A programmed gel system and its preparation method and application

Abstract

The application relates to the field of biotechnology, and discloses a programmed gel system as well as a preparation method and application thereof. The application integrates mature membrane-anchored exosomes, a programmed molecular chaperone protection system and an active oxygen response interpenetrating network gel in a systematic way to construct a multi-stage collaborative platform for active reinforcement of an exosome membrane structure, dynamic protection during a storage period and intelligent delivery at a lesion site. The platform provides a comprehensive and systematic solution to the core obstacles faced by therapeutic exosomes in clinical transformation, especially exosomes derived from mesenchymal stem cells with tissue repair functions.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a programmed gel system, its preparation method, and its applications. Background Technology

[0002] Exosomes are nanoscale vesicles secreted by cells, carrying bioactive molecules such as proteins, nucleic acids, and lipids from the source cell. They play important roles in cell communication, tissue repair, and immune regulation, and have become highly promising biopharmaceuticals for disease treatment and drug delivery. However, exosomes face challenges in their clinical translation: First, they have poor stability. Exosomes have a lipid bilayer membrane structure, which makes them susceptible to oxidative stress, enzymatic hydrolysis, changes in osmotic pressure, and mechanical forces during in vitro storage, transportation, or in vivo delivery, leading to membrane damage, leakage of contents, and loss of bioactivity, affecting their standardized preparation and long-term preservation. Second, existing preservation technologies have limitations. Conventional methods such as deep freezing (-80°C) can maintain activity for a short period, but suffer from freeze-thaw damage, high energy consumption, and inconvenience. While freeze-drying improves stability, it may cause protein denaturation and aggregation, and the reconstitution process is complex.

[0003] In recent years, encapsulating exosomes with materials such as hydrogels to enhance their stability has become a research hotspot. For example, there have been reports of loading exosomes onto hyaluronic acid or gelatin-based hydrogels. However, these systems mostly focus on physical sustained-release functions, and their ability to protect the activity of exosomes during long-term storage remains insufficient, especially lacking synergistic strategies to enhance the stability of exosomes at the molecular level. In addition, some studies have improved exosome stability through chemical modification (such as polyethylene glycolation) or the addition of cryoprotectants (such as trehalose), but single methods often have limited effects and are difficult to maintain activity for a long time under mild storage conditions. More importantly, existing systems usually fail to achieve intelligent linkage with the pathological microenvironment of specific diseases. For example, in lesions with continuous oxidative stress, such as chronic wounds (such as diabetic foot ulcers), an ideal delivery system should be able to sense and respond to changes in the microenvironment to achieve on-demand release of therapeutic agents.

[0004] Therefore, developing an integrated system that can achieve synergistic protection at multiple levels of molecules, vesicles, and formulations, and can intelligently release exosomes in response to pathological microenvironments (such as highly reactive oxygen species), is of urgent and important practical significance for promoting the clinical and industrial application of exosomes, especially exosomes with clear therapeutic functions (such as mesenchymal stem cell-derived exosomes), in the treatment of chronic intractable diseases. Summary of the Invention

[0005] This invention aims to overcome key bottlenecks in existing exosome preservation and delivery technologies by systematically integrating mature membrane anchoring materials, programmed molecular chaperone protection systems, and reactive oxygen species-responsive interpenetrating network gels to construct a multi-level synergistic platform that provides active reinforcement of exosome membrane structures, dynamic protection during storage, and intelligent delivery to lesion sites. This platform offers a comprehensive and systematic solution to the core obstacles faced by therapeutic exosomes (especially mesenchymal stem cell-derived exosomes with tissue repair functions) in clinical translation.

[0006] On one hand, the present invention provides a method for preparing a programmed gel system, the method comprising the following steps:

[0007] The steps for preparing membrane-anchored modified exosomes are as follows: First, the exosomes are modified using an amphiphilic lipid-polyethylene glycol conjugate, and then the modified product is purified to obtain the modified exosomes. The steps for preparing programmed exosomes are as follows: Modified exosomes are incubated with a static protective chaperone working solution. Through hydrophobic interactions and charge interactions, the protective chaperone is bound to the surface of the exosomes to obtain a primary loading mixture; the primary loading mixture is incubated with a dynamic release chaperone working solution to anchor the ROS-responsive module to the surface of the exosomes to obtain a secondary loading mixture; the secondary loading mixture is washed to obtain programmed exosomes. The steps for preparing a programmed exosome smart gel are as follows: the programmed exosomes are mixed with a smart gel precursor solution to obtain a mixture; the mixture is then cured using electromagnetic radiation to obtain a programmed gel system.

[0008] Furthermore, in the step of preparing membrane-anchored modified exosomes, the amphiphilic lipid-polyethylene glycol conjugate includes DSPE-PEG-COOH, DSPE-PEG-NH2, DSPE-PEG-NHS, DSPE-PEG-Mal, and / or DSPE-PEG-Biotin.

[0009] Furthermore, in the step of preparing programmed exosomes, the components of the static protective companion working solution include heat shock protein 70 or its functionally active fragment; the components of the dynamic release companion working solution include a fusion polypeptide of an exosome binding domain and a reactive oxygen species (ROS) response domain; the sequence of the fusion polypeptide is as shown in SEQ ID NO: 1, or the fusion polypeptide is a variant that has at least 80% identity with SEQ ID NO: 1 and retains the ROS response and exosome binding functions.

[0010] Furthermore, in the step of preparing programmed exosomes, the incubation conditions for the static protective companion working solution and the exosomes are: 4°C static incubation for 30-120 minutes; the incubation conditions for the primary loading mixture and the dynamic release companion working solution are: 4°C dark static incubation for 60-150 minutes.

[0011] Furthermore, in the step of preparing the programmed exosome smart gel, the components of the smart gel precursor solution include methacrylated gelatin, reactive oxygen species-responsive hyaluronic acid derivative, trehalose, and a photoinitiator.

