Exosome-loaded ros and ph dual-responsive hydrogel composite, and preparation method and application thereof

Abstract

This invention discloses a ROS- and pH-responsive hydrogel complex loaded with exosomes, its preparation method, and its applications, belonging to the field of biomedical engineering. The hydrogel complex of this invention comprises a ROS- and pH-responsive hydrogel carrier and exosomes loaded within the carrier. The hydrogel carrier is a three-dimensional network structure formed by dynamic covalent bonding between phenylboronic acid-modified hyaluronic acid and dopamine-modified gelatin. This hydrogel complex enables controlled release of exosomes, addressing the problems of short in vivo half-life, poor targeting, uncontrollable release, and inability to effectively address secondary damage cascade reactions associated with exosomes.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials, and more specifically, relates to a ROS and pH dual-responsive hydrogel composite loaded with exosomes, its preparation method and application. Background Technology

[0002] Spinal cord injury (SCI) is a severe central nervous system trauma that often leads to sensory and motor dysfunction or even permanent paralysis, placing a heavy burden on patients' families and society. Its pathological process includes primary mechanical injury and secondary injury. Secondary injury involves oxidative stress (excessive accumulation of reactive oxygen species), neuroinflammation, excitatory toxicity, and local acidosis, ultimately creating a microenvironment that inhibits axonal regeneration. Current clinical treatments mainly include surgical decompression, drug therapy, and rehabilitation training; however, these treatments have limited effectiveness and cannot effectively repair damaged nerve tissue or rebuild nerve function.

[0003] Human induced pluripotent stem cell-derived exosomes (hiPSC-Exos) are a cell-free therapy strategy. They can cross the blood-spinal cord barrier, carry neurotrophic factors and miRNAs, and have the potential to inhibit neuroinflammation, protect neurons, promote axonal growth and angiogenesis. At the same time, they avoid the ethical controversy of embryonic stem cells and the risk of immune rejection of allogeneic stem cells. However, direct injection of exosomes into the spinal cord injury site still has the following technical bottlenecks: (1) Exosomes have a short half-life in vivo and are easily cleared by the liver and spleen; (2) Exosomes lack targeting and are difficult to stay at the injury site and exert a sustained effect, resulting in low bioavailability; (3) The lipid bilayer membrane structure of exosomes is relatively fragile and has poor structural stability.

[0004] Therefore, developing an intelligent delivery system that can respond to high oxidative stress and acidic microenvironment after spinal cord injury, achieve on-demand release of exosomes, and synergistically exert anti-inflammatory, antioxidant, and neuroprotective effects is of great significance for promoting the recovery of neurological function. Summary of the Invention

[0005] To address the aforementioned deficiencies or improvement needs of existing technologies, this invention provides a ROS- and pH-responsive hydrogel complex of exosomes, its preparation method, and its applications. The hydrogel complex comprises a ROS- and pH-responsive hydrogel carrier and exosomes loaded within the carrier. The hydrogel carrier is a three-dimensional network structure formed by dynamic covalent bonding between phenylboronic acid-modified hyaluronic acid and dopamine-modified gelatin. This invention's hydrogel complex enables controlled release of exosomes, solving the problems of short in vivo half-life, poor targeting, uncontrollable release, and inability to effectively address secondary damage cascade reactions.

[0006] According to a first aspect of the present invention, a ROS and pH dual-responsive hydrogel complex loaded with exosomes is provided, comprising a ROS and pH dual-responsive hydrogel carrier and exosomes loaded within the carrier; The hydrogel carrier is a three-dimensional network structure formed by the cross-linking of phenylboronic acid-modified hyaluronic acid and dopamine-modified gelatin through dynamic covalent bonds. The dynamic covalent bonds are phenylboronic acid ester bonds formed by the complexation of phenylboronic acid groups on the side chains of phenylboronic acid-modified hyaluronic acid and catechol groups on the side chains of dopamine-modified gelatin.

[0007] Preferably, the exosomes are exosomes derived from human induced pluripotent stem cells or exosomes derived from mesenchymal stem cells.

[0008] Preferably, the exosomes derived from human induced pluripotent stem cells have a particle size of 30-150 nm and express exosome markers CD9, CD63, and CD81.

[0009] Preferably, the loading amount of exosomes in the complex is 50~70 μg / 500 μL.

[0010] According to another aspect of the present invention, a method for preparing the ROS and pH dual-responsive hydrogel complex loaded with exosomes is provided, comprising the following steps: (1) Hyaluronic acid was dissolved in a buffer solution, and an activator was added to activate the carboxyl group. Then, 3-aminophenylboronic acid was added to react. After dialysis and lyophilization, phenylboronic acid-modified hyaluronic acid was obtained. Gelatin was dissolved in a buffer solution, and an activator was added to activate the carboxyl groups. Then, dopamine hydrochloride was added to carry out a light-protected reaction. After dialysis and lyophilization, dopamine-modified gelatin was obtained. (2) Mix the exosomes with the phenylboronic acid-modified hyaluronic acid solution to obtain the phenylboronic acid-modified hyaluronic acid loaded with exosomes. Then add the dopamine-modified gelatin solution and crosslink in situ to form a gel by utilizing the dynamic reversible complexation reaction between the phenylboronic acid group and the catechol group, thus obtaining the ROS and pH dual-responsive hydrogel complex loaded with exosomes.

