DHEA polypeptide, sustained-release hydrogel, coated exosome delivery preparation and application

Abstract

The invention relates to the technical field of medicines, in particular to a DHEA polypeptide, a sustained-release hydrogel, a coated exosome delivery preparation and application. Assembling the DHEA on the heptapeptide to obtain a DHEA polypeptide; the amino acid sequence of the heptapeptide is FFGSSSR, and the DHEA is dehydroepiandrosterone. By introducing the FFGSSSR heptapeptide with high biocompatibility, in-vitro self-assembly encapsulation of the fat-soluble drug DHEA is realized, the fat-soluble drug DHEA is prepared into an amphiphilic preparation, and the problem that the DHEA is difficult to prepare is solved; according to the invention, the exosome is introduced, and the exosome has the effect of cooperatively regulating the retina microenvironment, so that the RIR injury is treated by multiple targets; when the exosome is obtained, the R28 cells are pretreated by utilizing BBR and an anoxic condition, so that not only is the paracrine effect of the R28 cells enhanced, but also the RIR injury treatment effect of the exosome is improved.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a DHEA polypeptide, a sustained-release hydrogel, an exosome-encapsulated delivery preparation and applications. Background Art

[0002] Retinal ischemia-reperfusion (RIR) injury is a pathological phenomenon in which retinal damage is aggravated instead of alleviated after the blood supply to the ischemic retinal tissue is restored. This lesion poses a huge challenge to the effective treatment of clinical eye diseases. RIR injury is a common cause of many retinal diseases, including acute glaucoma, retinal vascular occlusion, and retinopathy of prematurity. RIR injury causes the death of retinal ganglion cells (RGCs) and subsequent optic nerve degeneration, leading to vision loss and permanent blindness. Studies have confirmed that there are many factors affecting RIR injury, mainly including oxidative stress, immune and inflammatory responses. These different factors act alone or promote each other, forming a complex RIR regulatory network. Therefore, it is urgent to develop drugs that can be used for multi-target treatment of RIR injury.

[0003] Globally, at least 2.2 billion people have impaired near or far vision, of which at least 1 billion have visual impairment that could be prevented or needs to be treated. Glaucoma is one of the main causes of visual impairment and blindness worldwide. It is estimated that by 2030, the number of glaucoma patients worldwide will reach 9.54 million, and the number of patients is mainly concentrated in Asia. The current treatment strategies for RIR damage-related diseases such as glaucoma are mainly laser surgery, retinal imaging screening, and continuous or intermittent intraocular injections of drugs. However, these treatment strategies have problems such as greater eye damage, low patient compliance, and poor prognosis. In addition, multiple intraocular injections can easily lead to inflammation. Therefore, there is an urgent need for drugs that can be slowly released in the eye after a single injection.

[0004] Dehydroepiandrosterone (DHEA) is the most abundant circulating steroid hormone in humans and is produced by the adrenal glands, gonads, and brain. DHEA has previously been shown to bind to the nerve growth factor receptor tropomyosin-related kinase A (TrkA), thereby exerting neuroprotective effects. DHEA was found to reduce microglia-mediated inflammation in an acute lipopolysaccharide-induced neuroinflammation model in mice and in cultured microglia in vitro. In a 2012 study, it was shown that DHEA protects retinal neurons by alleviating excitotoxicity. Other studies have shown that DHEA has a beneficial therapeutic effect in NMDA-induced degeneration of retinal ganglion cells (RGCs). In a previous study, Zhao et al. reported a DHEA nanoparticle with a surface coupled to the RGC-targeting cholera toxin B domain (CTB), which enables the nanoparticle to be detected and accumulated in the RGC layer, resulting in effective and long-lasting protection of RGCs. As mentioned above, RIR damage leads to RGCs death and subsequent optic nerve degeneration, resulting in vision loss and permanent blindness. This shows that DHEA has great therapeutic potential in addressing the death of RGCs and optic nerve degeneration in RIR injury.

[0005] Many existing physicochemical, biopharmaceutical and pharmacokinetic barriers make it a challenge to successfully deliver drugs to the site of action. The use of drug delivery systems can overcome these difficulties by changing the solubility, targeting, metabolism, degradation and toxicity characteristics of the parent drug. Compared with other existing drug delivery systems (including liposomes and polymeric nanoparticles, etc.), supramolecular hydrogels show unique advantages. Among the candidate substances for forming supramolecular hydrogels, amino acids, peptides, nucleobases, sugars and their derivatives have been widely studied as important components of life systems. In particular, self-assembling peptide-based hydrogels have broad application prospects due to their ease of design and preparation, chemical diversity, good biocompatibility, bioactivity and biodegradability. Unlike polymer-based hydrogels that are strongly covalently cross-linked, supramolecular hydrogels are formed by various weak non-covalent interactions between hydrogels, including hydrophobic, hydrogen bonding, pp stacking, electrostatic and van der Waals interactions. Due to their high reversibility, multiple non-covalent interactions give supramolecular hydrogels injectability and rapid response to various external stimuli. At the same time, they have similar properties to the extracellular matrix, such as softness and bioactivity, and most of them are microporous and filled with water, making them excellent carriers for drug delivery.

[0006] Insulin-like growth factor-1 (IGF-1) is a growth factor with differentiation, anti-apoptosis and metabolic functions in the periphery, and has anti-inflammatory properties in the nervous system. Studies have reported that mice with inactivation of the gene product (IGF-1- / -) due to mutations in the IGF-1 gene will experience age-related vision loss, accompanied by structural changes in the first synapse of the retinal pathway. Nishida et al. reported that the c domain of IGF-1 can promote the healing of corneal epithelial damage. They screened a series of 12 amino acid GST fusion proteins and obtained mutants in which various amino acids were replaced by alanine. Their results showed that replacing any amino acid in the SSSR tetrapeptide sequence would result in loss of activity. Therefore, the SSSR tetrapeptide is essential for the biological activity of IGF-1. In order to simulate the physiological function of IGF-1 and achieve self-assembly, the present invention designed and synthesized the FFGSSSR polypeptide (IGF-1 mimetic peptide).

[0007] In recent years, the development of stem cell regenerative medicine has brought new hope for the treatment of fundus diseases. Many studies have shown that stem cells can promote the repair of damaged retinal cells through paracrine exosomes and cytokines. Exosomes are extracellular vesicles with a diameter of 30-150nm. They are responsible for cell-to-cell communication and play a role in the exchange of cellular substances and information. Exosome-transported miRNA promotes cell-to-cell communication through epigenetic regulation of receiving cells. In addition, when maternal cells are stimulated by the environment, the contents of secreted exosomes will change due to the remodeling of epigenetic chromatin and affect their biological effects. The use of cytokines, drugs, hypoxic conditions or physical factors before treatment can enhance the biological functions of mesenchymal stem cells and enhance their paracrine effects. After a large number of literature surveys, it was found that before obtaining exosomes, pre-treatment of stem cells with baicalin, tanshinone or ginsenoside Rg1 usually results in better therapeutic effects.

[0008] At present, there are no reports on the self-assembly of DHEA and IGF-1 mimetic peptides to form a hydrogel preparation for the treatment of RIR injury, nor are there any reports on the preparation of DHEA and exosomes for the multi-target treatment of RIR injury. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a DHEA polypeptide, a sustained-release hydrogel, an encapsulated exosome delivery preparation and applications.

[0010] In order to achieve the above object, the present invention provides the following technical solutions:

[0011] The present invention provides a DHEA polypeptide, wherein DHEA is assembled onto a heptapeptide to obtain the DHEA polypeptide;

[0012] The amino acid sequence of the heptapeptide is FFGSSSR, and the DHEA is dehydroepiandrosterone.

[0013] Preferably, the preparation method of the DHEA polypeptide comprises the following steps:

[0014] 1) mixing and stirring dehydroepiandrosterone, succinic anhydride, 4-dimethylaminopyridine and pyridine to obtain a capping group of DHEA;

[0015] 2) The end-capping group of DHEA obtained in step 1), the heptapeptide, O-benzotriazole-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and N,N-dimethylformamide are mixed and reacted to obtain DHEA polypeptide.

