Use of a silk fibroin nanoparticle hydrogel in treating pressure ulcer

Abstract

The application discloses application of a silk fibroin nanoparticle hydrogel in treatment of pressure sore ulcers and relates to the technical field of biochemistry.The core points of the technical solution include the following steps: S1, preparation of a silk fibroin solution;S2, preparation of nanoparticles;S3, production and preparation of milk fat globule epidermal growth factor VIII (MFG-E8) in exosomes;S4, purification of MFG-E8 protein;S5, preparation of silk fibroin nanoparticles coated with MFG-E8;S6, preparation of MFG-E8-silk fibroin nanoparticles crosslinked with NGR peptides; and S7, preparation of a collagen / silk fibroin hydrogel.The silk fibroin nanoparticle hydrogel is a non-invasive treatment method and is more easily accepted by patients in clinical application.In addition, the targeted nanoparticle treatment method for pressure sore sites can significantly improve the treatment effect of patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochemistry, more particularly, it relates to the application of silk fibroin nanoparticle hydrogel in treating pressure ulcer. BACKGROUND

[0002] The pathological mechanism of pressure ulcer is that due to the dysfunction of activity, the local skin blood vessels are long-term compressed, the blood circulation is blocked, and the tissue is necrotic after ischemia and hypoxia, resulting in pressure ulcer. The current treatment methods for pressure ulcer include relieving pressure, dressing protection, debridement, etc., and the main way to reduce damage and promote tissue or ulcer repair is to delay the progression of pressure ulcer through physical or surgical methods, and there is no research on the related mechanism of endothelial cell damage caused by ischemia and hypoxia after the occurrence of pressure ulcer, and there is no research on the role of endothelial cell-derived exosomes and the proteins carried by them in inhibiting the occurrence and development of pressure ulcer.

[0003] The treatment of pressure ulcer mainly includes removing necrotic tissue, and using wound dressing, compression bandage and electrospun scaffold and other tissue materials to keep the wound moist, promote tissue proliferation and improve the wound environment to promote healing, etc. The new three-dimensional knitted fabric can effectively reduce the expression of neutrophil elastase, while increasing the level of transforming growth factor, thereby promoting cell proliferation. In addition, techniques such as negative pressure suction can efficiently remove inflammatory secretions and necrotic tissue, significantly promoting tissue repair and healing. The application of debridement, skin flap transplantation and foam dressing can not only stimulate cell proliferation and migration, induce angiogenesis, but also further promote wound healing and enhance the mechanical stress bearing capacity of the skin around the wound. The mechanism related to the occurrence and treatment of pressure ulcer mainly focuses on repairing the wound, achieving antibacterial, wound acidification, enhancing microcirculation and cell stimulation. Embryonic stem cell-derived exosomes can activate the Nrf2 pathway, inhibit endothelial cell aging, restore vascular dysfunction caused by aging, and accelerate wound healing in mice. Atmospheric pressure cold plasma can achieve antibacterial, wound acidification, enhanced microcirculation and cell stimulation, thereby promoting tissue remodeling and endothelial cell proliferation to accelerate wound healing. Boswellia standardized extract exerts its potential for promoting wound healing by coordinating mechanisms, promoting the generation of growth factors and inhibiting cell apoptosis, increasing angiogenesis to accelerate the wound healing of pressure ulcer. During the occurrence of pressure ulcer, blood vessels play an important role; however, there is no research report on how to inhibit endothelial cell ferroptosis caused by hypoxia. Moreover, in the treatment research, there is no related report on the use of targeted nanoparticles to inhibit vascular damage at the pressure ulcer site.

[0004] Studies have shown that normal mature skin vascular endothelial cells express almost no or only low levels of CD13 molecules. However, in the research of this project, we first found that under long-term pressure hypoxia conditions, the expression of CD13 in endothelial cells (ECs) was significantly increased. NGR is a polypeptide containing an arginine-glycine-asparagine (Arg-Gly-Asn) motif, and is also a high-affinity ligand for CD13. Since the NGR peptide molecule contains amino (-NH2), it can catalyze the reaction with carboxyl (-COOH), and the surface of silk fibroin nanoparticles is also rich in amino (-NH2), which can also catalyze the reaction with carboxyl (-COOH). Therefore, by using the catalyst EDC and NHS, we successfully coupled NGR peptide with silk fibroin nanoparticles by using dicarboxylic acid polyvinyl alcohol (HOOC-PEG-COOH) as a crosslinking agent, so that the silk fibroin nanoparticles have the ability to specifically target the blood vessels of pressure ulcer sites. Based on this technology, the project developed a kind of vascular targeted nanoparticles, which can effectively inhibit the iron death of vascular endothelial cells caused by hypoxia, thereby improving and preventing the formation of pressure ulcers, and showing important clinical significance and social value. SUMMARY

[0005] To solve the above problems, the present application provides a pressure ulcer treatment scheme based on silk fibroin nanoparticle hydrogel. By mixing targeted nanoparticles with silk fibroin / collagen hydrogel, a sustained-release carrier for targeted treatment of pressure ulcers is developed. After the carrier is applied to the pressure skin, it not only can effectively absorb the exudate at the pressure ulcer site, but also can transport MFG-E8 in the targeted nanoparticles to the inside of the endothelial cells through the specific binding of NGR peptide and CD13 molecules on the surface of endothelial cells, thereby inhibiting the iron death of endothelial cells caused by hypoxia, reducing vascular damage, delaying and inhibiting the formation of pressure ulcers, and significantly reducing the occurrence of skin pressure ulcers.

