Application of ESC-Exos loaded with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores

Abstract

The present invention relates to the technical field of bedsores, and in particular to the application of ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores. This application is to introduce Ca-I-Ti cross-linked epidermal growth factor by electroporation of embryonic stem cell exosomes, and administer the drug through hydrogel microneedles to treat wound recovery and bacterial infection of bedsores. The application of ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores provided by the present invention is to bring ESC-Exos introduced with Ca-I-Ti and cross-linked epidermal growth factor to the deep part of necrotic tissue through nano-microneedles, and perform antibacterial treatment of bedsores through the bactericidal effect achieved by Ca-I-Ti to avoid antibiotic resistance, and then enter the necrotic tissue cells through ESC-Exos for intracellular repair, and promote vascular growth and tissue repair of necrotic tissue through epidermal growth factor, and finally perform long-term slow release through gel permeation administration to complete local repair.

Description

Technical Field

[0001] The present invention relates to the technical field of bedsores, and in particular to the application of ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores. Background Art

[0002] Bedsores, also known as pressure injuries, have become a major public health issue worldwide, with an incidence rate of 15% to 30% among hospitalized patients. This is especially true for long-term bedridden patients, the elderly, and individuals who have suffered severe trauma or stroke. These people suffer from ischemia and hypoxia of the skin and soft tissues due to long-term pressure, which in turn causes bedsores. The severity of bedsores not only affects the patient's quality of life, but also increases infection, hospitalization time, and medical expenses, placing a heavy burden on patients and their families.

[0003] Internationally, bedsores are listed as one of the five most common problems that cause serious harm to patients, and are also one of the most expensive complications. It has three major characteristics: high incidence, difficult treatment, and easy recurrence. Once complications occur in bedsore infection, the mortality rate can be as high as 50% even under hospitalization conditions. It is also one of the main causes of death in paraplegic patients. The bedsore-related mortality rate in elderly patients reaches 23% to 37%. After bedsores occur, the mortality rate of the elderly will increase by 4 times. If they are not cured, the mortality rate will increase by 6 times. Although there are many traditional treatment methods, they are often not ideal, and even due to the abuse of antibiotics during treatment, multidrug-resistant "super bacteria" are caused, which aggravates the condition. In order to improve the treatment effect, we need to find a more comprehensive and personalized treatment method to meet the needs of patients.

[0004] Traditional methods of treating bedsores mainly include pressure relief, debridement, antibacterial dressing, antibiotics and surgical treatment, but there are also many problems, as follows:

[0005] First, daily cleaning causes great pain to patients. The treatment process requires cleaning bedsores, removing dead skin, and bandaging. Each dressing change requires re-cleaning the wound. Medical staff usually rinse the wound with saline, and use a scalpel, chemical solution or medical maggots to remove dead tissue. Removing necrotic tissue is usually painless. Although necrotic tissue does not cause pain, the surrounding healthy tissue will produce severe pain, which is a huge pain for patients.

[0006] Secondly, the extensive use of antibiotics can cause bacterial resistance. The loss of the skin barrier function of bedsores and the exposure of the wound surface are very likely to cause infection, which can easily lead to the abuse of antibiotics. For superficial infections, antibiotics are applied to the skin of the affected area for treatment. If the infection has spread to deeper tissues, bones, or reaches the entire body through the blood, the doctor will also let the patient take orally or inject a large amount of antibiotics. Some more serious complications, such as osteomyelitis and nervous system infections, require long-term intravenous administration of large amounts of antibiotics. This greatly increases the incidence of bacterial resistance and may have serious consequences.

[0007] Finally, surgery has significant limitations. Deep bedsores are difficult to treat. Sometimes they need to be closed with skin grafts and flaps with skin and sometimes muscle. In these procedures, thicker healthy tissue with a good blood supply is surgically re-transplanted to the damaged area. Skin grafts are effective for larger, shallow bedsores. Flaps with skin and muscle are used to close bedsores over large bony areas, usually at the base of the spine, the hips, and the upper end of the femur. For some people, surgery can improve quality of life. But not everyone is a good candidate for this surgery, especially frail, malnourished older people, who are unfortunately the main group of people who get bedsores.

[0008] In recent years, stem cell exosomes have been widely used in various aspects such as cell repair and wound recovery. For example, MINasser et al. found that exosomes derived from mesenchymal stem cells and their microRNA play a good role in cardiac repair and regeneration. [Nasser MI,Masood M,Adlat S,Gang D,Zhu S,Li G,Li N,Chen J,Zhu P.Mesenchymal stem cell-derived exosome microRNA as therapy for cardiac ischemic injury.Biomed Pharmacother.2021Nov ; 143:112118] It has a wide range of effects, including promoting myocardial cell regeneration, promoting angiogenesis, regulating inflammation, anti-apoptosis, etc. It exhibits very excellent properties in the body's endogenous repair.

