Polypeptide and use thereof

Abstract

Provided are a polypeptide and the use thereof. The polypeptide has a sequence of KSLSLSLGPASLSLSLK. The polypeptide is prepared into a responsive polypeptide hydrogel, which encapsulates extracellular vesicles to form an extracellular vesicle hydrogel, thereby improving the retention of the extracellular vesicles at an administration site, controlling the release of the extracellular vesicles from the hydrogel, and further improving the utilization efficiency of the extracellular vesicles. The polypeptide hydrogel has a good biocompatibility, and the structure is less affected by changes in physiological or pathological environments. The polypeptide hydrogel is universal for different diseases and different administration sites, promotes the clinical transformation application of the extracellular vesicles, and is also expected to become a universal drug carrier for improving the utilization efficiency of delivered drugs.

Description

A polypeptide and its application Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the field of drug delivery systems. Background Art

[0002] Extracellular vesicles (EVs), as an emerging cell-free therapeutic approach, hold great potential for treating major diseases. Leveraging their stable lipid bilayer structure, EVs can transport and deliver bioactive molecules such as proteins and RNA between cells, thereby mediating intercellular signaling and exerting regulatory effects on receptor cells. After release, EVs can interact with the extracellular matrix and neighboring cells within the microenvironment, exerting paracrine effects, or be transported by bodily fluids to distant target cells, exerting long-range secretory effects. The biological information transmission and target cell regulatory functions of EVs enable them to play important roles in physiological and pathological processes such as antigen presentation, tumor growth and migration, and tissue repair, and thus hold great potential for clinical application. In recent years, EVs have garnered increasing attention and application in disease treatment and drug delivery. Natural stem cell-derived EVs have shown great potential in the treatment of cancer, tissue repair, inflammatory diseases, and neurological disorders, while engineered EVs have garnered significant attention as delivery vehicles for biomolecular therapeutics. However, short half-life, rapid clearance, and low retention at the site of administration are major challenges facing the clinical translation of extracellular vesicles. Depending on the target organ of the disease, the delivery method of extracellular vesicles varies. A search of the ClinicalTrails.gov clinical research website revealed that: exosomes (a type of extracellular vesicle) exoSTING developed by Codiak BioSciences was injected intratumorally (NCT04592484) to treat solid tumors; a study using extracellular vesicles as antiplatelet agents to treat acute myocardial infarction was administered orally (NCT02931045); a clinical study on mesenchymal stem cell (MSCs)-derived exosomes for the treatment of dry eye disease (NCT04213248) used exosomes dispersed in artificial tears for direct ocular administration; a clinical study on MSC exosomes for the treatment of acute stroke (NCT03384433) used exosomes for direct intracranial injection; and a clinical study on MSCs-derived extracellular vesicles for the treatment of Covid-19 (NCT04276987) used a method where the extracellular vesicles were atomized and actively inhaled by the patient.

[0003] As can be seen, current clinical research on MSC-derived extracellular vesicles primarily utilizes local administration. For neurological diseases, single local injections are often used, while research on chronic diseases requiring long-term and repeated administration is lacking. This may be due to two factors: First, compared to synthetic drugs, extracellular vesicles require lengthy preparation times, multiple steps, and significant capital expenditures, resulting in higher costs and a difficulty supporting long-term, intensive administration. Second, in practical applications, extracellular vesicle-based therapeutics face challenges such as short half-lives and rapid clearance. Due to opsonization and clearance by the mononuclear phagocyte system, the half-life of intravenously injected extracellular vesicles is only 1-6 hours. Cleared extracellular vesicles accumulate in the spleen and liver, resulting in rapid clearance by the innate immune system, limiting their application as effective therapeutics. Consequently, more targeted delivery of extracellular vesicles is needed to reduce waste during administration and in vivo, and to improve delivery efficiency. Research and development of delivery formulations tailored to the specific disease targets is necessary to meet the needs of more efficient clinical translation of extracellular vesicles.

[0004] To address the aforementioned extracellular vesicle delivery issues, researchers have developed novel extracellular vesicle delivery strategies utilizing scaffold materials. Combining gel materials with extracellular vesicles and performing local injection can enhance the retention of extracellular vesicles at the lesion site and maintain their stability, thereby improving the efficiency of extracellular vesicle delivery. Among these, hydrogels, three-dimensional structures with a certain viscosity formed by swelling polymer materials in water, are widely used for sustained drug release. However, conventional hydrogels, as local drug sustained-release systems, rely primarily on drug diffusion and material degradation for drug release. This release is slow and lacks controllability, often failing to achieve the desired effect of improving drug utilization efficiency. The microenvironments of normal human tissues and lesions differ significantly, so the development of environmentally responsive drug delivery systems based on the local physiological or pathological microenvironment at the administration site has attracted widespread attention in recent years. Responsive hydrogels can sense and respond to the microenvironment surrounding the gel (such as temperature, pH, light, ions, enzymes, pressure, electric and magnetic fields), resulting in changes such as assembly or degradation of the gel network structure. Although environmentally responsive hydrogels can degrade the hydrogel network based on external stimuli to control drug release, the molecular structure of the hydrogel needs to be designed accordingly according to the microenvironment of different administration sites or drug targets. Slight changes in the physiological or pathological environment may affect the effect of drug release, so the applicability is relatively small.

