CN115350318A - Chitosan/fibroin nanofiber drug-loaded multilayer film and preparation method thereof - Google Patents
Chitosan/fibroin nanofiber drug-loaded multilayer film and preparation method thereof Download PDFInfo
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Abstract
The invention provides a chitosan/fibroin nanofiber drug-loaded multilayer film and a preparation method thereof, relates to the technical field of biomedical materials, and aims to prepare a chitosan/fibroin nanofiber (CHI/SNF) multilayer film by taking electrostatic interaction as a driving force and obtain a BBR-CHI/SNF multilayer film by introducing natural antibacterial drug berberine (BBR). Preliminarily discusses the in vitro drug release behavior of the drug-loaded multilayer film and the antibacterial performance to staphylococcus aureus and pseudomonas aeruginosa. The test result shows that the drug loading of the multilayer film can be controlled by adjusting the number of layers of the film. The multilayer film has antibacterial adhesion and certain antibacterial performance, after the BBR is loaded, the antibacterial performance is improved through the synergistic effect of the BBR and CHI in the multilayer film, the growth of staphylococcus aureus and pseudomonas aeruginosa is inhibited, the inhibition rates of the staphylococcus aureus and the pseudomonas aeruginosa respectively reach 59.62 +/-4.28% and 51.65 +/-3.77%, and the multilayer film has a good application prospect in the field of surface antibacterial of biomedical materials.
Description
Technical Field
The invention relates to the technical field of biomedical materials, in particular to a chitosan/fibroin nanofiber drug-loaded multilayer film and a preparation method thereof.
Background
Biological biomedical materials are important components of medical instrument preparation materials, and are generally applied to the production and manufacture of various medical instruments, such as antibacterial polymer dressings, bone grafting materials, artificial skins and blood vessels, medical catheters and the like. However, the surface is very adherent to bacteria, and the adhesion of bacteria to each other by signal molecules leads to accumulation of the same type of bacteria, thereby forming a biofilm and causing bacterial infection. Therefore, it is of great importance to design an antimicrobial surface of a biomaterial that can prevent infection. In recent years, researchers have developed various methods for preparing surface antibacterial coatings, including hydrogel methods, initiated polymer brush methods, layer-by-layer self-assembly methods, and the like. Among them, the layer-by-layer self-assembly (LBL) technology is widely used for surface modification of biomedical materials because of the advantages of flexible bottom plate, simple operation, wide application, controllable thickness, etc., and is a technology for spontaneous formation of thin films by a layer-by-layer alternate deposition process driven by weak interaction force between chemical groups on the surface of a template and film-forming substances. This concept was first proposed in 1966 by Iler to produce a multilayer film structure by alternately adsorbing a substrate having a surface charged with an oppositely charged colloidal solution. The most commonly used material for LBL film formation is polyelectrolyte, and polyanionic electrolytes with negative charges and polycationic electrolytes with positive charges can self-assemble into a film on the surface of a template through electrostatic adsorption.
Chitosan (CHI) is a high molecular functional material with abundant sources, good permeability and adsorptivity, and has the effects of broad-spectrum antibiosis, hemostasis, wound healing promotion and the like. The CHI molecule contains a large amount of amino groups, the amino groups are protonated to enable the CHI to be positively charged, so that the adsorption effect on bacteria is generated, and then silk fibroin nanofiber (SNF) capable of inhibiting the growth of the bacteria through the interaction with negative charges on the surface of the bacteria is a natural polyanion compound existing in silk, and the natural polyanion compound has high beta-folding content and charge density, strong hydrophobicity, good water dispersibility, and better film forming property and mechanical property. The stable SNF is widely used as an assembly element or a reinforcing agent to prepare a functional material and composite materials CHI and SNF which are both natural high molecular materials, has no toxicity and good biocompatibility, and is an excellent layer-by-layer self-assembly membrane material.
