A live probiotic culture delivery hydrogel scaffold and methods of making and using the same

Abstract

This invention relates to the field of materials science and technology, providing a live probiotic culture and delivery hydrogel scaffold, its preparation method, and its applications. The method provided by this invention is simple to operate and offers significant potential for application. The prepared hydrogel scaffold, when used in medical dressings for treating infected skin wounds, exhibits characteristics such as ultra-efficient polymerization, bioprintability, and injectability. It also demonstrates significant antibacterial and repair effects on infected skin wound models, achieving in-situ curing and sealing of the wound with only 1 second of UV irradiation. The method provided by this invention ensures excellent survival rates, controllable colonization efficiency, and sufficient biocompatibility of the probiotics embedded within the hydrogel scaffold. The antibacterial strategy of this invention significantly inhibits the colonization of pathogenic microorganisms, reshapes the composition of the host's symbiotic microbiota, and is microenvironmentally friendly.

Description

Technical Field

[0001] This invention belongs to the field of materials science and technology, and particularly relates to a hydrogel scaffold for the culture and delivery of live probiotics, its preparation method, and its application. Background Technology

[0002] The skin is the largest and most important organ in the human body, but it is easily damaged due to injury, disease, or surgery, severely impairing its natural antibacterial ability and leading to wound infections. To prevent such complications, various antibacterial methods have been developed to inhibit the proliferation of harmful bacteria and accelerate wound healing. However, these antibacterial substances are likely to cause pathogens to develop resistance and may eradicate beneficial probiotics. Therefore, there is an urgent need to develop strategies that can inhibit the growth of harmful bacteria and harmonize the natural balance of the skin's microbiome.

[0003] Probiotic therapy is an emerging wound treatment method. Lactobacillus reuteri, in particular, can produce various antimicrobial molecules such as reuteri protein, organic acids, and ethanol, inhibiting the proliferation of pathogenic microorganisms, reshaping the composition of symbiotic bacteria, and balancing the host's immune system. Importantly, using Lactobacillus reuteri helps treat pathogens and eradicate bacteria without producing the common side effects associated with antibiotic treatment. However, the application of probiotics is severely limited due to their low viability, metabolic activity, and colonization efficiency during processing, storage, and transportation. Currently used wound hydrogel dressings for packaging and delivering various antimicrobial substances generally require a long cross-linking time (greater than 1 min), which may reduce the effectiveness of the encapsulated probiotics and significantly hinder ease of use. Therefore, it is essential to develop a highly efficient polymeric dressing solution that effectively protects the activity of probiotics. Simultaneously, droplet microfluidics can precisely produce uniform hydrogel microspheres, accurately controlling the probiotic loading density within the dressing. Applying rapid gelation reactions of alginate and calcium ions on a microfluidic platform can minimize the damage to probiotics in adverse environments such as hypoxia, cross-linking agents, or surfactant accumulation. Therefore, we propose a hydrogel scaffold for live probiotic culture delivery, its preparation method, and its applications. Summary of the Invention

[0004] The purpose of this invention is to provide a hydrogel scaffold for the culture and delivery of live probiotics, its preparation method, and its application, in order to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] A method for preparing a hydrogel scaffold for live probiotic culture delivery includes the following steps:

[0007] Step S1: Preparation of norbornene-modified gelatin: Dissolve 1-2 g of 5-norbornene-2-carboxylic acid in 10-40 mL of dichloromethane. After complete dissolution, add 2-3 g of N-hydroxysuccinimide and 3-4 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Stir at room temperature for 20-24 h to obtain a white mixture. Wash the mixture three times with an appropriate amount of saturated NaHCO3 solution and deionized water, respectively. Finally, dry the mixture with anhydrous MgSO4. After drying, rotary evaporate the solution to obtain a white solid product. Dissolve 1-2 g of gelatin in a mixture of 10-50 mL of DMF and water and stir until completely dissolved. Add 1-2 g of the white solid product and 60-100 µL of N,N-diisopropylethylamine. React at room temperature for 8-12 h. Dialyze the mixture in deionized water using a dialysis bag for 3-5 days. Freeze dry to obtain norbornene-modified gelatin.