[0005] Therefore, there is an urgent need for a more efficient drug delivery carrier to solve the delivery problem of extracellular vesicles, improve drug utilization efficiency, and better realize the potential of MSC-EVs in disease treatment.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a polypeptide, a polypeptide hydrogel and a responsive extracellular vesicle hydrogel and their applications. The polypeptide hydrogel is used as a drug carrier to improve the delivery efficiency of drugs, especially to solve the problem of low utilization efficiency of extracellular vesicles.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] A polypeptide, the polypeptide sequence is: KSLSLSLGPASLSLSLK (abbreviated as KPK polypeptide).

[0010] A polypeptide hydrogel is formed by mixing an isotonic solution of the polypeptide and a saline solution. The polypeptide hydrogel is a hydrogel in which KPK polypeptides spontaneously assemble into nanofibers in salt ions or in human tissue fluid, and further cross-link to form a porous network structure.

[0011] The concentration of the polypeptide hydrogel is 0.1w / v%-50w / v% mass volume concentration; preferably 0.1w / v%-10w / v% mass volume concentration; more preferably 0.1w / v%-2w / v% mass volume concentration; more preferably 1w / v% mass volume concentration.

[0012] As a preferred embodiment, the isotonic solution includes an aqueous solution of monosaccharides or disaccharides, and the monosaccharide or disaccharide isotonic solution includes one or more of sucrose, glucose, galactose, fructose, ribose, mannose, arabinose and xylose solutions; the salt solution includes phosphate buffer, physiological saline, borate buffer, tris-hydrochloric acid buffer solution and hydroxyethylpiperazineethanesulfonic acid buffer solution.

[0013] A responsive extracellular vesicle hydrogel comprises extracellular vesicles and the polypeptide hydrogel, wherein the polypeptide hydrogel wraps the extracellular vesicles and comprises an isotonic solution of a polypeptide with the sequence KSLSLSLGPASLSLSLK and a saline solution.

[0014] As a preferred embodiment, a responsive extracellular vesicle hydrogel is provided, wherein the extracellular vesicles are drug-entrapped. Extracellular vesicles can be used directly as drugs to treat diseases, or they can be drug-entrapped and used as delivery vehicles for these drugs. Extracellular vesicle hydrogels can enhance the drug delivery efficiency of their drug carriers, improve the retention of extracellular vesicles at the site of administration, and control the release of extracellular vesicles and drugs from the hydrogel, thereby improving drug utilization efficiency. Drugs that can be entrapped include small molecule drugs, peptides, proteins, and nucleic acid drugs.

[0015] The present invention also discloses the use of the polypeptide in preparing a polypeptide hydrogel. The hydrogel structure is less affected by changes in physiological or pathological environments and is universally applicable to different diseases and different administration sites.

[0016] The use of the polypeptide or the polypeptide hydrogel in the preparation of a drug carrier. The present invention experimentally found that the KPK polypeptide hydrogel has good biocompatibility. It was observed that primary neurons attached and grown on the KPK gel showed a morphology similar to that of neurons on a poly-L-lysine matrix, and there was no significant difference in the number of neurons, the length of neuronal protrusions, and the number of protrusion branches, suggesting that it can be used as a universal drug carrier to prolong the retention time of drugs. The drugs that can be encapsulated include extracellular vesicles, lipid nanoparticles, lipoproteins, small molecule drugs, polypeptides, proteins, nucleic acid drugs, cell therapy drugs, etc.

[0017] The polypeptide or polypeptide hydrogel is used to prepare a responsive extracellular vesicle hydrogel. The polypeptide hydrogel is used to encapsulate extracellular vesicles to form an extracellular vesicle hydrogel, thereby improving the retention of extracellular vesicles at the administration site and controlling the release of extracellular vesicles from the hydrogel, thereby improving the utilization efficiency of extracellular vesicles.

[0018] Responsive extracellular vesicle hydrogels are used in the preparation and treatment of skin damage and aging in dermatological diseases, promoting wound healing, preventing and treating hypertrophic scars, reducing skin pigmentation, treating acne, ophthalmic diseases such as dry eye and macular degeneration, respiratory system to fight and defend against chronic obstructive pulmonary disease, and treat acute respiratory distress syndrome, neurological diseases such as Alzheimer's disease, nerve cell repair in stroke, traumatic brain injury, gynecological promotion of endometrial repair, premature ovarian insufficiency and accelerated vaginal surgical wound repair, liver diseases such as liver fibrosis, chronic kidney disease, organ transplantation to treat graft-versus-host disease, osteoarthritis, and oral diseases to treat periodontitis, Alzheimer's disease, nerve cell repair in stroke, brain glioma, mucosal damage repair, diabetes and macular degeneration. Extracellular vesicles can be used to treat Alzheimer's disease, repair nerve cells in stroke, repair gliomas, repair mucosal injuries, and eye diseases such as diabetes and macular degeneration. Responsive extracellular vesicle hydrogels improve the retention of extracellular vesicles at the site of administration, for example, in the treatment of various eye diseases, including traumatic diseases, autoimmune diseases, neovascular diseases, and glaucoma. This increases their retention in the eye, suggesting that extracellular vesicle hydrogels have the potential to be used in areas such as mucosa to improve the retention of extracellular vesicles and their utilization efficiency; enhance the enrichment of extracellular vesicles in glioma sites; and prolong the duration of extracellular vesicle action in various mucosal and skin injury sites, such as the ear canal and vagina. Controlling the release of extracellular vesicles from the hydrogel, and thereby improving their utilization efficiency, can have a more significant therapeutic effect on diseases that can be treated with extracellular vesicles.

