An asymmetric dressing and method of making the same

Abstract

The application relates to an asymmetric dressing and a preparation method thereof, an antibacterial drug is loaded on a collagen material, the collagen material is provided with antibacterial performance, and the antibacterial performance is characterized; a growth factor IGF-2 is loaded on MOFs particles, and a core layer high polymer material is combined with the MOFs, so that the collagen material with a core-shell structure can induce fibroblast migration and promote tissue healing. The prepared collagen material is used to prepare an asymmetric medical dressing, a hydrophobic PCL / collagen-PCL blended high polymer material prepared by using an electrostatic spinning technology is used on the upper layer of the dressing, and a hydrophilic core-shell structure collagen material prepared by using an extrusion technology is used on the lower layer, so that the multifunctional asymmetric medical dressing is realized. The water absorption rate, the water retention rate, the coagulation property and the cell compatibility of the asymmetric dressing are tested and characterized, and corresponding function evaluation is carried out.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of dressings, and particularly relates to an asymmetric dressing and a preparation method thereof. BACKGROUND

[0002] With the gradual recognition of wound healing theory, the development of wound dressings has also undergone great changes. At present, wound dressings are expected to cover wounds and accelerate the healing process. Traditional dressings, also known as inert dressings (gauze, cotton pad and bandage), are the most widely used clinical dressings due to their low cost and simple manufacturing process. However, some shortcomings (such as difficulty in keeping the wound moist and easy adhesion to granulation tissue) limit their application. Today, medical dressings are gradually multifunctional, which not only provide a moist environment for wound healing, but also relieve pain and improve hypoxic or anoxic environment.

[0003] Fibrous dressing is one of the widely used medical dressing types, which has obvious advantages. It has significant structural similarity to ECM in terms of porosity, morphology and mechanical properties. In addition to structural similarity, the fibrous system can also be composed of inorganic and organic materials, providing the necessary growth environment for cells participating in the wound healing process. On the other hand, inspired by the structure of the epidermis and dermis of the skin, some researchers further improved the fibrous dressing into an asymmetric wetting dressing. For example, Dong-hui Liang et al. prepared a double-layer AgNPs / chitosan composite dressing, which has self-cleaning ability on one side, which can minimize the adhesion of water and pollutants on one side; and the other side has the ability to absorb exudates. Chun-ping Su et al. prepared a stearic acid modified starch / chitosan composite sponge with asymmetricity, which has a super-hydrophobic surface on one side to prevent water, blood and bacteria from penetrating; and the other side has a hydrophilic surface with optimal water absorption, blood absorption and coagulation ability. These asymmetric wetting dressings show good wound healing-promoting ability.

[0004] At present, how to improve the water absorption rate, water retention rate and coagulation property of the asymmetric dressing, and further effectively promote wound healing and improve healing quality is a technical problem that needs to be solved urgently. SUMMARY

[0005] The present application is provided to solve the technical problems mentioned in the background, and therefore a kind of asymmetric dressing and its preparation method are needed, antibacterial drugs are loaded on collagen material, collagen material has antibacterial performance, and antibacterial performance is characterized;IGF-2 is loaded on MOFs particle, and then core-shell structure collagen material is combined with MOFs, so that collagen material of core-shell structure can induce fibroblast migration and promote tissue healing.The prepared collagen material is used to prepare asymmetric medical dressing, wherein the upper layer of the dressing is hydrophobic PCL / collagen-PCL blend polymer material prepared by electrospinning technology, and the lower layer is hydrophilic core-shell structure collagen material prepared by extrusion technology, so as to realize multifunctional asymmetric medical dressing.The water absorption rate, water retention rate, coagulation and cell compatibility of the asymmetric dressing are tested and characterized, and corresponding function evaluation is made.

[0006] One of the purposes of the present application is to provide an asymmetric dressing, comprising a hydrophobic layer and a hydrophilic layer, the hydrophilic layer comprising a core layer CS / CLL and a shell layer ICFs, the core layer CS / CLL being dispersed with pZIF-8@IGF-2, the shell layer ICFs being loaded with mupirocin, and the hydrophobic layer being oriented nanofiber.

[0007] Further, the hydrophilic layer is an oriented nanofiber prepared by electrospinning technology.

[0008] Further, the hydrophobic layer is composed of an inner layer and an outer layer, wherein the inner layer is in contact with the hydrophilic layer.

[0009] Further, the inner layer is PCL / COL I nanofiber.

[0010] Further, the outer layer is PCL nanofiber.

[0011] The second purpose of the present application is to provide a method for preparing the asymmetric dressing as described above, comprising:

[0012] Preparation of the hydrophilic layer: prepare ICFs (Insoluble Collagen Fibers, ICFs) gel, and add mupirocin to obtain shell layer material; prepare CS / CLL, and add pZIF-8@IGF-2 to obtain core layer material, and prepare the hydrophilic layer by co-extrusion of the shell layer material and the core layer material.

