Composite for promoting wound healing

Abstract

The present invention relates to a composite for promoting wound healing, comprising: a substrate layer configured to absorb wound exudate and stop bleeding; and a probiotic loading layer configured to modulate a wound flora structure; the probiotic loading layer is deposited on the substrate layer. Based on a probiotic-antibacterial agent combination strategy, a medicament integrating two main functions of eradicating exogenous pathogenic bacteria and regulating and controlling wound flora is developed, and the medicament is applied to collaborative treatment of diabetic chronic wounds. Meanwhile, the technical bottleneck of solid-state storage and utilization of probiotics is broken through based on an electrostatic spinning technology. The medicament disclosed by the invention has the advantages and characteristics of self-supporting, tissue wet adhesion, hemostasis, seepage absorption, medicament slow release, stable storage, probiotic delivery, antifouling and the like, and a new carrier and a new scheme are provided for treatment of P.aeruginosa infected diabetic wounds.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and particularly to a complex for promoting wound healing. Background Art

[0002] The poor healing of diabetic wounds has become one of the most costly complications. The wound healing process in diabetic patients does not strictly follow the four stages of normal wound healing (hemostasis, inflammation, proliferation, remodeling). The disorder of glucose and lipid metabolism in the diabetic state brings a series of adverse effects to wound healing, including increased oxidative stress level, chronic inflammation, abnormal angiogenesis, tissue hypoxia, peripheral neuropathy, abnormal extracellular matrix, apoptosis, etc. In addition, the poor healing of diabetic wounds increases the chance of infection by bacteria and other microorganisms, and even develops into systemic infection, endangering life.

[0003] Currently, diabetic wounds are still a difficult point in clinical treatment, mainly relying on physical debridement, systemic or topical antibiotics, the use of dressings and active drugs, etc. to prevent infection and promote healing, but the effects are often very little. Therefore, there is an urgent need to find new targets and develop new technologies to effectively address this medical and health problem of treating diabetic wounds.

[0004] Recent studies have shown that the microbial flora coexisting in the human intestine, oral cavity, skin, etc. plays an important role in regulating the body functions and the occurrence and development of diseases. As the largest organ of the human body, the skin is colonized with a rich flora. The structure and evolution of the skin flora have an important impact on regulating the function of the body surface immune barrier and the wound healing process. The flora colonized and transplanted on the body surface has been proven to promote the healing of chronic wounds by regulating the immune response, improving the inflammatory response, promoting cell proliferation and epidermal remodeling, etc. Diabetic wounds often complicate the dysbiosis of the microbial flora at and around the wound, thereby inducing further infection and delaying healing, and the degree of flora abnormality is closely related to the course and condition of the disease.

[0005] The regulation of the wound flora in diabetes mainly relies on two approaches: molecular drugs and the transplantation of live bacteria (such as probiotics). Among them, the transplantation of live bacteria is more in line with the theory of human-microbe commensalism and has many advantages such as safety, high efficiency, economy, and good compliance. The transplantation of probiotics, such as Staphylococcus epidermidis and various Lactobacilli (Lactobacillus bulgaricus, Lactobacillus reuteri (L. reuteri), Lactobacillus casei, etc.), has made positive progress in promoting the healing of various wounds. However, there are still a large number of problems to be solved urgently in theory and technology in existing research, including: 1) The interaction between probiotics and wound flora, the regulation mechanism, and the impact on the development of the disease are not fully elaborated. Some research conclusions are even contradictory and need to be further clarified. 2) The technical implementation means of probiotic transplantation are outdated, lacking a stable, efficient, multifunctional, and productizable delivery platform. In particular, there is a lack of a solution that can be stored stably for a long time and "ready to use immediately". 3) Although probiotics can optimize the structure of the colonized flora, their effect of excluding exogenous pathogenic bacteria infection is poor. Diabetic wounds are prone to secondary infections, so relying solely on flora regulation is not enough to deal with complex infections. Summary of the Invention

[0006] In view of the technical problems existing in the prior art, the present invention provides a complex for promoting wound healing, including: a basal layer configured to absorb wound exudate and stop bleeding; and a probiotic-loaded layer configured to regulate the structure of the wound flora; the probiotic-loaded layer is deposited on the basal layer.