[0016] Preferably, in step 1), the volume ratio of the moles of dehydroepiandrosterone, the moles of succinic anhydride, the moles of 4-dimethylaminopyridine and pyridine is 1 mmol:2 mmol:0.2 mmol:10 mL;

[0017] The stirring time is 45h.

[0018] Preferably, in step 2), the volume ratio of the moles of the end-capping group of DHEA, the moles of the heptapeptide, the moles of O-benzotriazole-tetramethyluronium hexafluorophosphate, the moles of N,N-diisopropylethylamine and the moles of N,N-dimethylformamide is 1 mmol:0.5 mmol:1.5 mmol:2 mmol:10 mL;

[0019] The reaction time is 2h.

[0020] The present invention also provides a sustained-release hydrogel, characterized in that it contains the DHEA polypeptide described in the above technical solution, and the concentration of the DHEA polypeptide in the sustained-release hydrogel is 8 mmol / L.

[0021] Preferably, the method for preparing the sustained-release hydrogel comprises the following steps: ultrasonically dissolving the DHEA polypeptide and water, and heat-treating the DHEA polypeptide to obtain a sustained-release hydrogel;

[0022] The mass of the DHEA polypeptide and the volume ratio of water are 1 mg:108 μL.

[0023] The present invention also provides an encapsulated exosome delivery preparation, wherein each 216 μL of the encapsulated exosome delivery preparation contains 1 mg of DHEA polypeptide and 1.728 mg of exosomes, and the exosomes are derived from R28 cells.

[0024] Preferably, the method for preparing the encapsulated exosome delivery preparation comprises the following steps:

[0025] Mixing the DHEA polypeptide and water, and heat treating to obtain a clear solution;

[0026] Before the clear solution is gelled, it is mixed with exosomes to obtain an encapsulated exosome delivery preparation.

[0027] The present invention also provides the use of the DHEA polypeptide described in the above technical solution or the sustained-release hydrogel described in the above technical solution in the preparation of a drug for treating retinal ischemia-reperfusion injury.

[0028] The present invention also provides the use of the encapsulated exosome delivery preparation described in the above technical solution in the preparation of a drug for treating retinal ischemia-reperfusion injury.

[0029] Beneficial effects of the present invention:

[0030] (1) The present invention realizes in vitro self-assembly encapsulation of the fat-soluble drug DHEA by introducing the FFGSSSR heptapeptide (IGF-1 mimetic peptide) with high biocompatibility, and prepares the fat-soluble drug DHEA into an amphiphilic preparation, thereby solving the problem of DHEA being difficult to make into a drug;

[0031] (2) The present invention achieves the slow release of DHEA in cells by introducing the FFGSSSR heptapeptide (IGF-1 mimetic peptide) with high biocompatibility, thereby enhancing the anti-inflammatory and antioxidant activity of DHEA;

[0032] (3) The present invention introduces FFGSSSR heptapeptide (IGF-1 mimetic peptide) with high biocompatibility. The FFGSSSR heptapeptide can simulate the physiological function of IGF-1, thereby enhancing the effect of the hydrogel preparation in nourishing nerve cells and resisting cell apoptosis;

[0033] (4) The sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention can be used for inflammation caused by RIR damage, providing a new method for treating retinal inflammation, and can also be used for excessive reactive oxygen species caused by RIR damage, providing a new method for treating RIR diseases.

[0034] (5) The present invention introduces exosomes, which have the function of synergistically regulating the retinal microenvironment, thereby achieving multi-target treatment of RIR damage; when obtaining exosomes, R28 cells are pretreated with BBR and hypoxic conditions, which not only enhances the paracrine effect of R28 cells, but also improves the RIR damage treatment effect of exosomes;

[0035] (6) The sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention can be used for inflammation caused by RIR damage, providing a new method for the treatment of retinal inflammation, and can also be used for excessive reactive oxygen species caused by RIR damage, providing a new method for the treatment of RIR diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0037] Figure 1 is a transmission electron micrograph of the sustained-release DHEA polypeptide hydrogel preparation prepared in Example 1 of the present invention;

[0038] Figure 2 is a graph showing the detection result of the uptake of Cy5.5-DHEA polypeptide hydrogel by R28 cells in Example 1;

[0039] Figure 3 This is a graph showing the detection results of the inflammatory factor NO secreted by the cells of Example 1;

[0040] Figure 4 This is a graph showing the detection results of the inflammatory factor IL-1β secreted by the cells of Example 1;

[0041] Figure 5 is a graph showing the detection result of the intracellular ATP content in Example 1;

[0042] Figure 6 is a graph showing the detection result of the intracellular LDH content in Example 1;

[0043] Figure 7 is a graph showing the fluorescence detection result of intracellular reactive oxygen species in Example 1;

[0044] Figure 8 This is the result of HE staining of the retina of SD rats in Example 1;

[0045] Fig. 9 This is a graph showing the immunofluorescence detection result of SD rat retinal neural tubulin β-III-Tubulin in Example 1;

[0046] Fig.10 This is a diagram showing the immunofluorescence detection result of the glial cell-specific protein IBA1 in Example 1;

[0047] Fig.11 This is a diagram showing the immunofluorescence detection result of the glial cell-specific protein GFAP in Example 1;

[0048] Fig.12 is a graph showing the fluorescence detection result of the retina at day 7 in Example 1;

[0049] Fig.13 is a graph of the fluorescence detection result of the retina at day 14 in Example 1;

[0050] Fig.14 is the ERG detection result diagram of Example 1;

[0051] Fig.15 is a transmission electron micrograph of the sustained-release DHEA polypeptide hydrogel preparation prepared in Example 2 of the present invention;

[0052] Fig.16 This is a transmission electron microscopy image of the exosomes obtained in Example 2 of the present invention;

[0053] Fig.17 This is a transmission electron micrograph of the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared in Example 2 of the present invention;

[0054] Fig.18 This is a graph showing the detection results of the uptake of Cy5.5-labeled DHEA polypeptide hydrogel exosomes by R28 cells in Example 2;

[0055] Fig.19 This is a graph showing the detection results of the secretion of the inflammatory factor NO in each group of cells in Example 2;

[0056] Fig. 20 is a graph showing the results of measuring the intracellular ATP content in each group of cells in Example 2;

[0057] Fig.21 is a graph showing the results of fluorescence detection of reactive oxygen species in each group of cells in Example 2;

[0058] Fig. 22 2 is the HE staining result of retina of SD rats in each group in Example 2;

[0059] Fig.23 is a graph showing the immunofluorescence detection results of retinal neural tubulin β-III-Tubulin of each group of SD rats in Example 2;

[0060] Fig.24 This is a graph showing the immunofluorescence detection results of the glial cell-specific protein IBA1 of each group of SD rats in Example 2;

[0061] Fig.25 1 is a graph showing the immunofluorescence detection results of the glial cell-specific protein GFAP of each group of SD rats in Example 2;

[0062] Fig.26 1 is a graph showing the fluorescence detection results of the retina of the unmodeled SD rats and the modeled SD rats in Example 2 on the 7th day;

[0063] Fig. 27 It is a graph showing the fluorescence detection results of the retina of the non-modeled SD rats and the modeled SD rats in Example 2 on the 14th day. DETAILED DESCRIPTION

[0064] The invention provides a DHEA polypeptide, which is obtained by assembling DHEA onto a heptapeptide; the amino acid sequence of the heptapeptide is FFGSSSR, and the DHEA is dehydroepiandrosterone.

[0065] In the present invention, the preparation method of the DHEA polypeptide comprises the following steps:

[0066] 1) mixing and stirring dehydroepiandrosterone, succinic anhydride, 4-dimethylaminopyridine and pyridine to obtain a capping group of DHEA;

[0067] 2) The end-capping group of DHEA obtained in step 1), the heptapeptide, O-benzotriazole-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and N,N-dimethylformamide are mixed and reacted to obtain DHEA polypeptide.

