Method for the preparation of exosomes based on dynamic hydrogels and delivery system

Abstract

The application designs a preparation method and delivery system of exosomes based on dynamic hydrogel. The preparation method comprises the following steps: adding gelatin, acrylic group modified cyclodextrin and a photoinitiator into a buffer solution to prepare a precursor solution; mixing cells and the precursor solution, and performing photo-induced crosslinking to form a dynamic hydrogel encapsulating the cells; after culturing the dynamic hydrogel in a culture medium, collecting the culture medium, and separating and purifying the functionalized exosomes. The exosomes have high purity and better repair effect on cells. The dynamic hydrogel can further be used as a delivery system by encapsulating the exosomes, and the period-controllable slow-release delivery is further realized by introducing a polypeptide capable of combining with the exosomes into the dynamic hydrogel.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials and biological agents, and in particular, a method for preparing and delivering exosomes based on dynamic hydrogels. Background Technology

[0002] Exosomes are cellular secretions consisting of tiny vesicles enclosed in a cell membrane, ranging from 50 to 150 nm in size. These minute vesicles can be released into the extracellular environment through secretion and transmit information between cells. Exosomes hold great promise for clinical applications because they are naturally secreted by cells, possess repair and antioxidant functions, and do not induce immune responses. Furthermore, their membrane structure and nanoscale size make them suitable as drug delivery systems, capable of crossing the blood-brain barrier, and hold immense potential in the treatment of central nervous system diseases.

[0003] Currently, most exosomes are obtained through two-dimensional culture on cell culture dishes or suspension culture on microspheres. While there are reports of using hydrogel GelMA to culture cells and obtain exosomes, these cell culture methods cannot effectively simulate the growth and physiological functions of cells under in vivo physiological conditions, nor the stress changes during cell production. Due to varying mechanical stimuli experienced by cells, the function of exosomes obtained by these methods may be limited, and they are also difficult to use as biomarkers for more accurate drug efficacy assessment. Furthermore, exosomes have poor stability; direct injection into the body results in rapid metabolic clearance, significantly reducing therapeutic efficacy. Typically, after injection, exosome solutions diffuse rapidly in the body, making precise treatment difficult. Therefore, developing new methods for exosome preparation, especially functionally enhanced exosomes, remains a pressing issue. Existing technologies have reported loading exosomes onto hydrogels for in vivo delivery, but the sustained-release effect of exosomes in vivo is unstable, making it difficult to determine the appropriate release cycle. Summary of the Invention

[0004] The main objective of this invention is to overcome the problems existing in the background art described above, and to provide a method for preparing exosomes based on dynamic hydrogels, and a delivery system thereof. One objective of this application is to provide a method for preparing exosomes, comprising the following steps:

[0005] 1) Prepare a precursor solution by adding gelatin, acrylic-modified cyclodextrin and photoinitiator to a buffer solution; mix the cells and the precursor solution, and photoinitiate cross-linking to form a dynamic hydrogel encapsulating the cells;

[0006] 2) After culturing the dynamic hydrogel in a culture medium, the culture medium is collected, and exosomes are obtained by separation and purification.

[0007] After cells are added to the precursor solution, a three-dimensional hydrogel network is formed through photocrosslinking. This hydrogel network is a dynamically crosslinked hydrogel network, including host-guest interactions between gelatin and cyclodextrin, and covalent crosslinking interactions between acrylic acid cyclodextrins. During cell culture, this dynamically crosslinked network better mimics the physical properties of the extracellular matrix. By culturing cells in the dynamic hydrogel in three dimensions, paracrine signaling is stimulated, resulting in exosomes with better functional repair compared to those obtained in conventional two-dimensional cell culture media. Furthermore, the dynamic properties and physicochemical characteristics of the dynamic hydrogel, such as its dynamic and mechanical properties, can be controlled by adjusting the degree of substitution of acrylic acid groups and the molar ratio between the host (gelatin) and guest (cyclodextrin). This allows for the simulation of the physiological characteristics of the microenvironment of different tissues in vivo (brain, heart, liver, spleen, lungs, kidneys, cartilage, bone, tumors, etc.), regulating the cell culture environment, thereby obtaining functionally enhanced cell-secreting exosomes and preparing customized exosomes for specific tissue repair or disease treatment.

[0008] In some preferred embodiments, the buffer solution has a pH of 7-8, the precursor solution contains 5-15% (w / v) gelatin, 5-15% (w / v) acrylic acid-modified cyclodextrin, and 0.01-0.5% photoinitiator; in some preferred embodiments, the modified cyclodextrin has an acrylic acid substitution degree of 1.0-1.4. Acrylic acid groups refer to groups containing acrylic acid and its derivatives.

[0009] In some preferred embodiments, the photoinitiator initiates photocrosslinking in the blue light range, such as 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone (I-2959) in the 365 nm wavelength range, and ethyl 2,4,6-trimethylbenzoylphenylphosphonate (TPOL) and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP) in the 365-405 nm wavelength range. In some embodiments, the photoinitiator is preferably phenyl(2,4,6-trimethylbenzoyl)phosphate (LAP).

[0010] The method for preparing exosomes described in this application is applicable to exosomes secreted by all types of cells. In some embodiments, the cells are stem cells, such as mesenchymal stem cells, neural stem cells, adipose stem cells, umbilical cord blood stem cells, and bone marrow stem cells; in some embodiments, the culture medium is a serum-free culture medium; in some embodiments, the dynamic hydrogel is first cultured in a conventional culture medium, and then cultured in a serum-free culture medium. In some embodiments, the exosomes prepared by the method can be used to a) prepare pharmaceutical compositions for treating diabetes; or b) prepare formulations or compositions that lower blood glucose, repair insulin-producing cells, increase insulin expression, and improve insulin resistance.

[0011] Another object of this application is to provide an application of a dynamic hydrogel in the preparation of exosomes, wherein the dynamic hydrogel comprises a dynamic cross-linked network formed by gelatin and acrylic-modified cyclodextrin through host-guest interactions, and the modified cyclodextrin is photo-initiated covalently cross-linked.

[0012] Another object of this application is to provide an exosome delivery system comprising a dynamic hydrogel formed by host-guest interactions and covalent cross-linking of gelatin and acrylic-modified cyclodextrin, wherein the cyclodextrin is photo-initiated for covalent cross-linking. In some embodiments, the delivery system further comprises a polypeptide linked to the dynamic hydrogel that can bind to exosomes. By linking the polypeptide to the dynamic hydrogel, a sustained-release period with controllable release can be provided for the exosomes.

[0013] In some embodiments, the release cycle of exosomes can be controlled by adjusting the molar ratio of the peptides and exosomes incorporated into the dynamic hydrogel.

[0014] In some embodiments, the polypeptide is linked to the hydrogel via N-hydroxysuccinimide (NHS) modified with acrylic acid groups. The amino groups on the polypeptide undergo click chemistry with the modified NHS, which then covalently crosslinks with cyclodextrin modified with acrylic acid groups via a photo-initiated crosslinking reaction. By introducing polypeptide chains that can bind to exosomes into the dynamic hydrogel, the sustained-release effect of exosomes is greatly enhanced, and precise delivery of exosomes is achieved, preventing their diffusion and clearance in vivo, which would affect the delivery efficiency.