[0007] In some embodiments, the basal layer is loaded with a preparation targeting harmful bacteria in the wound.

[0008] In some embodiments, the harmful bacteria are iron-metabolizing bacteria.

[0009] In some embodiments, the basal layer is configured to slowly release the preparation.

[0010] In some embodiments, the preparation includes gallium ions.

[0011] In some embodiments, the basal layer includes hyaluronic acid or dopamine-grafted hyaluronic acid.

[0012] In some embodiments, the probiotic-loaded layer includes polyvinyl alcohol nanofibers.

[0013] In some embodiments, the probiotic-loaded layer includes probiotics selected from one or more of the following groups: Staphylococcus epidermidis, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus casei, yeast, Bacillus, Bifidobacterium, Clostridium butyricum.

[0014] Use of the complex as described above in the preparation of a medicament for promoting wound healing.

[0015] In some embodiments, the wound is a wound of a diabetic patient.

[0016] A medicament for promoting wound healing, comprising the complex as described in any one of the above.

[0017] The preparation method of the complex as described in any one of the above, comprising: preparing a base layer, including: obtaining dopamine-grafted hyaluronic acid; mixing dopamine-grafted hyaluronic acid with a preparation targeting harmful bacteria in the wound; freezing the mixed solution in an environment of -50 to -80 °C for 10 - 15 hours; and freeze-drying for 40 - 60 hours to obtain the base layer; and preparing a probiotic-loaded layer, including: obtaining a PVA solution; adding probiotics to the PVA solution to obtain a spinning solution; and obtaining the probiotic-loaded layer by electrospinning; wherein, the probiotic-loaded layer is deposited on the base layer.

[0018] Based on the probiotic-antibacterial agent combination strategy, this application develops a medicament integrating the two major functions of eliminating exogenous pathogenic bacteria and regulating the wound flora, and applies it to the synergistic treatment of diabetic chronic wounds. At the same time, based on the electrospinning technology, the technical bottleneck of the solid-state storage and utilization of probiotics is broken through. The medicament of this application has many advantageous characteristics such as self-supporting, tissue wet adhesion, hemostasis, exudate absorption, drug slow release, stable storage and delivery of probiotics, and anti-fouling, providing a new carrier and new solution for the treatment of diabetic wounds infected with P. aeruginosa. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Next, the preferred embodiments of the present invention will be further described in detail with reference to the drawings, wherein:

[0020] Figure 1 is the ultraviolet-visible absorption spectrum of the dopamine-grafted hyaluronic acid (HD) base layer material prepared according to an embodiment of the present application and hyaluronic acid (HA) as a comparison;

[0021] Figure 2 is a physical photograph of the GaHD / LRNF complex sample according to an embodiment of the present application;

[0022] Figure 3 is the scanning electron microscope image of the GaHD / LRNF complex prepared according to an embodiment of the present application; wherein Figure 3 A is the GaHD layer of the GaHD / LRNF complex according to an embodiment of the present application, Figure 3 B is the LRNF layer of the GaHD / LRNF complex according to an embodiment of the present application;

[0023] Figure 4The diameters of the inhibition zones of different complexes (HD, GaHD, GaHD / LRNF) according to an embodiment of the present application against P. aeruginosa;

[0024] Figure 5 The viable probiotic counts (A) during the continuous storage of the GaHD / LRNF complex and the growth curve (B) after 28 days of storage according to an embodiment of the present application;

[0025] Figure 6 The curve of the healing efficiency (%) of the GaHD / LRNF complex over time on a full-thickness skin wound model in mice according to an embodiment of the present application. Detailed implementation manners

[0026] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Apparently, the described embodiments are some but not all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] In the following detailed description, reference may be made to the accompanying drawings which form a part hereof, and in which are shown by way of illustration specific embodiments in which the application may be practiced. In the drawings, like reference numerals describe substantially similar components in different figures. The various specific embodiments of the present application have been described in sufficient detail below to enable those of ordinary skill in the art with relevant knowledge and technology to implement the technical solutions of the present application. It should be understood that other embodiments may be utilized or changes may be made to the embodiments of the present application.

