Probiotic gel and application thereof in treatment of diabetic complications

Abstract

The invention provides a probiotic gel and application thereof in treatment of diabetic complications, the probiotic gel comprises an engineering modified probiotic, the probiotic is Weissella, the Weissella is combined with a liposome containing deferoxamine through electrostatic adsorption, the surface of the Weissella has positive charges, and the Weissella has positive charges. The surface of the liposome is negatively charged. The engineering modified probiotics can release NO and deferoxamine at the same time and has a good treatment effect on oxidative stress and inflammation, the invention further provides the probiotic gel, the probiotic gel is successfully applied to treatment of diabetic complications, particularly treatment of diabetic foot ulcers, and the probiotic gel has a good application prospect. The probiotic gel can significantly promote angiogenesis and wound healing, has an anti-inflammatory effect, and has important significance in treatment of diabetic foot ulcer.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to a probiotic gel and an application thereof in the treatment of diabetic complications. Background Art

[0002] Diabetic foot ulcer (DFU) is a common and serious complication in diabetic patients, who are at risk of amputation. The amputation rate of DFU patients is 10 to 20 times higher than that of non-diabetic patients, mainly due to severe limb ischemia. Studies have shown that high blood sugar levels in diabetic ischemic limbs catalyze the production of a large number of toxic free radicals, which significantly inhibit the activity of hypoxia-inducible factor, thereby hindering transcription-mediated tissue repair, cell growth and proliferation.

[0003] Current angioplasty is often not ideal in restoring blood circulation in patients with ischemic foot ulcers. For example, although surgical bypass surgery is effective, it causes a lot of trauma, requires a long recovery time, and increases the risk of infection and thrombosis. In addition, although angioplasty is a less invasive option, it has a low efficacy. And although it is effective in specific cases of severe limb ischemia, it is not generally applicable to all patients (Beckman JA, Schneider PA, Conte MS. Advances in Revascularization for Peripheral Artery Disease: Revascularization in PAD. Circ Res. 2021 Jun 11; 128 (12): 1885-1912.).

[0004] Probiotic therapy has gained significant recognition in clinical research, especially the use of engineered bacteria to promote wound healing. Weissella cibaria (WC) is a probiotic used for oral health care, which is recognized for its anti-inflammatory and antibacterial properties. WC can synthesize NO locally, which helps regulate cardiovascular function and stimulate skin cell activation, thereby promoting angiogenesis (Chen HH, Fu FS, Chen QW, Zhang Y, Zhang XZ. Two-Pronged Microbe Delivery of Nitric Oxide and Oxygen for Diabetic Wound Healing. Nano Lett. 2023 Jun 28; 23 (12): 5595-5602.). However, the efficacy of WC alone in treating diabetic ischemic limbs is lower than the ideal standard, mainly because the high glucose levels in diabetic ischemic limbs catalyze the production of a large number of toxic free radicals. These free radicals significantly inhibit the activity of hypoxia-inducible factor, thereby hindering transcription-mediated tissue repair, cell growth and proliferation. Summary of the invention

[0005] In view of the deficiencies in the treatment of diabetic foot ulcers in the prior art, the present invention provides a probiotic gel and its application in the treatment of diabetic complications, specifically including an engineered probiotic, wherein the probiotic is Weissella, and the engineered probiotic combines deferoxamine with Weissella to catalyze the symbiotic relationship, and improves the oxidative stress in the ischemic microenvironment through the synergistic dual effects of NO and deferoxamine, stimulates the formation and proliferation of endothelial tubular structures, and then promotes local revascularization and wound healing in diabetic foot ulcers, and has good safety. Compared with the use of WC or deferoxamine alone, the effect is significantly enhanced, especially in promoting the migration of HUVECs, wound healing, and promoting local angiogenesis. The specific scheme is as follows:

[0006] The first aspect of the present invention provides an engineered probiotic, wherein the probiotic is Weissella, and Weissella is combined with liposomes containing deferoxamine through electrostatic adsorption.

[0007] The surface of the Weissella is positively charged. Preferably, the surface of the Weissella is modified by poly(Ethylene imine), poly(allylamine hydrochloride) or metal cations.

[0008] Preferably, the metal cations include one or more of zinc ions, calcium ions, copper ions, iron ions, manganese ions or magnesium ions.

[0009] The liposome surface is negatively charged. Preferably, the liposome contains one or more of poly(lactic-co-glycolic acid), poly(D,L-lactide-co-glycolide)-polyethylene glycol-carboxyl, methoxypolyethylene glycol poly(lactic-co-glycolic acid), polydopamine or indocyanine green-azide.

[0010] In a specific embodiment of the present invention, the surface of the Weissella is modified by polyallylamine hydrochloride, and the liposome contains poly(lactic-co-glycolic acid).

[0011] The hydrated particle size of the engineered Weissella is 100-150 nm.

[0012] The Weissella includes one or more of Weissella cibaria, Weissella viridescens, Weissella paramesenteroides, Weissella kandleri or Weissella hellenica.

[0013] In a specific embodiment of the present invention, the Weissella is Weissella cibaria.

[0014] The second aspect of the present invention provides a method for constructing the engineered probiotics described in the first aspect, the method comprising combining Weissella with liposomes containing deferoxamine by electrostatic adsorption.

[0015] Preferably, the positively charged Weissella spp. is mixed with liposomes containing deferoxamine in a solvent (eg, water), and then centrifuged (preferably at 4000 g-8000 g for 1-5 min) to obtain engineered Weissella spp. (WC@DPA).

[0016] Preferably, the construction method comprises:

[0017] Step 1: Modify Weissella to give it a positive surface charge;

[0018] Step 2: preparing liposomes containing deferoxamine; the surface of the liposomes is negatively charged;

[0019] Step 3: The Weissella with positive surface charge obtained in step 1 is mixed with the liposome with negative surface charge obtained in step 2, and the engineered Weissella (WC@DPA) is obtained after centrifugation.

[0020] The step 2 comprises:

[0021] S2.1: dissolving deferoxamine in polyvinyl alcohol solution;

[0022] S2.2: emulsifying the solution obtained in S2.1 with ethyl acetate containing poly(lactic acid-co-glycolic acid) copolymer;

[0023] S2.3: ultrasonically treat the mixture obtained in S2.2 to obtain a primary emulsion;

[0024] S2.4: transfer the primary emulsion into sucrose solution and then perform ultrasonic treatment;

[0025] S2.5: The mixture obtained in S2.4 was stirred at room temperature overnight to completely evaporate the ethyl acetate;

[0026] S2.6: Centrifuge the mixture obtained in S2.5, and wash the precipitate three times with deionized water to obtain liposomes.