[0006] The above technical purposes of the present application are achieved by the following technical scheme: a preparation method of silk fibroin nanoparticles, comprising the following steps:

[0007] S1. Preparation of silk fibroin solution;

[0008] S2. Preparation of nanoparticles;

[0009] S3. Production of milk fat globule epidermal growth factor VIII (MFG-E8) in exosomes;

[0010] S4. Purification of MFG-E8 protein;

[0011] S5. Preparation of MFG-E8 coated silk fibroin nanoparticles;

[0012] S6. Preparation of cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle.

[0013] The present application is further provided with a specific preparation method of the silk fibroin solution, comprising the following steps:

[0014] S1. Take 2g of silkworm cocoons with removed pupae, put into 1L water, add 5-10g of Na2CO3, so that the concentration of Na2CO3 is 0.5%-1%, then cook in water at 90-100℃ for 30-60min until the sericin is completely removed;

[0015] S2. Wash the silk fibroin with removed sericin in deionized water for 3-5 times, and then dry in an oven at 60℃;

[0016] S3. Prepare 50ml of ternary solution, the preparation method is: double distilled water, ethanol, and calcium chloride are 30.43ml: 19.57ml: 24g respectively;

[0017] S4. Add the silkworm cocoons to the ternary solution, and dissolve at 60±5℃ for 1-2h until the silk fibroin is completely dissolved into liquid; centrifuge at 4000rpm / min (3220g) for 10min, and collect the supernatant;

[0018] S5. Dialyze the supernatant in a dialysis bag with a molecular weight cut-off of 8000-14000 for 48h, after dialysis is completed, centrifuge at 4000rpm / min (3220g) for 10min, collect the supernatant, and measure the protein concentration.

[0019] The present application is further provided with a specific preparation method of the nanoparticle, comprising the following steps:

[0020] S1. Dilute the obtained silk fibroin solution to 10mg / ml (1%), add 5ml of acetone solution in a 15ml centrifuge tube, vortex the acetone on a vortex mixer, the volume ratio of the acetone solution to the 1% silk fibroin solution is 5:1, that is, add 1ml of 1% silk fibroin solution with a mass fraction of 1% to the acetone, after the dripping is completed, continue to vortex for 30-60s;

[0021] S2. After the vortex is completed, centrifuge at 6000rpm / min (3840g) for 10min, pour off the supernatant and collect the precipitate;

[0022] S3. Add 10ml of double distilled water for washing, centrifuge at 6000rpm / min (3840g) for 10min, pour off the supernatant and collect the precipitate to remove the residual acetone;

[0023] S4. Repeat step S3;

[0024] S5. Add 5ml of double distilled water in the precipitate, ultrasonic on ice, the condition is: ultrasonic time 2min, pause 2s ultrasonic 2s, amplitude 30%;

[0025] S6. After ultrasonic, centrifugal 10min at 6000rpm / min, obtain milky white supernatant;

[0026] S7. Continue to centrifugal 10min at 12000rpm / min, obtain nanoparticle precipitate, after freeze-drying, obtain nanoparticle.

[0027] The application is further provided with a specific production and preparation method of milk fat globule epidermal growth factor VIII (MFG-E8) in exosomes, comprising the following steps:

[0028] S1. Extract rat vascular endothelial primary cells, and culture to obtain exosomes secreted by the cells;

[0029] S2. Perform mass spectrum detection analysis on the exosomes, and the result shows that MFG-E8 is very rich in the exosomes;

[0030] S3. Construct HBLV-r-MFG-E8-3xFlag-Green-PURO transfection lentivirus;

[0031] S4. Culture CHO-s cells in an incubator at 37 DEG C and 5% CO2, and make the CHO-s cells adhere under static conditions, and then transfer the lentivirus into the adherent cells;

[0032] S5. After 3 days, add puromycin to select the CHO-s cells into which MFG-E8 is transferred;

[0033] S6. After the CHO-s cells containing green fluorescence reach about 80%, perform trypsin digestion, centrifugal 5min at 1000rpm / min after cell digestion, collect the cell precipitate, add a special culture medium for CHO-s cells to culture on a shaker at a speed of 150rpm / min, and make the CHO-s cells suspended culture;

[0034] S7. The cells grow in the culture medium, and after the culture medium changes from clear to turbid, centrifugal 5min at 1200rpm / min, and collect the CHO-s cell precipitate;

[0035] S8. Lyse the precipitated cells in a cell lysis solution containing a protease inhibitor PMSF on ice for 30-60min, and the volume fraction of the protease inhibitor PMSF is 1%;

[0036] S9. After lysis, ultrasonic, the condition is: ultrasonic time 8s, pause 2s ultrasonic 2s, amplitude 20%;

[0037] S10. 4℃, 12000 rpm / min, centrifugation for 10 min;

[0038] S11. Collect the supernatant containing MFG-E8 protein.

[0039] The application further provides a specific method for purifying MFG-E8 protein, comprising the following steps:

[0040] S1. Purify MFG-E8 protein by adding 20 ul of anti-Flag magnetic beads to 500 ul of protein supernatant, and incubate at room temperature for 2 h on a shaker;

[0041] S2. Magnetic separation, separate on a magnetic stand for 20 s, and remove the supernatant;

[0042] S3. Elution, add 100 ul of 3xFlag polypeptide to each 20 ul of magnetic bead suspension for elution, and incubate at room temperature for 30-60 min;

[0043] S4. Magnetic stand separation for 20 s, and the supernatant is the eluted Flag-tagged protein MFG-E8;

[0044] S5. Western blotting detection of the purified protein;

[0045] S6. Preparation and detection of MFG-E8 purified protein.

