CN118436622A - A method for preparing drug-loaded nanofiber membrane - Google Patents

Abstract

The invention discloses a method for preparing a drug-loaded nanofiber membrane, comprising the steps of uniformly dispersing drug powder in an aqueous solution of polyvinyl alcohol added with a surfactant to obtain an electrospinning precursor dispersion; using an electrostatic spinning method, the electrospinning precursor dispersion is made into a drug-loaded nanofiber membrane; nanocellulose and epichlorohydrin are dispersed in an aqueous solution of sodium hydroxide to prepare a crosslinking solution, the drug-loaded nanofiber membrane is immersed in the crosslinking solution for immersion crosslinking, and drying to obtain a crosslinked drug-loaded nanofiber membrane. The drug-loaded nanofiber membrane prepared in the invention has both high water absorption and low dissolution loss rate, and has good antibacterial effect, and exhibits good tensile properties under both dry and wet conditions.

Description

A method for preparing drug-loaded nanofiber membrane

Technical Field

The invention relates to the technical field of electrostatic spinning, and more specifically to a method for preparing a drug-loaded nanofiber membrane.

Background technique

The skin is the main barrier between the human body and the environment. It is constantly exposed to the outside world and is therefore easily damaged by different types of lesions. "Haodele" powder has a significant clinical effect in the antibacterial adjuvant treatment of various skin diseases and is one of the most widely used Mongolian medicines for the treatment of skin diseases.

Although "Haodele" powder has good antibacterial and antimicrobial effects, the traditional way of administration is to boil it and then take a medicinal bath, which is inconvenient to use and has a low drug utilization rate. Nanofibers have the advantages of small diameter, small pore size, large specific surface area, and controllable morphology. Due to their own characteristics, they are clinically manifested as having high air permeability and good pus absorption.

Currently, there is a method to make "Haodele" powder into drug-loaded nanofiber membranes, but when the drug-loaded nanofiber membranes are not cross-linked, the mechanical properties of the membranes are poor and they are prone to dissolution, resulting in unsatisfactory antibacterial effects. Therefore, a new method for preparing cross-linked drug-loaded nanofiber membranes is needed to obtain high-performance drug-loaded nanofiber membranes whose raw materials are harmless to the human body.

Summary of the invention

To this end, the technical problem to be solved by the present invention is to provide a method for preparing drug-loaded nanofiber membranes to improve the mechanical properties and antibacterial properties of dressings made of "Haodele" powder.

In order to solve the above technical problems, the present invention provides the following technical solutions:

A method for preparing a drug-loaded nanofiber membrane comprises the following steps:

(1) uniformly dispersing drug powder in an aqueous solution of polyvinyl alcohol to which a surfactant has been added to obtain an electrospinning precursor dispersion;

(2) using an electrospinning method to prepare a drug-loaded nanofiber membrane from an electrospinning precursor dispersion;

(3) Dispersing nanocellulose and epichlorohydrin in an aqueous solution of sodium hydroxide to prepare a crosslinking solution, immersing the drug-loaded nanofiber membrane in the crosslinking solution for immersion crosslinking, and drying to obtain a crosslinked drug-loaded nanofiber membrane. Preparing drug powder into drug-containing nanofibers can improve the efficacy of the drug and the convenience of use.

In the above-mentioned method for preparing drug-loaded nanofiber membrane, in step (1), the surfactant is sodium dodecyl sulfate, and the preparation method of the electrospinning precursor dispersion is to add polyvinyl alcohol and sodium dodecyl sulfate into ultrapure water, and stir at room temperature and heat and stir in sequence; after naturally cooling to room temperature, add drug powder and continue stirring to obtain the electrospinning precursor dispersion; heating and stirring help the polyvinyl alcohol to fully dissolve, and adding drug powder after cooling to room temperature is beneficial to protect the active ingredients in the drug powder from being destroyed by high temperature;

In step (2), the electrospinning precursor dispersion is loaded into a medical syringe, and the electrospinning precursor dispersion droplets discharged from the needle of the medical syringe are stretched into nanofibers by an electrostatic field, and the nanofibers are deposited on a receiving plate to form a drug-loaded nanofiber membrane;

In step (3), during the soaking cross-linking process, the cross-linking liquid is subjected to ultrasonic treatment, and the temperature of the cross-linking liquid is kept constant or gradually reduced. The cross-linking reaction can improve the mechanical strength of the drug-loaded nanofiber membrane, and at the same time improve its water absorption and dissolution properties to a certain extent, thereby improving the drug release capacity of the drug-loaded nanofiber membrane.

In the above-mentioned method for preparing drug-loaded nanofiber membrane, in step (3), the cross-linking time is 1 to 6 hours; during the cross-linking process, the temperature of the cross-linking liquid is 45 to 70°C and is kept constant; and the ultrasonic frequency is 25 to 30 kHz. Under appropriate temperature, ultrasound can accelerate the chemical reaction between the drug-loaded nanofiber membrane and the cross-linking liquid through its high-frequency vibration, and is conducive to the entanglement of nanocellulose with PVA fibers. This acceleration can shorten the cross-linking time and improve the cross-linking efficiency; ultrasound can maintain the uniform texture of the cross-linking liquid, which helps to avoid differences in the degree of cross-linking within the membrane, thereby improving the overall performance of the membrane.