[0013] Preparation of the hydrophobic layer: prepare a solution with PCL and COL I as solute and HFIP as solvent, and based on the solution, perform spinning according to the set electrospinning parameters to prepare the inner layer of the hydrophobic layer, configure PCL solution, and perform spinning on the basis of the inner layer according to the same electrospinning parameters to prepare the outer layer of the hydrophobic layer, wherein the electrospinning parameters include voltage, injection speed, receiving distance and drum rotation speed.

[0014] Further, the pZIF-8 is prepared by the following method:

[0015] Zn(CH3COO)2·H2O and 2-methylimidazole are dissolved in Tris-HCl solution, and a dopamine solution is added to the mixture to adjust the formation of pZIF-8 crystals.

[0016] Further, the pZIF-8@IGF-2 is prepared by the following method:

[0017] Zn(CH3COO)2·H2O and 2-methylimidazole are dissolved in 20 mL Tris-HCl solution, and IGF-2 is added to obtain pZIF-8@IGF-2.

[0018] Further, the mass ratio of the PCL to the COL I is 1:(0.6-1).

[0019] Further, the PCL solution concentration is 8%.

[0020] The application has the advantages that:

[0021] (1) DA is introduced in the process of ZIF-8 assembly, and pZIF-8 with regular dodecahedron morphology is successfully prepared by in-situ polymerization.

[0022] (2) The scratch test shows that 100 ng / mL IGF-2 has a significant migration effect on HDFs cells.

[0023] (3) pZIF-8 can be used as a good carrier for drug loading, and the loading rate is higher than that of ZIF-8. The release rate of TB in pZIF-8@TB is faster in 24 h, and then it is slow, which can last for 168 h. pZIF-8 degrades slowly in vitro, and the residual mass ratio is 87.33±4.13% after 336 h.

[0024] (4) In the hydrophilic layer, the minimum inhibitory concentration of mupirocin on Staphylococcus aureus is 0.128 mg / mL.

[0025] (5) In the hydrophobic layer, the optimal mixing ratio of PCL / COL is PCL 18.75 The water contact angle test proves that it can be used as a bridge layer between PCL and the hydrophilic layer, and the good mechanical properties of PCL 18.75 lay a good foundation for the toughness of the prepared dressing.

[0026] (6) The combination of the shell-core microporous extrusion technology and the electrospinning technology can prepare an asymmetric dressing with a hydrophobic outer layer, good air permeability, and anti-adhesion, a hydrophilic inner layer, good moisture absorption, and repair promotion, and the dressing has good cell compatibility. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Design idea diagram of asymmetric dressing of embodiments of the present application.

[0028] Figure 2 MOFs-SEM diagram of embodiments of the present application.

[0029] Figure 3 (A) MOFs-XRD diagram; (B) MOFs-FTIR diagram of embodiments of the present application.

[0030] Figure 4 Cell scratch healing degree diagram of embodiments of the present application.

[0031] Figure 5 Scratch distance quantitative diagram of each concentration of embodiments of the present application.

[0032] Figure 6 Mupirocin minimum inhibitory concentration (mg / mL) diagram of embodiments of the present application.

[0033] Figure 7 The left side is an antibacterial circle experiment diagram, and the right side is a different concentration antibacterial circle diameter distribution diagram.

[0034] Figure 8 (A) pZIF-8@Rhodamine-BSA fluorescence detection diagram; (B) TB standard curve; (C) ZIF-8@TB, pZIF-8@TB TB loading rate of embodiments of the present application.

[0035] Figure 9 (A) TB release curve diagram; (B) pZIF-8 degradation curve diagram of embodiments of the present application.

[0036] Figure 10 (A) PCL-Ⅰ collagen composite film after coomassie brilliant blue staining of embodiments of the present application, a: PCL 6.25 ; b: PCL 12.25 ; c: PCL 18.75 ; d: PCL 25 ; e: PCL 50 ; f: PCL 100 ; (B) water contact angle test.

[0037] Figure 11 (A) scanning electron microscope diagram of the outer side (OHL pho ) and the inner side (IHL pho ) of the hydrophobic layer of embodiments of the present application; (B) OHL pho , IHL pho fiber orientation angle distribution interval diagram from left to right

[0038] Figure 12 Water contact angle of the outer hydrophobic layer (OHL pho ) and the inner hydrophobic layer (IHL pho ) of the embodiment of the present application

[0039] Figure 13 Water absorption and water retention of the HL phi and the HL pho of the embodiment of the present application

[0040] Figure 14 Coagulation performance test of the HL phi and the HL pho of the embodiment of the present application

[0041] Figure 15 In vitro cell compatibility test of the asymmetric dressing of the embodiment of the present application: the left side represents cell live-dead staining; the right side represents cell skeleton staining. DETAILED DESCRIPTION

[0042] The following part of the embodiments is only for better illustrating the present application, but the content of the present application is not limited to the application in the embodiments. Therefore, the skilled in the art can make non-essential improvements and adjustments to the embodiments according to the above content of the present application and apply them to other embodiments, which are still within the protection scope of the present application.