[0068] The present invention preferably mixes and stirs dehydroepiandrosterone, succinic anhydride, 4-dimethylaminopyridine and pyridine to obtain a capping group of DHEA. In the present invention, the volume ratio of the mole of dehydroepiandrosterone, the mole of succinic anhydride, the mole of 4-dimethylaminopyridine and the volume ratio of pyridine is preferably 1mmol:2mmol:0.2mmol:10mL. In the present invention, the stirring time is preferably 45h, preferably stirring at room temperature. The present invention preferably concentrates in vacuo to remove most of the solvent after stirring, adds an appropriate amount of 2M hydrochloric acid, filters by suction, takes the filter cake, and obtains a capping group containing DHEA.

[0069] The present invention preferably reacts the obtained DHEA end-capping group, heptapeptide, O-benzotriazole-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and N,N-dimethylformamide after mixing to obtain DHEA polypeptide. In the present invention, the volume ratio of the mole of the end-capping group of DHEA, the mole of the heptapeptide, the mole of O-benzotriazole-tetramethyluronium hexafluorophosphate, the mole of N,N-diisopropylethylamine and N,N-dimethylformamide is preferably 1mmol:0.5mmol:1.5mmol:2mmol:10mL. In the present invention, the reaction time is preferably 2h, and the reaction is preferably carried out at room temperature. The present invention does not specifically limit the synthesis method of the heptapeptide, and those skilled in the art can synthesize it according to the conventional method of synthesizing amino acids.

[0070] The present invention provides a sustained-release hydrogel, containing the DHEA polypeptide described in the above technical scheme, wherein the concentration of the DHEA polypeptide in the sustained-release hydrogel is 8mmol / L. In the present invention, the preparation method of the sustained-release hydrogel preferably comprises the following steps: ultrasonically dissolving the DHEA polypeptide and water, and obtaining a sustained-release hydrogel after heat treatment; the mass ratio of the DHEA polypeptide to the volume of water is 1mg:108μL. In the present invention, the sustained-release hydrogel is colorless, transparent, and hydrogel-like. In the present invention, the water is preferably double distilled water. The present invention has no special limitation on the method of ultrasonic dissolution, as long as it can be dissolved. The present invention has no special limitation on the conditions of the heat treatment, as long as the solution can be treated to be completely transparent.

[0071] The present invention also provides an exosome delivery preparation, each 216 μL of exosome delivery preparation contains 1 mg DHEA polypeptide and 1.728 mg exosomes, and the exosomes are derived from R28 cells. In the present invention, the preparation method of the exosome delivery preparation preferably includes the following steps: mixing the DHEA polypeptide and water, heat treating, and obtaining a clear solution; before the clear solution is gelled, mixing with exosomes to obtain an exosome delivery preparation. In the present invention, the water is preferably double distilled water. The present invention does not specifically limit the conditions of the heat treatment, and the solution can be treated to be completely transparent. In the present invention, the method for obtaining exosomes preferably includes: resuscitating R28 cells, then culturing for 2 days with a cell culture medium containing berberine (10 μmol / L), and then subjecting to hypoxia for 12 hours, collecting the supernatant of the R28 cells after hypoxia, and obtaining exosomes by differential centrifugation, transferring the cell supernatant to a centrifuge tube, centrifuging at 300g for 5 minutes, collecting the supernatant, and centrifuging at 2100g and 10000g for 30 minutes. The cell supernatant after centrifugation is filtered through a 0.2 μm sterile filter membrane and collected in an ultracentrifuge tube, accurately weighed and balanced. After transferring the ultracentrifuge tube to an ultracentrifuge, centrifuge at 100000g for 70 minutes, resuspend the precipitate in PBS, and repeat the above steps to obtain exosomes.

[0072] The present invention also provides the use of the DHEA polypeptide described in the above technical solution or the sustained-release hydrogel described in the above technical solution in the preparation of a drug for treating retinal ischemia-reperfusion injury. The present invention has no special restrictions on the dosage form and preparation method of the drug, and those skilled in the art can make conventional settings.

[0073] The present invention also provides the use of the encapsulated exosome delivery preparation described in the above technical solution in the preparation of a drug for treating retinal ischemia-reperfusion injury. The present invention has no special limitations on the dosage form and preparation method of the drug, and those skilled in the art can make routine settings.

[0074] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0075] Example 1

[0076] 1. Preparation of sustained-release DHEA polypeptide hydrogel preparation

[0077] 1. Preparation of end-capping groups containing DHEA

[0078] 1.0 mmol DHEA, 2.0 mmol succinic anhydride and 0.2 mmol 4-dimethylaminopyridine (DMAP) were dissolved in 10 mL pyridine, stirred at room temperature (25°C) for 45 h, and concentrated in vacuo to remove most of the solvent. An appropriate amount of 2 M hydrochloric acid was added, and the mixture was filtered and the filter cake was taken to obtain a capping group containing DHEA, which was recorded as compound 1.

[0079] The molecular formula of DHEA is C 19 H 28 O2, molecular weight is 288.43, structural formula is as follows:

[0080]

[0081] 2. Synthesis of IGF-1 mimetic peptide on a solid phase synthesizer

[0082] The IGF-1 mimetic peptide provided by the present invention is a heptapeptide with an amino acid sequence of FFGSSSR.

[0083] (1) Loading and processing resin

[0084] 0.5 mmol 2-Cl-Trt resin was loaded into a solid phase synthesizer, 10 mL anhydrous dichloromethane (DCM) was added, and the mixture was placed on a shaker and shaken for 5 min to allow the 2-Cl-Trt resin to fully swell. The DCM was then pressed out of the solid phase synthesizer containing the 2-Cl-Trt resin using an ear bulb.

[0085] (2) Connect the first amino acid to the resin

[0086] 0.75mmol Fmoc-protected phenylalanine (F) was dissolved in 10mL anhydrous DCM, 0.75mmol N,N-diisopropylethylamine (DIEPA) was added, and the mixture was transferred to the above-mentioned solid phase synthesizer, and 0.75mmol DIEPA was added. The mixture was reacted at room temperature for 1h. After the reaction, the reaction solution in the solid phase synthesizer was removed with an ear bulb, and then washed with anhydrous DCM for 5 times (10mL DCM each time, and the washing time was 1min). Then, 20mL of a mixed solution of anhydrous DCM, DIEPA and methanol in a volume ratio of 17:1:2 was added, and the mixture was reacted at room temperature for 10min to obtain a resin connected with phenylalanine (F), which was recorded as SA-F.

[0087] (3) Synthesis of FF dipeptide

[0088] After SA-F was prepared on the solid phase synthesizer, the reaction solution in the solid phase synthesizer was removed with an ear bulb, and the mixture was first washed with anhydrous DCM for 5 times (10 mL of DCM each time, and the washing time was 1 min), and then washed with N,N-dimethylformamide (DMF) for 5 times (10 mL of DMF each time, and the washing time was 1 min), and then 10 mL of DMF containing 20% ​​(v / v) piperidine was added, and the mixture was reacted at room temperature for 25 min, and then washed with DMF for 5 times (10 mL of DMF each time, and the washing time was 1 min), 1.0 mmol of Fmoc-protected phenylalanine (F), 1.5 mmol of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 2.0 mmol of DIEPA and 10 mL of DMF were mixed evenly to obtain a mixed solution, and the mixed solution was added to the above solid phase synthesizer, and reacted at room temperature for 2 h to obtain FF dipeptide, which was recorded as SA-FF.

