CN114855365A

CN114855365A - Drug-loaded metal-organic framework composite electrostatic spinning fiber membrane and preparation method and application thereof

Info

Publication number: CN114855365A
Application number: CN202210377159.XA
Authority: CN
Inventors: 方立明; 尹磊; 汤绮雯; 柯琦; 赖姗姗; 范鹏辉; 苏健裕
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2022-08-05

Abstract

The invention discloses a drug-loaded metal organic framework composite electrostatic spinning fibrous membrane as well as a preparation method and application thereof. The preparation method comprises the following steps: (1) adding ZIF-8 nano particles loaded with dimethyloxalyl glycine into hexafluoroisopropanol, adding gelatin and polycaprolactone after ultrasonic dispersion, and fully stirring to obtain an electrospinning solution; (2) and (2) filling the electrospinning solution obtained in the step (1) into an injector, and preparing the drug-loaded metal-organic framework composite electrospinning fibrous membrane by an electrospinning method. In the invention, ZIF-8 not only can be used as an antibacterial agent to exert antibacterial performance, but also can be used as a carrier of the DMOG to regulate the release rate of the DMOG together with a Gel-PCl fibrous membrane. The composite fiber membrane also has good water vapor transmission rate, can simulate extracellular matrix, has good antibacterial performance and biocompatibility, can promote the migration of endothelial cells, the in vitro vascularization and other performances, and has good application prospect in the field of chronic wound healing difficulty.

Description

Drug-loaded metal-organic framework composite electrostatic spinning fiber membrane and preparation method and application thereof

Technical Field

The invention relates to the field of biomedical materials, in particular to a drug-loaded metal-organic framework composite electrostatic spinning fiber membrane and a preparation method and application thereof.

Background

The skin is the largest organ of the human body and plays an important role in regulating the metabolism of the human body and resisting external invasion, however, various pathological or accidental accidents easily cause skin wounds, and when the wounds are hindered from normal healing by factors such as bacterial infection and the like, the wounds are easily transformed into wounds which are difficult to heal, so that serious economic loss is brought to the human society, and the bacterial infection and insufficient new blood vessels are the two main obstacles for healing the wounds which are difficult to heal by tissues.

However, with the continuous development of human research on wound healing, the requirements of wound dressings are increased, and traditional dressings such as gauze and the like cannot meet the complex requirements of modern wound healing theory on wound dressings due to insufficient self functionality and difficulty in combination with bioactive components.

The nanofiber membrane prepared by the electrostatic spinning technology has the characteristics of high specific surface area and high porosity. In addition, nanofibers prepared by electrospinning can well mimic extracellular matrix (ECM), which is advantageous for proliferation of epithelial cells and neogenesis of tissue. Thus, the electrospinning process has unique advantages in the field of making wound dressings.

Furthermore, electrospinning to make wound dressings allows some active ingredient (usually an antibacterial or a repair-promoting drug) to be added to the fibers to impart functionality. This is difficult to achieve with conventional dressings. In the research of Emily et al, two electro-spinning dressings loaded with Ciprofloxacin (CIP) are prepared by a co-spinning method and a physical adsorption method respectively. (Emily Buck, Vimal Maisuria, Nathalie Tufenkji, and Marta Cerrtuti ACS Applied Bio Materials 20181 (3),627-635) however, the introduction and more frequent use of antibiotics in antibacterial therapy has triggered the development and rapid development of antibiotic resistance in microorganisms, as methicillin-resistant Staphylococcus aureus (MRSA) has developed multiple resistance to all types of antibiotics, which has become an urgent issue in the treatment of bacterial infections of wounds.

Dimethoxalylglycine (DMOG) is a small molecule drug for promoting angiogenesis, is a competitive inhibitor of proline 4-hydroxylase (PHDs), and plays a role in stabilizing HIF-1 alpha by reducing the decomposition of HIF-1 alpha which is an important gene in the process of wound healing, thereby promoting angiogenesis. Kong et al prepared a dimethyloxalylglycine-nanosilicate fiber membrane (publication No. CN112755241A) by ordinary co-spinning. However, the fiber membrane loaded with DMOG prepared by the blend spinning method may cause burst release of the drug during actual use, leading to premature inactivation of the drug and potential safety risks. Therefore, it is significant to adopt a proper slow-release means for the DMOG to meet the requirement of long-term controlled release of repair-promoting drugs in the wound healing process. Xu and the like adopt ZIF-67 as a carrier, prepare DMOG-loaded ZIF-67 nanoparticles by an adsorption method, then mix the DMOG-loaded ZIF-67 nanoparticles with gelatin and levorotatory polylactic acid, and prepare the nanofiber dressing containing the DMOG-loaded ZIF-67 complex by an electrostatic spinning method, so that good slow-release effect on the DMOG can be realized, and angiogenesis is promoted. However, the dressing does not have antibacterial performance and cannot meet various requirements of the complex environment of the wound on the wound dressing.