[0019] A hydrogel, as used herein, refers to a hydrophilic, three-dimensional structure with a certain viscosity formed by swelling natural or synthetic polymers in water through chemical or physical crosslinking. Hydrogels formed from proteins and polypeptides have excellent biocompatibility. Polypeptides can typically be prepared through self-assembly. Under specific conditions, they utilize intermolecular non-covalent forces, such as ionic bonds, hydrophobic forces, hydrogen bonds, and van der Waals forces, to spontaneously aggregate into nanofibers with a highly ordered internal structure, ultimately forming a hydrogel.

[0020] In a preferred embodiment of the present invention, the polypeptide hydrogel is prepared by dissolving the KPK polypeptide in a sucrose or mannitol aqueous solution to form a KPK polypeptide sucrose or mannitol solution. After the polypeptide is fully dissolved to form a clear, viscous solution, the pH is adjusted to neutral and the solution is mixed with an equal volume of phosphate buffer or saline to form a transparent KPK polypeptide hydrogel. The polypeptide hydrogel of the present invention is prepared by mixing a sucrose solution of the KPK polypeptide with a phosphate buffer, or by mixing a mannitol solution of the KPK polypeptide with saline, or may be prepared by other conventional methods.

[0021] In a preferred embodiment of the present invention, the extracellular vesicle hydrogel is prepared by vortex mixing KPK polypeptide hydrogel and mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) to form MSC-EVs-GEL. It can also be prepared by other conventional methods.

[0022] In the present invention, "polypeptide hydrogel", "KPK polypeptide hydrogel", "KPK polypeptide gel", "KPK gel", and "KPK hydrogel" all refer to a hydrogel containing KPK polypeptide formed by mixing the KPK polypeptide isotonic solution and saline solution.

[0023] In the present invention, "MSC-EVs-GEL", "MSC-EVs gel", "MSC-EVs hydrogel", "MSC-EVs polypeptide gel", "MSC-EVs polypeptide hydrogel", and "extracellular vesicle hydrogel" all refer to KPK polypeptide hydrogels that encapsulate extracellular vesicles.

[0024] The advantages of this invention lie in the fact that it designs polypeptides to form EVs-responsive polypeptide hydrogels that encapsulate extracellular vesicles to form the extracellular vesicle hydrogel, thereby enhancing the retention of extracellular vesicles at the site of administration and controlling the release of extracellular vesicles from the hydrogel, thereby improving the utilization efficiency of extracellular vesicles. This polypeptide hydrogel has good biocompatibility, its structure is less affected by changes in physiological or pathological environments, and is universally applicable to different diseases and different administration sites, promoting the clinical application of extracellular vesicles and potentially becoming a universal drug carrier, improving the utilization efficiency of delivered drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1. Construction and characterization of MSC-EV hydrogels. (a) Morphology of KPK and KK hydrogels. (b) Electron microscopy images of MSC-EVs and BV2-EVs, scale bar: 100 nm. (c) Circular dichroism analysis of KPK and KK hydrogels. (d) AFM images of KPK and KK hydrogels. (e) TEM image of KPK hydrogel, scale bar: 200 nm. (f) SEM image of KPK hydrogel. (g) Degradation of KPK hydrogel by MSC-EVs. KPK hydrogels were incubated with PBS (Con), MSC-EV solution (MSC-EVs), MSC-EV solution containing 20 μM SP-13786 (MSC-EVs+SP), 20 μM talabostat mesylate (MSC-EVs+TM), or trypsin at 37°C for 3 days. The remaining weight of the KPK hydrogels was monitored daily. (h) Cryo-TEM observation of KPK hydrogel degradation by MSC-EVs. KPK hydrogels were incubated with PBS or 0.5 mg / ml MSC-EVs at 37°C for 48 hours. The marked white arrows indicate the punctate morphology of degraded KPK hydrogels. Scale bar: 100 nm. (i) Morphology of MSC-EVs gel observed under scanning electron microscopy. Scale bar, 100 nm. (j) Distribution of PKH26 (red)-labeled MSC-EVs in MSC-EVs gels observed under fluorescence confocal microscopy. Scale bar, 10 μm. (k) Degradation of MSC-EVs gels was measured by rheometry. G', elastic modulus; G", viscous modulus.

[0026] Figure 2. MSC-EVs can be released from KPK hydrogels and promote neuronal growth. (a) Fluorescence confocal microscopy observation of BV2 cell uptake of MSC-EVs released from MSC-EVs-GEL. MSC-EV gels were loaded into transwell chambers (1 μm pore size) and incubated with BV2 cells seeded in 24-well plates for 24 hours. PKH26 (red) labels the membrane of MSC-EVs, and CFSE (green) labels the contents of MSC-EVs. DAPI (blue) indicates cell nuclei. Scale bar, 25 μm. (b) Morphological observation of the effect of KPK hydrogels on neuronal growth. Morphology of mouse primary neurons grown on polylysine-coated (PLL) or KPK hydrogels (KPK) for 6 days. TUBB3 (red) labels neuronal processes, and DAPI (blue) labels cell nuclei. Scale bar, 200 μm. The number of neurons (c), total neurite length (d), and number of branch points (e) were analyzed using the high-content NeuronalProfiling program. n = 3.