[0012] Furthermore, in the step of preparing the programmed exosome smart gel, the electromagnetic radiation includes ultraviolet light; the wavelength of the ultraviolet light is 355-375 nm, and the intensity is 3-10 mW / cm². 2 The irradiation time is 20-120 seconds.

[0013] On the other hand, the present invention provides a programmed gel system, which is prepared by the above-described preparation method; The programmed gel system includes programmed exosomes, an interpenetrating network gel matrix, and trehalose; The programmed exosomes are anchored and modified with an amphiphilic lipid-polyethylene glycol conjugate, and the programmed exosomes are linked with a static protection chaperone and a dynamic release chaperone. The interpenetrating network gel matrix comprises an interpenetrating first network and a second network; the first network is obtained by crosslinking methacryloyl gelatin; the second network is obtained by crosslinking reactive oxygen species-responsive hyaluronic acid derivatives; the interpenetrating network gel matrix encapsulates the programmed exosomes; The trehalose is dispersed in the interpenetrating network gel matrix.

[0014] On the other hand, the present invention provides the application of the above-described programmed gel system or the programmed gel system prepared by the above-described preparation method in the preparation of a medicament for treating chronic refractory wounds.

[0015] Furthermore, the chronic, non-healing wounds include diabetic foot ulcers, pressure ulcers, and / or venous ulcers.

[0016] On the other hand, the present invention provides an exosome preservation kit, the components of which include an amphiphilic lipid-polyethylene glycol conjugate, a static protection partner working solution, a dynamic release partner working solution, and a smart gel precursor solution.

[0017] Furthermore, the amphiphilic lipid-polyethylene glycol conjugates include DSPE-PEG-COOH, DSPE-PEG-NH2, DSPE-PEG-NHS, DSPE-PEG-Mal, and / or DSPE-PEG-Biotin.

[0018] Furthermore, the static protective companion working solution comprises heat shock protein 70 or a functionally active fragment thereof; the dynamic release companion working solution comprises a fusion polypeptide of an exosome binding domain and a reactive oxygen species (ROS) responsive domain; the sequence of the fusion polypeptide is as shown in SEQ ID NO: 1, or the fusion polypeptide is a variant having at least 80% identity with SEQ ID NO: 1 and retaining ROS responsive and exosome binding functions.

[0019] Furthermore, the components of the smart gel precursor solution include methacrylamide gelatin, reactive oxygen species-responsive acrylamide hyaluronic acid derivatives, trehalose, and photoinitiators.

[0020] The technical solution of this invention has the following advantages: 1. Overcome the inherent instability of exosome membranes: Enhance the resistance of exosome membranes to oxidative stress and hydrolytic degradation at the molecular level, and solve the problems of leakage and inactivation of their contents during storage; 2. Improve the limitations of existing single-protection modes for vectors: Change the current passive strategy of relying on physical encapsulation or simple additive protection, and build an intelligent integrated system that can implement multi-level, active and synergistic protection of exosomes; 3. Achieve long-term stable preservation under mild conditions: Establish a stabilization scheme that can efficiently maintain the biological activity of exosomes for an ultra-long period (≥12 months) under conventional cold chain conditions (2-8℃), replacing the dependence on ultra-low temperature or complex freeze-drying processes; 4. Provide an integrated solution covering storage and delivery: Develop a formulation platform that combines ultra-long storage stability with intelligent response release function to achieve effective protection of exosomes during storage and controllable release in application scenarios. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 This is a flowchart of the preparation process for the programmed exosome intelligent gel formulation (i-ExoGel); Figure 2 The results of NTA and membrane potential characterization for membrane anchoring modification of exosomes are shown in the figure, where a is the particle size result of NTA detection, b is the result of NTA detection recovery rate, and c is the result of membrane potential characterization. Figure 3 This is a flowchart of the programmed exosome preparation process; Figure 4 The figures show the results of programmed exosome NTA and nanoflow cytometry characterization, where a represents the particle size of NTA detection, b represents the NTA recovery rate, and c represents the loading efficiency verification results. Figure 5 This is a graph showing the results of testing the long-term preservation performance of i-ExoGel compared to three control groups; Figure 6 The graph shows the release results of i-ExoGel, where a is the in vitro cumulative release curve in a ROS environment containing H2O2 and a normal PBS environment, and b is the release results of different experimental groups in a ROS environment containing H2O2. Figure 7 This is a diagram showing the effect of i-ExoGel programmed gel loaded with umbilical cord mesenchymal stem cell exosomes in Example 6 on skin wound repair. In the diagram, a is the wound photos and healing curves of each group, b is the level of reactive oxygen species (H2O2) in the wound edge tissue, c is the histological morphology observed by HE staining, and d is the expression changes of key inflammatory factors TNF-α, growth factor VEGF and antioxidant gene Nrf2 in the wound tissue analyzed by qPCR and other techniques. Detailed Implementation

[0023] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0024] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0025] Materials used in this application: I. Core Biomaterials and Reagents 1. Cell source and culture: Cells: Human umbilical cord mesenchymal stem cells (hUMSC), catalog number PCS-500-010 (ATCC).

[0026] Culture medium: Mesenchymal stem cell culture medium, product number PT-3001 (Lonza); Exosome-free culture medium (for exosome collection): Exosome-free fetal bovine serum, catalog number EXO-FBS-50A-1 (System Biosciences), was prepared with DMEM / F12 basal medium at a final concentration of 10% (v / v). That is, the exosome-free serum and DMEM / F12 basal medium were mixed at a volume ratio of 1:9 (v / v) and then filtered through a 0.22 μm filter membrane for sterilization before use.

[0027] 2. Exosome isolation and purification: Materials: SEC column for purification: HiPrep M16 / 60 Sephacry IMS-500HR, catalog number 28935606 (Cytiva); PBS buffer, catalog number 10010023 (Gibco); BCA protein quantification kit, catalog number 23225 (ThermoFisher).