[0015] The selection of the polypeptide chain is broad and can be optimized for different delivery sites or exosome delivery functions, thus having wide applications. For example, in some embodiments, the polypeptide includes, but is not limited to, CP05 and its derivative peptides, CP06 and its derivative peptides, CP07 peptide and its derivative peptides, and integrin-related binding peptides. In some preferred embodiments, the polypeptide chain is additionally linked with a glycine (G) as the first N-terminus, that is, a glycine is added before the N-terminus of the polypeptide peptide segment.

[0016] Another object of this application is to provide a method for preparing an exosome delivery system, comprising:

[0017] 1) Mix gelatin and acrylic acid-modified cyclodextrin in a buffer solution, add a photoinitiator, and prepare a precursor solution;

[0018] 2) Mix the precursor solution and exosome solution, and then irradiate with light to initiate cross-linking and form a hydrogel.

[0019] In some embodiments, the process further includes premixing an acrylic-modified N-hydroxysuccinimide with a polypeptide at 4°C as a replenishing solution, then adding the replenishing solution to an exosome solution and mixing to obtain an exosome mixture, which is then added to the precursor solution and photo-initiated to form a sustained-release dynamic hydrogel; wherein the polypeptide can bind to the exosomes.

[0020] In some embodiments, the polypeptide includes, but is not limited to, CP05 and its derivative peptides, CP06 and its derivative peptides, CP07 and its derivative peptides, and integrin-related binding peptides. In some preferred embodiments, the polypeptide chain is additionally linked with a glycine (G) as the first N-terminus. In some embodiments, the polypeptide chain is PPFLMLLKGSTR, PPFLMLFKSPKR, or PPFLMLFKSPKG; in some preferred embodiments, the molar ratio of the modified NHS to the polypeptide ranges from (5:1) to (1:5), for example, optionally 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5, and the concentration of the modified NHS or polypeptide in the supplemental solution is 0.04-1.5 mg / mL.

[0021] Another objective of this application is to provide an application of a dynamic hydrogel system in the preparation and delivery of exosomes. Specifically, exosomes are obtained by three-dimensionally culturing cells using a dynamic hydrogel, and then the hydrogel system is used to deliver the exosomes.

[0022] In some embodiments, the dynamic hydrogel system is formed via photocrosslinking initiated by a precursor solution comprising gelatin and acrylic acid-modified cyclodextrin. The dynamic hydrogel is formed through host-guest interactions between aromatic residues on the gelatin and the acrylic acid-modified cyclodextrin, and through photocrosslinking between carbon-carbon double bonds on the acrylic acid residues. This dynamic hydrogel can mimic the extracellular matrix environment, and using it for three-dimensional cell culture yields functionally enhanced exosomes. Subsequently, the exosomes are encapsulated in the hydrogel and injected into the delivery site, enabling precise sustained-release drug delivery at the therapeutic site.

[0023] Another object of this application is to provide an exosome kit comprising a precursor solution including gelatin, acrylic acid-modified cyclodextrin, and a photoinitiator; the precursor solution is mixed with cells, and photo-initiated cross-linking is performed to prepare a dynamic hydrogel for preparing functionally enhanced exosomes. After obtaining the exosomes, the precursor solution is added again, and photo-initiated cross-linking is performed to form a dynamic hydrogel for injection into specific areas for sustained-release delivery.

[0024] As a further step, the kit includes a replenishment solution comprising an exosome-binding peptide and an acrylate-modified N-hydroxysuccinimide. In some embodiments, the peptide includes, but is not limited to, CP05 and its derivative peptides, CP06 and its derivative peptides, CP07 and its derivative peptides, and integrin-associated binding peptides. In some preferred embodiments, the peptide chain is additionally linked with a glycine (G) as the first N-terminal nucleotide.

[0025] In some embodiments, after obtaining exosomes, a supplementary solution is added and mixed, followed by the addition of a precursor solution. Photoinitiated cross-linking forms a dynamic hydrogel, which is then injected into a specific site for sustained-release delivery, with a release period of over 14 days. In some embodiments, the release period of the exosome delivery can be controlled by adjusting the ratio of the supplementary solution to the exosomes. The kit utilizes a precursor solution to achieve the preparation of functionally enhanced exosomes and controlled-release delivery, resulting in good therapeutic effects while being convenient and efficient to use.

[0026] The beneficial technical effects of this application are as follows:

[0027] 1. Dynamic hydrogels can alter their dynamic and mechanical properties by adjusting their components, thus mimicking the physiological characteristics of different tissue microenvironments in vivo. This allows for the regulation of the composition of exosomes secreted by cultured cells, enabling the preparation of customized exosomes for specific tissue repair or disease treatment. Exosomes cultured in mesenchymal stem cells show more than three times the blood glucose-lowering effect compared to exosomes cultured using traditional two-dimensional methods. Combined with a sustained-release dynamic gel delivery system, blood glucose control is extended to over 14 days.

[0028] 2. The extraction process of exosomes obtained by the method of this application using differential centrifugation is more efficient than extraction methods based on two-dimensional or traditional three-dimensional culture. Because the cultured cells are encapsulated in a dynamic hydrogel, larger molecules such as cell debris, apoptotic bodies, and protein aggregates permeate into the cell culture medium at a lower rate and in greater quantities than exosomes. Under similar differential centrifugation conditions, the exosomes obtained by the method of this application have higher purity.

[0029] 3. Simple operation: It can achieve both exosome acquisition and delivery using a single hydrogel system, simplifying the types of reagents and making the operation easy to learn.

[0030] 4. Controllable delivery cycle: Depending on the treatment cycle, different exosome release curves can be achieved by changing the concentration of the supplemental solution, thus achieving the effect of long-term treatment with a single dose.

[0031] 5. Precision: Hydrogels can be injected to cover irregular wounds or directly injected into the lesion to achieve precise treatment. Attached Figure Description

[0032] Figure 1 Scanning electron microscopy (SEM) image of the exosomes prepared in Example 1 (left image), and nanoparticle tracking analysis (NTA) technique.

[0033] To characterize the distribution of exosomes (right figure).

[0034] Figure 2 The fluorescence intensity of exosomes encapsulated in hydrogels changes over time.

[0035] Figure 3 A schematic diagram illustrating the injectability of a sustained-release dynamic hydrogel encapsulating exosomes.

[0036] Figure 4 The viability of damaged β cells was determined after incubation for 72 hours with normal exosomes and functionally enhanced exosomes, respectively.

[0037] Figure 5 The left figure shows the changes in insulin expression in rats after injection with encapsulated exosome hydrogel, and the right figure shows the quantitative data of insulin expression fluorescence intensity.

[0038] Figure 6 Normalized quantitative data of insulin expression from Western blot (WB) (all exosome concentrations were 100 μg / mL).

[0039] Figure 7 Normalized quantitative data of phosphorylation of insulin receptor substrate IRS-1 (exosome concentrations were all 100 μg / mL).

[0040] Figure 8 Changes in blood glucose levels in diabetic rats after injection of exosomes.

[0041] Figure 9 IPGTT was tested on day 7 after diabetic rats received encapsulated exosome hydrogel injection.

[0042] Figure 10 IPGTT results of diabetic rats on day 14 after injection of encapsulated exosome hydrogel. Figure 11 Cell viability was determined by CCK-8 assay after three days of co-culturing MSCs obtained by different methods with damaged β cells. Detailed Implementation

[0043] The following provides a detailed description of some embodiments of the present invention. It should be emphasized that the following examples are merely exemplary and should not be considered as limiting the scope and application of the present invention.