[0011] Preferably, the phenylboronic acid-modified hyaluronic acid solution has a mass-volume fraction of 1-3%; the dopamine-modified gelatin solution has a mass-volume fraction of 8-12%; and the volume mixing ratio of the phenylboronic acid-modified hyaluronic acid solution to the dopamine-modified gelatin solution is 1:(1-3).

[0012] Preferably, in step (1), the activator is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0013] Preferably, the exosomes are exosomes derived from human induced pluripotent stem cells or exosomes derived from mesenchymal stem cells.

[0014] According to another aspect of the present invention, the use of a ROS and pH dual-responsive hydrogel complex loaded with exosomes in the preparation of a drug for repairing spinal cord injury is provided.

[0015] Preferably, the complex is used to enable on-demand release of exosomes in response to ROS and acidic pH in the spinal cord injury microenvironment, and to scavenge reactive oxygen species, regulate microglia polarization, inhibit the secretion of pro-inflammatory factors, and promote the release of anti-inflammatory factors.

[0016] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: (1) This invention constructs a composite system combining ROS / pH dual-response hydrogel and exosomes, which fully leverages the dual advantages of precise delivery of intelligent responsive materials and multi-target therapy of stem cell exosomes. It solves the bottleneck problems in the prior art, such as short residence time of exosomes in vivo, uncontrollable release, and inability to effectively cope with complex pathological microenvironments, and provides a brand-new technical solution and idea for the preparation of spinal cord injury drugs.

[0017] (2) The HA-PBA / Gel-DA hydrogel system constructed in this invention has a crosslinking core of phenylboronic acid ester dynamic covalent bonds, which simultaneously exhibit ROS-responsive and pH-responsive characteristics. Under normal physiological conditions (low ROS, neutral pH), the hydrogel structure is stable and the exosome retention rate is high. When implanted into the spinal cord injury lesion, the hydrogel can sense two key pathological signals of the local microenvironment—high concentration of reactive oxygen species and acidic pH—leading to the breakage of phenylboronic acid ester bonds and relaxation of the network structure, thereby enabling on-demand and continuous release of exosomes. In the high ROS / acidic microenvironment of spinal cord injury, on-demand release of exosomes is achieved, promoting the transformation of microglia to the M2 phenotype, inhibiting pro-inflammatory factors, and promoting the release of anti-inflammatory factors. This "intelligent response" characteristic avoids the problems of drug burst release or release lag in traditional delivery systems, matching the release kinetics of exosomes with the spatiotemporal patterns of pathological evolution after spinal cord injury, precisely targeting the peak of inflammation and the secondary injury period, and significantly improving treatment efficiency.

[0018] (3) The hydrogel carrier of this invention has therapeutic effects itself and forms a synergistic effect with exosomes. On the one hand, the phenylboronic acid group, as an electron acceptor, can actively remove excess reactive oxygen species in the damaged area and directly reduce the damage of oxidative stress to neurons and oligodendrocytes. On the other hand, the hiPSC-Exos released in response carries a variety of miRNAs and neurotrophic factors, which can inhibit the excessive activation of microglia, promote their transformation from a pro-inflammatory phenotype to an anti-inflammatory phenotype, and reduce the levels of inflammatory factors such as TNF-α and IL-6. This dual-action mode of "material active anti-oxidation + exosome immune regulation" realizes the synergistic intervention of multiple pathological pathways such as oxidative stress, neuroinflammation, and excitotoxicity after spinal cord injury, and effectively blocks the cascade amplification effect of secondary injury.

[0019] (4) The hydrogel composite of the present invention provides multiple favorable conditions for spinal cord injury repair. First, the three-dimensional porous network structure of the hydrogel (pore size up to 50-200 μm) simulates the natural extracellular matrix, providing space for the migration of endogenous cells and the extension of axons, while inhibiting excessive deposition of glial scars. Second, after the release of exosomes, the cellular communities such as neurons around the injured area activate cell survival signaling pathways by delivering bioactive molecules, significantly reducing the neuronal apoptosis rate. At the same time, the hydrogel of the present invention has good injectability, which allows the composite to perfectly fit the irregular spinal cord injury cavity and achieve complete filling of the injured area.

[0020] (5) The preparation process of this invention is mild and free of toxic reagents, which greatly ensures the activity of exosomes, has good biocompatibility, and has broad prospects for clinical translation. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of the gel complex of the present invention.

[0022] Figure 2 The Fourier transform infrared spectrum and nuclear magnetic resonance hydrogen spectrum of HA-PBA and Gel-DA described in this invention are shown.

[0023] Figure 3 The rheological test results for the hydrogel described in this invention show its rapid gelation ability and suitable mechanical strength. Figure 3 In the diagram, A represents the time scan result, and B represents the frequency scan result.