[0009] Cheng et al. found that exosomes derived from embryonic stem cells can "rejuvenate" endothelial cells through related pathways, use endogenous mechanisms to resist oxidation, promote angiogenesis at the wound site, and improve cell damage caused by ischemia-reperfusion and nutritional deficiencies caused by vascular damage. [Chen B,Sun Y,Zhang J,Zhu Q,Yang Y,Niu X,Deng Z,Li Q,Wang Y.Human embryonic stem cell-derived exosomes promote pr essure ulcer healing in aged mice by rejuvenating senescent endothelial cells.Stem Cell Res Ther.2019May 21 ; 10(1):142] .

[0010] The main cause of bedsores is reperfusion injury caused by long-term pressure. Free radicals damage cells and then blood vessels, leading to malnutrition and tissue necrosis, which gradually develop into ulcers. Exosomes have excellent angiogenesis and intracellular repair effects, and are expected to become an ideal drug for bedsore treatment.

[0011] To this end, the application of ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores was designed to provide another technical solution to the above technical problems. Summary of the invention

[0012] Based on this, it is necessary to provide the application of ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores in order to solve the technical problems raised in the above-mentioned background technology.

[0013] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0014] Application of ESC-Exos in the treatment of bedsores.

[0015] According to the use of claim 1, the application method is to introduce Ca-I-Ti into the ESC-Exos and cross-link EGF.

[0016] A nano-microneedle for treating bedsores is prepared from ESC-Exos cross-linked with EGF and introduced with Ca-I-Ti.

[0017] The preparation method of nano-microneedles for treating bedsores comprises the following steps:

[0018] 1) injecting a mixed solution a into a customized polydimethylsiloxane microneedle array mold, wherein the mixed solution a is a sustained-release outer layer needle polymer solution;

[0019] 2) centrifuging the microneedle array mold obtained in step 1) at 5000 rpm for 10 minutes, taking it out after it is completely filled, and scraping off the excess mixed solution a;

[0020] 3) placing the microneedle array mold obtained in step 2) in a silica gel desiccator and drying at room temperature for 2 h;

[0021] 4) taking out the microneedle array mold obtained in step 3), and injecting a mixed solution b, wherein the mixed solution b is a fast-dissolving inner layer needle body polymer solution;

[0022] 5) centrifuging the microneedle array mold obtained in step 4) at 5000 rpm for 10 minutes, taking it out after it is completely filled, and scraping off the excess mixed solution b;

[0023] 6) placing the microneedle array mold obtained in step 5) in a silica gel desiccator and drying at room temperature for 2 h;

[0024] 7) injecting the mixed solution c into the microneedle array mold obtained in step 6), centrifuging at 4000 rpm for 5 minutes, and taking it out after forming a backing, wherein the mixed solution c is a backing polymer solution;

[0025] 8) placing the microneedle array mold obtained in step 7) in a silica gel desiccator and drying at room temperature for 2 h;

[0026] 9) The hydrogel microneedles are prepared through steps 1) to 8).

[0027] As a preferred embodiment of the method for preparing the nano-microneedle for treating bedsores provided by the present invention, the method for preparing the mixed solution a is as follows:

[0028] The cellulose material, high molecular weight fibrin, protective agent and drug are placed in a centrifuge according to a ratio of 0.2-:1-10:5-20:0.1-50, centrifuged at v1000-10000rpm, the centrifugation time is 1-30min; the drying time is 1-12h.

[0029] As a preferred embodiment of the method for preparing the nano-microneedle for treating bedsores provided by the present invention, the method for preparing the mixed solution b is as follows:

[0030] The water-soluble high molecular polymer and the drug are placed in a centrifuge according to a ratio of 10-100:5-50, the centrifugal speed is 1000-10000rpm, the centrifugal time is 1-30min; the drying time is 1-12h.

[0031] As a preferred embodiment of the method for preparing the nano-microneedle for treating bedsores provided by the present invention, the mixed solution c is a backing polymer solution, and its preparation method is as follows:

[0032] PVA and gelatin were mixed in a ratio of 6:1, frozen at -40°C to -10°C for one day, thawed at room temperature for 1-2 hours, and repeated 3-5 times. Hyaluronic acid was added to obtain a backing polymer solution c.

[0033] As a preferred embodiment of the method for preparing the nano-microneedles for treating bedsores provided by the present invention, the drug is ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF.

[0034] As a preferred embodiment of the method for preparing the nano-microneedle for treating bedsores provided by the present invention, the drug is ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF, and the steps are as follows:

[0035] A. Exosomes were resuspended in electroporation buffer and electroporated using a 0.4 cm cuvette with aluminum electrodes on a GenePulser II electroporation system with a capacitive extender.