[0008] Step S2: Preparation of thiol-modified gelatin: Dissolve 2-3 g of dimethyl 3,3-dithiopropionate and 2-3 mL of hydrazine hydrate in 20-50 mL of methanol, stir overnight at room temperature, filter the resulting suspension to obtain a white solid, and wash three times with methanol and deionized water respectively; dissolve 1-2 g of gelatin in 100-300 mL of deionized water, stir to dissolve, add 0.5-1 g of white solid, adjust the pH of the resulting solution to 4-5, then add 0.5-0.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and after 2-4 h, raise the pH of the solution to 7; then mix 4-5 g of dithiothreitol with the solution, stir to dissolve, and adjust the pH of the solution to 8-9; after reacting the solution at room temperature for 1 day, adjust the pH to 3-4.5; transfer the solution to a dialysis bag containing 0.1 mol / L... -1 Dialysis with NaCl in an acidic dialysis buffer with a pH of 3–4.5 for 1–2 days, followed by dialysis with 0.3 mmol / mL solution. -1 Dialyze in an aqueous HCl solution for 1-2 days, then dialyze in deionized water for 1-2 days, and freeze-dry to obtain thiol-modified gelatin;

[0009] Step S3: Fabrication of microfluidic chip;

[0010] Step S4: Prepare the microfluidic dispersed phase and continuous phase solution:

[0011] Aqueous phase: The hydrogel precursor solution of the dispersed phase specifically includes an aqueous solution of 1-2% sodium alginate, 25-50 mM calcium EDTA, and Lactobacillus reuteri at a certain density;

[0012] Oil phase: The continuous phase solution specifically comprises mineral oil containing 4-6% by volume SPAN 80 and 0.05-0.1% by volume acetic acid;

[0013] Step S5: Preparation of gel microspheres loaded with live probiotics: The aqueous phase and oil phase are loaded into syringes and attached to an injection pump. The syringes are connected to a microfluidic chip via polyethylene tubing. The injection pump controls the flow rates of the aqueous and oil phases separately, with flow rates of 100~1000 µL / h for the aqueous and oil phases, respectively. -1 and 200~2000 µL h -1 After a certain production period, the generated gel microspheres were collected and washed with mineral oil, PBS solution containing 0.5-2% TWEEN 20, and PBS solution by centrifugation to obtain gel microspheres loaded with live Lactobacillus reuteri.

[0014] Step S6: Prepare a hydrogel scaffold for live probiotic culture delivery, including:

[0015] Step S61: Prepare an aqueous solution of norbornene gelatin containing 6-20% by mass of the gelatin obtained in step S1;

[0016] Step S62: Prepare an aqueous solution of thiol gelatin obtained in step S2 containing 0.05-0.1% by mass of the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 6-20% by mass of the photoinitiator.

[0017] The solutions obtained in steps S61 and S62 are mixed in a certain proportion, and then the gel microspheres loaded with live Lactobacillus reuteri obtained in step S5 are added to obtain a hydrogel scaffold for live probiotic culture delivery.

[0018] Furthermore, in steps S1 and S2, the molecular weight of the gelatin is 5-10 W, and the gelatin is pigskin gelatin or fish skin gelatin; the molecular weight cutoff of the dialysis bag is 8000-15000 Da.

[0019] Furthermore, in step S3, the angle between the dispersed phase channel and the continuous phase channel of the microfluidic chip is 55~70°.

[0020] Furthermore, in step S4, the Lactobacillus reuteri contained in the dispersed phase is replaced with probiotics, cells, or biological functional factors that fight harmful bacteria.

[0021] Furthermore, in step S4, the method for preparing calcium ethylenediaminetetraacetate is as follows:

[0022] After mixing calcium chloride and ethylenediaminetetraacetic acid in a 1:1 molar ratio, the pH of the mixed solution was adjusted to 7.4.

[0023] Furthermore, in step S5, when the flow rate of the oil phase is 1000 µL / h... -1 The size of the obtained gel microspheres ranged from 50 to 300 µm.

[0024] A live probiotic culture delivery hydrogel scaffold prepared according to the method.

[0025] Application of a live probiotic culture delivery hydrogel scaffold in the preparation of medical dressings for treating infected skin wounds.

[0026] Compared with the prior art, the beneficial effects of the present invention are:

[0027] 1. The method provided by this invention is simple to operate and has great potential. The prepared hydrogel scaffold is applied to medical dressings for treating infected skin wounds. It has the characteristics of ultra-high efficiency polymerization, bioprintability and injectability. It also has significant antibacterial and repair effects on infected skin wound models. It can be cured in situ and seal the wound with only 1 second of ultraviolet irradiation.