Disclosure of Invention
According to the invention, firstly, a chitosan/fibroin nanofiber (CHI/SNF) multilayer film is prepared by a layer-by-layer self-assembly technology and taking electrostatic interaction as a driving force, and then an antibacterial natural drug berberine (BBR) is loaded into the multilayer film (figure 1) by a physical adsorption method, so that the BBR-CHI/SNF multilayer film is obtained. The surface of the multilayer film has antibacterial adhesion and has the synergistic antibacterial function of CHI and BBR. Experiments show that the multilayer film can control the drug loading rate by adjusting the film thickness, and has anti-adhesion and growth inhibition effects on staphylococcus aureus (S. Aureus, gram positive) and pseudomonas aeruginosa (P. Aeruginosa, gram negative).
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention discloses a drug-loaded multilayer film, which is prepared by taking antibacterial chitosan and fibroin nanofiber as main materials, preparing the antibacterial chitosan and fibroin nanofiber into a multilayer film by a layer-by-layer self-assembly film-forming technology, then carrying out chemical crosslinking, and finally loading a natural antibacterial drug berberine.
The second aspect of the invention discloses a specific preparation method of a drug-loaded multilayer film, which comprises the following steps:
s1, cleaning a treatment substrate by using an instrument;
s2, immersing the substrate into a Chitosan (CHI) solution for 10-15min, and cleaning for three times;
s3, subsequently immersing the substrate into a fibroin nanofiber (SNF) solution for 10-15min, and cleaning for three times to form a CHI/SNF double layer;
s4, repeating the immersion and washing operations in steps S2 and S3 on the CHI/SNF double layer obtained in S3 to obtain (SNF/CHI) n (n represents the number of bilayers formed) multilayer film structures.
S5, preparing S4 (SNF/CHI) n Carrying out crosslinking treatment on the multilayer film structure to obtain a crosslinked multilayer film;
s6, soaking the crosslinked multilayer film prepared in the S5 in a berberine (BBR) solution for 1-1.5h, and then cleaning the multilayer film to remove the unloaded drug to obtain the drug-loaded multilayer film.
Preferably, the apparatus in step S1 is a plasma cleaning apparatus, so that the silicon wafer and the glass slide are negatively charged.
Preferably, the substrate in step S1 includes a silicon wafer and a glass slide.
Preferably, the concentration of the CHI solution in the step S2 is 1-1.5mg/mL.
Preferably, the concentration of the SNF solution in the step S3 is 1-1.5mg/mL.
More preferably, the step S5 of crosslinking treatment specifically comprises: (SNF/CHI) n Immersing the multilayer film structure into 0.05-0.06mol/L morpholine ethanesulfonic acid (MES) solution containing 0.2-0.3 mol/L1-ethyl- (3-dimethyl aminopropyl) carbonyl diimine (EDC) and 5-6mmol/L LN-hydroxysuccinimide (NHS) for 20min, cross-linking in 2.5% glutaraldehyde solution for 40-50min after cleaning, and rinsing with deionized water three times.
Preferably, the concentration of the BBR solution of step S6 is 1-1.5mg/mL.
The third aspect of the invention discloses a preparation process of fibroin nanofiber (SNF):
a1, placing the silkworm cocoons into a degumming solution, boiling for 25-35min, and washing to obtain degummed silk;
a2, dissolving the degummed silk in a sodium hydroxide/urea mixed solution, freezing for 11-13h, stirring for 10-20min, and repeating the operation for 3-5 times to obtain a silk solution;
and A3, filling the silk solution into a dialysis bag, placing the dialysis bag into deionized water for dialysis for 3-4 days, carrying out ultrasonic centrifugation on the solution after dialysis, taking supernatant, placing the supernatant into a freezer dryer for precooling at-20 ℃ to-25 ℃, then placing the supernatant into the freezer dryer for freeze-drying for 48-50 hours to obtain white powder SNF, and storing the white powder SNF in a refrigerator at 4 ℃ for later use.
Preferably, the degumming solution in step A1 is 0.4-0.6% (w/v) of sodium bicarbonate solution.
