CN117899006A - Composite hydrogel loaded with disulfiram nano vesicles and application of composite hydrogel in preparation of diabetic skin wound healing drugs - Google Patents
Composite hydrogel loaded with disulfiram nano vesicles and application of composite hydrogel in preparation of diabetic skin wound healing drugs Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/145—Amines having sulfur, e.g. thiurams (>N—C(S)—S—C(S)—N< and >N—C(S)—S—S—C(S)—N<), Sulfinylamines (—N=SO), Sulfonylamines (—N=SO2)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- Engineering & Computer Science (AREA)
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Abstract
The invention belongs to the technical field of biological medicines, and discloses a composite hydrogel loaded with disulfiram nano vesicles. The invention uses disulfiram and PLGA copolymer to form the core of nano medicine, uses mesenchymal stem cell membrane to coat the disulfiram and PLGA copolymer to synthesize nano vesicle, and mixes the nano vesicle with hydrogel to prepare the composite hydrogel loaded with the disulfiram nano vesicle. The nano particles can regulate immune inflammation, promote cell proliferation and play a role in promoting wound healing; the biomembrane of the outer layer of the nano-drug can enhance the biological concealment of the nano-particles to prevent the nano-particles from being rapidly cleared in vivo, and simultaneously, the extracellular vesicles from the mesenchymal stem cells can exert the tissue repair function similar to that of the mesenchymal stem cells and synergistically exert the function of promoting wound healing; the hydrogel changes the traditional injection administration mode, plays roles of moisturizing, ventilating and slowly releasing medicines, and promotes wound healing.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and in particular relates to a composite hydrogel loaded with disulfiram nano vesicles and application thereof in preparing a medicine for healing diabetic skin wound.
Background
Diabetic foot refers to the damage of skin and deep tissues far away from ankle joints of diabetics, frequent infection and/or arterial occlusion of lower limbs with different degrees, and serious patients involve muscle and bone tissues, which is one of complications of long-term diabetics. As diabetes is changed into epidemic disease from rare diseases in China, the prevalence rate of diabetic foot is obviously increased, and the prevalence rate of diabetic foot is as high as 8.1% for diabetics over 50 years old in China. It is estimated that Zhong Jiuyou diabetic patients amputate every 20 seconds worldwide, with a annual mortality rate of up to 11% for diabetic foot ulcers and a mortality rate of up to 22% for amputees. Therefore, diabetic foot is one of the main causes of disability and death of diabetics, causes a heavy burden to society, and is an urgent need to solve the serious public health problem. The contents of modern diabetic foot wound care comprise debridement, dressing for promoting wound surface wet healing, decompression, blood vessel assessment and management, infection treatment, blood sugar control and the like. Although much research has focused on accelerating the wound healing process, no satisfactory treatment is currently available.
Recent studies have indicated that, due to metabolic and biochemical abnormalities in the body, the proinflammatory neutrophil pathway in diabetics is enhanced, mainly by Neutrophil Extracellular Traps (NETs) formation, extracellular ROS production and pro-inflammatory cytokine production up-regulated, and excessive accumulation of these substances persists inflammation, causing tissue damage and delayed wound healing. The newly discovered effects of disulfiram in inhibiting GSDMD pore formation and thus NETs may provide new therapeutic indications for the reuse of disulfiram to treat a number of diseases caused or exacerbated by excessive inflammation.
Extracellular Vesicles (EVs) are cell-derived membrane structures, have natural lipid bilayer membrane structures, can be used as ideal drug carriers for delivering drugs to required target sites, have functions of immunoregulation, inflammation inhibition, cell proliferation promotion, angiogenesis and the like, and are widely applied to skin injuries.
In recent years, the combination of EVs with hydrogel biomaterials such as chitosan, alginate, and the like has become a hot spot of research, and hydrogels encapsulating EVs can improve the durability and stability of EVs release at a wound site without affecting the structural and functional integrity of EVs. The biocompatible hydrogel can realize noninvasive and stable delivery of a large number of EVs to a target site, and has good moisture retention and air permeability, so that the biocompatible hydrogel becomes an ideal choice of wound dressing.