[0024] Figure 4 The image shown is a scanning electron microscope image of the hydrogel carrier described in this invention, revealing that the hydrogel has a three-dimensional porous network structure.

[0025] Figure 5 For the biocompatibility detection of the hydrogel carrier described in this invention, Figure A is a statistical graph of cell viability detected by the CCK-8 assay; Figure 5In the diagram, B represents the fluorescence image of live and dead cells, where green fluorescence represents live cells and red fluorescence represents dead cells. Figure 5 C in the figure represents the quantitative result of live and dead staining.

[0026] Figure 6 This is a diagram showing the identification results of hiPSC-Exos as described in this invention. Figure 6 In the diagram, A represents the particle size distribution of the nanoparticles as analyzed by tracking. Figure 6 B in the image is a diagram of the morphology of exosomes observed by transmission electron microscopy. Figure 6 In the figure, C represents the results of Western blotting detection of exosome markers CD63, CD9, and CD81.

[0027] Figure 7 This shows the distribution of exosomes in the hydrogel described in this invention.

[0028] Figure 8 This describes the in vitro response of the hydrogel described in this invention to release exosomes. Figure 8 In this context, A represents the cumulative release rate of exosomes under different conditions; Figure 8 B in the figure represents the cumulative release of exosomes under different conditions, showing that the release rate of exosomes under the conditions of the spinal cord injury microenvironment was significantly higher than that in the PBS group.

[0029] Figure 9 This is a graph illustrating the in vitro anti-inflammatory effect of the complex described in this invention—regulation of microglia polarization. Figure 9 In the image, A represents the immunofluorescence pattern; Figure 9 B in the graph represents the quantitative statistical analysis of fluorescence intensity.

[0030] Figure 10 The in vitro anti-inflammatory effect of the complex described in this invention was demonstrated by measuring the secretion level of inflammatory factors. ELISA was used to detect the levels of pro-inflammatory factors TNF-α and IL-6 in the cell culture supernatant of each group. Figure 10 (A and B in the text) and anti-inflammatory factors IL-10, IL-4 ( Figure 10 The secretion concentrations of C and D in the body. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0032] The present invention provides a ROS / pH dual-responsive hydrogel complex loaded with exosomes. The complex consists of a ROS / pH dual-responsive hydrogel carrier and exosomes uniformly loaded within the three-dimensional network of the carrier. The exosomes are uniformly dispersed in the hydrogel carrier through physical embedding.

[0033] The ROS / pH dual-responsive hydrogel carrier is a three-dimensional network structure formed by dynamic covalent cross-linking of hyaluronic acid modified with phenylboronic acid (HA-PBA) and gelatin modified with dopamine (Gel-DA).

[0034] In some embodiments, the exosomes are exosomes derived from human induced pluripotent stem cells or mesenchymal stem cells. The exosomes derived from human induced pluripotent stem cells have a particle size of 30-150 nm and express CD9, CD63, and CD81 exosome marker proteins. In some embodiments, the grafting rate of phenylboronic acid in the HA-PBA is 15-30%; and the grafting rate of dopamine in the Gel-DA is 10-25%. A grafting rate that is too low results in insufficient cross-linking points and insufficient gel strength; a grafting rate that is too high may lead to an overly dense gel, affecting exosome release.

[0035] In some embodiments, the exosome loading is 50-80 μg / 500 μL.

[0036] Figure 1 This is a flowchart illustrating the preparation process of the gel complex of the present invention. The present invention also provides a method for preparing a ROS / pH dual-responsive hydrogel loaded with exosomes, mainly comprising the following steps: (1) Synthesis of phenylboronic acid modified hyaluronic acid: Hyaluronic acid was dissolved in PBS buffer, the carboxyl group was activated by adding an activator, and then 3-aminophenylboronic acid was added to react. After dialysis and lyophilization, HA-PBA was obtained. (2) Synthesis of dopamine-modified gelatin: Dissolve gelatin in PBS buffer, add activator to activate carboxyl groups, then add dopamine hydrochloride for light-protected reaction, and obtain Gel-DA by dialysis and lyophilization; (3) Dissolve the HA-PBA and Gel-DA in phosphate buffer solution to obtain HA-PBA and Gel-DA solutions respectively; (4) Mix the exosomes with the HA-PBA solution obtained in step (3) to obtain the HA-PBA mixture loaded with exosomes as component A; the Gel-DA solution obtained in step (3) is component B; (5) Mix component A and component B, and crosslink them in situ to form a gel by utilizing the dynamic reversible complexation reaction between the phenylboronic acid group and the catechol group, so as to obtain the ROS / pH dual-response hydrogel complex loaded with exosomes.

[0037] In some embodiments, the molecular weight of the hyaluronic acid in step (1) is 50kDa-150kDa.

[0038] In some embodiments, the activator in steps (1) and (2) is EDC·HCl and NHS.

[0039] In some embodiments, the amount of 3-aminophenylboronic acid added in step (1) is 1 to 2 times the amount of hyaluronic acid.

[0040] In some embodiments, the dialysis in step (1) uses a dialysis bag with a molecular weight cutoff of 3.5 kDa and is dialyzed in deionized water for 48 to 96 hours to ensure complete removal of reaction byproducts.