[0028] 3. Programmed modification of exosomes: Membrane anchoring and coupling reagents: DSPE-PEG(2000)-COOH, catalog number 880151 (Avanti PolarLipids); 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), catalog number E6383 (Sigma-Aldrich); N-hydroxysuccinimide (NHS), catalog number 130672 (Sigma-Aldrich); MES buffer (0.1 M, pH 6.0), catalog number M2933 (Sigma-Aldrich).

[0029] Bioactive molecules: Recombinant human HSP70 protein, catalog number HY-P74877 (MCE); dynamic release chaperone: ROS-responsive peptide (sequence: CLLPGAKKCGGGSGGGSYFFYGMRRYFFL, SEQ ID NO: 1), which can be chemically synthesized; protectant: trehalose, catalog number T9531 (Sigma-Aldrich), for preparing PBS containing 5% (w / v) trehalose.

[0030] 4. Intelligent gel matrix synthesis: Materials: Photoinitiators: Irgacure 2959, catalog number 410896 (Sigma-Aldrich); LAP, catalog number 900889 (Sigma-Aldrich).

[0031] Synthetic raw materials: GelMA: GeIMA (Type A, 60-80% methacrylamide), catalog number GMP-GELMA-60A (EFL, Engineering For Life); ROS-responsive hyaluronic acid derivatives: sodium hyaluronate (molecular weight 100-200 kDa), catalog number H5388 (Sigma-Aldrich); cystamine dihydrochloride, catalog number D1410 (Sigma-Aldrich).

[0032] i-ExoGel precursor preparation and cross-linking: Purified programmed exosomes, GelMA, and HA-SS-AC were co-dissolved in PBS containing 0.1% (w / v) photoinitiator. The mixture was then subjected to 365 nm UV light (intensity 5-10 mW / cm²). 2 Irradiate for 30-60 seconds to form in-situ cross-linked i-ExoGel.

[0033] 5. Animal experimental materials: Animal model: spontaneous type II diabetic db / db mice (8 weeks old, male) (strain number 000642, Jackson Lab) to establish a skin defect wound model; Tissue analysis reagents: RNA extraction kit 74104 (Qiagen), reactive oxygen species (H2O2) detection kit, catalog number D6883 (Sigma-Aldrich).

[0034] II. Main Instruments and Equipment Ultracentrifuge: Optima XE-90 with Type 70 Ti rotor (Beckman Coulter).

[0035] Size exclusion chromatography system: ÄKTA pure 25 (Cytiva).

[0036] Nanoparticle tracking and analysis instrument: NanoSight NS300 (Malvern Panalytical).

[0037] Rheometer: Discovery HR-2 (TA Instruments).

[0038] UV crosslinker: OmniCure® S1500 (Excelitas Technologies), or 365nm LED point light source system III. General Instructions Unless otherwise specified, all chemical reagents are of analytical grade or higher purity and are commercially available. All buffer solutions were prepared with ultrapure water and sterilized by filtration through a 0.22 μm filter. All cell culture-related procedures were performed under aseptic conditions. Animal experiments were conducted in accordance with relevant ethical guidelines and with prior approval.

[0039] This application provides a smart programmed gel system for long-term stable preservation and controllable delivery of exosomes, and the flowchart of its preparation method is shown below. Figure 1 .

[0040] Example 1: Preparation and characterization of membrane-anchored modified exosomes 1. Exosome isolation: Human umbilical cord mesenchymal stem cells (hUMSCs) were cultured in a standard cell culture incubator at 37°C, 5% CO2, and saturated humidity using mesenchymal stem cell-specific medium (PT-3001 (Lonza)) until the cell density reached 80%. Then, the medium was replaced with exosome-free medium and cultured under the same conditions (37°C, 5% CO2) for 48 hours. The cell culture medium was collected. The cell culture medium was then centrifuged sequentially at 400g for 10 min, 2,000g for 20 min, and 10,000g for 30 min. The supernatant was retained after each centrifugation to remove cell debris and precipitate, yielding the centrifuged supernatant. The supernatant after centrifugation was filtered through a 0.22 μm filter membrane to obtain a clear cell culture supernatant. Subsequently, following the guidelines of the International Society for Extracellular Vesicles (MISEV2018), the clear cell culture supernatant was purified by size exclusion chromatography (for details, see: Théry C, Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of Extracellular Vesicles. 2018;7(1):1535750). Specifically, the exosome purification process used a HiPrep S-500 HR column with pre-cooled 1× PBS (pH 7.4) as the mobile phase to perform size exclusion chromatography on the clear cell culture supernatant. The exosome fraction corresponding to the first major UV absorption peak was collected. To obtain high-purity exosomes and perform buffer replacement, the exosome fraction was transferred to a 100 kDa ultrafiltration centrifuge tube and concentrated to a volume ≤500 µL by centrifugation at 4°C and 4,000 g. Then, 5 mL of fresh, pre-chilled 1× PBS was added, and the mixture was centrifuged again under the same conditions. This "dilution-concentration" washing step was repeated twice to thoroughly remove residual salts and any soluble proteins that might be co-purified from the chromatographic mobile phase, resulting in purified exosome fractions. Nanoparticle tracking analysis (NTA) was then used to determine the particle concentration and size distribution of the exosomes in the washed fraction, and the particle size was adjusted to 1×10⁻⁶ particles with 1× PBS. 11 particles / mL, to obtain an exosome suspension.