Disclosure of Invention

In order to overcome the problems, the invention provides a drug-loaded metal organic framework composite electrostatic spinning fibrous membrane with antibacterial and DMOG slow release functions, and a preparation method and application thereof. The preparation method comprises the steps of preparing DMOG-loaded ZIF-8 particles through a one-step method, ultrasonically dispersing DMOG @ ZIF-8 in a solvent, adding gelatin and polycaprolactone, fully stirring to prepare an electrospinning solution, and preparing the drug-loaded metal-organic framework composite electrospinning fibrous membrane through an electrospinning method. ZIF-8 is used as a carrier of the DMOG while being used as a non-antibiotic antibacterial agent, and can carry out slow release on the DMOG so as to prolong the release period of the DMOG. By combining the electrostatic spinning technology, the prepared nanofiber membrane creates conditions for better applying the medicine-carrying ZIF-8 nanoparticles to the field of wound healing. The drug-loaded metal organic framework composite electrostatic spinning fiber membrane provided by the invention can simultaneously meet the requirements of infection resistance in the wound healing process and slow release of an angiogenesis promoting drug DMOG so as to better promote the angiogenesis effect, and has a good application prospect in the field of wound repair.

The object of the present invention is achieved by the following technical means.

A preparation method of a drug-loaded metal organic framework composite electrostatic spinning fiber membrane comprises the following steps:

(1) adding the drug-loaded metal organic framework into hexafluoroisopropanol, adding gelatin and polycaprolactone after uniform dispersion, and fully stirring to obtain an electrospinning solution; the drug-loaded metal-organic framework is ZIF-8 nano particles loaded with dimethyloxalyl glycine;

(2) and (2) filling the electrospinning solution obtained in the step (1) into an injector, and preparing the drug-loaded metal-organic framework composite electrospinning fibrous membrane by an electrospinning method.

Preferably, the addition amount of the drug-loaded metal organic framework in the electrospinning liquid is 3-5 mg/mL.

Preferably, the addition amount of the drug-loaded metal organic framework in the electrospinning solution is 3 mg/mL.

Preferably, the mass ratio of the gelatin to the polycaprolactone in the step (1) is 4:6-6: 4; the mass-volume ratio of the total mass of the gelatin and the polycaprolactone to the mass of the hexafluoroisopropanol is 0.1-0.14 g/mL.

Preferably, the mass ratio of the gelatin to the polycaprolactone in the step (1) is 1: 1; the mass-volume ratio of the total mass of the gelatin and the polycaprolactone to the mass of the hexafluoroisopropanol is 0.12 g/mL.

Preferably, the molecular weight of the polycaprolactone is 8 ten thousand.

Preferably, the stirring time of the step (1) is not less than 72 hours.

Preferably, the preparation of the drug-loaded metal-organic framework in the step (1) comprises the following steps:

adding zinc nitrate hexahydrate into anhydrous methanol, adding dimethyloxalglycine and 2-methylimidazole into the anhydrous methanol together, performing ultrasonic treatment to fully dissolve the two solutions respectively, pushing the mixed solution of dimethyloxalglycine and 2-methylimidazole into the anhydrous methanol solution of zinc nitrate hexahydrate by using a micro-injection pump, fully stirring, centrifuging the generated white drug-loaded nanoparticles, washing and drying to obtain the drug-loaded metal-organic framework.