[0046] The application further provides a specific preparation method of silk fibroin nanoparticles coated with MFG-E8, comprising the following steps:

[0047] S1. Dilute the obtained silk fibroin solution to 20 mg / ml (2%), and add 500 ul of purified MFG-E8 protein (0.93 mg / ml) to 500 ul of 2% silk fibroin solution, so that the silk fibroin concentration is 1%;

[0048] S2. Add 5 ml of acetone solution to a 15 ml centrifuge tube, and vortex the acetone on a vortex mixer. The volume ratio of the acetone solution to the silk fibroin solution containing MFG-E8 purified protein is 5:1, that is, 1 ml of MFG-E8-silk fibroin solution in S1 is added dropwise to the acetone, and after the dropwise addition is completed, vortex for another 30-60 s;

[0049] S3. After vortexing, centrifuge at 6000 rpm / min (3840 g) for 10 min, and discard the supernatant to collect the precipitate;

[0050] S4. Add 10 ml of double-distilled water for washing, centrifuge at 6000 rpm / min (3840 g) for 10 min, discard the supernatant to collect the precipitate, and remove the residual acetone;

[0051] S5. Repeat step S3;

[0052] S6. Add 5ml of double distilled water in the precipitate, perform ultrasonic on ice, with the condition that ultrasonic time is 3min, pause 3s ultrasonic 2s, amplitude 30%;

[0053] S7. After ultrasonic, centrifuge at 3000rpm / min for 10min, obtain milky white supernatant;

[0054] S8. Continue to centrifuge the milky white supernatant at 12000rpm / min for 10min, obtain nanoparticle precipitate, after freeze-drying, obtain MFG-E8 coated silk fibroin nanoparticles;

[0055] S9. Detect the MFG-E8 coated silk fibroin nanoparticles.

[0056] The application further provides a specific preparation method of the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticles, comprising the following steps:

[0057] S1. Take the MFG-E8 coated silk fibroin nanoparticles (produced from 500ul of 2% silk fibroin solution and 500ul of purified MFG-E8 protein (0.93mg / ml)), add 1ml of double distilled water to dissolve the nanoparticles;

[0058] S2. Add 10mg of EDC, 50mg of HOOC-PEG-COOH and 50mg of NGR peptide into 1ml of the nanoparticle solution, react at room temperature on a shaking table for 0.5-1h;

[0059] S3. Further add 10mg of NHS, react at room temperature on a shaking table overnight;

[0060] S4. The next day, centrifuge the nanoparticle solution at 6000rpm / min for 10min, discard the supernatant, and obtain the preparation of cross-linked NGR peptide MFG-E8-silk fibroin nanoparticles.

[0061] The application further provides a collagen / silk fibroin hydrogel preparation method of the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticles, comprising the following steps:

[0062] S1. Concentrate the extracted silk fibroin solution in 10%-15% polyvinyl alcohol 20000 solution for 48h, so that the final concentration of the silk fibroin solution is 8%-10%;

[0063] S2. Take 0.24g of carbomer, dissolve in 1ml of double distilled water;

[0064] S3. Take polyvinyl alcohol 2g dissolved in 10ml of double distilled water, so that its mass fraction is 20%;

[0065] S4. Take 800ul of 1mol / L NaOH solution into the carbomer solution, stir evenly, and then add 800ul of 20% polyvinyl alcohol;

[0066] S5. Take 8%-10% silk fibroin solution 4ml, add to the carbomer solution and stir evenly;

[0067] S6. Take 2ml of 1% collagen solution, and add 200ul of 1mol / L NaOH and mix evenly, then add to the carbomer solution;

[0068] S7. Take the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle solution dissolved in 200ul of double distilled water, and add to the carbomer solution and stir evenly;

[0069] S8. Adjust the PH value to 6-7;

[0070] S9. Place in a humid environment for 12-24h until it is cross-linked into glue;

[0071] S10. Detect the nanoparticles in the collagen / silk fibroin hydrogel.

[0072] In summary, the present application has the following beneficial effects:

[0073] The silk fibroin nanoparticle hydrogel of the present application can be more recognized by patients as a non-invasive treatment method in clinical application, and targeted nanotherapy can improve the treatment effect of patients. In subsequent application research, we can carry out corresponding clinical application research on the basis of animal experiments, and after application transformation, not only can provide a new treatment method for pressure ulcer patients, but also will produce huge social and economic value. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 is the mass spectrometry detection analysis diagram of the exosome in the embodiment of the present application;

[0075] Figure 2 is the MFG-E8 purified protein detection diagram in the embodiment of the present application;

[0076] Figure 3 is the coated MFG-E8 silk fibroin nanoparticle detection diagram in the embodiment of the present application;

[0077] Figure 4 is the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle detection diagram in the embodiment of the present application;

[0078] Figure 5 Figure 1 is a cell uptake experiment diagram of the MFG-E8-silk fibroin nanoparticle crosslinked with NGR peptide in an embodiment of the present application;

[0079] Figure 6 Figure 2 is a schematic diagram of the collagen hydrogel in an embodiment of the present application;

[0080] Figure 7 Figure 3 is an electron microscope detection diagram of the collagen hydrogel in an embodiment of the present application;

[0081] Figure 8 Figure 4 is a storage modulus G' and loss modulus G" detection diagram of the collagen hydrogel in an embodiment of the present application;