[0044] The functionally enhanced exosomes described in this application refer to exosomes obtained by conventional two-dimensional and three-dimensional culture methods that possess enhanced abilities to regulate cell state and function, such as improving cell viability and enhancing cell repair capabilities. The enhanced function may also refer to lowering blood glucose, increasing insulin expression, improving insulin resistance, reducing inflammatory responses, promoting angiogenesis, inhibiting fibrosis, and enhancing immune function.

[0045] Unless otherwise stated, all reagents used in this application are commercially available products.

[0046] Example 1

[0047] 1.1 Preparation of Standard Dynamic Hydrogels

[0048] Synthesis of Acrylic Acid-Modified β-Cyclodextrin (AC-β-CD): 10 g of β-CD and 7 mL of triethylamine were added to 150 mL of dimethylformamide, and the mixture was cooled to 0 °C. 5 mL of acrylic acid was added to the mixture, and the mixture was stirred continuously at 0 °C for 12 hours. The mixture was then filtered to remove the precipitate, yielding a clear solution. This solution was concentrated by rotary evaporation, and the concentrated solution was slowly poured into 10 times its volume of acetone solution to obtain the precipitated crude modified β-CD product. The degree of acryloyl substitution of the modified β-CD was calculated by NMR. Using this method, modified β-CD with a degree of substitution between 1.0 and 1.4 was prepared by varying the ratio of cyclodextrin to acrylic acid. In this example, Ac-β-CD with degrees of substitution of 1.0, 1.1, 1.2, 1.3, and 1.4 were synthesized. Adjusting the degree of acrylic acid substitution in modified cyclodextrin can regulate the mechanical properties of dynamic hydrogels. Within this range, the storage modulus of the hydrogel is 0.8-3 kPa. As the degree of substitution increases, the storage modulus increases accordingly, thereby matching the growth environment of different cells and optimizing the function of cultured cells. Furthermore, the mechanical strength of the hydrogel also affects the release rate of its encapsulated exosomes.

[0049] Preparation of the precursor solution for the standard dynamic hydrogel: The precursor solution of the hydrogel mainly consists of gelatin and modified β-CD. A photoinitiator (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, LAP, TCI) is added and dissolved in PBS. The precursor solution forms a dynamic hydrogel under light irradiation. In this example, precursor solutions with different proportions of gelatin and modified CD were prepared, wherein the gelatin content ranged from 5% w / v to 10% w / v, the modified β-CD from 8% w / v to 15% w / v, and the LAP from 0.01% w / v.

[0050] According to the test results, when the ratio of the pre-body solution is within the above range, the resulting dynamic hydrogel has good injectability and adhesion; the hydrogel has moderate strength, is suitable for the growth of bone marrow mesenchymal stem cells, is non-cytotoxic and degradable.

[0051] Human tissues possess dynamic stress relaxation properties, a characteristic lacking in most traditional hydrogel materials. Dynamic hydrogels can highly mimic the extracellular matrix of human cells. Their highly dynamic three-dimensional network structure not only exhibits cell-adaptive properties, allowing them to adjust their network structure as cells grow, but also, as discovered in this application, provides mechanical stimulation to cells in ways that traditional hydrogels cannot, promoting cell proliferation and regulating cell secretion, thereby increasing exosome production and obtaining customized, functionally enhanced exosomes.

[0052] Cells within living organisms are constantly subjected to various forms of mechanical stimulation (such as the rigidity of the extracellular matrix and stress relaxation). The dynamic hydrogel described in this application possesses tunable stress relaxation and mechanical properties, enabling it to provide cells with diverse mechanical environments. Cells can sense these mechanical stimuli and transduce these signals into biochemical signals, thereby activating a series of intracellular responses and ultimately influencing cell differentiation, migration, and other functions. Unlike traditional three-dimensional hydrogel culture, which only provides support and protection for cells, this application discovers that dynamic hydrogels can provide a large number of mechanical stimulation signals to promote exosome secretion. Currently, many physical and chemical stimulation methods exist to increase exosome secretion rates and yields. Studies have shown that physical stimulation can significantly increase exosome production, especially mechanical stimulation, which can increase secretion values ​​by approximately 150 times compared to traditional two-dimensional culture. However, excessive mechanical stimulation can cause excessive cellular stress, affecting cell growth. The dynamic gel developed in this project possesses tunable stress relaxation and mechanical properties, mimicking the mechanical characteristics of the extracellular matrix in the human body, providing cells with appropriate mechanical stimulation, promoting exosome generation while maintaining high cell viability.

[0053] Furthermore, the mechanical properties of dynamic hydrogels can be modulated over a wide range by altering host-guest interactions or covalent cross-linking. Therefore, by adjusting the component ratios of dynamic hydrogels, the microenvironmental characteristics of different tissues in vivo can be precisely simulated, matching the matrix stiffness and mechanical properties of the native microenvironment in which different cells reside. This provides different cells with mechanical stimuli similar to their native microenvironment, enabling the construction of biomimetic three-dimensional culture models in vitro, resulting in customized exosomes with different functions to meet the therapeutic needs of various diseases.

[0054] Based on the above experiments, the inventors selected a standard dynamic hydrogel formulation ratio for subsequent testing (standard dynamic hydrogel). The ratio was: 8% w / v gelatin, 10% w / v modified β-CD, and 0.05% w / v LAP, dissolved in phosphate buffer (PBS) at 37°C. The standard dynamic hydrogel was then obtained by exposing it to 405nm visible light for 5 minutes with a wavelength power of 5-20W.

[0055] 1.2 Obtaining exosomes from cells cultured in dynamic hydrogels

[0056] Cells were encapsulated using a standard dynamic hydrogel. Primary bone marrow mesenchymal stem cells (MSCs) extracted from rats at 2 weeks gestation were used in the test. MSCs were first mixed with a precursor solution of the standard dynamic hydrogel, with a concentration of 5 × 10⁻⁶ MSCs per 50 μl of hydrogel precursor solution. 5 A hydrogel encapsulating cells was obtained by using the photocuring method described in 1.1.

[0057] The encapsulated MSCs were cultured in a complete medium (α-MEM medium (Gibco) supplemented with 16.7% fetal bovine serum (Sigma-Aldrich), 1% L-glutamine (Gibco), and 1% penicillin-streptomycin solution (Gibco)) for two days. After the MSCs were fully activated, they were cultured in a serum-free medium (Lonza X-Vivo20 for testing) for 24 hours. The serum-free medium was collected, and three-dimensional exosomes were obtained by differential centrifugation. The differential centrifugation was set at 4°C, centrifuging at 500g for 5 minutes to collect the supernatant, centrifuging at 2000g for 5 minutes to collect the supernatant, centrifuging at 10000g for 30 minutes to collect the supernatant, and finally centrifuging at 100000g for 70 minutes to collect the precipitate, which was the desired exosome. The experimental results of characterizing the exosomes by electron microscopy and nanoparticle tracking analysis (NTA) are shown in the figures below. Figure 1 As shown.

[0058] Comparison of exosomes prepared by dynamic hydrogels with other methods for obtaining exosomes

[0059] This experiment investigated the efficiency of obtaining exosomes from mesenchymal stem cells (MSCs) using conventional two-dimensional culture, GelMA culture, and dynamic hydrogel culture. In the GelMA hydrogel, the solid content of GelMA was 8%. MSCs were encapsulated using GelMA at a density of 500,000 cells per 50 μL of gel. Exosomes were obtained using differential centrifugation in α-MEM complete medium (α-MEM + 20% fetal bovine serum + 1% streptomycin / penicillin + 1% L-glutamine).