[0027] Figure 3. MSC-EV gel enhances nasal retention and brain delivery of MSC-EVs after intranasal administration. Mice were intranasally administered with DiD-labeled MSC-EVs or MSC-EV-GEL and observed using in vivo CT and fluorescence microscopy. (a) Nasal retention. (b) Semi-quantitative analysis of nasal retention. (c) Brain distribution of MSC-EVs and MSC-EV-GEL after intranasal administration. Mice were intranasally administered with DiD-labeled MSC-EVs or MSC-EV-GEL. Brain tissue was collected 1 hour, 6 hours, and 6 days after administration and imaged using an in vivo fluorescence imaging system. (d) Semi-quantitative analysis of brain distribution of MSC-EVs and MSC-EV gel after intranasal administration. (e) Distribution of DiD-labeled MSC-EVs (red) in the nasal cavity and olfactory bulb of mice 6 days after intranasal administration of MSC-EV-GEL. Scale bar, 50 μm. (f) Distribution of DiD-labeled MSC-EVs (red) in the mouse cortex 6 days after intranasal administration of MSC-EVs-GEL. Astrocytes, microglia, and neurons were labeled with anti-GFAP, IBA1, and NeuN antibodies (green), respectively. Scale bar, 25 μm. Data are mean ± SD, n = 3.

[0028] Figure 4. MSC-EVs-GEL more effectively rescues memory impairment in 5×FADAD mice. 5×FAD mice were intranasally administered KPK blank hydrogel (5×FAD+KPK), MSC-EVs (5×FAD+MSC-EVs), and MSC-EVs gel (5×FAD+MSC-EVs-GEL). Wild-type littermates were administered weekly for 5 weeks. Morris water maze testing was then performed, and Barnes maze testing was performed at week 6. af, MWM results: (a) latency in the navigation test, (b) number of platform passes in the spatial exploration test, (c) percentage of time spent swimming in the platform quadrant in the spatial exploration test, (d) percentage of distance spent swimming in the platform quadrant in the spatial exploration test, (e) swimming speed in the spatial exploration test, and (f) typical swimming trajectories in the spatial exploration test. Data are mean ± SD; n = 6–7. (g) latency during the acquisition training phase; (h) number of incorrect hole entries in the test test. Data are mean ± SD, n = 7-8.

[0029] Figure 5. MSC-EVs-GEL more effectively alleviates neuronal damage and promotes neurogenesis in 5× FADA mice. (a) Golgi staining of cortical neurons from 5× FADA mice treated intranasally with KPK blank hydrogel, MSC-EVs, and MSC-EVs-GEL for 5 weeks. Scale bar, 50 μm. (b) Nice staining of the cortex, dentate gyrus, hippocampus, CA1, CA2, and CA3. Scale bar, 50 μm. (c) BrdU staining of the SGZ of 5× FADA mice and quantitative results (e). Scale bar, 25 μm. (d) Proliferation of neuronal stem cells and neurogenesis in the subgranular layer of the dentate gyrus of the mouse hippocampus. (e) Quantitative results of Golgi staining of cortical neurons. (f) Quantitative results of BrdU staining of the SGZ of 5× FADA mice. (g) Quantitative analysis of neuronal stem cell proliferation and neurogenesis in the subgranular layer of the dentate gyrus of the mouse hippocampus using ImageJ. Scale bar, 40 μm.

[0030] Figure 6. Effects of MSC-EVs-GEL on astrocytes and microglia in 5×FAD mice. Five weeks after intranasal administration of KPK hydrogel, MSC-EVs, and MSC-EVs-GEL to 5×FAD and WT mice, respectively, (a-C) GFAP immunostaining reveals astrocytes in the mouse brain. (a) Astrocyte morphology in the hippocampus and cortex of the mouse brain; quantitative analysis of the area of ​​GFAP-positive staining in the hippocampus (b) and cortex (c). (e-D) Effects of MSC-EVs-GEL on microglia in 5×FAD mice. IBA1 immunostaining reveals microglia in the mouse brain. (d) Microglia morphology in the hippocampus and cortex of the mouse brain; quantitative analysis of the area of ​​IBA1-positive staining in the hippocampus (e) and cortex (f). n = 6-8, scale bar, 100 μm.

[0031] Figure 7. MSC-EV gel exhibits good safety after 5 weeks of intranasal administration. ab, Nasal mucosal morphology of 5×FAD mice. HE staining of the nasal septum and nasal concha (a) and neuron-specific enolase expression (b). Scale bar, 50 μm. (c) HE staining of major organs in treated 5×FAD and WT mice. Scale bar, 50 μm.

[0032] Figure 8. Peptide gel enhances EV accumulation in gliomas. In vivo imaging (a) and quantitative data (b) 5 hours after intranasal administration of DiO-labeled MSC-EVs-GEL to a glioma model mouse orthotopically inoculated with human glioblastoma cells (U87MG cell line). In vivo imaging (c) and images of excised brain tissue (d) 5 hours after intranasal administration of DiO-labeled MSC-EVs-GEL to an orthotopically inoculated glioma model mouse.

[0033] Figure 9. Peptide hydrogels enhance ocular retention of MSC-EVs. In vivo imaging (a) and quantitative data (b) of DiO-labeled MSC-EVs-GEL administered to C57 mice 4 hours after ocular administration. DETAILED DESCRIPTION

[0034] The technology of the present invention is described in detail below in conjunction with specific embodiments. It should be understood that the following specific embodiments are only intended to help those skilled in the art understand the present invention, and are not intended to limit the present invention.