Preferably, the volume mass volume ratio of the zinc nitrate hexahydrate to the anhydrous methanol is 0.4 g: 10-15mL is preferably 0.4: 10 (g/mL); the mass ratio of dimethyloxalyl glycine to 2-methylimidazole is 1: 9-2: 9, preferably 2: 9.

preferably, the drug-loading rate of the drug-loading metal organic framework is 15-25 wt%

Preferably, the stirring temperature does not exceed 40 ℃, preferably 25 ℃; the stirring time is 12-24h, preferably 12 h; the rotation speed of the centrifugation is 8000-12000rpm, and is preferably 10000 rpm; the drying temperature is 40-50 deg.C, and the drying time is not less than 12 h.

Preferably, the electrostatic spinning in the step (2) has the spinning voltage of 9kV to 14kV, the temperature of 15 ℃ to 30 ℃, the relative humidity of 40 percent to 60 percent, the feeding speed of 1 mL/h to 2mL/h, the receiving distance of 10 cm to 15cm and the roller rotating speed of 100rpm to 400 rpm.

Preferably, the electrostatic spinning in the step (2) has the spinning voltage of 12kV, the temperature of 15-30 ℃, the relative humidity of 40-60%, the feeding speed of 1.2mL/h and the roller rotating speed of 200 rpm.

The drug-loaded metal-organic framework composite electrostatic spinning fiber membrane prepared by the preparation method.

The application of the drug-loaded metal-organic framework composite electrostatic spinning fiber membrane in preparation of dressings.

Compared with the prior art, the invention has the following advantages and beneficial effects:

(1) the composite fiber membrane has good antibacterial and slow-release effects on angiogenesis promoting medicine DMOG. Under the preferable addition amount, the compound has obvious antibacterial effect on both escherichia coli and staphylococcus aureus, and can obviously prolong the release period of the DMOG.

(2) The composite fiber film has good water vapor transmission rate, and is close to the ideal water vapor transmission rate of the antibacterial dressing; the diameters of the fibers are mostly distributed in the range of 400-500 nm, so that the ECM can be well simulated, and the growth of cells is facilitated.

(3) The composite nanofiber has good biocompatibility under the condition of the optimal addition amount, and can promote the migration of endothelial cells, in-vitro vascularization and other performances.

Drawings

FIG. 1a and FIG. 1b are respectively a scanning electron microscope topography of ZIF-8 and DMOG @ ZIF-8 obtained in example 1 of the present invention.

FIG. 2 is a scanning electron micrograph of the fiber films obtained in example 2 with different amounts of DMOG @ ZIF-8 added (the amount of DMOG @ ZIF-8 added is 0(A)1(B)2(C)3(D)5(E) unit: mg/mL).

FIGS. 3a and 3B are graphs showing the results of the antibacterial activity of the fiber membrane obtained in example 3 according to the present invention against Escherichia coli and Staphylococcus aureus in different amounts of DMOG @ ZIF-8 (the amount of DMOG @ ZIF-8 added is 0(A)1(B)2(C)3(D)5(E) unit: mg/mL).

FIGS. 4a, 4b and 4c are graphs of zinc ion release curves, cytotoxicity test graphs and cell death staining graphs of different concentrations of zinc ions on HUVECs of the composite fiber membranes with different DMOG @ ZIF-8 addition amounts obtained in example 4 of the present invention, respectively.

Fig. 5 is a graph of the cumulative release of DMOG from two different fiber membranes loaded with DMOG in different ways, obtained in example 5 of the present invention.

FIG. 6 is a graph showing the effect of different component fibrous membranes on the promotion of cell proliferation in example 6 of the present invention.

FIG. 7 is a graph showing the effect of different components on the promotion of cell migration of HUVECs in example 7 of the present invention.

FIG. 8 is a graph showing the effect of GP (a), ZGP (b) and DZGP (c) fiber membranes on the in vitro vascularization of HUVECs in example 8.

FIG. 9 is a graph showing the water vapor transmission rate test results of the fiber membranes of different compositions in example 9 of the present invention.

Fig. 10 is a schematic view of the DZGP fiber membrane antibacterial and angiogenesis promotion of the present invention.

Detailed Description

The following examples further illustrate embodiments of the present invention, but the embodiments of the present invention are not limited thereto.

The schematic diagram of the DZGP fiber membrane antibacterial and angiogenesis promoting effect of the present invention is shown in FIG. 10.