[0027] The amplitude of the ultrasound is 10%-50%, preferably 15%-30%, for example 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

[0028] The ultrasonic time in the ultrasonic treatment is 80s-200s, preferably 100s-150s, for example 80s, 90s, 100s, 120s, 130s, 140s, 150s, 160s, 170s, 180s, 190s, 200s.

[0029] The concentration of polyvinyl alcohol in S2.1 is 1%-5%, preferably 2%-5%, for example 1%, 2%, 2.4%, 2.8%, 3%, 3.5%, 4%, 4.5%, 5%.

[0030] The concentration of the poly(lactic acid-co-glycolic acid) copolymer in ethyl acetate in S2.2 is 1%-5%, preferably 2%-4%, for example 1%, 2%, 3%, 4%, 5%.

[0031] The concentration of sucrose in S2.4 is 10%-60%, preferably 15%-50%, for example 10%, 20%, 30%, 40%, 50%, 60%.

[0032] The centrifugal force of the centrifugation in the S2.6 is 5000g-15000g, preferably 8000g-12000g, for example 5000g, 6000g, 7000g, 8000g, 9000g, 10000g, 11000g, 12000g, 13000g, 14000g, and 15000g.

[0033] The centrifugation time in the S2.6 is 5 min-20 min, preferably 8 min-15 min, for example 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, and 20 min.

[0034] In a specific embodiment of the present invention, the step 2 is:

[0035] S2.1: Dissolve deferoxamine in 2.8% polyvinyl alcohol solution;

[0036] S2.2: emulsify the solution obtained in S2.1 with ethyl acetate containing 3% of poly(lactic acid-co-glycolic acid) copolymer;

[0037] S2.3: The mixture obtained in S2.2 was sonicated at 25% amplitude for 120 s to obtain a primary emulsion;

[0038] S2.4: The primary emulsion was transferred to a 20% sucrose solution and then sonicated for 120 s;

[0039] S2.5: The mixture obtained in S2.4 was stirred at room temperature overnight to completely evaporate the ethyl acetate;

[0040] S2.6: The mixture obtained in S2.5 was centrifuged at 9000 g for 10 min, and the precipitate was washed three times with deionized water to obtain liposomes.

[0041] The centrifugal force of the centrifugation in the step three is 4000g-8000g. Preferably, the centrifugal force of the centrifugation is 5000g-7000g, for example, 4000g, 4500g, 5000g, 5500g, 6000g, 6500g, 7000g, 7500g, 8000g.

[0042] The centrifugation time in step three is 1 min-5 min. Preferably, the centrifugation time is 2 min-4 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min.

[0043] The third aspect of the present invention provides a probiotic gel, wherein the probiotic gel comprises the engineered probiotics described in the first aspect or the engineered probiotics obtained by the construction method described in the second aspect.

[0044] The probiotic gel comprises one or more of polyvinyl alcohol, polyacrylamide, sodium alginate, chitosan, hydroxyethyl methacrylate, hyaluronic acid or its salt, carboxymethyl cellulose or gelatin, preferably hyaluronic acid.

[0045] In a specific embodiment of the present invention, the probiotic gel is WC@DPA encapsulated with 5-20% (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20%) hyaluronic acid.

[0046] The fourth aspect of the present invention provides a method for preparing the probiotic gel described in the third aspect, the preparation method comprising mixing the engineered probiotics described in the first aspect or the engineered probiotics obtained by the construction method described in the second aspect with one or more of polyvinyl alcohol, polyacrylamide, sodium alginate, chitosan, hydroxyethyl methacrylate, hyaluronic acid or its salt, carboxymethyl cellulose or gelatin.

[0047] Preferably, the mixing includes stirring mixing.

[0048] Preferably, the mixing includes mixing at room temperature.

[0049] The preparation method comprises removing bubbles from the mixture obtained after mixing, preferably by ultrasonic removal of the bubbles from the mixture.

[0050] The concentration of the engineered probiotics is 10 4 -10 12 CFU / mL, preferably 10 6 -10 10 CFU / mL, e.g. 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL, 10 11 CFU / mL, 10 12 CFU / mL.

[0051] In a specific embodiment of the present invention, the concentration of the probiotics is 10 8 CFU / mL.

[0052] In a specific embodiment of the present invention, the preparation method is to mix the engineered probiotics described in the first aspect or the engineered probiotics obtained by the construction method described in the second aspect with hyaluronic acid or a salt thereof.

[0053] In a specific embodiment of the present invention, the preparation method is to mix positively charged probiotics (preferably PAH-modified) with DPA and hyaluronic acid or its salt.

[0054] The concentration of the probiotics is 10 4 -10 12 CFU / mL, preferably 10 6 -10 10 CFU / mL, e.g. 10 4 CFU / mL, 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL, 10 9 CFU / mL, 10 10 CFU / mL, 10 11 CFU / mL, 10 12 CFU / mL.

[0055] The molar amount of DPA is 0.1-5 μM, for example, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5 μM.

[0056] The concentration of the hyaluronic acid or its salt is 5-15 mg / mL, preferably 8-12 mg / mL, for example 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL.

[0057] Preferably, the concentration of the hyaluronic acid or its salt is 10 mg / mL.

[0058] The fifth aspect of the present invention provides an application of the engineered probiotic described in the first aspect, the engineered probiotic obtained by the construction method described in the second aspect, the probiotic gel described in the third aspect, or the probiotic gel obtained by the preparation method described in the fourth aspect in the preparation of drugs for promoting wound healing, promoting angiogenesis, anti-inflammation, or treating diabetic complications.

[0059] The wounds include chronic wounds and / or non-chronic wounds.

[0060] Preferably, the chronic wound comprises a diabetic foot ulcer.

[0061] Preferably, the non-chronic wounds include one or more of trauma, burns, scalds, pressure sores, ulcers or dermatitis.

[0062] In a specific embodiment of the present invention, the wound is a diabetic foot ulcer.

[0063] The diabetic complications include diabetic foot ulcers.

[0064] The sixth aspect of the present invention provides a medicine, which comprises the engineered probiotics described in the first aspect, the engineered probiotics obtained by the construction method described in the second aspect, the probiotic gel described in the third aspect, or the probiotic gel obtained by the preparation method described in the fourth aspect, and pharmaceutically acceptable excipients.

