CN111317865A

CN111317865A - Preparation method of double-layer antibacterial composite film

Info

Publication number: CN111317865A
Application number: CN201811542836.9A
Authority: CN
Inventors: 江晓红; 李贝贝; 何纯; 肖金涛; 周兵兵; 许正巍
Original assignee: Nanjing University of Science and Technology
Current assignee: Nanjing University of Science and Technology
Priority date: 2018-12-17
Filing date: 2018-12-17
Publication date: 2020-06-23

Abstract

The invention discloses a preparation method of a double-layer antibacterial composite film. The method adopts a low-power electron beam deposition technology to prepare the double-layer antibacterial composite film with the bottom layer of polylactic acid and ciprofloxacin with the mass ratio of 1:1 and the upper layer of polyurethane and ciprofloxacin with the mass ratio of 1: 1. The antibacterial film prepared by the invention has strong binding force with a substrate, good compactness, controllable film thickness, good antibacterial performance and excellent slow release performance, can realize slow release antibacterial performance for 10 days, and can be used for transplanting artificial prostheses to eliminate local inflammatory reaction.

Description

Preparation method of double-layer antibacterial composite film

Technical Field

The invention belongs to the technical field of preparation of antibacterial films, and relates to a preparation method of a double-layer antibacterial composite film.

Background

Polyurethanes (PU) are polymers containing urethane groups (-NHCOO-) in the backbone of the polymer structure, and are generally prepared by stepwise addition polymerization of polyisocyanates, polyol polymers, or aromatic diamines. Because the polyurethane contains strongly polar urethane groups, different thermoset polyurethane and thermoplastic polyurethane products can be prepared by adjusting the ratio of NCO/OH. The polyurethane has the advantages of good biocompatibility, biodegradation, biostability, hemagglutination resistance and antibacterial property, excellent mechanical property, wear resistance, easy processing and forming, and the like, and is applied to a plurality of biomedical fields such as artificial heart valves, vascular coatings, drug controlled release, and the like.

Polylactic acid (PLA) has good thermal stability, no pollution in the production process, good biocompatibility and degradability, certain antibacterial property and wide application, and is an ideal green high polymer material when being used for packaging materials, injection molding, biological medicine and the like.

Ciprofloxacin (CIP) has a wide antibacterial action, and has a good antibacterial effect on escherichia coli, staphylococcus aureus, pseudomonas aeruginosa and the like.

Nguyen et al adopt a coaxial electrospinning technology to prepare a polylactic acid/chitosan composite nanofiber material, and an antibacterial experiment on escherichia coli shows that the polylactic acid/chitosan composite nanofiber material can completely inhibit the growth of bacteria in the first 12 hours, but then the bacteria start to gradually grow, and the antibacterial persistence is poor (Leshunjiang et al. polylactic acid-based antibacterial material research progress [ J ] application chemical industry, 2014,43(5):916 918.). An acrylic resin-based vancomycin membrane material is prepared by adopting an electrostatic dry powder coating method by the Jing Han and the like, and the sustained-release degradation is carried out, so that the medicament is accumulated and released for 6 days to achieve the total release (Jing Han, the effective.

The principle of the low-power electron beam deposition technology is that electrons are accelerated after passing through an accelerating electric field to form electron beams, the electron beams bombard the surface of a target material, the electrons can quickly lose energy, kinetic energy is converted into heat energy, and the target material is quickly heated and evaporated. Electron beam evaporation is the main method for preparing high-melting-point and high-purity films, and has the advantages of wide power regulation range and convenient use.

Disclosure of Invention

The invention aims to provide a preparation method of a double-layer antibacterial composite film with strong bonding force between the film and a substrate and good compactness. The method adopts a low-power electron beam method to prepare the double-layer antibacterial composite film at room temperature, so that PLA and PU have enough energy to carry ciprofloxacin drug molecules to deposit on a substrate, and the method has low power to protect the ciprofloxacin drug molecules from being damaged.

The technical scheme for realizing the purpose of the invention is as follows:

the preparation method of the double-layer composite antibacterial film comprises the following specific steps:

step 1, uniformly mixing ciprofloxacin powder and polylactic acid, placing the mixture in a reaction chamber, adjusting the distance between a target and a clean substrate to be 20-30 cm, vacuumizing, coating, setting the working current to be 6-7A and the working voltage to be 0.8-1.2 kV, and preparing the polylactic acid-based ciprofloxacin antibacterial film by adopting an electron beam deposition technology;

and 2, uniformly mixing the ciprofloxacin and the polyurethane, adjusting the distance between the target and a clean substrate to be 20-30 cm, vacuumizing, coating, setting the working current to be 7-8A and the working voltage to be 0.9-1.3 kV, and depositing a polyurethane-based ciprofloxacin antibacterial film on the polylactic acid-based ciprofloxacin antibacterial film by adopting an electron beam deposition technology to obtain the double-layer composite antibacterial film.