Example 1 preparation of drug-loaded metal-organic framework composite electrospun fiber membrane

Preparing ZIF-8 nano particles: taking 400mg of zinc nitrate hexahydrate in 10mL of anhydrous methanol, taking 1g of 2-methylimidazole in 15mL of anhydrous methanol, carrying out ultrasonic treatment to accelerate dissolution, loading the mixed solution of the 2-methylimidazole by using a syringe, adding the mixed solution into the anhydrous methanol solution of the zinc nitrate hexahydrate at the push speed of 0.3mL/min under the control of a micro-injection pump, magnetically stirring for 12 hours, centrifuging at the rotating speed of 10000rpm, washing and precipitating twice by using the anhydrous methanol, and carrying out vacuum drying at the temperature of 40 ℃ for 24 hours to obtain the ZIF-8 nano particles.

Preparing DMOG-loaded ZIF-8 nanoparticles: taking 400mg of zinc nitrate hexahydrate in 10mL of anhydrous methanol, taking 0.2g of DMOG and 1g of 2-methylimidazole in 15mL of anhydrous methanol, carrying out ultrasonic treatment to accelerate dissolution, loading a mixed solution of the DMOG and the 2-methylimidazole by using an injector, adding the mixed solution into an anhydrous methanol solution of the zinc nitrate hexahydrate at a push speed of 0.3mL/min under the control of a micro-injection pump, magnetically stirring for 12 hours, centrifuging at a rotating speed of 10000rpm, washing and precipitating twice by using the anhydrous methanol, and carrying out vacuum drying for 24 hours at 40 ℃ to obtain the DMOG @ ZIF-8 nanoparticles.

(II) preparing an electrospinning solution: taking 0, 10, 20, 30 and 50mg of DMOG @ ZIF-8 nano particles in 10mL of hexafluoroisopropanol respectively, carrying out ultrasonic dispersion treatment for 10min by using an ultrasonic crusher to fully disperse the nano particles, weighing 0.6g of gelatin and 0.6g of polycaprolactone, adding the gelatin and the polycaprolactone into the suspension, and carrying out magnetic stirring for 72 hours to obtain an electrospinning solution.

(III) preparing composite fibers by electrostatic spinning: loading the electrospinning solution into an injector, setting the pushing speed of a micro injection pump to be 1.2mL/h, the voltage to be 12kV, the receiving distance to be 12cm, and carrying out electrostatic spinning on the spinning solution under the condition that the rotating speed of a collecting roller is 200rpm to obtain the drug-loaded metal-organic framework composite electrostatic spinning fiber membrane.

Example 2 scanning Electron microscope Observation of drug-loaded Metal-organic framework composite electrospun fiber Membrane

ZIF-8 and DMOG @ ZIF-8 were prepared as in example 1. And (3) putting ZIF-8 and DMOG @ ZIF-8 in absolute ethyl alcohol, performing ultrasonic dispersion, then dropwise adding the mixture onto a titanium sheet, performing gold spraying treatment for 60s, and observing the morphology and the particle size of the nanoparticles under a high-power scanning electron microscope. As shown in the figure 1a and the figure 1b, the particle size distribution of ZIF-8 and DMOG @ ZIF-8 is respectively 70-120 nm and 90-150 nm.

The drug-loaded metal-organic framework composite electrospun fiber membrane was prepared as in example 1. Attaching the fiber membrane sample on the conductive adhesive, spraying gold for 60s by using a gold spraying instrument, and observing by using a tungsten filament scanning electron microscope. As shown in FIG. 2, the diameter distribution of the component fibers is concentrated between 200 and 600nm, the average diameter is about 450nm, and the fibers can well imitate extracellular matrix.

Example 3 characterization of antibacterial Properties of drug-loaded Metal-organic framework composite electrospun fiber Membrane