[0036] B allows the ions in the suspension to diffuse into the exosomes through the pores to obtain ESC-Exos introduced with Ca-I-Ti;

[0037] C. The ESC-Exos obtained in step B and EGF are mixed in a buffer solution, and a cross-linking agent is added. The ESC-Exos physically cross-linked with EGF are obtained by high-speed centrifugation.

[0038] It can be seen without a doubt that the above-mentioned technical solution of the present application can definitely solve the technical problem to be solved by the present application.

[0039] At the same time, through the above technical solutions, the present invention has at least the following beneficial effects:

[0040] 1. The application of ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores provided by the present invention is to bring the ESC-Exos introduced with Ca-I-Ti and the cross-linked epidermal growth factor (EGF) to the deep part of the necrotic tissue through nano-microneedles, and the bactericidal effect achieved by Ca-I-Ti is used to fight against bedsores and avoid antibiotic resistance. Then, embryonic stem cell exosomes (ESC-Exos) enter the necrotic tissue cells for intracellular repair, and epidermal growth factor is used to promote vascular growth and tissue repair of necrotic tissue. Finally, gel permeation administration is used for long-term slow release to complete local repair.

[0041] 2. The present invention uses a gel microneedle patch to administer drugs, which can reduce the pain of debridement for patients, and treat wound recovery and bacterial infection of bedsores. At the same time, the antibacterial effect of Ca-I-Ti metal ions can avoid antibiotic resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0043] Figure 1 The pathogenesis of bedsores in the prior art;

[0044] Figure 2 A schematic diagram of the dilemma of conventional treatment of bedsores in the prior art;

[0045] Figure 3 A schematic diagram of the functions of stem cell exosomes in the prior art;

[0046] Figure 4 Schematic diagram of the existing ESC-Exos that enhanced angiogenesis and improved endothelial cell senescence at wound sites in aged mice;

[0047] Figure 5 A schematic diagram of the microneedle preparation process in the prior art;

[0048] Figure 6 This is a schematic diagram of the regulatory mechanism of ESC regulating Nrf2 of the present invention;

[0049] Figure 7 This is a schematic diagram of the EGF of the present invention promoting rapid healing of wound surface;

[0050] Figure 8 This is a schematic diagram of an electron microscope photograph of the EGF nanospheres of the present invention;

[0051] Fig. 9 Schematic diagram of the mechanism of action of antibacterial ions of the present invention;

[0052] Fig.10 Schematic diagram of electron microscope photograph of microneedle taken for preliminary experiment of the present invention;

[0053] Fig.11 This is a schematic diagram of the working principle of the hydrogel microneedle of the present invention;

[0054] Fig.12 This is a schematic diagram of the determination of characteristic molecules on the surface of embryonic stem cell exosomes of the present invention;

[0055] Fig.13 This is a schematic diagram of an electron microscope photograph of the exosomes of the present invention;

[0056] Fig.14 It is a schematic diagram of the preliminary experiment control of the present invention;

[0057] Fig.15 This is a schematic diagram of a microneedle patch using 10% hyaluronic acid as a matrix in a preliminary experiment of the present invention;

[0058] Fig.16 The figure is a schematic diagram of the preparation of the bedsore rat model of the present invention. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0060] In order to enable those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0061] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features and technical solutions in the embodiments may be combined with each other.

[0062] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0063] Reference Figure 6-Figure 16 , Application of ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF in the treatment of bedsores.

[0064] 1. Data retrieval

[0065] 1.1. ESC-Exos (embryonic stem cell exosomes) enhance angiogenesis and improve endothelial cell senescence at the wound site in mice

[0066] An important cause of bedsores is tissue necrosis caused by ischemia-reperfusion, and the corresponding tissue ischemia cannot be relieved. The blood vessel density at the wound is low, and angiogenesis is considered to be a key factor in wound recovery. In the ESC-Exos (embryonic stem cell exosomes) study of the wound recovery mechanism, the blood vessel density of elderly mice using exosomes was significantly higher than that of the control group, and even comparable to that of young mice. This is undoubtedly very good news, which is very suitable for the causes of bedsores and the population of patients (most of whom are elderly people).

[0067] 1.1.1. ESC-Exos rejuvenate senescent endothelial cells by activating Nrf2

[0068] Increased oxidative stress with age is an important feature of endothelial cell senescence. D-gal treatment caused an increase in reactive oxygen species (ROS) levels in HUVECs, while incubation with ESC-Exos almost eliminated this effect. In addition, the levels of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured, and the results showed that D-gal treatment significantly increased MDA levels and reduced the activities of these antioxidant-related molecules. In contrast, ESC-Exos treatment significantly reversed these changes. The results indicate that ESC-Exos are able to reduce oxidative stress by enhancing the activity of the endogenous antioxidant system.