[0041] In some embodiments, the gelatin in step (2) is derived from cold-water fish skin or pig skin, with a Bloom index of 150-300.

[0042] In some embodiments, the light-avoidance reaction time in step (2) is 12 to 24 hours.

[0043] In some embodiments, the dialysis in step (2) uses a dialysis bag with a molecular weight cutoff of 8 to 10.4 kDa and is dialyzed in deionized water for 48 to 96 hours to ensure complete removal of reaction byproducts.

[0044] In some embodiments, the mass-volume fraction of the HA-PBA solution in step (3) is 1-3%; and the mass-volume fraction of the Gel-DA solution is 8-12%.

[0045] In some embodiments, the volume ratio of HA-PBA to Gel-DA solution is 1:(1~3).

[0046] The present invention also provides the application of the gel complex in the preparation of a drug for repairing spinal cord injury, wherein the complex enables the on-demand release of exosomes in response to the high reactive oxygen species and acidic pH in the spinal cord injury microenvironment, and synergistically promotes spinal cord injury repair by scavenging reactive oxygen species, regulating microglia polarization, inhibiting the secretion of pro-inflammatory factors, and promoting the release of anti-inflammatory factors.

[0047] The following are specific examples.

[0048] Example 1 This embodiment provides a method for preparing phenylboronic acid-modified hyaluronic acid and dopamine-modified gelatin, the specific steps of which are as follows: (1) Preparation of phenylboronic acid-modified hyaluronic acid (HA-PBA), the specific preparation steps are as follows: 1.0 g of hyaluronic acid was dissolved in 150 mL of PBS buffer. Then, 0.57 g of N-hydroxysuccinimide, 1.57 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.67 g of 3-aminophenylboronic acid were added to the solution, and the pH was adjusted to 6. The reaction was then stirred at 25 °C for 48 h. After the reaction, the mixture was dialyzed with deionized water for 3 days, with the dialysate changed every 8 hours. The hyaluronic acid was then lyophilized to obtain HA-PBA.

[0049] (2) Preparation of dopamine-modified gelatin (Gel-DA), the specific preparation steps are as follows: 4.0 g of gelatin was added to 100 mL of PBS buffer and dissolved thoroughly by stirring at 40 °C. Then, 2.0 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.2 g of N-hydroxysuccinimide were added, and the reaction was carried out for 1 hour. After the reaction was complete, 4.0 g of dopamine was added, and the reaction was continued at 40 °C in the dark for 24 hours. After the reaction was completed, the mixture was dialyzed with deionized water for 3 days, with the dialysate changed every 8 hours. The gel-DA was obtained by lyophilization after dialysis.

[0050] (3) Characterization of the synthesized HA-PBA and Gel-DA: A small amount of sample was taken for Fourier transform infrared spectroscopy and nuclear magnetic resonance hydrogen spectroscopy analysis.

[0051] This embodiment successfully prepared phenylboronic acid-modified hyaluronic acid and dopamine-modified gelatin. Firstly, the results of FTIR showed that ( Figure 2 The spectrum of HA-PBA (A) at 1470 cm⁻¹ - ¹ and 1370cm - A new absorption peak appeared at ¹, which is attributed to the skeletal vibration of the benzene ring and the BO stretching vibration of the phenylboronic acid group, respectively. These newly appearing characteristic peaks confirm that the phenylboronic acid group was successfully grafted onto hyaluronic acid. Simultaneously, in the 1H NMR spectrum of HA-PBA (…),… Figure 2 In HA-PBA, a new characteristic peak appears in the δ 7.2–7.8 ppm range, attributed to the aromatic protons on the benzene ring of the phenylboronic acid group. By comparing the integrated areas of the benzene ring proton peak and the HA acetyl proton peak, the grafting rate of phenylboronic acid in HA-PBA was calculated to be 25.5%. Similarly, in the FTIR of Gel-DA, a new characteristic peak appears at 1282 cm⁻¹. - ¹A new absorption peak appeared, which was attributed to the CO stretching vibration of catechol, confirming that dopamine molecules were successfully grafted onto gelatin. Figure 2 (C). Simultaneously, in the hydrogen NMR spectrum of Gel-DA ( Figure 2A new characteristic peak appeared in Gel-DA in the δ 6.6-6.9 ppm range, attributed to the aromatic protons on the benzene ring of the dopamine catechol group. These aromatic proton peaks also confirmed the success of the modification. The grafting rate of dopamine in Gel-DA was calculated to be 18.1%. Therefore, this example successfully synthesized phenylboronic acid-modified hyaluronic acid and dopamine-modified gelatin for subsequent experiments.

[0052] Example 2 This embodiment provides a method for preparing a ROS / pH dual-responsive hydrogel, and the specific preparation steps are as follows: (1) Preparation of phenylboronic acid-modified hyaluronic acid (HA-PBA), the specific preparation steps are as follows: 1.0 g of hyaluronic acid was dissolved in 300 mL of MES buffer. Then, 0.57 g of N-hydroxysuccinimide, 1.57 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 0.67 g of 3-aminophenylboronic acid were added to the solution, and the pH was adjusted to 6. The reaction was then stirred at 25 °C for 48 h. After the reaction, the mixture was dialyzed with deionized water for 3 days, with the dialysate changed every 8 hours. The hyaluronic acid was then lyophilized to obtain HA-PBA.

