Responsive release microcapsules, methods of making and using the same

Abstract

The present application relates to the technical field of wound care, and in particular to a responsive release microcapsule and a preparation method and application thereof.The preparation method comprises the following steps: S1, dissolving polylactic acid-glycolic acid copolymer in an organic solvent, adding an emulsifier, and ultrasonic emulsification to form a water-in-oil emulsion; S2, mixing a polyphenol compound, elastin, vegetable fat and a probiotic bacteria suspension to form a four-component mixed solution, and slowly adding the four-component mixed solution to the water-in-oil emulsion, continuing ultrasonic emulsification to form a multiple emulsion, evaporating and removing the organic solvent to obtain a four-component active ingredient mixed solution; S3, dissolving PDMAEMA in deionized water, adding a crosslinking agent and stirring, slowly adding the four-component active ingredient mixed solution, crosslinking reaction and freeze-drying to obtain the responsive release microcapsule.The microcapsule can release corresponding active ingredients according to the pH value change in different stages of wound healing, effectively shorten the wound healing time, improve the healing quality, and reduce scar formation.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of wound care, in particular to a responsive release microcapsule and a preparation method and application thereof. BACKGROUND

[0002] Wound healing is a complex physiological process composed of multiple stages. In the hemostasis stage, damaged blood vessels rapidly contract, and platelets respond immediately by aggregating to form a platelet plug. At the same time, coagulation factors are activated to convert fibrinogen into fibrin, which interweaves into a network to reinforce the thrombus, thereby effectively stopping bleeding. Subsequently, in the inflammation reaction stage, immune cells such as macrophages and neutrophils are attracted to the wound site by chemotactic factors and rapidly migrate to the wound site. Macrophages phagocytize pathogens and necrotic tissue, and release inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). These mediators, on the one hand, initiate the healing process, and on the other hand, if released in excess, can lead to excessive inflammation, which has a negative impact on wound healing. Then in the cell proliferation stage, fibroblasts proliferate and synthesize extracellular matrix such as collagen and fibronectin, and endothelial cells form new blood vessels to provide nutrients and oxygen for wound healing. Finally, in the tissue remodeling stage, the newly synthesized collagen fibers are continuously adjusted and arranged to enhance the strength and elasticity of the tissue at the wound site.

[0003] In the actual healing process, many problems seriously hinder the normal recovery of the wound. Infection is a very prominent problem. Pathogenic bacteria such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa prefer the warm, humid, and nutrient-rich environment of the wound, and multiply in large numbers. They not only consume the necessary nutrients for wound healing, but also secrete toxins, such as the α-hemolysin produced by Staphylococcus aureus, which can destroy cell integrity and trigger a strong inflammatory response, causing the wound to become red, swollen, and painful, with increased exudation, and in severe cases, systemic infections such as sepsis, greatly delaying the healing process. According to statistics, about 20%-30% of chronic wounds have varying degrees of infection.

[0004] Slow healing is also a common problem. For example, in diabetic patients, long-term high blood sugar causes damage to vascular endothelial cells, thickening of the blood vessel wall, and narrowing of the lumen, affecting blood supply to the wound site and hindering the transport of oxygen and nutrients. At the same time, the high blood sugar environment suppresses the function of immune cells, reducing the body's ability to fight infection, greatly prolonging the healing time of the wound, and even developing into a chronic non-healing wound. In addition, the depth and area of the wound, as well as the patient's age, nutritional status, etc., all have a significant impact on the healing rate. Large deep burn wounds often take months to heal, causing great pain to the patient.

[0005] Scar formation is also a big trouble for patients. When the inflammatory response is excessive or the tissue repair is unbalanced during the wound healing process, the fibroblasts proliferate excessively, synthesize too much collagen and arrange disorderly, leading to excessive proliferation of scar tissue. This not only affects the appearance of the skin, but also may cause skin dysfunction, such as scar contracture at the joint site which can limit joint movement, causing double burden to patients in life and psychology.

[0006] Traditional wound care products have obvious limitations. Ordinary gauze only relies on physical barrier effect to block foreign matter from contacting the wound, and cannot provide active ingredients to promote healing, and has weak effect on infection prevention and healing acceleration. Dressings containing antibacterial ingredients, such as silver ion-containing dressings, can inhibit bacterial growth, but silver ions have a high oxidation state and are easy to form complexes with many substances, which will change the stability and affect cell proliferation, migration and differentiation, interfering with the normal wound healing process. Moreover, traditional products are difficult to release corresponding active ingredients according to the physiological needs of wounds at different healing stages, and cannot achieve personalized treatment.

[0007] With the progress of medical technology and the improvement of people's health needs, there is an urgent need for a new type of wound care product that can simultaneously resist bacteria, reduce inflammation, promote tissue repair and regulate the microecological environment to accelerate wound healing and improve healing quality. SUMMARY

[0008] The purpose of the present application is to provide a responsive release microcapsule and its preparation method and application.

[0009] The first object of the present application is to provide a preparation method of a responsive release microcapsule, comprising the following steps:

[0010] S1, dissolving polylactic acid-glycolic acid copolymer in an organic solvent, adding an emulsifier, and ultrasonic emulsification to form a water-in-oil emulsion;

[0011] S2, mixing polyphenolic compounds, elastin, vegetable fat and probiotic bacteria suspension to form a four-component mixed solution, and slowly adding it to the water-in-oil emulsion formed in step S1, continuing ultrasonic emulsification to form a multiple emulsion, and then removing the organic solvent by evaporation to obtain a four-component active ingredient mixed solution;

[0012] S3, dissolving polymethyl methacrylate dimethylaminoethyl ester in deionized water, adding a crosslinking agent and stirring uniformly, slowly dropping the four-component active ingredient mixed solution obtained in step S2, and crosslinking to form microcapsules, which are washed and freeze-dried to obtain the responsive release microcapsule.