[0069] 1.1.2. ESC-Exos downregulate Keap1 expression by transferring miR-200a, thereby activating Nrf2 in senescent endothelial cells

[0070] Keap1 is a negative regulator of Nrf2, and its expression is upregulated with age. Chronic D-gal treatment led to increased Keap1 expression in HUVECs, while ESC-Exos treatment reduced Keap1 expression in aged HUVECs, indicating that ESC-Exos may activate Nrf2 by inhibiting Keap1. In addition, miRNAs in exosomes can be transferred to recipient cells and regulate their functions by regulating gene expression. miR-200a was highly enriched in ESC-Exos, and after ESC-Exos incubation, the level of miR-200a increased in aged HUVECs, consistent with the downregulation of Keap1. Dual luciferase reporter experiments further verified that miR-200a can target the 3′-UTR of Keap1 mRNA. These results indicate that miR-200a is able to regulate the expression of Keap1 by transferring into HUVECs.

[0071] In summary, embryonic stem cell exosomes can repair damaged cells in an endogenous manner. ESC-Exos treatment effectively rejuvenates endothelial cell senescence and promotes anti-oxidation and angiogenesis, thereby enhancing wound healing.

[0072] 1.2. Surface growth factors

[0073] If the wound can be quickly healed and exposed, subsequent infection can be effectively avoided. Combined with the internal repair function of stem cell exosomes, it can have a significant effect on the overall recovery of the wound. Epidermal growth factor (EGF) is a growth factor that can accelerate wound healing. It plays a key role in wound healing by regulating cell survival, proliferation, migration and differentiation. In rat models, EGF has also been shown to protect fibroblasts from oxidative stress by clearing toxic oxidative products that have already formed during the acute wound healing phase. At the same time, the various excellent mechanisms of EGF are summarized as follows.

[0074] (1) Increased fibroblast proliferation and enhanced procollagen mRNA expression;

[0075] (2) Activate myofibroblast proliferation and collagen deposition to increase wound contraction;

[0076] (3) EGF regulates the production of free radicals by keratinocytes, stimulating cell proliferation and inflammation resolution after wounding;

[0077] (4) Accelerate gene transcription to enhance the expression of antioxidant defense enzymes, thereby protecting cells from the effects of superoxide radicals.

[0078] EGF has the function of promoting cell proliferation, migration and differentiation, and is effective in promoting rapid wound recovery. It also has certain effects on the inflammatory recovery of bedsores and anti-oxidation.

[0079] 1.3. Application of Ca-I-Ti antimicrobial ions to improve antibiotic resistance

[0080] While promoting overall wound healing, effective antibacterial properties cannot be ignored. It is estimated that the abuse of antibiotics will cause the continued emergence of drug resistance, and antibiotic-resistant infections will cause more than 10 million deaths each year. Therefore, there is an urgent need to find new materials to replace antimicrobial agents to alleviate or solve the imminent bacterial resistance crisis. Bedsores are often resistant to antibiotics in clinical practice, so new antibacterial materials are needed. Although there have been related studies on antibacterial ions such as Ag and Cu in clinical practice, their long-term biological toxicity cannot be ignored. Norimasa Ikeda et al. found that [keda N,Fujibayashi S,Yamaguchi S,Goto K,Otsuki B,Kawai T,Shimizu T,Okuzu Y,Masamoto K,Shimizu Y,Takaoka Y,Matsud a S.Bioactivity and antibacterial activity of iodine -containing calcium titanate against implant-associated infection.Biomater Adv.2022Jul ; 138:212952] The invented Ca-I-Ti nanomaterials. The antibacterial advantages of Ca-l-Ti ions over traditional antibacterial ions such as Cu, Ag, and pure Ti are as follows:

[0081] (1) Iodine-containing calcium titanate 5 (Ca-l-Ti) showed no obvious cytotoxicity.

[0082] (2) In in vivo biomechanical tests, Ca-I-Ti exhibited excellent tissue compatibility.

[0083] (3) Compared with pure titanium, Staphylococcus aureus was reduced by 95% on Ca-I-Ti in a rat model.

[0084] Antimicrobial ions are selectively introduced into exosomes using electroporation, and arrive at damaged tissues with exosomes, improving the targeting of drugs. Exosomes have a lipid bilayer membrane structure that can stably encapsulate and deliver antimicrobial ions to target cells or tissues. Through the natural fusion process of exosomes, antimicrobial ions can efficiently enter the interior of cells and exert antimicrobial effects.

[0085] 1.4. Hydrogel microneedle drug delivery system

[0086] The formation of biofilm in patients with bedsores is another major obstacle that affects the bactericidal efficiency of traditional antibiotics. Traditional drug delivery methods are difficult to penetrate, and drugs can only be used after cutting and debridement, which also causes great pain to patients. The microneedle transdermal drug delivery system can penetrate the barrier without affecting the pain receptors, improving drug delivery efficiency and patient compliance. At the same time, in the treatment of bedsores, it can penetrate the biofilm well to reach the deep tissue that needs treatment, achieve targeted treatment, and relatively closed wounds can reduce the risk of subsequent infection.