Preferably, the ratio of sodium hydroxide to urea in step A2 is 1.3-1.5:1.
compared with the prior art, the invention has the following remarkable advantages and effects:
(1) The invention discloses a BBR-CHI/SNF multilayer film which is prepared by taking CHI and SNF as raw materials, depositing on the surface of a silicon wafer by using an LBL technology and taking electrostatic interaction as a driving force to form the CHI/SNF multilayer film, and loading an antibacterial drug BBR. With S.aureus and P.aeruginosa as research objects, the invention proves that the BBR-CHI/SNF multilayer film has good antibacterial adhesion and bacteriostatic effects, and discovers that the BBR and CHI/SNF multilayer film has a synergistic antibacterial effect, so that the inhibition rates of the BBR-CHI/SNF multilayer film on S.aureus and P.aeruginosa reach 59.62 +/-4.28% and 51.65 +/-3.77% respectively.
(2) Characterization of the multilayer film using UV-vis and SEM revealed that the growth formation process of the multilayer film was uniform, the surface of the film was rough and had a rich porous structure. In the research of the in-vitro drug simulated release behavior of the drug-carrying multilayer film, the invention discovers that the multilayer film has the property of quick release. The drug loading capacity of the multilayer film is positively correlated with the number of layers of the multilayer film, and the drug loading capacity can be regulated and controlled by controlling the number of layers of the multilayer film. The preparation method of the membrane is simple and easy to implement, meets the requirement of large-scale batch production, the performance of the membrane is easy to regulate and control, and the antibacterial performance of the membrane is expected to be further improved through subsequent optimization design, so that the membrane can be applied to the preparation of biomedical materials with good surface antibacterial performance.
Drawings
FIG. 1 is a schematic diagram of the preparation of a drug-loaded multilayer film of the present invention;
FIG. 2a shows (CHI/SNF) 20 SEM image of multilayer film, FIG. 2b is (CHI/SNF) 30 SEM image of multilayer filmAn image;
FIG. 3a shows the present invention (CHI/SNF) 20 Graph of absorbance of multilayer film at different wavelengths, FIG. 3b is a graph of the present invention (CHI/SNF) n A line graph of absorbance of the multilayer film at a wavelength of 310 nm;
FIG. 4a shows the present invention (CHI/SNF) n Graph of multilayer film drug release, FIG. 4b is the present invention (CHI/SNF) n A histogram of drug release of the multilayer film at different drug loadings;
FIGS. 5a and d are SEM images of adhesion of blank silicon wafer to Staphylococcus aureus and Pseudomonas aeruginosa, and FIGS. b and e are (CHI/SNF) 30 Adhesion SEM images of multilayer film to Staphylococcus aureus and Pseudomonas aeruginosa, and panels c and f are BBR-Supported (CHI/SNF) 30 An adhesion SEM image of the multilayer film to staphylococcus aureus and pseudomonas aeruginosa;
FIG. 6 shows BBR-loaded (CHI/SNF) according to the invention 30 Histogram of inhibition of staphylococcus aureus and pseudomonas aeruginosa.
Detailed Description
The following describes embodiments of the present invention in further detail with reference to the examples and the accompanying drawings. The following examples and figures are illustrative of the present invention and are not intended to limit the scope of the invention.
The main raw materials are as follows:
CHI, degree of deacetylation: 75-85% from sigma aldrich trade, inc; the silk is purchased from northwest silkworm bases; BBR, standard, purity: 98% from Shanghai Michelin Biochemical technology, inc.; BBR, assay control, purity: 98% from Doctoroma Biotechnology, inc.; LB broth, agar, purchased from Qingdao Haibo Biotech limited; s. aureus (ATCC 25923), p.aeruginosa (CMCC 10104), available from solebao scientific co, beijing; PBS buffer, purchased from Biotechnology engineering (Shanghai) Co., ltd; the other reagents are all common reagents in the market.
Preparation of fibroin nano-fiber
Cutting silkworm cocoon into pieces, adding into 0.5% (w/v) sodium bicarbonate solution, boiling for 30min, washing with deionized water, and repeating twice to obtain degummed silk. It was dissolved in sodium hydroxide/urea (sodium hydroxide: urea = 1.39) solution, frozen for 12h, stirred for 15min and repeated for three days. Putting the obtained solution into a dialysis bag, dialyzing in deionized water for 3 days, ultrasonically centrifuging the dialyzed solution, taking supernatant, pre-cooling at-20 ℃, freeze-drying in a freeze dryer for 48h to obtain white powder SNF, and storing in a refrigerator at 4 ℃.