[0028] 2. The method provided by this invention can ensure that the probiotics embedded in the hydrogel scaffold have excellent survival rate, controllable colonization efficiency and sufficient biosafety.

[0029] 3. The antibacterial strategy of the present invention can significantly inhibit the colonization of pathogenic microorganisms and reshape the composition of the symbiotic microbiome in the host, and is microenvironmentally friendly. Attached Figure Description

[0030] Figure 1 In the image, (a) shows the NMR spectra of norbornene gelatin (GelNB) and mercapto gelatin (GelSH) obtained by modifying gelatin as the main component; (b) shows a schematic diagram of the microfluidic chip structure; (c) shows a photograph of the gel microspheres obtained by microfluidic chip production (the embedded image is a high-magnification microscope image); (d) shows the size range of gel microspheres produced under different aqueous and oil phase flow rates; and (e) shows the reaction diagram of the dressing after ultraviolet irradiation.

[0031] Figure 2 In the diagram, (a) is a schematic diagram of the hydrogel scaffold being extruded; (b) is a schematic diagram of the hydrogel scaffold being injected; and (c) is a schematic diagram of the 3D printing of the hydrogel scaffold.

[0032] Figure 3 In the images, (a) shows colony images of Staphylococcus aureus and Escherichia coli after co-culturing with the control group, a sterile hydrogel scaffold (GelNBSH), and a sterile hydrogel scaffold (GelNBSH-L), respectively; (b) shows images of the inhibition zones of the sterile hydrogel scaffold and the sterile hydrogel scaffold; (c) shows scanning electron microscope images of Staphylococcus aureus and Escherichia coli after co-culturing with the control group, a sterile hydrogel scaffold, and a sterile hydrogel scaffold, respectively (the bacteria at the arrows show obvious collapse); and (d) shows confocal images of live Lactobacillus reuteri loaded with microspheres in the dressing after 1 day and 3 days of culture.

[0033] Figure 4In the image, (a) shows representative digital photographs of Staphylococcus aureus-infected mouse back wounds after treatment with the control group, pure hydrogel dressing, and live probiotic dressing on days 0, 2, 4, 7, 10, and 14, respectively; (b) is an overlay image of the wound changes corresponding to (a). Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0036] An embodiment of the present invention provides a method for preparing a hydrogel scaffold for live probiotic culture delivery, comprising the following steps:

[0037] Step S1: Preparation of norbornene-modified gelatin: Dissolve 1-2 g of 5-norbornene-2-carboxylic acid in 10-40 mL of dichloromethane. After complete dissolution, add 2-3 g of N-hydroxysuccinimide and 3-4 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Stir at room temperature for 20-24 h to obtain a white mixture. Wash the mixture three times with an appropriate amount of saturated NaHCO3 solution and deionized water, respectively. Finally, dry the mixture with anhydrous MgSO4. After drying, rotary evaporate the solution to obtain a white solid product. Dissolve 1-2 g of gelatin in a mixture of 10-50 mL of DMF and water (volume ratio 1:1) and stir until completely dissolved. Add 1-2 g of the white solid product and 60-100 µL of N,N-diisopropylethylamine. React at room temperature for 8-12 h. Dialyze the mixture in deionized water using a dialysis bag for 3-5 days. Freeze-dry the gelatin to obtain norbornene-modified gelatin and store it at low temperature.

[0038] Step S2: Preparation of thiol-modified gelatin: Dissolve 2-3 g of dimethyl 3,3-dithiopropionate and 2-3 mL of hydrazine hydrate in 20-50 mL of methanol, stir overnight at room temperature, filter the resulting suspension to obtain a white solid, and wash three times with methanol and deionized water respectively; dissolve 1-2 g of gelatin in 100-300 mL of deionized water (temperature above room temperature), stir to dissolve, add 0.5-1 g of white solid, adjust the pH of the resulting solution to 4-5 with 0.1 mol mL⁻¹ HCl aqueous solution, then add 0.5-0.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, after 2-4 h, raise the pH of the solution to 7 with 0.1 mol mL⁻¹ NaOH aqueous solution; then mix 4-5 g of dithiothreitol with the solution, stir to dissolve, and adjust the pH of the solution to 8-9; react the solution at room temperature for 1 hour. After 3 days, adjust the pH to 3-4.5; transfer the solution to a dialysis bag containing 0.1 mol / mL. -1 Dialysis with NaCl in an acidic dialysis buffer with a pH of 3–4.5 for 1–2 days, followed by dialysis with 0.3 mmol / mL solution. -1 Dialyze in an aqueous HCl solution (excluding NaCl) for 1-2 days, then dialyze in deionized water for 1-2 days, freeze-dry to obtain thiol-modified gelatin, and store at low temperature;