[0060] The number of bone marrow mesenchymal stem cells encapsulated in the standard dynamic hydrogel is consistent with the number of bone marrow mesenchymal stem cells in two-dimensional culture, and exosomes are obtained using differential centrifugation. The conventional two-dimensional culture medium formulation is: α-MEM medium (Gibco) supplemented with 20% fetal bovine serum (Sigma-Aldrich), 1% L-glutamine (Gibco), and 1% penicillin-streptomycin solution (Gibco).

[0061] The protein content of exosomes obtained by the above method was determined using the BCA method, and the number of exosome particles was detected using a nanoparticle tracer analyzer (NTA).

[0062] BCA assay: The BCA protein quantification kit from Beyotime was used for the assay. The concentration gradient curve was in the range of 0.1-1 mg / mL, and the R value of the concentration gradient curve was <0.99. Data were obtained in triplicate for each protein sample.

[0063] For NTA detection, control exosomes and enhanced exosomes were first balanced to the same concentration, then diluted 1000 times, vortexed thoroughly, and injected into the sample cell. Measurement was started when only 10-20 particles were visible in a single field of view in the observation camera. The instrument was Nanosight NS300, and the process was repeated three times.

[0064] The results showed that, using the BCA method, the protein concentration of exosomes prepared by dynamic hydrogels at the same concentration volume was approximately 2.5 times and 1.5 times that obtained by two-dimensional culture methods and GelMA culture methods, respectively. NTA analysis also revealed that, within the same confidence interval, exosomes obtained by dynamic hydrogels adjusted to the same concentration had a higher particle count, and the proportion of particles in the 90-130 nm range was higher in the generated images. The three-dimensional polymer network of the dynamic hydrogel restricts the release of larger cell debris and other impurities from the dynamic gel, while exosomes with a size of 50-150 nm are released more easily. Therefore, the purity of the exosomes subsequently isolated is higher than that obtained when cells are in direct or partial contact with the culture medium. These experiments demonstrate that exosomes obtained by dynamic hydrogels not only have higher yields but also higher purity compared to exosomes obtained by other two-dimensional and three-dimensional methods.

[0065] Example 2

[0066] 2.1 Preparation method of sustained-release dynamic hydrogel

[0067] Preparation of methacrylic acid-modified N-hydroxysuccinimide (MMA-NHS)

[0068] 86 mg of methacrylic acid (MMA) and 115 mg of N-hydroxysuccinimide (NHS) were dissolved in 10 mL of tetrahydrofuran (THF) at 5 °C. 206 mg of N,N'-dicyclohexylcarbodiimide (DCC) was added, and the mixture was stirred continuously for 24 hours. The reaction solution was filtered under vacuum to remove the precipitate. Ten times the volume of the solution of ice-cold diethyl ether was added, and the precipitate was collected by vacuum filtration. The precipitate was dissolved in THF, and recrystallized by adding ice-cold diethyl ether. This step was repeated three times. The collected product was dried in a vacuum oven to a solid state.

[0069] MMA-NHS can couple with the amino groups on peptides via click chemistry to form MMA-NHS-peptides. Subsequently, the carbon-carbon double bonds provided by the MMA groups can undergo visible light-initiated free radical polymerization with the carbon-carbon double bonds on acrylic acid-modified β-CD. A precursor solution is prepared by adding gelatin, modified β-CD, and a photoinitiator to a buffer solution. After adding the MMA-NHS-peptide, a sustained-release dynamic hydrogel containing the peptide can be formed by illumination.

[0070] Polypeptide synthesis

[0071] 1.0 g of 2-chlorotriphenylphosphine resin (containing 1 mmol of chlorine per kilogram) was added to a solid-phase synthesis tube and swollen with 10 mL of dry dichloromethane (DCM) for 1 hour. After removing the solvent, 3 equivalents of Fmoc-Arg(Pbf)-OH (3.0 mmol), 6 equivalents of N,N-diisopropylethylamine (DIPEA, 6.0 mmol), and 10 mL of dry N,N-dimethylformamide (DMF) were added to the resin. The resulting resin-containing solution was reacted at room temperature for 2 hours. After the reaction, the resin was washed three times alternately with DMF. To proceed with the next amino acid coupling reaction, the resin was treated with 10 mL of DMF containing 20% ​​piperidine at room temperature for 15 minutes to remove the Fmoc groups. Then, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU, 3 equivalents), 1-hydroxybenzotriazole (HOBT, 3 equivalents), and DIPEA (6 equivalents) were added to DMF to couple Fmoc-lys-OH (1 equivalent) to the resin for 1 hour. This process was repeated until the last proline (P) was added, followed by an additional glycine (G) as the first N-terminal molecule. After washing, the resin was treated with 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% water for 2 hours to cleave the peptide and remove side-chain protecting groups. After filtration, the peptide was precipitated with a large amount of cold water, and centrifuged to obtain the crude product. Finally, the crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC), and the synthesis was verified by quadrupole time-of-flight mass spectrometry (TOF-MS).

[0072] Using the experimental methods described above, the inventors synthesized an integrin-related binding peptide (peptide segments PPFLMLLKGSTR / PPFLMLFKSPKR / PPFLMLFKSPKG, representing three corresponding peptide sequences from humans, rats, and mice, respectively; the second rat-related peptide segment is used later). This sequence belongs to a segment within the LG3 domain of laminin α3, a chain-spherical domain. This sequence enables cell adhesion, diffusion, and binding to integrin α3β1. Integrin binding sites exist on the membrane structure of exosomes, thus enabling anchoring. Furthermore, due to the presence of multiple amino groups on the peptide chain side groups, a minimally sterically hindered amino acid, glycine G, was added before this peptide segment to ensure the click reaction occurs primarily at the peptide terminus.

[0073] Preparation of supplementary solution and sustained-release dynamic hydrogel :

[0074] The dynamic hydrogel formulations of this embodiment are shown in Table 1.

[0075] Table 1. Formulations of standard dynamic hydrogels and sustained-release dynamic hydrogels (1 mL system):

[0076]

[0077] This experiment investigated the effect of varying the final concentrations of MMA-NHS and peptides in the supplemental solution on the sustained-release effect. In this group of experiments, the final concentration of exosomes in the system was controlled at 1 mg / mL. The final concentration of MMA-NHS in the control group was 0.12 mg / mL, and the final concentration of the peptide solution was 1 mg / mL (ensuring a molar ratio of MMA-NHS to peptides of 1:1). The proportions of the experimental groups are shown in Table 2 below.

[0078] Table 2

[0079]

[0080] The four experimental groups and the control group were combined. After inserting exosomes into the DIO fluorescent probe, the sustained release of exosomes was observed under a laser confocal microscope over a four-week period. Compared with the control group, the sustained release effect of ratio 1 and ratio 2 was not as good as that of the control group, while the effect of ratio 3 and ratio 4 was slightly better than that of the control group, but the effect was not significant.