[0043] The present application will be further described in combination with the drawings of the specification.

[0044] Embodiment 1

[0045] In this embodiment, pZIF-8@IGF-2 is dispersed in the core layer CS / CLL, and mupirocin is loaded in the shell layer ICFs. The oriented porous fiber hydrophilic layer (Hydrophilic Layer, HL phi ) is prepared by using the self-developed shell-core microporous extrusion device to simulate the structure of the dermis layer, so as to have the functions of absorbing exudate and promoting cell migration. The hydrophobic layer is prepared by using electrospinning technology to prepare oriented nanofibers to simulate the epidermis, so as to have the functions of resisting bacterial adhesion, air permeability and avoiding excessive dehydration of the wound surface. In order to improve the bonding force between the hydrophilic layer and the hydrophobic layer, the hydrophobic layer (Hydrophobic Layer, HL pho ) is designed as two layers: inner layer (Inner Hydrophobic Layer, IHL pho) with the hydrophilic layer, PCL / COL I nanofibers, outer layer (OHL pho ) with the external environment, PCL nanofibers. The design idea is as shown in Figure 1

[0046] The experimental materials are shown in Table 1.

[0047] Table 1. Experimental materials

[0048] The experimental instruments are shown in Table 2. Table 2. Experimental instruments Preparation and characterization of the dressing: Preparation of the hydrophilic layer (HL phi ) (1) Preparation of MOFs particles ① Preparation of ZIF-8

[0049] Dissolve 0.1M Zn(CH3COO)2·H2O and 1.6M 2-methylimidazole in 100mL Tris-HCl solution (pH=8.5) and stir at room temperature. After 3h of reaction, centrifuge the mixture (10000rpm, 10min) and wash with deionized water three times. Finally, collect the precipitate, freeze-dry to obtain ZIF-8.

[0050] ② Preparation of pZIF-8

[0051] Dissolve 0.1M Zn(CH3COO)2·H2O and 1.6M 2-methylimidazole in 100mL Tris-HCl solution (pH=8.5), stir for 1min, then add 0.5mg / mL dopamine (DA) solution to the mixture to adjust the formation of pZIF-8 crystals, and stir at room temperature. After 3h of reaction, centrifuge the mixture (10000rpm, 10min) and wash with deionized water three times. Finally, collect the precipitate, freeze-dry to obtain pZIF-8.

[0052] (2) Characterization of MOFs particles

[0053] ① SEM

[0054] Disperse an appropriate amount of MOFs particles in anhydrous ethanol by ultrasonic dispersion, take 5μL and drop on a silicon wafer, dry and then treat with gold spraying for 60s, and observe and record the photograph by SEM.

[0055] ② XRD

[0056] ​After grinding and passing the dried ZIF-8 through a 200-mesh sieve, it was applied to a sample stage, taking care to keep the sample and the edges of the sample stage at the same level, and measured using an X-ray powder diffractometer. Test conditions: scan angle 5°-60°, speed 5° / min.

[0057] 3) FTIR

[0058] 1-2 mg ICFs sample, cut into small pieces, mixed with 200 mg KBr powder, ground in an agate mortar and pressed into a pellet (16 MPa, 2 min). The spectrum was recorded in the range of 4000-400 cm -1 .

[0059] (3) Drug concentration primary screening

[0060] 1) IGF-2 promotes fibroblast (HDFs) migration

[0061] IGF-2 was diluted in PBS and added to the growth medium to make different concentration gradients: 50 ng / mL, 100 ng / mL, 150 ng / mL, 200 ng / mL. HDFs mode cells, 1.0 x 10 4 cells / cm 2 were seeded in a 24-well plate and added with growth medium (10% FBS, 100 U / mL penicillin-streptomycin dissolved in DMEM medium). When the cells reached 80% confluence, a scratch was made using a 200 μL pipette tip, and washed twice with PBS. The growth medium containing different concentrations of IGF-2 described above was added. Photographs were taken at 0 h, 12 h and 24 h using an inverted microscope, and semi-quantitative analysis was performed using ImageJ software.

[0062] 2) Mupirocin minimum inhibitory concentration (MIC)

[0063] 2 mg mupirocin was taken and added to 7.8 mL of fresh liquid medium, and mixed by blowing. The mupirocin concentration was 0.256 mg / mL, 1 mL was taken and added to a 48-well plate, three parallel samples were set up, and the concentration was diluted to 0.25 μg / mL using a two-fold dilution method. Each well was added with 1 x 10 6 CFU / mL of bacterial solution, and cultured for 16-24 h. The minimum inhibitory concentration was finally observed and photographed for analysis.