[0087] 2. Construction Technology

[0088] The epidermal growth factor was cross-linked with embryonic stem cell exosomes (introduced with Ca-I-Ti by electroporation) and administered via hydrogel microneedles to treat wound recovery of bedsores and bacterial infections in rats to determine its therapeutic effect.

[0089] 2.1. Tool selection

[0090] Microneedles, nano-microspheres and stem cell exosomes, which are currently the hot spots in medical research, are selected as the main tools and research objects. Ca-I-Ti embryonic stem cell exosomes combined with epidermal growth factor nano-microneedles are made. It has high targeting and effectiveness and can be accurately delivered to damaged tissues. The internal repair effect of exosomes and the repair effect of epidermal growth factor can well restore tissue activity, rebuild a good environment, resist oxidation, and restore vascular density. At the same time, electroporation is used to implant Ca-I-Ti into exosomes to reach the deep surface of the wound and kill some bacteria that are difficult to reach by common routes.

[0091] 2.2. Construction method

[0092] 2.2.1 Embryonic stem cell exosomes combined with epidermal growth factor nanospheres as therapeutic drugs

[0093] As vesicle-like substances secreted by stem cells, embryonic stem cell exosomes can fuse into cells in necrotic tissues and produce internal repair effects on damaged cells. Various active substances help rebuild blood vessels and restore wound surfaces, avoiding the problem that traditional treatments cannot improve the ischemic environment. Exosomes also have a good effect in reducing ROS and promoting angiogenesis.

[0094] At the same time, epidermal growth factor is effective in promoting rapid wound healing. The two are combined to treat bedsores, treating damaged tissues internally to promote reconstruction, and quickly healing nearby wounds to avoid infection. The preliminary experimental results are good.

[0095] 2.2.2 Electroporation technology to introduce Ca-l-Ti ions into exosomes and administer drugs with microneedles

[0096] Antibacterial measures for bedsore wounds are of utmost importance. The new antibacterial ion Ca-l-Ti is introduced into exosomes by electroporation. The new antibacterial ion has no biological toxicity and good bactericidal effect. At the same time, the microneedle drug delivery system is used, and the antibacterial ion can reach the deep tissue with the exosomes for more thorough antibacterial and treatment. It avoids the problem of large-scale abuse of antibiotics and difficulty in treating deep tissue infections in traditional solutions. The microneedle drug delivery system can penetrate the biofilm well to reach the deep tissue that needs treatment, achieving targeted treatment. Relatively closed wounds can reduce the risk of subsequent infection.

[0097] 2.2.3 Double-layer microneedles achieve burst release followed by sustained release

[0098] In order to reduce the pain of patients having to change dressings repeatedly, a double-layer microneedle with sustained release (publication number CN202111018682.5) was referenced, which can keep the wound site in a therapeutic concentration for a long time.

[0099] 3. Preparation of Ca-I-Ti

[0100] 3.1 Preparation of Ca-I-Ti solid

[0101] Preparation material: Commercially pure titanium [ISO5832-2] (purity greater than 99.5%)

[0102] NaOH solution (5mol / L)CaCl2 (0.5mol / L)ICl3 (0.01mol / L)

[0103] 1) Sterilization, cleaning and drying of prepared materials

[0104] Sterilization: Ti was sterilized in ethylene oxide gas (800mg / L-1000mg / L), temperature 55-60℃, relative humidity 60-80% for 6 hours.

[0105] Cleaning and drying: The sterilized Ti was cleaned in an ultrasonic cleaner with acetone, 2-ethanol, and pure water for 30 seconds and then dried.

[0106] 2) Cutting of Ti

[0107] Cut Ti into small pieces (or buy small pieces of Ti directly)

[0108] 3) Heat treatment of Ti

[0109] Soak Ti in 5 mol / L NaOH solution at 60°C for 24 hours

[0110] Then, Ti was immersed in a 0.1 mol / L CaCl2 solution at 40°C for 24 h and kept shaking.

[0111] Finally, Ti was heated to 600℃ and maintained for 1h, and then stored in hot water.

[0112] 4) Introduction of I

[0113] The Ti treated in 3) was placed in a 0.01 mol / L ICl3 solution at 80°C for 24 hours to obtain a Ca-I-Ti sample.

[0114] 3.2 Crushing of Ca-I-Ti materials

[0115] The obtained sample is placed in a high-speed ball mill (or other crushing machine) and crushed to 0.1 micron level to prepare a suspension.