Disclosure of Invention
In order to solve the problems in the prior art, the invention uses disulfiram and D, L-lactic acid-co-glycolic acid (PLGA) copolymer to form the core of the nano-drug, uses mesenchymal stem cell membrane to coat the disulfiram and the D, L-lactic acid-co-glycolic acid (PLGA) copolymer to design and synthesize the nano-vesicle, and mixes the nano-vesicle with hydrogel to prepare the composite hydrogel loaded with the disulfiram nano-vesicle. Aims to solve the following problems: 1) The disulfiram nano vesicle prepared by combining disulfiram and mesenchymal stem cell extracellular vesicles participates in regulating wound immunoinflammation, promoting cell proliferation and solving the problem of tissue injury caused by continuous aggravation of chronic inflammation at a diabetic wound; 2) By utilizing the characteristics of moisture retention and ventilation of the hydrogel and improving the release of the medicine, the limitation of quick removal of intravenous injection of the medicine is solved. So that the prepared composite hydrogel loaded with the disulfiram nanometer vesicles achieves the purposes of improving the immune environment of wounds, accelerating the healing of the wounds and treating diabetic skin wounds and other traumas.
In order to achieve the above purpose, the present invention provides the following technical solutions:
A composite hydrogel comprising nanoparticles of core-shell structure and a gel component, the core consisting of disulfiram and D, L-lactic-co-glycolic acid (PLGA) copolymer; the shell is a mesenchymal stem cell membrane; the gel component is sodium alginate.
Preferably, the diameter of the nanoparticle is 120-200 nm.
Preferably, the mass ratio of the poly (D, L-lactic acid-co-glycolic acid) to the disulfiram is 10:0.5 to 3.
Preferably, the mixing mass ratio of the mesenchymal stem cell membrane (shell) to the PLGA drug-loaded nanometer (core) is 0.125-0.5:1.
Preferably, the mass ratio of the bionic mesenchymal stem cell membrane nano drug-loaded particles to the sodium alginate hydrogel is 5:1.
The invention also claims a preparation method of the composite hydrogel, which comprises the following steps:
S1, extracting mesenchymal stem cell membranes;
S2, synthesis of PLGA drug-loaded nano-cores: adding poly (D, L-lactic acid-co-glycolic acid) and drug disulfiram, mixing, and magnetically stirring to obtain the final product;
S3, mixing the mesenchymal stem cell membrane obtained in the step S1 with the PLGA drug-loaded nano-inner core obtained in the step S2, and performing ultrasonic treatment to obtain bionic mesenchymal stem cell membrane nano drug-loaded particles;
s4, synthesizing sodium alginate hydrogel: mixing sodium alginate in water, mixing and dissolving in a shaking table at 4 ℃ overnight, adding calcium chloride, and fully and uniformly mixing to obtain sodium alginate hydrogel;
s5, fully mixing the bionic mesenchymal stem cell membrane nano drug-loaded particles in the step S3 with the sodium alginate hydrogel in the step S4 at the temperature of 4 ℃.
Preferably, the magnetic stirring condition in the step S2 is 300-500 rpm, the temperature is 4-25 ℃ and the time is 4-48 h.
Preferably, the condition of the water bath ultrasonic wave in the step S3 is that the power is 50W-100W, the temperature is 4-25 ℃ and the time is 3-30 minutes.
The composite hydrogel loaded with the bionic mesenchymal stem cell membrane nano drug-loaded particles can regulate immunity, promote cell proliferation and angiogenesis, and play a role in promoting wound healing; in addition, the disulfiram wrapped by the bionic nanoparticle inner core can reduce the generation of NETs aiming at neutrophils at the wound surface of the diabetic skin and promote wound healing. The composite hydrogel of the bionic loading mesenchymal stem cell membrane nano drug-loaded particles can inhibit neutrophils from being stimulated and activated to generate NETs in a hyperglycemic environment, simultaneously regulate inflammatory reaction along with the expression change of inflammatory factors, and can be used for preparing drugs for reducing the content of inflammatory factors at wounds of diabetics.
Compared with the prior art, the invention has the beneficial effects that:
The invention provides a bionic mesenchymal stem cell membrane nano drug-loaded particle, the surface of a biological membrane of which can enhance the biological concealment of the nano particle and prevent the nano particle from being rapidly cleared in vivo; the drug-loaded particles can inhibit the generation of NETs by neutrophil stimulation, reduce tissue damage caused by accumulation of NETs, and play an immunoregulation role in the healing treatment of diabetic skin wound surfaces; the extracellular vesicles derived from the mesenchymal stem cells can play a tissue repair function similar to that of the mesenchymal stem cells, and can play a role in promoting wound healing in a synergistic manner. The bionic mesenchymal stem cell membrane nano drug-loaded particles prepared by the invention are simple to synthesize, the raw materials are biological membranes and degradable PLGA, and the bio-compatibility is high, so that the bionic mesenchymal stem cell membrane nano drug-loaded particles are suitable for the treatment of diabetic skin wound surfaces.