[0013] Further, the mass-volume ratio of the polyphenol compound, elastin, vegetable fat, and probiotic bacteria suspension is (1.5-2.5) g:(2.5-3.5) g:(3-5) ml:(0.8-1.2) ml. The concentration of the probiotic bacteria suspension is 1.0 x 10 9 ~1.0 x 10 10 CFU / ml.

[0014] Lactobacillus acidophilus can secrete organic acids such as lactic acid, reduce the local pH value of the wound, and create an acidic environment to inhibit the growth of harmful bacteria. At the same time, the antigen components on its surface can bind to the surface receptors of immune cells, regulate the immune system, and promote wound healing. Short-chain fatty acids such as acetic acid and propionic acid produced by Bifidobacterium can provide energy for cells around the wound and promote cell growth; the bacteriocins produced have antibacterial activity and can inhibit the reproduction of harmful bacteria.

[0015] Further, the polyphenol compound includes one or more of gallic acid and epigallocatechin-3-gallate.

[0016] High-purity polyphenol substances such as gallic acid and epigallocatechin-3-gallate (EGCG) are selected. The phenolic hydroxyl group in the molecular structure of gallic acid has strong antibacterial and antioxidant capacity. The phenolic hydroxyl group can bind to proteins and lipids on the bacterial cell membrane, destroy the integrity of the cell membrane, and effectively inhibit the growth of pathogenic bacteria such as Staphylococcus aureus and Escherichia coli. At the same time, it can provide hydrogen atoms to combine with free radicals, scavenge free radicals generated during the wound healing process, and reduce oxidative stress damage. EGCG promotes the proliferation of fibroblasts and the expression of elastin genes by regulating intracellular mitogen-activated protein kinase (MAPK) signaling pathways, thereby accelerating wound healing.

[0017] Further, the vegetable fat includes one or more of olive oil and macadamia nut oil.

[0018] Olive oil is rich in monounsaturated fatty acids such as oleic acid and vitamin E. Oleic acid can penetrate into the stratum corneum of the skin, fill the intercellular lipid gap, and form a protective film on the surface of the wound, reducing water evaporation. It also has anti-inflammatory effects by inhibiting the release of inflammatory mediators. Vitamin E, as a strong antioxidant, can neutralize free radicals and protect cells from oxidative damage, promoting wound healing. Macadamia nut oil is rich in oleic acid and palmitoleic acid, which can participate in the metabolism of skin cells, regulate cell physiological functions, promote cell metabolism, accelerate wound healing, and reduce scar formation.

[0019] Further, the probiotic bacteria are Lactobacillus acidophilus and Bifidobacterium.

[0020] Lactobacillus acidophilus can secrete organic acids such as lactic acid, reduce the local pH value of the wound, create an acidic environment to inhibit the growth of harmful bacteria. At the same time, the antigen components on its surface can bind to the surface receptors of immune cells to regulate the immune system and promote wound healing. Short-chain fatty acids such as acetic acid and propionic acid produced by Bifidobacterium can provide energy for cells around the wound and promote cell growth; the bacteriocins produced have antibacterial activity and can inhibit the reproduction of harmful bacteria.

[0021] Further, in step S1, the organic solvent is dichloromethane, and the emulsifier is polyvinyl alcohol; the mass-volume ratio of the polylactic acid-glycolic acid copolymer, the emulsifier and the organic solvent is (1-1.5) g:(0.5-0.8) g:(10-15) ml.

[0022] Further, in step S2, the mass ratio of the quaternary mixed solution to the water-in-oil emulsion is (0.8-1.2):(2.5-3.5).

[0023] Further, in step S3, the mass ratio of the polydimethylaminoethyl methacrylate, the crosslinking agent and the quaternary system active ingredient mixed solution is (2-2.5) g:(0.1-0.15) g:(10-15) ml.

[0024] Further, the crosslinking agent is glutaraldehyde or N,N'-methylene bisacrylamide.

[0025] The second object of the present application is to provide a responsive release microcapsule prepared by the preparation method described above.

[0026] The second object of the present application is to provide a wound care patch comprising the responsive release microcapsule described above.

[0027] Further, the wound care patch comprises a hydrophilic polyurethane layer, a smart delivery layer and a waterproof layer arranged from inside to outside, and the smart delivery layer comprises the responsive release microcapsule.

[0028] The wound care patch is composed of three layers of structure, the inner layer is a wound contact layer, which adopts a hydrophilic polyurethane material. The material contains a large number of hydrophilic groups such as hydroxyl and amino groups in its molecular structure, has good biocompatibility and water absorption. It can absorb wound exudate, maintain a moist environment for the wound, and is beneficial to cell migration and proliferation, and its microporous structure allows the slow release of active ingredients to the wound surface. The middle layer is a smart delivery layer, which contains the responsive release microcapsule. The outer layer is a waterproof layer, which adopts a breathable polyurethane film with a compact molecular structure, which can prevent external bacteria and moisture from entering, while having certain air permeability to ensure gas exchange in the wound area and maintain the normal physiological environment of the wound.

[0029] In use, the wound care patch is directly attached to the surface of the cleaned wound, and the wound is in the inflammation stage, the microcapsules in the responsive release system preferentially release polyphenols and probiotics, inhibit inflammation and bacterial growth; as the wound heals, the pH value decreases, and the system releases elastin and vegetable oil, promoting tissue repair. Patients can replace the wound care patch regularly according to the wound healing condition according to the product instruction. Generally, the wound care patch is replaced every 1-2 days during the inflammation stage, and the replacement interval can be extended to 3-5 days as the wound heals.

[0030] Further, the smart delivery layer further comprises graphene.

[0031] Further, the aqueous polyurethane layer contains silver nanoparticles and / or sodium hyaluronate.

