidatively modified natural polysaccharide B in step 2 is at least one of aldehyde-modified hyaluronic acid, aldehyde-modified chitosan, or aldehyde-modified sodium alginate, preferably aldehyde-modified hyaluronic acid.
[0011] Furthermore, the aldehyde-modified hyaluronic acid is obtained by oxidizing hyaluronic acid with an oxidizing agent. The molecular weight of the hyaluronic acid is 100-300 kDa, and the degree of oxidation is 10%-30%.
[0012] Furthermore, in the layer-by-layer self-assembly technology described in step 3, the polyelectrolyte pair used for encapsulation includes a positively charged polyelectrolyte and a negatively charged polyelectrolyte; the positively charged polyelectrolyte is preferably chitosan, polylysine, or polyethyleneimine; the negatively charged polyelectrolyte is preferably sodium alginate, hyaluronic acid, or polyglutamic acid.
[0013] Furthermore, the preparation of the microencapsulated exosomes in step 3 includes: immersing or mixing adipose-derived stem cell exosomes sequentially in chitosan solution and sodium alginate solution, alternately encapsulating them in 1-10 layers, preferably 3-5 layers, through electrostatic adsorption, and then adding calcium ion solution for solidification to obtain chitosan / sodium alginate microencapsulated exosomes.
[0014] Furthermore, the particle size of the microencapsulated exosomes described in step 3 is 200-500 nm.
[0015] Furthermore, the mixing described in step 4 is carried out at a low temperature of 4-10°C; the solution concentration of the amination temperature-responsive polymer A is 1-10 wt%, and the solution concentration of the oxidatively modified natural polysaccharide B is 0.5-5 wt%; the concentration of the microencapsulated exosomes in the final hydrogel is 50-500 μg / mL based on the amount of exosome protein; and the pH of the mixing system is adjusted to 6.8-7.4.
[0016] The application of the biomimetic thermosensitive hydrogel loaded with adipose-derived stem cell exosomes prepared according to any one of claims 1-9 in the preparation of drugs or medical dressings for repairing skin damage, promoting wound healing or treating ischemic diseases.
[0017] The technical effects and advantages of this invention are as follows: This invention achieves the following beneficial effects by constructing a composite network composed of thermosensitive synthetic polymers and natural polysaccharides, and introducing microencapsulation technology to encapsulate adipose-derived stem cell exosomes: First, the hydrogel system exists in an injectable sol state at low temperatures and rapidly solidifies into a gel at body temperature through the synergistic effect of thermally induced physical cross-linking and Schiff base dynamic covalent bonds, exhibiting excellent thermosensitive properties and self-healing ability. Second, the microencapsulated shell effectively avoids the loss of exosome activity during gel preparation and the initial in vivo environment, and achieves long-term sustained release of exosomes through a multi-level barrier structure of "gel-microcapsule," delaying their in vivo clearance rate. Furthermore, the composition and structure of this composite hydrogel, to a certain extent, mimic the characteristics of the natural extracellular matrix, providing a suitable three-dimensional microenvironment for cell adhesion and proliferation. Animal experimental results show that the experimental group using this hydrogel outperformed the control group using naked exosomes or unloaded exosome-free gels in terms of wound healing rate, re-epithelialization process, and neovascularization density. Attached Figure Description
[0018] Figure 1 This is a flowchart of the method of the present invention. Detailed Implementation
[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to specific embodiments. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.
[0020] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the raw materials and reagents used in the embodiments are all commercially available products.
[0021] General methods for the isolation and purification of adipose-derived stem cell exosomes (ADSC-Exos): Human adipose-derived mesenchymal stem cells (fDMSCs) from passages 3-6 were seeded in DMEM / F12 medium containing 10% fetal bovine serum (FBS, after ultracentrifugation to remove exosomes) and incubated at 37°C. Incubate in an incubator. When cell confluence reaches 70-80%, discard the culture medium, wash twice with PBS, and replace with DMEM / F12 medium without exosome serum for another 48 hours. Collect the cell culture supernatant and extract exosomes using differential ultracentrifugation: centrifuge at 300×g for 10 minutes at 4°C to remove dead cells; collect the supernatant and centrifuge at 2000×g for 20 minutes to remove cell debris; collect the supernatant and centrifuge at 10000×g for 30 minutes to remove large vesicles; collect the supernatant and ultracentrifuge at 100000×g for 70 minutes, discard the supernatant, resuspend the precipitate in PBS, and wash once more by ultracentrifugation at 100000×g for 70 minutes. The resulting precipitate is the purified exosome. Resuspend in an appropriate amount of PBS, determine the protein concentration using a BCA protein quantification kit, and store at -80°C for later use.