[0028] The present invention proposes a new scheme for the combined use of probiotics and antibacterial agents to treat chronic diabetic wounds. Pseudomonas aeruginosa (P. aeruginosa) is one of the main pathogenic bacteria invading skin wounds. Gallium (Ga) is an antibacterial agent targeting bacterial iron metabolism, which has a lower risk of drug resistance and is safer to use compared to antibiotics. The present invention selects a gallium ion antibacterial agent sensitive to P. aeruginosa and the probiotic L. reuteri with antibacterial and flora-regulating activities, and prepares a complex GaHD / LRNF based on electrospun nanofibers through a layer-by-layer assembly technique.

[0029] In the present application, "HD" refers to dopamine-grafted hyaluronic acid.

[0030] In the present application, "HA" refers to hyaluronic acid, which is used as a control to compare the differences in the structures, functions, etc. between HD and HA.

[0031] In the present application, "LR" refers to Lactobacillus reuteri (L. reuteri).

[0032] In the present application, "PVA" refers to polyvinyl alcohol.

[0033] In the present application, "LRNF" refers to Lactobacillus reuteri (L. reuteri)-poly(vinyl alcohol) nanofibers (PVA NF). After dispersing Lactobacillus reuteri into polyvinyl alcohol, the mixed solution is spun, stretched, etc. to obtain a polyvinyl alcohol nanofibrous structure, which encapsulates a spindle-shaped structure.

[0034] In the present application, "GaHD / LRNF" refers to a complex obtained by loading gallium ions onto dopamine-grafted hyaluronic acid and then depositing nanofibers encapsulating the probiotic Lactobacillus reuteri thereon. It has the effects of improving the bacterial structure of wounds and promoting wound healing.

[0035] In the present application, "harmful bacteria" refers to bacteria that may invade skin wounds, which can cause difficult wound healing, biological infections of wounds, and even systemic biological infections, etc. In some embodiments, harmful bacteria include but are not limited to: Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus, Escherichia coli, Vibrio vulnificus, Streptococcus hemolyticus, Escherichia coli, Clostridium, Clostridium tetani, etc.

[0036] In the present application, "probiotics" refers to bacteria that conform to the theory of human-microbe commensalism and have characteristics such as safety, high efficiency, economy, and good compliance. They can promote wound healing, improve the microbial structure at the wound site, promote wound healing, and reduce biological infections. In some embodiments, probiotics include but are not limited to: Staphylococcus epidermidis, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus casei, yeast, Bacillus, Bifidobacterium, Clostridium butyricum.

[0037] In the present application, the "agar diffusion method" is also called the diffusion method. It is a method that uses the diffusion of the drug to be tested in an agar plate to inhibit the growth of bacteria around it, forming a transparent circle, that is, an inhibition zone, and determines the antibacterial titer of the drug to be tested according to the size of the inhibition zone.

[0038] The present invention provides a complex for promoting wound healing, comprising: a basal layer configured to absorb wound exudate and stop bleeding; and a probiotic-loaded layer configured to regulate the wound flora structure; the probiotic-loaded layer is deposited on the basal layer.

[0039] In some embodiments, the base layer loads a preparation that targets harmful bacteria in the wound. In some embodiments, the preparation includes gallium ions. In some embodiments, the harmful bacteria are iron-metabolizing bacteria. In some embodiments, the base layer is configured to slowly release the preparation. In some embodiments, the base layer is dopamine-grafted hyaluronic acid (Hyaluronic acid-dopamine, HD) with tissue wet adhesion properties, where HD is prepared by crosslinking hyaluronic acid and dopamine hydrochloride through an EDC / NHC crosslinking reaction. Gallium nitrate (Ga) is loaded in HD to form a GaHD layer, which plays roles in support, adhesion, hemostasis, absorption of wound exudate, and slow release of antibacterial agents.

[0040] In some embodiments, the probiotic-loaded layer includes poly(vinyl alcohol) nanofibers (PVA NF), which encapsulate probiotics and play roles in storing, protecting, and delivering probiotics.