[0041] 2. Exosome Membrane Anchoring Modification: Exosome membrane anchoring modification employs an aqueous micelle insertion method based on liposome technology. This method utilizes the self-assembly and spontaneous insertion of amphiphilic lipid molecules into the lipid bilayer in aqueous solution, effectively maintaining the integrity and bioactivity of the exosome membrane structure. The specific procedure is as follows: First, DSPE-PEG(2000)-COOH is dissolved in chloroform and then dried using nitrogen to form a lipid film. The lipid film is added to 1×PBS buffer preheated to 60℃ and sonicated in a water bath until the solution is clear, preparing a 1 mM micelle stock solution. The micelle stock solution is then filtered through a 0.22 µm filter membrane for sterilization, yielding the filtered micelle stock solution. 100 µL of the purified exosome suspension (particle concentration: 1×10⁻⁶) is taken... 11 Add 2-5 µL of filtered micelle stock solution to obtain particles / mL, and bring the volume to 110-120 µL with 1×PBS to obtain a mixture. Incubate the mixture at 37°C in the dark with gentle shaking (300 rpm) for 2 hours. After incubation, purify the product using an ultrafiltration centrifuge tube with a molecular weight cutoff of 100 kDa: wash with 500 µL of 1×PBS for 10 minutes each time at 4°C and 14,000g, repeating this process 3 times to completely remove uninserted free micelles. Resuspend the final precipitate in an appropriate amount of 1×PBS buffer to obtain PEGylated exosomes (Exo-PEG).

[0042] 3. Physical characterization of exosome membrane anchoring modification (nanoparticle tracking analysis, NTA): To evaluate the effect of DSPE-PEG-COOH membrane anchoring modification on the physical properties of exosomes, the particle size distribution and concentration of exosomes before and after modification were determined using NTA technology. The operation followed the standardized method of this technique (see: Filipe V, Hawe A, Jiskoot W. Critical evaluation of nanoparticle tracking analysis (NTA) by NanoSight for the measurement of nanoparticles and proteinaggregates. Pharmaceutical Research. 2010, 27(5): 796-810), and referred to the guidelines of the International Society for Extracellular Vesicles [Théry C, et al. J Extracell Vesicles. 2018;7(1):1535750]. Using a NanoSight NS300 analyzer, the Exo-PEG sample and the control exosomes that underwent the same incubation and purification process but without the addition of DSPE-PEG-COOH were diluted with sterile 1×PBS to make their particle concentration reach the optimal detection range of the NTA instrument (usually 10). 7 -10 9 (particles / mL). The Brownian motion trajectories of particles were captured at a constant flow rate, and the hydrodynamic diameter and concentration of the particles were calculated using software analysis. Results are as follows: Figure 2 As shown, the average particle size and main distribution peak (~120 nm) of the modified exosomes (Exo-PEG) are basically consistent with those of the control exosomes. Figure 2 (a) demonstrates that the membrane anchoring modification process did not cause significant exosome aggregation or particle size changes. For example... Figure 2 Figure b shows that the particle recovery rate of Exo-PEG (defined as the ratio of the final total number of Exo-PEG particles to the final total number of control exosome particles) is greater than 85%, indicating that the membrane anchoring modification process is mild, can efficiently maintain the structural integrity of exosomes, and has a high recovery rate, which meets the requirements for subsequent formulation preparation.

[0043] 4. Zeta potential analysis: PEGylated exosomes (Exo-PEG) and control exosomes were analyzed using a Malvern Zetasizer Nano ZS potentiometer. Results are as follows: Figure 2As shown in c, the zeta potential of the control exosome was -15.2 ± 1.5 mV, consistent with its typically negatively charged surface (typical range: -10 to -30 mV). After modification with DSPE-PEG-COOH, the zeta potential of Exo-PEG shifted significantly negatively, increasing to -28.3 ± 2.1 mV (p<0.01), an absolute increase of approximately 13.1 mV. This change is consistent with the reported negative increase in zeta potential after anionic PEG (such as PEG-COOH) modification of nanoparticles (Suk JS, et al. Adv Drug Deliv Rev. 2016;99(Pt A):28-51). This result directly demonstrates that the DSPE-PEG-COOH molecule has successfully inserted into the lipid bilayer of exosomes through its hydrophobic DSPE end, exposing its negatively charged carboxyl group (-COOH) on the exosome surface, thereby achieving efficient membrane anchoring modification.

[0044] Example 2: Preparation and Characterization of Programmed Exosomes This embodiment is based on the programmed design concept of "anchoring-protection-response". Building upon the existing membrane anchoring reinforcement, it performs a two-stage sequential loading of molecular chaperones onto exosomes to construct programmed exosomes with both "long-term stability" and "intelligent response" characteristics. The operation process is as follows: Figure 3 As shown.

[0045] 1. Prepare the working solution: Static protection working solution: Recombinant human HSP70 protein (HY-P74877, MCE) was diluted to 1 mg / mL with PBS buffer containing 5% (w / v) trehalose (i.e., the final concentration of trehalose was 50 mg / mL). Dynamic release chaperone working solution: The ROS-responsive peptide CLPGAKKCGGGSGGGSYFFYGMRRYFFL (SEQ ID NO: 1) was synthesized, wherein the N-terminal sequence CLPGAKKC is a CD63-targeting cyclic peptide, and the C-terminal sequence YFFYGMRRYFFL is the reactive oxygen species (ROS) response core. This peptide was prepared into a 1 mM stock solution using sterile, antioxidant-free PBS buffer (pH 7.4). Before use, it was diluted to the final concentration required for the reaction.

[0046] 2. Primary loading (construction of a static protective layer): Take 200 µL of exosome (Exo-PEG) suspension modified with DSPE-PEG-COOH membrane anchoring (concentration: 1×10⁻⁶). 11Add 10 µL of static protective chaperone working solution (recombinant human HSP70, concentration 1 mg / mL), mix gently to obtain mixture A, in which the final concentration of HSP70 is 50 µg / mL. Incubate mixture A at 4°C for 60 minutes to obtain the primary loading mixture. This step aims to utilize the molecular chaperone activity of HSP70 to form a stable "molecular barrier" on the surface of exosomes through nonspecific hydrophobic interactions, thereby enhancing the stability of exosomes in complex environments and resisting thermal aggregation and enzymatic degradation.