[0039] Step S3: Fabrication of the microfluidic chip: Following silicon substrate cleaning, photoresist spin coating, UV exposure mask, plasma etching, polydimethylsiloxane (PDMS) and crosslinking agent mixing and infusion, curing, demolding, PDMS and substrate surface oxygen plasma activation, and channel bonding, a microfluidic chip is obtained (see David A. Weitz et al. Small, 1702955, 14, (2018)). The microchannel structure design is as follows... Figure 1 As shown in (b), the overall width and height of the channel are both approximately 200 μm. The delivery channel, after the dispersed phase is cut by the continuous phase, can have its width appropriately increased and is wavy to buffer the flow turbulence and stabilize the droplet shape. Furthermore, the angle between the dispersed phase channel and the continuous phase channel is 55–70°.

[0040] Step S4: Prepare the microfluidic dispersed phase and continuous phase solution:

[0041] Aqueous phase: The hydrogel precursor solution of the dispersed phase specifically includes an aqueous solution of 1-2% sodium alginate, 25-50 mM calcium EDTA, and Lactobacillus reuteri at a certain density;

[0042] The preparation method of calcium ethylenediaminetetraacetate is as follows: calcium chloride and ethylenediaminetetraacetic acid are mixed in a molar ratio of 1:1, and the pH of the mixed solution is adjusted to 7.4;

[0043] Oil phase: The continuous phase solution specifically includes mineral oil (CAS No. 8020-83-5) containing 4-6% by volume of SPAN 80 and 0.05-0.1% by volume of acetic acid.

[0044] Step S5: Preparation of gel microspheres loaded with live probiotics: All microfluidic devices were cleaned with alcohol and then sterilized by UV irradiation for 2 hours before use. All required solutions were sterilized by filtration (filter pore size 200 nm) before adding live Lactobacillus reuteri. Subsequently, the aqueous and oil phases were separately loaded into syringes and attached to the syringe pump. The syringes were connected to the microfluidic chip via polyethylene tubing. The syringe pump independently controlled the flow rates of the aqueous and oil phases, which were 100–1000 µL / h, respectively. -1 and 200~2000 µL h -1 (The flow rate of the oil phase is generally higher than that of the water phase.) After a certain period of production, the generated gel microspheres are collected and mineral oil, PBS solution containing 0.5-2% TWEEN 20 (by volume), and PBS solution are added and centrifuged to wash the microspheres to obtain gel microspheres loaded with live Lactobacillus reuteri.

[0045] Step S6: Prepare a hydrogel scaffold for live probiotic culture delivery, including:

[0046] Step S61: Prepare an aqueous solution of norbornene gelatin containing 6-20% by mass of the gelatin obtained in step S1;

[0047] Step S62: Prepare an aqueous solution of thiol gelatin obtained in step S2 containing 0.05-0.1% by mass of the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 6-20% by mass of the photoinitiator.

[0048] The solutions obtained in steps S61 and S62 are mixed in a certain proportion, and then the gel microspheres loaded with live Lactobacillus reuteri obtained in step S5 are added (the volume of the gel microspheres accounts for about 1 / 10 to 1 / 3 of the total volume after mixing) to obtain a hydrogel scaffold for live probiotic culture delivery.

[0049] In this embodiment of the invention, preferably, in step S3, the width and height of the delivery channel can be appropriately increased or decreased, which helps to increase or decrease the size of the resulting microdroplets when the flow rate of the two-phase solution remains constant. In step S6, the mixing ratio of the gel microspheres and the modified gelatin solution can be adjusted according to application requirements.

[0050] In a preferred embodiment of the present invention, in steps S1 and S2, the molecular weight of the gelatin is 5-10 w, and the gelatin is pigskin gelatin or fish skin gelatin; the molecular weight cutoff of the dialysis bag is 8000-15000 Da.