[0064] 3) Effective concentration of mupirocin loaded on oriented porous fibers

[0065] After freeze-drying of the oriented porous fibers, the antibacterial circle experiment was performed, with the following steps:

[0066] Take 1 mL of the cultured Staphylococcus aureus liquid, add it to 9 mL of liquid medium, and mix it gently. Then take 1 mL of the liquid and spread it on the solid medium in a culture dish. Shake it gently and remove the excess liquid. Cut a 5 mm diameter circle from the dressing and place it on the solid medium. Place the culture dish upside down in the incubator for 12 hours. Take a picture and analyze the size of the inhibition zone using Image J software.

[0067] (4) Model drug loading and release

[0068] Rhodamine-BSA was used as the model drug. 0.1M Zn(CH3COO)2·H2O and 1.6M 2-methylimidazole were dissolved in 20mL Tris-HCl solution (pH=8.5), and 10μg Rhodamine-BSA was added and stirred for 1min. The subsequent steps were the same as the minimum inhibitory concentration of Mupirocin, and the resulting product was denoted as pZIF-8@Rhodamine-BSA. An appropriate amount of pZIF-8@Rhodamine-BSA was added to PBS solution and ultrasonicated for 10min to disperse uniformly. It was then added dropwise to a glass slide and covered with a cover glass for observation under an inverted fluorescence microscope and photography.

[0069] Further, with trypan blue (TB) as the model drug, the preparation steps were the same as above (10μg Rhodamine-BSA was replaced by 100μg TB), and the resulting product was denoted as pZIF-8@TB, which was used for subsequent encapsulation efficiency, drug release, and degradation performance tests.

[0070] ① Standard curve

[0071] 1mg of trypan blue was weighed and added to 10mL of PBS (pH=7.4) to make a 100μg / mL trypan blue standard solution. The maximum absorbance was measured under a spectrophotometer by scanning the full wavelength. Five groups of trypan blue concentration gradients were set: 10μg / mL, 20μg / mL, 50μg / mL, 80μg / mL, and 100μg / mL, and a standard curve was drawn.

[0072] ② Encapsulation efficiency

[0073] A solution of trypan blue with an initial concentration of 100μg / mL was prepared and added to the ZIF-8 solution. After stirring for 3h, ZIF-8@TB was prepared. After centrifugation at 10000rpm for 10min, 200μL of supernatant was taken to detect the concentration of trypan blue C1 under an enzyme marker. The adsorption value of trypan blue in ZIF-8 was calculated as C X :

[0074]

[0075] ③ Drug release

[0076] Take 1 mg of ZIF-8@TB as raw material for subsequent release test, add 1 mL of PBS (pH = 7.4) solution, disperse ZIF-8@TB in the solution, light shaking in 37℃, 120 rpm shaking table, a total of 10 groups of sampling time points (1h, 2h, 4h, 8h, 12h, 1d, 2d, 4d, 7d, 14d), three parallel samples in each group, take out 200 μL supernatant at different time intervals and test the release absorbance at 583 nm wavelength with enzyme label instrument.

[0077] ④ In vitro degradation

[0078] Take 1 mg of pZIF-8@TB and disperse it in 1 mL of PBS (pH = 7.4), set four sampling times, 1d, 3d, 7d, and 14d. Six parallel samples are set for each sampling time. Record the initial weight, vacuum dry after each sampling time, weigh, and draw the degradation time-mass curve to analyze the degradation performance.

[0079] (5) Preparation of pZIF-8@IGF-2

[0080] The preparation method is the same as the above (4) mode drug loading and release, and the product is denoted as pZIF-8@IGF-2.

[0081] (6) Preparation of HL phi

[0082] Prepare 10 mL of ICF gel, and at the same time, based on the conditions of ③ in the mode drug loading and release, add mupirocin; prepare 10 mL of CS / CLL solution, and at the same time, add 0.022 g of pZIF-8@IGF-2.

[0083] After the shell core layer material is synchronously extruded, received and freeze-dried, it is manually woven into a 10 mm x 10 mm sample dressing.

[0084] Preparation of hydrophobic layer (HL pho )

[0085] (1) PCL / COLⅠ blending ratio exploration

[0086] Prepare a composite film by casting PCL and COLⅠ solution according to Table 3 with HFIP as solvent on a polytetrafluoroethylene plate.

[0087] Table 3. PCL-COLⅠ blending solution ratio table

[0088]

[0089] Note: PCL X The subscript x represents the mass percentage of collagen relative to PCL​

[0090] ①Uniformity test

[0091] The film was cut into 10 mm x 10 mm sample, immersed in coomassie brilliant blue dye for 30 s, then washed repeatedly with deionized water, and placed under an optical microscope for photography.