[0047] 3. Secondary Loading (Installation of Dynamic Response Switch): Add an appropriate amount of dynamic release chaperone working solution to the primary loading mixture obtained in the previous step to obtain mixture system B. The final concentration of the ROS-responsive peptide in mixture system B is 5 μM (total volume 200 μL). Incubate mixture system B at 4°C in the dark for 90 minutes to obtain the secondary loading mixture (i.e., programmed exosome crude product). During this process, the peptide specifically binds to the membrane protein CD63 through its N-terminal CD63-targeting cyclic peptide, thereby anchoring the C-terminal ROS-responsive module to the exosome surface.

[0048] 4. Purification, Volume Adjustment, and Quantification: Transfer the secondary loading mixture to a 100 kDa ultrafiltration centrifuge tube with a molecular weight cutoff. Centrifuge at 4°C and 4000g, discard the filtrate, and add 500 μL of pre-cooled PBS buffer containing 5% (w / v) trehalose (i.e., a final trehalose concentration of 50 mg / mL) to the concentrate. Centrifuge and wash again. Repeat this washing step once. Finally, adjust the volume of the purified exosomes to 100 μL using the same buffer to obtain a purified programmed exosome (i-Exosome) suspension. After preparation and volume adjustment to 100 µL using this procedure, the particle concentration of the purified programmed exosome (i-Exosome) suspension is approximately (1.5 ± 0.3) × 10⁻⁶. 11 particles / mL.

[0049] 5. Characterization and Validation: Particle size and concentration were determined by NTA assay, and the results are as follows. Figure 4 The average particle size and distribution peak (~120 nm) of the programmed exosomes (i-Exosome) were basically consistent with those of the modified exosomes (Exo-PEG), and their particle number recovery rate (defined as the ratio of the total number of i-Exosome particles to the total number of Exo-PEG particles used) was >90%. Figure 4 (b). The above results indicate that the entire loading and purification process is stable and efficient, and can obtain high concentrations of programmed exosomes with high recovery rates.

[0050] 6. Loading Efficiency Validation (using FITC-labeled derivatives): To quantitatively evaluate the loading efficiency of the dynamic release chaperone (ROS-responsive peptide), a labeling working solution with a working concentration of 5 μM was prepared using a peptide covalently linked to the N-terminus of fluorescein isothiocyanate (FITC) for parallel experiments. Specifically, the experimental group (i-Exosome-FITC) underwent two-stage loading of membrane-anchored modified exosomes (Exo-PEG) according to the procedure described in Example 2. In the second-stage loading step, the FITC-labeled working solution replaced the original dynamic release chaperone working solution, and the labeled sample was purified. The control group (Exo-PEG) underwent the exact same incubation and purification process but without the addition of any dynamic release chaperone working solution. The purified samples from both groups were analyzed using nanoflow cytometry (488 nm excitation, 525 / 40 nm detection, side scattering gating, ≥10,000 particles per sample). The results are as follows: Figure 4 As shown in Figure c, the experimental group exhibited a significant FITC positive signal, demonstrating that the two-stage loading strategy can achieve efficient and specific anchoring of the dynamic release chaperone, and the process has good repeatability.

[0051] Example 3: Preparation and solidification of programmed exosome smart gel This embodiment aims to encapsulate the programmed exosomes with both stability and responsiveness prepared in Example 2 into an interpenetrating network gel with reactive oxygen species (ROS) responsiveness, thereby constructing a terminal formulation platform that integrates "long-term storage stability" and "pathological microenvironment-triggered release".

[0052] 1. Raw material synthesis: Synthesis of GelMA (methacrylamide gelatin): Gelatin was dissolved in 0.25 M carbonate buffer (pH 9.0) at 50°C to prepare a 10% (w / v) solution (i.e., a final gelatin concentration of 100 mg / mL). Then, under continuous stirring and at 50°C, methacrylic anhydride was slowly added dropwise at a ratio of 0.8 mL per gram of gelatin. After reacting for 3 hours, the reaction solution was dialyzed against deionized water at 40-50°C for 5-7 days (molecular weight cutoff 12-14 kDa). After lyophilization, GelMA was obtained. 1 H-NMR analysis showed that its degree of substitution was approximately 80%.

[0053] Synthesis of HA-SS-AC (Reactive Oxygen Response Acrylamide Hyaluronic Acid): Step 1: Synthesis of HA-SS-NH2. First, sodium hyaluronate (100 kDa) was dissolved in 0.1 M MES buffer (pH 5.5) to prepare a solution with a final concentration of 10 mg / mL. EDC and NHS were added sequentially, at amounts of 2 and 5 times the molar amount of the repeating unit of hyaluronic acid (each unit contains one carboxyl group), respectively. The mixture was stirred at room temperature for 30 minutes to obtain an activated solution. Subsequently, cystamine dihydrochloride was added to the activated solution at an amount of 1.2 times the molar amount of the repeating unit of hyaluronic acid. The reaction was continued at room temperature for 12 hours. After the reaction was completed, mixture A was obtained. Mixture A was transferred to a dialysis bag with a molecular weight cutoff (MWCO) of 3.5 kDa and dialyzed against deionized water for 3 days. After lyophilization, a white solid precipitate was obtained, which is the intermediate product HA-SS-NH2. Step 2: Synthesis of HA-SS-AC. The HA-SS-NH2 solid was redissolved in PBS, and the pH was adjusted to 8.5 with NaOH solution. Acrylic anhydride was slowly added dropwise under ice bath conditions, the amount being 10 times the molar amount of cystamine used in Step 1. The reaction was carried out at 4°C for 6 hours. After the reaction, mixture B was obtained. NaCl was added to mixture B to a final concentration of 5% (w / v), followed by 3 volumes of pre-cooled anhydrous ethanol. A white flocculent precipitate was observed to form. The precipitate was collected by centrifugation, washed with 75% ethanol, and redissolved in deionized water. The precipitate was then transferred to a dialysis bag with a MWCO of 12-14 kDa and dialyzed against deionized water for 2 days (changing the water 3-4 times during this period). Finally, the lyophilized solid was obtained as the final product, HA-SS-AC.