[0035] Example 1. Synthesis of polypeptide

[0036] The present invention is designed to synthesize the KPK polypeptide sequence (SEQ ID NO 1: KSLSLSLGPASLSLSLK) by solid phase synthesis, and the KK peptide (SEQ ID NO 2: KSLSLSLSLSLSLK) with no response characteristics is used as a negative control. The polypeptide is synthesized by solid phase synthesis or liquid phase synthesis with a purity of more than 95%. The specific method of solid phase synthesis is as follows: amino acids are connected to chloromethyl polystyrene resin, and the amino protecting group is deaminated under the protection of trifluoroacetic acid. It is then cut by hydrogen fluoride, precipitated in an ether ice bath, dissolved in acetonitrile and then rotary evaporated, and further purified using a preparative high-performance liquid phase acetonitrile water system.

[0037] Example 2. Preparation of smart responsive polypeptide release hydrogel

[0038] Dissolve the KPK polypeptide in a 0.22 μM filtered 298 mM sucrose solution to prepare a 2-200 mg / ml KPK polypeptide sucrose solution. Allow the solution to swell at room temperature until the polypeptide is fully dissolved, forming a clear, viscous solution. Adjust the pH to 7 using 1N NaOH using pH paper. Mix a defined volume of the KPK polypeptide sucrose solution with an equal volume of PBS to form a transparent KPK hydrogel (Figure 1a).

[0039] KK peptide was dissolved in a 0.22 μM filtered 298 mM sucrose solution to prepare a 2-200 mg / ml KK peptide sucrose solution. Dissolution of KK formed a clear, non-viscous aqueous solution. The pH was adjusted to 7 using 1N NaOH and pH paper. A specific volume of the KK peptide sucrose solution was mixed with an equal volume of PBS. The solution remained clear, and no hydrogel formed (Figure 1a).

[0040] Construction of MSC-EVs-GEL: The above KPK hydrogel was prepared by ultracentrifugation, polyethylene glycol precipitation or extracellular vesicle preparation kit (Capture TMThe MSC-EVs prepared by Extracellular Vesicle Isolation Kit (Code No. 635741, Takara) were vortexed and mixed evenly to form MSC-EVs-GEL, so that the extracellular vesicles were encapsulated by polypeptide hydrogel and stored at 4°C.

[0041] Example 3. Characterization of smart responsive polypeptide release hydrogel

[0042] MSC-EVs were prepared by ultrahigh-speed centrifugation. Dynamic light scattering determined the particle size distribution of MSC-EVs to be 114.0 ± 10.9 nm with a zeta potential of -41.4 ± 1.9 mV; BV2-EVs to be 128.4 ± 9.2 nm with a zeta potential of -42.3 ± 1.5 mV. Transmission electron microscopy revealed that both MSC-EVs and BV2-EVs exhibited a classic saucer-like structure with a particle size of approximately 100 nm (Figure 1b).

[0043] The secondary structure of the self-assembled hydrogel was detected by circular dichroism spectroscopy (Figure 1c). The KPK hydrogel showed a typical β-sheet conformation, while the KK sequence did not. Atomic force microscopy of the nanoscale morphology of the peptide after mixing with PBS revealed that the KK peptide aggregated into particles of approximately 1 nm in size but did not assemble into fibers (Figure 1d), while the KPK peptide self-assembled into long, curly fibrous structures with a diameter of approximately 1.65 nm. Transmission electron microscopy and scanning electron microscopy results confirmed that KPK can self-assemble into slender fibers, and the fibers are further cross-linked to form a three-dimensional network structure (Figure 1e, f).

[0044] To determine the degradation properties of KPK hydrogels in the presence of MSC-EVs, KPK hydrogels were co-incubated with MSC-EVs, and the wet weight of the gels was monitored daily. The negative control PBS did not cause significant weight changes in the KPK hydrogels, while the positive control trypsin produced significant weight loss at all time points tested (Figure 1g). Cryo-transmission electron microscopy analysis further confirmed the degradation of KPK gels after treatment with MSC-EVs (Figure 1h). After incubation with MSC-EVs for 48 hours at 37°C, some KPK fibers disintegrated and degraded into round fragments (indicated by arrows in the figure. Figure 1h). These results indicate that MSC-EVs can degrade KPK hydrogels.

[0045] MSC-EVs-GEL was prepared by adding equal volumes of MSC-EVs in PBS to a 20 mg / ml KPK sucrose solution. Scanning electron microscopy (SEM) revealed that the MSC-EVs hydrogel was bound to the KPK gel without causing morphological damage (Figure 1i). Confocal microscopy revealed that PKH26-labeled MSC-EVs were uniformly dispersed in the KPK hydrogel as very uniform dots, without significant aggregation (Figure 1j). Oscillatory stress sweep analysis was used to determine the storage modulus G′ and loss modulus G″ of KPK hydrogel and MSC-EVs gel. The results showed that at 4°C, MSC-EVs gel incubated for 24 h showed a G′ of 27 Pa, slightly lower than the G′ of KPK hydrogel (36 Pa). At 37°C, after incubation for the same time, the G′ of MSC-EVs gel decreased significantly to 14 Pa (Figure 1k). The above results indicate that MSC-EVs can destroy the structural integrity of MSC-EVs-GEL and induce changes in the rheological properties of KPK hydrogel, and this degradation activity is temperature-dependent.