[0081] The final exosome concentration was varied: In this experiment, the molar ratio of MMA-NHS to peptide in the supplemental solution was controlled at 1:1. The final exosome concentration in the control group was 1 mg / mL. The final exosome concentrations in the experimental groups were 0.25 mg / mL, 0.5 mg / mL, 1.5 mg / mL, and 2 mg / mL. The four experimental groups and the control group were combined, and after inserting exosomes into a DIO fluorescent probe, the sustained-release of exosomes was observed under a laser confocal microscope over a two-week period. Compared with the control group, the low-concentration exosome group showed a similar sustained-release rate, while the high-concentration exosome group exhibited a burst release of exosomes, with excessively rapid initial release. This suggests that a final exosome concentration of 1-1.5 mg / mL is more suitable at this supplemental solution concentration.

[0082] Experimental results show that when the ratio of MMS-NHS to peptide is greater than 1:1, the release period is longer (more than 10 days). When the ratio is less than 1:1, the release rate of exosomes increases, but is still less than that of the standard dynamic hydrogel without added peptides (5 days). In this example, the concentration of MMS-NHS was 0.04-1.5 mg / mL.

[0083] In subsequent efficacy verification examples, an equimolar ratio of NHS and peptides was selected to allow the NHS to react with the N-terminus of the peptides while preserving the intact chain segment to perform its function, thus constructing a sustained-release dynamic hydrogel. By adjusting the molar ratio of NHS to peptides and the proportion of exosomes added, a controllable sustained-release period was achieved.

[0084] Preparation of sustained-release functionally enhanced exosomes by hydrogel encapsulation

[0085] An exosome mixture was prepared by mixing a supplemental solution (containing 1 mg / ml of modified NHS and peptides) with an exosome solution (2.5 mg / ml), with a volume ratio of 1:3. The exosome mixture was incubated at room temperature for 2 hours, then mixed at 37°C with the precursor solution and exosome mixture at a volume ratio of 3:2 (precursor solution:exosome mixture). Blue light initiation was then applied for 5 minutes to form a sustained-release dynamic hydrogel.

[0086] 2.2 Test of sustained-release effect of sustained-release dynamic hydrogel

[0087] After extracting functionally enhanced exosomes according to the method in Example 1, the exosomes were co-incubated with 3,3'-dioctadecyloxacarbonylchloride (DIO, a green fluorescent probe for cell membranes, from Beyotime Biotechnology) at 37°C for 20 minutes. Then, they were centrifuged at 4°C and 100,000 g for 70 minutes. The precipitate was collected, and the probe-inserted exosomes were encapsulated using the encapsulation method described in 2.1. The fluorescence intensity at different days was recorded using an LSM-880 laser confocal microscope (see Example 1). Figure 2The standard dynamic hydrogel was obtained by directly mixing the precursor solution and exosome solution and then initiating a photocrosslinking reaction. The sustained-release dynamic hydrogel was obtained by mixing the precursor solution and the supplement solution and then initiating crosslinking. The fluorescence spectrum below shows the quantitative fluorescence statistics, which show that the standard dynamic hydrogel releases most of the exosomes within 5 days, while the sustained-release dynamic hydrogel takes two weeks to reach the same fluorescence intensity.

[0088] An exosome mixture was prepared by mixing the supplementary solution with the exosome solution, and then a precursor solution was added to prepare a dynamic hydrogel. The molar ratio of peptide to exosome in the exosome mixture was tested at 3:1, 2:1, 1:1, 1:2, and 1:3 (final exosome concentration 1 mg / ml). Experimental results showed that all the above ratios produced a sustained-release effect. When the molar ratio of peptide to exosome was 1:3, the sustained-release period was approximately 14 days. Increasing the proportion of peptide gradually extended the sustained-release period. When the ratio was less than 1:1, the extension of the sustained-release period became less significant. After the exosome mixture was left at room temperature for 2 hours, it was mixed at 37°C with a volume ratio of precursor solution to exosome mixture of 4:1, 3:1, 2:1, 3:2, and 1:1, and then subjected to blue light initiation for 5 minutes to form a sustained-release dynamic hydrogel. Experimental results showed that when the proportion of the exosome mixture was constant, a ratio greater than 4:1 between the precursor solution and the exosome mixture reduced the release rate of the dynamic hydrogel.

[0089] Conventional hydrogel drug delivery systems have gel pores much larger than the diameter of exosomes. After implantation into the human body, exosomes are rapidly and massively released due to diffusion of concentration gradients and rapid exchange of substances with the surrounding environment. However, the dynamic hydrogel drug delivery system of this application incorporates peptides to anchor exosomes, allowing them to be slowly released as the hydrogel swells and degrades, thus achieving a long-term therapeutic effect.

[0090] Figure 3 This study demonstrates the injectability of a sustained-release dynamic hydrogel encapsulating exosomes. The hydrogel was stained pink with rhodamine and loaded into a syringe, allowing it to be injected through a 1 ml needle. It also exhibits customizable shape and adhesion, illustrating that the sustained-release dynamic hydrogel encapsulating exosomes can achieve precise local injection therapy.

[0091] Example 3

[0092] This embodiment investigates the enhancing function of exosomes prepared by dynamic hydrogels in cells.

[0093] 3.1 Experimental Methods

[0094] This experiment used functionally enhanced exosomes obtained from primary bone marrow mesenchymal stem cells cultured in hydrogel 3D as described in Example 1. Exosomes obtained from conventionally cultured bone marrow mesenchymal stem cells (MSCs) in 2D culture served as a control group. The culture medium formulation for the control group was: α-MEM medium (Gibco) supplemented with 20% fetal bovine serum (Sigma-Aldrich), 1% L-glutamine (Gibco), and 1% penicillin-streptomycin solution (Gibco). PBS solution containing the exosomes was added to the cell culture medium for incubation. The therapeutic effect in the cells was tested using the functionally enhanced exosomes prepared according to the method of Example 1. The treatment concentrations for the control group and the functionally enhanced exosome group were 10, 25 μg / mL, 50 μg / mL, 100 μg / mL, and 125 μg / mL.

[0095] Injury protocol for pancreatic β-cells (Oricell, INS-1 rat insulinoma cell line): Cells were treated with a mixture of 10 μmol / L streptozotocin (STZ, Beyotime Biotechnology, a commonly used drug for modeling type 2 diabetes) in standard culture medium (RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin-streptomycin solution (Gibco)) for 8 hours. Under these conditions, cell proliferation decreased by 25%, and reactive oxygen species (ROS) were upregulated by 28%, mimicking the injury state of pancreatic β-cells in the late stage of type 2 diabetes.

[0096] CCK-8 assay: Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology) was used to detect cell viability in two groups of pancreatic β cells after injury and treatment. Assay procedure: 10 μL of CCK-8 solution was added to each well of both injured and treated β cells. The cells were incubated for 45 minutes, and absorbance was measured at 450 nm. Cell proliferation was compared between the experimental groups by comparing absorbance values; higher absorbance indicates a higher cell count, meaning more cells proliferated within the same time period, i.e., faster cell proliferation.

[0097] 3) ROS Detection: The reactive oxygen species (ROS) detection kit from Beyotime Biotechnology was used to detect the damaged state of pancreatic β-cell lines. Detection procedure: The culture medium of the treated pancreatic β-cells was replaced with 10 μmol / L DCFH-DA (a probe that is non-fluorescent itself but can be hydrolyzed by intracellular esterases to generate DCFH after entering the cell. DCFH cannot permeate the cell membrane, and intracellular ROS can oxidize the non-fluorescent DCFH to generate fluorescent DCF. The level of intracellular ROS can be determined by detecting the intensity of green fluorescence). The cells were incubated at 37°C for 20 minutes, washed three times with serum-free culture medium to remove any DCFH-DA that had not entered the cells, and the intensity of green fluorescence was observed using an Invitrogen EVOS M5000 fluorescence imaging system.