[0092] ②Water contact angle test

[0093] The film was cut into 10 mm x 10 mm sample, pasted on a glass slide. A 5 μL drop of deionized water was added to the film surface using a syringe perpendicular to the sample surface. When the water droplet was stable on the surface for about 1 s, the image was captured. Each sample was repeated 3-5 different areas, and the contact angle was statistically analyzed.

[0094] ③Mechanical tensile property test

[0095] The film was cut into 30 mm x 10 mm sample, and the test program was set to the middle effective distance of 10 mm, and the tensile rate of 10 mm / min. The sample was stretched until it broke, and the displacement-force curve was recorded in real time. The mechanical properties of the film were characterized by parameters such as breaking strength, breaking elongation and Young's modulus.

[0096] (2) Electrospinning preparation of HL pho

[0097] Based on the optimized ratio of PCL and COL I solution, the electrospinning parameters were set as follows: voltage 30 kV, injection speed 0.004 mm / s, receiving distance 22 cm, and drum speed 2000 rpm. The inner layer of the hydrophobic layer IHL was prepared by spinning. pho .

[0098] Further, an 8% PCL solution was prepared, and the outer layer of the hydrophobic layer OHL was prepared by spinning with the same parameters based on the inner layer. pho The inner and outer layers of the film were tested for static water contact angle and SEM.

[0099] HL phi and HL pho performance comparison

[0100] Coagulation performance test:

[0101] 100 μL of blood (containing 10% sodium citrate) was dropped on HL phi and HL phoThe sample surface was immersed at 37°C for 5 minutes, then gently rinsed with 50 mL of distilled water to remove uncoagulated red blood cells. Three replicates were set up for each sample. The absorbance was measured at 540 nm using a hemoglobin assay kit and a UV spectrophotometer. The hemoglobin content (HC) in the sample was calculated according to the following formula.

[0102] HC(g / U=(OD-OD o )×367.7

[0103] Where OD0 is the blank absorbance value and OD is the absorbance value of the sample being tested.

[0104] Preparation and Cell Compatibility Testing of Asymmetric Dressings

[0105] HL ph o Cut into 10mm × 10mm splines; in HL phi A uniform coating of 1% chitosan solution was applied to the surface, and HL was then applied. ph O-strips were applied to the surface and dried to obtain an asymmetric dressing. To preliminarily assess the cytotoxicity of this dressing, it was immersed in 2 mL of growth medium for 24 h. HDF cells were then cultured at 2 × 10⁻⁶ cells / mL. 4 Cells were seeded per well in 24-well plates. After cell adhesion, the culture medium was replaced with 600 μL of leaching solution, and the plates were incubated for 24 h. One portion of the samples was stained according to the live / dead staining kit instructions. The other portion of the samples was fixed with paraformaldehyde and then stained with rhodamine-phalloidin to stain the cytoskeleton F-actin. The samples were observed and photographed using an inverted fluorescence microscope.

[0106] Experimental results:

[0107] Hydrophilic layer (HL) phi The characterization of )

[0108] (1) Morphological and structural characterization of MOFs

[0109] like Figure 2 As shown, the surface morphology of ZIF-8 and pZIF-8 was observed by SEM. The synthesized ZIF-8 and pZIF-8 are regular dodecahedral crystals with a particle size distribution between 150 nm and 300 nm, which is consistent with the morphology of ZIF-8 synthesized in the literature.

[0110] Furthermore, XRD and FTIR were used to characterize the structures of ZIF-8 and pZIF-8. Figure 3As shown, (A) is the XRD curve of MOFs particles, ZIF-8 and pZIF-8 were prepared according to the above method, the XRD curves of ZIF-8 and pZIF-8 are basically consistent, and the XRD peaks are consistent with the ZIF-8 in the literature, indicating that the introduction of DA does not destroy the crystal structure of ZIF-8. The FTIR results are as follows Figure 3 As shown in (B), a sharp strong peak appears at 420 cm- 1 , which corresponds to the stretching vibration peak of Zn-N, which is the characteristic peak of ZIF-8; the sharp peaks at 692 cm- 1 , 755 cm- 1 , 1174 cm- 1 , 1307 cm- 1 all belong to the bending vibration peaks of imidazole ring; the peaks at 1420 cm- 1 , 1457 cm- 1 all are the stretching vibration peaks of imidazole ring; the peaks at 994 cm- 1 , 1144 cm- 1 are the characteristic stretching vibration peaks of imidazole C-N; the peak at 1581 cm- 1 is attributed to the characteristic stretching vibration peak of imidazole C=N. The infrared peak values of pZIF-8 are shown in the figure, which are basically consistent with ZIF-8.