Drawings
FIG. 1 is a schematic diagram of the preparation of DSF-EP and its application in the repair of diabetic skin wounds;
FIG. 2 is a representation of DSF-EP wherein the exosome marker protein of FIG. 2A; FIG. 2B is the absorbance of DSF-EP in different solutes; FIG. 2C is a particle size distribution plot; FIG. 2D is a potential distribution diagram;
FIG. 3 is a graph of DSF-EP promoting diabetic wound healing, wherein FIG. 3A is a graph of wound healing at various times; FIG. 3B is a graph showing the change in healing area over time for different groups;
FIG. 4 shows the reduction of inflammatory factor expression by DSF-EP. FIGS. 4A through 4E represent IL-1α, IL-1β, IL-6, TNF- α, IL-18, respectively.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The preparation was carried out under conventional conditions or conditions recommended by the manufacturer, where no specific conditions were noted in the examples. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1: preparation of DSF-EP comprising the steps of:
1. mesenchymal stem cell membrane extraction
(1) MSC cells (about 10 8) up to 70% -80% confluence were collected, centrifuged at 1000g for 5min, washed 3 times with 1 XPBS and the final cell pellet resuspended in 3ml hypotonic buffer (1 mmol/L NaHCO 3 0.2mmol/L EDTA, 1 mmol/LPMSF); lysis overnight at 4 ℃;
(2) Loading the cell heavy suspension in the step (1) into a Dounce Dunn homogenizer, placing the homogenizer on ice, and grinding the cells for 20 times;
(3) Taking the heavy suspension in the step (2), centrifuging at 3200 Xg for 5 minutes to remove large fragments, and collecting supernatant;
(4) Taking 20000g of the supernatant in (3), centrifuging for 25 minutes, discarding the precipitate, collecting the supernatant, centrifuging for 100000g×35 minutes, discarding the supernatant after centrifugation, collecting the white precipitate (i.e., cell membrane), and re-suspending with 500 μl of 1×pbs;
(5) Taking the heavy suspension in the step (4) to detect the content of the membrane protein.
In order to verify the feasibility of the bionic mesenchymal stem cell nanoparticle, the inventor characterizes the surface membrane protein of the bionic mesenchymal stem cell nanoparticle and related receptor proteins thereof. The inventor firstly detects the surface protein of the bionic giant mesenchymal stem cell nano-particle, through protein electrophoresis detection, the experimental result is shown as figure 2A, the band of the bionic giant mesenchymal stem cell nano-particle is consistent with the band of the mesenchymal stem cell exosome, and is different from the band of the mesenchymal stem cell lysate protein, the surface of the bionic giant mesenchymal stem cell nano-particle is covered with the mesenchymal stem cell lysate protein, thus proving the feasibility of utilizing the bionic mesenchymal stem cell membrane to play the role of promoting wound repair.
2. Synthesis of PLGA drug-loaded nanokernel
(1) Disulfiram is dissolved in 1ml of acetone according to the mass concentration ratio of 4:1;
(2) Adding poly (D, L-lactic acid-co-glycolic acid) into the mixture (1), wherein the mass concentration ratio of the medicine to the poly (D, L-lactic acid-co-glycolic acid) is 3:10;
(3) Rapidly pumping the mixed solution in the step (2) into 3ml ddH 2 O rotating at high speed by using a 1ml syringe;
(4) And (3) naturally evaporating the mixed solution in the step (3) on a stirrer for 4 hours (completely evaporating the acetone), so as to obtain the PLGA drug-loaded nano-inner core.
3. Preparation and evaluation of bionic mesenchymal stem cell membrane nano drug-loading system
The key point of the construction system of the bionic mesenchymal stem cell nano-particle is that PLGA nano-inner cores stably support surface membrane materials, the inventor sets and compares the absorbance of drug-loaded nano-particles in different solutes, and determines whether the membrane mesenchymal stem cell membrane nano-drug-loaded system is successfully prepared or not by detecting the absorbance of the drug-loaded nano-particles in water, oil and 1xPBS and comparing the stability of the materials under different mass ratios.
(1) Mixing the mesenchymal stem cell membrane extract in the first step and the PLGA drug-loaded nanometer core liquid in the second step together (the mass ratio of the membrane protein to the PLGA nanometer particles is 1:2);
(2) And (3) performing ultrasonic treatment on the mixture in the step (1) in a water bath with the power of 100W for 3min to obtain the bionic mesenchymal stem cell membrane nano drug-loaded particles.
(3) Dissolving the nano particles prepared in the step (2) in water, oil and 1xPBS respectively, fully stirring and standing for 10min at room temperature, and detecting absorbance.