[0116] 3.3 Exosome conductivity method to import TI-I-CA ions

[0117] The technology creates small pores in the exosome membrane by applying an electric field to exosomes suspended in a conductive solution. The current disrupts the phospholipid bilayer of the exosome, resulting in the formation of temporary pores. Ions in the suspension can diffuse into the interior of the exosomes through the pores. The integrity of the exosome membrane is then restored after the drug loading process.

[0118] Exosomes were resuspended in electroporation buffer and electroporated using a 0.4 cm cuvette with aluminum electrodes on a GenePulser II electroporation system with a capacitive extender (Bio-Rad, Hercules, CA, USA). The volume of the electroporated sample ranged from 200 to 450 μL (containing up to 100 μg of total exosome protein) and was set to 400 V and 125 μF.

[0119] 3.4 Cross-linking of EGF and exosomes

[0120] Cross-linking step Isolation and purification of exosomes Exosomes are extracted from cell culture supernatants, usually using ultracentrifugation or commercial extraction kits.

[0121] The preparation of EGF may use EGF of higher purity, which may be recombinant EGF.

[0122] The prepared EGF and exosomes were mixed in an appropriate buffer and a crosslinking agent was added. A high-speed centrifugation method was used to promote the formation of hydrogen bonds and hydrophobic bonds to form physical crosslinks (EGF-hydrogel-exosomes (containing Ca-I-Ti)).

[0123] 4. Microneedle patch preparation and treatment methods

[0124] 4.1 Preparation of related microneedle patches

[0125] 4.1.1 Solution preparation

[0126] Sustained-release outer needle polymer solution a: The mass ratio of cellulose material, high molecular weight fibrin, protective agent and drug is (0.2-2): (1-10): (5-20): (0.1-50) centrifugation v1000~10000rpm, centrifugation time is 1~30min; drying time is 1~12h

[0127] Preferably, the cellulose material is carboxymethyl cellulose; the polymer fibrous protein is silk fibroin; the protective agent is trehalose; and the drugs are Ca-I-Ti exosomes and epidermal growth factor.

[0128] Fast-dissolving inner layer needle body polymer solution b: water-soluble high molecular polymer / drug mass ratio (10-100): (5-50) centrifugal speed is 1000-10000rpm, centrifugal time is 1-30min; drying time is 1-12h.

[0129] Preferably, the water-soluble high molecular polymer is hyaluronic acid, and the drug is Ca-I-Ti exosomes and epidermal growth factor.

[0130] The mass fraction of the backing polymer solution c is 10-80%; the centrifugal speed is 1000-5000 rpm, the centrifugal time is 1-10 minutes; the drying time is 24-72 hours. Solution configuration: PVA / gelatin (mass ratio is 6:1), frozen at -40°C to -10°C for one day, thawed at room temperature for 1-2 hours, repeated 3-5 times, hyaluronic acid is added to obtain the backing polymer solution c.

[0131] 4.1.2 Microneedle fabrication

[0132] First, the mixed solution a was injected into the customized polydimethylsiloxane (PDMS, Sylgard184) microneedle array mold, and the PDMS microneedle array mold was centrifuged at 5000rpm for 10 minutes. After it was completely filled, it was taken out and the excess premixed solution was scraped off. The PDMS microneedle array mold was placed in a silica gel dryer and dried at room temperature for 2 hours. The PDMS microneedle array mold was taken out and injected with the mixed solution b. Then, the PDMS microneedle array mold was centrifuged at 5000rpm for 10 minutes. After it was completely filled, it was taken out and the excess premixed solution was scraped off. The PDMS microneedle array mold was placed in a silica gel dryer and dried at room temperature for 2 hours. Finally, the mixed solution c was injected into the PDMS microneedle array mold and centrifuged at 4000rpm for 5 minutes to form a backing and then taken out. The PDMS microneedle array mold was placed in a silica gel dryer and dried at room temperature for 24 hours.

[0133] 4.2 Construction of bedsore model rats

[0134] After 72 healthy SD rats were adaptively raised for 1 week, they were fasted and deprived of water for 8 hours before the experiment. After the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3mL / 100g), the buttocks on both sides of the midline of the back were depilated with a depilatory agent (8gNas+20mL ethanol+80mL water). The skin preparation area was 4cm×4cm. The surgical area was disinfected with 5% povidone iodine. A 2cm incision was made near the head end of the left buttocks of the rats. The fascia and tissue were bluntly separated with forceps, and a sterile iron sheet was placed under the muscle. The incision was sutured with 4-0 Dexon polyglycolic acid thread, and then sutured with 5% povidone iodine to prevent infection. After the operation, each rat was intramuscularly injected with 0.2mL of penicillin for three days. On the 4th day after the operation, a round magnet was placed outside the rat to exert pressure on the muscle and skin. After each pressure application for 2 hours, the magnet was removed and blood perfusion was performed for 0.5 hours. This was one cycle. The rats were removed after 5 consecutive cycles every day. Repeat the cycle early the next morning.