[0065] The pharmaceutically acceptable excipients include one or more of excipients, diluents, adhesives, lubricants, humectants, emulsifiers, preservatives, antioxidants, buffers, antibacterial agents, disintegrants, surfactants, suspending agents, suspending aids, solubilizers, thickeners, stabilizers, sweeteners and flavors.

[0066] The medicament can be used to treat humans or non-human animals, such as non-human mammals.

[0067] The drug can be administered by any suitable route, such as enteral administration (e.g. oral) or parenteral administration (e.g. intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.).

[0068] The drug can be in any suitable dosage form, such as a dosage form for gastrointestinal administration or a dosage form for parenteral administration, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nasal drops, sublingual tablets, suppositories, aerosols, effervescent tablets, pills, gels, etc.

[0069] The various dosage forms of the drug can be prepared according to conventional production methods in the pharmaceutical field.

[0070] The drug may contain 0.01-99.5% (specifically, 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) of the engineered probiotics described in the first aspect or the engineered probiotics obtained by the construction method described in the second aspect or the probiotic gel described in the third aspect or the probiotic gel obtained by the preparation method described in the fourth aspect in a weight ratio.

[0071] The drug has one or more effects of promoting wound healing, promoting angiogenesis or anti-inflammation or treating diabetic complications.

[0072] The wounds include chronic wounds or non-chronic wounds.

[0073] According to a seventh aspect of the present invention, a liposome containing deferoxamine is provided, wherein the surface of the liposome is negatively charged.

[0074] S2.1: dissolving deferoxamine in polyvinyl alcohol solution;

[0075] S2.2: emulsifying the solution obtained in S2.1 with ethyl acetate containing poly(lactic acid-co-glycolic acid) copolymer;

[0076] S2.3: ultrasonically treat the mixture obtained in S2.2 to obtain a primary emulsion;

[0077] S2.4: transfer the primary emulsion into sucrose solution and then perform ultrasonic treatment;

[0078] S2.5: The mixture obtained in S2.4 was stirred at room temperature overnight to completely evaporate the ethyl acetate;

[0079] S2.6: Centrifuge the mixture obtained in S2.5, and wash the precipitate three times with deionized water to obtain liposomes.

[0080] The amplitude of the ultrasound is 10%-50%, preferably 15%-30%, for example 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

[0081] The ultrasound time is 80s-200s, preferably 100s-150s, for example 80s, 90s, 100s, 120s, 130s, 140s, 150s, 160s, 170s, 180s, 190s, 200s.

[0082] The concentration of polyvinyl alcohol in S2.1 is 1%-5%, preferably 2%-5%, for example 1%, 2%, 2.4%, 2.8%, 3%, 3.5%, 4%, 4.5%, 5%.

[0083] The concentration of the poly(lactic acid-co-glycolic acid) copolymer in ethyl acetate in S2.2 is 1%-5%, preferably 2%-4%, for example 1%, 2%, 3%, 4%, 5%.

[0084] The concentration of sucrose in S2.4 is 10%-60%, preferably 15%-50%, for example 10%, 20%, 30%, 40%, 50%, 60%.

[0085] The centrifugal force of the centrifugation in the S2.6 is 5000g-15000g, preferably 8000g-12000g, for example 5000g, 6000g, 7000g, 8000g, 9000g, 10000g, 11000g, 12000g, 13000g, 14000g, and 15000g.

[0086] The centrifugation time in the S2.6 is 5 min-20 min, preferably 8 min-15 min, for example 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, and 20 min.

[0087] In a specific embodiment of the present invention, the step 2 is:

[0088] S2.1: Dissolve deferoxamine in 2.8% polyvinyl alcohol solution;

[0089] S2.2: emulsify the solution obtained in S2.1 with ethyl acetate containing 3% of poly(lactic acid-co-glycolic acid) copolymer;

[0090] S2.3: The mixture obtained in S2.2 was sonicated at 25% amplitude for 120 s to obtain a primary emulsion;

[0091] S2.4: The primary emulsion was transferred to a 20% sucrose solution and then sonicated for 120 s;

[0092] S2.5: The mixture obtained in S2.4 was stirred at room temperature overnight to completely evaporate the ethyl acetate;

[0093] S2.6: The mixture obtained in S2.5 was centrifuged at 9000 g for 10 min, and the precipitate was washed three times with deionized water to obtain liposomes.

[0094] In the eighth aspect of the present invention, a method for treating diabetic foot ulcers is provided, the method comprising applying to an individual in need an effective amount of the engineered probiotics described in the first aspect, the engineered probiotics obtained by the construction method described in the second aspect, the probiotic gel described in the third aspect, the probiotic gel obtained by the preparation method described in the fourth aspect, the drug described in the sixth aspect, or the liposome described in the seventh aspect.

[0095] The ninth aspect of the present invention provides a method for promoting wound healing or angiogenesis or anti-inflammation, the method comprising applying an effective amount of the engineered probiotics described in the first aspect, the engineered probiotics obtained by the construction method described in the second aspect, the probiotic gel described in the third aspect, the probiotic gel obtained by the preparation method described in the fourth aspect, the drug described in the sixth aspect, or the liposome described in the seventh aspect to an individual in need.

[0096] The tenth aspect of the present invention provides a method for treating diabetic complications, the method comprising applying an effective amount of the engineered probiotics described in the first aspect, the engineered probiotics obtained by the construction method described in the second aspect, the probiotic gel described in the third aspect, the probiotic gel obtained by the preparation method described in the fourth aspect, the drug described in the sixth aspect, or the liposome described in the seventh aspect to an individual in need.

[0097] The term "pharmaceutically acceptable" as used herein refers to a substance that neither significantly irritates an organism nor inhibits the biological activity and properties of the active substance of the administered product.

[0098] As used herein, "treating" means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease after the disease has begun to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders.

[0099] The "effective amount" of the present invention refers to the amount or dosage of the WC@DPA or drug of the present invention that provides the desired treatment after being administered to an individual or an organ in a single or multiple doses.

[0100] The "individual" described in the present invention can be a human or a non-human mammal, or a cell, tissue or organ of a human or a non-human mammal. The non-human mammal can be a wild animal, a zoo animal, an economic animal, a pet, an experimental animal, etc. Preferably, the non-human mammal includes but is not limited to pigs, cattle, sheep, horses, donkeys, foxes, raccoon dogs, minks, camels, dogs, cats, rabbits, mice (such as rats, mice, guinea pigs, hamsters, gerbils, chinchillas, squirrels) or monkeys, etc.