[0032] Design mechanism of the responsive release microcapsules of the application

[0033] (1) Shell material: pH-responsive mechanism of PDMAEMA

[0034] ① Acidic environment (pH < 7.0)

[0035] - Protonation of tertiary amine groups (-N(CH)) → formation of positively charged -N+(CH3)2H

[0036] - Weakening of molecular chain electrostatic repulsion + strengthening of hydrophobic interaction → shell shrinkage and crinkling, pore closure, and release inhibition.

[0037] ② Basic environment (pH > 7.0)

[0038] - Deprotonation of tertiary amine groups → molecular chain neutralization, reduced hydrophilicity.

[0039] - Hydrophobic segment aggregation → shell swelling or partial degradation, forming a porous structure and accelerating release.

[0040] (2) Release trigger conditions and functions of the four active ingredients

[0041]

[0042] Response logic of the responsive release microcapsules of the application at each stage of wound healing

[0043] (1) Inflammation stage (0-3 days, pH 7.5-9.0)

[0044] ① Microenvironment characteristics

[0045] - Bacterial proliferation (such as Staphylococcus aureus) metabolizes ammonia, raising the local pH.

[0046] - Secretion of a large amount of inflammatory factors (IL-6, TNF-α).

[0047] ②Microcapsule response

[0048] - PDMAEMA shell deprotonation → swelling (relaxation) to form micropores.

[0049] ③ Preferential release of polyphenols (e.g. gallic acid):

[0050] - Inhibit bacterial biofilm, neutralize free radicals (anti-inflammatory).

[0051] ④ Gradual release of probiotics (e.g. Lactobacillus acidophilus): Release mechanism: only in the alkaline environment (pH > 6.0) caused by infection, the content is released.

[0052] - Secretion of bacteriocins, competitive inhibition of pathogenic bacteria colonization.

[0053] (2) Proliferation phase (3-14 days, pH 6.5-7.4)

[0054] ① Microenvironment characteristics:

[0055] - Inflammation is reduced, and the pH gradually decreases to neutral.

[0056] - Fibroblasts and capillaries begin to proliferate.

[0057] ② Microcapsule response:

[0058] - PDMAEMA partial protonation → shell moderate relaxation.

[0059] ③ Release of elastin:

[0060] - Provide raw materials for collagen synthesis, accelerate granulation tissue formation.

[0061] ④ Slow release of plant oils (e.g. olive oil):

[0062] - Rich in unsaturated fatty acids, promote epidermal regeneration.

[0063] (3) Maturation phase (> 14 days, pH 4.5-6.5)

[0064] ① Microenvironment characteristics:

[0065] - The skin pH returns to normal weak acidity (acidic shell reconstruction).

[0066] - Collagen remodeling, scar formation.

[0067] ② Microcapsule response:

[0068] - PDMAEMA complete protonation → shell tight closure.

[0069] ③ Residual oil continues to release:

[0070] - Maintain wound moist, reduce scar contracture.

[0071] Wound care patch synergistic release mechanism design (PDMAEMA and hydrophilic polyurethane material)

[0072] (1) Release kinetics regulation

[0073]

[0074]

[0075] (2) Synergistic application mechanism

[0076] (1) Infection period response (alkaline pH)

[0077] - Steps:

[0078] 1. PDMAEMA outer layer deprotonation, hydrophobic micropore opening.

[0079] 2. Alkaline environment triggers PU inner layer carboxyl ionization or dynamic bond breakage.

[0080] 3. Polyphenols diffuse through PDMAEMA micropores, and PU synchronously dissolves to release probiotics.

[0081] (2) Healing period protection (acidic / neutral pH)

[0082] - Steps:

[0083] 1. PDMAEMA protonation contraction, closing the release channel.

[0084] 2. PU maintains structural stability, slowly releases oil moisturizing.

[0085] The beneficial effects of the present application are as follows:

[0086] The microcapsule shell of the present application adopts polydimethylaminoethyl methacrylate (PDMAEMA) as a pH-sensitive polymer material. The polyphenols, elastin, vegetable oil and probiotics in the quaternary system synergize to promote wound healing from multiple aspects such as antibacterial and anti-inflammatory, promoting tissue repair, regulating microecological environment, etc. The responsive release microcapsule plays a key role in this process, which can accurately release the corresponding active ingredients according to the pH value changes in different stages of wound healing. The tertiary amine groups on the PDMAEMA molecular chain are protonated in an acidic environment, making the microcapsule shell positively charged and in a contracted state; in an alkaline environment, the tertiary amine groups are deprotonated, the molecular chain is stretched, and the microcapsule shell swells or degrades. During the inflammatory period of the wound, the pH value is usually alkaline, and the microcapsule gradually releases polyphenols and probiotics in an alkaline environment, plays an antibacterial and anti-inflammatory role, and timely inhibits inflammation and kills pathogenic bacteria to create a good environment for wound healing; as the wound heals, the pH value gradually returns to normal, and the microcapsule releases elastin and vegetable oil to promote tissue repair and regeneration, effectively shortening the wound healing time, improving the healing quality, and reducing scar formation. Through clinical experiments, it is verified that the wound healing time of patients using the wound care patch of the present application is shortened by an average of 3-5 days, and the scar area is reduced by about 30%-40%.

[0087] The wound care patch is designed to automatically release active ingredients according to changes in the wound environment, reducing the complex operation process of wound care for patients. At the same time, the product has good biocompatibility and air permeability, can provide a comfortable healing environment for the wound, and significantly reduces the discomfort of patients during use. DETAILED DESCRIPTION

[0088] The following is a specific embodiment of the present application, which further describes the technical solutions of the present application, but the present application is not limited to these embodiments.

[0089] Preparation of bacterial suspension: the probiotics were inoculated in sterilized MRS liquid medium, cultured at 37℃ on a shaking table for 20h, centrifuged at 4000r / min for 10min to collect the bacterial bodies, washed twice with sterile normal saline (0.9% NaCl), resuspended to prepare a bacterial suspension with a concentration of 1.0×10 9 ~1.0×10 10 CFU / ml.