[0082] Figure 9 Figure 5 is a schematic diagram of the silk fibroin hydrogel in an embodiment of the present application;

[0083] Figure 10 Figure 6 is an electron microscope detection diagram of the silk fibroin hydrogel in an embodiment of the present application;

[0084] Figure 11 Figure 7 is a storage modulus G' and loss modulus G" detection diagram of the silk fibroin hydrogel in an embodiment of the present application;

[0085] Figure 12 Figure 8 is a schematic diagram of the collagen / silk fibroin hydrogel in an embodiment of the present application;

[0086] Figure 13 Figure 9 is an electron microscope detection diagram of the collagen / silk fibroin hydrogel in an embodiment of the present application;

[0087] Figure 14 Figure 10 is a storage modulus G' and loss modulus G" detection diagram of the collagen / silk fibroin hydrogel in an embodiment of the present application;

[0088] Figure 15 Figure 11 is a nanoparticle detection diagram in the collagen / silk fibroin hydrogel in an embodiment of the present application;

[0089] Figure 16 Figure 12 is an immunofluorescence detection diagram of the expression of MFG-E8 and CD13 in the hypoxic site of pressure sores in an embodiment of the present application;

[0090] Figure 17 Figure 13 is an immunohistochemical detection diagram of the expression of MFG-E8 and CD13 in the hypoxic site of pressure sores in an embodiment of the present application;

[0091] Figure 18 Figure 14 is a targeting tissue blood vessel detection diagram of the MFG-E8-silk fibroin nanoparticle crosslinked with NGR peptide in an embodiment of the present application;

[0092] Figure 19 Figure 15 is a skin ulcer repair and statistical analysis diagram of an animal in an embodiment of the present application;

[0093] Figure 20 This is a diagram showing the ulcer repair status of an animal detected by Masson staining in an embodiment of the present invention. Detailed Implementation

[0094] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0095] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. The invention will now be described in detail with reference to the embodiments.

[0096] Example:

[0097] like Figures 1-20 As shown, the preparation method of silk fibroin nanoparticles includes the following steps:

[0098] S1. Preparation of silk fibroin solution;

[0099] S2. Preparation of nanoparticles;

[0100] S3. Production and preparation of milk fat globule epidermal growth factor VIII (MFG-E8) in exosomes;

[0101] S4.MFG-E8 protein purification;

[0102] S5. Preparation of silk fibroin nanoparticles coated with MFG-E8;

[0103] S6. Preparation of MFG-E8-silk fibroin nanoparticles with cross-linked NGR peptides.

[0104] The specific preparation method of silk fibroin solution includes the following steps:

[0105] S1. Take 2g of silkworm cocoons with the pupae removed, put them in 1L of water, add 5-10g of Na2CO3 to make the Na2CO3 concentration 0.5%-1%, and then boil them in water at 90-100℃ for 30-60 minutes until the sericin is completely removed.

[0106] S2. Wash the sericin-free silk fibroin in deionized water 3-5 times, and then dry it in an oven at 60℃;

[0107] S3. Prepare 50 ml of ternary solution, preparation method: double distilled water, ethanol, calcium chloride are 30.43 ml: 19.57 ml: 24 g respectively;

[0108] S4. Add cocoon to the ternary solution, dissolve at 60±5℃ for 1-2h until the silk fibroin is completely dissolved into liquid; centrifuge at 4000 rpm / min (3220g) for 10 min, collect the supernatant;

[0109] S5. Dialyze the supernatant in a dialysis bag with a molecular weight cut-off of 8000-14000 for 48h, after dialysis, centrifuge at 4000 rpm / min (3220g) for 10 min, collect the supernatant, and determine the protein concentration.

[0110] The specific preparation method of the nanoparticles comprises the following steps:

[0111] S1. Dilute the obtained silk fibroin solution to 10 mg / ml (1%), add 5 ml of acetone solution in a 15 ml centrifuge tube, vortex the acetone on a vortex mixer, the volume ratio of acetone solution to 1% silk fibroin solution is 5:1, that is, 1 ml of 1% silk fibroin solution is added dropwise into acetone, after the dropwise addition is completed, continue to vortex for 30-60s;

[0112] S2. After vortexing, centrifuge at 6000 rpm / min (3840g) for 10 min, discard the supernatant and collect the precipitate;

[0113] S3. Add 10 ml of double distilled water for washing, centrifuge at 6000 rpm / min (3840g) for 10 min, discard the supernatant and collect the precipitate to remove the residual acetone;

[0114] S4. Repeat step S3;

[0115] S5. Add 5 ml of double distilled water to the precipitate, and perform ultrasonic treatment on ice, with the conditions being: ultrasonic treatment time 2 min, pause 2 s, ultrasonic treatment 2 s, amplitude 30%;

[0116] S6. After ultrasonic treatment, centrifuge at 6000 rpm / min for 10 min to obtain a milky white supernatant;

[0117] S7. Continue to centrifuge the milky white supernatant at 12000 rpm / min for 10 min to obtain a nanoparticle precipitate, which is freeze-dried to obtain the nanoparticles.

