CN114177359B - Antibacterial coating and preparation method and application thereof - Google Patents
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Abstract
The invention provides an antibacterial coating and a preparation method and application thereof. The antibacterial coating can be loaded with at least one drug, not only can realize the synergistic effect between the drugs and reduce the generation of drug resistance, but also can play a role in drug slow release through PLGA microspheres and prolong the drug release time so as to maintain the minimum bactericidal concentration time of the drug and prolong the catheter blockage time.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an antibacterial coating as well as a preparation method and application thereof.
Background
Urinary catheters are a common medical device in clinical use, and are tubular devices that are inserted into the bladder cavity, primarily through the urinary tract, for the purpose of draining urine and irrigating the bladder. The urinary catheter used clinically at present mainly comprises silicone rubber, latex (including siliconized latex urinary catheter), polyvinyl chloride and the like, urinary tract infection caused by the urinary catheter is mainly caused by that external bacteria can be in a proper environment to adhere and propagate due to long-time built-in of the urinary catheter, so that a biological membrane is formed, the drug resistance of the bacteria is enhanced, and the bacteria are difficult to remove. Urinary catheter-associated urinary tract infections are the most common cause of iatrogenic urinary tract infections, with incidence rates accounting for 80% of urinary tract infections. At present, the most direct method for solving urinary tract infection is to frequently replace a catheter to avoid long-time built-in, but certain limitation exists in application.
The long-term use of indwelling catheters increases the risk of patient infection with CAUTI (Catheter-Associated Urinary Tract infection), and it is therefore necessary to develop an antibacterial and bacteriostatic coating. The prior antibacterial modification of the surface of the catheter is mainly divided into physical modification and chemical modification. The physical modification mainly comprises material surface modification, which inhibits the initial attachment of bacteria and enables the catheter surface to be easier for urethra insertion through the catheter coating, and reduces the secondary injury of patients. The main representatives of this method are antifouling coatings, which do not kill microorganisms directly, but prevent bacteria from attaching to the surface, thus forming a biofilm. The mechanism of the antifouling material comprises spatial repulsion, electrostatic repulsion and low surface energy to prevent dirt from attaching to the surface of the catheter, so as to prevent planktonic bacteria from forming a conditioning film and inhibit the formation of a biological film. Chemical modifications primarily include the use of various antimicrobial substances including functional groups, polymers, antibiotics, bacteriophages, antimicrobial peptides, and the like. Currently, the biological bactericidal paint clinically tested is mainly antibiotic, and many other medicines (triclosan, chlorhexidine, nitric oxide, enzyme, antibacterial peptide and the like) are currently in the research stage and have great potential.
Current drug loading inhibits bacterial membrane formation and is prone to drug resistance. The mechanisms of the bacteria for generating drug resistance mainly comprise: (1) the drug-resistant strain is used for acting on the antibacterial drug by synthesizing certain inactivating enzyme, so that the antibacterial activity of the drug-resistant strain is lost; (2) the target site of drug action is changed; (3) alterations in cell wall permeability and active efflux mechanisms. The method for inhibiting the bacterial biofilm is mainly based on different reagents and loading modes, and has less concern on the release curve of a single drug, namely the release of the drug cannot be controlled. The main reason is that the current antibacterial coating has a single composition, and currently, the antibiotic coating applied to clinic releases most of the drugs in a short time when the antibiotic coating carries the drugs, so that the drug concentration cannot maintain the minimum bactericidal concentration for a long time, and the generation of the drug resistance of bacteria is easily promoted.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. To this end, the invention proposes, in a first aspect, an antimicrobial coating having a lubricating effect and an antimicrobial effect.
The second aspect of the invention provides a preparation method of the antibacterial coating.
A third aspect of the present invention provides a silicone rubber comprising the above-described antibacterial coating layer.
The fourth aspect of the invention provides a preparation method of the silicon rubber.
According to a first aspect of the present invention, an antibacterial coating is provided, which comprises a modified chitosan (CHI-C) coating and polylactic-co-glycolic acid (PLGA) microspheres, wherein the PLGA microspheres are loaded on the surface of the modified chitosan coating.