[0041] In some embodiments, the probiotic-loaded layer includes probiotics selected from one or more of the following groups: Staphylococcus epidermidis, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus casei, yeast, Bacillus, Bifidobacterium, Clostridium butyricum.

[0042] Use of the complex as described above in any one of the preceding paragraphs in the preparation of a medicament for promoting wound healing. In some embodiments, the wound is a wound of a diabetic patient.

[0043] A medicament for promoting wound healing, comprising the complex as described above in any one of the preceding paragraphs.

[0044] A method for preparing the complex as described above in any one of the preceding paragraphs, comprising: preparing a base layer, including: obtaining dopamine-grafted hyaluronic acid; mixing the dopamine-grafted hyaluronic acid with a preparation that targets harmful bacteria in the wound; freezing the mixed solution in an environment of -50 to -80 °C for 10 - 15 hours; and freeze-drying for 40 - 60 hours to obtain the base layer; and preparing a probiotic-loaded layer, including: obtaining a PVA solution; adding probiotics to the PVA solution to obtain a spinning solution; and obtaining the probiotic-loaded layer by electrospinning; wherein, the probiotic-loaded layer is deposited on the base layer.

[0045] The GaHD / LRNF complex of the present application has antibacterial activity against P. aeruginosa and the activity of improving the skin wound flora structure.

[0046] The preparation method of the complex of the present application will be described in detail below through examples, and the promoting effect of the complex on wound healing will be verified.

[0047] Example 1 Preparation of the base layer GaHD

[0048] According to an embodiment of the present application, 600 mg of hyaluronic acid was taken, 60 ml of water and 60 μL of concentrated hydrochloric acid were added, and the mixture was stirred for 2 h to dissolve it completely. After dissolution, 320 mg of EDC and 200 mg of NHS were weighed, and the two were poured into a beaker and stirred for another 30 min to dissolve. Subsequently, 320 mg of dopamine hydrochloride was weighed and poured into the beaker, and the reaction was stirred in the dark for 6 h. The solution was collected and placed in a dialysis bag (cut-off molecular weight 3000 D), and dialyzed in 1500 mL of ultrapure water for 48 h, with the water changed every 12 h. After dialysis, the solution was taken out and frozen in a -60 °C refrigerator for 12 h. Subsequently, it was placed in a freeze dryer and freeze-dried for 48 h to obtain a white sponge-like product (Hyaluronic acid-dopamine, hereinafter referred to as HD), which was packed into a self-sealing bag and stored in a 4 °C refrigerator.

[0049] As Figure 1 shown, compared with the original hyaluronic acid, the ultraviolet-visible absorption spectrum of the obtained product shows a characteristic absorption peak of the dopamine ortho-diphenol structure at 280 nm, indicating that dopamine molecules have been successfully grafted onto the hyaluronic acid backbone. Alone, HA does not have tissue adhesion. Dopamine monomers contain catechol groups and can adhere to the surface of various substrates (including biological tissues) through various forces.

[0050] To prepare GaHD, 200 mg of HD solid was redissolved in 20 mL of ultrapure water, 8 mg of gallium nitrate was weighed, and added to the HD solution with stirring to mix well. Subsequently, it was poured into a plastic petri dish with a diameter of 90 mm and placed in a -60 °C refrigerator for 12 h, and then freeze-dried for 48 h to obtain the GaHD substrate layer.

[0051] Example 2 Preparation and Characterization of GaHD / LRNF Complex

[0052] According to an embodiment of the present application, 500 mg of PVA1799 was weighed, 5 mL of ultrapure water was added, and after swelling in a 95 °C water bath for 1 h, it was transferred to a 95 °C water bath and stirred for 3 h to dissolve it completely. After cooling to room temperature, a PVA solution was obtained. LR was cultured in MRS medium at 37 °C for 12 h until the logarithmic growth phase (concentration about 10 9 CFU / mL). 2 mL of the bacterial solution was taken, centrifuged at 7000 rpm for 3 min, the supernatant was discarded, PBS was added, and then centrifuged at 7000 rpm for 3 min again. The PBS was discarded, and 2 mL of the PVA solution was added to obtain the LR / PVA spinning solution. A 5 mL syringe was taken, 3 mL of the spinning solution was drawn, placed on a portable electrospinning machine, and the spinning conditions were set as follows: voltage 10 kV, feeding speed 1.0 mL / min, receiving distance 15 cm, and receiving time 1 h. Using GaHD as the receiving substrate, the spinning solution undergoes processes such as stretching, solvent evaporation, and curing in the electrostatic field, and finally nanofibers are deposited on the surface of GaHD.