Preparing a drug-loaded metal organic framework composite electrostatic spinning fiber membrane according to the method of example 1, cutting the fiber membranes with different addition amounts of DMOG @ ZIF-8 with the mass of 20mg into 1mLPBS, adding diluted escherichia coli or staphylococcus aureus into the fiber membranes, and enabling the final concentration of the bacteria to be 10 ⁵ And (3) CFU/mL, after co-culturing the material and the bacteria for 4 hours, uniformly diluting the culture solution to a proper multiple by ultrasonic oscillation, taking 50mL of the liquid to perform plate coating on a solid agar culture medium, culturing the agar culture medium after plate coating at 37 ℃ overnight, and observing the colony number on the plate. When the addition amount of DMOG @ ZIF-8 is more than or equal to 1mg/mL, the composite fiber membrane has obvious effect on escherichia coliThe antibacterial effect of the composite fiber membrane on staphylococcus aureus is not high when the addition amount is less than 3mg/mL (see figure 3a), the antibacterial effect of the composite fiber membrane on staphylococcus aureus is gradually enhanced along with the increase of the addition amount of DMOG @ ZIF-8 (see figure 3b), and the antibacterial effect on escherichia coli and staphylococcus aureus is good when the addition amount of DMOG @ ZIF-8 is 3mg/mL and 5 mg/mL. Therefore, in order to ensure that the composite fiber membrane has good antibacterial effect on the two most representative gram-negative bacteria and gram-positive bacteria, the addition amount of DMOG @ ZIF-8 is not less than 3 mg/mL.

Example 4 detection of Zinc ion Release and verification of Zinc ion toxicity of drug-loaded Metal-organic framework composite electrospun fiber Membrane

(II) Zinc ion Release test

Composite fiber membranes with different DMOG @ ZIF-8 addition amounts are prepared according to the method of example 1, and 100mg of the composite fiber membranes with different components are weighed, wherein the DMOG @ ZIF-8 addition amounts are 0mg/mL,1mg/mL,2mg/mL,3mg/mL and 5 mg/mL. Added to 5mL of PBS, the culture device was incubated at 37 ℃ and 100rpm in a constant temperature shaker, and 1mL of each of the liquids was removed from the culture device at different sampling time points and supplemented with 1mL of fresh PBS. The collected liquid taken out at different time periods is measured for the concentration of zinc ions by a Zincon indicator method and is used for making a release curve, as shown in fig. 4 a. (the mass-to-volume ratios of the materials and PBS in the release test were maintained as in the antimicrobial test in order to evaluate the toxicity of the zinc ion release concentration on cells in the composite fiber membranes at different DMOG @ ZIF-8 addition levels in the case of uniform dosage later.)

And secondly, verifying the toxicity of zinc ions, namely directly determining the antibacterial performance of the composite fiber membrane by the release level of the zinc ions in the composite fiber membrane, however, the cytotoxicity is caused by the zinc ions with too high concentration, and verifying the toxicity of the zinc ions with different concentrations on umbilical vein endothelial cells (HUVECs) in order to evaluate the potential cytotoxicity of different components DMOG @ ZIF-8 by combining the release curves of the zinc ions in the components.

After the cells are planted in a 24-well plate for culture and adherence, the culture medium is removed, and the ECM culture medium containing zinc ions with different concentrations is added, wherein the zinc ion concentrations are respectively 0, 2.5, 5, 10, 15 and 25 mu g/mL. After 24h of culture, the viability of the cells was determined with CCK-8 (see FIG. 4 b). Cell viability was observed using a live-dead staining assay (see FIG. 4 c).

From the CCK-8 results, when the concentration of zinc ions is less than or equal to 15 mug/mL, the activity of HUVEc cells is not affected, and when the concentration of zinc ions reaches 25 mug/mL, the activity of the cells is only 45 percent of that of a control group, and obvious cytotoxicity is shown. Therefore, when the concentration of zinc ions is greater than 15 mug/mL, the vitality of cells is gradually reduced, and according to the release curve of zinc ions in FIG. 4a, the concentration of zinc ions in the composite fiber membrane with the addition amount of DMOG @ ZIF-8 of 5mg/mL is more than 15mg/mL within less than 24h, and the rapid increase of the concentration of zinc ions in a short time may cause the generation of cytotoxicity, and according to the results of example 3 and this example, the optimum addition amount of DMOG @ ZIF-8 can be determined to be 3mg/mL, and under the addition amount, DMOG @ ZIF-8-Gel-PCL has a good antibacterial effect on Escherichia coli and Staphylococcus aureus, can avoid the generation of severe cytotoxicity, and provides a precondition for the further promotion of cell behavior.