[0098] 4) Immunofluorescence staining: Pancreatic β-cells were fixed overnight at 4°C with 4% paraformaldehyde solution, permeabilized with 0.25% Triton-X for 25 minutes, and blocked with PBS containing 3% bovine serum albumin and 0.1% Tween 20 for 1 hour. Insulin monoclonal antibody was used as the primary antibody (Proteintech, 1:500), and a green fluorescent mouse antibody was used as the secondary antibody (Proteintech, 1:500). Cell nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI, Beyotime Biotechnology, 1:500), and the cytoskeleton was stained with phalloidin (Cytoskeleton, 1:500). Observation and imaging were performed using an LSM-880 laser confocal microscope. This example also included immunofluorescence staining of the key pancreatic β-cell markers Glut2 / Glut4 to investigate the cell repair effect of exosomes.

[0099] 5) Western Blot Experiment: Pancreatic β cells were lysed using RIPA lysis buffer at 100 μL / well on ice for 30 minutes, centrifuged at 12000 rpm for 10 minutes, and the supernatant was collected. After determining the protein concentration using the BCA method, protein loading buffer was added, and the cells were incubated at 95°C for 10 minutes. The protein sample preparation was then complete. Subsequently, SDS-PAGE gels were prepared, and the protein sample and marker were added to the loading wells respectively. Electrophoresis was performed at 80V for 20 minutes and at 120V for 1 hour. The bands on the gel were transferred to a PVDF membrane using a semi-dry transfer method, and blocked with Yakult rapid blocking buffer for 20 minutes. The membrane was then incubated with the corresponding primary antibody overnight at 4°C, followed by incubation with the corresponding secondary antibody at room temperature for 1 hour. The membrane was then developed using a Bio-RAD imaging system.

[0100] This embodiment uses STZ-induced damaged β-cells to investigate the therapeutic effect of exosomes on damaged cells. Damaged β-cells were incubated for 72 hours with different concentrations of normal exosomes (control group) and functionally enhanced exosomes, respectively. Cell viability was then tested using the CCK-8 assay. Figure 4 The experimental data for the 100 μg / mL exosome group are shown; this example examines the immunofluorescence staining data of insulin, in which... Figure 5 The intensity of green in the left image reflects the amount of insulin expression, while the right image shows quantitative data on the fluorescence intensity of insulin expression. Immunofluorescence staining experiments showed that the key markers of pancreatic β cells, Glut2 / Glut4, also increased significantly after exosome treatment. Figure 6 This is normalized quantitative statistical data from Western blot results. It can be seen that the functionally enhanced exosomes prepared in this application, as measured by CCK-8, showed better cell viability in damaged β cells at concentrations of 50 μg / mL and 100 μg / mL. Furthermore, immunofluorescence staining and Western blot experiments showed that the functionally enhanced exosomes had better therapeutic effects than the control exosomes extracted from two-dimensional cell culture, including a higher increase in insulin expression. Figure 6 The insulin receptor substrate IRS-1 is phosphorylated to a higher degree. Figure 7 The expression of glucose transporter receptor proteins Glut2 and Glut4 was increased. IRS is a substrate of an activated pancreatic islet receptor tyrosine kinase, containing a dozen tyrosine residues that can be phosphorylated. Phosphorylated IRS exposes binding sites for numerous signal transduction chaperones. Insulin resistance inhibits IRS tyrosine phosphorylation, affecting insulin signal transduction and leading to diabetes. Therefore, increased IRS tyrosine phosphorylation indicates the restoration of cellular insulin sensitivity, effectively improving insulin resistance and restoring cellular function. This demonstrates that the functionally enhanced exosomes of this application not only unilaterally stimulate insulin expression but also inhibit apoptosis and repair pancreatic islet cells, showcasing the more comprehensive therapeutic capabilities of the exosomes obtained through hydrogel three-dimensional culture.

[0101] Example 4

[0102] This embodiment investigates the therapeutic effects of functionally enhanced exosomes prepared and delivered using dynamic hydrogels in animal experiments.

[0103] This embodiment examines the effect of applying the functionally enhanced exosomes obtained from the hydrogel system prepared by the method of Example 2 and the sustained-release effect of encapsulated exosomes in the treatment of type 2 diabetes.

[0104] Type 2 diabetes modeling: Rats were fed a high-fat diet for 30 days, then injected with streptozotocin (30 mg per kg body weight) for 5 consecutive days. After 3 days of observation, random blood glucose was measured for 3 consecutive days. If the daily blood glucose was >16.7 mM, an intraperitoneal glucose tolerance test (IPGTT) was performed. A passing result on the IPGTT was considered a successful modeling.

[0105] IPGTT test: After fasting for 12 hours, the fasting blood glucose of rats was measured by intraperitoneal injection of 50% glucose solution (2g per kg body weight). Blood glucose values ​​were then measured at three time points: 40, 80 and 120 minutes, and a curve was plotted.

[0106] Exosome injection therapy: 1 mg / mL of functionally enhanced exosomes (in PBS solution) were encapsulated in a sustained-release dynamic hydrogel, wherein the volume ratio of precursor solution: supplement solution: exosome solution was 6:1:3.

[0107] In type 2 diabetic rats, 500-700 μL of hydrogel (the specific injection volume depended on the rat's body weight) was injected orally into the pancreas to achieve a dose of exosomes of 1 mg per kg of body weight. Five experimental groups were designed: a normal control group (healthy rats), a diabetic group (untreated type 2 diabetic rats), a sustained-release dynamic hydrogel treatment group (type 2 diabetic rats injected with sustained-release dynamic hydrogel), an exosome treatment group (type 2 diabetic rats treated with enhanced exosomes), and a sustained-release exosome treatment group (type 2 diabetic rats treated with extended-release dynamic hydrogel-encapsulated exosomes). The normal control and diabetic groups received the same volume of saline. Blood glucose levels were monitored in rats for two weeks following the single exosome injection. The results are as follows: Figure 8 As shown. Figure 8 This is a summary graph of the average random blood glucose data measured at different time points for each rat in five groups during a two-week treatment period. Throughout the two-week cycle, the blood glucose level in the sustained-release exosome treatment group remained at a low level, while in contrast, the exosome treatment group showed a decrease in blood glucose as early as the day after injection, but the blood glucose level rose to a hyperglycemic state as the treatment cycle progressed. Figure 9 and Figure 10 The IPGTT was tested on day 7 and day 14, respectively. This experiment further demonstrates that sustained-release dynamic hydrogel in situ injection into the pancreas has a long-term therapeutic effect on type 2 diabetes, can enhance the local organ-specific sustained-release function of exosomes, and control blood glucose in type 2 diabetic rats within a two-week test period.

[0108] Example 5

[0109] This embodiment investigates the regulatory effect of dynamic hydrogels on exosomal proteins secreted by cells.