[0053] As Figure 2 shown, the surface of the obtained complex is a white fibrous structure. As Figure 3 shown, scanning electron microscope pictures indicate that the HD basal layer and the LRNF layer have different structures. The HD layer is a porous layered structure ( Figure 3 A), and the LRNF layer is a fibrous structure ( Figure 3 B), with a fiber diameter of about 300 μm, and a spindle-like structure is wrapped therein, which is the LR bacterium. The above results indicate that the complex can be successfully prepared by this layer-by-layer assembly method.

[0054] Example 3 Antibacterial Activity of the GaHD / LRNF Complex

[0055] According to an embodiment of the present application, the antibacterial activity of the complex GaHD / LRNF was evaluated by the antibacterial zone method. P. aeruginosa was cultured in LB medium at 37 °C for 8 h until the logarithmic growth phase (concentration about 10 9 CFU / mL). Take 1 mL of the bacterial solution, centrifuge at 7000 rpm for 3 min, discard the supernatant, add PBS, then centrifuge at 7000 rpm for 3 min again, discard the PBS, and re-add 1 mL of PBS, and pipette to resuspend. Take 10 μL of the resuspended bacterial solution and add it to 1 mL of PBS to make the concentration about 10 7 CFU / mL. Take 100 μL of this bacterial solution and evenly coat it on the LB agar culture plate with glass beads. There are 3 kinds of experimental samples, namely the HD film, the GaHD film and the GaHD / LRNF film. The samples were made into circular discs with a diameter of 5 mm using a 5 mm puncher, placed on the agar culture plate coated with P. aeruginosa, and covered with a 7 mm Oxford cup around the discs. The plate was inverted and statically cultured at 37 °C for 24 h, and then taken out to measure the diameter of the antibacterial zone.

[0056] As Figure 4 shown, the HD film has no antibacterial activity against P. aeruginosa, and both the GaHD film and the GaHD / LRNF film show antibacterial activity against P. aeruginosa, and the antibacterial zone diameter of the latter is larger than that of the former, indicating that gallium nitrate and LR have a synergistic inhibitory effect on P. aeruginosa.

[0057] Example 4 Storage Stability of the Complex GaHD / LRNF

[0058] According to an embodiment of the present application, the prepared complex GaHD / LRNF is stored in a refrigerator at 4°C. On the 0th day, 14th day, 21st day, and 28th day respectively, 3 pieces of composite membranes with an area of 1 cm * 1 cm are cut, shaken and fully dissolved in 1 mL of PBS, and then 100 μL is taken and evenly coated on an MRS agar culture plate with glass beads. The plate is inverted and cultured statically at 37°C for 48 h and then taken out, and the number of colonies is counted. Based on the number of colonies on the 0th day, the survival effect is evaluated. In addition, on the 28th day, after the sample is dissolved in PBS, 10 μL is taken and added to 100 μL of MRS medium in a 96-well plate, and 3 wells are made in parallel. The plate is placed in an enzyme-linked immunosorbent assay (ELISA) reader and cultured with shaking at 37°C for 24 h, and the absorbance OD at 600 nm is continuously measured. 600 , and a growth curve is plotted.

[0059] As Figure 5 shown in 6 A, after the complex is stored for 28 days, the number of viable probiotic bacteria in it does not decrease significantly and remains at about 10 2 CFU / cm Figure 5 . As shown in B, after the complex is stored for 28 days, the probiotic bacteria in it can still grow and proliferate normally. The above data indicate that the complex GaHD / LRNF has excellent storage stability.