[0090] The elastin used in the embodiment is the recombinant elastin disclosed in Chinese patent CN117551184B.

[0091] Example 1

[0092] Raw material preparation: accurately weigh 3g of gallic acid with a purity of ≥98%, 2g of EGCG with a purity of ≥95%, 5g of elastin, 8g of olive oil, 5g of macadamia nut oil, and 1.0×10 10CFU.

[0093] Responsive release microcapsule preparation: 1 g of polylactic acid-glycolic acid copolymer (PLGA) was dissolved in 10 mL of dichloromethane, 0.5 g of polyvinyl alcohol (PVA) was added as an emulsifier, and emulsified under the condition of ultrasonic power 200 W for 10 minutes to form a water-in-oil (W / O) emulsion. Subsequently, the mixed polyphenol, elastin, vegetable fat and probiotic solution was slowly dropped into, and continued to be emulsified at 200 W power for 15 minutes to obtain a multiple emulsion (W / O / W). Dichloromethane was removed by evaporation at room temperature in a fume hood to obtain a mixed solution of active ingredients of the quaternary system;

[0094] 2 g of poly (dimethylamino ethyl methacrylate) (PDMAEMA) was dissolved in 20 mL of deionized water, 0.1 g of N,N'-methylene bisacrylamide (MBA) was added as a crosslinking agent and stirred uniformly. 20 ml of the mixed solution of active ingredients of the quaternary system was slowly dropped into, and reacted at 60°C for 4 hours to form microcapsules by crosslinking reaction. After washing the microcapsules with deionized water for 3 times, they were dried in a freeze dryer for 48 hours for standby use.

[0095] Wound care patch assembly: a hydrophilic polyurethane material was made into an inner layer contact wound layer with a thickness of 0.5 mm; a responsive release microcapsule was constructed as a smart delivery layer as an intermediate layer; a breathable hydrophobic polyurethane film was made into an outer layer waterproof layer with a thickness of 0.1 mm.

[0096] Through the hot pressing process, the three-layer structure was compounded at 120°C and 5 MPa pressure to obtain a wound care patch.

[0097] Example 2

[0098] Raw material preparation: 2 g of gallic acid with purity ≥98%, 3 g of EGCG with purity ≥95%, 4 g of elastin, 6 g of olive oil, 4 g of macadamia nut oil, and 0.8×101 CFU of Lactobacillus acidophilus and Bifidobacterium, respectively.

[0099] Responsive release microcapsule preparation: using a similar method as in Example 1, adjusting the ratio of PLGA to emulsifier, dissolving 1.2 g of PLGA in 12 mL of dichloromethane, and adding 0.6 g of polyvinyl alcohol (PVA). By optimizing the ultrasonic emulsification time and power, a W / O emulsion was prepared by ultrasonic emulsification at 220 W power for 12 minutes. The mixed polyphenol, elastin, vegetable fat and probiotic solution was slowly dropped into, and emulsified at 220 W power for 18 minutes to obtain a multiple emulsion (W / O / W). Dichloromethane was removed by evaporation at room temperature in a fume hood to obtain a mixed solution of active ingredients of the quaternary system.

[0100] Change the ratio of PDMAEMA and crosslinking agent, dissolve 2.2 g of PDMAEMA in 22 mL of deionized water, add 0.12 g of N,N'-methylene bisacrylamide (MBA), adjust the crosslinking density of the microcapsule, slowly drop 20 mL of the active ingredient mixed solution of the quaternary system, react at 60°C for 4 hours, and form microcapsules through crosslinking reaction. After washing the microcapsules with deionized water for 3 times, dry them in a freeze dryer for 48 hours for standby.

[0101] Wound care patch assembly: add 0.2 g of sodium hyaluronate as a moisturizing agent to the inner layer that contacts the wound layer to improve the moisturizing performance of the product and prolong the time of keeping the wound moist. The rest is the same as Example 1.

[0102] Example 3

[0103] Raw material preparation: weigh 4 g of gallic acid with a purity of ≥98%, 1.5 g of EGCG with a purity of ≥95%, 6 g of elastin, 10 g of olive oil, 6 g of macadamia nut oil, and 1.2 x 101 CFU of Lactobacillus acidophilus and Bifidobacterium, respectively.

[0104] Responsive release microcapsule preparation: soybean phospholipid is selected instead of polyvinyl alcohol as an emulsifier, and the ultrasonic emulsification time and power are adjusted. Emulsify for 15 minutes at an ultrasonic power of 250 W to form a W / O emulsion, slowly drop the mixed polyphenol, elastin, vegetable oil and probiotic solution, and emulsify for 20 minutes at a power of 280 W to obtain a multiple emulsion (W / O / W). Evaporate dichloromethane at room temperature in a fume hood to remove the active ingredient mixed solution of the quaternary system.

[0105] Wound care patch assembly: mix a small amount of silver nanoparticles into the inner layer that contacts the wound layer to enhance the antibacterial performance of the product. The inhibition zone diameter of the inner layer containing silver nanoparticles against Staphylococcus aureus reaches 15 mm. The rest is the same as Example 1.

[0106] Example 4

[0107] Raw material preparation: weigh 1.5 g of gallic acid with a purity of ≥98%, 3.5 g of EGCG with a purity of ≥95%, 4.5 g of elastin, 7 g of olive oil, 5 g of macadamia nut oil, and 0.9 x 101 CFU of Lactobacillus acidophilus and Bifidobacterium, respectively.

[0108] Responsive release microcapsule preparation: replace the crosslinking agent N,N'-methylene bisacrylamide with glutaraldehyde. The rest is the same as Example 1.

[0109] Wound care patch assembly: add a small amount of graphene to the middle intelligent delivery layer to enhance the sensor signal conduction performance. The rest is the same as Example 1.