[0053] (2) Preparation of dopamine-modified gelatin (Gel-DA), the specific preparation steps are as follows: 4.0 g of gelatin was added to 100 mL of PBS buffer and dissolved thoroughly by stirring at 40 °C. Then, 2.0 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.2 g of N-hydroxysuccinimide were added, and the reaction was carried out for 1 hour. After the reaction was complete, 4.0 g of dopamine was added, and the reaction was continued at 40 °C in the dark for 24 hours. After the reaction was completed, the mixture was dialyzed with deionized water for 3 days, with the dialysate changed every 8 hours. The gel-DA was obtained by lyophilization after dialysis.

[0054] (3) Preparation of ROS / pH dual-responsive hydrogel, the specific preparation steps are as follows: Dissolve HA-PBA at 2% (w / v) in PBS buffer, then dissolve Gel-DA at 10% (w / v) in PBS buffer. Then mix HA-PBA and Gel-DA at a ratio of 1:3 and perform dynamic covalent cross-linking to obtain ROS / pH dual-responsive HG hydrogel.

[0055] (4) Take a small amount of hydrogel for scanning electron microscopy observation, rheological testing and evaluation of injectability.

[0056] The rheological test results of the ROS / pH dual-response hydrogel obtained in this example are as follows: Figure 3 As shown, where Figure 3Figure A shows the time-scan results of the hydrogel, illustrating the dynamic changes in its storage modulus and damage modulus over time. It can be seen that the storage modulus of the hydrogel is consistently higher than the damage modulus, indicating its rapid gelation capability. Furthermore, the stabilized storage modulus is around 100 Pa, which highly matches the mechanical properties of natural spinal cord tissue. This mechanical compatibility is crucial for preventing mechanical mismatch between the implant and surrounding tissues and reducing secondary injuries. Figure 3 B in the graph represents the frequency scan of the hydrogel, showing the trends of G′ and G″ as a function of frequency (0.1–10 Hz) under 1% strain. The results show that G′ is consistently higher than G″ across the entire detection frequency range, and G′ remains relatively stable without being sensitive to frequency changes, indicating that the hydrogel possesses good structural stability and resistance to deformation, enabling it to maintain its intact gel state in the complex mechanical environment of vivo. This result provides assurance for the long-term existence and functional maintenance of the hydrogel in vivo. Meanwhile, as… Figure 4 As shown, the hydrogel exhibits a typical three-dimensional porous structure after freeze-drying, with a uniform pore size distribution and a pore size of up to 100 μm in the largest part. This provides ample space for the loading of exosomes, ensuring high loading capacity and high encapsulation efficiency. It also facilitates the diffusion of nutrients and the removal of metabolic waste, maintains the biological activity of the loaded material, and provides a favorable microenvironment for the subsequent release of exosomes and cell ingrowth.

[0057] Biocompatibility testing of ROS / pH dual-responsive hydrogels includes the following steps: (1) The prepared hydrogel was placed in SH-SY5Y cell complete culture medium and extracted at 37°C and 5% CO2 for 24 hours. The supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization to obtain the hydrogel extract.

[0058] (2) Human neuroblastoma cells (SH-SY5Y) were cultured in complete culture medium and routinely cultured in a 37°C, 5% CO2 incubator. Cells were passaged every 2-3 days, and cells in the logarithmic growth phase were used for experiments.

[0059] (3) SH-SY5Y cells were divided into groups of 3×10 3 Cells were seeded at a density of 1 cell / well in 96-well plates. The experimental group was cultured in hydrogel extract medium, while the control group was cultured in complete medium. Cell viability was assessed using the CCK-8 assay after 1–3 days of culture. 10 μL of CCK-8 solution was added to each well, gently vortexed to mix, and incubated for another hour. The absorbance at 450 nm was then measured using a microplate reader.

[0060] (4) Similarly, SH-SY5Y cells were seeded onto confocal culture dishes at a seeding density of 1×10⁻⁶. 4Cells were cultured per dish to ensure full adhesion. After adhesion, the original culture medium was discarded. Fresh complete culture medium was added to the control group, while hydrogel extraction buffer was added to the experimental group. Cells were then cultured for another 24 hours. Cell viability was then observed using live / dead cell staining, and fluorescence images of the stained cells were captured using a confocal microscope.

[0061] The ROS / pH dual-responsive hydrogel prepared in this embodiment exhibits good biocompatibility. Figure 5 As shown. Compared with the blank control group, after culturing for 24, 48, and 72 hours, the cell viability of the hydrogel extract group remained above 90%, with no significant difference from the control group, indicating that the extract of this material has no cytotoxicity and good biocompatibility. Figure 5 (A) Figure 5 The results of live / dead staining with B showed that almost no dead cells were observed in the hydrogel group, and the cell survival rate was comparable to that of the control group, with no significant difference. This further confirms that the dual-responsive hydrogel has excellent cell compatibility. These results fully demonstrate that the ROS / pH dual-responsive hydrogel prepared in this invention is non-cytotoxic, has good biocompatibility, can provide a safe growth microenvironment for nerve cells, and has the potential to be used as a material for spinal cord injury repair.