[0089] (4) Synthesis of FFGSSSR heptapeptide

[0090] After SA-FF was prepared on a solid phase synthesizer, the operation of step (3) was repeated 5 times, and the Fmoc-protected phenylalanine (F) was replaced with Fmoc-protected glycine (G), Fmoc-protected serine (S), Fmoc-protected serine (S), Fmoc-protected serine (S), and Fmoc-protected arginine (R) in equal amounts, to obtain the FFGSSSR heptapeptide, i.e., the IGF-1 mimetic peptide, denoted as SA-FFGSSSR. The molecular formula of SA-FFGSSSR is C 39 H 54 N 10 O 14 , molecular weight is 886.92, structural formula is as follows:

[0091]

[0092] 3. Assemble DHEA and IGF-1 mimetic peptide

[0093] After SA-FFGSSSR is prepared on a solid phase synthesizer, the operation of step (3) is repeated once, and an equal amount of Fmoc-protected phenylalanine (F) is replaced with compound 1 (containing a DHEA blocking group) to obtain DHEA-FFGSSSR, i.e., a DHEA-IGF-1 mimetic peptide, denoted as DHEA polypeptide.

[0094] The molecular formula of DHEA-FFGSSSR is C 58 H 80 N 10 O 15 , molecular weight is 1157.33, structural formula is as follows:

[0095]

[0096] 4. Remove the DHEA polypeptide from the solid phase synthesizer and make it into powder

[0097] After the DHEA polypeptide is prepared on the solid phase synthesizer, the solid phase synthesizer is washed 5 times with DMF (10 mL of DMF each time, washing time 1 min), and then washed 5 times with anhydrous DCM (10 mL of DCM each time, washing time 1 min), and then 10 mL of a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water (H2O) in a volume ratio of 38:1:1 is added, and the reaction is carried out at room temperature for 30 min. The product is cut off from the 2-Cl-Trt resin, and the solvent is removed by vacuum concentration to obtain a crude DHEA polypeptide, which is separated and purified by high performance liquid chromatography (HPLC), and freeze-dried to obtain a DHEA polypeptide powder.

[0098] 5. Make DHEA polypeptide powder into hydrogel

[0099] Accurately weigh 1 mg of DHEA polypeptide powder (prepared in step 4) and place it in a vial, add 108 μL of double distilled water and ultrasonically dissolve it, slowly heat the bottom of the vial with an alcohol lamp and slowly shake the vial until the DHEA polypeptide powder is completely dissolved, stop heating after observing that the solution is completely clear, let it stand, and obtain a sustained-release DHEA polypeptide hydrogel preparation, which is recorded as DHEA polypeptide hydrogel. The hydrogel preparation is colorless, transparent, and hydrogel-like, and the concentration of DHEA polypeptide is 8 mmol / L.

[0100] The transmission electron microscopy image of the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention is shown in Figure 1 .Depend on Figure 1 It can be seen that the hydrogel preparation has regular nanofibers, and these nanofibers are interwoven to form a network structure.

[0101] 2. Exploring the therapeutic effect of sustained-release DHEA polypeptide hydrogel preparation on RIR injury at the cellular level

[0102] IGF-1 mimetic peptide solution: The FFGSSSR heptapeptide (SA-FFGSSSR) was synthesized on a solid phase synthesizer using the same method as above, and the FFGSSSR heptapeptide was cut off from the resin, and the IGF-1 mimetic peptide powder was prepared by vacuum concentration, HPLC separation and purification, and lyophilization. The IGF-1 mimetic peptide powder was dissolved in water to prepare an IGF-1 mimetic peptide solution with a concentration of 8 mmol / L for standby use.

[0103] DHEA solution: Dissolve DHEA powder in dimethyl sulfoxide (DMSO) to prepare a DHEA solution with a concentration of 8 mmol / L for standby use.

[0104] 1. Cellular uptake experiment

[0105] 0.11mmol DHEA polypeptide powder (prepared in step 4), 0.1mmol amino-reactive fluorescent dye (Cy5.5-NHS) and 0.2mmol DIEPA were dissolved in 2mL anhydrous DMF, stirred overnight at room temperature in the dark, and monitored by liquid chromatography-mass spectrometry (LCMS). After the reaction was completed, HPLC was used for separation and purification, and freeze-dried to obtain fluorescently labeled DHEA polypeptide powder. The fluorescently labeled DHEA polypeptide powder was made into a hydrogel using the same method as step 5, which was recorded as Cy5.5-DHEA polypeptide hydrogel.

[0106] Rat retinal progenitor cells (R28 cells) were inoculated in a 2 cm cell confocal dish and incubated overnight to allow the cells to adhere to the wall. Cy5.5-DHEA polypeptide hydrogel (final concentration 20 μmol / L) was added to the culture medium. The culture medium containing Cy5.5-DHEA polypeptide hydrogel was washed away after 2 h, 4 h, 8 h, 12 h, and 24 h, respectively. After cell fixation, permeabilization, and DAPI labeling of cell nuclei, a laser confocal microscope was used to observe the uptake of Cy5.5-DHEA polypeptide hydrogel by R28 cells over time.

[0107] The results of the detection of the uptake of Cy5.5-DHEA polypeptide hydrogel by R28 cells are shown in Figure 2 .Depend on Figure 2 It can be seen that: as time goes by, the Cy5.5-DHEA polypeptide hydrogel is successfully taken up by R28 cells, and the uptake is time-dependent, which indicates that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention has good biocompatibility and R28 cell uptake ability.

[0108] 2. Anti-inflammatory activity study

[0109] Primary macrophages extracted from mouse bone marrow were inoculated in 24-well plates and divided into control group, model group, non-drug-loaded group, DHEA group and DHEA polypeptide hydrogel group. After incubation overnight, the IGF-1 mimetic peptide solution, DHEA solution and DHEA polypeptide hydrogel prepared above were added to the non-drug-loaded group, DHEA group and DHEA polypeptide hydrogel group, respectively (the final concentrations of IGF-1 mimetic peptide, DHEA and DHEA polypeptide were all 20 μmol / L) for pretreatment for 2 h. Then, lipopolysaccharide (LPS) was added to the model group, non-drug-loaded group, DHEA group and DHEA polypeptide hydrogel group to establish an inflammatory model. The control group did not receive any treatment. After 24 h, the total nitric oxide (NO) detection kit and interleukin-1β (IL-1β) detection kit were used to detect the contents of inflammatory factors NO and IL-1β secreted by cells in the cell supernatant of each group.

[0110] The detection results of inflammatory factors NO and IL-1β secreted by cells in each group are shown in Figure 3 and Figure 4 .

[0111] Depend on Figure 3 and Figure 4 It can be seen that compared with the model group, the addition of DHEA polypeptide hydrogel significantly inhibited the production of inflammatory factors NO and IL-1β. This shows that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention has good anti-inflammatory effect.

[0112] 3. Research on the protection ability of retinal cells

[0113] R28 cells were seeded in a 96-well plate and divided into a non-drug-loaded group, a DHEA group and a DHEA polypeptide hydrogel group. After incubation overnight, the non-drug-loaded group was replaced with a whole culture medium containing different concentrations of IGF-1 mimetic peptide for pretreatment for 24 hours, the DHEA group was replaced with a whole culture medium containing different concentrations of DHEA for pretreatment for 24 hours, and the DHEA polypeptide hydrogel group was replaced with a whole culture medium containing different concentrations of DHEA polypeptide hydrogel for pretreatment for 24 hours. After pretreatment, each group replaced the sugar-free culture medium and placed the 96-well plate in a hypoxic chamber (simulating an ischemic and hypoxic environment to establish a RIR injury model). After incubation at 37°C for 12 hours, the cells were taken out and the contents of adenosine triphosphate (ATP) and lactate dehydrogenase (LDH) in the cells of each group were detected.

[0114] The results of the detection of intracellular ATP and LDH content in each group are shown in Figure 5 and Figure 6 .Depend on Figure 5 and Figure 6It can be seen that the presence of DHEA polypeptide hydrogel significantly inhibited the phenomenon of decreased intracellular ATP content after the establishment of the RIR injury model, and significantly reduced the intracellular LDH content, and the two therapeutic effects were dose-dependent. This shows that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention exhibits good retinal cell protection ability in vitro.