In the above-mentioned preparation method of drug-loaded nanofiber membrane, in step (3), the cross-linking time is 1 to 6 hours; at the beginning of immersion cross-linking, the temperature of the cross-linking liquid is 45 to 70°C, and the temperature of the cross-linking liquid decreases by 2 to 2.5°C every 1 hour; the ultrasonic frequency is 25 to 30kHz. Too high a temperature may cause a large amount of degradation of nanocellulose in an alkaline environment, or fracture, increase the brittleness of the membrane, reduce flexibility and ductility, and affect the tensile strength and elongation at break of the membrane; and too high a temperature may cause the cross-linking reaction rate to be too fast, resulting in uneven cross-linking degree inside the PVA membrane; when the cross-linking temperature is too low, the speed of the cross-linking reaction decreases, which may lead to insufficient cross-linking, which will reduce the water resistance, solvent resistance and other properties of the membrane; the method of cooling cross-linking can ensure uniform and sufficient cross-linking while allowing the membrane to maintain a certain degree of flexibility and ductility.

In the above-mentioned method for preparing drug-loaded nanofiber membrane, in step (3), the cross-linking time is 5 hours; when the immersion cross-linking begins, the temperature of the cross-linking liquid is 60°C, and the temperature of the cross-linking liquid decreases by 2.5°C every hour; the ultrasonic frequency is 25kHz.

In the above-mentioned method for preparing drug-loaded nanofiber membrane, in step (3), the mass volume ratio of the drug-loaded nanofiber membrane to the cross-linking liquid is 0.005-0.02 g/mL. Too much cross-linking agent will lead to excessive cross-linking reaction, making the connection between PVA molecular chains too tight, which will increase the hardness of the membrane and reduce its flexibility; insufficient cross-linking agent will reduce the tensile strength, elongation at break and impact resistance of the membrane. The membrane may become easily deformed and damaged.

The above-mentioned method for preparing the drug-loaded nanofiber membrane, the method for preparing the cross-linking liquid is: nanocellulose and epichlorohydrin are added to a sodium hydroxide aqueous solution with a pH of 10 to 11 in sequence and stirred evenly; the volume ratio of the sodium hydroxide aqueous solution to epichlorohydrin is 100: (3 to 5), and the mass volume ratio of the nanocellulose to the sodium hydroxide aqueous solution is 4 to 6 g/100 mL; the diameter of the nanocellulose is 10 to 50 nm, and the length is 0.5 to 3 μm. Nanocellulose swells in a sodium hydroxide aqueous solution with a pH of 10 to 11, and its structure becomes soft and loose, and its water retention capacity increases; the cross-linking reaction of epichlorohydrin and PVA needs to be carried out under alkaline conditions. When the amount of epichlorohydrin nanocellulose is within this dosage range, it can improve the water retention and mechanical strength of the PVA film, and at the same time, the nanocellulose is not easy to form more crystals or aggregates inside the PVA film, resulting in a decrease in the flexibility of the PVA film; when the diameter and length of the nanocellulose are within this range, it can better cooperate with the micropores in the PVA fiber membrane and entangle the PVA spinning. When used for cooling cross-linking, the use of this nanocellulose is conducive to making the membrane obtain good water absorption and flexibility.

In the above-mentioned method for preparing drug-loaded nanofiber membrane, in step (1), the stirring time at room temperature is 0.5 to 1 h, the heating temperature during heating and stirring is 80 to 85°C, and the stirring time is 3 to 4 h; the stirring time after adding the drug powder is continued for 12 to 14 h; in the electrospinning precursor dispersion, the mass ratio of polyvinyl alcohol to drug powder is 20:(1 to 5), the mass fraction of polyvinyl alcohol is 8wt% to 9wt%, and the mass fraction of sodium dodecyl sulfate is 0.15wt% to 0.25wt%.

In the above-mentioned method for preparing drug-loaded nanofiber membrane, in step (2), the inner diameter of the medical syringe needle is 0.5-0.7 mm, the distance between the receiving plate and the needle is 13-15 cm, the speed at which the medical syringe discharges the electrospinning precursor dispersion is 0.5-0.6 mL·h ^-1 , the spinning voltage is 14-16 kV, the ambient temperature is 28-30° C., the ambient humidity is 31%-35%, and the spinning time is 4.5-5.5 h.