[0110] Comparative Example 1 (without polyphenolic compounds)

[0111] Raw material preparation: 5 g of elastin, 8 g of olive oil, 5 g of macadamia oil, and 1 x 101 CFU of Lactobacillus acidophilus and Bifidobacterium were weighed.

[0112] Responsive release microcapsules were prepared using the same method as in Example 1, and a wound care patch was assembled.

[0113] Comparative Example 2 (without elastin)

[0114] Raw material preparation: 3 g of gallic acid (purity ≥ 98%), 2 g of EGCG (purity ≥ 95%), 8 g of olive oil, 5 g of macadamia oil, and 1 x 101 CFU of Lactobacillus acidophilus and Bifidobacterium were weighed.

[0115] Responsive release microcapsules were prepared using the same method as in Example 1, and a wound care patch was assembled.

[0116] Comparative Example 3 (without vegetable oil)

[0117] Raw material preparation: 3 g of gallic acid (purity ≥ 98%), 2 g of EGCG (purity ≥ 95%), 5 g of elastin (molecular weight < 1000 Da), and 1 x 101 CFU of Lactobacillus acidophilus and Bifidobacterium were weighed. 10

[0118] Responsive release microcapsules were prepared using the same method as in Example 1, and a wound care patch was assembled.

[0119] Comparative Example 4 (without probiotics)

[0120] Raw material preparation: 3 g of gallic acid (purity ≥ 98%), 2 g of EGCG (purity ≥ 95%), 5 g of elastin (molecular weight < 1000 Da), 8 g of olive oil, and 5 g of macadamia oil were weighed.

[0121] Responsive release microcapsules were prepared using the same method as in Example 1, and a wound care patch was assembled.

[0122] Comparative Example 5 (traditional care ingredients)

[0123] Raw material preparation: 5 g of petrolatum, 2 g of zinc oxide, 1 g of silver sulfadiazine, and 10 g of glycerin were weighed.

[0124] Care patch preparation:

[0125] (1) Raw material preparation: petrolatum, zinc oxide, silver sulfadiazine, and glycerin were accurately weighed, and non-woven fabric was prepared as a carrier material.

[0126] ​(2) Melt the Vaseline: Put the weighed Vaseline into a clean container, place it in a constant temperature water bath at 60-80°C, and continuously stir it with a glass rod or an electric stirrer until the Vaseline is completely melted, ensuring uniform heating.

[0127] (3) Mix the raw materials: Slowly pour the zinc oxide and silver sulfadiazine into the melted Vaseline, continue stirring for 15-30 minutes to ensure uniform mixing of all ingredients, and add glycerol and stir until well mixed.

[0128] (4) Coating and shaping: Use a doctor blade coater to evenly coat the mixture onto non-woven fabric, controlling the thickness to be 0.5mm, and then cut it into the appropriate size and shape.

[0129] (5) Drying and solidification: Place the coated care patch in a 45°C oven for 1-2 hours to complete the solidification process, obtaining a traditional wound care patch.

[0130] Application Example 1 Animal Experiment

[0131] 1. Experimental animals and grouping

[0132] (1) Rat experiment: Select 60 healthy SD rats with a body weight of 200-250g. Randomly divide them into three groups: experimental group 1 (using the wound care patch prepared in Example 1), experimental group 2 (using the wound care patch prepared in Example 2), and the control group (using commercially available non-woven fabric medical sterile dressing), with 20 rats in each group.

[0133] (2) Mouse experiment: Select another 80 healthy C57BL / 6 mice with a body weight of 18-22g. Randomly divide them into four groups: two groups correspond to the wound care patches of Example 3 and Example 4, each with 20 mice; one group is Comparative Example 5 (traditional care ingredient care patch), with 20 mice; and one group is the blank control group, with 20 mice, which only undergoes wound treatment without using a care patch.

[0134] (3) Rabbit experiment: Select 30 healthy New Zealand white rabbits with a body weight of 2-2.5kg. Randomly divide them into three groups: one group uses the care patch of Example 1, one group uses the care patch of Example 2, and one group uses commercially available non-woven fabric medical sterile dressing as a control, with 10 rabbits in each group.

[0135] 2. Wound model establishment

[0136] (1) Rat wound model: After disinfecting the rat's back with iodophor, use sterile surgical instruments to create a 1cm diameter circular full-thickness skin defect wound, simulating human wound conditions. The surgical process strictly follows aseptic operation principles to avoid infection.

[0137] (2) Mouse wound model: Similarly, disinfect the mouse's back with iodophor and create a 0.5cm diameter circular full-thickness skin defect wound.

[0138] (3) Rabbit wound model: On both sides of the back of the rabbit, after disinfection with iodophor, a circular full-thickness skin defect wound with a diameter of 2 cm was made.

[0139] 3. Experimental process

[0140] (1) Rat experimental process: After the wound was made, the rats in each group were treated immediately. The rats in experimental group 1 and experimental group 2 were respectively pasted with the wound care patches prepared in example 1 and example 2 at the wound site. The rats in the control group were pasted with commercially available non-woven fabric medical sterile dressing at the wound site. The wound healing of the rats was observed every day, including the degree of swelling, exudate, and infection, and the records were made.

[0141] (2) Mouse experimental process: The mice in the corresponding example 3 and example 4 groups were respectively pasted with the corresponding care patches; the mice in comparative example 5 group were pasted with traditional care ingredient care patches; the mice in the blank control group were not treated with dressing at the wound site. The wound of the mice was observed every day, and the wound change was recorded, such as whether there was pus, healing trend, etc.

[0142] (3) Rabbit experimental process: The rabbits in the group using the care patches of example 1 and example 2 were respectively pasted with the corresponding care patches at the wound site, and the rabbits in the control group were pasted with commercially available ordinary wound dressing at the wound site. The wound of the rabbits was checked every day, and attention was paid to whether there were abnormalities such as inflammation aggravation and healing stagnation.