Preparation and characterization of CHI/SNF multilayer films
Firstly, a plasma cleaning instrument is used for treating the silicon wafer and the glass slide, so that the surfaces of the silicon wafer and the glass slide have hydrophilicity and are negatively charged. Then, the silicon wafer and the glass slide are immersed in CHI solution with the concentration of 1mg/mL for 10min and washed with deionized water for three times, wherein the time is 2min, 1min and 1min respectively. The wafer was then immersed in a SNF solution at a concentration of 1mg/mL for 10min, again through three washing steps, to form a bilayer. The same soaking and rinsing procedures were performed on the above materials according to the required number of layers, the assembly process was repeated and monitored using UV-vis, and finally obtained (SNF/CHI) n (n represents the number of bilayers formed) multilayer film structures. The multilayer film was then immersed in a 0.05mol/L morpholine ethanesulfonic acid (MES) solution containing 0.2mol/LEDC and 5mmol/LNHS for 20min, washed, crosslinked in a 2.5% glutaraldehyde solution for 40min, and rinsed three times with deionized water. Characterization was then performed by Scanning Electron Microscopy (SEM)
Example 1: characterization by Scanning Electron Microscope (SEM)
And (3) observing the thickness change of the coating in the self-assembly process of the substrate layer by adopting UV-VIS, wherein the wavelength scanning range is 190-900nm, and the scanning speed is 2nm.
The surface morphology of the CHI/SNF multilayer films was studied by SEM. We have successfully prepared (CHI/SNF) on silicon wafer substrates 20 (FIG. 1 (c)) and (CHI/SNF) 30 The multilayer film (fig. 1 (d)), as can be seen from the figure, the multilayer films with different layers all show high rough and porous surface structures, the increase of the layers makes the film surface rough and irregular, and the intricate fiber structure forms a plurality of fine pores, and the porous structure is the key of the multilayer film for adsorbing the medicine.
Example 2: measurement of BBR detection wavelength
Precisely weighing 25mg of BBR reference substance in a 25mL volumetric flask, ultrasonically dissolving, and adding deionized water to dilute to a scale as a stock solution. 1.5mL of the stock solution was accurately measured and placed in a 50mL volumetric flask, a BBR-free solution was used as a blank, a full-wavelength scan was performed using UV-vis, and 345nm was selected as the peak of absorbance detection of BBR, as shown in FIG. 2 (a), (CHI/SNF) n (n =0,4,8, 12, 16, 20) the absorption value of the multilayer film gradually increases with every four layers assembly. Furthermore, at 310nm (FIG. 2 (b)), the absorbance of the CHI/SNF multilayer film increased linearly with the number of layers (R) 2 = 0.912), indicating that the growth process of the multilayer film thickness is relatively uniform.
And (3) putting the 0.5mLBBR stock solution into a 50mL volumetric flask, adding deionized water to a constant volume, and shaking up. The solution was measured out by 0mL, 0.5mL, 1.25mL, 2.5mL, 5mL, 7.5mL, 10mL, 15mL, placed in a 50mL volumetric flask, and then deionized water was added to the flask to a constant volume and shaken up. The absorbance A was measured at 345nm and the concentration C was linearly regressed with A to give the standard curve equation: y =0.0595x-0.0037 2 =0.995, indicating that the BBR is in a concentration range of 0-3. Mu.g/mL, and the two are in a good linear relationship.
Example 3: CHI/SNF multilayer film to BBR loading and in vitro release
And soaking the multilayer film in a BBR standard solution of 1mg/mL for 1h, and then washing the multilayer film with deionized water to remove the unloaded drug to obtain the drug-loaded multilayer film. The BBR-CHI/SNF multilayer film was immersed in 10mL of release medium in 1 XPBS buffer and incubated at 37 ℃. Multiple time points were set up and 1mL of PBS buffer containing BBR was collected at each time point (while 1mL of fresh PBS was supplemented to the release medium) and BBR in vitro cumulative release experiments were measured using UV-vis.