In the above-mentioned method for preparing drug-loaded nanofiber membrane, the drug powder is "Haodele" powder. In step (1), the stirring time at room temperature is 0.5h, the heating temperature during heating and stirring is 85°C, and the stirring time is 3h; the stirring time after adding the drug powder is continued for 12h; in the electrospinning precursor dispersion, the mass ratio of polyvinyl alcohol to drug powder is 20:5, the mass fraction of polyvinyl alcohol is 8wt%, and the mass fraction of sodium dodecyl sulfate is 0.2wt%;

In step (2), the inner diameter of the needle of the medical syringe is 0.6 mm, the distance between the receiving plate and the needle is 15 cm, the speed of the medical syringe discharging the electrospinning precursor dispersion is 0.6 mL·h ^-1 , the spinning voltage is 16 kV, the ambient temperature is 30° C., the ambient humidity is 35%, and the spinning time is 5 h;

The preparation method of the cross-linking liquid is as follows: nanocellulose and epichlorohydrin are added to a sodium hydroxide aqueous solution with a pH of 10 in sequence and stirred evenly, wherein the volume ratio of the sodium hydroxide aqueous solution to epichlorohydrin is 100:5, and the mass volume ratio of the nanocellulose to the sodium hydroxide aqueous solution is 5 g/100 mL; the diameter of the nanocellulose is 10 to 50 nm, and the length is 0.5 to 3 μm;

In step (3), the cross-linking time is 5 hours; when the immersion cross-linking begins, the temperature of the cross-linking liquid is 60°C, and the temperature of the cross-linking liquid decreases by 2.5°C every hour; the ultrasonic frequency is 25kHz; and the mass volume ratio of the drug-loaded nanofiber membrane to the cross-linking liquid is 0.01g/mL.

The technical solution of the present invention achieves the following beneficial technical effects:

1. The present invention uses epichlorohydrin EPI and nanocellulose CNF as crosslinking agents. Compared with traditional glutaraldehyde crosslinking, the crosslinking method used in the present invention has a higher crosslinking efficiency. When the volume ratio of EPI to sodium hydroxide solution in the crosslinking liquid is 5:100, the mass volume ratio of CNF to sodium hydroxide solution is 5g/100mL, and the mass volume ratio of the drug-loaded nanofiber membrane to the crosslinking liquid is 0.01g/mL, the crosslinking reaction can be fully carried out. At the same time, 25kHz ultrasonic crosslinking is used to ensure that the crosslinking liquid always maintains a uniform texture during the crosslinking process. The crosslinking is carried out by a cooling crosslinking method with an initial temperature of 60°C, a cooling rate of 2.5°C/h, and a crosslinking time of 5h, and a crosslinked drug-loaded nanofiber membrane with greater tensile strength, better water absorption, and better anti-dissolution properties is obtained. The length of the CNF used in the present invention is 0.5 to 3 μm, which is relatively long. It will swell under alkaline conditions, and the structure becomes soft and loose, which can form good entanglement with the drug-loaded nanofibers. The cross-linking liquid prepared by cellulose of this length and epichlorohydrin is used for ultrasonic immersion cross-linking, which can improve the mechanical strength of the drug-loaded nanofiber membrane while allowing the cross-linked drug-loaded nanofiber membrane to still retain a certain water absorption, which is beneficial for the cross-linked drug-loaded nanofiber membrane to absorb tissue fluid exuded from the wound, and is beneficial for the drug in the cross-linked drug-loaded nanofiber membrane to enter the tissue fluid and diffuse in the wound with the tissue fluid, thereby enhancing the antibacterial effect of the cross-linked drug-loaded nanofiber membrane.

2. The cooling cross-linking method adopted in the present invention makes the cross-linking process more accurate and controllable. Compared with the constant temperature cross-linking, the cooling cross-linking with an initial temperature of 60°C, a cooling rate of 2.5°C/h, and a cross-linking time of 5h in the present invention ensures that epichlorohydrin and polyvinyl alcohol fully react and reduce the solubility of the film. It prevents the cross-linking reaction from being too fast, causing the drug-loaded nanofiber membrane to quickly harden and become brittle, and also prevents a large amount of nanocellulose from being degraded, resulting in the cross-linked drug-loaded nanofiber membrane. The flexibility is too low, which is conducive to obtaining a more ideal tensile breaking stress and elongation at break. The water absorption rate of the drug-loaded nanofiber membrane obtained by cooling and cross-linking for 5 hours can reach 751%, while the solubility rate is only 12%. This shows that the drug-loaded nanofiber membrane can quickly absorb body fluids when treating open wounds, and can remain stable in a humid environment, and because the water absorption of the drug-loaded nanofiber membrane is improved, its drug release ability is also improved.

3. The cooling cross-linked drug-loaded nanofiber membrane exhibits good tensile properties under both dry and wet conditions. The tensile stress at break and the elongation at break are significantly improved compared to the uncross-linked drug-loaded nanofiber membrane, making the cross-linked drug-loaded nanofiber membrane more suitable for fitting wounds at joints and able to resist stretching caused by joint movement.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram of the operation steps for preparing rabbit skin tissue slices according to an embodiment of the present invention;

FIG2 is a graph showing the average diameter of the cross-linked drug-loaded fibers at different cross-linking times according to an embodiment of the present invention;

FIG3 is a morphology diagram of the drug-loaded nanofiber membrane after immersion in PBS according to an embodiment of the present invention;

FIG4a is a slice diagram of rabbit skin tissue in the cross-linked drug-loaded nanofiber membrane group in multiple intact skin stimulation experiments in an embodiment of the present invention;

FIG4b is a slice of rabbit skin tissue in the non-cross-linked drug-loaded nanofiber membrane group in multiple intact skin stimulation experiments in an embodiment of the present invention;

FIG4c is a slice diagram of rabbit skin tissue in the pure PVA fiber group in multiple intact skin stimulation experiments in an embodiment of the present invention;

FIG4d is a diagram of skin tissue sections of rabbits in the blank gauze group in multiple intact skin stimulation experiments in an embodiment of the present invention.