[0062] Comparative Example 1 Except for changing the volume ratio of HA-PBA to Gel-DA to 3:1, the remaining steps were the same as in Example 2. The resulting hydrogel took longer than 20 minutes to form, and its mechanical strength was insufficient, failing to meet the requirements of subsequent experiments.

[0063] Example 3 This embodiment provides a method for preparing a ROS / pH dual-responsive hydrogel loaded with exosomes derived from human induced pluripotent stem cells. The specific preparation steps are as follows: (1) Extraction of exosomes from human induced pluripotent stem cells: Human induced pluripotent stem cell lines were cultured, and then the cell supernatant after culture in serum-free medium was collected. Exosomes were separated by differential centrifugation: centrifugation at 300×g for 10 minutes at 4℃ to remove live cells and collect the supernatant; centrifugation at 2000×g for 10 minutes at 4℃ to discard dead cells and collect the supernatant; centrifugation at 10,000×g for 30 minutes at 4℃ to remove apoptotic bodies, membrane fragments and organelles and collect the supernatant; centrifugation at 100,000×g for 70 minutes at 4℃ and aspirate the supernatant; 1 mL of ice-cold DPBS was added to resuspend the precipitate; centrifugation at 100,000×g for 70 minutes at 4℃ and aspirate the supernatant; 1 mL of PBS was added to resuspend the precipitate and collected in one centrifuge tube, aliquoted into EP tubes and stored at -80℃.

[0064] (2) Identification of exosomes derived from human induced pluripotent stem cells: A small amount of exosome suspension was taken for nanoparticle tracking analysis, and then the morphology of exosomes was observed by transmission electron microscopy. Finally, the exosome marker proteins CD63, CD9 and CD81 were tested by Western Blot.

[0065] (3) Take the HA-PBA prepared in Example 1 and dissolve it in PBS buffer to obtain a 3% (w / v) HA-PBA solution.

[0066] (4) Take the hiPSC-Exos prepared in step (1) and add it to the above HA-PBA solution. Gently blow it evenly so that the concentration of HA-PBA drops to 2% (w / v) to obtain the HA-PBA mixture loaded with exosomes (component A).

[0067] (5) Take the Gel-DA prepared in Example 1 and dissolve it in PBS buffer to obtain a 10% (w / v) Gel-DA solution (component B).

[0068] (6) Mix component A and component B together at a volume ratio of 1:3 to make the total volume of the hydrogel 500 μL and the content of exosomes 50 μg. Let the mixture stand at room temperature for 3-5 minutes to form a hydrogel complex through in-situ cross-linking by utilizing the dynamic reversible reaction between the phenylboronic acid group and the hydroxyl group.

[0069] (7) The hydrogel complex obtained in step (6) is a ROS / pH dual-responsive hydrogel loaded with exosomes derived from human induced pluripotent stem cells, denoted as Exos@HA-PBA / Gel-DA.

[0070] The exosomes derived from human induced pluripotent stem cells obtained in this example showed a concentrated particle size distribution as determined by NTA analysis. Figure 6 As shown in Figure A, the main peak is located at 70 μm, which is consistent with the typical particle size range of exosomes (30-150 nm), and the particle concentration of exosomes is [missing value]. Transmission electron microscopy (TEM) Figure 6 In section B), observation reveals that the exosomes exhibit a typical cup-shaped vesicle structure with intact membranes and uniform size, indicating that the extraction process did not damage the integrity of the exosomes. Figure 6 As shown in Figure C, Western blotting analysis revealed positive expression of CD63, CD9, and CD81 in exosomes, consistent with the protein marker characteristics of exosomes. These results confirm the successful acquisition of high-purity, structurally intact human induced pluripotent stem cell-derived exosomes, which can be used for subsequent experiments.

[0071] The distribution of exosomes in ROS / pH dual-responsive hydrogels loaded with exosomes derived from human induced pluripotent stem cells was evaluated. The specific preparation steps are as follows: (1) Exosome fluorescent labeling: Take the hiPSC-Exos suspension prepared in step (1) above and operate according to the PKH26 dye kit to finally obtain PKH26 labeled exosomes (PKH26-Exos).

[0072] (2) Set up the following two groups for comparative observation: Experimental group: Following the steps (4) to (6) above, PKH26-labeled exosomes were used to replace unlabeled exosomes to prepare hydrogel complexes loaded with PKH26-Exos. The entire preparation process was carried out in the dark.

[0073] Control group: Blank hydrogel complex (without exosomes) was prepared using the same method and served as a negative control to exclude interference from the autofluorescence of the hydrogel matrix.