The fibroin nano-fiber with the fibril structure reserved enables the CHI/SNF multilayer film to have a porous structure, and BBR serving as a small molecule drug is loaded into the multilayer film in a physical adsorption mode. Loading the drug in this physical method is in principle more advantageous than chemical methods because no covalent bond formation is required and the loading and release of the drug is reversible. The BBR-CHI/SNF multilayer film was placed in PBS buffer at the same osmotic pressure and temperature as human body for analysis of drug release characteristics. Drawing (A)And 3, the research result of the CHI/SNF multilayer film drug release behavior is shown. As can be seen from FIG. 3 (a), the drug is released in PBS in a diffusion manner, the drug loading capacity of the CHI/SNF multilayer film increases with the increase of the number of the layers of the CHI/SNF multilayer film, however, the drug release curves of the CHI/SNF multilayer films with different drug loading contents are basically consistent, which indicates that the CHI/SNF multilayer films all have the phenomenon of rapid drug release. In FIG. 3 (b), (CHI/SNF) 20 And (CHI/SNF) 30 The drug loading rates of the drug carriers are respectively 5.42 +/-0.15 mu g/cm 2 And 7.49. + -. 0.31. Mu.g/cm 2 ,(CHI/SNF) 30 The drug loading capacity is (CHI/SNF) 20 The ratio is 1.38 times, which shows that the drug loading capacity can be effectively improved by increasing the number of layers of the multilayer film, and therefore, the drug loading capacity can be regulated and controlled by changing the number of layers of the multilayer film.
Example 4: evaluation of anti-bacterial adhesion Capacity of BBR-CHI/SNF multilayer film
2 sterile shake tubes were added with 5mLLB medium, and a single colony was picked from agar plated with S.aureus and P.aeruginosa mother liquor, respectively, and cultured at 37 ℃ for 16 hours at 200 rpm. A24-well cell culture plate was prepared, and 1mL of bacterial suspension of S.aureus and P.aeruginosa (10. Sup. Th.) was prepared 5 CFU/mL), adding BBR- (CHI/SNF) 30 Multilayer film (1 cm. Times.1 cm), (CHI/SNF) 30 The blank silicon wafers of the multilayer film and the unmodified multilayer film are incubated for 12h at 37 ℃. The medium was removed, and loose and floating bacteria were washed off with sterile PBS, repeated 3 times, and fixed with 4% paraformaldehyde. The cells were then washed 3 more times with PBS and dried before observing bacterial adhesion using a scanning electron microscope.
We evaluated the anti-adhesion ability of the multilayer films to s.aureus and p.aeruginosa by SEM characterization, and as a result, as shown in fig. 5 (a-c) and 5 (d-f), respectively, significant bacteria adhered to the surface of the blank silicon wafer substrate: s. aureus generates significant aggregation on the surface of the blank silicon wafer substrate, and p.aeruginosa forms a biofilm on the surface of the blank silicon wafer substrate. Significantly different from the blank silicon wafer base plate surface, whether S.aureus or P.aeruginosa, in (CHI/SNF) 30 Multilayer film and BBR- (CHI/SNF) 30 The number of bacteria on the surface of the multilayer film is small, and the phenomenon of bacterial adhesion hardly occurs. This is achieved byThe results show that the multilayer film has good antibacterial adhesion capability and can effectively inhibit the generation of bacterial aggregation or the formation of bacterial biofilms on the surface of the material.
Example 5: antibacterial performance test of BBR-CHI/SNF multilayer film
The experiment is divided into BBR- (CHI/SNF) 30 Multilayer film group, (CHI/SNF) 30 Multilayer membrane group, free BBR group, corresponding material was added to sterile 96-well cell culture plates. Aureus suspension (10. Mu.L.) was added to each well 5 CFU/mL), using 100 μ L of bacterial liquid and 100 μ LLB culture medium as positive control and 200 μ LLB culture medium as blank control, placing 96-well plate in 37 deg.C constant temperature incubator, culturing for 24 hr, taking out multilayer film, and measuring OD with microplate reader 600 The inhibition ratio is calculated by the following formula.
As for the inhibition rate of p.aeruginosa, it was calculated by changing s.aureus to p.aeruginosa with the rest of the experimental conditions unchanged and repeating the above procedure.