[0051] In a preferred embodiment of the present invention, in step S4, the *Lactobacillus reuteri* strain numbered ATCC6475 is cultured in MRS broth before being added to the dispersed phase at a temperature of 37 °C, and the loading density is adjustable according to the application scenario. The *Lactobacillus reuteri* contained in the dispersed phase can be replaced with other probiotics, cells, or biological functional factors that fight harmful bacteria.

[0052] In a preferred embodiment of the present invention, in step S5, when the flow rate of the oil phase is 1000 µL / h... -1 The size of the obtained gel microspheres ranged from 50 to 300 µm.

[0053] A live probiotic culture delivery hydrogel scaffold prepared according to the method.

[0054] Application of a live probiotic culture delivery hydrogel scaffold in the preparation of medical dressings for treating infected skin wounds.

[0055] The specific application is as follows: the dressing is loaded with a syringe and squeezed onto the wound surface, and then polymerized in situ on the wound by short-term ultraviolet light irradiation (not exceeding 1 second), for antibacterial and repair treatment of infected wounds.

[0056] In this embodiment of the invention, preferably, the dressing containing a live probiotic culture delivery hydrogel scaffold can be further refined into a tissue-like structure using 3D printing technology.

[0057] See Figure 1 (a) The spectrum shows the characteristic peaks corresponding to the modified functional groups, indicating successful modification. See also Figure 1 (c) As can be seen, the gel microspheres are of uniform size. See also Figure 1 (e) The dressing solidifies instantaneously in less than 1 second after being exposed to ultraviolet light.

[0058] See Figure 2 (a) Demonstrates the extrudability of the hydrogel scaffold. See also Figure 2 (b) Demonstrates the injectability of the hydrogel scaffold. See also Figure 2 (c) demonstrates the printability of the hydrogel scaffold.

[0059] See Figure 3 The results showed that the live probiotic culture delivery hydrogel scaffold significantly enhanced antibacterial activity and ensured the activity of the probiotics.

[0060] See Figure 4 The results showed that the live probiotic culture-delivered hydrogel scaffold had excellent repair effects on infected wounds.

[0061] Example 1: A method for preparing a hydrogel scaffold for live probiotic culture delivery, comprising the following steps:

[0062] Step S1: Dissolve 2 g of 5-norbornene-2-carboxylic acid in 40 mL of dichloromethane. After complete dissolution, add 2.24 g of N-hydroxysuccinimide and 3.58 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and stir at room temperature for 1 day. The resulting white mixture is washed three times with appropriate amounts of saturated NaHCO3 solution and deionized water, and finally dried with anhydrous MgSO4. After drying, the solution is rotary evaporated to obtain a white solid product. Dissolve 2 g of gelatin in 50 mL of a mixture of DMF and water (volume ratio 1:1) and stir until completely dissolved. Add 2 g of the above white product and then add 0.1 mL of N,N-diisopropylethylamine. After reacting at room temperature for 12 h, dialyze through a 15000 Da dialysis bag in deionized water for 5 days. Finally, freeze-dry to obtain norbornene-modified gelatin and store at -80 °C.

[0063] Step S2: Preparation of thiol-modified gelatin: Dissolve 2.4 g of dimethyl 3,3-dithiopropionate and 2.5 mL of hydrazine hydrate in 50 mL of methanol and stir overnight at room temperature. Filter the resulting suspension to obtain a white solid, and wash three times with methanol and deionized water, respectively. Dissolve 2 g of gelatin in 300 mL of deionized water (>37 ℃), stir to dissolve, and then add 1 g of the above white solid. Use 0.1 mol / L... -1 The pH of the resulting solution was adjusted to 4.50 with an aqueous HCl solution, and then 0.6 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide was added. After 4 h, 0.1 mol / mL... -1 The pH of the solution was raised to 7 using an aqueous NaOH solution. Then, 4.5 g of dithiothreitol was mixed with this solution, stirred until dissolved, and the pH was adjusted to 8. After reacting at room temperature for 1 day, the pH was adjusted to 4. The solution was then transferred to a dialysis bag and subjected to a reaction containing 0.1 mol / L NaOH. -1 The solution was thoroughly dialyzed for 2 days in an acidic dialysis buffer containing NaCl at pH 4, followed by 0.3 mmol / mL... -1 Dialyze the gelatin in an aqueous HCl solution (without NaCl) for 2 days, then dialyze it in deionized water for 2 days. Freeze-dry the gelatin to obtain thiol-modified gelatin and store it at -80 °C.