[0074] (3) The two prepared hydrogel composites were placed in confocal culture dishes and observed immediately using a laser confocal microscope. The excitation wavelength was 551 nm and the emission wavelength was 567 nm. Z-stack images of different depths of the hydrogel were acquired. Images of the control group were acquired using the same parameters to determine the background fluorescence level.

[0075] In this embodiment, the exosomes in the ROS / pH dual-responsive hydrogel complex loaded with exosomes derived from human induced pluripotent stem cells are uniformly distributed. Figure 7 As shown, PKH26-labeled exosomes exhibit a uniform and diffuse distribution throughout the three-dimensional hydrogel network, with no obvious local aggregation or deposition. Furthermore, the exosomes are evenly distributed across different depth layers of the hydrogel, indicating that no sedimentation or stratification occurred during the cross-linking process. No red fluorescence signal was observed in the control group under the same conditions, ruling out matrix interference. This uniform distribution characteristic lays an important foundation for the subsequent controlled release of exosomes and their exertion of biological functions.

[0076] Example 4 Preparation and in vitro release evaluation of ROS / pH dual-responsive hydrogels loaded with exosomes derived from human induced pluripotent stem cells, including the following steps: (1) ROS / pH dual-responsive hydrogel complex loaded with hiPSC-Exos was prepared according to the method described in Example 3. Each hydrogel had a volume of 500 μL and contained 50 μg of exosomes. Three parallel samples were set up for each group for release experiments.

[0077] (2) Place the hydrogel in centrifuge tubes with different release media, and set up 3 parallel groups for each group: normal group: pH 7.4 PBS; damage simulation group: pH 6.0 PBS + 1mM H2O2.

[0078] (3) Place the centrifuge tubes in a constant temperature shaker at 37℃ and rotate at 100 rpm. Take out all the released medium at preset time points (1, 2, 4, 6, 8, 10, 12, 24, 36, 48h) and add an equal volume of fresh medium.

[0079] (4) The CD63 protein content in the release medium was detected using a CD63 ELISA kit, and the release amount of exosomes was characterized by the expression level of the exosome-specific marker CD63.

[0080] (5) Calculate the cumulative release rate based on the cumulative release of CD63 at each time point: Cumulative release rate (%) = (total amount of CD63 released up to this time point / initial total amount of CD63 in exosomes in hydrogel) * 100%. The initial total amount of CD63 in exosomes in hydrogel will be obtained by measuring an equal amount of exosomes (50 μg) using the same ELISA kit.

[0081] The results of this embodiment demonstrate that the ROS / pH dual-responsive hydrogel complex loaded with hiPSC-Exos constructed in this invention exhibits excellent pathological microenvironment responsiveness characteristics: such as... Figure 8 As shown, under normal physiological conditions (pH 7.4), the hydrogel structure is stable, and exosome release is slow, with a cumulative release rate of only 30.65% after 48 hours. This indicates that under normal conditions, the hydrogel network structure is stable, the phenylboronic ester bonds remain intact, and it has good encapsulation and retention capabilities for exosomes. However, under simulated spinal cord injury microenvironment (pH 6.0 + 1 mM H2O2), the hydrogel responds rapidly, and the exosome release rate is significantly accelerated, exhibiting obvious responsive release characteristics, with a cumulative release rate of 70.63% after 48 hours. The results indicate that under simulated spinal cord injury microenvironment conditions, the hydrogel responds rapidly, enabling on-demand and continuous release of exosomes, precisely targeting the pathological process at the injury site. This release characteristic allows the exosome release kinetics to match the peak periods of inflammatory response and oxidative stress after spinal cord injury, potentially achieving "intelligent delivery" in vivo, maximizing the neuroprotective and immunomodulatory effects of exosomes, and providing a precise drug delivery platform for spinal cord injury repair.

[0082] Example 5 The preparation method and in vitro anti-inflammatory effect evaluation of the human ROS / pH dual-responsive hydrogel complex loaded with exosomes derived from induced pluripotent stem cells are as follows: (1) A hydrogel complex was prepared according to Example 3, and the prepared hydrogel was placed in mouse microglia (BV2) complete culture medium and extracted at 37°C and 5% CO2 for 24 hours. The supernatant was collected and filtered through a 0.22 μm filter membrane for sterilization to obtain the hydrogel extract.

[0083] (2) Culture mouse microglia in a complete culture medium.

[0084] (3) BV2 cells were divided into two groups of 2 × 10⁻⁶ cells. 4 Seeds were planted at a density of 100 cells / well in 24-well plates and confocal culture dishes and incubated for 12 hours.

[0085] (4) After the cells adhered to the wall, they were treated according to the following groups: Control group: Routine culture, without any stimulation. Model group: supplemented with fresh complete culture medium and lipopolysaccharide (LPS) (100 ng / mL). Blank gel group: Add blank gel extract and LPS (100 ng / mL) Free exosomes group: Add complete culture medium containing free exosomes and LPS (100 ng / mL). Hydrogel complex group: hydrogel complex extract and LPS (100 ng / mL) were added. All groups were cultured for 24 hours for subsequent testing. LPS was mixed thoroughly with the appropriate culture medium or extract before addition.