The multilayer film is intended for contact sterilization, so this experiment was performed by contacting the bacterial suspension with BBR- (CHI/SNF) 30 And (4) directly co-incubating the multilayer films to test the antibacterial performance. As can be seen in FIG. 5, free BBRs and (CHI/SNF) 30 Although the multilayer films all have certain bacteriostatic effects, the inhibition rates are not high, wherein the inhibition rates of free BBR on S.aureus and P.aeruginosa are respectively only 29.65 +/-3.01% and 25.06 +/-0.53%. And BBR is loaded into (CHI/SNF) 30 After lamination, BBR- (CHI/SNF) 30 The multilayer film shows a remarkably enhanced bacteriostatic effect: the inhibition rates of S.aureus and P.aeruginosa reached 59.62 + -4.28% and 51.65 + -3.77%, respectively, the above results show that BBR adsorbed in the multilayer film has a synergistic antibacterial effect with the film itself, thereby allowing BBR- (CHI/SNF) 30 The multilayer film shows a relatively ideal bacteriostatic effect.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The chitosan/fibroin nanofiber drug-loaded multilayer film is characterized by comprising the following preparation methods:
s1, cleaning a treatment substrate by using an instrument;
s2, immersing the substrate into a Chitosan (CHI) solution for 10-15min, and cleaning for three times;
s3, subsequently immersing the substrate into a fibroin nanofiber (SNF) solution for 10-15min, and cleaning for three times to form a CHI/SNF double layer;
s4, repeating the immersion and washing operations in the steps S2 and S3 on the CHI/SNF bilayer prepared in S3 to obtain (SNF/CHI) n (n represents the number of bilayers formed) multilayer film structures.
S5, preparing S4 (SNF/CHI) n Carrying out crosslinking treatment on the multilayer film structure to obtain a crosslinked multilayer film;
s6, soaking the crosslinked multilayer film prepared in the S5 in a berberine (BBR) solution for 1-1.5h, and then cleaning the multilayer film to remove the unloaded drug to obtain the drug-loaded multilayer film.
2. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 1, wherein the instrument in step S1 is a plasma cleaning instrument, such that a silicon wafer and a glass slide are negatively charged.
3. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 1, wherein the substrate in step S1 comprises a silicon wafer and a glass slide.
4. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 1, wherein the concentration of the CHI solution in the step S2 is 1-1.5mg/mL.
5. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 1, wherein the concentration of the SNF solution in step S3 is 1-1.5mg/mL.
6. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 1, wherein the step S5 of cross-linking treatment specifically comprises the following steps: (SNF/CHI) n The multilayer film structure is immersed in 0.05-0.06mol/L morpholine ethanesulfonic acid (MES) solution containing 0.2-0.3 mol/L1-ethyl- (3-dimethylaminopropyl) carbodiimides (EDC) and 5-6mmol/L LN-hydroxysuccinimide (NHS) for 20min, and after washing, crosslinked in 2.5% glutaraldehyde solution for 40-50min, and rinsed three times with deionized water.
7. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 1, wherein the concentration of the solution of step S6BBR is 1-1.5mg/mL.
8. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 1, wherein the silk fibroin nanofiber (SNF) is prepared by the following process:
a1, placing the silkworm cocoons into a degumming solution, boiling for 25-35min, and washing to obtain degummed silk;
a2, dissolving the degummed silk in a sodium hydroxide/urea mixed solution, freezing for 11-13h, stirring for 10-20min, and repeating the operation for 3-5 times to obtain a silk solution;
and A3, filling the silk solution into a dialysis bag, placing the dialysis bag into deionized water for dialysis for 3-4 days, carrying out ultrasonic centrifugation on the solution after dialysis, taking supernatant, placing the supernatant into a freezer dryer for precooling at-20 ℃ to-25 ℃, then placing the supernatant into the freezer dryer for freeze-drying for 48-50 hours to obtain white powder SNF, and storing the white powder SNF in a refrigerator at 4 ℃ for later use.
9. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 8, wherein the degumming solution in step A1 is 0.4-0.6% (w/v) aqueous sodium bicarbonate.
10. The chitosan/silk fibroin nanofiber drug-loaded multilayer film of claim 8, wherein the ratio of sodium hydroxide to urea in step A2 is 1.3-1.5:1.
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