[0054] 2. Preparation of the smart gel precursor solution: Weigh 50.0 mg of methacrylamide gelatin (GelMA), 20.0 mg of reactive oxygen species-responsive acryloylamide hyaluronic acid (HA-SS-AC), 50.0 mg of trehalose, and 0.5 mg of photoinitiator Irgacure 2959, and dissolve them together in an appropriate amount of phosphate buffered saline (PBS, pH 7.4). Then, use PBS to precisely adjust the total volume of the mixture to 1.0 mL, and filter it through a 0.22 µm pore size filter for sterilization to obtain a clear and homogeneous smart gel precursor working solution. The final concentrations of each component in the smart gel precursor working solution are as follows: GelMA 5% (w / v), HA-SS-AC 2% (w / v), trehalose 5% (w / v), and Irgacure 2959 0.05% (w / v). The prepared working solution should be stored on ice in the dark and used within a short period of time.

[0055] 3. Mixing of Programmed Exosomes and Gel Precursor: In a clean bench, place 200 μL of the programmed exosome (i-Exosome) suspension prepared in Example 2 and 800 μL of the smart gel precursor working solution prepared in Example 3 into the same sterile 1.5 mL EP tube. Gently pipette and mix thoroughly, avoiding the introduction of air bubbles. This step aims to ensure uniform molecular-level dispersion of the exosomes in the cross-linked network precursor. After mixing, immediately place the centrifuge tube containing the mixture on ice to protect it from light and inhibit premature activation of the photoinitiator. Complete the subsequent dispensing and photocuring steps within 10 minutes.

[0056] 4. Dispensing and Photocrosslinking Curing: Rapidly dispense 100 μL of the "exosome-gel precursor mixture" obtained in the previous step into 48-well cell culture plates. Immediately place the culture plates in a 365 nm UV crosslinking chamber at 5 mW / cm². 2 Irradiate the gel under light intensity for 60-90 seconds until the liquid in the wells completely transforms into a non-flowing, transparent gel. Carefully remove the formed hydrogel discs (approximately 6 mm in diameter and 2 mm thick) using a sterile spatula, transferring each gel to an individual storage tube containing 1 mL of sterile stabilizing solution (PBS containing 0.1% (w / v) gentamicin). The final formulation—the intelligent programmed exosome hydrogel (i-ExoGel)—is stored at 4°C protected from light for subsequent experiments.

[0057] Example 4: Validation of the long-term stability of the formulation This embodiment aims to systematically evaluate the ability of i-ExoGel to maintain the bioactivity of exosomes under conventional cold chain storage conditions, and to reveal the synergistic stabilizing effect of "programmed engineering" and "smart gel carrier" by setting up a multi-dimensional control group.

[0058] 1. Experimental Design: Four groups of samples were set up and stored at 4℃ in the dark (except for the frozen control). The specific groups are as follows: Experimental Group 1 (Exo-PEG, membrane anchoring only): The Exo-PEG suspension (from step 2 of Example 1) was frozen at -80℃. Experimental Group 2 (Gel carrier control Exo-PEG Gel, membrane anchoring + ordinary gel): An equal amount of Exo-PEG was mixed with the smart gel precursor (without ROS-responsive elements) and solidified to obtain Exo-PEG Gel, which was stored at 4℃ to evaluate the effect of simple gel encapsulation. Experimental Group 3 (Programmed modification control, i-Exosome, membrane anchoring + programmed modification): Exo-PEG was programmed modified according to Example 2 to obtain i-Exosome suspension, which was stored in PBS containing 5% trehalose at 4℃ to evaluate the effect of programmed modification in solution. Experimental Group 4 (i-ExoGel complete system, i-ExoGel, membrane anchoring + programmed modification + smart gel): Exo-PEG was sequentially modified by programmed modification in Example 2 and encapsulated with smart gel in Example 3 to obtain i-ExoGel, which was stored at 4°C.

[0059] 2. Sample processing: Samples were taken at predetermined time points (months 0, 3, 6, 9, and 12). For gel samples (experimental groups 2 and 4), the gel matrix was thoroughly degraded by gentle digestion with 1 mg / mL collagenase I solution at 37°C for 2 hours to release the encapsulated exosomes. Subsequently, the exosomes from all groups were purified by size exclusion chromatography.

[0060] 3. Core Evaluation Indicators and Experimental Methods: The in vitro tubular formation ability of human umbilical vein endothelial cells (HUVECs) was used as the core indicator for evaluating exosome bioactivity. Exosome samples from experimental groups 1-4 were co-cultured with cells to obtain co-culture systems. Before preparing the co-culture systems, the total number of exosomes in the exosome samples from experimental groups 1-4 was normalized to ensure that the number of exosomes added to the cell co-culture system was the same for each group. The total length of tubular structures in the co-culture systems of different experimental groups was quantitatively calculated. With the tubular formation ability of the sample at month 0 as 100%, samples were taken at months 0, 3, 6, 9, and 12, and the relative activity retention rate at each time point was calculated. Results are as follows: Figure 5 After 12 months of storage, the exosome activity retention rate of experimental group 4 (i-ExoGel) was 66%, which was significantly higher than that of the other three groups.

[0061] Example 5: Validation of reactive oxygen species responsiveness and delivery efficiency of i-ExoGel (a) Verification of intelligent release characteristics (e.g.) Figure 6 a) This embodiment aims to verify the smart release characteristics of the i-ExoGel formulation, namely, that it remains stable under normal conditions, but can rapidly release encapsulated exosomes in a microenvironment with increased reactive oxygen species (ROS) simulating lesions.