[0022] Example 1 1. Amination-modified thermosensitive polymer Synthesis (1) Synthesis of carboxyl-terminated P(NIPAM-co-AAm)-COOH: NIPAM (10.17 g, 90 mmol), AAM (0.71 g, 10 mmol), mercaptoacetic acid (0.46 g, 5 mmol), and azobisisobutyronitrile (AIBN, 0.16 g, 1 mmol) were dissolved in 100 mL of anhydrous tetrahydrofuran. The mixture was deoxygenated by purging with nitrogen for 30 minutes and then stirred in an oil bath at 65 °C for 24 hours. After the reaction was complete, the mixture was concentrated by rotary evaporation, precipitated with a large amount of diethyl ether, filtered, and dried under vacuum to obtain the carboxyl-terminated copolymer P(NIPAM-co-AAm)-COOH. The molecular weight determined by GPC was approximately [missing value]. .
[0023] (2) Amination modification: Dissolve 1.0 g of the above copolymer in 50 mL of LMES buffer (0.1 M, pH = 5.5), add 0.3 g of EDC and 0.18 g of NHS, and activate at room temperature for 30 minutes. Then slowly add 0.5 g of hexamethylenediamine (dissolved in 5 mL of LMES buffer) and stir at room temperature for 24 hours. Transfer the reaction solution to a dialysis bag (MWCO3500) and dialyze with deionized water for 3 days, changing the water every 6 hours. Freeze-dry the dialysate to obtain a white flocculent solid. .
[0024] 2. Synthesis of Aldehyaluronic Acid (OHA) Dissolve 1.0 g of sodium hyaluronate (HA, molecular weight 200 kDa) in 100 mL of deionized water and stir until completely dissolved. Add 0.5 g of sodium periodate and stir in the dark for 4 hours (room temperature). Add 2 mL of ethylene glycol to terminate the reaction and continue stirring for 1 hour. Transfer the reaction solution to a dialysis bag (MWCO 8000-14000) in the dark and dialyze with deionized water for 3 days, changing the water every 6 hours. Freeze-dry the dialysate to obtain a white, spongy solid, OHA. The degree of aldehyde substitution was determined to be approximately 20% by hydroxylamine titration.
[0025] 3. Preparation of microencapsulated exosomes (MC@Exos) Prepare a 0.5 mg / mL chitosan solution (dissolved in 1% acetic acid solution, pH adjusted to 5.5 with NaOH) and a 1.0 mg / mL sodium alginate solution (dissolved in 0.9% NaCl solution). Dilute the purified ADSC-Exos to 200 μg / mL (based on exosome protein) with PBS. At 4°C, take 1 mL of exosome suspension and slowly add it dropwise to 10 mL of chitosan solution, stirring magnetically for 15 minutes; then add 20 mL of sodium alginate solution and continue stirring for 15 minutes; centrifuge at 10000×g for 15 minutes to collect the precipitate, and wash twice with PBS. Repeat the above "chitosan-sodium alginate" adsorption process 3 times (i.e., a total of 3 layers are coated). Finally, add 10 mL of 55 mM sodium alginate solution. The solution was cured for 20 minutes, centrifuged at 10000×g for 10 minutes, and washed twice with PBS to obtain microencapsulated exosomes MC@Exos, which were then resuspended in 1 mL of PBS for later use. Dynamic light scattering (DLS) measurements showed that the particle size increased from approximately 100 nm to approximately 320 nm after microencapsulation, and the Zeta potential changed from negative (-15 mV) to positive (+20 mV) and then back to negative (-18 mV), proving that the multilayer membrane encapsulation was successful.
[0026] 4. Preparation of biomimetic thermosensitive composite hydrogels Will Prepare a 5wt% solution using PBS pre-chilled at 4°C, and prepare a 3wt% solution of OHA using PBS pre-chilled at 4°C. Mix 1 mL of MC@Exos suspension (containing approximately 200 μg of exosomal protein) with 1 mL of OHA solution in an ice bath until homogeneous. While stirring in an ice bath, slowly add this mixture dropwise to a final volume of 2 mL. Add the solution while gently stirring. Adjust the pH to 7.4 with a trace amount of 0.1M NaOH solution to obtain the precursor solution of the biomimetic thermosensitive hydrogel loaded with MC@Exos (labeled Gel-1). This precursor solution is a flowing liquid at 4°C, and rapidly forms a gel upon heating to 37°C.
[0027] Example 2 The molar ratio of NIPAM to AAM in P(NIPAM-co-AAm) was adjusted to 85:15, i.e., 9.61 g (85 mmol) of NIPAM and 1.07 g (15 mmol) of AAM, with the remaining synthesis conditions the same as in Example 1. The resulting amination-modified thermosensitive polymer was labeled P2. Hydrogels were prepared according to steps 2-4 of Example 1, with OHA, MC@Exos, and their ratios remaining unchanged, to obtain hydrogel Gel-2.