(4) Synthesis of sodium alginate hydrogel: mixing sodium alginate in water, mixing and dissolving in a 4-degree shaking table overnight, adding calcium chloride, and fully and uniformly mixing to obtain sodium alginate hydrogel;
(5) Fully mixing the bionic mesenchymal stem cell membrane nano drug-loaded particles of (2) with the sodium alginate hydrogel of (4) at 4 ℃.
As can be seen from fig. 2B, there is a difference in absorbance after the biomimetic mesenchymal stem cell membrane nanoparticle and the single disulfiram are dissolved in water, oil and 1xPBS, respectively, and DSF-EP absorbance is reduced in PBS, indicating that degradation of the nanoparticle encapsulated with the biomimetic mesenchymal stem cell membrane is reduced. As can be seen from fig. 2C, the synthesized bionic mesenchymal stem cell membrane nanoparticle is relatively uniform under an electron microscope, has a particle size of about 150nm, and has an obvious complete bilayer structure on the surface, which proves that the nanoparticle is covered by a biological membrane. The hydration particle size of the single PLGA nano particle is about 180nm, the potential is-35 mV, the potential of the single membrane vesicle is about 140nm, the potential is-40 mV, after the membrane wraps the PLGA inner core, compared with the single membrane, the hydration particle size is reduced to 150nm, the potential is reduced to-30 mV, the side surface proves that the membrane is modified on the surface of the PLGA nano inner core, and the preparation of the membrane-wrapped nano particle is stable (shown in figure 2C and figure 2D).
Example 2DSF-EP promotes wound healing in diabetic mice skin, the experimental procedure is as follows:
(1) Construction of diabetic mouse model
① C57 randomly selected 3-4 week male mice are adaptively bred for one week
② High fat and high sugar feeding for 6-8 weeks, measuring weight and fasting blood glucose level
③ The mice are fasted and not forbidden for water for over night (more than 12 hours), the blood sugar level of the mice is weighed and measured, calculated according to the number of animals and injection dosage, streptozotocin is placed at room temperature and protected from light for 10 minutes to defrost thoroughly, the required STZ (freeze-dried powder) is weighed and placed into a dry and light-proof sterile bottle, and the bottle is wrapped by aluminum foil or tinfoil paper;
④ The citrate buffer and STZ-containing bottles were placed in an ice bath in advance and brought together into the animal house. The STZ was dissolved by adding an appropriate amount of a citric acid buffer at a concentration of 1% (w/v) and 10mg/ml, and the solution was sufficiently dissolved. Injecting the corresponding volume of STZ into the abdominal cavity according to the empty stomach weight of the animal, and finishing the injection within 30 minutes;
⑤ And continuing to feed with high sugar, detecting blood sugar level on the 2 nd, 3 rd, 5 th and 7 th days after administration, and determining that the mice with the continuous two fasting blood sugar levels of more than 11.1mmol/L are diabetic mice.
(2) Construction of diabetic mouse skin injury model
① Selecting qualified diabetic mice in the step (1), weighing and anesthetizing the mice, and shaving back fur;
② Each mouse had a circular wound of 1X 1cm in size on the back
③ Each group of mice was respectively given PBS, PLGA, exo-PLGA, DSF (disulfiram), DSF-EP 100ul hydrogel wound site smeared, and wound closure was observed with days 0, 3, 6, 9, 12, 15 after wound formation.
The wound healing of each group of mice is seen in fig. 3A, and the wound healing area statistics of fig. 3B shows that DSF-EP group (composite hydrogel) mice heal at the highest rate and have statistical significance, demonstrating that DSF-EP (composite hydrogel) can promote skin wound healing of diabetic mice.
Example 3DSF-EP inhibits neutrophil NETs production and action on inflammatory factors as follows:
(1) Isolation of bone marrow neutrophils from diabetic mice
① Taking tibia femur of a diabetic mouse, blowing out cells in bone marrow by using normal saline and fixing the volume to 5mL;
② 4mL of neutrophil extraction kit A was taken in a 15mL centrifuge tube, and 2mL of C was carefully added dropwise to the upper layer. At this time, the liquid level separation is visible between the liquid A and the liquid C, and the mixture of bone marrow cells and physiological saline is dripped into the uppermost layer, and 950g is centrifuged for 20min;
③ Separating the liquid after centrifugation, transferring the separated liquid layer containing the neutrophils into a new 15mL centrifuge tube, adding physiological saline to 12mL, centrifuging for 10min at 950g, and discarding the supernatant;
④ Adding 1ml of erythrocyte lysate for 10min, stopping with normal saline, centrifuging at 1800rpm for 5min, and discarding the supernatant;
⑤ Physiological saline or serum-free medium weight, counts;
(2) Blocking neutrophil stimulation by DSF-EP to produce NETs
① Primary neutrophil plating: neutrophils in good condition were plated in 12-well plates, 5×10 5 cells per well, 1mlRIPA medium was added, and incubated at 37 ℃ for 1h after 1ul pbs, PMA (1 mg/ml) stimulus was added, respectively;
② DSF-EP treatment of primary neutrophils: 100ulPBS, PLGA, DSF, EXO-PLGA and DSF-EP are added into each hole, and incubated for 3 hours at 37 ℃;
③ The culture supernatant was collected ② and stored at-30 ℃.