Detailed ways

1. Preparation of drug-loaded nanofiber membrane

Polyvinyl alcohol (PVA) is a water-soluble polymer that is non-toxic and harmless to the human body and has good biocompatibility. It is the preferred material for medical dressings. In this embodiment, when the electrospinning method is used to prepare the drug-loaded nanofiber membrane, it is first necessary to use "Haodele" powder (hereinafter referred to as M) and PVA to prepare an electrospinning precursor dispersion.

Use a balance to accurately weigh 8wt% PVA and 0.2wt% sodium dodecyl sulfate SDS and add them to ultrapure water. Stir at room temperature for 0.5h, then put them in a magnetic stirring water bath and stir at 85℃ for 3h to obtain 8wt% (mass fraction) PVA aqueous solution. When the temperature drops to room temperature, add "Haodele" powder to the solution according to PVA:M＝20:0, PVA:M＝20:1, PVA:M＝20:2, PVA:M＝20:3, PVA:M＝20:4 and PVA:M＝20:5 (mass ratio) and stir them magnetically at room temperature for 12h to ensure that the "Haodele" powder and PVA solution are fully mixed and uniform to obtain electrospinning precursor dispersion.

The prepared electrospinning precursor dispersion was loaded into a 5mL medical syringe. The outer diameter of the syringe needle was 0.9mm and the inner diameter was 0.6mm. It was ensured that there were no bubbles in the syringe. Then the syringe was placed in the propeller. The aluminum foil was cut into a size of 20cm×20cm as a receiving plate. The distance between the receiving plate and the syringe needle was 15cm. The needle was aligned with the center of the receiving plate, and the propulsion speed of the propeller (i.e., the speed at which the medical syringe discharges the electrospinning precursor dispersion) was adjusted to 0.6mL·h ^-1 , the spinning voltage was 16kV, the spinning time was 5h, the ambient humidity was 35%, and the ambient temperature was 30℃. Under these conditions, the droplets at the needle were pulled and stretched to the receiving plate under the action of the electric field, the solvent evaporated in the air, and the fibers accumulated on the receiving plate to form a drug-loaded nanofiber membrane. The drug-loaded nanofiber membrane was collected, placed in a blast drying oven for 2h, and then put into a sealed bag and marked for use.

2. Morphological characterization of drug-loaded nanofiber membranes

Scanning electron microscope (SEM) was used to observe the fiber morphology of drug-loaded nanofiber membranes with different PVA:M mass ratios. 50 fibers were selected under the electron microscope, and the fiber diameter was measured. The obtained fiber diameter data was used to calculate the average fiber diameter using Excel. and standard deviation SD.

According to the fiber morphology, combined with environmental parameters (precursor solution viscosity, surface tension, conductivity, voltage of the spinning process, spinning distance, ambient temperature and humidity), the final electrospinning precursor dispersion dosing ratio PVA:M=20:5 (mass ratio) is appropriate. If this ratio is exceeded, there will be obvious insoluble matter in the solution. At this ratio, the diameter of the fiber in the drug-loaded nanofiber membrane is 255.4±42.9nm, which is larger than the fiber diameter of other ratios and contains more drugs.

3. Cross-linking of drug-loaded nanofiber membranes

In this embodiment, nanocellulose (CNF, manufactured by Xinyuhong, item number xyh001) with a diameter of 10 to 50 nm and a length of 0.5 to 3 μm and epichlorohydrin (EPI) were used to cross-link the drug-loaded nanofiber membrane (PVA:M=20:5).

Prepare a NaOH solution with a pH of 10, add 5 g of CNF and 5 mL of EPI to every 100 mL of NaOH solution, stir evenly, and obtain a crosslinking solution. Divide the above-mentioned drug-loaded nanofiber membranes into several groups, and immerse the drug-loaded nanofiber membranes in a container of crosslinking solution while adding EPI, according to the mass volume ratio of the drug-loaded nanofiber membrane to the crosslinking solution of 0.01 g/mL, and allow the crosslinking solution to submerge the membranes.

The container containing the crosslinking liquid and the drug-loaded nanofiber membrane was placed in an ultrasonic water bath preheated to 60°C, and the temperature of the ultrasonic water bath was lowered by 2.5°C every hour (i.e., cooling crosslinking was performed), and another group of constant temperature ultrasonic water baths at 60°C were set up as a control (constant temperature crosslinking). The ultrasonic frequency was 25kHz, and the immersion crosslinking time was 1h, 2h, 3h, 4h, 5h and 6h, respectively. After the crosslinking was completed, the membrane surface was repeatedly rinsed with distilled water, dried at room temperature, and then placed in a blast drying oven for 12h to obtain a crosslinked drug-loaded nanofiber membrane.