[0086] (5) After 24 hours of culture, BV2 cells seeded in confocal culture dishes were subjected to immunofluorescence staining.

[0087] (6) After culturing for 24 hours, the cell culture supernatant from each group of 24-well plates was collected, centrifuged at 1000g for 10 minutes at 4℃ to remove cell debris, and the supernatant was aliquoted and stored at -80℃ for later use. Subsequently, the concentrations of pro-inflammatory factors TNF-α and IL-6 and anti-inflammatory factors IL-4 and IL-10 in the supernatant were detected using an ELISA kit.

[0088] The hydrogel complex obtained in this embodiment was systematically evaluated for its in vitro anti-inflammatory effect using the hydrogel extraction method combined with immunofluorescence staining and ELISA detection. Firstly, the results of immunofluorescence staining showed ( Figure 9 In the A group, the inducible nitric oxide synthase (iNOS) (M1 marker) of the complex group showed only weak red fluorescence, and quantitative analysis showed that the fluorescence intensity was significantly lower than that of the model group. Figure 9 (B in the text). Arginase-1 (Arg-1) (M2 marker) shows a distinct green fluorescence in the complex. Figure 9 A in the text), quantitative analysis shows ( Figure 9 The fluorescence intensity in group B was significantly higher than that in the model group. This indicates that the hydrogel complex can effectively promote the transformation of microglia to the anti-inflammatory M2 phenotype. ELISA detection showed... Figure 10The secretion concentrations of TNF-α and IL-6 in the complex group were significantly lower than those in the model group, while the concentrations of IL-4 and IL-10 were significantly increased, indicating a significant improvement in the inflammatory microenvironment. In summary, the ROS / pH dual-responsive hydrogel complex loaded with hiPSC-Exos described in this invention can effectively regulate microglial cell polarization, inhibit the secretion of pro-inflammatory factors, and promote the production of anti-inflammatory factors, with significantly better effects than free exosomes. It exhibits good in vitro anti-inflammatory activity, providing solid experimental evidence for its in vivo application in spinal cord injury repair.

[0089] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A ROS and pH dual-responsive hydrogel complex loaded with exosomes, characterized in that, Includes a ROS and pH dual-responsive hydrogel carrier and exosomes loaded within the carrier; The hydrogel carrier is a three-dimensional network structure formed by the cross-linking of phenylboronic acid-modified hyaluronic acid and dopamine-modified gelatin through dynamic covalent bonds. The dynamic covalent bonds are phenylboronic acid ester bonds formed by the complexation of phenylboronic acid groups on the side chains of phenylboronic acid-modified hyaluronic acid and catechol groups on the side chains of dopamine-modified gelatin.

2. The ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in claim 1, characterized in that, The exosomes are exosomes derived from human induced pluripotent stem cells or mesenchymal stem cells.

3. The ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in claim 2, characterized in that, The exosomes derived from human induced pluripotent stem cells have a particle size of 30-150 nm and express exosome markers CD9, CD63, and CD81.

4. The ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in claim 1, characterized in that, The loading amount of exosomes in the complex is 50~70 μg / 500 μL.

5. The method for preparing the ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Hyaluronic acid was dissolved in a buffer solution, and an activator was added to activate the carboxyl group. Then, 3-aminophenylboronic acid was added to react. After dialysis and lyophilization, phenylboronic acid-modified hyaluronic acid was obtained. Gelatin was dissolved in a buffer solution, and an activator was added to activate the carboxyl groups. Then, dopamine hydrochloride was added to carry out a light-protected reaction. After dialysis and lyophilization, dopamine-modified gelatin was obtained. (2) Mix the exosomes with the phenylboronic acid-modified hyaluronic acid solution to obtain the phenylboronic acid-modified hyaluronic acid loaded with exosomes. Then add the dopamine-modified gelatin solution and crosslink in situ to form a gel by utilizing the dynamic reversible complexation reaction between the phenylboronic acid group and the catechol group, thus obtaining the ROS and pH dual-responsive hydrogel complex loaded with exosomes.

6. The method for preparing the ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in claim 5, characterized in that, The phenylboronic acid-modified hyaluronic acid solution has a mass-volume fraction of 1-3%; the dopamine-modified gelatin solution has a mass-volume fraction of 8-12%; and the volume mixing ratio of the phenylboronic acid-modified hyaluronic acid solution to the dopamine-modified gelatin solution is 1:(1-3).

7. The method for preparing the ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in claim 5, characterized in that, In step (1), the activator is N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride.

8. The method for preparing the ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in claim 5, characterized in that, The exosomes are exosomes derived from human induced pluripotent stem cells or mesenchymal stem cells.

9. The use of the ROS and pH dual-responsive hydrogel complex loaded with exosomes as described in any one of claims 1-4 in the preparation of a drug for repairing spinal cord injury.

10. The application as described in claim 9, characterized in that, The complex is used to enable on-demand release of exosomes in response to ROS and acidic pH in the spinal cord injury microenvironment, and to scavenge reactive oxygen species, regulate microglia polarization, inhibit the secretion of pro-inflammatory factors, and promote the release of anti-inflammatory factors.

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