[0143] 4. Detection index and method

[0144] (1) Wound area measurement

[0145] Rat: On the 3rd day, the 7th day, the 10th day and the 14th day of the experiment, the wound of the rat was photographed using a digital camera, the wound area was measured by image analysis software, and the wound healing rate was calculated. Wound healing rate = (initial wound area - measured wound area) / initial wound area x 100%.

[0146] Mouse: On the 2nd day, the 5th day, the 8th day and the 12th day of the experiment, the wound of the mouse was photographed using a high-precision image acquisition device, the wound area was measured using professional image analysis software, and the healing rate was calculated.

[0147] Rabbit: On the 5th day, the 10th day, the 15th day and the 20th day of the experiment, the wound of the rabbit was photographed, and the wound area was measured and the healing rate was calculated with the aid of an image analysis system.

[0148] (2) Histopathological examination

[0149] Rats: On the 7th and 14th day of the experiment, 5 rats were randomly selected from each group, and the wound and surrounding tissues were fixed, sectioned, stained, and other treatments were performed. Histopathological changes were observed under a microscope, including inflammatory cell infiltration, granulation tissue formation, and epithelialization degree. Image analysis software was used to measure the thickness of the epidermis, the density of collagen fibers in the dermis, and other indicators to evaluate the quality of wound healing.

[0150] Mice: On the 6th and 10th day of the experiment, 5 mice were randomly selected from each group, and the wound tissue was fixed, sectioned, hematoxylin-eosin (H&E) stained, and Masson stained. The presence of inflammatory cells, fibroblasts, and collagen fibers was observed to evaluate the progress of wound healing.

[0151] Rabbits: On the 10th and 15th day of the experiment, 3 rabbits were randomly selected from each group, and the wound and surrounding tissues were fixed and sectioned. Immunohistochemical staining was used to detect the expression of vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1) in the wound tissue to analyze the mechanism of wound healing.

[0152] (3) Inflammatory factor detection

[0153] Rats: Enzyme-linked immunosorbent assay (ELISA) was used to detect the content of inflammatory factors tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the wound tissue of rats. On the 1st, 3rd, and 7th day of the experiment, wound tissue samples were collected, and tissue homogenate was extracted. The ELISA kit operation steps were followed to detect the absorbance at 450 nm wavelength using a microplate reader. The content of inflammatory factors was calculated according to the standard curve.

[0154] Mice: Western blot was used to detect the protein expression level of inflammatory factors TNF-α, IL-1β, and others in the wound tissue of mice. On the 1st, 3rd, and 5th day of the experiment, wound tissue samples were collected, and total protein was extracted. SDS-PAGE electrophoresis, membrane transfer, blocking, primary and secondary antibody incubation, and other operations were performed. Finally, the band gray value was detected by a chemiluminescence imaging system to analyze the expression changes of inflammatory factors.

[0155] Rabbits: Real-time fluorescent quantitative PCR (qPCR) technology was used to detect the mRNA expression level of inflammatory factors TNF-α, IL-6, and others in the wound tissue of rabbits. On the 3rd, 7th, and 10th day of the experiment, wound tissue samples were collected, and total RNA was extracted. After reverse transcription to cDNA, qPCR amplification was performed. The relative expression of inflammatory factor mRNA was calculated according to the Ct value.

[0156] (4) Microbial detection

[0157] Rat: On the 5th and 10th day of the experiment, the wound surface secretions of rats were collected with sterile cotton swabs, which were placed in sterile normal saline for elution, and the eluate was used for bacterial culture. The eluate was inoculated on blood agar, MacConkey agar and other culture media, and cultured at 37°C for 24-48 hours. After that, the colony morphology was observed and gram staining, biochemical identification and other methods were used to determine the types and quantities of pathogenic bacteria in wound infection.

[0158] Mouse: On the 4th and 8th day of the experiment, microbial detection was performed on the wounds of mice. High-throughput sequencing technology was used to extract total microbial DNA from wound secretions, and 16S rRNA gene was amplified by PCR. After library construction, sequencing analysis was performed to understand the changes in wound microbial community structure and diversity.

[0159] Rabbit: On the 7th and 12th day of the experiment, rabbit wound secretions were collected and detected using a microbial rapid detection kit. By detecting the enzyme activities of β-glucuronidase, catalase and other enzymes in the secretions, it was quickly determined whether there was bacterial infection in the wound and the degree of infection.

[0160] (5) Skin mechanical property test

[0161] Rat: After the wound completely healed (about 21 days), 5 rats were selected from each group, and the wound and surrounding normal skin tissue were taken to make standard tensile specimens. Electronic universal material testing machine was used for tensile test, and the tensile speed was set at 1 mm / min. The mechanical properties such as breaking strength and breaking elongation of the skin were recorded to evaluate the recovery of skin strength after wound healing.

[0162] Rabbit: After the wound healed (about 28 days), the mechanical properties of the skin at the wound site were tested. Non-invasive skin elasticity tester was used to deform the skin by negative pressure suction, and the elasticity parameters such as R2 (elastic recovery rate) and R5 (elastic recovery ratio) were measured to evaluate the elasticity recovery of the skin after wound healing.

[0163] 5. Experimental results

[0164] (1) Animal experiment: rat

[0165]

[0166] (2) Animal experiment: mouse

[0167]

[0168]

[0169] (3) Animal experiment: rabbit

[0170]

[0171]

[0172] Application Example 2

[0173] (1) Purpose of the experiment: This application example aims to explore the specific effects and synergies of polyphenolic compounds, elastin, vegetable fat and probiotics in the wound healing process through comparative examples and control examples, so as to verify the effectiveness and advantages of the responsive release microcapsules and wound care patches in the present application.