[0028] Example 3 The number of microencapsulation layers was adjusted to 5 layers, that is, the "chitosan-sodium alginate" adsorption process was repeated 5 times, and the remaining steps were the same as in Example 1, to obtain hydrogel Gel-3.
[0029] Comparative Example 1 (Direct Loading of Naked Exosomes) Hydrogels were prepared according to steps 1, 2, and 4 of Example 1, but microencapsulation was not performed in step 3. Instead, the same amount of naked ADSC-Exos (200 μg protein) was directly mixed with OHA solution and then mixed with P(NIPAM-co-AAm)-NH2 solution to prepare hydrogel Gel-D1.
[0030] Comparative Example 2 (Comparison of commercially available temperature-sensitive materials) A 25 wt% aqueous solution of poloxamer 407 (P407) was prepared (dissolved at 4 °C). Naked ADSC-Exos (200 μg protein) was mixed into 1 mL of P407 solution under ice bath conditions to obtain hydrogel Gel-D2. This material can also form a gel at 37 °C.
[0031] Comparative Example 3 (blank gel without exosomes) Hydrogels were prepared according to steps 1, 2, and 4 of Example 1, but in step 3, an equal volume of PBS was used instead of the MC@Exos suspension to obtain a blank gel, Gel-D3, which does not contain exosomes.
[0032] Performance Tests and Results 1. Determination of gelation time The gelation time of each hydrogel precursor solution at 37℃ was determined using the inverted test tube method. 2 mL of sample was placed in a 4 mL vial and placed in a 37℃ constant temperature water bath. The vial was tilted every 10 seconds, and the time required for the liquid surface to stop flowing was observed. Each sample was measured three times, and the average value was taken. The results are shown in Table 1.
[0033] 2. Rheological property testing An Anton Paar MCR302 rheometer was used with a parallel plate rotor (25 mm in diameter) and a 1 mm gap. The hydrogel precursor solution was loaded onto a plate preheated to 4 °C and heated from 4 °C to 50 °C at a rate of 1 °C / min. The test frequency was 1 Hz, the strain was 1%, and the storage modulus was recorded. ) and loss modulus ( The change with temperature. Record the temperature at 37℃. The values are shown in Table 1.
[0034] 3. Investigation of in vitro release behavior The in vitro release curves of exosomes were determined using dialysis. 1 mL of each hydrogel precursor solution (Gel-1, Gel-2, Gel-3, Gel-D1, Gel-D2) was placed in a dialysis bag (MWCO 300 kDa), sealed, and then immersed in a centrifuge tube containing 20 mL of PBS (pH 7.4). The tubes were incubated at 37°C with a constant-temperature shaker (100 rpm). At predetermined time points (1, 3, 6, 12 hours; 1, 3, 5, 7, 10, 14 days), all release media were collected and an equal volume of fresh PBS was added. The exosome protein content in the release media was determined using the BCA method, and the cumulative release percentage was calculated. Each group had three replicates. The results are shown in Table 1 (only the cumulative release rates on days 7 and 14 are listed in the table).
[0035] 4. Animal experiment: Full-thickness skin defect model in diabetic mice (1) Model establishment: Male C57BL / 6 mice (8 weeks old, weighing 20-25g) were induced to develop a diabetic model by intraperitoneal injection of streptozotocin (STZ, 50mg / kg / d, for 5 consecutive days). A blood glucose level ≥16.7mmol / L was considered a successful model. The diabetic mice were randomly divided into 5 groups (n=10 per group): PBS control group, Gel-D3 group (blank gel), Gel-D1 group (naked exosome gel), Gel-D2 group (poloxam exosome group), and Gel-1 group (bionic hydrogel group of this invention). After anesthetizing the mice, a full-thickness skin defect with a diameter of 1.0cm was created on each side of the spine.
[0036] (2) Administration: The PBS group was given 100 μL of PBS; the gel group was given 100 μL of the corresponding hydrogel precursor solution (applied to the wound and rapidly gelled at 37°C). Cover with sterile dressing and fix with tape.
[0037] (3) Observation indicators: The size of the wound was photographed and recorded on postoperative days 0, 3, 7, and 14. The wound area was calculated using ImageJ software, and the healing rate was calculated using the formula "healing rate (%) = (initial area - area on day n) / initial area × 100%". Mice were sacrificed on day 14, and wound tissue was taken for H&E staining and Masson staining to observe the new epithelium and collagen deposition. The results are shown in Table 2.
[0038] Table 1 Comparison of physicochemical properties of different hydrogels
[0039] Note: Data is mean ± SD, n=3.
[0040] Table 2 Comparison of wound healing rates among different groups of mice (%, mean ± SD, n=10)
[0041] Note: * indicates p<0.05 compared to the Gel-D1 group.