[0101] The "and / or" described in the present invention includes all combinations of items connected by the term, and each combination should be deemed to have been listed separately herein. For example, "A and / or B" includes "A", "A and B" and "B". For another example, "A, B and / or C" includes "A", "B", "C", "A and B", "A and C", "B and C" and "A and B and C".

[0102] Beneficial Effects

[0103] (1) The present invention provides an engineered probiotic, wherein the probiotic is Weissella, specifically Weissella cibaria (WC), and the surface of the engineered WC is combined with a liposome containing deferoxamine to form WC@DPA. Experimental verification shows that preparing deferoxamine in the form of liposomes does not affect the release of deferoxamine, and the modification of WC by liposomes does not affect the normal growth and function of WC.

[0104] (2) WC@DPA exerts dual effects by releasing NO and deferoxamine. The engineered WC@DPA was prepared into a gel, and the therapeutic effect of WC@DPA gel was verified in vivo and in vitro.

[0105] A: In in vitro experiments, human skin fibroblasts were cultured under high sugar and low oxygen conditions to simulate the microenvironment of diabetic foot ulcers. After treatment with WC@DPA gel, the proliferation and migration of skin fibroblasts were promoted, and angiogenesis was promoted. In the scratch experiment, the wound area was significantly reduced after treatment with WC@DPA gel, indicating that WC@DPA gel has the function of promoting angiogenesis and wound healing.

[0106] B: In the in vivo experiment, STZ was used to induce diabetic mice, and a circular wound with a diameter of 10 mm was made on the back of the mice. The therapeutic effect of WC@DPA gel on the wound was monitored. The results showed that after treatment with WC@DPA gel, the ROS level of mice was reduced, the level of pro-inflammatory factors was reduced, the level of anti-inflammatory factors was increased, the vascular density of mice was enhanced, and the wound area showed a more complete epidermis and abundant granulation tissue, indicating that WC@DPA gel can reduce local tissue inflammation and oxidative stress in diabetic mice, promote wound healing, and has good safety.

[0107] (3) Using diabetic mice with ischemic injury in the lower limbs as a research model for diabetic foot ulcers, after treatment with WC@DPA gel, the mice showed recovery of healthy muscle morphology and increased capillary density. The limbs of the treated mice showed a significant decrease in the level of pro-inflammatory factors and a significant increase in the level of anti-inflammatory factors, that is, WC@DPA gel converted persistent inflammation into an acute inflammatory response that is more conducive to healing, and accelerated the ability of vascular reconstruction on the 5th day after treatment.

[0108] (4) WC@DPA gels possess unique properties, such as eliminating harmful hydroxyl radicals, rebalancing the oxidative state, and remodeling the chronic inflammatory landscape of DFU. In addition, WC inherently maintains the continuous production of NO, promoting local delivery to ischemic areas, which is particularly beneficial for enhancing blood circulation in the lower limbs. Experimental evidence strongly suggests that the dual effects of DFO liposomes and NO-releasing engineered WC significantly improve cell survival, migration, and oxidative stress management, and accelerate the reconstruction of angiogenesis and muscle regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1 : Schematic diagram of the preparation of WC@DPA and its gel and the promotion of revascularization after DFU limb ischemia injury. WC@DPA gel releases NO and DFO into the ischemic area to relieve oxidative stress and chronic inflammation, where PLAG stands for poly(lactic-co-glycolic acid), DFO stands for deferoxamine, DES-D stands for thin film hydration ultrasound, WC stands for Weissella cibaria, and PAH stands for polyallylamine hydrochloride.

[0110] Figure 2 : Effects of different WC concentrations on the fluorescence intensity of NO-specific probe DAR-1.

[0111] Figure 3 :NO release of different concentrations of WC, where a is the Griess determination standard curve for NO detection, and b is the Griess determination of NaNO in different concentrations of WC 2 concentration.

[0112] Figure 4 : Scavenging effects of different concentrations of DFO and DPA on hydroxyl radicals.

[0113] Figure 5 : Hydration particle size distribution of liposomes (DPA).

[0114] Figure 6 : Zeta potential change during the preparation process of WC@DPA.

[0115] Figure 7 : Representative TEM images of DPA, WC, and WC@DPA from left to right.

[0116] Figure 8 : Colony images of WC and WC@DPA.

[0117] Fig. 9 : The number of bacteria per plate of WC and WC@DPA.

[0118] Fig.10 : Fluorescence intensity of NO-specific probe DAR-1 in WC, WC@DPA and WC@DPA.

[0119] Fig.11 : DFO release curves of DPA and WC@DPA.

[0120] Fig.12 : Schematic diagram of WC@DPA gel promoting wound healing.

[0121] Fig.13 :Use H 2 O 2Representative graphs of DCFH-DA probe fluorescence intensity in stimulated RAW264.7 macrophages.

[0122] Fig.14 :Use H 2 O 2 Quantitative analysis of DCFH-DA probe fluorescence intensity in stimulated RAW264.7 macrophages, ***p<0.001.

[0123] Fig.15 : Representative fluorescence images of M1 / M2 levels in different groups.

[0124] Fig.16 : Relative density of M1 / M2 levels in different groups.

[0125] Fig.17 : The expression levels of IL-6 in RAW264.7 macrophages stimulated by LPS in different groups, **p<0.01.

[0126] Fig.18 :The expression levels of IL-10 in RAW264.7 macrophages stimulated by LPS in different groups, ***p<0.001.

[0127] Fig.19 : Representative images of HSFs in different groups under 33 mM glucose and 6 h of hypoxia, where the scale bar is 100 μm, and Hypoxia+HG represents hypoxia+high glucose conditions.

[0128] Fig. 20 : Cell viability analysis of HSFs in different groups under 33 mM glucose and 6 h hypoxia, scale bar is 100 μm, ***p<0.001.

[0129] Fig.21 : Representative images of angiogenesis in HUVECs, where the scale bar is 100 μm, and Hypoxia+HG represents hypoxia+high glucose conditions.

[0130] Fig. 22 : Quantitative analysis of vessel length in HUVECs, where the scale bar is 100 μm, Hypoxia+HG represents hypoxia+high glucose conditions, ***p<0.001.

[0131] Fig.23 : Representative images of transwell migration assay performed on HUVECs. Scale bar is 100 μm, where Hypoxia+HG indicates hypoxia+high glucose conditions.