In some embodiments of the present invention, the polylactic acid-glycolic acid copolymer microspheres have a diameter of 30 to 50 μm.
In some preferred embodiments of the present invention, the mass ratio of the modified chitosan coating layer to the polylactic acid-glycolic acid copolymer microspheres is (35-55): 1.
in some more preferred embodiments of the present invention, the antibacterial coating further comprises at least one drug supported on the polylactic acid-glycolic acid copolymer microspheres.
In some more preferred embodiments of the present invention, the drug loading rate of the polylactic acid-glycolic acid copolymer microspheres is 10-13% of the mass of the polylactic acid-glycolic acid copolymer microspheres.
In some more preferred embodiments of the present invention, the modified chitosan coating is an acylated modified chitosan coating. The reagent used for acylation modification is selected from any one of dihydroxyphenyl propionic acid and dimethylformamide.
In some more preferred embodiments of the invention, the drug is selected from at least one of an antibiotic, a molecular pump inhibitor, an enzyme, a bacteriophage.
According to a second aspect of the present invention, there is provided a method for preparing an antibacterial coating, comprising the steps of: adding the polylactic acid-glycolic acid copolymer microspheres into the modified chitosan solution, and stirring to obtain the polylactic acid-glycolic acid copolymer microspheres.
In some preferred embodiments of the present invention, the rotation speed of the stirring is 500rpm to 1000rpm.
According to a third aspect of the invention, a silicone rubber is provided, which comprises a dopamine-modified silicone rubber and the antibacterial coating, wherein the antibacterial coating is coated on the surface of the dopamine-modified silicone rubber.
In some embodiments of the invention, the dopamine modified silicone rubber is prepared by soaking silicone rubber in a dopamine hydrochloride solution.
According to a fourth aspect of the present invention, there is provided a method for preparing the above silicone rubber, comprising the steps of:
s1: soaking the silicone rubber in a dopamine hydrochloride solution to prepare dopamine modified silicone rubber;
s2: and coating the antibacterial coating on the surface of the dopamine modified silicone rubber.
In some embodiments of the invention, the concentration of the dopamine hydrochloride solution in S1 is 1.0 to 3.5mg/mL.
In some preferred embodiments of the present invention, in S1, the soaking time is 2 to 5 hours.
In some more preferred embodiments of the present invention, the preparation method of the silicone rubber further comprises cleaning the silicone rubber before S1, wherein the cleaning is performed by using a mixed solution of ammonia and hydrogen peroxide; more preferably, the mixed solution is a mixture of ammonia water with a volume concentration of 30% and hydrogen peroxide according to a volume ratio of (1-1.5): 1 are mixed.
The invention has the beneficial effects that:
1. the antibacterial coating has an excellent lubricating effect, and the CHI-C coating can greatly reduce the hydrophilic angle on the surface of the silicon rubber, so that the hydrophilicity of the surface of the silicon rubber is increased, a barrier for inhibiting nonspecific protein adsorption is established, the surface friction of the silicon rubber can be effectively reduced, and the damage of a contact surface caused by the friction of the silicon rubber is reduced.
2. The antibacterial coating has excellent antibacterial effect, the PLGA microspheres can load at least one drug, the synergistic effect among the drugs can be realized, the generation of drug resistance is reduced, the PLGA microspheres can play a drug slow-release effect, the drug release time is prolonged, the minimum bactericidal concentration time of the drug is maintained, and the catheter blockage time can be prolonged.
3.3. After the dopamine on the surface of the silicon rubber is modified, the antibacterial coating is more easily adhered to the surface of the silicon rubber, and the antibacterial coating and the silicon rubber are combined more stably.
Drawings
The invention is further described with reference to the following figures and examples, in which:
FIG. 1 is a scanning electron microscope of PLGA microspheres of the present invention.
FIG. 2 is a scanning electron microscope cross-sectional view of the antibacterial coating of example 1 of the present invention.