[0046] Transwell experiments were further applied to explore whether MSC-EVs released from MSC-EVs-GEL can be absorbed by brain cells. BV2 microglia were used as a cell model. The MSC-EVs membrane and contents were labeled with PKH26 and carboxyfluorescein succinimidyl ester (CFSE), respectively. MSC-EVs-GEL was added to the Transwell chamber, and BV2 cells were cultured at the bottom of a 24-well plate. After 24 hours of co-incubation, fluorescence imaging analysis showed that MSCs-EVs released from MSC-EVs-GEL were successfully absorbed by BV2 cells (Figure 2a). In addition, PKH26 and CFSE signals were found to colocalize in the cells, which means that MSC-EVs were released from MSC-EVs-GEL and internalized by BV2 cells in an intact form.

[0047] At the same time, it was found that KPK gel has good biocompatibility, because primary neurons attached and grown on KPK gel were observed to show a morphology similar to that of neurons on poly-L-lysine matrix (Figure 2b), and there was no significant difference in the number of neurons, process length and number of process branches (Figure 2c-e). In addition, it can be seen from Figure 2b that the neuronal processes are significantly distinguished from the bottom of the well plate at the boundary of the KPK hydrogel. This is because the primary neurons in the uncoated area at the bottom of the well plate cannot adhere to the wall and grow, suggesting that KPK hydrogel has the potential to promote nerve adhesion and growth matrix. Adding nerve growth factor to KPK hydrogel significantly promotes the increase in the length of neuronal processes and the number of process branches, suggesting that KPK hydrogel can be used as a universal drug carrier.

[0048] Example 4. MSC-EVs-GEL enhances nasal retention and brain entry of MSC-EVs after intranasal administration

[0049] Mucociliary clearance results in a half-life of the liquid formulation in the nasal cavity of only 15-20 minutes. To determine the retention of MSC-EVs gel in the nasal cavity, MSC-EVs were labeled with DiD, and MSC-EVs-GEL or MSC-EVs solution was administered intranasally to nude mice. CT / fluorescence images of the nasal cavity were collected at different time points after administration. The results showed that at all time points after treatment, the retention of MSC-EVs-GEL in the nasal cavity was significantly higher than that of MSC-EVs solution (Figure 3a). Compared with MSC-EVs gel, the clearance rate of MSC-EVs solution was significantly higher, especially in the first 6 hours (Figure 3b).

[0050] The amount and pathway of MSC-EVs entering the brain were examined 1 hour, 6 hours, and 6 days after administration. Compared with the MSC-EV solution, the amount of MSC-EVs entering the brain was significantly increased after treatment with MSC-EVs gel (Figure 3c-d). Even 6 days after intranasal administration of MSC-EVs-GEL, strong fluorescence signals of MSC-EVs were still found near the basal layer of the nasal mucosal epithelium (Figure 3e). Fluorescence signals of MSC-EVs were also observed in the olfactory bulb area near the nasal mucosa. In the cortex, MSC-EVs were mostly co-localized with neurons, but were sparsely distributed in microglia (Figure 3f). A small amount of DiD fluorescence signals appeared near astrocytes at the edge of cerebral blood vessels (Figure 3f), indicating that MSC-EVs may be cleared from the brain through paravascular pathways.

[0051] Example 5. MSC-EVs-GEL more effectively improves cognitive dysfunction in 5×FADAD model mice

[0052] Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading form of dementia. In our previous study, intranasal administration of MSC-EVs every two days to AD model mice effectively improved cognitive impairment. MSC-derived extracellular vesicles have been shown to promote angiogenesis, inhibit inflammation, inhibit apoptosis, and promote tissue regeneration. In the nervous system, they also promote axonal regeneration and neurogenesis. Therefore, mesenchymal stem cell-derived extracellular vesicles have great potential for the treatment of neurological diseases. Previous results showed that MSC-EV hydrogels retained significantly longer in the nasal cavity than MSC-EV solutions, and brain entry of MSC-EVs persisted for at least six days after administration. Therefore, we administered MSC-EV-GEL intranasally to 5×FAD mice, a classic transgenic mouse model of AD, once weekly for five weeks. Following administration, the animals were tested using the Morris water maze to assess spatial learning and memory. On the fourth day of the positional navigation experiment, the latency of 5×FAD mice in the MSC-EVs gel group was significantly reduced compared with 5×FAD mice treated with KPK blank gel (Figure 4a). In the spatial exploration experiment, the swimming speeds of the different groups were similar, but the percentage of time and distance in the target quadrant of 5×FAD mice treated with MSC-EVs-GEL was much longer than that of 5×FAD mice treated with KPK blank gel or MSC-EVs (Figure 4b-f).

[0053] The Barnes maze test was used to evaluate spatial learning and memory in a dry environment. During the 6 consecutive days of acquisition training, the latency of mice in all groups decreased. Compared with mice treated with KPK blank gel, mice in the MSC-EVs gel group had significantly reduced latency to reach the escape hole and the number of errors before reaching the escape hole (Figure 4g-h). Most importantly, compared with mice treated with MSC-EVs solution, model mice treated with MSC-EVs-GEL made significantly fewer errors in the exploration test (Figure 4h). These results indicate that MSC-EVs-GEL effectively enhances the effect of MSC-EVs in improving spatial learning and memory function in 5×FAD mice.