[0111] (2) Preliminary screening of drug concentration

[0112] ① IGF-2 promotes fibroblast migration

[0113] In order to determine whether IGF-2 affects the migration of HDFs cells, the scratch test was used to preliminarily screen the effective concentration of IGF-2 on the migration of HDFs cells. As shown in Figure 4 , IGF-2 can promote the migration of HDFs cells. At 0h, the scratch distance of all concentrations is basically the same; after 12h, the scratch distance begins to differ, and all concentrations produce scratch distances of different lengths, among which 100ng / mL is more obvious; after 24h, the scratch distance of all concentrations is shortened again, and the distance of 100ng / mL is significantly shorter than that of other concentrations, and the distance of each concentration is semi-quantitatively analyzed as shown in Figure 5 . These results show that 100ng / mL IGF-2 has a significant migration effect on HDFs cells.

[0114] ② Minimum inhibitory concentration (MIC) of mupirocin

[0115] As shown in Figure 6The minimum inhibitory concentration of mupirocin is shown, using Staphylococcus aureus as the experimental bacteria, and using the double dilution method. From left to right, the concentrations are: 0.128 mg / mL, 0.64 mg / mL, 0.32 mg / mL, 0.16 mg / mL, 0.08 mg / mL, 0.04 mg / mL, 0.02 mg / mL, and 0.01 mg / mL. The results show that the minimum inhibitory concentration is 0.128 mg / mL. When the concentration is close to the minimum inhibitory concentration, mupirocin has an inhibitory effect on the growth of Staphylococcus aureus. The closer the concentration is to the minimum inhibitory concentration, the lower the turbidity of the solution.

[0116] ③Effective concentration of oriented porous fiber loaded mupirocin

[0117] Based on the minimum inhibitory concentration of ②, four gradients are set: MIC, 3xMIC, 6xMIC, and 9xMIC, as shown in Figure 7 The four concentration gradients all have antibacterial effects. The diameters of the antibacterial circles of the four concentration gradients are: 1.53 cm ± 0.21 cm, 1.70 cm ± 0.16 cm, 1.79 cm ± 0.14 cm, and 1.88 cm ± 0.14 cm. As the antibacterial concentration increases, the antibacterial effect also gradually increases, but there is no significant difference. Considering factors such as preparation cost, in the future, the minimum inhibitory concentration of collagen fiber material will be mixed to prepare asymmetric medical dressings.

[0118] (3) Model drug loading

[0119] TB and rhodamine-BSA are used as model drugs, as shown in Figure 8 where A is the fluorescence detection of rhodamine-labeled BSA protein loaded onto pZIF-8. The left side is the blank control group, and the right side is pZIF-8@rhodamine-BSA.

[0120] In the fluorescence microscope, red fluorescence can be observed, indicating that the model drug BSA protein is successfully loaded onto pZIF-8. Figure 8 In (B), the standard curve of TB is shown, with a correlation coefficient of 0.9975, indicating good feasibility and linear relationship. Figure 8 In (C), the encapsulation efficiency of TB loaded onto ZIF-8 and pZIF-8 is shown. The loading rate of ZIF-8@TB is 75.15% ± 1.67%, and the loading rate of pZIF-8@TB is 95.52% ± 0.80%. Therefore, after adding DA to ZIF-8, the loading efficiency of the model drug is significantly improved, which also helps the loading of IGF-2 on pZIF-8.

[0121] (4) Release and degradation

[0122] As shown in Figure 9As shown in (A), TB will undergo a drug burst release within 24 hours, possibly due to a large amount of TB adsorbed on or immediately below the dressing surface and released suddenly. After 24 hours, the release tends to be gradual and in a sustained-release state, but it still has the drug release effect. After 168 hours, the drug release tends to be stable, indicating that the effective drug release time is about 168 hours.

[0123] Furthermore, we examined the in vitro degradation behavior of pZIF-8 and plotted the curves as shown below. Figure 9 As shown in Figure (B), after 336 hours, the residual mass ratio of pZIF-8 was 87.33 ± 4.13%, indicating that pZIF-8 degrades slowly in vitro. The degradation mechanism may involve metal ions in ZIF-8 exchanging electrons with DA, causing electrons from DA to be transferred to ZIF-8. This process alters the chemical structure of DA, reducing its stability and thus promoting its degradation.

[0124] Hydrophobic layer (HL) pho The representation of )

[0125] (1) Exploration of PCL / COLⅠ blending ratio

[0126] ① Blending uniformity test and water contact angle test

[0127] Because polycaprolactone (PCL) molecules lack reactive functional groups, the bonding force between electrospun PCL films and oriented collagen fiber layers would theoretically be weak if they were directly bonded. Therefore, we propose to prepare HLpho as a gradient composition with an outer PCL layer and an inner PCL / COLⅠ layer, using the PCL / COLⅠ layer as a bridge between the hydrophobic and hydrophilic layers. The uniformity of the mixture of COLⅠ and PCL components directly affects the mechanical properties of the material.

[0128] Therefore, Coomassie Brilliant Blue staining solution, with its ability to rapidly and visually stain collagen, was used to stain composite membranes. Under a microscope, the uniformity of the mixture of COLⅠ and PCL could be directly observed through the color distribution. Figure 10 As shown in (A): PCL 12.25 Composite membranes and PCL 18.75 The collagen distribution of the composite membrane was significantly more uniform than that of the other groups.