FIG. 3 is a scanning electron microscope cross-sectional view of the antibacterial coating of comparative example 1 of the present invention.
FIG. 4 is a scanning electron microscope surface image of the antibacterial coating of example 1 of the present invention.
FIG. 5 is a scanning electron microscope surface image of the antibacterial coating of comparative example 1 of the present invention.
FIG. 6 is an IR spectrum of a silicone rubber without an antibacterial coating applied thereon and a silicone rubber obtained in comparative example 1.
FIG. 7 is a graph showing the antibacterial effect of different silicone rubbers according to test example 2 of the present invention on Proteus mirabilis.
FIG. 8 is a graph showing the antibacterial effect of various silicone rubbers according to test example 2 of the present invention against Staphylococcus aureus.
FIG. 9 is a graph showing the results of cell biocompatibility experiments in test example 3 of the present invention, in which different types of silicone rubber were co-cultured with cells for 1 day (left panel) and 3 days (right panel).
Detailed Description
The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.
In the following examples or comparative examples, the silicone rubber was treated as follows:
cleaning the silicon rubber: taking out the cut silicon rubber, washing with deionized water, and mixing the prepared ammonia water with the volume concentration of 30% and hydrogen peroxide according to the proportion of 1:1 (v/v), adding the silicon rubber into the mixed solution, stirring at a constant speed for 10min, taking out, and air-drying by using nitrogen.
Surface dopamine modification treatment of silicone rubber: taking out the air-dried silicone rubber, taking 2mg/mL dopamine hydrochloride solution, adding the silicone rubber, and completely soaking for 3h.
The scanning electron micrograph of the PLGA microspheres used in the examples or comparative examples of the present invention is shown in fig. 1, and it can be seen from fig. 1 that the diameters of the PLGA microspheres are about 30 μm to 50 μm.
Example 1
The embodiment prepares the silicone rubber coated with the drug-loaded microsphere coating, and the specific process is as follows:
s1: preparation of CHI-C solution: chitosan (130kDa, 3.25mmol) and dihydroxyphenylpropionic acid (HCA, 6.49 mmol) were dissolved in PBS (50mL, pH = 5). EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 6.49 mmol) was then added to the mixture, stirred at room temperature for 12 hours and after extensive dialysis (Mw: 3500) the product was stored in a centrifuge tube.
S2: preparing an antibacterial coating: taking 1.8g of CHI-C solution, adding 20mg of triclosan-loaded PLGA microspheres and 20mg of chlorhexidine-loaded PLGA microspheres, and stirring on a magnetic stirrer at the stirring speed of 500-1000 rpm;
s3: preparation of silicon rubber: spin-coating the mixed solution on the surface of silicon rubber, and drying in an oven at 50 ℃.
Example 2
The embodiment prepares the silicone rubber coated with the drug-loaded microsphere coating, and the specific process is as follows:
s1: preparation of CHI-C solution: chitosan (130kDa, 3.25mmol) and dihydroxyphenylpropionic acid (HCA, 3.25 mmol) were dissolved in PBS (50mL, pH = 5). EDC (3.25 mmol) was then added to the mixture, stirred at room temperature for 12 hours, and after extensive dialysis (Mw: 3500), the product was stored in centrifuge tubes.
S2: preparing an antibacterial coating: adding 40mg of triclosan-loaded PLGA microspheres into 1.8g of CHI-C solution, and stirring on a magnetic stirrer at the stirring speed of 500-1000 rpm;
s3: and (3) spin-coating the mixed solution on the surface of the silicon rubber, and drying in an oven at 50 ℃ to obtain the silicon rubber CHI-C + TMS.
Example 3
The embodiment prepares the silicone rubber coated with the drug-loaded microsphere coating, and the specific process is as follows:
s1: preparation of CHI-C solution: chitosan (130kda, 3.25mmol) and dihydroxyphenylpropionic acid (HCA, 9.75 mmol) were dissolved in PBS (50ml, ph = 5). EDC (9.75 mmol) was then added to the mixture, stirred at room temperature for 12 hours, and after extensive dialysis (Mw: 3500), the product was stored in centrifuge tubes.