[0060] Example 5 Therapeutic effect of the complex GaHD / LRNF

[0061] According to an embodiment of the present application, the method for establishing a diabetic mouse model is as follows: 30 female Balb / c mice are intraperitoneally injected with streptozotocin (STZ) at a dose of 180 mg / kg. Mice with a blood glucose > 300 mg / dL measured 2 days later are considered to have successfully established the model (hereinafter referred to as diabetic mice). The diabetic mice are anesthetized by intraperitoneal injection of sodium pentobarbital, and a full-thickness skin wound (about 5 mm in diameter) is made on their backs with forceps and surgical scissors.

[0062] Thereafter, the mice are randomly divided into 5 groups, and their wounds are given a single P. aeruginosa infection (10 μL, 10 10 CFU / mL). The 5 groups of mice are treated with dressings including the following components respectively: 1) blank (no dressing is used); 2) HD; 3) GaHD; 4) LRNF; 5) GaHD / LRNF. Subsequently, the mice are placed in a unified environment for feeding. The degree of wound healing is continuously monitored on the 0th, 3rd, 7th, 14th, and 21st days.

[0063] Figure 6The therapeutic effect of the GaHD / LRNF complex according to an embodiment of the present application on a full-thickness skin wound model in mice, that is, the curve of the healing efficiency (%) changing with time. Where the wound healing efficiency = [(initial wound area - remaining wound area at the time of detection) / initial wound area]*100%. As Figure 6 shown, the wound healing efficiency of the HD group was the same as that of the blank group, that is, there was no healing effect on the wound; when GaHD and LRNF were used alone, they both had a certain healing effect on the wound; the GaHD / LRNF had the best healing effect on the wound, and the healing effect at 21 days could reach about 90%.

[0064] Wound swab samples were collected on the 7th, 14th, and 21st days for 16S rDNA sequencing to analyze the bacterial community composition. The mice were sacrificed on the 21st day, and the wound tissues were collected for pathological sectioning and H&E staining to evaluate the effect of the treatment in each group on tissue remodeling.

[0065] The above embodiments are only for illustrating the present invention and are not intended to limit the present invention. Those of ordinary skill in the relevant technical field can make various changes and modifications without departing from the scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the disclosure of the present invention.

Claims

1. A complex for promoting wound healing, comprising: A basal layer configured to absorb wound exudate and stop bleeding; and A probiotic-loaded layer configured to regulate the wound flora structure; The probiotic-loaded layer is deposited on the basal layer.

2. The composite according to claim 1, wherein the basal layer is loaded with a preparation targeting harmful bacteria in the wound.

3. The composite according to claim 2, wherein the harmful bacteria are iron-metabolizing bacteria.

4. The composite according to claim 2, wherein the basal layer is configured to slowly release the preparation.

5. The composite according to claim 2, wherein the preparation comprises gallium ions.

6. The composite according to claim 1, wherein the basal layer comprises hyaluronic acid or dopamine-grafted hyaluronic acid.

7. The composite according to claim 1, wherein the probiotic-loaded layer comprises polyvinyl alcohol nanofibers.

8. The composite according to claim 1, wherein the probiotic-loaded layer comprises probiotics selected from one or more of the following groups: Staphylococcus epidermidis, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus casei, Saccharomyces cerevisiae, Bacillus, Bifidobacterium, Clostridium butyricum.

9. Use of the composite according to any one of claims 1-8 in the preparation of a medicament for promoting wound healing.

10. The use according to claim 9, wherein the wound is a wound of a diabetic patient.

11. A medicament for promoting wound healing, comprising the composite according to any one of claims 1-8.

12. A method for preparing the composite according to any one of claims 1-8, comprising: Preparing a basal layer, comprising: Obtaining dopamine-grafted hyaluronic acid; Mixing dopamine-grafted hyaluronic acid with a preparation targeting harmful bacteria in the wound; Freezing the mixed solution in an environment of -50 to -80 °C for 10-15 hours; and Freeze-drying for 40-60 hours to obtain the basal layer; and Preparing a probiotic-loaded layer, comprising: Obtaining a PVA solution; Adding probiotics to the PVA solution to obtain a spinning solution; and [[ID= ​

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