4. Morphological characterization of cross-linked drug-loaded nanofiber membranes

(1) Scanning electron microscopy (SEM)

The fiber morphology in the cross-linked drug-loaded nanofiber membrane (PVA:M=20:5) was observed using a scanning electron microscope, and the average diameter and standard deviation of the fibers in the cross-linked drug-loaded nanofiber membrane at different cross-linking times were compared. The method was the same as the scanning electron microscope observation method of the drug-loaded nanofiber membrane mentioned above.

(2) Water absorption and dissolution rate test

The main function of "Haodele" powder is antibacterial and antibacterial. Sometimes it is applied to open wounds. Such wounds generally have tissue fluid exudation, which needs to be absorbed in time and maintain a good air permeability environment to facilitate wound recovery. This requires that the wound dressing has a certain water absorption and the dissolution loss rate should be low. In order to obtain the optimal cross-linking time, water absorption experiments and dissolution loss experiments were carried out. The drug-loaded nanofiber membranes with different cross-linking times were cut into 2cm×2cm squares and weighed. Three parallel experiments were performed and the average value was taken and recorded as w1. Then, after soaking in phosphate buffer solution (PBS) for 24 hours, the fiber membrane was taken out and the excess water on the surface was absorbed with filter paper until no obvious water stains were seen. The weight was weighed and recorded as w2. Finally, it was dried in a freeze drying oven for 12 hours and weighed as w3. Calculate the water absorption rate and dissolution loss rate. The calculation method is formula (1) and formula (2);

5. Scanning electron microscopy (SEM) analysis of drug-loaded nanofiber membrane

According to the SEM photo analysis, before cross-linking, the average diameter of the fibers in the drug-loaded nanofiber membrane (PVA:M=20:5) was 255.4±42.9nm. Figure 2 is a graph of the average diameter of the drug-loaded fibers after cross-linking at different cross-linking times, and Table 1 is the relevant data. As shown in Figure 2, cross-linking increases the diameter of the fibers in the drug-loaded nanofiber membrane, and as the cross-linking time increases, the average diameter of the fibers also increases. The method of using constant temperature cross-linking can obtain thicker fibers faster than the method of using cooling cross-linking, and the mechanical strength of the fiber membrane composed of thicker fibers will be higher.

Table 1 Average fiber diameter and standard deviation at different crosslinking times

6. Water absorption and dissolution rate test of cross-linked drug-loaded nanofibers

The results of water absorption and dissolution rate tests of the drug-loaded nanofiber membranes with cooling crosslinking time of 1h, 2h, 3h, 4h, 5h and 6h are shown in Table 2, and the results of constant temperature crosslinking are shown in Table 3. After crosslinking, the hydrophilic groups of the drug-loaded nanofiber membranes are reduced, the fiber membranes become dense, and the water resistance increases. Since nanocellulose itself has a certain hydrophilicity, after crosslinking of nanocellulose, PVA and EPI, the water absorption performance of the drug-loaded nanofiber membrane is improved to a certain extent, and the dissolution rate will decrease, so the performance of the crosslinked drug-loaded nanofiber membrane is relatively ideal.

Comparing the water absorption rate and dissolution loss rate of the drug-loaded nanofiber membrane cross-linked at constant temperature and cross-linked at reduced temperature under the cross-linking time of 5h, it can be seen that the dissolution loss rates of the two are relatively close, but the water absorption rate of the drug-loaded nanofiber membrane obtained by cooling cross-linking is higher, and the higher water absorption rate is conducive to the absorption of pus by the drug-loaded nanofiber membrane. The water absorption rate of the cross-linked drug-loaded nanofiber membrane at constant temperature cross-linking for 4h is close to that at cooling cross-linking for 5h, but the dissolution loss rate of the drug-loaded nanofiber membrane at constant temperature cross-linking for 4h is higher than that of the drug-loaded nanofiber membrane at cooling cross-linking for 5h. In summary, the water absorption rate and dissolution loss rate of the drug-loaded nanofiber membrane at cooling cross-linking for 5h are more ideal.

Table 2 Water absorption and dissolution rate of drug-loaded nanofiber membranes cross-linked at different cross-linking times

1h 2h 3h 4h 5h 6h Water absorption 502% 891% 843% 798% 751% 736% Dissolution rate 45.5% 36.7% 27.6% 15.3% 12.0% 11.8%

Table 3 Water absorption and dissolution rate of constant temperature cross-linked drug-loaded nanofiber membranes at different cross-linking times

1h 2h 3h 4h 5h 6h Water absorption 504% 871% 809% 759% 723% 672% Dissolution rate 49.7% 32.2% 19.1% 14.7% 11.3% 9.5%

The uncrosslinked and 5h cooling crosslinked drug-loaded fiber membranes were cut into 1×1cm sizes, and then phosphate buffer solution PBS was taken with a rubber-tipped dropper and dropped on the drug-loaded nanofiber membranes. After soaking for 3 hours, the morphological characteristics of the drug-loaded fiber membranes were observed. Figure 3 shows the experimental results. It can be seen from the figure that the uncrosslinked drug-loaded fiber membranes dissolved immediately when they met PBS, and the drug-loaded nanofiber membranes obtained by cooling crosslinking did not shrink significantly, and basically maintained the same morphology, indicating that the water resistance of the drug-loaded nanofiber membranes crosslinked by cooling was better than that of the uncrosslinked drug-loaded nanofiber membranes.