[0115] 4. Research on the generation of reactive oxygen species in retinal cells

[0116] R28 cells were seeded in a 2 cm confocal dish and divided into a control group (without any treatment), a model group, a non-drug-loaded group, a DHEA group and a DHEA polypeptide hydrogel group. After incubation overnight, the non-drug-loaded group was replaced with a full culture medium containing an IGF-1 mimetic peptide (final concentration of 20 μmol / L) for pretreatment for 24 h, the DHEA group was replaced with a full culture medium containing DHEA (final concentration of 20 μmol / L) for pretreatment for 24 h, and the DHEA polypeptide hydrogel group was replaced with a full culture medium containing DHEA polypeptide hydrogel (DHEA polypeptide final concentration of 20 μmol / L) for pretreatment for 24 h. After the pretreatment, the model group, the non-drug-loaded group, the DHEA group and the DHEA polypeptide hydrogel group were all replaced with a sugar-free culture medium and the confocal dish was placed in a hypoxic chamber. After incubation at 37°C for 12 h, it was taken out, the sugar-free culture medium was discarded, and the cells were washed with phosphate buffer. The generation of reactive oxygen species in each group was detected using a reactive oxygen detection kit, and the detection results were photographed using a confocal microscope.

[0117] The fluorescence detection results of intracellular reactive oxygen species in each group are shown in Figure 7 .Depend on Figure 7 It can be seen that compared with the control group, the intracellular reactive oxygen level in the model group was significantly increased, while after treatment with DHEA polypeptide hydrogel, the intracellular reactive oxygen level was significantly reduced. This shows that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention can significantly inhibit the intracellular reactive oxygen level.

[0118] 3. To investigate the therapeutic effect of sustained-release DHEA polypeptide hydrogel preparation on RIR injury at the animal level, the RIR injury model of SD rats was established using the anterior chamber pressurization method for later use.

[0119] 1. Retinal HE staining

[0120] The modeled SD rats were divided into a model group, a non-drug-loaded group and a DHEA polypeptide hydrogel group. The IGF-1 mimetic peptide solution prepared previously was injected into the vitreous of the SD rats in the non-drug-loaded group, and the DHEA polypeptide hydrogel group was injected into the vitreous of the SD rats in the DHEA polypeptide hydrogel group. The injection dose was 4 μL. The SD rats in the model group did not receive any treatment. After 7 days, the SD rats in each group were killed, and the eyeballs were removed and fixed, paraffin-embedded, and hematoxylin-eosin stained in sequence. The stained sections were placed under a microscope to observe the retinal structure and morphology.

[0121] The results of HE staining of SD rat retina are shown in Figure 8 .Depend on Figure 8 It can be seen that the retinal layers of the SD rats in the model group became thinner and the retinal ganglion cells were lost; compared with the SD rats in the model group, the retinal morphology and thickness of the SD rats in the DHEA polypeptide hydrogel group were well restored after the vitreous injection of DHEA polypeptide hydrogel. This shows that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention successfully inhibited the apoptosis of retinal cells after ischemia-reperfusion and improved the functional state of the retina.

[0122] 2. Optic nerve cell protection experiment

[0123] The modeled SD rats were divided into a model group, a non-drug-loaded group and a DHEA polypeptide hydrogel group. The IGF-1 mimetic peptide solution prepared above was injected into the vitreous of the SD rats in the non-drug-loaded group, and the DHEA polypeptide hydrogel prepared above was injected into the vitreous of the SD rats in the DHEA polypeptide hydrogel group. The injection dose was 4 μL. The SD rats in the model group were not treated in any way. After 7 days, the SD rats in each group were killed, and the eyeballs were taken out. After sucrose dehydration, 4% paraformaldehyde fixation and cryoembedding treatment, frozen sections were made with a thickness of 8 μm. The retinal neural tubulin β-III-Tubulin was fluorescently stained and the cell nucleus was labeled with DAPI. The fluorescence intensity was observed using a laser confocal microscope.

[0124] The results of immunofluorescence detection of retinal tubulin β-III-Tubulin in SD rats in each group are shown in Fig. 9 .Depend on Fig. 9 It can be seen that compared with the SD rats in the model group, the fluorescence intensity of retinal neural tubulin β-III-Tubulin in the SD rats in the DHEA polypeptide hydrogel group significantly increased after the DHEA polypeptide hydrogel was administered. This indicates that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention has a good protective effect on optic nerve cells after retinal ischemia-reperfusion occurs.

[0125] 3. Retinal inflammation inhibition experiment

[0126] The frozen sections of the retina obtained in the previous optic nerve cell protection experiment were taken, and the glial cell-specific proteins IBA1 and GFAP were fluorescently stained, and the cell nuclei were labeled with DAPI, and the fluorescence intensity was observed using a laser confocal microscope.

[0127] The results of immunofluorescence detection of glial cell-specific proteins IBA1 and GFAP are shown in Fig.10 and Fig.11 .Depend on Fig.10 and Fig.11 It can be seen that the fluorescence intensity of glial cell-specific proteins IBA1 and GFAP in the SD rats in the model group increased significantly, and glial cells were activated under ischemia-reperfusion; compared with the SD rats in the model group, the fluorescence of glial cell-specific proteins IBA1 and GFAP in the SD rats in the DHEA polypeptide hydrogel group was significantly reduced after the DHEA polypeptide hydrogel was administered, indicating that the activation and proliferation of glial cells were inhibited, and the inflammatory response was inhibited. This shows that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention has a good ability to inhibit retinal inflammation in vivo.

[0128] 4. Retinal drug uptake experiment

[0129] The Cy5.5-DHEA polypeptide hydrogel prepared above was injected into the vitreous of unmodeled SD rats and modeled SD rats at a dose of 4 μL. The eyeballs were removed on the 7th and 14th days, respectively. The retinas were fixed and flattened. The retinas were soaked in methanol and the fluorescence of the retinal pigment epithelium was observed using a laser confocal microscope.

[0130] On the 7th day, the results of retinal fluorescence detection were shown in Fig.12 ; On the 14th day, the results of retinal fluorescence detection were shown in Fig.13 .Depend on Fig.12 and Fig.13 It can be seen that: on the 7th day, the fluorescence intensity of the retina was the strongest, whether it was the SD rats after modeling (modeling eyes) or the SD rats without modeling (normal eyes), indicating that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention can be absorbed by the retina for a long time. Until the 14th day, red fluorescence can still be detected on the retina of the modeling eye and the normal eye, indicating that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention has good biocompatibility and long-term sustained release.

[0131] 5. ERG detection

[0132] The SD rats after modeling were divided into a model group, a non-drug-loaded group, and a DHEA polypeptide hydrogel group. 24 hours after modeling, the vitreous of the SD rats in the non-drug-loaded group was injected with the previously prepared IGF-1 mimetic peptide solution, and the vitreous of the SD rats in the DHEA polypeptide hydrogel group was injected with the previously prepared DHEA polypeptide hydrogel, with an injection dose of 4 μL. The SD rats in the model group were not treated in any way. After 7 days, ERG detection was performed on the SD rats in each group. All experimental SD rats were dark-adapted for more than 12 hours before ERG detection. Before the start of ERG detection, all experimental SD rats were anesthetized by intraperitoneal injection of 1% pentobarbital (3 mL / kg) and 50% isoflurane (250 μL / kg). Under dim red light irradiation, the pupils were dilated with tropicamide eye drops. After the SD rats were deeply anesthetized, stainless steel subcutaneous needle electrodes were implanted subcutaneously as ground electrodes (tail) and reference electrodes (under a single eyelid). Gold electrodes were used as experimental electrodes. The experimental electrodes were gently placed on the surface of the cornea of ​​the SD rats. During the experiment, 0.1% sodium hyaluronate eye drops were continuously dripped on the gold electrodes to keep the cornea moist. All experimental SD rats were placed on a hot platform maintained at 37°C. Under dark adaptation conditions, the intensity of white light stimulation was initially adjusted to 0.01 cd.s / m -2 、3.0cd.s / m -2 and 10.0cd.s / m -2 , both eyes detect simultaneously to obtain a-wave, b-wave and oscillatory potential (Ops) signals.