Example 5 sustained release characterization of drug-loaded metal-organic framework composite electrospun fiber membranes on DMOG

Preparing DMOG-loaded ZIF-8 nanoparticles: 400mg of zinc nitrate hexahydrate is taken in 10mL of anhydrous methanol, 0.2g of DMOG and 1g of 2-methylimidazole are taken in 15mL of anhydrous methanol, ultrasonic treatment is carried out to accelerate dissolution, the mixed solution of DMOG and 2-methylimidazole is added into the anhydrous methanol solution of zinc nitrate hexahydrate by a syringe at the push speed of 0.3mL/min under the control of a micro-injection pump, magnetic stirring is carried out for 12h, centrifugation is carried out at the rotating speed of 10000rpm, the precipitate is washed twice by anhydrous methanol, and vacuum drying is carried out for 24h at the temperature of 40 ℃.

Preparing an electrospinning solution: and (3) taking 30mg of DMOG @ ZIF-8 nano particles into 10mL of hexafluoroisopropanol, carrying out ultrasonic dispersion treatment for 10min by using an ultrasonic crusher to fully disperse the nano particles, weighing 0.6g of gelatin and 0.6g of polycaprolactone, adding the gelatin and the polycaprolactone into the suspension, and carrying out magnetic stirring for 72 hours to obtain an electrospinning solution.

Preparing composite fibers by electrostatic spinning: loading the electrospinning solution into an injector, setting the pushing speed of a micro-injection pump to be 1.2mL/h, the voltage to be 12kV, the receiving distance to be 12cm, and carrying out electrostatic spinning on the spinning solution at the rotating speed of a collecting roller of 200rpm to obtain the composite nanofiber, wherein the label of the composite nanofiber is DMOG @ ZIF-8-Gel-PCL.

Preparation of control sample: and (3) taking 10mg of DMOG into 10mL of hexafluoroisopropanol, treating for 10min by using an ultrasonic cleaning instrument to accelerate dissolution, weighing 0.6g of gelatin and 0.6g of polycaprolactone into the suspension, and magnetically stirring for 72 hours to obtain the electrospinning solution. And loading the electrospinning solution into an injector, setting the pushing speed of a micro-injection pump to be 1.2mL/h, the voltage to be 12kV, the receiving distance to be 12cm, and carrying out electrostatic spinning on the spinning solution at the rotating speed of a collecting roller to obtain the composite nanofiber, wherein the composite nanofiber is marked as DMOG-Gel-PCL.

Respectively cutting DMOG @ ZIF-8-Gel-PCL (DZGP) and DMOG-Gel-PCL (DGP) into small square samples with the same area, and respectively recording the weights of the small square samples. Each group of samples was soaked in 5mL of PBS solution (pH 7.4) and incubated at 150rpm in a constant temperature shaker at 37 ℃. At different time collection points, 1mL of broth was removed from both sets of samples simultaneously and the culture apparatus was supplemented with 1mL of fresh PBS solution.

And measuring the content of the DMOG in the collected culture solution by using a high performance liquid chromatograph. The cumulative release profiles of DMOG in fibers for the two different DMOG loading regimes are shown in figure 5. It can be seen from the accumulated release curve that in the DMOG-Gel-PCl fiber component prepared by the common blend spinning method, the DMOG release shows the phenomenon of burst release within the earliest few days and then rapidly reaches the platform stage, while in the DZGP component, the DMOG shows the characteristic of long-term slow release, can be continuously and slowly released for more than 12 days, shows good slow release effect, and can avoid excessive burst release of the DMOG in the initial hemostasis stage and inflammation stage (probably within the first 3 days of the wound healing cycle) of wound healing.

Example 6 characterization of the ability of drug-loaded metal-organic framework composite electrospun fibrous membrane to promote cell proliferation

(1) Preparing a DMOG @ ZIF-8-Gel-PCL (hereinafter referred to as DZGP) composite nanofiber with the addition amount of 3mg/mL of DMOG @ ZIF-8 according to the method in the embodiment 5; meanwhile, under the same other conditions, preparing ZIF-8-Gel-PCL (hereinafter referred to as ZGP) composite nano-fibers with the addition of ZIF-8 of 3 mg/mL; meanwhile, Gel-PCL (hereinafter, abbreviated as GP) containing neither DMOG @ ZIF-8 nor ZIF-8 was prepared as a control under otherwise identical conditions.

(2) Material sterilization: cutting fibers of three components of Gel-PCL, ZIF-8-Gel-PCL and DMOG @ ZIF-8-Gel-PCL into a round sheet with the diameter of 14mm, attaching the round sheet to the bottom of a 24-pore plate, fixing the round sheet by using a sterile silicone rubber gasket, keeping the pore plate cover open, and sterilizing for 24 hours under the irradiation of ultraviolet light of a super clean bench.