7. Test of dry and wet tensile properties of cooling cross-linked drug-loaded nanofiber membrane

The drug-loaded nanofiber membrane should have a certain tensile strength and ductility. When the drug-loaded nanofiber membrane is tightly attached to the wound at the joint, it will be stretched during the joint movement. If the tensile and ductility of the drug-loaded nanofiber membrane are poor, it may be torn, causing the wound to be exposed. In addition, the drug-loaded nanofiber membrane will absorb the tissue fluid of the wound, so it needs to maintain a certain tensile strength and ductility when wet.

The dry tensile properties and wet tensile properties of the drug-loaded nanofiber membranes (PVA:M=20:5) were tested respectively for the uncrosslinked drug-loaded nanofiber membranes, the drug-loaded nanofiber membranes crosslinked at constant temperature for 4h, and the drug-loaded nanofiber membranes crosslinked at reduced temperature for 5h. When testing the dry tensile properties, the tensile stress at break and the elongation at break (i.e., the growth rate of the length when the sample is broken) were tested at a tensile rate of 1mm/min; when testing the wet tensile properties, the sample was immersed in distilled water at 25℃ for 24h and then tested, and the tensile stress at break and the elongation at break were also tested.

Table 4 shows the test results. As shown in Table 4, compared with the uncrosslinked drug-loaded nanofiber membrane, the dry tensile properties and wet tensile properties of the crosslinked drug-loaded nanofiber membrane after constant temperature crosslinking for 4 hours and after cooling crosslinking for 5 hours are improved, and the improvement is more obvious when cooling crosslinking. When cooling crosslinking for 5 hours, the flexibility and ductility of the drug-loaded nanofiber membrane are relatively better, which is manifested in a relatively higher elongation at break.

Table 4 Test results of tensile properties of drug-loaded nanofiber membranes

8. Skin irritation test of drug-loaded nanofiber membrane

(1) Single complete skin irritation test

Referring to the skin irritation test in the 2017 edition of the national standard (GB/T 16886) for the biological evaluation of medical devices, three Japanese large-eared white rabbits weighing 2.5 kg were selected, regardless of gender, and purchased from the Changyang Xishan Breeding Farm in Beijing [SCXK (Beijing) 2016-0007] to test the skin irritation of cross-linked drug-loaded nanofiber membranes (cross-linked at reduced temperature for 5 hours, PVA:M = 20:5) and uncross-linked drug-loaded nanofiber membranes (PVA:M = 20:5) on white rabbits, and un-drug-loaded PVA fiber membranes and gauze were used as controls. During the test, the drug-loaded nanofiber membranes to be tested were soaked with physiological saline, and the drug-loaded nanofiber membranes to be tested were attached to different parts of the back of the white rabbits after depilation, and fixed with medical tape for 4 hours. After 4 hours, the substance was removed and the affected area was cleaned with warm water. After 1 hour, 24 hours, 48 hours and 72 hours, the skin reaction of the rabbits was observed and scored according to Tables 5 and 6. After scoring, the results were calculated according to formula (3) and then statistically analyzed.

Table 5 Skin irritation response scoring criteria

Table 6 Evaluation criteria for skin irritation intensity

Points evaluate 0～0.4 Very mild irritation 0.5～1.9 Mild irritant 2～4.9 Moderate irritation 5-8 Severe irritation

(2) Multiple complete skin irritation tests

After the single intact skin irritation experiment was completed, the single intact skin irritation experiment was repeated twice, that is, multiple intact skin irritation experiments, and the experimental method of each repetition was the same as the method of the above-mentioned single intact skin irritation experiment. 72 hours after the end of the last single intact skin irritation experiment, the rabbit was killed, and its skin tissue was taken to make slices, and stained with HE (hematoxylin-eosin), and the skin tissue was observed under a microscope. The relevant experimental steps are shown in Figure 1.

9. Analysis of the results of the drug-loaded nanofiber membrane skin irritation experiment

Table 7 shows the scoring results of a single complete skin irritation experiment, and Tables 8 and 9 show the scoring results of multiple complete skin irritation experiments. As shown in Table 7, there was no erythema and edema in the pure PVA fiber group and the blank gauze group, and the average reaction value was 0 points, which was non-irritating; but in the single complete skin irritation experiment, when observed 24h and 48h after administration, the cross-linked drug-loaded nanofiber membrane group and the uncross-linked drug-loaded nanofiber membrane group showed slight redness at the administration site. At 72h, the erythema disappeared, and there was only slight edema on the rabbit skin. At 72h, the average reaction values of the cross-linked drug-loaded nanofiber membrane group and the uncross-linked drug-loaded nanofiber membrane group were 0.17 and 0.33 (P<0.5), respectively. The average reaction value of the cross-linked drug-loaded nanofiber membrane group was less than that of the uncross-linked drug-loaded nanofiber membrane group, indicating that the cross-linked drug-loaded nanofiber membrane was extremely mildly irritating to the rabbit skin.