[0064] Step S3: Fabrication of the microfluidic chip: Following silicon substrate cleaning, photoresist spin coating, UV exposure mask application, plasma etching, polydimethylsiloxane (PDMS) and crosslinking agent (mixed in a 10:1 ratio) infusion, curing, demolding, PDMS activation with oxygen plasma on the substrate surface, and channel bonding, a microfluidic chip is obtained. Before being cut by the continuous phase, the pore width and height are both 200 μm. After cutting, the transport pore width is 250 μm, while the height remains unchanged, and it exhibits a wavy shape to buffer the turbulent flow and stabilize the droplet shape. Furthermore, the angle between the dispersed phase channel and the continuous phase channel is 60°.

[0065] Step S4: Prepare the microfluidic dispersed phase and continuous phase solution:

[0066] Aqueous phase: The dispersed phase of the hydrogel precursor solution specifically includes 2% sodium alginate (by mass), 50 mM calcium ethylenediaminetetraacetate (EDTA), and a solution with a density of 5.0 × 10⁻⁶. 8 mL -1 An aqueous solution of Lactobacillus reuteri;

[0067] The method for preparing calcium ethylenediaminetetraacetic acid is to mix calcium chloride and ethylenediaminetetraacetic acid in a 1:1 molar ratio and then adjust the pH to 7.4.

[0068] Oil phase: The continuous phase solution specifically comprises mineral oil containing 6% by volume SPAN 80 and 0.1% by volume acetic acid.

[0069] Step S5: All microfluidic devices were cleaned with alcohol and then sterilized by UV irradiation for 2 hours before use. All required solutions were sterilized by filtration (filter pore size 200 nm) before adding live Lactobacillus reuteri. Subsequently, the dispersed and continuous phase solutions were separately loaded into syringes and attached to the syringe pump. The syringes were connected to the microfluidic chip via polyethylene tubing, and the flow rates of both phases were individually controlled by the syringe pump. The flow rates of the aqueous and oil phases were fixed at 150 µL / h. -1 and 1000µL h -1 Gel microspheres with a size of 150 ± 5 µm were prepared. After a period of production, the generated gel microspheres were collected and washed three times by centrifugation with mineral oil, PBS solution containing 1% TWEEN 20, and PBS solution, respectively, to obtain gel microspheres loaded with live Lactobacillus reuteri.

[0070] Step S6: Prepare a hydrogel scaffold for live probiotic culture delivery, including:

[0071] Step S61: Prepare an aqueous solution of norbornene gelatin containing 10% by mass of the gelatin obtained in step S1;

[0072] Step S62: Prepare an aqueous solution of thiol gelatin obtained in step S2 containing 0.1% by mass of the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 10% by mass of the photoinitiator.

[0073] The solutions obtained in steps S61 and S62 are mixed in equal proportions, and then the gel microspheres loaded with live Lactobacillus reuteri obtained in step S5 (the volume of the gel microspheres accounts for about 1 / 5 of the total volume after mixing) are added to obtain a hydrogel scaffold for live probiotic culture delivery.

[0074] The live probiotic culture delivery hydrogel scaffold was used in the preparation of medical dressings for treating infected skin wounds. The dressing was loaded with a syringe and squeezed onto the wound surface. It was then polymerized in situ in the wound by brief ultraviolet light irradiation (not exceeding 1 second) for antibacterial and repair treatment of infected wounds.