[0118] The specific production and preparation method of milk fat globule epidermal growth factor VIII (MFG-E8) in exosomes comprises the following steps:

[0119] S1. Extract rat vascular endothelial primary cells, and culture them to obtain exosomes secreted by the cells;

[0120] S2. Mass spectrometry analysis of exosomes showed that MFG-E8 was very rich in exosomes;

[0121] S3. Construct HBLV-r-MFG-E8-3xFlag-Green-PURO transfection lentivirus;

[0122] S4. Cultivate hamster ovary cells (CHO-s cells) in an incubator at 37°C and 5% CO2. Make the CHO-s cells adhere under static conditions, and then transfer the lentivirus after adhesion;

[0123] S5. After 3 days, add puromycin to select CHO-s cells with MFG-E8;

[0124] S6. After the green fluorescent CHO-s cells reach about 80%, perform trypsin digestion. After the cells are digested, centrifuge at 1000 rpm / min for 5 min, collect the cell precipitate, add a special culture medium for CHO-s cells, and culture on a shaker at 150 rpm / min to suspend the CHO-s cells;

[0125] S7. The cells grow in the culture medium. After the culture medium becomes turbid, centrifuge at 1200 rpm / min for 5 min to collect the CHO-s cell precipitate;

[0126] S8. Lyse the precipitated cells in a cell lysis solution containing protease inhibitor PMSF on ice for 30-60 min. The volume fraction of protease inhibitor PMSF is 1%;

[0127] S9. After lysis, perform ultrasonic treatment with the following conditions: ultrasonic time 8 s, pause 2 s, ultrasonic 2 s, and amplitude 20%;

[0128] S10. Centrifuge at 12000 rpm / min for 10 min at 4°C;

[0129] S11. Collect the supernatant containing MFG-E8 protein.

[0130] The specific method for purifying MFG-E8 protein includes the following steps:

[0131] S1. Purchase Flag-tag protein immunoprecipitation kit (magnetic bead method)

[0132] S2. According to the method, add 20 ul of anti-Flag magnetic beads to 500 ul of protein supernatant to purify MFG-E8 protein. Incubate at room temperature for 2 h on a shaker;

[0133] S3. Magnetic separation, separate on a magnetic stand for 20 s, and remove the supernatant;

[0134] S4. Elution, 100ul of 3xFlag polypeptide was added to each 20ul magnetic bead suspension for elution, incubated at room temperature for 30-60 minutes;

[0135] S5. Magnetic stand separation for 20s, supernatant was 3xFlag tagged protein MFG-E8 eluted;

[0136] S6. Western blotting detection was performed on the purified protein;

[0137] S7. Preparation and detection of MFG-E8 purified protein.

[0138] The specific preparation method of the MFG-E8 coated silk fibroin nanoparticle includes the following steps:

[0139] S1. The obtained silk fibroin solution was diluted to 20mg / ml (2%), and 500ul of purified MFG-E8 protein was added to 500ul of 2% silk fibroin solution, so that the silk fibroin concentration was 1%;

[0140] S2. 5ml of acetone solution was added to a 15ml centrifuge tube, and the acetone was vortexed on a vortex mixer. The volume ratio of acetone solution to silk fibroin solution containing MFG-E8 purified protein was 5:1, that is, 1ml of MFG-E8-silk fibroin solution of S1 was added dropwise to acetone, and after the dropwise addition was completed, vortexing was continued for 30-60s;

[0141] S3. After vortexing, centrifuge at 6000rpm / min (3840g) for 10min, discard the supernatant and collect the precipitate;

[0142] S4. Add 10ml of double distilled water for washing, centrifuge at 6000rpm / min (3840g) for 10min, discard the supernatant and collect the precipitate to remove the residual acetone;

[0143] S5. Repeat step S3;

[0144] S6. Add 5ml of double distilled water to the precipitate, and perform ultrasonic on ice, with the conditions of ultrasonic time 3min, pause 3s ultrasonic 2s, amplitude 30%;

[0145] S7. After ultrasonic, centrifuge at 3000rpm / min for 10min, and obtain the milky white supernatant;

[0146] S8. Continue to centrifuge the milky white supernatant at 12000rpm / min for 10min, obtain the nanoparticle precipitate, and freeze-dry to obtain the MFG-E8 coated silk fibroin nanoparticle;

[0147] S9. Detection of MFG-E8 coated silk fibroin nanoparticle.

[0148] The specific preparation method of the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle includes the following steps:

[0149] S1. Purchase NGR peptide, dicarboxylic acid polyethylene glycol (HOOC-PEG-COOH), and EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide), NHS (N-hydroxysuccinimide)

[0150] S2. Take the MFG-E8 coated silk fibroin nanoparticle (produced from 500 ul of 2% silk fibroin solution and 500 ul of purified MFG-E8 protein), add 1 ml of double distilled water to dissolve the nanoparticle;

[0151] S3. Add 10 mg of EDC, 50 mg of HOOC-PEG-COOH, and 50 mg of NGR peptide to the 1 ml nanoparticle solution, and react at room temperature on a shaker for 0.5-1 h;

[0152] S4. Add another 10 mg of NHS, and react at room temperature on a shaker overnight;

[0153] S5. The next day, centrifuge the nanoparticle solution at 6000 rpm / min for 10 min, discard the supernatant, and obtain the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle preparation precipitate the nanoparticle.

[0154] The cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle is detected.

[0155] The cell uptake experiment of the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle is performed.

[0156] The collagen / silk fibroin hydrogel preparation method includes the following steps:

[0157] S1. Take one rat, anesthetize with sodium pentobarbital, and then execute by cervical dislocation.

[0158] S2. Take off the tail, soak in 75% alcohol for 5 min, and then wash with PBS salt solution or normal saline for 2-3 times.

[0159] S3. Peel off the skin to expose the white collagen of the tail.