[0062] 1. Establishing a release model: Take the i-ExoGel sample prepared in Example 4 (each sample contains approximately 1×10⁻⁶ g / L). 9 The particles exosomes were placed in dialysis bags containing 1 mL of release medium (MWCO: 300 kDa).

[0063] 2. The release media were divided into two groups: PBS (control group); PBS containing 100 μM H2O2 (experimental group, simulating the pathological level of ROS microenvironment).

[0064] 3. Experimental Method: The dialysis bag was immersed in a container containing a large amount of the corresponding release medium and incubated in a shaker (100 rpm) at 37°C. All external media were collected at 0, 3, 6, 12, and 24 hours, and replaced with fresh media. The exosome content in each collected medium was quantitatively determined using a highly sensitive CD63 exosome surface marker ELISA kit, and the cumulative release percentage was calculated.

[0065] 4. Experimental results: such as Figure 6 As shown in Figure a, i-ExoGel exhibits different release kinetics in the two media. In the experimental group containing 100 μM H2O2, exosome release is rapid, with a cumulative release rate exceeding 70% after 24 hours, demonstrating significant ROS-responsive release characteristics. In contrast, in the control group containing ordinary PBS, release is extremely slow, with a cumulative release rate of less than 30% after 24 hours, demonstrating the gel's good stability under normal physiological conditions.

[0066] (II) Comparison of delivery efficiency of different formulations under pathological ROS conditions (e.g.) Figure 6 (b) This section aims to simulate a uniform high-ROS pathological microenvironment and systematically compare the differences in release kinetics of four different exosome formulations under ROS stimulation in Example 4, thereby evaluating their delivery efficiency.

[0067] 1. Establishing a release model: Four groups of samples stored for 0 months (fresh) in Example 4 were tested in parallel: Experimental group 1 (Exo-PEG, membrane anchoring only); Experimental group 2 (gel carrier control, Exo-PEG Gel, membrane anchoring + ordinary gel); Experimental group 3 (programmed modification control, i-Exosome, membrane anchoring + programmed modification); Experimental group 4 (i-ExoGel complete system, i-ExoGel, membrane anchoring + programmed modification + smart gel). Each group was guaranteed to contain approximately 1×10⁻⁶ samples. 9Particle exosomes were placed in dialysis bags containing 1 mL of release medium (MWCO: 300 kDa).

[0068] 2. Release medium: PBS medium containing 100 μM H2O2 is used uniformly to simulate the pathological ROS microenvironment.

[0069] 3. Experimental Method: The dialysis bag was immersed in a container containing a large amount of the corresponding release medium and incubated in a shaker (100 rpm) at 37°C. All external media were collected at 0, 3, 6, 12, and 24 hours, and replaced with fresh media. The exosome content in each collected medium was quantitatively determined using a highly sensitive CD63 exosome surface marker ELISA kit, and the cumulative release percentage was calculated.

[0070] 4. Experimental results, such as Figure 6 Under uniform 100 μM H2O2 stimulation, the four formulations exhibited distinctly different release kinetics. The i-ExoGel group showed the fastest and most complete release curve, with a 24-hour cumulative release rate significantly higher than all other groups (p<0.05). This indicates that i-ExoGel not only achieves intelligent release, but its gel matrix and programmed modifications also effectively protect exosomes from inactivation in the harsh pathological microenvironment.

[0071] The results of this embodiment ( Figure 6 The results show that i-ExoGel possesses excellent ROS-responsive smart release characteristics. In simulated pathological high ROS environments, compared with other formulations, it can achieve more efficient exosome delivery and more complete biological function protection, providing key evidence for its targeted therapeutic application in ROS-elevated lesions such as ischemia and inflammation.

[0072] Example 6: Application of i-ExoGel in Diabetic Skin Wound Repair This embodiment aims to evaluate the therapeutic effect of a smart gel loaded with programmed exosomes (i-ExoGel) on refractory diabetic skin ulcers.

[0073] 1. Animal model: Eight-week-old male db / db mice were used to create a full-thickness skin defect with a diameter of 6 mm on their backs.

[0074] 2. Experimental Grouping and Treatment (n=6): Animals were randomly divided into the following four groups, the design of which directly corresponds to the key controls for in vitro functional validation: Group 1 (i-ExoGel Treatment Group): The wound was covered with i-ExoGel prepared according to Example 3 (complete system: membrane anchoring + programmed + smart gel). Group 2 (Exo-PEG Suspension, Membrane Anchoring Only): An equal volume of membrane-anchored exosome (Exo-PEG) suspension was injected locally into the wound (prepared as in Example 1, only membrane anchoring, without programmed and gelation processes). Group 3: Programmed Exosome Suspension Group: An equal volume of programmed exosome (i-Exosome) suspension (dissolved in PBS containing 5% trehalose) was injected locally into the wound, only programmed, without gel protection and sustained release. Group 4: Positive Drug Control Group: Commercially available recombinant epidermal growth factor gel (rhEGF Gel) was applied to the wound as a reference for the efficacy of commonly used clinical drugs. All interventions were performed on days 0, 3, and 6 after modeling, with 50 μL of gel or an equivalent amount of drug / solution applied to each wound each time.

[0075] 3. Comprehensive evaluation of efficacy during the study: dynamic tracking of wound area changes, calculation of healing rate and plotting of time-healing curves; collection of wound edge tissue and quantitative detection of reactive oxygen species (H2O2) levels to reveal the oxidative stress state of the wound microenvironment; full-thickness wound tissues were collected on the 7th day of treatment (proliferative phase), paraffin-embedded, sectioned and HE-stained to observe histological morphology; and qPCR and other techniques were used to analyze the expression changes of key inflammatory factors TNF-α, growth factor VEGF and antioxidant gene Nrf2 in the wound tissue.