[0132] Fig.24: Quantitative analysis of the transwell migration experiment performed on HUVECs, the scale bar is 100 μm, where Hypoxia+HG represents hypoxia+high glucose conditions, ***p<0.001.

[0133] Fig.25 : Representative images of HUVECs migration, scale bar is 100 μm, where Hypoxia+HG indicates hypoxia+high glucose conditions.

[0134] Fig.26 : Quantitative analysis of HUVECs migration, the scale bar is 100 μm, where Hypoxia+HG represents hypoxia+high glucose conditions, **p<0.01.

[0135] Fig. 27 : Body weight and blood glucose concentration of mice in different groups, where a is body weight and b is blood glucose concentration.

[0136] Fig.28 : Schematic diagram of the chronic wound treatment process in diabetic mice.

[0137] Fig.29 : Representative images of wound areas of different groups on days 0, 4, 8 and 14, n=6.

[0138] Fig.30 : Graphs showing wound healing areas of different groups on days 0, 4, 8 and 14, n=6.

[0139] Fig.31 : The number of days required for 50% wound healing in different groups, n=6, ***p<0.001.

[0140] Fig.32 : DCFH-DA staining and CD31 immunohistochemical analysis of different groups on day 6, n=3.

[0141] Fig.33 : Quantitative analysis of DCFH-DA in wound areas of different groups on day 6, n=3, ***p<0.001.

[0142] Fig.34 : Quantitative analysis of the average microvessel density in different groups on day 14, n=3, *p<0.05, ***p<0.001.

[0143] Fig.35 : H&E and Masson images of wound tissues in different groups on day 14, n=3.

[0144] Fig.36 : H&E images of wound areas in different groups on days 3 and 6, scale bar is 100 μm.

[0145] Fig.37 : Granulation tissue thickness of different groups on days 3 and 6, n=3.

[0146] Fig.38 : Masson staining images of wound areas in different groups on days 3 and 6, scale bar is 100 μm.

[0147] Fig.39 : Quantification of collagen deposition in different groups on days 3 and 6.

[0148] Fig.40 : Granulation tissue thickness in different groups on day 14, n=3, *p<0.05, **p<0.01.

[0149] Fig.41 : Quantification of collagen deposition in different groups on day 14, n=3, ***p<0.001.

[0150] Fig.42 : Quantitative analysis of epidermal thickness in different groups on day 14, n=3, **p<0.01.

[0151] Fig.43 : The expression levels of IL-6 in wound tissues of different groups on day 6, *p<0.05.

[0152] Fig.44 : On the 6th day, the expression levels of IL-10 in wound tissues of different groups, **p<0.01, ***p<0.001.

[0153] Fig.45 : H&E staining images of mouse heart, liver, spleen, lung and kidney sections.

[0154] Fig.46 : Serum biochemical parameters of mice in different groups, n=3.

[0155] Fig.47 : Routine hematological test indicators of mice in different groups, n=3.

[0156] Fig.48 : Schematic diagram of the treatment process of ischemic limbs in diabetic mice, where LDF refers to laser Doppler perfusion image.

[0157] Fig.49 : Representative laser Doppler perfusion images of ischemic hindlimbs of non-diabetic mice, untreated group, WC@DPA gel-treated, DPA gel-treated, and WC gel-treated on days 0, 2, 6, 10, and 14.

[0158] Fig.50 : Quantitative analysis of blood perfusion in the hindlimb by the ischemic / non-ischemic ratio.

[0159] Fig.51 : H&E staining images of non-ischemic limbs and ischemic limbs in different groups.

[0160] Fig.52 : H&E staining images of non-ischemic limbs and ischemic limbs in different groups, scale bar is 100 μm.

[0161] Fig.53 : Immunofluorescence images of dystrophin in nonischemic and ischemic limbs of different groups.

[0162] Fig.54 : Immunofluorescence images of dystrophin in non-ischemic and ischemic limbs of different groups, scale bar is 100 μm.

[0163] Fig.55 : Quantitative analysis of dystrophin in non-ischemic and ischemic limbs of different groups, *p<0.05.

[0164] Fig.56 : Quantitative analysis of CD31-positive endothelial cells in the blood vessels of the hind limbs of different groups.

[0165] Fig.57 : Immunofluorescence images of CD31-positive endothelial cells in the blood vessels of the hind limbs of different groups.

[0166] Fig.58 : Immunofluorescence images of CD31-positive endothelial cells in the blood vessels of the hind limbs of different groups, scale bar is 100 μm. DETAILED DESCRIPTION

[0167] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0168] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0169] Unless otherwise specified, the reagents, materials, and instruments used in the following examples can be obtained from commercial sources.

[0170] The present invention provides an engineered Weissella, specifically taking Weissella cibaria (WC) as an example, WC can locally synthesize nitric oxide (NO), promote delivery to ischemic areas, regulate cardiovascular function, stimulate skin fibroblast activation, and promote angiogenesis. The present invention combines liposomes containing deferoxamine (DPA) with WC to obtain WC@DPA, and integrates WC@DPA into hyaluronic acid gel to obtain a potent WC@DPA gel.

[0171] Taking diabetic foot ulcer (DFU) as an example, the WC@DPA gel has a significant effect in the treatment of DFU, such as eliminating harmful hydroxyl radicals, rebalancing the oxidative state and the chronic inflammatory pattern of DFU. The WC@DPA gel releases NO and DFO into the ischemic area, relieves oxidative stress and chronic inflammation, improves cell survival and migration, and promotes angiogenesis, muscle regeneration and revascularization (see Figure 1 ).

[0172] The starting strain of Weissella used in the following examples is Weissella cibaria, which comes from Henan BNCC Microbial Strain Engineering Technology Research Center. It is also recorded in the document Kang MS, Lee DS, Lee SA, Kim MS, Nam SH. Effects of probiotic bacterium Weissella cibaria CMU on periodontal health and microbiota: a randomised, double-blind, placebo-controlled trial. BMC Oral Health. 2020 Sep 2; 20(1): 243.

[0173] 1. Reagents used in the examples

[0174] Polyallylamine hydrochloride (PAH, Mw: 17500 Da), hyaluronic acid (HA), poly(lactic-co-glycolic acid) (PLGA) and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich.