[0054] Example 6. MSC-EVs gel more effectively alleviates neuronal damage and promotes neurogenesis

[0055] Synaptic plasticity refers to changes in the number, shape, and function of synapses caused by prolonged neural activity. It is fundamental to learning and memory and is crucial for brain function. A reduction in synaptic number and altered synaptic plasticity are key pathological manifestations of AD. To evaluate the effects of MSC-EVs-GEL on synaptic changes, blank KPK hydrogel, MSC-EVs, and MSC-EVs-GEL were intranasally administered to 5×FAD and WT mice for 5 weeks. Golgi staining of dendritic spines in the mouse brains was performed. The study showed that dendritic spine density in cortical neurons of 5×FAD animals was significantly reduced compared to WT mice, but was largely restored (increased by 35.3%) after MSC-EVs-GEL treatment (Figures 5a and 5e), suggesting that MSC-EVs-GEL can alter cognitive function by affecting dendritic spine density. Furthermore, the effect of MSC-EVs gel on neuronal cell body morphology was assessed by Nissl staining (Figure 5b). Nissl bodies are neuronal structures that reflect neuronal activity and function. The damaged neuronal cell bodies were darkly stained and shrunken, leaving gaps between neurons, while normal neurons were lightly stained blue and arranged side by side. The results showed that the Nissl bodies in the cortex and dentate gyrus regions, and the CA2 and CA3 regions of the hippocampus of the MSC-EVs-GEL-treated model mice were significantly more orderly and stained lighter than those in the KPK blank gel-treated mice. The neuronal morphology and intercellular spaces returned to normal.

[0056] Newborn neurons have significant plasticity and can improve learning and cognitive functions. Therefore, the next step was to investigate whether MSC-EVs-GEL affects the number of newborn neurons in the subgranular layer of the hippocampal dentate gyrus of 5×FAD mice. BrdU and doublecorxin (DCX) are used as markers of cell proliferation and immature neurons, respectively (Figures 5c and 5f). Compared with KPK blank gel, MSC-EVs gel MSC-EVs-GEL increased BrdU-positive cells by 262% and DCX-positive area by 369% (Figures 5d and 5g). In addition, MSC-EVs-GEL showed stronger neurogenesis ability than MSC-EVs liquid preparations, with an increase of 2.90 times and 1.84 times in BrdU-positive cells and DCX-positive area, respectively (Figures 5f and 5g).

[0057] In addition to neuroprotection and neurogenesis, MSC-EVs have also been shown to influence inflammatory responses. Astrocyte activation in AD, also known as reactive gliosis, is a common phenomenon in the AD pathology, manifested by astrocyte proliferation and increased GFAP expression. GFAP staining results showed that the GFAP-positive area in the cortex of 5×FAD mice treated with MSC-EVs-GEL was significantly reduced by 14.7% compared to that in 5×FAD mice treated with KPK (Figures 6a-c). This suggests that MSC-EVs-GEL has a certain inhibitory effect on cortical astrocyte proliferation and activation. Microglia are immune cells inherent to the central nervous system. Microglial activation plays a crucial role in neuronal damage during AD. First, we investigated the effect of MSC-EVs-GEL administration on microglia in the brains of model mice by labeling microglia with IBA1. Results showed that neither MSC-EVs-GEL nor MSC-EVs administration significantly affected microglia in the hippocampus of 5×FAD mice. However, compared with the KPK-administered group, the amount of cortical microglia in the MSC-EVs-GEL group decreased by 16.5% ( Figure 6 d-e ).

[0058] Example 7. Safety evaluation of polypeptide hydrogel

[0059] Peptide hydrogels require long-term administration, and safety is paramount. KPK peptides were formulated into a 20 mg / ml sucrose solution. A certain volume of KPK peptide solution was taken and mixed with phosphate buffer to form a transparent KPK hydrogel. The above KPK hydrogel was mixed with MSC-EVs prepared by ultracentrifugation to prepare MSA-EVs-GEL. After 5 weeks of intranasal administration, major organs were harvested and sectioned to preliminarily evaluate the safety of nasal administration of KPK hydrogel. Nasal HE staining analysis showed that the mucosa on both sides of the nasal septum and nasal concha were intact in all experimental groups (Figure 7a), indicating that the nasal administration is safe. Neuron-specific enolase antibody staining showed that neurons in the nasal mucosa showed a deep staining continuous structure with uniform thickness (Figure 7b). In the heart, liver, spleen, and kidney tissues of each treatment group, myocardial fibers were normal, hepatocytes were not turbid, the splenic white pulp and red pulp structure were clear, and the renal glomeruli and renal tubules were normal in structure, with no microscopic morphological changes (Figure 7c).

[0060] Example 8. Peptide gel enhances the enrichment of EVs in brain glioma sites

[0061] Gliomas are a type of intracranial malignant tumor with a high degree of primary disease, and their five-year mortality rate ranks third among all types of tumors. Due to the invasive growth characteristics of glioma cells, surgical resection usually requires adjuvant drug therapy. Although some new therapeutic drugs have been developed and marketed, due to the heterogeneity and progressive nature of tumors, coupled with the efficiency limitations of intracerebral drug delivery, there is an urgent need to develop drugs that can effectively enter the brain and achieve glioma intervention. Therefore, MSC-EVs-GEL, which can be administered intranasally to increase brain entry, has great potential for the treatment of glioma. KPK polypeptide was formulated into a 20mg / ml mannitol solution. A certain volume of KPK polypeptide solution was taken and mixed with physiological saline to form a transparent KPK hydrogel, to which DiO-labeled MSC-EVs were added and mixed to prepare MSC-EVs-GEL. After DiO-labeled MSC-EVs-GEL was intranasally administered to glioma model mice in which human malignant glioblastoma cells (U87MG cell line) were inoculated in situ in the brain, in vivo imaging showed that the amount of MSC-EVs-GEL entering the brain was significantly higher than that of MSC-EVs (Figure 8a, b). At the same time, MSC-EVs and MSC-EVs-GEL were respectively injected into the brain tumor of the orthotopic glioma model mouse for 5 hours. The retention of MSC-EVs-GEL in the brain was 1.28 times that of the MSC-EVs group. The above results show that polypeptide gel increases the amount of MSC-EVs entering the brain in the glioma model.