[0160] S4. Take off the tail collagen, cut it into 1-3 mm 3 size, and wash with PBS salt solution or normal saline once.

[0161] S5. Centrifuge at 4000 rpm / min (3220 g) for 5 min, and discard the supernatant.

[0162] S6. Add 100 ml of acetic acid solution with a volume fraction of 0.5%-1% to the precipitate.

[0163] S7. Dissolve for 48 h on a shaker at 4°C.

[0164] S8. After complete dissolution, centrifuge at 8000 rpm / min for 10 min at 4°C, and collect the supernatant.

[0165] S9. Salting-out of the supernatant in 10% NaCl solution for 10 min, and a large amount of flocculent collagen is observed in the 10% NaCl solution.

[0166] S10. Centrifuge the precipitated collagen at 8000 rpm / min for 10 min at 4°C, and collect the collagen precipitate.

[0167] S11. Add 50-100 ml of 0.1 mmol / L hydrochloric acid solution to dissolve the collagen precipitate.

[0168] S12. Freeze-dry the dissolved collagen solution, and store it at -80°C for a long time.

[0169] Method for preparing collagen hydrogel

[0170] Dissolve 100-150 mg of collagen powder in 10 ml of 0.1 mmol / L hydrochloric acid solution, add an appropriate amount of 1 mol / L NaOH to make the pH value of the collagen solution neutral, crosslink at 37°C, and form collagen hydrogel after 5-10 min, and detect the characteristics of the hydrogel.

[0171] Method for preparing silk fibroin hydrogel, comprising the following steps:

[0172] S1. Concentrate the extracted silk fibroin solution in 10%-15% polyvinyl alcohol 20000 solution for 48 h, so that the final concentration of the silk fibroin solution is 8%-10%.

[0173] S2. Dissolve 0.24 g of carbomer in 3 ml of double-distilled water.

[0174] S3. Dissolve 2 g of polyvinyl alcohol in 10 ml of double-distilled water to make the mass fraction 20%.

[0175] S4. Add 800 ul of 1 mol / L NaOH solution to the carbomer solution, stir uniformly, and then add 800 ul of 20% polyvinyl alcohol.

[0176] S5. Add 4 ml of 8%-10% silk fibroin solution, and stir uniformly.

[0177] S6. Adjust the pH value to 6-7.

[0178] S7. Place in a humid environment for 12-24h until it crosslinks into a gel.

[0179] S8. Test the hydrogel properties.

[0180] A collagen / silk fibroin hydrogel preparation method, comprising the following steps:

[0181] S1. Concentrate the extracted silk fibroin solution in a 10%-15% polyvinyl alcohol 20000 solution for 48h to make the final concentration of the silk fibroin solution 8%-10%.

[0182] S2. Take 0.24g of carbomer and dissolve it in 1ml of double distilled water.

[0183] S3. Take 2g of polyvinyl alcohol and dissolve it in 10ml of double distilled water to make the mass fraction 20%.

[0184] S4. Take 800ul of 1mol / L NaOH solution and add it to the carbomer solution, stir evenly, then add 800ul of 20% polyvinyl alcohol.

[0185] S5. Add 4ml of 8%-10% silk fibroin solution and stir evenly.

[0186] S6. Take 2ml of 1% collagen solution and mix 200ul of 1mol / L NaOH evenly, then add it to the carbomer solution.

[0187] S7. Adjust the pH value to 6-7.

[0188] S8. Place in a humid environment for 12-24h until it crosslinks into a gel.

[0189] S9. Test the collagen / silk fibroin hydrogel.

[0190] The application also proposes a collagen / silk fibroin hydrogel preparation method of MFG-E8-silk fibroin nanoparticle loaded with crosslinked NGR peptide, comprising the following steps:

[0191] S1. Concentrate the extracted silk fibroin solution in a 10%-15% polyvinyl alcohol 20000 solution for 48h to make the final concentration of the silk fibroin solution 8%-10%;

[0192] S2. Take 0.24g of carbomer and dissolve it in 1ml of double distilled water.

[0193] S3. Take 2g of polyvinyl alcohol and dissolve it in 10ml of double distilled water to make the mass fraction 20%.

[0194] S4. Take 800ul of 1mol / L NaOH solution into the carbomer solution, stir evenly, and then add 800ul of 20% polyvinyl alcohol;

[0195] S5. Take 4ml of 8%-10% silk fibroin solution, add it to the carbomer solution and stir evenly;

[0196] S6. Take 2ml of 1% collagen solution, mix 200ul of 1mol / L NaOH, and then add it to the carbomer solution;

[0197] S7. Take the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle solution in 200ul of double distilled water, and add it to the carbomer solution and stir evenly;

[0198] S8. Adjust the PH value to 6-7;

[0199] S9. Place it in a humid environment for 12-24h until it is cross-linked into glue;

[0200] S10. Detect the nanoparticles in the collagen / silk fibroin hydrogel.

[0201] Detection of MFG-E8 and CD13 expression in hypoxic parts of pressure ulcers.

[0202] Detection of cross-linked NGR peptide MFG-E8-silk fibroin nanoparticles targeting tissue blood vessels.

[0203] Animal experiments are carried out, and the repair of animal skin ulcers and Masson staining for detecting ulcer repair are analyzed.