[0175] 2′,7-dichlorofluorescein diacetate (DCFH-DA), cell viability detection kit and total nitric oxide assay kit were purchased from Biotechnology Research Institute; hydroxyl free radical scavenging ability assay kit was purchased from Sangon Biotech Co., Ltd.; mouse IL-6 / IL-10 ELISA kit was from Elaright Biotech Co., Ltd.

[0176] 2. Statistical analysis methods involved in the embodiments

[0177] Data are presented as individual measurements, and bars represent group mean ± SD. Statistical significance was assessed by one-way ANOVA. p < 0.05 was considered statistically significant.

[0178] Example 1: Preparation and characterization of WC@DPA

[0179] 1.1 Preparation of DPA

[0180] Liposome (DPA) was prepared by double emulsion solvent diffusion method. First, DFO (20 mg) was dissolved in 1 mL of 2.8% polyvinyl alcohol (PVA) solution, and then emulsified with 3 mL of ethyl acetate containing 3% PLGA. The mixture was ultrasonically treated for 120 seconds with 25% amplitude using a probe ultrasonic generator to form a primary emulsion, and the emulsion was then transferred to a 20% sucrose solution of 10 mL and subjected to secondary ultrasonic treatment for 120 seconds. Subsequently, the mixture was stirred at room temperature overnight to promote complete evaporation of ethyl acetate. Liposome (DPA) was collected by centrifugation at 9000 g for 10 minutes and washed 3 times with deionized water.

[0181] 1.2. Synthesis of WC@DPA

[0182] First, the surface of WC was modified with biocompatible polyallylamine hydrochloride (PAH) to make it positively charged. Then 1 mL of PAH-modified WC solution was mixed with 2 mg of DPA (1 mg / mL) in ultrapure water and gently shaken for 60 seconds. The resulting solution was centrifuged at 6000g for 3 minutes to obtain WC@DPA.

[0183] 1.3 Preparation of WC@DPA gel

[0184] WC@DPA gel consists of 1×10 8 CFU / mL PAH-modified WC, 2μM DPA, and 10mg / mL hyaluronic acid were mixed, mixed at room temperature by a magnetic stirrer, and bubbles were removed by ultrasound to obtain WC@DPA gel. That is, 10% hyaluronic acid was used to encapsulate WC, DPA, or WC@DPA.

[0185] 1.4. NO detection

[0186] The NO-sensitive fluorescent probe DAR-1 (20 μM) was added to the WC culture solution, WC@DPA culture solution or WC@DPA gel, and then cultured in MRS medium at 37°C with shaking. The NO fluorescence intensity was measured using a microplate reader (excitation wavelength of 566 nm and emission wavelength of 586 nm). In addition, the NO concentration was examined using the Griess assay.

[0187] 1.5. Hydroxyl radical detection

[0188] Prepare different concentrations of DFO in a 10 mL volumetric flask and use the corresponding volume of distilled water as a blank control. First, add 1 mL of o-phenanthroline (5 mM), 4 mL of pH 7.4 phosphate buffer solution and 1 mL of 5 mM FeSO 4 The solution was then adjusted to 10 mL with methanol. The absorbance of the superoxide anion radical system was monitored at a wavelength of 320 nm using an ultraviolet spectrophotometer every 20 minutes.

[0189] The same method was used to detect the hydroxyl radical scavenging ability of liposomes (DPA).

[0190] 1.6 Test results

[0191] To evaluate the ability of WC to produce NO, the DAR-1 fluorescent probe was used, combined with the results of Griess analysis. The data showed that the concentration of 1×10 8 CFU / mL WC produced the highest amount of NO ( Figure 2 and Figure 3 ). Therefore, this optimal concentration was selected for subsequent experiments. The scavenging ability of DFO on hydroxyl radicals was verified by hydroxyl radical detection test. When the concentration of DFO was 4 μM, the hydroxyl radicals were significantly reduced. When the concentration of DFO was 2 μM, the scavenging ability of hydroxyl radicals was the strongest. The scavenging ability of liposomes (DPA) on hydroxyl radicals was detected, and it was found that there was no significant difference in the scavenging ability of DFO and liposomes (DPA) on hydroxyl radicals ( Figure 4 ). The hydrated particle size of WC@DPA at the optimal concentration is 122.4nm ( Figure 5 ).

[0192] The zeta value of the engineered bacteria WC@DPA is -7.04mV ( Figure 6 ), transmission electron microscopy (TEM) showed that WC initially had a smooth surface, and after the addition of liposomes (DPA), the WC surface became obviously granular ( Figure 7 ). These results confirm the effective synthesis of DPA and WC.

[0193] To verify the growth activity of WC, WC and WC@DPA were incubated in MRS medium for 24 h. The results showed that the addition of liposomes did not hinder the growth of WC ( Figure 8 and Fig. 9 ). In addition, comparative analysis of NO generation showed no significant difference between WC@DPA, WC@DPA gel, and unmodified WC, suggesting that the specific modification of the liposomes as well as the gel formulation did not impair the function of WC ( Fig.10). In order to study the effect of engineering modification on DFO release, WC@DPA and DPA were placed in PBS solution. Fig.11 It can be seen that the DFO release was 20% in the first 12 hours, and then about 50% in 48 hours. After more than 72 hours, the drug release curve gradually became consistent. The initial rapid release phase may be due to the diffusion of DFO through the outer layer of the liposomes, while the extended release phase indicates that the liposome matrix continues to leak out. There is no significant difference in the DFO release between WC@DPA and DPA.

[0194] Example 2: WC@DPA inhibits oxidative stress and anti-inflammation

[0195] By exposing RAW264.7 macrophages to hydrogen peroxide (H 2 O 2 ), and then co-cultured them with WC@DPA gel using the Transwell system to observe the in vitro anti-inflammatory effect of WC@DPA gel ( Fig.12 ).

[0196] 2.1. Intracellular ROS detection

[0197] RAW264.7 macrophages were cultured at 5 × 10 4 The cells were seeded at a density of 100 cells / well in the lower chamber of a 24-well Transwell system. The control group was cultured in basal 1640 medium at 37°C, and the experimental group was cultured with 100 μM H 2 O 2 After incubation for 1 hour, the cells were divided into 4 groups, including H 2 O 2 The first group was cultured in basal 1640 medium at 37°C, and the other three groups were inoculated with DPA gel, WC gel, and WC@DPA gel in the upper chamber of a 24-well Transwell system and maintained at 37°C. After 24 hours, all cell groups were incubated with the DCFH-DA fluorescent probe for 30 minutes at room temperature in the dark and imaged using a confocal laser scanning microscope (FV1000, Olympus). The experiment showed that WC@DPA gel could reduce H 2 O 2 Induced oxidative stress ( Fig.13 and Fig.14 ).