[0110] To verify the effect of the dynamic hydrogel of this application in regulating cell behavior, the efficacy of bone marrow mesenchymal stem cells (MSCs) encapsulated in the dynamic hydrogel and cultured in two-dimensional and conventionally encapsulated MSCs in GelMA hydrogels in the treatment of damaged β cells was investigated. The experimental methods are as follows:

[0111] MSC and β-cell co-culture experiment: A total of five groups were designed: normal β-cell group, untreated damaged β-cell group after streptozotocin (STZ) treatment group, MSC treatment group using two-dimensional culture (STZ-β-cell+MSC), MSC treatment group using GelMA encapsulation group (STZ-β-cell+MSC@GelMA), and MSC treatment group using dynamic gel GelCD encapsulation group (STZ-β-cell+MSC@GelCD).

[0112] The co-culture experiment design for MSCs and β cells was as follows: The GelMA hydrogel contained 8% GelMA, while the GelCD hydrogel formulation consisted of 8% gelatin and 10% modified CD. MSCs were encapsulated using GelMA and GelCD separately at a density of 500,000 cells per 50 μL gel. They were initially cultured separately for two days in α-MEM complete medium (α-MEM + 20% fetal bovine serum + 1% streptomycin / penicillin + 1% L-glutamine) to allow the dynamic mechanical properties of the gel to fully stimulate the MSCs. After β cells were treated with STZ to create damaged β cells, the GelCD / GelMA encapsulating MSCs were co-cultured with the damaged β cells in 0.4 μm pore size transwell chambers. Simultaneously, 500,000 MSCs loaded onto fresh transwell chambers were co-cultured with the damaged β cells (these MSCs belonged to the same batch as the MSCs encapsulated two days prior), creating an MSC treatment group. After three days of co-culture, subsequent experiments including CCK-8 assays, qPCR, and immunofluorescence staining were performed.

[0113] In CCK-8 assays, under the same conditions, the MSC treatment group encapsulated in dynamic hydrogels was able to restore damaged β cells to 77% of their normal cell viability, a significant 15% improvement over the other two treatment groups. Figure 11(As shown). In subsequent qPCR experiments, regarding gene expression in MSCs co-cultured with damaged β cells, MSC@GelMA and MSC@GelCD showed significantly higher expression of Il-6, CNTF, TGF-β, and adiponectin in MSC@GelCD compared to the other two groups. These four genes have been reported in the literature for successfully treating type 2 diabetes. Therefore, it is believed that the paracrine function of MSCs stimulated by the unique mechanical properties of the dynamic hydrogel is fully activated, enabling them to secrete more cellular secretions with therapeutic effects on diabetes—something difficult to achieve with conventional hydrogels. Therefore, the dynamic hydrogel of this application plays a prominent role in regulating cell secretion.

[0114] This experiment further investigated the qPCR experiment of functionally enhanced exosomes obtained by dynamic hydrogels. The specific method is as follows:

[0115] 1. RNA Extraction: Add 1 mL of Trizol reagent (Novazia) to normal β cells, damaged β cells, control exosome treatment group, and enhanced exosome treatment group, respectively, and react on ice for 20 minutes. Add 200 μL of chloroform (pre-cooled on ice), invert and mix about 60 times (until pinkish-white), and let stand on ice until separation occurs (upper layer milky white, lower layer pinkish-purple). Centrifuge at 12000 rpm, 4°C for 15 minutes, collect the supernatant (about 500 μL), add an equal volume of isopropanol (pre-cooled on ice), gently invert and mix, let stand on ice for 10 minutes to precipitate RNA. Centrifuge at 12000 rpm, 4°C for 10 minutes. A white precipitate will appear at this point. Discard the supernatant in a laminar flow hood. Add 900 μL of... Use 75% ethanol (prepared fresh and pre-cooled on ice) to gently blow up the precipitate, centrifuge at 7500 rpm and 4°C for 5 minutes, discard the supernatant, and repeat twice; remove the ethanol, place the centrifuge tube in a clean bench and blow for 5 minutes, add 20 μL of DEPC water, and place in a 60°C water bath for 10 minutes to dissolve the RNA precipitate.

[0116] 2. Reverse transcription: RNA concentration was measured using a Thermo Fisher NanoDrop micro spectrophotometer. Reverse transcription was performed with 2 μL of reverse transcriptase (Takara) and 500 ng of RNA per 10 μL system to obtain cDNA.

[0117] 3. qPCR experiment: Each reaction well contains 20 μL of reaction system, and the volumes of each component are shown in Table 3.

[0118] Table 3

[0119] reagents volume Rapid Taq Enzyme Mix (Takara) 10μL DEPC Water (Biyuntian) 7.2μL cDNA 2μL qPCR Primer-F 0.4μL qPCR Primer-R 0.4μL

[0120] The reaction process is shown in Table 4.

[0121] Table 4

[0122] 95℃ 5 minutes 95℃ 15 seconds 60℃ 15 seconds 72℃ 15 seconds (return to 95℃ for 15 seconds, then expand for 30 and 40 cycles respectively) 4℃ ∞

[0123] The primers used for qPCR detection in this embodiment included insulin, Pdx1, Nlrp3, Caspase3, Glut2, and Glut4. Pdx1 is the pancreatic-duodenal homology cassette 1, and its activation promotes the expression of important pancreatic β-cell genes such as insulin, somatostatin, and glucokinase. Nlrp3 is a nucleotide-binding oligomerization domain-like receptor protein belonging to the inflammasome family. Caspase3 is caspase 3, a protease that can cause DNA cleavage and promote apoptosis. Glut2 and Glut4 are two glucose transporter receptor proteins. The experiments showed that the enhanced exosome treatment group exhibited the highest expression of insulin and Pdx1 genes, demonstrating that enhanced exosomes can restore the ability of β-cells to secrete insulin. The lowest expression of Nlrp3 and Caspase3 genes indicates that the addition of enhanced exosomes improved the inflammatory microenvironment of damaged β-cells. The highest expression of Glut2 and Glut4 genes indicates that enhanced exosomes also improved β-cell function and promoted the recovery of β-cell viability.

[0124] Proteomics analysis of exosomes

[0125] Sample preparation: Frozen exosome samples were removed, added to liquid nitrogen, and thoroughly ground. Pre-cooled acetone containing 10% trichloroacetic acid was added at a 1:10 ratio, and the mixture was precipitated at -20°C for 1 hour. The precipitate was then centrifuged at 15000g for 15 minutes at 4°C, collected, and pre-cooled acetone was added. The mixture was precipitated at -20°C for 1 hour, and this process was repeated once. The precipitate was then centrifuged at 15000g for 15 minutes at 4°C, collected, and left exposed for approximately 5 minutes until dry. The dried precipitate was added to phenol solution and mixed until evenly dispersed. An equal volume of phenol-Tris-hydrochloric acid (7.5) saturated solution was added, and the mixture was stirred at 4°C for 30 minutes. The mixture was centrifuged at 5000g for 30 minutes, and the upper phenol layer was collected. Five volumes of pre-cooled 0.1M ammonium acetate-methanol solution were added, and the mixture was precipitated at -20°C for 1 hour. The precipitate was then centrifuged at 10000g for 10 minutes at 4°C, and the precipitate was collected. Five volumes of pre-cooled methanol were then added, and the mixture was stirred gently. The mixture was centrifuged at 10000g for 10 minutes at 4°C, and the precipitate was collected. Repeat the steps twice with acetone instead of methanol to thoroughly remove methanol. Centrifuge at 10000g for 10 minutes at 4℃ and collect the precipitate. Dissolve the dried powder in the sample lysis buffer and incubate at 30℃ in a constant temperature water bath to fully dissolve the protein. Centrifuge the solution at 15000g for 15 minutes at room temperature, collect the supernatant, and centrifuge again to collect the supernatant to thoroughly remove insoluble impurities. The supernatant is the total protein solution of the tissue. Determine the protein concentration, aliquot, and store at -80℃ for later use.