[0204] Mechanism of action: Based on the key proteins carried in exosomes, the application develops a kind of nanoparticles targeting blood vessels, which can inhibit the iron death of vascular endothelial cells caused by hypoxia, and achieve the prevention and improvement of pressure ulcers. Specifically, we first extract endothelial cell-derived exosomes, and determine the important protein MFG-E8 carried by them through mass spectrometry analysis; then we use CHO-s cells to produce and purify MFG-E8 protein, and then prepare silk fibroin nanoparticles wrapped with MFG-E8. After the occurrence of pressure ulcers, the expression of CD13 on vascular endothelial cells increases significantly due to ischemia and hypoxia. Based on this phenomenon, we use NGR peptide which can specifically bind to CD13 on vascular endothelial cells to synthesize targeted nanoparticles. Further, by cross-linking the amino group (-NH2) of NGR peptide with the amino group (-NH2) of silk fibroin nanoparticles through HOOC-PEG-COOH, we successfully constructed the cross-linked targeted nanoparticles of NGR peptide and silk fibroin nanoparticles, which can accurately target ischemic and hypoxic blood vessels and effectively inhibit the occurrence of endothelial cell mitochondrial autophagy and iron death.

[0205] Collagen hydrogel can promote cell growth due to its high water content and large pore structure, while silk fibroin hydrogel is characterized by high strength, low water content, and various amino acids and polypeptides that promote cell proliferation. Based on the characteristics of collagen hydrogel and silk fibroin hydrogel, we mixed the two in a certain proportion to prepare silk fibroin / collagen hydrogel, thereby better retaining the advantages of the two hydrogels. After constructing an animal model, we mixed the above-mentioned targeted nanoparticles with the prepared silk fibroin / collagen hydrogel to construct a sustained-release carrier for releasing silk fibroin nanoparticles that inhibit mitochondrial autophagy and ferroptosis. The sustained-release carrier is applied to the surface of the skin to implement corresponding targeted therapy, and the therapeutic effect is analyzed and evaluated.

[0206] The specific embodiments are only used to illustrate the present application, and are not intended to limit it. Those skilled in the art can make non-creative modifications to the embodiments according to actual needs after reading the specification, and as long as the content falls within the scope of the claims of the present application, it is protected by the patent law.