[0076] 4. Experimental results are as follows Figure 7 The i-ExoGel treatment group showed significant advantages in many aspects: the wound closure speed was significantly faster, and the healing rate on day 5 and day 10 was significantly higher than that of other groups; histological examination showed that epithelial regeneration was earlier and more complete, the density of new blood vessels was higher, and the collagen fibers were more orderly arranged; molecular detection confirmed that the treatment could effectively downregulate pro-inflammatory factors and upregulate the expression of pro-angiogenic and antioxidant proteins.

[0077] The above results collectively demonstrate that the i-ExoGel system can achieve precise and synergistic therapeutic intervention targeting the unique pathological microenvironment of diabetic wounds (high oxidative stress, persistent inflammation, and vascular dysfunction), significantly promoting the repair of refractory wounds and providing solid preclinical experimental evidence for its clinical translation into chronic wounds such as diabetic foot ulcers.

[0078] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A method for preparing a programmed gel system, characterized in that, The preparation method includes the following steps: The steps for preparing membrane-anchored modified exosomes are as follows: First, the exosomes are modified using an amphiphilic lipid-polyethylene glycol conjugate, and then the modified product is purified to obtain the modified exosomes. The steps for preparing programmed exosomes are as follows: The modified exosomes are incubated with a static protective chaperone working solution. Through hydrophobic interactions and charge interactions, the protective chaperone is bound to the surface of the exosomes to obtain a primary loading mixture. The primary loading mixture is incubated with a dynamic release chaperone working solution to anchor the ROS response module to the surface of the exosomes to obtain a secondary loading mixture. The secondary loading mixture was washed to obtain programmed exosomes; The steps for preparing a programmed exosome smart gel are as follows: the programmed exosomes are mixed with a smart gel precursor solution to obtain a mixture; the mixture is then cured using electromagnetic radiation to obtain a programmed gel system.

2. The preparation method according to claim 1, characterized in that, In the step of preparing membrane-anchored modified exosomes, the amphiphilic lipid-polyethylene glycol conjugate includes DSPE-PEG-COOH, DSPE-PEG-NH2, DSPE-PEG-NHS, DSPE-PEG-Mal and / or DSPE-PEG-Biotin.

3. The preparation method according to claim 1, characterized in that, In the process of preparing programmed exosomes, the static protective chaperone working solution comprises heat shock protein 70 or a functionally active fragment thereof; the dynamic release chaperone working solution comprises a fusion polypeptide of an exosome binding domain and a reactive oxygen species (ROS) responsive domain; the sequence of the fusion polypeptide is as shown in SEQ ID NO: 1, or the fusion polypeptide is a variant having at least 80% identity with SEQ ID NO: 1 and retaining ROS responsiveness and exosome binding function.

4. The preparation method according to claim 1, characterized in that, In the process of preparing programmed exosomes, the incubation conditions for the static protective companion working solution and the exosomes are: 4°C static incubation for 30-120 minutes; the incubation conditions for the primary loading mixture and the dynamic release companion working solution are: 4°C dark static incubation for 60-150 minutes.

5. The preparation method according to claim 1, characterized in that, In the step of preparing programmed exosome smart gel, the components of the smart gel precursor solution include methacrylated gelatin, reactive oxygen species-responsive hyaluronic acid derivative, trehalose, and a photoinitiator.

6. The preparation method according to claim 1, characterized in that, In the step of preparing programmed exosome smart gels, the electromagnetic radiation includes ultraviolet light; the wavelength of the ultraviolet light is 355-375 nm, and the intensity is 3-10 mW / cm². 2 The irradiation time is 20-120 seconds.

7. A programmed gel system, characterized in that, The programmed gel system is prepared by the preparation method according to any one of claims 1-6; The programmed gel system includes programmed exosomes, an interpenetrating network gel matrix, and trehalose; The programmed exosomes are anchored and modified with an amphiphilic lipid-polyethylene glycol conjugate, and the programmed exosomes are linked with a static protection chaperone and a dynamic release chaperone. The interpenetrating network gel matrix comprises an interpenetrating first network and a second network; the first network is obtained by crosslinking methacryloyl gelatin; the second network is obtained by crosslinking reactive oxygen species-responsive hyaluronic acid derivatives; the interpenetrating network gel matrix encapsulates the programmed exosomes; The trehalose is dispersed in the interpenetrating network gel matrix.

8. The use of the programmed gel system of claim 7 or the programmed gel system prepared by the preparation method of any one of claims 1-6 in the preparation of a medicament for treating wounds.

9. The application according to claim 8, characterized in that, The wound includes chronic, non-healing wounds; the chronic, non-healing wounds include diabetic foot ulcers, pressure ulcers, and / or venous ulcers.

10. An exosome preservation kit, characterized in that, The kit comprises an amphiphilic lipid-polyethylene glycol conjugate, a static protection working solution, a dynamic release working solution, and a smart gel precursor solution. Preferably, the amphiphilic lipid-polyethylene glycol conjugate includes DSPE-PEG-COOH, DSPE-PEG-NH2, DSPE-PEG-NHS, DSPE-PEG-Mal and / or DSPE-PEG-Biotin; Preferably, the static protective companion working solution comprises heat shock protein 70 or a functionally active fragment thereof; the dynamic release companion working solution comprises a fusion polypeptide of an exosome binding domain and a reactive oxygen species (ROS) responsive domain; the sequence of the fusion polypeptide is as shown in SEQ ID NO: 1, or the fusion polypeptide is a variant having at least 80% identity with SEQ ID NO: 1 and retaining ROS responsive and exosome binding functions; Preferably, the components of the smart gel precursor solution include methacrylamide gelatin, reactive oxygen species-responsive acrylamide hyaluronic acid derivative, trehalose, and a photoinitiator.

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