[0075] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. A method for preparing a hydrogel scaffold for live probiotic culture delivery, characterized in that, Includes the following steps: Step S1: Preparation of norbornene-modified gelatin: Dissolve 1-2 g of 5-norbornene-2-carboxylic acid in 10-40 mL of dichloromethane. After complete dissolution, add 2-3 g of N-hydroxysuccinimide and 3-4 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide. Stir at room temperature for 20-24 h to obtain a white mixture. Wash the mixture three times with an appropriate amount of saturated NaHCO3 solution and deionized water, respectively. Finally, dry the mixture with anhydrous MgSO4. After drying, rotary evaporate the solution to obtain a white solid product. Dissolve 1-2 g of gelatin in a mixture of 10-50 mL of DMF and water and stir until completely dissolved. Add 1-2 g of the white solid product and 60-100 µL of N,N-diisopropylethylamine. React at room temperature for 8-12 h. Dialyze the mixture in deionized water using a dialysis bag for 3-5 days. Freeze dry to obtain norbornene-modified gelatin. Step S2: Preparation of thiol-modified gelatin: Dissolve 2-3 g of dimethyl 3,3-dithiopropionate and 2-3 mL of hydrazine hydrate in 20-50 mL of methanol, stir overnight at room temperature, filter the resulting suspension to obtain a white solid, and wash three times with methanol and deionized water respectively; dissolve 1-2 g of gelatin in 100-300 mL of deionized water, stir to dissolve, add 0.5-1 g of white solid, adjust the pH of the resulting solution to 4-5, then add 0.5-0.8 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and after 2-4 h, raise the pH of the solution to 7; then mix 4-5 g of dithiothreitol with the solution, stir to dissolve, and adjust the pH of the solution to 8-9; after reacting the solution at room temperature for 1 day, adjust the pH to 3-4.5; transfer the solution to a dialysis bag containing 0.1 mol / L... -1 Dialysis with NaCl in an acidic dialysis buffer (pH 3–4.5) for 1–2 days, followed by dialysis with 0.3 mmol / L solution. -1 Dialyze in an aqueous HCl solution for 1-2 days, then dialyze in deionized water for 1-2 days, and freeze-dry to obtain thiol-modified gelatin; Step S3: Fabrication of microfluidic chip; Step S4: Prepare the microfluidic dispersed phase and continuous phase solution: Aqueous phase: The hydrogel precursor solution of the dispersed phase specifically includes 1-2% sodium alginate, 25-50mM calcium ethylenediaminetetraacetate, and an aqueous solution of Lactobacillus reuteri at a certain density; Oil phase: The continuous phase solution specifically comprises mineral oil containing 4-6% by volume SPAN 80 and 0.05-0.1% by volume acetic acid; Step S5: Preparation of gel microspheres loaded with live probiotics: The aqueous phase and oil phase are loaded into syringes and attached to an injection pump. The syringes are connected to a microfluidic chip via polyethylene tubing. The injection pump controls the flow rates of the aqueous and oil phases separately, with the flow rates of the aqueous and oil phases being 100~1000 µL / h, respectively. -1 and 200~2000µL h -1 After a certain production period, the generated gel microspheres were collected and washed with mineral oil, PBS solution containing 0.5-2% TWEEN 20, and PBS solution by centrifugation to obtain gel microspheres loaded with live Lactobacillus reuteri. Step S6: Prepare a hydrogel scaffold for live probiotic culture delivery, including: Step S61: Prepare an aqueous solution of norbornene gelatin containing 6-20% by mass of the gelatin obtained in step S1; Step S62: Prepare an aqueous solution of thiol gelatin obtained in step S2 containing 0.05-0.1% by mass of the photoinitiator phenyl (2,4,6-trimethylbenzoyl) lithium phosphate and 6-20% by mass of the photoinitiator. The solutions obtained in steps S61 and S62 are mixed in a certain proportion, and then the gel microspheres loaded with live Lactobacillus reuteri obtained in step S5 are added to obtain a hydrogel scaffold for live probiotic culture delivery.

2. The method for preparing a live probiotic culture delivery hydrogel scaffold according to claim 1, characterized in that, In steps S1 and S2, the molecular weight of the gelatin is 5-10w, and the gelatin is pigskin gelatin or fish skin gelatin; the molecular weight cutoff of the dialysis bag is 8000-15000 Da.

3. The method for preparing a live probiotic culture delivery hydrogel scaffold according to claim 2, characterized in that, In step S3, the angle between the dispersed phase channel and the continuous phase channel of the microfluidic chip is 55~70°.

4. The method for preparing a live probiotic culture delivery hydrogel scaffold according to claim 3, characterized in that, In step S4, the method for preparing calcium ethylenediaminetetraacetate is as follows: calcium chloride and ethylenediaminetetraacetic acid are mixed in a 1:1 molar ratio, and the pH of the mixed solution is adjusted to 7.

4.

5. The method for preparing a live probiotic culture delivery hydrogel scaffold according to claim 1, characterized in that, In step S5, when the flow rate of the oil phase is 1000 µL / h -1 The size of the obtained gel microspheres ranges from 50 to 300 µm.

6. A live probiotic culture delivery hydrogel scaffold prepared by the method according to any one of claims 1-5.

7. The use of the live probiotic culture delivery hydrogel scaffold according to claim 6 in the preparation of medical dressings for treating infected skin wounds.

Images