[0198] 2.2 Anti-inflammatory effect of WC@DPA gel in vitro

[0199] To evaluate the anti-inflammatory effect of WC@DPA gel on lipopolysaccharide (LPS)-treated RAW264.7 macrophages, RAW264.7 macrophages were cultured at 5×10 4Cells were seeded at a density of 100 cells / well in the lower chamber of a 24-well Transwell system. WC@DPA gel, WC gel, and DPA gel were added to the upper chamber of the Transwell system. After incubation, cells were stained with F4 / 80 (Biolegend, Cat. No. 123109), CD86 (Biolegend, Cat. No. 159203), and CD206 (Biolegend, Cat. No. 141707) antibodies and imaged using a confocal laser scanning microscope.

[0200] The polarization of macrophages into pro-inflammatory (M1-like) or anti-inflammatory (M2-like) phenotypes determines the healing process of chronic wounds. The study found that WC@DPA gel can effectively polarize M1 macrophages stimulated by lipopolysaccharide (LPS) into M2 macrophages, while CD86 expression is reduced and CD206 expression is increased ( Fig.15 and Fig.16 ), and also demonstrated that WC@DPA gel can effectively reduce the level of pro-inflammatory cytokine IL-6 and increase the level of anti-inflammatory cytokine IL-10 ( Fig.17 and Fig.18 ), indicating that WC@DPA gel has anti-inflammatory properties.

[0201] Example 3: WC@DPA promotes the proliferation of skin fibroblasts

[0202] 3.1. WC@DPA gel promotes the proliferation of skin fibroblasts

[0203] Co-culturing human skin fibroblasts (HSFs) under high glucose and hypoxia conditions can replicate the microenvironment of diabetic foot ulcers. HSFs were cultured in a medium containing 33 mM glucose (5 × 10 4 / well), exposed to 1% O 2 The cells were incubated for 6 hours. WC@DPA gel, DPA gel or WC gel were added to the upper chamber of the Transwell system. Calcein-AM staining was used to assess the viability of HSFs, and confocal laser scanning microscopy was used for imaging. ImageJ software was used for quantitative analysis of the results.

[0204] The live cell staining test confirmed that both the WC@DPA gel group and the DPA gel group promoted the proliferation of HSFs ( Fig.19 and Fig. 20 ).

[0205] 3.2. WC@DPA gel promotes angiogenesis of HUVECs

[0206] Human umbilical vein endothelial cells (HUVECs) were cultured at 5×10 4Cells / well were seeded in the lower chamber of a Matrigel-coated culture plate containing 33 mM glucose and incubated in 1% O 2 After culturing for 6 hours, WC@DPA gel, DPA gel or WC gel were added to the upper chamber of the Matrigel-coated culture plate, and the angiogenesis process was observed and imaged using an optical microscope. The results showed that the number of connection nodes and the total length of blood vessels increased ( Fig.21 and Fig. 22 ), indicating that co-culture with WC@DPA gel can promote angiogenesis.

[0207] 3.3 WC@DPA gel promotes the migration of HUVECs

[0208] HUVECs were cultured at 1×10 4 Cells / well were seeded in the upper chamber of the Matrigel-coated culture plate, and DPA gel, WC gel or WC@DPA gel was added to the lower chamber. HUVECs that migrated to the bottom surface were stained with 0.5% crystal violet solution for 1 hour, and then imaged and quantitatively analyzed using an optical microscope. The results showed that WC@DPA gel had a positive effect on the migration function of HUVECs ( Fig.23 and Fig.24 ).

[0209] In addition, HUVECs were cultured at 1×10 5 The density of cells / well was subcultured in the lower chamber. A sterile p200 pipette tip was used to scratch and then washed with PBS to remove the detached cells. The scratch test further confirmed the migration function of WC@DPA gel in promoting HUVECs ( Fig.25 ). Under the dual stress of high glucose and hypoxia, the scratch wound area was significantly reduced when co-cultured with WC@DPA gel, indicating that wound healing was improved ( Fig.26 ).

[0210] In summary, these in vitro experiments indicate that WC@DPA gel not only promotes the formation and growth of endothelial cells, but also facilitates the proliferation of human skin fibroblasts and the migration response of epidermal cells, thereby promoting the wound healing process.

[0211] Example 4: WC@DPA promotes wound healing in diabetic mice

[0212] 4.1 Evaluation of the diabetic wound healing model

[0213] Animal experiments were conducted using Balb / C male mice (weighing about 25 grams), specifically: diabetic mice were intraperitoneally injected with 100 mg / kg streptozotocin (STZ) once a day for two days. Animal experiments were conducted in accordance with the ethical guidelines of the Animal Ethics Committee of Hubei University (Animal Ethics Number: 20240022). The mice were randomly divided into five groups: acute wound group (Non-DM), diabetic chronic wound group (DM-Control), diabetic chronic wound group given DPA gel (DM-DPA), diabetic chronic wound group given WC gel (DM-WC), and diabetic chronic wound group given WC@DPA gel (DM-WC@DPA). All mice were housed under a 12-hour light-dark cycle, and the wounds were photographed every other day to monitor the progress of wound healing, and the images were analyzed using ImageJ software. After 14 days of treatment, all mice were sacrificed and skin tissues were collected for evaluation, including hematoxylin and eosin (H&E) staining, Masson's trichrome staining, enzyme-linked immunosorbent assay for IL-6 and IL-10, and immunohistochemistry / immunofluorescence staining for CD31 / DCFH-DA.

[0214] The results showed that STZ-induced diabetic mice showed typical symptoms, including weight loss and increased blood sugar ( Fig. 27 A precise full-thickness circular wound with a diameter of 10 mm was made in the central area of ​​the back of the mouse. The healing process was monitored by regular photographic recordings and DPA gel, WC gel or WC@DPA gel ( Fig.28 ), the healing rate of diabetic mice was significantly slower compared with the non-diabetic group, however, the wounds treated with WC@DPA gel showed the fastest healing progress ( Figure 29-Figure 31 ).

[0215] In addition, on the 6th day after treatment, the ROS level in the skin tissue was evaluated using DCFH-DA staining, and it was found that the ROS level in diabetic mice was significantly higher. Compared with the non-diabetic group, both DPA gel and WC@DPA gel treatment effectively reduced the ROS level ( Figure 32-33 In addition, CD31 immunohistochemical analysis showed that the vascular density of WC@DPA gel-treated mice was enhanced on day 14 of treatment ( Fig.34 ).