[0129] Furthermore, static water contact angle test results show (e.g.) Figure 10 In the (B) group, when COLⅠ was added to PCL, the water contact angle of all materials was <90°, indicating hydrophilicity. Furthermore, the water contact angle decreased slowly with increasing COLⅠ content in PCL. 12.25A significant decrease was observed in the group. The results indicate that the addition of COLⅠ increases the hydrophilicity of the composite membrane surface, which may be beneficial for its interaction with HL. phi connect.

[0130] ② Mechanical tensile test

[0131] In a hybrid system, the content of components has a significant impact on the mechanical properties of the blend. Therefore, we conducted mechanical tensile tests on the composite film. As shown in Table 4, within a certain range, with the increase of collagen content, the stress of the PCLCOLⅠ composite film increases, and the corresponding strain increases. 18.75 The stress and strain in the group reached their maximum values, with a maximum stress of 19.85 ± 1.81 MPa and a maximum strain of 1364 ± 172%. Within this range, PCL 6.25 PCL 12.5 PCL 18.75 There were no significant differences in the elastic modulus, yield stress, and yield strain among the three groups. However, when the collagen content exceeded 18.75%, the tensile stress and strain of the PCL-COLⅠ composite film showed a significant decrease.

[0132] Considering factors such as the mixing uniformity of PCL-COLⅠ, static water contact angle, and mechanical properties, we selected PCL. 18.75 Mixing ratio for subsequent IHL pho Preparation of .

[0133] Table 4 Mechanical and tensile properties of PCL-COLⅠ composite film

[0134]

[0135] Note: Experimental data are expressed as mean ± standard deviation (mean ± SD), n ≥ 3

[0136] In the table, "\" indicates that the data did not exist or was invalid during the actual measurement process.

[0137] (2) Preparation of HL by electrospinning pho

[0138] HL was observed using SEM. pho outer layer (OHL) pho ) and inner layer (IHL) pho ).like Figure 11 As shown in (A), the fibers prepared by electrospinning technology have a small diameter, uniform distribution, and good orientation. Semi-quantitative analysis results show (as shown in...) Figure 11 (B) shows that IHL pho ,OHL pho The fiber orientation distribution of both is concentrated between -20° and 20°, indicating that both have obvious fiber orientation, while IHLpho The number of fiber orientation between -20°-40° and 20°-40° is significantly higher than OHL pho , OHL pho The overall orientation is significantly better than IHL pho .

[0139] Further, the static water contact angle of the inner and outer layers of the material was tested, and the results showed (as shown in Figure 12 ), the water contact angles of OHL pho and IHL pho were 119.0°±3.5° and 74.87°±3.7°, respectively, showing the characteristics of hydrophobic outer layer and hydrophilic inner layer, indirectly indicating that IHL pho can promote the effective combination of HL phi and OHL pho .

[0140] Performance comparison between HL phi and HL pho

[0141] (1) Water absorption rate and water retention rate

[0142] As shown in Figure 13 , the results of water absorption rate (835.5%±79.9% vs 691.7%±133.6%) and water retention rate (641.2%±52.4% vs 107.3%±30.9%) both showed a trend that HL phi was higher than HL pho , indicating that the dressing showed asymmetric wetting performance, and the inner layer had excellent ability to absorb exudate.

[0143] (2) Coagulation performance test

[0144] In addition, the in vitro coagulation performance of HL phi and HL pho was detected by hemoglobin test kit. The results showed (as shown in Figure 14 ), the hemoglobin content of HL phi was significantly higher than that of HL pho , which could reach 606.13±99.93 g / L, indicating that the layer had good coagulation ability. Based on the previous water retention, water absorption rate and coagulation test, the results showed that in the asymmetric dressing, HL phi would play a major role in absorbing exudate and hemostasis.

[0145] Preparation of asymmetric dressing and cell compatibility test

[0146] Live-dead staining can directly reflect the initial cytotoxicity of the material (red for dead cells, green for normal adherent and spread living cells). As shown in Figure 15 ​As shown, there are only a few dead cells in the field of view, and the live cells appear spindle-shaped.

[0147] Further observation of the cytoskeleton morphology of HDFs (as shown in FIG. 6) showed that the cells adhered and spread well, and F-actin assembled well. Figure 15 The above results preliminarily indicated that the asymmetric dressing had good cell compatibility.

[0148] The main achievements of the present application are as follows:

[0149] (1) Based on the current extrusion equipment, the optimized key parameters of extrusion are as follows: collagen concentration 6%, extrusion needle size 17G, core layer component SA (5% CLL), and shell-core layer extrusion rate 3 mm / s. After optimization of the parameters, the results of SEM observation, measurement analysis of pore size and fiber orientation, and water absorption and water retention tests showed that the oriented porous collagen fiber prepared by using the self-developed shell-core microporous extrusion device had high orientation, complete porous structure, and high water absorption and water retention. This indicated that the shell-core microporous extrusion device can be used to prepare oriented porous collagen fiber.