Preferably, the substrate is selected from a titanium sheet or a silicon sheet.

Preferably, the mass ratio of the polylactic acid to the ciprofloxacin is 1: 1.

Preferably, the mass ratio of the polyurethane to the ciprofloxacin is 1: 1.

Preferably, when the vacuum pumping is carried out, the vacuum degree reaches 6 × 10^-3～8×10^-3Pa。

Compared with the prior art, the invention has the following advantages:

(1) the electron beam evaporation particles have large kinetic energy, and the prepared antibacterial film has strong binding force with the substrate and good compactness;

(2) when the electron beam is evaporated, the thickness of the film can be measured in the deposition process according to a film thickness meter, so that the film forming thickness can be conveniently controlled;

(3) the prepared double-layer antibacterial composite film material has good antibacterial performance, the slow release time can reach 10 days, and the double-layer antibacterial composite film material can be used for transplanting artificial prostheses to eliminate local inflammatory reaction;

(4) the material is evaporated under the vacuum condition, so that the prepared material can be prevented from being polluted and oxidized, and the designable capacity of the prepared material is strong.

Drawings

FIG. 1 is a graph showing the antibacterial effect of a composite film (PLA: CIP/PU: CIP) against Staphylococcus aureus when the substrates are Ti and Si sheets, respectively, wherein FIG. 1(A) is a Ti sheet; FIG. 1(B) the substrate is a Si wafer;

FIG. 2 is an X-ray photoelectron spectrum of a composite film (PLA: CIP/PU: CIP) when a substrate is a Si sheet;

FIG. 3 is a graph showing the antibacterial effect of the double-layered antibacterial composite film after sustained release for 1d, 2d and 3d when the substrate is a Ti sheet;

FIG. 4 is a graph showing the antibacterial effect of the double-layer antibacterial composite film after sustained release for 4d, 5d and 6d when the substrate is a Ti sheet;

FIG. 5 is a graph showing the antibacterial effect of the double-layered antibacterial composite film after sustained release for 7d and 8d when the substrate is a Ti sheet;

FIG. 6 is a graph showing the antibacterial effect of the double-layered antibacterial composite film after 9d and 10d of slow release when the substrate is a Ti sheet;

FIG. 7 is a graph showing the antibacterial effect of the PLA-based ciprofloxacin single-layer film after sustained release for 1d, 2d and 3d when the substrate is a Ti sheet;

FIG. 8 is a graph showing the antibacterial effect of a PLA-based ciprofloxacin single-layer film when the substrate is a Ti sheet and the target mass ratios are 1:1 and 2:1, respectively;

fig. 9 is a graph showing the cumulative release concentration of the antibacterial ingredient in the PLA-based ciprofloxacin single-layer film at target mass ratios of 1:1 and 2:1, respectively.

Detailed Description

The invention is further illustrated by the following examples and figures.

Example 1

Respectively taking 3 titanium sheets and silicon sheets with the thickness of 1 x 1cm, placing the titanium sheets and the silicon sheets in ethanol, ultrasonically cleaning for 15min, then washing with water, repeating the cleaning steps for 3 times, sealing the small beaker filled with the titanium sheets and the silicon sheets by using a preservative film, and placing the beaker into a muffle furnace for drying. Placing the dried titanium sheet and the dried silicon sheet in a cavity for electron beam deposition, fixing a substrate by using a clamp, and then depositing a PLA-based ciprofloxacin antibacterial film, wherein the method comprises the following specific steps:

(1) PLA-based ciprofloxacin antibacterial film

Placing the target material with the mass ratio (PLA: CIP ═ 1:1) into a reaction chamber for electron beam deposition, and respectively vacuumizing by using a mechanical pump and a molecular pump until the vacuum degree reaches 6 × 10^-3～8×10^-3Pa. Turning on a power supply for depositing the film, adjusting the working current to 6-7A, controlling the working voltage to 0.8-1.2 kV, and when the vacuum degree is 10^-2When Pa is needed, the plasma substance between the target material and the substrate can be seen to transfer from the target material to the substrate through the observation window, whether the deposition is finished or not can be judged through the change of the film thickness on the film thickness meter, when the film thickness does not change any more, the deposition is finished, at the moment, the current and the voltage are slowly reset to zero, 10min is waited, after the temperature of the vacuum chamber is reduced to the room temperature, the power supply of the instrument is turned off, and the film coating is finished.