[0062] Example 9. Polypeptide hydrogel prolongs the ocular action time of EVs

[0063] In the field of ophthalmology, extracellular vesicles can be used to treat various eye diseases, including traumatic diseases, autoimmune diseases, neovascular diseases, and glaucoma. For example, extracellular vesicles can be used to deliver drugs or mRNA that promote cell regeneration and reduce inflammation, and can also be used to deliver anti-angiogenic and neuroprotective drugs. However, due to the washout of tears in the eyes, the residence time of extracellular vesicles in the eyes is short after administration, and the utilization rate of extracellular vesicles is limited. Therefore, the retention of extracellular vesicles in the eyes can be increased by using extracellular vesicle hydrogel preparations, thereby increasing the bioavailability of EVs. After DiO-labeled MSC-EVs-GEL was administered to C57 mice by eye drops for 4 hours, in vivo imaging showed that the ocular retention of MSC-EVs-GEL was significantly higher than that of MSC-EVs (Figure 9), suggesting that MSC-EVs gel can improve the bioavailability of MSC-EVs in the eyes.

[0064] Example 10. Polypeptide hydrogel prolongs the duration of action of EVs in the ear canal, vagina, and skin injury sites

[0065] The above results show that the hydrogel formulation prolongs the residence time of EVs in the eye, suggesting that hydrogels may be used in sites such as mucosa to enhance the retention of extracellular vesicles and improve their utilization efficiency. In a mouse model of secretory otitis media, ovalbumin combined with the immune adjuvant alumina was injected intraperitoneally. Five hours after intrathecal administration of MSC-EVs-GEL, the retention of MSC-EVs-GEL in the damaged ear canal was significantly higher than that of MSC-EVs, reaching 3.4 times that of the MSC-EVs group.

[0066] Vaginitis model preparation: 10-week-old female ICR mice were subcutaneously injected with 0.05 mL of an oil containing 0.1 mg of estradiol benzoate three times every two days. Then, 20 μL of a Candida albicans suspension (containing approximately 50,000 Candida albicans spores) was inoculated intravaginally. Four days after modeling, MSC-EVs-GEL was applied intravaginally for 5 hours. The intravaginal retention of MSC-EVs-GEL and MSC-EVs was measured, showing that the MSC-EVs-GEL group retained 2.85 times more than the MSC-EVs group.

[0067] In the rat full-thickness skin defect wound model, 4 hours after MSC-EVs-GEL administration, the amount of MSC-EVs-GEL retained in the skin injury site was 2.3 times that of the MSC-EVs group.

[0068] The above results indicate that the hydrogel formulation prolongs the duration of action of EVs in various mucosal and skin sites.

[0069] The above description is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that The polypeptide sequence is: KSLSLSLGPASLSLSLK.

2. A polypeptide hydrogel, characterized in that: The polypeptide hydrogel is formed by mixing an isotonic solution of the polypeptide according to claim 1 and a saline solution.

3. The polypeptide hydrogel according to claim 2, characterized in that: The isotonic solution includes one or more of sucrose, glucose, galactose, fructose, ribose, mannose, arabinose and xylose solutions, and the salt solution includes one or more of phosphate buffer, physiological saline, borate buffer, tris(hydroxymethyl)aminomethane-hydrochloric acid buffer solution and hydroxyethylpiperazineethanesulfonic acid buffer.

4. A responsive extracellular vesicle hydrogel, characterized in that: It comprises extracellular vesicles and the polypeptide hydrogel according to claim 2, wherein the polypeptide hydrogel encapsulates the extracellular vesicles.

5. The responsive extracellular vesicle hydrogel according to claim 4, characterized in that: The extracellular vesicles encapsulate drugs, which include small molecule drugs, polypeptides, proteins and nucleic acid drugs.

6. Use of the polypeptide according to claim 1 in preparing polypeptide hydrogel.

7. Use of the polypeptide according to claim 1 or the polypeptide hydrogel according to claim 2 in the preparation of a drug carrier.

8. Use of the polypeptide according to claim 1 or the polypeptide hydrogel according to claim 2 in preparing a responsive extracellular vesicle hydrogel.

9. Use of the responsive extracellular vesicle hydrogel according to claim 4 or 5 in the preparation of drugs for treating skin damage and aging, promoting wound healing, preventing and treating hypertrophic scars, reducing skin pigmentation, acne, dry eyes, macular degeneration, fighting and defending against chronic obstructive pulmonary disease, acute respiratory distress syndrome, Alzheimer's disease, nerve cell repair, traumatic brain injury, promoting endometrial repair, premature ovarian insufficiency, vaginal surgical wound repair, liver fibrosis, chronic kidney disease, graft-versus-host disease, osteoarthritis, and periodontitis.

Images