Claims

1. A method for preparing a silk fibroin nanoparticle hydrogel, characterized by: The hydrogel is a collagen / silk fibroin hydrogel loaded with cross-linked NGR peptide, silk fibroin nanoparticles wrapped MFG-E8, and the preparation method of the hydrogel comprises the following steps: S1. Preparation of silk fibroin solution; S101. Take 2g of silkworm cocoons with removed silkworm pupae, put them into 1L water, add 5-10g Na2CO3 to make the concentration of Na2CO3 0.5%-1%, then cook in 90-100℃ water for 30-60min until the sericin is completely removed; S102. Wash the silk fibroin removed of sericin in deionized water for 3-5 times, and then dry in an oven at 60℃; S103. Prepare 50ml of ternary solution, the preparation method is as follows: double distilled water, ethanol, and calcium chloride are 30.43ml, 19.57ml, and 24g respectively; S104. Add the silkworm cocoons to the ternary solution, and dissolve at 60±5℃ for 1-2h until the silk fibroin is completely dissolved into liquid; centrifuge at 4000rpm / min for 10min, and collect the supernatant; S105. Dialyze the supernatant in a dialysis bag with a molecular weight cutoff of 8000-14000 for 48h, after dialysis, centrifuge at 4000rpm / min for 10min, collect the supernatant, and determine the protein concentration; S2. Preparation of nanoparticles; S201. Dilute the obtained silk fibroin solution to 10mg / ml, add 5ml of acetone solution in a 15ml centrifuge tube, and vortex the acetone on a vortex mixer, the volume ratio of acetone solution to 1%silk fibroin solution is 5:1, that is, 1ml of 1% silk fibroin solution is added dropwise into acetone, after the dropwise addition is completed, continue to vortex for 30-60s; S202. After vortexing, centrifuge at 6000rpm / min for 10min, pour off the supernatant and collect the precipitate; S203. Add 10ml of double distilled water for washing, centrifuge at 6000rpm / min for 10min, pour off the supernatant and collect the precipitate to remove the residual acetone; S204. Repeat step S203; S205. Add 5ml of double distilled water to the precipitate, and perform ultrasonic treatment on ice, the conditions are as follows: ultrasonic time 2min, pause 2s, ultrasonic 2s, amplitude 30%; S206. After ultrasonic treatment, centrifuge at 6000rpm / min for 10min, and obtain the milky white supernatant; S207. Continue to centrifuge the milky white supernatant at 12000rpm / min for 10min, obtain the nanoparticle precipitate, and freeze-dry to obtain the nanoparticles; S3. Production and preparation of milk fat globule epidermal growth factor VIII (MFG-E8) in exosomes; S301. Extract rat vascular endothelial primary cells, and culture them to obtain the exosomes secreted by the cells; S302. Perform mass spectrometry detection and analysis on the exosomes, and the results show that MFG-E8 is rich in the exosomes; S303. Construct HBLV-r-MFG-E8-3xFlag-Green-PURO transfection lentivirus; S304. Culture CHO-s cells in a 37℃, 5% CO2 incubator, make the CHO-s cells adhere under static conditions, and then transfer the lentivirus into the adherent cells; S305.3 days later, add puromycin to select CHO-s cells into which MFG-E8 is transferred; S306.After the CHO-s cells containing green fluorescence reach about 80%, perform trypsin digestion, centrifuge the digested cells at 1000 rpm / min for 5 min, collect the cell precipitate, and culture the cells in a special medium for CHO-s cells on a shaker at a speed of 150 rpm / min to suspend the culture of the CHO-s cells; S307.Centrifuge the cells at 1200 rpm / min for 5 min after the culture solution changes from clear to turbid, and collect the CHO-s cell precipitate; S308.Centrifuge the cells at 1200 rpm / min for 5 min after the culture solution changes from clear to turbid, and collect the CHO-s cell precipitate; S309.After lysis, perform ultrasonication under the following conditions: ultrasonication time 8 s, pause 2 s, ultrasonication 2 s, and amplitude 20%; S310.Centrifuge at 12000 rpm / min at 4°C for 10 min; S311.Collect the supernatant containing MFG-E8 protein; S4.Purification of MFG-E8 protein; S5.Preparation of MFG-E8-coated silk fibroin nanoparticle; S501.Dilute the obtained silk fibroin solution to 20 mg / ml, and add 500 ul of purified MFG-E8 protein to 500 ul of 2% silk fibroin solution to make the silk fibroin concentration 1%; S502.Add 5 ml of acetone solution to a 15 ml centrifuge tube, and vortex the acetone on a vortex mixer. The volume ratio of the acetone solution to the silk fibroin solution containing the purified MFG-E8 protein is 5:1, that is, 1 ml of MFG-E8-silk fibroin solution in S501 is added dropwise to the acetone, and after the dropwise addition is completed, vortex for 30-60 s; S503.After vortexing, centrifuge at 6000 rpm / min for 10 min, discard the supernatant, and collect the precipitate; S504.Add 10 ml of double-distilled water to wash, centrifuge at 6000 rpm / min for 10 min, discard the supernatant, collect the precipitate, and remove the residual acetone; S505.Repeat steps S503 and S504; S506.Add 5 ml of double-distilled water to the precipitate, and perform ultrasonication on ice under the following conditions: ultrasonication time 3 min, pause 3 s, ultrasonication 2 s, and amplitude 30%; S507.After ultrasonication, centrifuge at 3000 rpm / min for 10 min, and obtain the milky-white supernatant; S508.Continue to centrifuge the milky-white supernatant at 12000 rpm / min for 10 min to obtain the nanoparticle precipitate, freeze-dry the precipitate, and obtain the MFG-E8-coated silk fibroin nanoparticle; S6.Preparation of MFG-E8-silk fibroin nanoparticle cross-linked with NGR peptide; S601.Take the MFG-E8-coated silk fibroin nanoparticle, and add 1 ml of double-distilled water to dissolve the nanoparticle; S602. Add 10 mg EDC, 50 mg HOOC-PEG-COOH and 50 mg NGR peptide into 1 ml nanoparticle solution, and react for 0.5-1 h at room temperature on a shaker; S603. Add 10 mg of NHS again, and react overnight at room temperature on a shaker; S604. Centrifuge the nanoparticle solution at 6000 rpm / min for 10 min the next day, discard the supernatant, and obtain the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticle precipitate; S7. Preparation of collagen / silk fibroin hydrogel rich in cross-linked NGR peptide MFG-E8-silk fibroin nanoparticles; S701. Concentrate the extracted silk fibroin solution in 10%-15% polyvinyl alcohol 20000 solution for 48 h, so that the final concentration of the silk fibroin solution is 8%-10%; S702. Take carbomer 0.24 g, dissolve in 1 ml double distilled water to prepare solution A; S703. Take polyvinyl alcohol 2 g, dissolve in 10 ml double distilled water, and the mass fraction is 20%; S704. Take 800 ul of 1 mol / L NaOH solution, add to solution A, stir uniformly, then add 800 ul of 20% polyvinyl alcohol, and prepare solution B; S705. Take 4 ml of 8%-10% silk fibroin solution, add to solution B, stir uniformly, and prepare solution C; S706. Take 2 ml of 1% collagen solution, add 200 ul of 1 mol / L NaOH solution, mix uniformly, and then add to solution C to prepare solution D; S707. Take the cross-linked NGR peptide MFG-E8-silk fibroin nanoparticles, dissolve in 200 ul of double distilled water, and add to solution D to stir uniformly to prepare solution E; S708. Adjust the pH value of solution E to 6-7; S709. Place in a humid environment for 12-24 h until it is cross-linked into a glue; S710. Detect the nanoparticles in the collagen / silk fibroin hydrogel.

2. The method of claim 1, wherein: The specific method for purifying the MFG-E8 protein comprises the following steps: S1. According to 500 ul protein supernatant, add 20 ul anti-Flag magnetic bead suspension to purify the MFG-E8 protein by magnetic bead method, and incubate at room temperature on a shaker for 2 h; S2. Magnetic separation, separate on a magnetic stand for 20 s, and remove the supernatant; S3. Elution, add 100 ul of 3xFlag polypeptide to each 20 ul magnetic bead suspension for elution, and incubate at room temperature for 30-60 min; S4. Magnetic stand separation for 20 s, and the supernatant is the eluted 3xFlag-tagged protein MFG-E8; S5. Western blotting detection of the purified protein; S6. Preparation and detection of MFG-E8 purified protein.

3. A collagen / silk fibroin hydrogel rich in cross-linked NGR peptide MFG-E8-silk fibroin nanoparticles prepared based on the method of claim 1 or 2.

4. Use of a collagen / silk fibroin hydrogel enriched in cross-linked NGR peptide- MFG-E8-silk fibroin nanoparticles as claimed in claim 3 for the preparation of a product for the treatment of pressure sore ulcers.

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