(2) Deposited PU-based ciprofloxacin antibacterial film

Taking out the used target material, replacing the target material with the mass ratio (PU: CIP ═ 1:1) in a vacuum chamber, and respectively vacuumizing by using a mechanical pump and a molecular pump until the vacuum degree reaches 6 × 10^-3～8×10^-3Pa. And (3) turning on a power supply for depositing the film, adjusting the working current to 7-8A, controlling the working voltage to 0.9-1.3 kV, when the film thickness on the film thickness measuring instrument begins to increase, the film begins to deposit, and the film thickness does not change any more, namely the deposition is finished, slowly returning the current and the voltage to zero at the moment, waiting for 10min, reducing the temperature of the vacuum chamber to room temperature, turning off the power supply of the instrument, and finishing the film coating of the double-layer composite film.

(3) Sustained release of composite membranes

And (3) taking 10 composite membranes, respectively soaking the composite membranes in physiological saline for 1d, 2d, 3d, 4d, 5d, 6d, 7d, 8d, 9d and 10d, and then taking out the composite membranes for an antibacterial experiment.

(4) Culture of Staphylococcus aureus

Weighing 0.5g of yeast extract powder, 1.0g of peptone and 1.0g of NaCl, dissolving in a beaker by using deionized water, dropwise adding a small amount of NaOH solution until the pH value is adjusted to 7, then conducting drainage by using a glass rod, pouring the solution into a 100mL conical flask, fixing the volume, shaking up, taking three clean small test tubes, taking 5.00mL of solution in each small test tube by using a liquid-moving gun, sealing the openings of the test tubes by using tinfoil, and then putting the small test tubes into a high-pressure steam sterilization pot for sterilization.

After sterilization, placing the test tube, the inoculating loop and the lighter in a clean bench, irradiating for 20min with an ultraviolet lamp, blowing for 2min after irradiation, wearing disposable rubber gloves, taking out staphylococcus aureus strains, igniting an alcohol lamp, wiping hands and the clean bench with alcohol cotton, placing the inoculating loop on the outer flame of the alcohol lamp for burning until the inoculating loop is red, rotating and burning the part of the inoculating loop rod, placing the test tube opening containing the staphylococcus aureus on the alcohol lamp for burning, then stretching the inoculating loop into the test tube containing the staphylococcus aureus, taking a proper amount of strains to inoculate in a liquid culture medium, wrapping with tinfoil, tying with a rubber band, repeating the steps for three times until 3 test tubes are completely inoculated, extinguishing the alcohol lamp, closing the clean bench, placing the inoculated staphylococcus aureus in a shaking table at 30 ℃, culturing for 12 h.

(5) Research on antibacterial performance of double-layer composite film

Weighing 0.5g of yeast extract powder, 1.0g of peptone, 1.0g of NaCl and 1.6g of agar powder, dissolving the yeast extract powder in a beaker by using deionized water, adjusting the pH value to 7, fixing the volume of a 100mL conical flask, shaking uniformly, sealing the opening of the conical flask by using a sealing film, and putting a culture dish, a pair of tweezers, a pipette tip and a liquid culture medium which are wrapped by newspaper into a high-pressure steam sterilization pot for sterilization.

After sterilization, putting the culture dish, the pipette tip and the lighter into an ultra-clean workbench, irradiating for 20min by using an ultraviolet lamp, blowing for 2min by using a fan, selecting one of three cultured test tubes of staphylococcus aureus with the best effect, pouring the test tube into a conical flask, fully vibrating and shaking uniformly; the prepared double-layer composite antibacterial film is placed in the center of a culture dish, one surface of a composite film material coated film faces upwards, 5.0mL of culture medium is transferred to the culture dish by a liquid transfer gun, air bubbles are avoided as much as possible in the transfer process, the transfer process is rapid, and the culture medium is prevented from being solidified. Finally, the inoculated culture medium is placed in an incubator at 30 ℃ for culturing for 24 hours, the picture is shown in figure 1 after being taken out, and the inhibition zones show that the double-layer composite membranes with different substrates have good antibacterial effects on staphylococcus aureus, the diameter of the inhibition zone is 31mm when the Ti sheet is taken as the substrate in figure 1(A), the diameter of the inhibition zone is 34mm when the Si sheet is taken as the substrate in figure 1(B), and the antibacterial effects of the membrane materials of different substrates are similar, which shows that the binding force of the membrane materials and the substrate is stronger and the stable antibacterial effect is achieved.