[0133] The results of ERG testing are shown in Fig.14 .Depend on Fig.14 It can be seen that the a wave and b wave disappeared in the ERG waveform of the model group, while the a wave and b wave can be significantly restored in the DHEA polypeptide hydrogel group. This shows that the sustained-release DHEA polypeptide hydrogel preparation prepared by the present invention can significantly restore the damaged retinal function.

[0134] Example 2

[0135] 1. Preparation of sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation

[0136] 1. Preparation of end-capping groups containing DHEA

[0137] 1.0 mmol DHEA, 2.0 mmol succinic anhydride and 0.2 mmol 4-dimethylaminopyridine (DMAP) were dissolved in 10 mL pyridine, stirred at room temperature (25°C) for 45 h, and concentrated in vacuo to remove most of the solvent. An appropriate amount of 2 M hydrochloric acid was added, and the mixture was filtered and the filter cake was taken to obtain compound 1, which contained a capping group of DHEA.

[0138] 2. Solid phase synthesis of IGF-1 mimetic peptide using Fmoc method

[0139] The IGF-1 mimetic peptide provided by the present invention is a heptapeptide with an amino acid sequence of FFGSSSR.

[0140] (1) Loading and processing resin

[0141] 0.5 mmol 2-Cl-Trt resin was loaded into a solid phase synthesizer, 10 mL anhydrous dichloromethane (DCM) was added, and the mixture was placed on a shaker and shaken for 5 min to allow the 2-Cl-Trt resin to fully swell. The DCM was then pressed out of the solid phase synthesizer containing the 2-Cl-Trt resin using an ear bulb.

[0142] (2) Connect the first amino acid to the resin

[0143] 0.75mmol Fmoc-protected phenylalanine (F) was dissolved in 10mL anhydrous DCM, 0.75mmol N,N-diisopropylethylamine (DIEPA) was added, and the mixture was transferred to the above-mentioned solid phase synthesizer, and 0.75mmol DIEPA was added. The mixture was reacted at room temperature for 1h. After the reaction, the reaction solution in the solid phase synthesizer was removed with an ear bulb, and then washed with anhydrous DCM for 5 times (10mL DCM each time, and the washing time was 1min). Then, 20mL of a mixed solution of anhydrous DCM, DIEPA and methanol in a volume ratio of 17:1:2 was added, and the mixture was reacted at room temperature for 10min to obtain a resin connected with phenylalanine (F), which was recorded as SA-F.

[0144] (3) Synthesis of FF dipeptide

[0145] After SA-F was prepared on the solid phase synthesizer, the reaction solution in the solid phase synthesizer was removed with an ear bulb, and the mixture was first washed with anhydrous DCM for 5 times (10 mL of DCM each time, and the washing time was 1 min), and then washed with N,N-dimethylformamide (DMF) for 5 times (10 mL of DMF each time, and the washing time was 1 min), and then 10 mL of DMF containing 20% ​​(v / v) piperidine was added, and the mixture was reacted at room temperature for 25 min, and then washed with DMF for 5 times (10 mL of DMF each time, and the washing time was 1 min), 1.0 mmol of Fmoc-protected phenylalanine (F), 1.5 mmol of O-benzotriazole-tetramethyluronium hexafluorophosphate (HBTU), 2.0 mmol of DIEPA and 10 mL of DMF were mixed evenly to obtain a mixed solution, and the mixed solution was added to the above solid phase synthesizer, and reacted at room temperature for 2 h to obtain FF dipeptide, which was recorded as SA-FF.

[0146] (4) Synthesis of FFGSSSR heptapeptide

[0147] After SA-FF was prepared on the solid phase synthesizer, the operation of step (3) was repeated 5 times, and the Fmoc-protected phenylalanine (F) was replaced with equal amounts of Fmoc-protected glycine (G), Fmoc-protected serine (S), Fmoc-protected serine (S), Fmoc-protected serine (S), and Fmoc-protected arginine (R) in sequence to obtain the FFGSSSR heptapeptide, i.e., the IGF-1 mimetic peptide, denoted as SA-FFGSSSR.

[0148] 3. Assemble DHEA and IGF-1 mimetic peptide on a solid phase synthesizer

[0149] After SA-FFGSSSR is prepared on a solid phase synthesizer, the operation of step (3) is repeated once, and an equal amount of Fmoc-protected phenylalanine (F) is replaced with compound 1 (containing a DHEA blocking group) to obtain DHEA-FFGSSSR, i.e., a DHEA-IGF-1 mimetic peptide, denoted as DHEA polypeptide.

[0150] 4. Remove the DHEA polypeptide from the solid phase synthesizer and make it into powder

[0151] After the DHEA polypeptide is prepared on the solid phase synthesizer, the solid phase synthesizer is washed 5 times with DMF (10 mL of DMF each time, washing time 1 min), and then washed 5 times with anhydrous DCM (10 mL of DCM each time, washing time 1 min), and then 10 mL of a mixed solution of trifluoroacetic acid (TFA), triisopropylsilane (TIS) and water (H2O) in a volume ratio of 38:1:1 is added, and the reaction is carried out at room temperature for 30 min. The product is cut off from the 2-Cl-Trt resin, and the solvent is removed by vacuum concentration to obtain a crude DHEA polypeptide, which is separated and purified by high performance liquid chromatography (HPLC), and freeze-dried to obtain a DHEA polypeptide powder.

[0152] Accurately weigh 1 mg of DHEA polypeptide powder and place it in a vial, add 108 μL of double distilled water and ultrasonically dissolve it, slowly heat the bottom of the vial with an alcohol lamp and slowly shake the vial until the DHEA polypeptide powder is completely dissolved, stop heating after observing that the solution is completely clear, let it stand, and obtain a sustained-release DHEA polypeptide hydrogel preparation, recorded as DHEA polypeptide hydrogel.

[0153] The DHEA polypeptide hydrogel is colorless, transparent, and in the form of a hydrogel. The concentration of the DHEA polypeptide is 8 mmol / L. The transmission electron microscopy image is shown in FIG. Fig.15 .Depend on Fig.15 It can be seen that the DHEA polypeptide hydrogel has regular nanofibers, and these nanofibers are interwoven to form a network structure.

[0154] 5. Obtaining exosomes

[0155] Berberine (BBR) is a traditional Chinese medicine that can be isolated and purified from Coptis chinensis, and has a history of safe clinical application for many years. In recent years, in vivo studies of human retinal Müller cells and in vitro studies of rats have found that BBR can protect retinal Müller cells from damage in diabetic patients. Chen et al. further reported that in diabetic rats, BBR attenuated the apoptosis of retinal Müller cells through the AMPK / mTOR signaling pathway. The present invention will use BBR to pretreat rat retinal progenitor cells (R28 cells) to further enhance the therapeutic effect of exosomes.

[0156] R28 cells were revived and then cultured in a cell culture medium containing berberine (10 μmol / L) for 2 days. They were then subjected to hypoxia treatment for 12 h. The supernatant of the hypoxic R28 cells was collected and exosomes were obtained by differential centrifugation.

[0157] The exosomes were resuspended in 1.2 mL PBS, and the concentration of exosomes was 16 g / L (BCA method). The transmission electron microscopy image is shown in Fig.16 .Depend on Fig.16 It can be seen that the obtained exosomes have a typical cup-shaped structure.

[0158] 6. Encapsulating exosomes with DHEA peptide hydrogel

[0159] Accurately weigh 1 mg of the DHEA polypeptide powder prepared in step 4, place the DHEA polypeptide powder in a vial, add 108 μL of double distilled water and ultrasonically dissolve it, slowly heat the bottom of the vial with an alcohol lamp and slowly shake the vial until the DHEA polypeptide powder is completely dissolved, stop heating after observing that the solution is completely clear, set aside and wait for the temperature to drop, and slowly add 108 μL of the exosomes (containing 1.728 mg of exosomes) resuspended in PBS obtained in step 5 before gelation, gently shake to evenly disperse the exosomes, and let it stand until the hydrogel is formed to obtain a sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation (each 216 μL of hydrogel contains 1 mg of DHEA polypeptide powder and 1.728 mg of exosomes), which is recorded as DHEA polypeptide hydrogel exosomes.