[0126] Total protein quantification: The concentration of this sample was determined according to the BCA method. Protein reacts with Cu under alkaline conditions. 2+ Complexation, and Cu 2+ Reduced to Cu + BCA and Cu + They combine to form a stable purple-blue complex, which has a high light absorption value at 562 nm and is proportional to the protein concentration, thus allowing for the determination of protein concentration.

[0127] Sample testing: 20 μg of each sample was taken and separated by 12% SDS-PAGE electrophoresis. The separated gel was stained with Coomassie Brilliant Blue and scanned with an Image Scanner in grayscale mode at 300 dpi.

[0128] Protein reduction alkylation and enzymatic digestion: Protein concentration in the supernatant was determined using a 500M triethylammonium bicarbonate (TEMB) kit. 100 μg of each sample was then transferred to a new tube and adjusted to 100 μL with 8M urea. 11 μL of 1M dithiothreitol (DTT) was added and incubated at 37°C for 1 hour. The sample was then transferred to a 10K ultrafiltration tube (Millipore) and centrifuged at 14000g for 10 minutes. 120 μL of 55 mM iodoacetamide was added, and the mixture was incubated at room temperature in the dark for 20 minutes. After three consecutive centrifugations with 100 mM TEAB to replace the urea in the ultrafiltration tube, the sample was digested overnight with a 1:50 solution of trypsin (Promega).

[0129] Liquid chromatography-mass spectrometry (LC-MS / MS) was used for analysis: Each sample was suspended in 30 μL of solvent (C: 0.1% formic acid aqueous solution; D: acetonitrile solution containing 0.1% formic acid) and separated by nano-LC, followed by online electrospray ionization tandem mass spectrometry analysis. The experiment was performed on an Easy-nLC 1000 system (Thermo Fisher Scientific, MA, USA), connected to a Q-Exactive mass spectrometer (Thermo Fisher Scientific, MA, USA) equipped with an online nano-electrospray ionization source. 10 μL of peptide sample was loaded at a flow rate of 10 μL / min onto a trapping column (Thermo Scientific Acclaim PepMap C18, 100 μm x 2 cm), followed by linear gradient separation in an analytical column (Acclaim PepMap C18, 75 μm x 15 cm): 3% D to 32% D within 120 min. The column was equilibrated under initial conditions for 10 min. The column flow rate was controlled at 300 nL / min, and the electrospray voltage was 2 kV. Analysis of the data showed that most fractions were separated with an efficiency of approximately 70%, and no spray interruptions occurred, indicating that the gradient setting was ideal.

[0130] Proteomics analysis identified 5003 proteins under the Peptide Threshold 1.0% FDR and 1 Unique Peptide standard, which is within the normal range. The total number of spectra and their quality accuracy were both normal and reliable, and the spectra identification rate was normal. The statistical significance assessment of quantitative differentially expressed proteins was normal. Differential protein analysis was performed using bioinformatics to analyze the protein families to which the upregulated and downregulated proteins belonged.

[0131] In this embodiment, bone marrow mesenchymal cells (MSCs) were loaded onto a dynamic hydrogel. Cytoskeletal proteins were enriched using a cytoskeleton enrichment kit. Proteomics sequencing revealed several differentially expressed proteins, including myomicrotubule-associated protein 14 (MTMR14), fibrinolytic acid, inhibitor of metalloproteinases 2, mitochondrial fusion protein 1, type IV collagenase, FH1 / FH2 domain protein 1, and ATP-dependent RNA helicase. A search of the KEGG functional database showed that MTMR14 is involved in triphosphate metabolism, and triphosphates are involved in exosome secretion. Real-time quantitative PCR (qPCR) and immunofluorescence staining further demonstrated that the dynamic hydrogel promotes exosome secretion by regulating triphosphate metabolism. Therefore, the dynamic hydrogel of this application can regulate cellular exosome secretion through its rich mechanical properties and mechanical self-adaptability, representing a non-engineered, natural regulatory mechanism.

[0132] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A method for preparing an exosome delivery system, comprising: i) Mix gelatin and acrylic-modified cyclodextrin in a buffer solution, add a photoinitiator, and prepare a precursor solution; ii) N-hydroxysuccinimide modified with acrylic acid groups is premixed with peptides as a supplementary solution. The supplementary solution is then added to exosomes and mixed to obtain an exosome mixture. This mixture is then added to the precursor solution, and photo-induced cross-linking is used to form a sustained-release dynamic hydrogel as an exosome delivery system. The polypeptide is a polypeptide that can bind to the exosome, and the polypeptide is one or more of CP05 and its derivative peptides, CP06 and its derivative peptides, CP07 peptide and its derivative peptides, and integrin-related binding peptides.

2. The method as described in claim 1, characterized in that, The exosomes were prepared by the following method: 1) Mix the cells and precursor solution, and photo-initiated cross-linking forms a dynamic hydrogel encapsulating the cells; 2) After culturing the dynamic hydrogel in a culture medium, collect the culture medium and separate and purify the exosomes.

3. The method as described in claim 2, characterized in that, The cells are stem cells, and the exosomes obtained from the cells are used for one of the following a)-b): a) Preparation of a pharmaceutical composition for treating diabetes; b) Prepare formulations or compositions that can lower blood glucose, repair insulin cells, increase insulin expression, and improve insulin resistance.

4. The method according to any one of claims 1-3, characterized in that, A glycine G is added to the N-terminus of the polypeptide.

5. The method as described in any one of claims 1 or 3, characterized in that, The release cycle of the exosomes can be controlled by adjusting the ratio of the supplemental fluid to the exosomes.

6. An exosome delivery system obtained by the method according to any one of claims 1-5, characterized in that, The delivery system comprises a dynamic hydrogel formed by host-guest interactions and covalent cross-linking of gelatin and acrylic acid-modified cyclodextrin, wherein the cyclodextrin is photo-initiated for covalent cross-linking. The delivery system further comprises a polypeptide bound to exosomes and attached to the dynamic hydrogel, wherein the polypeptide is linked by acrylic acid-modified N-hydroxysuccinimide to provide sustained-release of exosomes with a controllable release period.

7. The application of the delivery system as described in claim 6 in the delivery of exosomes.

8. An exosome kit for use in the delivery system of claim 6, characterized in that, The kit includes a precursor solution and a supplement solution. The precursor solution includes gelatin, acrylate-modified cyclodextrin, and a photoinitiator. The supplement solution includes an exosome-binding peptide and acrylate-modified N-hydroxysuccinimide. The precursor solution contains 5-15% by weight (v / v) gelatin, 5-15% by weight (v / v) acrylate-modified cyclodextrin, and 0.01-0.5% by weight (v / v) photoinitiator. The exosome-binding peptide is one or more of CP05 and its derivative peptides, CP06 and its derivative peptides, CP07 and its derivative peptides, and integrin-related binding peptides.

9. The reagent kit as described in claim 8, characterized in that, Add a glycine G to the N-terminus of the polypeptide.

10. The kit as described in claim 8 or 9, characterized in that, The release cycle of the exosomes can be controlled by adjusting the ratio of the supplemental fluid to the exosomes.

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