[0216] To further explore the therapeutic effect of WC@DPA gel, skin samples from each treatment group were collected on days 3, 6, and 14 after treatment for histological analysis. The epidermal integrity of the diabetic control group was reduced, and granulation tissue formation was reduced ( Figure 35-Figure 39 ). In contrast, the epidermis in the area treated with WC@DPA gel was more intact and had more abundant granulation tissue ( Figure 40-Figure 42 ).

[0217] The anti-inflammatory potential of WC@DPA gel was further investigated by measuring the levels of inflammatory mediators by ELISA. The IL-6 level was increased in the diabetic control group, which may hinder wound healing ( Fig.43 Treatment with WC@DPA gel not only reduced the expression of pro-inflammatory chemokines such as IL-6, but also increased the level of anti-inflammatory IL-10 ( Fig.44 ), indicating that WC@DPA gel is effective in reducing inflammation and angiogenesis. These findings suggest that WC@DPA gel has the potential to alleviate local tissue inflammation and oxidative stress.

[0218] 4.2 Safety Assessment

[0219] On the 14th day of treatment, blood was collected from mice for routine hematological tests. Complete blood count (CBC) included the measurement of white blood cells (WBC), red blood cells (RBC), and platelets (PLT). Serum biochemical parameters such as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were evaluated. For histopathological analysis: Mice were euthanized, and the main organs of the mice (heart, liver, spleen, lung, kidney) were taken and fixed in 4% paraformaldehyde (PFA), followed by hematoxylin and eosin (H&E) staining.

[0220] Histological examination results showed that WC@DPA had no adverse effects on the normal physiological functions of mice ( Fig.45 In addition, routine blood tests (WBC, PLT, RBC) and blood biochemical examinations of inflammation, liver and kidney function indicators showed that WC@DPA did not cause any significant changes ( Fig.46 and Fig.47 ).

[0221] Example 5: WC@DPA promotes revascularization of hindlimb ischemia

[0222] The use of diabetic mice with lower limb ischemic injury as a diabetic foot ulcer (DFU) model is a recognized standard and helps to understand the basic mechanisms of the disease.

[0223] Model establishment: When establishing the limb ischemia model, surgery was performed on the left hind limb of diabetic mice. The skin incision was sutured after ligation, and gel was applied to the site. Real-time blood flow in both hind limbs was detected every two days using the moorFLPI imaging system (Moor instruments). After 14 days of treatment, all mice were sacrificed and limb tissues were collected for evaluation, including hematoxylin and eosin (H&E) staining, polymerase chain reaction (PCR) array, and immunofluorescence staining of anti-dystrophin and CD31.

[0224] To evaluate the revascularization effect of WC@DPA gel in ischemic injury, diabetic mice with unilateral hindlimb ischemic injury were treated with DPA gel, WC gel, and WC@DPA gel ( Fig.48 ). Laser Doppler perfusion imaging showed a gradual decrease in blood flow in the untreated group, in stark contrast to the WC@DPA-treated group, where blood flow recovery was significantly enhanced ( Figure 49-Figure 50 ). H&E staining highlighted severe muscle bundle damage in diabetic controls, while WC@DPA gel-treated mice showed restoration of healthy muscle morphology ( Fig.51 and Fig.52 ), which was different from the observations in the WC and DPA groups. Subsequent dystrophin staining emphasized the important role of WC@DPA gel in promoting muscle fiber repair, as evidenced by the increase in dystrophin-positive fibers compared with the DPA gel and WC gel groups ( Figure 53-Figure 55 ). Cross-sectional analysis of muscle fibers showed that fiber size was reduced in the ischemic limb compared with the non-ischemic limb in all experimental groups. Notably, mice treated with WC@DPA gel showed intact and regular fiber morphology, and WC@DPA gel had a protective or repairing effect relative to the DPA gel and WC gel treatment groups. On day 7, the density of CD31-positive capillaries in the WC@DPA gel group was significantly higher than that in the DPA gel and WC gel groups, highlighting the pro-angiogenic effect of WC@DPA ( Figure 56-Figure 58 ).

Claims

1. An engineered probiotic, characterized in that: The probiotics are Weissella, and the Weissella is combined with the liposome containing deferoxamine through electrostatic adsorption.

2. The engineered probiotic according to claim 1, characterized in that: The surface of the Weissella is positively charged, and the surface of the liposome is negatively charged.

3. The engineered probiotic according to claim 1, characterized in that: The Weissella includes one or more of Weissella cibaria, Weissella viridescens, Weissella paramesenteroides, Weissella kandleri or Weissella hellenica.

4. A method for constructing the engineered probiotic according to any one of claims 1 to 3, characterized in that: The construction method comprises: Step 1: Modify Weissella to give it a positive surface charge; Step 2: preparing liposomes containing deferoxamine; the surface of the liposomes is negatively charged; Step 3: Mix the Weissella with positive surface charge obtained in step 1 with the liposome with negative surface charge obtained in step 2, and obtain engineered Weissella after centrifugation.

5. A probiotic gel, characterized in that: The probiotic gel comprises the engineered probiotics according to any one of claims 1 to 3 or the engineered probiotics obtained by the construction method according to claim 4.

6. The probiotic gel according to claim 5, characterized in that The probiotic gel comprises one or more than two of polyvinyl alcohol, polyacrylamide, sodium alginate, chitosan, hydroxyethyl methacrylate, hyaluronic acid or its salt, carboxymethyl cellulose or gelatin.

7. Use of the engineered probiotics according to any one of claims 1 to 3, the engineered probiotics obtained by the construction method according to claim 4, or the probiotic gel according to any one of claims 5 to 6 in the preparation of drugs for promoting wound healing, promoting angiogenesis, anti-inflammation, or treating diabetic complications.

8. The use according to claim 7, characterized in that: The wounds include chronic wounds and / or non-chronic wounds.

9. The use according to claim 8, characterized in that: The chronic wounds include diabetic foot ulcers; The non-chronic wounds include one or more of trauma, burns, scalds, pressure sores, ulcers or dermatitis.

10. A drug, characterized in that The medicine comprises the engineered probiotics according to any one of claims 1 to 3, or the engineered probiotics obtained by the construction method according to claim 4, or the probiotic gel according to any one of claims 5 to 6, and pharmaceutically acceptable excipients.

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