The molecular formula of ciprofloxacin is C₁₇H₁₈FN₃O₃The molecular formula of the polylactic acid is (C)₃H₄O₂)_nThe polyurethane is a polymer with a main chain containing-NHCOO-repeating structural unit, and the XPS spectrum in figure 2 shows that the double-layer antibacterial composite film contains C, O, N, F four elements, so that the film components can be preliminarily judged to contain the polyurethane, polylactic acid and ciprofloxacin, and the antibacterial effect in figure 1 shows that ciprofloxacin medicine molecules in the film components are not changed through electron beam deposition.

The slow release effect of the composite antibacterial film is shown in fig. 3-6, and generally speaking, the inhibition zone shows the trend of firstly decreasing, then increasing and then decreasing. The diameters of the antibacterial rings from day 1 to day 6 are respectively 12mm, 13mm, 11mm, 10mm, 9mm and 8mm, the diameters of the antibacterial rings are in a decreasing trend, because ciprofloxacin drug molecules are released into the slow-release liquid along with the prolonging of the soaking time, the effective components in the membrane material are gradually reduced, and the diameters of the antibacterial rings are in a decreasing trend. The method is characterized in that the transition process is carried out from day 6 to day 7, the inhibition zone is increased from small to larger than that of the first day, the inhibition mechanism of the double-layer composite film is different from that of the single-layer composite film, the inhibition effect from day 1 to day 6 is the antibacterial effect of the PU-based ciprofloxacin film on the upper layer, the PU-based antibacterial film on the upper layer is completely released from day 7, the inhibition effect from day 7 to day 10 is the antibacterial effect of the PLA-based ciprofloxacin film on the lower layer, the diameters of the inhibition zones from day 7 to day 10 are respectively 15mm, 13mm, 11mm and 8mm, and the double-layer antibacterial composite film has great advantages compared with the single-layer film in the conventional sustained release process.

Comparative example 1

The comparative example is a PLA-based ciprofloxacin single-layer film with the target mass ratio of 1:1, the deposition process is the same as that of the PLA-based ciprofloxacin single-layer film at the bottom layer in example 1, the film material with the diameter of 7mm is respectively soaked in normal saline for 1 day, 2 days and 3 days, the film material is taken out to be used for an antibacterial experiment, the slow-release antibacterial effect is shown in figure 7, the diameters of antibacterial rings for 3 days are respectively 17mm, 14mm and 13mm, the slow release is always maintained for about 3 days, and the slow-release antibacterial duration of the double-layer antibacterial composite film is greatly prolonged compared with that of the single-layer PLA-based ciprofloxacin antibacterial film.

Comparative example 2

The film preparation process of the comparative example is basically the same as that of the PLA-based ciprofloxacin film at the bottom layer in example 1, and the only difference is that the mass ratio of polylactic acid to ciprofloxacin in the target raw material is 2: 1. The membrane materials with the target mass ratio of 1:1 and 2:1 are respectively subjected to an antibacterial test, the antibacterial effect is shown in figure 8, and the diameters of antibacterial rings of the membrane materials with the target mass ratio of 1:1 and 2:1 are respectively 40mm and 35 mm. And respectively soaking the membrane materials in the two proportions in physiological saline for 1 to 3 days, taking out, measuring the absorbance of the slow release solution by using an ultraviolet spectrophotometer, and calculating the cumulative release concentration of the ciprofloxacin by using a standard curve. As can be seen from FIG. 9, the accumulative release concentration of the PLA-based ciprofloxacin membrane material with the mass ratio of 1:1 is far higher than that of the membrane material with the mass ratio of 2:1, the accumulative release concentration reaches 2 mug/mL after three days of slow release, and as can be seen from the analysis of FIG. 8 and FIG. 9, the antibacterial and slow release effects of the membrane material with the target mass ratio of 1:1 are better than those of the membrane material with the mass ratio of 2:1, so that the membrane material with the mass ratio of 1:1 is selected as the bottom layer membrane of the double-layer antibacterial composite membrane.

Claims

1. The preparation method of the double-layer composite antibacterial film is characterized by comprising the following specific steps:

2. The method of claim 1, wherein the substrate is selected from a titanium sheet and a silicon sheet.

3. The preparation method according to claim 1, wherein the mass ratio of the polylactic acid to the ciprofloxacin is 1: 1.

4. The method according to claim 1, wherein the mass ratio of the polyurethane to the ciprofloxacin is 1: 1.

5. The method of claim 1, wherein the vacuum degree is 6 × 10^-3～8×10^-3Pa。