(3) DSF-EP culture supernatants inhibited macrophage secretion of inflammatory factors as follows:
① Primary megagranulocyte plating: fresh primary macrophages were plated in 24-well plates, each well containing 3X 105 neutrophils, with a culture volume of 400. Mu.l;
② Centrifuging 18000g of the incubation liquid in the step (2) for 15min to obtain a supernatant, adding the supernatant into a ① macrophage pore plate, and supplementing a culture medium to make the final volume of each pore of the culture liquid be 500 μl and the serum content be 10%;
③ After the macrophages in ② are incubated for 2 hours at 37 ℃, the macrophages are incubated for 2 hours at 37 ℃ after LPS is added;
④ The supernatant from ③ wells was discarded, cells were collected by adding 200ul of Trizol to each well for 5min, and cellular RNA was extracted and then assayed for inflammatory factor expression.
As shown in FIG. 4, the activation degree of macrophages after being treated by DSF-EP culture supernatant is obviously reduced, the DSF-EP culture supernatant can inhibit the macrophages from secreting inflammatory factors, the IL-6 and IL-1β expressed by macrophages in a DSF-EP culture supernatant treatment group are obviously reduced, and the potential of DSF-EP in anti-inflammatory treatment at the diabetic skin wound surface is revealed, thereby representing the feasibility of the invention in the diabetic skin wound surface.
The above examples of the present invention are only for clearly illustrating the technical solution of the present invention, and are not limited to the specific embodiments of the present invention. Any modification, equivalent replacement, improvement, etc. that comes within the spirit and principle of the claims of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A composite hydrogel comprising nanoparticles of core-shell structure and a gel component, characterized in that the core consists of disulfiram and PLGA copolymer; the shell is a mesenchymal stem cell membrane; the gel component is sodium alginate.
2. The composite hydrogel of claim 1, wherein the nanoparticles have a diameter of 120-200 nm.
3. The composite hydrogel of claim 1, wherein the mass ratio of PLGA to disulfiram is 10:0.5 to 3.
4. The composite hydrogel of claim 1, wherein the shell-core material is mixed in a mass ratio of 0.125-0.5:1.
5. The composite hydrogel of claim 1, wherein the mass ratio of the core-shell structured nanoparticles to sodium alginate is 5:1.
6. A method of preparing the composite hydrogel of claim 1, comprising the steps of:
S1, extracting mesenchymal stem cell membranes;
S2, synthesis of PLGA drug-loaded nano-cores: mixing PLGA and disulfiram, and stirring;
S3, mixing the mesenchymal stem cell membrane obtained in the step S1 with the PLGA drug-loaded nano-inner core obtained in the step S2, and performing ultrasonic treatment to obtain bionic mesenchymal stem cell membrane nano drug-loaded particles;
s4, synthesizing sodium alginate hydrogel: sodium alginate is mixed in water to prepare sodium alginate hydrogel;
S5, mixing the bionic mesenchymal stem cell membrane nano drug-loaded particles obtained in the step S3 with the sodium alginate hydrogel obtained in the step S4 at the temperature of 4 ℃.
7. The method of preparing a composite hydrogel according to claim 6, wherein the magnetic stirring in step S2 is performed at 300-500 rpm, at 4-25℃for 4-48 hours.
8. The method for preparing composite hydrogel according to claim 6, wherein the power of the water bath ultrasound in step S3 is 50W to 100W, the temperature is 4 ℃ to 25 ℃ and the time is 3 minutes to 30 minutes.
9. Use of the composite hydrogel according to any one of claims 1 to 5 for the preparation of a medicament for diabetic foot or diabetic skin injury.
10. The use according to claim 9, wherein the composite hydrogel is used to promote healing of diabetic foot or diabetic skin wound, reduce diabetic foot or diabetic skin wound healing time, reduce neutrophil nes expression at diabetic foot or diabetic skin injury wound, modulate IL-1 α, IL-1 β and/or IL-6 at immunosuppressive wound.
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