[0135] Construction of rats with stage I bedsores: 1 cycle. If observed with the naked eye: the skin is intact, the pressure area appears red and does not fade; compared with the surrounding tissue, the area may be painful or the skin temperature is elevated. Pathological observation: local surface squamous epithelium is lost, the residual squamous epithelium is thinned, the epidermis has local blister ulcers, the epidermis has local focal neutrophil aggregation, the dermis has a large number of lymphocytes infiltrated, and the surface gluteus maximus striations are blurred and shallow. The model is successfully constructed.

[0136] Construction of rats with stage II bedsores: After 5 cycles, if observed with the naked eye: the skin color of the pressure part becomes dark red, the surrounding area is red and swollen, there is no bleeding or pus, and there is obvious pain when pressing the local skin; pathological observation: most of the epidermis is lost, multifocal ulcers are formed, deep into the dermis, the dermis has a moderate amount of lymphocyte infiltration, the dermis collagen fibers are degenerated, there are more inflammatory cells infiltration, more inflammatory cells infiltration can be seen in the muscle fibers, the muscle fibers are obviously edematous, the interstitium is widened, some muscle fibers are broken, some striated muscles disappear, the muscle fibers are atrophied, and the sarcoplasm is unevenly stained.

[0137] The model is built successfully

[0138] Construction of rats with stage III bedsores: After 20 cycles, if the skin at the pressure point becomes darker and harder, there is no bleeding and no pain when punctured, and the fat is exposed after the black skin on the surface is cleaned, but muscles, tendons or bones are not visible. Pathological observation: the surface squamous epithelium is lost, the dermis is thinned, the dermis is infiltrated with a large number of lymphocytes and granulocytes, the local ulcers are deep into the muscle layer, some gluteus maximus muscles are atrophied and fibrotic, and the deep muscles are infiltrated with a large number of lymphocytes and monocytes. The model is successfully constructed.

[0139] Construction of rats with stage IV bedsores: After 30 cycles, if the following observations are made by naked eye: the edge of the iron sheet is red, swollen, eroded, with exudate or bleeding, which may be accompanied by necrosis, the skin is painless when pierced with a needle, the iron sheet is exposed or slips off, the skin becomes hard or turns into a black eschar, and bones, tendons or muscles are exposed. Pathological observations: the epidermis, dermis and gluteus maximus disappear, the deep muscle has multifocal inflammatory necrosis, accompanied by suppurative inflammation, some interstitial edema and bleeding, and large areas of fibrosis. The model is successfully constructed.

[0140] 4.3 Grouping and treatment of bedsore model rats

[0141] Group A consisted of SD rats treated with primary bedsores, with wounds treated with saline dressings;

[0142] Group B consisted of SD rats with first-stage bedsores, which underwent traditional surgical debridement;

[0143] Group C consisted of SD rats treated with a first-stage bedsore and were treated with a hydrogel microneedle patch;

[0144] Group D consisted of SD rats treated with stage II bedsores, with wounds treated with saline dressings;

[0145] Group E consisted of SD rats treated with stage II bedsores, which underwent conventional surgical debridement;

[0146] Group F consisted of SD rats treated with stage II bedsores and were treated with hydrogel microneedle patches;

[0147] Group G consisted of SD rats treated with stage III bedsores and the wounds were treated with saline dressings; Group H consisted of SD rats treated with stage III bedsores;

[0148] Group I consisted of SD rats treated with stage III bedsores and underwent conventional surgical debridement;

[0149] Group J consisted of SD rats treated with stage III bedsores and treated with hydrogel microneedle patches;

[0150] Group K consisted of SD rats treated with stage IV bedsores, with wounds treated with saline dressings;

[0151] Group L consisted of SD rats treated with stage IV bedsores and underwent conventional surgical debridement;

[0152] Group M consisted of SD rats with stage IV bedsores treated with hydrogel microneedle patches.

[0153] All wounds were treated for staphylococcal infection.

[0154] 4.4 Observation of relevant results

[0155] 4.4.1 Macroscopic wound healing assessment: On days 0, 7, and 14 after intervention, digital cameras were used to photograph and record wound healing conditions. ImageJ software was used to accurately measure the wound closure area at each observation time point and calculate the wound healing rate. The expression is as follows:

[0156] Wound healing rate (%) = (initial wound area - wound area at a specific time) / initial wound area × 100%

[0157] 4.4.2 Morphological changes of wound tissue: On the 7th and 14th days after intervention, 3 mice were randomly selected from each group, and the wound tissue was obtained after isoflurane inhalation anesthesia, and the wound length was accurately calculated using ImageJ software. In order to ensure the integrity of the tissue structure, the tissue samples were fixed in 40g / L formaldehyde solution for 48h, then dehydrated, paraffin-embedded, and sliced. The slice thickness was 5μm, and hematoxylin-eosin and Masson staining were used to comprehensively evaluate the pathological characteristics of the wound tissue.