[0042] Results Analysis: As shown in Table 1, the biomimetic thermosensitive hydrogels (Gel-1, Gel-2, and Gel-3) prepared in this invention can all rapidly gel at body temperature and possess high storage modulus, indicating good mechanical properties. The gelation time and modulus can be finely adjusted by regulating the NIPAM / AAm ratio. Increasing the number of microencapsulated layers (Gel-3) further delays the release rate of exosomes. In contrast, the gels directly loaded with naked exosomes (Gel-D1 and Gel-D2) exhibit significant burst release, with a release rate exceeding 60% within 7 days and approaching 90% within 14 days.
[0043] Table 2 shows the results of animal experiments. Mice treated with the hydrogel (Gel-1) of this invention showed significantly faster wound healing than other control groups, with a healing rate of 94.5% 14 days after surgery. Histological staining also showed that the newly formed tissue was rich in blood vessels and had neatly arranged collagen, indicating that the hydrogel can effectively promote the repair of chronic diabetic wounds.
[0044] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.
[0045] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.
Claims
1. A method for preparing a biomimetic thermosensitive hydrogel loaded with adipose-derived stem cell exosomes, characterized in that, Includes the following steps: Step 1: Prepare amination-modified temperature-responsive polymer A; Step 2: Prepare oxidatively modified natural polysaccharide B; Step 3: Preparation of adipose stem cell exosome microcapsules: Using layer-by-layer self-assembly technology, polyelectrolyte multilayer membranes are alternately wrapped on the surface of adipose stem cell exosomes to form microencapsulated exosomes; Step 4: Preparation of biomimetic temperature-sensitive composite hydrogel: The amination-modified temperature-responsive polymer A obtained in Step 1 and the oxidized modified natural polysaccharide B obtained in Step 2 are dissolved and then mixed with the microencapsulated exosomes obtained in Step 3. Through temperature-induced physical cross-linking and the synergistic effect of dynamic covalent bonds formed between amine and aldehyde groups, the mixture is solidified into a gel.
2. The preparation method according to claim 1, characterized in that, The amination-modified temperature-responsive polymer A in step 1 is a poly(N-isopropylacrylamide) copolymer with amine groups introduced into the end groups or side chains, preferably an amination-modified poly(N-isopropylacrylamide-co-acrylamide).
3. The preparation method according to claim 2, characterized in that, In the poly(N-isopropylacrylamide-co-acrylamide), the molar ratio of N-isopropylacrylamide to acrylamide is (85-95):(15-5).
4. The preparation method according to claim 1, characterized in that, The oxidatively modified natural polysaccharide B in step 2 is at least one of aldehyde-modified hyaluronic acid, aldehyde-modified chitosan, or aldehyde-modified sodium alginate, preferably aldehyde-modified hyaluronic acid.
5. The preparation method according to claim 4, characterized in that, The aldehyde-modified hyaluronic acid is obtained by oxidizing hyaluronic acid with an oxidizing agent. The molecular weight of the hyaluronic acid is 100-300 kDa and the degree of oxidation is 10%-30%.
6. The preparation method according to claim 1, characterized in that, In the layer-by-layer self-assembly technology described in step 3, the polyelectrolyte pairs used for encapsulation include a positively charged polyelectrolyte and a negatively charged polyelectrolyte; the positively charged polyelectrolyte is preferably chitosan, polylysine, or polyethyleneimine; the negatively charged polyelectrolyte is preferably sodium alginate, hyaluronic acid, or polyglutamic acid.
7. The preparation method according to claim 6, characterized in that, Step 3 describes the preparation of microencapsulated exosomes, which involves immersing or mixing adipose-derived stem cell exosomes sequentially into chitosan solution and sodium alginate solution, encapsulating them alternately in 1-10 layers, preferably 3-5 layers, through electrostatic adsorption, and then adding calcium ion solution for solidification to obtain chitosan / sodium alginate microencapsulated exosomes.
8. The preparation method according to claim 1, characterized in that, The particle size of the microencapsulated exosomes described in step 3 is 200-500 nm.
9. The preparation method according to claim 1, characterized in that, The mixing in step 4 is carried out at a low temperature of 4-10℃; the solution concentration of the amination temperature-responsive polymer A is 1-10wt%, and the solution concentration of the oxidatively modified natural polysaccharide B is 0.5-5wt%; the concentration of the microencapsulated exosomes in the final hydrogel is 50-500μg / mL based on the amount of exosome protein; the pH of the mixing system is adjusted to 6.8-7.
4.
10. The use of the biomimetic thermosensitive hydrogel loaded with adipose-derived stem cell exosomes prepared by any one of claims 1-9 in the preparation of drugs or medical dressings for repairing skin damage, promoting wound healing or treating ischemic diseases.