(3) Cell viability assay: HUVEc cells were seeded on the fibrous membrane at a density of 20000 per well. The cell viability was measured by CCK-8 method on

days

1, 3 and 5 of the culture, respectively, and the results are shown in FIG. 6.

As can be seen from FIG. 6, the proliferation of all three fiber membranes, GP, ZGP and DZGP, was very good, the number of cells increased with the increase of the culture time, and the results at day 5 showed that HUVECs grown on the DZGP fraction had better proliferation capacity than other fractions.

Example 7 characterization of HUVEc cell migration promoting ability of drug-loaded metal-organic framework composite electrospun fiber membrane

Three kinds of fibrous membranes, Gel-PCL, ZIF-8-Gel-PCL (ZIF-8 added in an amount of 3mg/mL), and DMOG @ ZIF-8-Gel-PCL (DMOG @ ZIF-8 added in an amount of 3mg/mL) were prepared, respectively, according to the method described in example 6.

The material was treated and sterilized by the method of step (2) in example 6.

The transfer well method is used for representing the migration condition of HUVEc cells, and the specific method comprises the following steps: transwell was placed in a 24-well plate containing the material (fibrous membranes in the lower Transwell chamber), 400. mu.L of ECM medium was added to the lower chamber, HUVEc cells were seeded at 50000/well in the upper Transwell chamber, and the cell suspension volume in the upper chamber was finally brought to 200. mu.L.

Transferring the 24-well plate to a constant temperature incubator at 37 ℃ and 5% CO ₂ The culture was carried out for 10 hours. The chambers were removed and the medium was aspirated off the chamber, transferred to a new 24-well plate, and cells were fixed for 1 hour by adding 4% paraformaldehyde solution. Adding into the mixture at a concentration of 0.1%Cells were stained with crystal violet solution for 30 minutes and the Transwell chamber was washed three times with PBS. The HUVECs above the septum of the chamber were gently scraped off with a sterile cotton swab, and the HUVECs that had migrated below the chamber were observed brightly with an inverted fluorescence microscope and photographed for counting the number of migrated cells. As shown in fig. 7. From the results of fig. 7, the migration number of HUVECs in the DZGP experimental group is significantly greater than that of the GP group and the DZGP group, which shows that the DMOG-loaded ZIF-8 composite electrospun fiber membrane has good ability to promote the migration of HUVECs cells.

Example 8 characterization of HUVEc cell in vitro tube formation promoting ability of drug-loaded metal-organic framework composite electrospun fibrous membrane

Three kinds of fiber membranes were prepared in the same manner as in example 6, respectively, and the materials were treated and sterilized in the same manner as in step (2) in example 6.

The Matrigel which had been previously dispensed and stored at-20 ℃ was taken out and placed in a refrigerator at 4 ℃ overnight to be thawed. The 48-well plate and the tip for the experiment were pre-cooled on ice, Matrigel was added to the 48-well plate in an amount of 100. mu.L per well, and gently shaken to uniformly spread the Matrigel over the bottom of the plate. The well plate with the matrigel spread thereon was transferred to a constant temperature incubator, and was incubated at 37 ℃ for 2 hours to solidify it, and then taken out. HUVEc cells were added to Matrigel-plated wells at a density of 50000/well. After incubation for 10 hours, the medium was aspirated and the number of nodes was counted by photographing the vascularization in each well under the bright field of an inverted fluorescence microscope. As shown in fig. 8.

From the results shown in fig. 8, in the DZGP experimental group, both the length of the tube and the number of the formed closed-loop circles are more than those of the GP group and the ZGP group, which indicates that the in vitro angiogenesis condition of HUVECs in the DZGP experimental group is significantly better than that of the GP group and the ZGP group, and the ZIF-8 composite electrospun fiber membrane loaded with DMOG has good capability of promoting in vitro angiogenesis of HUVECs, which indicates that the composite fiber membrane dressing can reasonably release the DMOG while resisting infection to promote the generation of blood vessels at wounds, and has good application potential in wound treatment.