[0160] The DHEA polypeptide hydrogel exosomes are colorless, transparent, and hydrogel-like. The transmission electron microscopy image is shown in Fig.17 .Depend on Fig.17 It can be seen that in the DHEA polypeptide hydrogel exosomes, nanofibers and exosomes exist simultaneously.

[0161] 2. Exploring the therapeutic effect of sustained-release DHEA peptide hydrogel-encapsulated exosome delivery preparation on RIR injury at the cellular level

[0162] IGF-1 mimetic peptide solution: The FFGSSSR heptapeptide (SA-FFGSSSR) was synthesized on a solid phase synthesizer using the same method as above, and the FFGSSSR heptapeptide was cut off from the resin, and the IGF-1 mimetic peptide powder was prepared by vacuum concentration, HPLC separation and purification, and lyophilization. The IGF-1 mimetic peptide powder was dissolved in water to prepare an IGF-1 mimetic peptide solution with a concentration of 8 mmol / L for standby use.

[0163] DHEA solution: Dissolve DHEA powder in dimethyl sulfoxide (DMSO) to prepare a DHEA solution with a concentration of 8 mmol / L for later use.

[0164] 1. Cellular uptake experiment

[0165] Dissolve 0.11 mmol DHEA polypeptide powder (prepared in step 4), 0.1 mmol amino-reactive fluorescent dye (Cy5.5-NHS) and 0.2 mmol DIEPA in 2 mL anhydrous DMF, stir overnight at room temperature in the dark, monitor the reaction by LCMS, and after the reaction is completed, separate and purify by HPLC, and lyophilize to obtain Cy5.5 labeled DHEA polypeptide powder. 1 mg of Cy5.5-labeled DHEA polypeptide powder was placed in a vial, 108 μL of double distilled water was added and ultrasonically dissolved, the bottom of the vial was slowly heated with an alcohol lamp and the vial was slowly shaken until the Cy5.5-labeled DHEA polypeptide powder was completely dissolved, and the heating was stopped after the solution was observed to be completely clear, and the solution was set aside to wait for the temperature to drop. Before gelation, 108 μL of exosomes (containing 1.728 mg of exosomes) resuspended in PBS obtained in step 5 was slowly added thereto, and the exosomes were gently shaken to evenly disperse the exosomes, and the solution was allowed to stand until the hydrogel was formed to obtain a fluorescently labeled sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation, which was recorded as Cy5.5-labeled DHEA polypeptide hydrogel exosomes.

[0166] Take 1 mL of exosomes resuspended in PBS, add 100 μL of DiI staining working solution, mix well, incubate at 37 °C in the dark for 30 min, and collect DiI-labeled exosomes by ultracentrifugation.

[0167] R28 cells were seeded in a 2 cm cell confocal dish and incubated overnight to allow the cells to adhere to the wall. Cy5.5-labeled DHEA polypeptide hydrogel exosomes and DiI-labeled exosomes were added (final concentration was 20 μmol / L), respectively. The culture medium containing Cy5.5-labeled DHEA polypeptide hydrogel exosomes or the culture medium containing DiI-labeled exosomes were washed away after 4 h, 8 h, 24 h, and 48 h, respectively. After cell fixation, permeabilization, and DAPI labeling of cell nuclei, laser confocal microscopy was used to observe the cell uptake of Cy5.5-labeled DHEA polypeptide hydrogel exosomes and DiI-labeled exosomes over time.

[0168] The results of the uptake of Cy5.5-labeled DHEA polypeptide hydrogel exosomes and DiI-labeled exosomes by R28 cells are shown in Fig.18 .Depend on Fig.18 It can be seen that: as time goes by, Cy5.5-labeled DHEA polypeptide hydrogel exosomes are successfully taken up by R28 cells and show a time-dependent uptake, which indicates that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention has good biocompatibility and cell uptake ability.

[0169] 2. Anti-inflammatory activity study

[0170] Primary macrophages extracted from mouse bone marrow were inoculated in 24-well plates and divided into a control group, an LPS group, a non-drug-loaded group, and a DHEA polypeptide hydrogel exosome group. After incubation overnight, the IGF-1 mimetic peptide solution prepared previously (final concentration of 20 μmol / L) was added to the non-drug-loaded group for pretreatment for 2 h, and the DHEA polypeptide hydrogel exosome group was added with the DHEA polypeptide hydrogel exosome solution prepared previously (final concentration of 20 μmol / L) for pretreatment for 2 h. Then, lipopolysaccharide (LPS) was added to the LPS group, the non-drug-loaded group, and the DHEA polypeptide hydrogel exosome group to establish an inflammatory model. The control group did not receive any treatment. After 24 h, the total nitric oxide (NO) detection kit was used to detect the content of the inflammatory factor NO secreted by the cells in the supernatant of each group of cells.

[0171] The results of the detection of the secretion of inflammatory factor NO in each group are shown in Fig.19 .Depend on Fig.19 It can be seen that compared with the lipopolysaccharide-stimulated inflammation model group (LPS group), the addition of DHEA polypeptide hydrogel exosomes significantly inhibited the production of inflammatory factor NO. This shows that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention has good anti-inflammatory effect.

[0172] 3. Research on the protection ability of retinal cells

[0173] R28 cells were seeded in a 96-well plate and divided into a control group, a non-drug-loaded group and a DHEA polypeptide hydrogel exosome group. After incubation overnight, the non-drug-loaded group was replaced with a full culture medium containing an IGF-1 mimetic peptide (final concentration of 20 μmol / L) for pretreatment for 24 h, and the DHEA polypeptide hydrogel exosome group was replaced with a full culture medium containing DHEA polypeptide hydrogel exosomes (final concentration of 20 μmol / L) for pretreatment for 24 h. After the pretreatment, the control group, the non-drug-loaded group and the DHEA polypeptide hydrogel exosome group were replaced with a sugar-free culture medium and the 96-well plate was placed in a hypoxic chamber (simulating an ischemic hypoxic environment to establish a RIR injury model). After incubation at 37°C for 12 h, the 96-well plate was taken out and the content of adenosine triphosphate (ATP) in the cells of each group was detected.

[0174] The results of the determination of intracellular ATP content in each group are shown in Fig. 20 .Depend on Fig. 20 It can be seen that the presence of DHEA polypeptide hydrogel exosomes significantly inhibited the phenomenon of decreased intracellular ATP content after the establishment of the RIR injury model. This shows that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention exhibits good retinal cell protection ability in vitro.

[0175] 4. Research on the generation of reactive oxygen species in retinal cells

[0176] R28 cells were seeded in a 2 cm confocal dish and divided into a control group, a model group, a non-drug-loaded group, a DHEA group and a DHEA polypeptide hydrogel exosome group. After incubation overnight, the non-drug-loaded group was replaced with a full culture medium containing an IGF-1 mimetic peptide (final concentration of 20 μmol / L) for pretreatment for 24 h, the DHEA group was replaced with a full culture medium containing DHEA (final concentration of 20 μmol / L) for pretreatment for 24 h, and the DHEA polypeptide hydrogel exosome group was replaced with a full culture medium containing DHEA polypeptide hydrogel (DHEA polypeptide final concentration of 20 μmol / L) for pretreatment for 24 h. After the pretreatment, all groups except the control group were replaced with sugar-free culture medium and the dish was placed in a hypoxic chamber. After incubation at 37°C for 12 h, the dish was taken out, the sugar-free culture medium was discarded, and the cells were washed with phosphate buffer. The generation of reactive oxygen species in the cells of each group was detected using a reactive oxygen detection kit, and the detection results were photographed using a confocal microscope.

[0177] The results of reactive oxygen species fluorescence detection of cells in each group are shown in Fig.21 .Depend on Fig.21 It can be seen that compared with the control group, the level of intracellular reactive oxygen species in the model group was significantly increased, while after treatment with DHEA polypeptide hydrogel exosomes, the level of reactive oxygen species was significantly reduced. This indicates that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention can significantly inhibit the level of intracellular reactive oxygen species.