[0150] (2) During the assembly of ZIF-8, DA was introduced, and pZIF-8 with regular dodecahedron morphology was successfully prepared by in-situ polymerization. pZIF-8 can be used as a good carrier for drug loading, and its loading rate is higher than that of ZIF-8. The release rate of TB in pZIF-8@TB is faster in the first 24 h, and then it is slow and can last for 168 h. pZIF-8 degrades slowly in vitro, and the residual mass ratio after 336 h is 87.33 ± 4.13%.

[0151] (3) The scratch test showed that 100 ng / mL IGF-2 had a significant migration effect on HDFs, and the minimum inhibitory concentration of mupirocin on Staphylococcus aureus was 0.128 mg / mL.

[0152] (4) The optimal mixing ratio of PCL / COL is PCL 18.75 The water contact angle test proved that it can be used as a bridge layer between PCL and the hydrophilic layer, and the good mechanical properties of PCL 18.75 also lay a good foundation for the toughness of the prepared dressing.

[0153] (5) The combination of the shell-core microporous extrusion technology and the electrospinning technology can prepare an asymmetric dressing with a hydrophobic, breathable, and anti-adhesion outer layer and a hydrophilic, moisture-absorbing, and repair-promoting inner layer, and the dressing has good cell compatibility.

[0154] The above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the description of the present application.

Claims

1. An asymmetric dressing characterized in that, The hydrophobic layer and the hydrophilic layer, the hydrophilic layer comprising a core layer SA / CLL in which pZIF-8@IGF-2 is dispersed and a shell layer ICFs in which mupirocin is loaded, the hydrophobic layer being oriented nanofiber; wherein SA is sodium alginate, and CLL is cellulose. The pZIF-8 is prepared by the following method: Zn(CH3COO)2·H2O and 2-methyl imidazole are dissolved in a Tris-HCl solution, and a dopamine solution is added to the mixture to regulate the formation of pZIF-8 crystals.

2. The asymmetric dressing of claim 1, wherein, The hydrophobic layer is an oriented nanofiber prepared by electrospinning technology.

3. The asymmetric dressing of claim 1, wherein, The hydrophobic layer is composed of an inner layer and an outer layer, wherein the inner layer is in contact with the hydrophilic layer.

4. The asymmetric dressing of claim 3, wherein, The inner layer is PCL / COL I nanofiber.

5. The asymmetric dressing of claim 4, wherein, The outer layer is PCL nanofiber.

6. A method of producing an asymmetric dressing according to any one of claims 1 to 5, characterized in that, The hydrophobic layer is prepared by the following method: PCL and COL I are used as solutes, and HFIP is used as a solvent to prepare a solution, based on the solution, electrospinning is performed according to the set electrospinning parameters to prepare the inner layer of the hydrophobic layer, a PCL solution is prepared, and electrospinning is performed based on the inner layer with the same electrospinning parameters to prepare the outer layer of the hydrophobic layer, the electrospinning parameters including voltage, injection speed, receiving distance, and drum rotation speed. The mass ratio of the PCL to the COL I is 1:(0.6-1). The concentration of the PCL solution is 8%. The hydrophobic layer and the hydrophilic layer, the hydrophilic layer comprising a core layer SA / CLL in which pZIF-8@IGF-2 is dispersed and a shell layer ICFs in which mupirocin is loaded, the hydrophobic layer being oriented nanofiber; wherein SA is sodium alginate, and CLL is cellulose.

7. The method of claim 6, wherein, The pZIF-8 is prepared by the following method: Zn(CH3COO)2·H2O and 2-methyl imidazole are dissolved in a Tris-HCl solution, and a dopamine solution is added to the mixture to regulate the formation of pZIF-8 crystals.

8. The method of claim 6, wherein, The hydrophobic layer is an oriented nanofiber prepared by electrospinning technology. The hydrophobic layer is composed of an inner layer and an outer layer, wherein the inner layer is in contact with the hydrophilic layer. The inner layer is PCL / COL I nanofiber. The outer layer is PCL nanofiber. The hydrophobic layer is prepared by the following method: PCL and COL I are used as solutes, and HFIP is used as a solvent to prepare a solution, based on the solution, electrospinning is performed according to the set electrospinning parameters to prepare the inner layer of the hydrophobic layer, a PCL solution is prepared, and electrospinning is performed based on the inner layer with the same electrospinning parameters to prepare the outer layer of the hydrophobic layer, the electrospinning parameters including voltage, injection speed, receiving distance, and drum rotation speed. The mass ratio of the PCL to the COL I is 1:(0.6-1). The concentration of the PCL solution is 8%.

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