[0158] 4.4.3 Kits for detecting the contents of SOD, MDA, IL-1α, and TNF-α After anesthesia, 5 mL of abdominal aorta blood was collected from each group of rats, centrifuged at 3000 r / min for 15 min, and the serum was separated and divided into centrifuge tubes and placed in a -80°C refrigerator for use. According to the reagent instructions, the water-soluble tetrazolium salt method was used to detect the content of SOD, the microplate method was used to detect the content of MDA, and the enzyme-linked immunosorbent (ELISA) kit was used to detect the content of IL-1α and TNF-α.

[0159] 4.4.4 Testing of antibacterial effect

[0160] Detect the sterilization rate of common bacterial infections and common antibiotic-resistant bacteria respectively

[0161] 4.4.5 Detection of changes in related factors in tissues

[0162] 4.5 Expected Results

[0163] The effect is good, with obvious effects on wound healing and related indicators, killing 95% or even more of the infectious bacteria.

[0164] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the present invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. Application of ESC-Exos in the treatment of bedsores.

2. The use according to claim 1, characterized in that: The application method is to introduce Ca-I-Ti into the ESC-Exos and cross-link EGF.

3. A nano-microneedle for treating bedsores, characterized in that: The nano-microneedles are prepared by cross-linking EGF and introducing Ca-I-Ti into ESC-Exos.

4. A method for preparing nano-microneedles for treating bedsores, characterized in that: The following steps are involved: 1) injecting a mixed solution a into a customized polydimethylsiloxane microneedle array mold, wherein the mixed solution a is a sustained-release outer layer needle polymer solution; 2) centrifuging the microneedle array mold obtained in step 1) at 5000 rpm for 10 minutes, taking it out after it is completely filled, and scraping off the excess mixed solution a; 3) placing the microneedle array mold obtained in step 2) in a silica gel desiccator and drying at room temperature for 2 h; 4) taking out the microneedle array mold obtained in step 3), and injecting a mixed solution b, wherein the mixed solution b is a fast-dissolving inner layer needle body polymer solution; 5) centrifuging the microneedle array mold obtained in step 4) at 5000 rpm for 10 minutes, taking it out after it is completely filled, and scraping off the excess mixed solution b; 6) placing the microneedle array mold obtained in step 5) in a silica gel desiccator and drying at room temperature for 2 h; 7) injecting the mixed solution c into the microneedle array mold obtained in step 6), centrifuging at 4000 rpm for 5 minutes, and taking it out after forming a backing, wherein the mixed solution c is a backing polymer solution; 8) placing the microneedle array mold obtained in step 7) in a silica gel desiccator and drying at room temperature for 2 h; 9) The hydrogel microneedles are prepared through steps 1) to 8).

5. The method for preparing nano-microneedles for treating bedsores according to claim 4, characterized in that: The preparation method of the mixed solution a is as follows: The cellulose material, high molecular weight fibrin, protective agent and drug are placed in a centrifuge according to a ratio of 0.2-:1-10:5-20:0.1-50, centrifuged at v1000-10000rpm, the centrifugation time is 1-30min; the drying time is 1-12h.

6. The method for preparing nano-microneedles for treating bedsores according to claim 4, characterized in that: The preparation method of the mixed solution b is as follows: The water-soluble high molecular polymer and the drug are placed in a centrifuge according to a ratio of 10-100:5-50, the centrifugal speed is 1000-10000rpm, the centrifugal time is 1-30min; the drying time is 1-12h.

7. The method for preparing nano-microneedles for treating bedsores according to claim 4, characterized in that: The mixed solution c is a backing polymer solution, and its preparation method is as follows: PVA and gelatin were mixed in a ratio of 6:1, frozen at -40°C to -10°C for one day, thawed at room temperature for 1-2 hours, and repeated 3-5 times. Hyaluronic acid was added to obtain a backing polymer solution c.

8. The method for preparing nano-microneedles for treating bedsores according to claim 5 or claim 6, characterized in that: The drug is ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF.

9. The method for preparing nano-microneedles for treating bedsores according to claim 8, characterized in that: The drug is ESC-Exos introduced with Ca-I-Ti and cross-linked with EGF, and the steps are as follows: A. Exosomes were resuspended in electroporation buffer and electroporated using a 0.4 cm cuvette with aluminum electrodes on a GenePulser II electroporation system with a capacitive extender. B allows the ions in the suspension to diffuse into the exosomes through the pores to obtain ESC-Exos introduced with Ca-I-Ti; C. The ESC-Exos obtained in step B and EGF are mixed in a buffer solution, and a cross-linking agent is added. The ESC-Exos physically cross-linked with EGF are obtained by high-speed centrifugation.

Images