[0178] 3. To investigate the therapeutic effect of sustained-release DHEA peptide hydrogel-encapsulated exosome delivery preparation on RIR injury at the animal level

[0179] The anterior chamber pressurization method was used to establish the retinal ischemia-reperfusion injury model in SD rats.

[0180] 1. Retinal HE staining

[0181] The modeled SD rats were divided into a model group, a non-drug-loaded group and a DHEA polypeptide hydrogel exosome group.

[0182] The IGF-1 mimetic peptide solution was injected into the vitreous of the SD rats in the non-drug-loaded group, and the DHEA polypeptide hydrogel exosomes (i.e., the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention) were injected into the vitreous of the SD rats in the DHEA polypeptide hydrogel exosome group. The injection dose was 4 μL. The SD rats in the model group were not treated in any way. After 7 days, the SD rats in each group were killed, the eyeballs were removed, and after fixation, paraffin embedding, and hematoxylin-eosin staining, the stained sections were placed under a microscope to observe the retinal structure and morphology.

[0183] The results of HE staining of retina of SD rats in each group are shown in Fig. 22 .Depend on Fig. 22 It can be seen that the retinal layers of the SD rats in the model group became thinner and the retinal ganglion cells were lost; compared with the model group, after the vitreous injection of DHEA polypeptide hydrogel exosomes, the morphology and thickness of the retina of the SD rats were well restored. This shows that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention successfully inhibited the apoptosis of retinal cells after ischemia-reperfusion and improved the functional state of the retina.

[0184] 2. Optic nerve cell protection experiment

[0185] The modeled SD rats were divided into a model group, a non-drug-loaded group and a DHEA polypeptide hydrogel exosome group.

[0186] IGF-1 mimetic peptide solution was injected into the vitreous of SD rats in the non-drug group, DHEA polypeptide hydrogel exosomes were injected into the vitreous of SD rats in the DHEA polypeptide hydrogel exosomes group, and the SD rats in the model group were not treated in any way. After 7 days, the SD rats in each group were killed, and the eyeballs were removed. After sucrose dehydration, 4% paraformaldehyde fixation, and cryoembedding, frozen sections were made with a thickness of 8 μm, and retinal neural tubulin β-III-Tubulin was fluorescently stained, and the cell nuclei were labeled with DAPI. The fluorescence intensity was observed using a laser confocal microscope.

[0187] The results of immunofluorescence detection of retinal tubulin β-III-Tubulin in each group of SD rats are shown in Fig.23 .Depend on Fig.23 It can be seen that compared with the model group, after administration of DHEA polypeptide hydrogel exosomes, the fluorescence intensity of retinal neural tubulin β-III-Tubulin of SD rats increased significantly. This shows that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention has a good protective effect on optic nerve cells after retinal ischemia-reperfusion occurs.

[0188] 3. Retinal inflammation inhibition experiment

[0189] The frozen sections of the retina obtained in the previous optic nerve cell protection experiment were taken, and the glial cell-specific proteins IBA1 and GFAP were fluorescently stained, and the cell nuclei were labeled with DAPI, and the fluorescence intensity was observed using a laser confocal microscope.

[0190] The immunofluorescence detection results of glial cell-specific proteins IBA1 and GFAP in each group of SD rats are shown in Fig.24 and Fig.25 .Depend on Fig.24 Fig.25 It can be seen that the fluorescence intensity of glial cell-specific proteins IBA1 and GFAP in the SD rats in the model group increased significantly, and glial cells were activated under ischemia-reperfusion; compared with the model group, after the administration of DHEA polypeptide hydrogel exosomes, the fluorescence of glial cell-specific proteins IBA1 and GFAP in SD rats decreased significantly, indicating that the activation and proliferation of glial cells in SD rats was inhibited, and the inflammatory response was inhibited. This shows that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention has a good ability to inhibit retinal inflammation in vivo.

[0191] 4. Retinal drug uptake experiment

[0192] Cy5.5-labeled DHEA polypeptide hydrogel exosomes were injected into the vitreous of unmodeled SD rats (normal eye group), and Cy5.5-labeled DHEA polypeptide hydrogel exosomes were injected into the vitreous of modeled SD rats (modeled eye group). The injection dose was 4 μL. The eyeballs were taken on the 7th and 14th days, and the retinas were fixed and flattened. After the retinas were soaked in methanol, the fluorescence of the retinal pigment epithelium was observed using a laser confocal microscope.

[0193] The fluorescence detection results of the retina of SD rats without modeling and SD rats with modeling on the 7th and 14th days are shown in Fig.26 and Fig. 27 .Depend on Fig.26 and Fig. 27It can be seen that: at the 7th day, the fluorescence intensity of the modeling eye group was the strongest, indicating that the DHEA polypeptide hydrogel exosomes can be absorbed by the retina for a long time, and red fluorescence can still be detected in the modeling eye group until the 14th day. This shows that the sustained-release DHEA polypeptide hydrogel-encapsulated exosome delivery preparation prepared by the present invention has good biocompatibility and long-term sustained release.

[0194] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A DHEA polypeptide, characterized in that Assembling DHEA onto the heptapeptide to obtain the DHEA polypeptide; The amino acid sequence of the heptapeptide is FFGSSSR, and the DHEA is dehydroepiandrosterone.

2. The DHEA polypeptide according to claim 1, characterized in that The preparation method of the DHEA polypeptide comprises the following steps: 1) mixing and stirring dehydroepiandrosterone, succinic anhydride, 4-dimethylaminopyridine and pyridine to obtain a capping group of DHEA; 2) The end-capping group of DHEA obtained in step 1), the heptapeptide, O-benzotriazole-tetramethyluronium hexafluorophosphate, N,N-diisopropylethylamine and N,N-dimethylformamide are mixed and reacted to obtain DHEA polypeptide.

3. The DHEA polypeptide according to claim 2, characterized in that In the step 1), the volume ratio of the moles of dehydroepiandrosterone, the moles of succinic anhydride, the moles of 4-dimethylaminopyridine and pyridine is 1 mmol:2 mmol:0.2 mmol:10 mL; The stirring time is 45h.

4. The DHEA polypeptide according to claim 2, characterized in that In the step 2), the volume ratio of the moles of the end-capping group of DHEA, the moles of the heptapeptide, the moles of O-benzotriazole-tetramethyluronium hexafluorophosphate, the moles of N,N-diisopropylethylamine and N,N-dimethylformamide is 1 mmol:0.5 mmol:1.5 mmol:2 mmol:10 mL; The reaction time is 2h.

5. A sustained-release hydrogel, characterized in that: Containing the DHEA polypeptide according to any one of claims 1 to 4, the concentration of the DHEA polypeptide in the sustained-release hydrogel is 8 mmol / L.

6. The sustained-release hydrogel according to claim 5, characterized in that The preparation method of the sustained-release hydrogel comprises the following steps: ultrasonically dissolving the DHEA polypeptide and water, and heat-treating the DHEA polypeptide to obtain the sustained-release hydrogel; The mass of the DHEA polypeptide and the volume ratio of water are 1 mg:108 μL.

7. A delivery preparation for encapsulated exosomes, characterized in that: Each 216 μL encapsulated exosome delivery formulation contained 1 mg DHEA polypeptide and 1.728 mg exosomes derived from R28 cells.

8. The encapsulated exosome delivery preparation according to claim 7, characterized in that The method for preparing the encapsulated exosome delivery preparation comprises the following steps: Mixing the DHEA polypeptide and water, and heat treating to obtain a clear solution; Before the clear solution is gelled, it is mixed with exosomes to obtain an encapsulated exosome delivery preparation.

9. Use of the DHEA polypeptide according to any one of claims 1 to 4 or the sustained-release hydrogel according to claim 5 in the preparation of a drug for treating retinal ischemia-reperfusion injury.

10. Use of the encapsulated exosome delivery preparation according to claim 7 or 8 in the preparation of a drug for treating retinal ischemia-reperfusion injury.

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