[0174] (2) 60 healthy SD rats weighing between 200-250g were selected and randomly divided into six groups, corresponding to Example 1, Example 2, Control Example 1 (without polyphenolic compounds), Control Example 2 (without elastin), Control Example 3 (without vegetable fat), and Control Example 4 (without probiotics), with 10 rats in each group. After disinfecting the back of the rats with iodophor, a 1cm diameter circular full-thickness skin defect wound was created using sterile surgical instruments to simulate human wound conditions, and the surgical process strictly followed aseptic operation principles to avoid infection. After the wound was created, the rats in each group were treated accordingly. The rats in Example 1 and Example 2 had the corresponding wound care patches prepared in the examples applied to their wounds; the rats in Control Examples 1-4 had the corresponding care patches prepared with the missing ingredients applied to their wounds. The rats' wound healing was observed daily, including the degree of swelling, exudate, and signs of infection, and records were kept.

[0175] (3) Experimental results

[0176]

[0177]

[0178] (4) Analysis of experimental results

[0179] Wound healing rate: The healing rates of Examples 1 and 2 at each time point were higher than those of the control examples, indicating that the complete four-component system can effectively accelerate wound healing. Control Example 1 lacks polyphenolic compounds, which has insufficient antibacterial and anti-inflammatory ability, delaying healing; Control Example 2 lacks elastin, which lacks tissue repair materials, limiting the healing rate; Control Example 3 lacks vegetable fat, affecting skin moisturizing and barrier repair; Control Example 4 lacks probiotics, which cannot effectively regulate the microecology, resulting in a lower healing rate than the examples.

[0180] Histopathology: The example presents a good state of tissue repair, with less inflammatory cell infiltration, and good condition of granulation tissue and collagen fibers. Comparative Example 1 has more inflammatory cells and less granulation tissue, due to the lack of polyphenolic compounds to effectively inhibit inflammation; Comparative Example 2 has abnormal fibroblasts and collagen fibers, due to the lack of collagen; Comparative Example 3 has poor epidermal repair and barrier function, related to the lack of plant lipids; Comparative Example 4 has slow clearance of inflammatory cells and a microecological imbalance, which is the result of the lack of probiotics. Inflammatory factor detection: In the example, the inflammatory factors TNF-a and IL-6 decreased significantly at 3 days and 7 days, indicating that the four-component system can effectively reduce inflammation. Comparative Example 1 has a slow decrease in inflammatory factors due to the lack of polyphenolic compounds; Comparative Examples 2, 3, and 4 also have insufficient inflammatory regulation due to the lack of components, resulting in relatively high levels of inflammatory factors.

[0181] Microbial detection: The example has fewer pathogenic bacteria, reflecting the good antibacterial and microecological regulation ability of the four-component system. Comparative Example 1 has more pathogenic bacteria and low community diversity, which is due to the lack of polyphenolic compounds, which reduces the antibacterial ability; Comparative Examples 2, 3, and 4 also cannot effectively inhibit the growth of pathogenic bacteria and regulate the microecology due to the lack of components.

[0182] Skin mechanical properties: The breaking strength and elongation at break of the example are more optimal, indicating good wound healing quality. Comparative Examples 1-4 have less skin strength and elasticity recovery due to the lack of components, further illustrating the importance of each component in restoring skin properties after wound healing.

[0183] Application Example 3 Human Experiment

[0184] 1. Preliminary preparation

[0185] (1) Ethical approval and recruitment planning: Strictly follow the medical ethics norms, submit the experimental plan to the relevant ethics committee, and obtain the formal approval document. Based on the experimental needs, develop a detailed volunteer recruitment plan, and recruit through multiple channels such as hospitals and communities.

[0186] (2) Establishment of screening criteria: Clearly define the inclusion criteria, only recruit volunteers aged 18-60 years old, ensure their physical health, no immune system diseases, diabetes, and other underlying diseases that interfere with wound healing, and have not used drugs that affect wound healing in the recent period. At the same time, limit the wound type to superficial trauma, such as abrasions and small area cuts, with a wound area of 2-5 square centimeters.

[0187] (3) Volunteer screening and determination: Through strict health checks and detailed medical history inquiries, screen the registered volunteers. Finally, 120 qualified volunteers participate in the experiment.

[0188] 2. Group implementation

[0189] (1) Randomization: Using a random number table, 120 volunteers were randomly divided into three groups: experimental group 1 (using the wound care patch prepared in Example 1), experimental group 2 (using the wound care patch prepared in Example 2), and the control group (using commercially available non-woven fabric medical sterile dressing), each group of 40 people, to ensure the randomness and balance of the grouping, and to reduce the bias between groups.

[0190] (2) Informed consent: Before the experiment, the volunteers were informed of the purpose, process, potential risks and benefits of the experiment. After the volunteers fully understood, they signed the informed consent form.

[0191] 3. Intervention implementation

[0192] (1) Wound treatment: The wound of the volunteer was cleaned by professional medical staff, and the wound was carefully flushed with physiological saline to completely remove dirt, foreign matter, etc. on the surface of the wound, and then gently wiped with sterile gauze.

[0193] (2) Use of care patch: According to the grouping, the wound of the volunteers in experimental group 1 and experimental group 2 was respectively pasted with the corresponding wound care patch prepared in the example, and the wound of the volunteers in the control group was pasted with commercially available non-woven fabric medical sterile dressing. At the same time, the volunteers were informed that they needed to keep the wound clean during the experiment, avoid strenuous exercise that could cause the wound to open, prevent the wound from getting wet, and replace the care patch according to the specified time, usually every 2-3 days, and adjust as appropriate according to the wound exudation.

[0194] 4. Data collection and evaluation

[0195] (1) Wound healing time record: From the use of the care patch, the volunteers or their family members were arranged to observe the wound condition daily, and accurately record the time when the wound completely healed, that is, the time when the wound surface scab fell off, the skin basically recovered flat, and there were no symptoms such as redness, exudation, etc.