The scoring results of multiple complete skin irritation experiments are shown in Tables 8 and 9. The scoring results show that at 1h and 24h after the second administration, the administration sites of the cross-linked drug-loaded nanofiber membrane group and the non-cross-linked drug-loaded nanofiber membrane group were slightly reddened. At 48h, the skin of the cross-linked drug-loaded nanofiber membrane group returned to normal, and at 72h, the skin of the non-cross-linked drug-loaded nanofiber membrane group returned to normal. The average reaction values of the pure PVA fiber and blank gauze groups were both 0 points, which were non-irritating. After the third administration, the average reaction values of all components were 0 points, that is, they all showed non-irritation.

Table 7 Scoring results of single complete skin irritation test

Table 8 Total score of multiple complete skin irritation tests

Table 9 Average response values of multiple complete skin irritation test scores

After multiple dosing and the last observation, the rabbit skin tissue at the dosing site was sliced and subjected to histopathological analysis. Figure 4a and Figure 4b are the results of the skin slices of the cross-linked drug-loaded nanofiber membrane group and the uncross-linked drug-loaded fiber membrane group, respectively. Figure 4c shows the skin slices of the pure PVA fiber group, and Figure 4d shows the skin slices of the blank gauze group. The results showed that the rabbit skin of the two drug-loaded fiber groups was not damaged, and no infiltration of granulocytes and lymphocytes was observed in the dermis, and no signs of inflammation were shown. The thickness of the epidermis and dermis of the skin was equal to that of the pure PVA fiber group and the blank gauze group. The results show that cross-linked drug-loaded nanofiber membranes, uncross-linked drug-loaded nanofiber membranes, and pure PVA membranes do not cause damage to rabbit skin.

10. Antibacterial experiment of drug-loaded nanofiber membrane

The inhibition zone method was used to observe whether the drug-loaded nanofiber membrane had an antibacterial effect, and then its antibacterial performance was compared with that of the original drug.

First, MH solid culture medium and LB liquid culture medium were prepared, and then LB liquid culture medium was used to resuscitate and culture Staphylococcus aureus. Finally, the revived Staphylococcus aureus was inoculated onto the MH solid culture medium plate using the dilution coating plate method, and a PVA film soaked in 1% hydrogen peroxide (numbered 1, pure film mass was 0.25 g), 0.25 g pure PVA film (numbered 2), 50 mg "Haodele" powder (numbered 3), 0.25 g cooling cross-linked drug-loaded nanofiber membrane (cooling cross-linked for 5 h, PVA:M=20:5, drug content was considered to be 50 mg, numbered 4) and 0.25 g constant temperature cross-linked drug-loaded nanofiber membrane (constant temperature cross-linked for 4 h, PVA:M=20:5, drug content was considered to be 50 mg, numbered 5) were added to the surface of the plate, respectively. The plates were cultured together overnight, and an antibacterial experiment was carried out. The antibacterial properties of the test materials were compared by measuring the diameter of the inhibition zone.

Table 10 shows the diameter of the inhibition zone on the MH solid plate. The results show that the "Haodele" powder has a good antibacterial effect on Staphylococcus aureus. The cross-linked drug-loaded nanofibers can release the "Haodele" powder and have obvious antibacterial and antibacterial effects on Staphylococcus aureus, while pure PVA fibers have no antibacterial effect. The diameter of the inhibition zone of the drug-loaded nanofiber membrane cross-linked by cooling is greater than that of the drug-loaded nanofiber membrane cross-linked by constant temperature. This may be because the water absorption of the drug-loaded nanofiber membrane cross-linked by cooling is better, and the drugs in the membrane are more likely to enter the water and diffuse to the surface of the culture medium with water molecules.

Table 10 Diameter of inhibition zone and amount of Mongolian medicine in each group

Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims (10)

1. The preparation method of the drug-loaded nanofiber membrane is characterized by comprising the following steps of:

(1) Uniformly dispersing the medicine powder in an aqueous solution of polyvinyl alcohol added with a surfactant to obtain an electrospinning precursor dispersion liquid;

(2) Preparing a drug-loaded nanofiber membrane from the electrospinning precursor dispersion liquid by using an electrospinning method;

(3) Dispersing nano cellulose and epichlorohydrin in aqueous solution of sodium hydroxide to prepare crosslinking liquid, immersing the drug-loaded nanofiber membrane in the crosslinking liquid for soaking crosslinking, and drying to obtain the crosslinking drug-loaded nanofiber membrane.

2. The method for preparing a drug-loaded nanofiber membrane according to claim 1, wherein in the step (1), the surfactant is sodium dodecyl sulfate, and the preparation method of the electrospinning precursor dispersion liquid comprises the steps of adding polyvinyl alcohol and sodium dodecyl sulfate into ultrapure water, and sequentially stirring at normal temperature and heating; naturally cooling to room temperature, adding medicine powder, and continuously stirring to obtain an electrospinning precursor dispersion liquid;

In the step (2), the electrospinning precursor dispersion liquid is filled into a medical injector, and the electrospinning precursor dispersion liquid droplets discharged from the needle head of the medical injector are stretched into nanofibers by utilizing an electrostatic field, so that the nanofibers are piled on a receiving plate to form a drug-loaded nanofiber membrane;

in the step (3), in the soaking and crosslinking process, ultrasonic treatment is carried out on the crosslinking liquid, and the temperature of the crosslinking liquid is kept constant or gradually reduced.