Example 9 air permeability measurement of drug-loaded metal-organic framework composite electrospun fiber membranes

Taking three penicillin bottles with the diameter of 1.8cm, adding 15mL of ultrapure water respectively, and shearing to obtain a section with the area of 3 multiplied by 3cm ² The bottle mouth is covered by the fiber membrane of each component, and a gap is not left between the fiber membrane and the upper edge of the bottle mouth by using a double-sided adhesive tape. The starting mass of the devices of each experimental group was recorded,

placing the devices at 25 ℃ under the condition that the humidity is 50% -60%, weighing and recording the mass of each device every 1h, calculating the mass reduction quantity delta m between time points, drawing a delta m-delta t graph by taking delta t as an abscissa and delta m as an ordinate, calculating the slope, and calculating the slope through a formula:

water vapor transmission rate (slope. times.24)/area (unit: g/m) ² /day)

And calculating the air permeability of each component fiber membrane.

The results are shown in FIG. 9, and the water vapor transmission rates of GP, ZGP and DZGP fiber membranes are calculated by the formula to be 2713.57, 2720.35 and 2723.66g/m respectively ² Day approaches the optimal range of water vapor transmission rate for wound dressings (2000-2500 g/m) ² Day), indicating that the composite fiber film has good water vapor transmission rate, which is one of the excellent properties as an ideal wound dressing.

Claims

1. The preparation method of the drug-loaded metal-organic framework composite electrostatic spinning fiber membrane is characterized by comprising the following steps:

(1) adding the drug-loaded metal organic framework into hexafluoroisopropanol, after uniformly dispersing, adding gelatin and polycaprolactone, and fully stirring to obtain an electrospinning solution; the drug-loaded metal-organic framework is ZIF-8 nano particles loaded with dimethyloxalyl glycine;

(2) and (2) filling the electrospinning solution obtained in the step (1) into a syringe, and preparing the drug-loaded metal-organic framework composite electrospun fiber membrane by using an electrospinning method.

2. The preparation method of the drug-loaded metal-organic framework composite electrospun fiber membrane of claim 1, wherein the addition amount of the drug-loaded metal-organic framework in the electrospinning solution is 3-5 mg/mL.

3. The preparation method of the drug-loaded metal-organic framework composite electrospun fiber membrane according to claim 2, wherein the addition amount of the drug-loaded metal-organic framework in the electrospinning solution is 3 mg/mL.

4. The preparation method of the drug-loaded metal-organic framework composite electrospun fiber membrane according to any one of claims 1 to 3, wherein the mass ratio of the gelatin to the polycaprolactone in the step (1) is 4:6 to 6: 4; the mass-volume ratio of the total mass of the gelatin and the polycaprolactone to the mass of the hexafluoroisopropanol is 0.1-0.14 g/mL.

5. The preparation method of the drug-loaded metal-organic framework composite electrospun fiber membrane according to claim 4, wherein the mass ratio of the gelatin to the polycaprolactone in the step (1) is 1: 1; the mass-volume ratio of the total mass of the gelatin and the polycaprolactone to the mass of the hexafluoroisopropanol is 0.12 g/mL.

6. The preparation method of the drug-loaded metal-organic framework composite electrospun fiber membrane according to any one of claims 1-3, characterized in that the stirring time in step (1) is not less than 72 h; the electrostatic spinning in the step (2) has the spinning voltage of 9kV-14kV, the temperature of 15-30 ℃, the relative humidity of 40-60%, the feeding speed of 1-2mL/h, the receiving distance of 10-15cm and the rotating speed of a roller of 100-400 rpm.

7. The preparation method of the drug-loaded metal-organic framework composite electrospun fiber membrane of claim 1, wherein the preparation of the drug-loaded metal-organic framework in the step (1) comprises the following steps:

8. The preparation method of the drug-loaded metal-organic framework composite electrospun fiber membrane of claim 7, wherein the volume-to-volume ratio of zinc nitrate hexahydrate to anhydrous methanol is 0.4 g: 10-15 mL; the mass ratio of dimethyloxalglycine to 2-methylimidazole is 1: 9-2: 9; the stirring time is 12-24 h; the drug-loading amount of the drug-loading metal-organic framework is 15-25 wt%.

9. A drug-loaded metal-organic framework composite electrospun fiber membrane prepared by the preparation method of any one of claims 1-8.

10. The use of the drug-loaded metal-organic framework composite electrospun fiber membrane of claim 9 in the preparation of a dressing.