[0196] (2) Healing quality evaluation: One week after the wound healed, a professional doctor who had undergone unified training was organized to evaluate the quality of wound healing using the visual analog scale (VAS). The evaluation content covers indicators such as scar size, color, flatness, etc. The VAS score is full marks 10 points, and the lower the score, the better the healing quality, such as no obvious scar, similar color to the surrounding skin, high flatness of the wound.

[0197] (3) Patient satisfaction survey: After the wound healed, the patients' satisfaction with the use of the care patch was collected through a questionnaire survey. The questionnaire content covers the comfort, adhesion, pain relief effect of the care patch, etc. Satisfaction is divided into three levels: very satisfied, satisfied, and dissatisfied. Volunteers select the corresponding options according to their own use experience, and set up an opinion feedback column to facilitate volunteers to make suggestions for improvement of the care patch.

[0198] 5. Quality control:

[0199] (1) During the experiment, arrange a special person to call back the volunteers every 3 days, and visit them once a week. Carefully check the wound healing, observe whether the use of nursing stickers is standardized, such as whether the position of the nursing stickers is accurate, whether the replacement frequency meets the requirements, etc. For volunteers with abnormal conditions such as wound infection (such as aggravated redness and swelling of the wound, purulent secretion, fever), allergy (such as skin rash, papules, severe itching), etc., immediately arrange them to go to the hospital for treatment by experienced doctors, and record the occurrence time, symptom performance, treatment measures and subsequent recovery of abnormal conditions in detail.

[0200] (2) Regularly organize consistency training for doctors participating in evaluation, invite industry experts to give lectures. The training content includes interpretation of wound healing evaluation standard, standard operation of VAS score, common problem solving method, etc. After the training, the doctors are examined through case examination, simulation evaluation, etc. to ensure that the doctors' understanding and execution of the evaluation standard are highly unified, effectively reduce human error, and ensure the accuracy and reliability of the experimental data.

[0201] 6. Experimental results

[0202]

[0203] From the experimental results of application examples 1-2, in general, the intelligent wound nursing stickers containing quaternary systems have obvious advantages in promoting wound healing, improving healing quality, reducing inflammatory response, regulating microbial community, and improving patient satisfaction compared to traditional nursing ingredient nursing stickers and ordinary wound dressings, and have good application prospects.

[0204] The above is not involved, which is applicable to the prior art.

[0205] Although some specific embodiments of the present application have been described in detail through examples, those skilled in the art should understand that the above examples are only for illustration, not for limiting the scope of the present application, and those skilled in the art can make various modifications or supplements to the described specific embodiments or replace them with similar ways, but will not deviate from the direction of the present application or exceed the scope defined by the appended claims. Those skilled in the art should understand that any modification, equivalent replacement, improvement, etc. made according to the technical essence of the present application to the above embodiments should be included in the protection scope of the present application.

Claims

1. A method for preparing responsive release microcapsules, characterized in that, Includes the following steps: S1. Dissolve the polylactic acid-glycolic acid copolymer in an organic solvent, add an emulsifier, and ultrasonically emulsify to form a water-in-oil emulsion; S2. A quaternary mixed solution is formed by mixing polyphenolic compounds, elastin, vegetable oils, and probiotic suspensions. This solution is then slowly added dropwise to the water-in-oil emulsion formed in step S1. Ultrasonic emulsification is continued to form a double emulsion. The organic solvent is then removed by evaporation to obtain a quaternary system active ingredient mixed solution. The polyphenolic compounds are gallic acid and EGCG; the vegetable oils are olive oil and macadamia nut oil; and the probiotics are Lactobacillus acidophilus and Bifidobacterium. S3. Dissolve poly(dimethylaminoethyl methacrylate) in deionized water, add a crosslinking agent and stir until homogeneous, then slowly drip the quaternary active ingredient mixture solution obtained in step S2 into the solution. The crosslinking reaction forms microcapsules, which are then washed and freeze-dried to obtain the responsive release microcapsules.

2. The preparation method according to claim 1, characterized in that, The mass-to-volume ratio of polyphenolic compounds, elastin, vegetable oils, and probiotic suspension was (1.5-2.5) g:(2.5-3.5) g:(3-5) ml:(0.8-1.2) ml, and the concentration of the probiotic suspension was 1.0 × 10⁻⁶. 9 ~1.0×10 10 CFU / ml.

3. The preparation method according to claim 1, characterized in that, In step S1, the organic solvent is dichloromethane, and the emulsifier is polyvinyl alcohol; the mass-volume ratio of the polylactic acid-glycolic acid copolymer, the emulsifier, and the organic solvent is (1-1.5) g:(0.5-0.8) g:(10-15) ml. In step S2, the mass ratio of the quaternary mixed solution to the water-in-oil emulsion is (0.8-1.2):(2.5-3.5).

4. The preparation method according to claim 1, characterized in that, In step S3, the crosslinking agent is N,N'-methylenebisacrylamide, and the mass-volume ratio of the mixed solution of poly(dimethylaminoethyl methacrylate), N,N'-methylenebisacrylamide and the quaternary active ingredient is (2-2.5) g:(0.1-0.15) g:(10-15) ml.

5. A responsive release microcapsule prepared by the preparation method according to any one of claims 1-4.

6. A wound care dressing, characterized in that, Including the responsive release microcapsules as described in claim 5.

7. The wound care dressing as described in claim 6, characterized in that, The wound care patch comprises, from the inside out, a hydrophilic polyurethane layer, a smart delivery layer, and a waterproof layer, wherein the smart delivery layer includes responsive release microcapsules.

8. The wound care dressing as described in claim 7, characterized in that, The smart delivery layer also includes graphene.

9. The wound care dressing as described in claim 7, characterized in that, The hydrophilic polyurethane layer contains silver nanoparticles and / or sodium hyaluronate.

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