3. The method for preparing a drug-loaded nanofiber membrane according to claim 2, wherein in the step (3), the crosslinking time is 1-6 hours; in the crosslinking process, the temperature of the crosslinking liquid is 45-70 ℃ and kept constant; the ultrasonic frequency is 25-30 kHz.

4. The method for preparing a drug-loaded nanofiber membrane according to claim 2, wherein in the step (3), the crosslinking time is 1-6 hours; when the soaking crosslinking begins, the temperature of the crosslinking liquid is 45-70 ℃, and the temperature of the crosslinking liquid is reduced by 2-2.5 ℃ every 1 h; the ultrasonic frequency is 25-30 kHz.

5. The method for preparing a drug-loaded nanofiber membrane according to claim 4, wherein in the step (3), the crosslinking time is 5 hours; when the soaking crosslinking starts, the temperature of the crosslinking liquid is 60 ℃, and after each 1 hour, the temperature of the crosslinking liquid is reduced by 2.5 ℃; the ultrasonic frequency was 25kHz.

6. The method for producing a drug-loaded nanofiber membrane according to claim 3, wherein in the step (3), the mass-to-volume ratio of the drug-loaded nanofiber membrane to the crosslinking liquid is 0.005 to 0.02g/mL.

7. The method for preparing the drug-loaded nanofiber membrane according to claim 2, wherein the preparation method of the crosslinking solution is that the nanocellulose and the epichlorohydrin are sequentially added into a sodium hydroxide aqueous solution with the pH value of 10-11 and are uniformly stirred; the volume ratio of the sodium hydroxide aqueous solution to the epichlorohydrin is 100: (3-5), the mass volume ratio of the nano cellulose to the sodium hydroxide aqueous solution is 4-6 g/100mL; the diameter of the nano cellulose is 10-50 nm, and the length is 0.5-3 mu m.

8. The method for preparing a drug-loaded nanofiber membrane according to claim 2, wherein in the step (1), the stirring time at normal temperature is 0.5-1 h, the heating temperature during heating and stirring is 80-85 ℃, and the stirring time is 3-4 h; adding the medicine powder and continuing stirring for 12-14 h; in the electrospinning precursor dispersion liquid, the mass ratio of polyvinyl alcohol to drug powder is 20: (1-5), the mass fraction of the polyvinyl alcohol is 8-9 wt%, and the mass fraction of the sodium dodecyl sulfate is 0.15-0.25 wt%.

9. The method for preparing a drug-loaded nanofiber membrane according to claim 2, wherein in the step (2), the inner diameter of a needle of a medical injector is 0.5-0.7 mm, the distance between a receiving plate and the needle is 13-15 cm, the speed of discharging an electrospinning precursor dispersion liquid by the medical injector is 0.5-0.6 ml.h ^-1, the spinning voltage is 14-16 kV, the ambient temperature is 28-30 ℃, the ambient humidity is 31-35%, and the spinning time is 4.5-5.5 h.

10. The method for preparing a drug-loaded nanofiber membrane according to claim 8 or 9, wherein the drug powder is "haodele" powder, and in the step (1), the stirring time at normal temperature is 0.5h, the heating temperature during heating and stirring is 85 ℃, and the stirring time is 3h; adding the medicine powder, and continuing stirring for 12 hours; in the electrospinning precursor dispersion liquid, the mass ratio of polyvinyl alcohol to drug powder is 20:5, the mass fraction of the polyvinyl alcohol is 8wt%, and the mass fraction of the sodium dodecyl sulfate is 0.2wt%;

in the step (2), the inner diameter of the needle of the medical injector is 0.6mm, the distance between the receiving plate and the needle is 15cm, the speed of the medical injector for discharging the electrospun precursor dispersion liquid is 0.6 mL.h ^-1, the spinning voltage is 16kV, the ambient temperature is 30 ℃, the ambient humidity is 35%, and the spinning time is 5h;

The preparation method of the crosslinking liquid comprises the steps of sequentially adding the nano cellulose and the epichlorohydrin into a sodium hydroxide aqueous solution with the pH of 10, and uniformly stirring, wherein the volume ratio of the sodium hydroxide aqueous solution to the epichlorohydrin is 100:5, the mass volume ratio of the nano cellulose to the sodium hydroxide aqueous solution is 5g/100mL; the diameter of the nano cellulose is 10-50 nm, and the length is 0.5-3 mu m;

In the step (3), the crosslinking time is 5h; when the soaking crosslinking starts, the temperature of the crosslinking liquid is 60 ℃, and after each 1 hour, the temperature of the crosslinking liquid is reduced by 2.5 ℃; the ultrasonic frequency is 25kHz; the mass volume ratio of the drug-loaded nanofiber membrane to the crosslinking liquid is 0.01g/mL.