S2: preparing an antibacterial coating: taking 1.8g of CHI-C solution, adding 40mg of chlorhexidine-loaded PLGA microspheres, and stirring on a magnetic stirrer at the stirring speed of 500-1000 rpm;
s3: and (3) spin-coating the mixed solution on the surface of the silicon rubber, and drying in an oven at 50 ℃ to obtain the silicon rubber CHI-C + CMS.
Example 4
The silicone rubber coated with the microsphere coating is prepared in the embodiment, and is different from the embodiment 1 in that the microspheres do not carry medicine, and the specific process is as follows:
s1: preparation of CHI-C solution: chitosan (130kDa, 3.25mmol) and dihydroxyphenylpropionic acid (HCA, 6.49 mmol) were dissolved in PBS (50mL, pH = 5). EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 6.49 mmol) was then added to the mixture, stirred at room temperature for 12 hours and after extensive dialysis (Mw: 3500) the product was stored in a centrifuge tube.
S2: preparing an antibacterial coating: adding 40mg of PLGA microspheres into 1.8g of CHI-C solution, and stirring on a magnetic stirrer at the stirring speed of 500-1000 rpm;
s3: preparing silicon rubber: and (3) spin-coating the mixed solution on the surface of the silicon rubber, and drying in an oven at 50 ℃ to obtain the silicon rubber CHI-C.
Comparative example 1
The comparative example prepares the silicone rubber coated with the coating, and the difference from the example 1 is that no PLGA microspheres are loaded, and the specific process is as follows:
preparing a coating: taking a certain amount of CHI-C solution, coating the solution on the surface of the silicon rubber by adopting a spin coating method, and drying in an oven at 50 ℃.
The cross-sectional views of the antibacterial coatings of example 1 and comparative example 1 are shown in fig. 2 and fig. 3, and the surface views are shown in fig. 4 and fig. 5, respectively, by scanning the silicone rubbers of example 1 and comparative example 1 through electron microscopy.
Comparing fig. 2 and fig. 3, it can be seen that the coatings prepared in example 1 and comparative example 1 can be stably attached to the surface of the silicone rubber, comparing fig. 2 and fig. 3, fig. 4 and fig. 5, the silicone rubber surface coating prepared in example 1 is also loaded with a plurality of microspheres,
test example 1
Infrared scanning analysis was performed on the silicone rubber without the antibacterial coating and the silicone rubber with the antibacterial coating (CHI-C) obtained in comparative example 1, and the results are shown in fig. 6, respectively.
As can be seen from FIG. 6, the FTIR spectrum of the CHI-C conjugate was 3300cm -1 Signals at (2) were assigned to tensile oscillations of the hydroxyl and amino groups at 1735cm in the spectrum -1 The peak at (b) is assigned to the carbon-based tensile vibration. The amide bands are respectively 1649cm -1 And 1560cm -1 A peak appears at 3009cm -1 The peak appears, which shows that there is C-H binding on the benzene ring, which proves that dopamine is successfully transplanted to CHI.
Test example 2
The silicone rubber without the coating and the silicone rubbers prepared in examples 1 to 4 were subjected to ultraviolet sterilization treatment, and then cultured in broth containing proteus mirabilis bacterial liquid and staphylococcus aureus bacterial liquid for 15 days, and the antibacterial effects thereof were observed periodically, as shown in fig. 7 and 8, respectively. Among them, the silicone rubber with no coating layer applied was designated as sample 1, the silicone rubber obtained in comparative example 1 was designated as sample 2, and the silicone rubbers obtained in examples 1 to 3 were designated as sample 3, sample 4, and sample 5, respectively.
As can be seen from fig. 7 and 8, the drug-loaded microsphere composite coating has a good antibacterial effect, and still shows an excellent inhibition effect on bacterial growth after co-culture for 15 days, and it can be found from the figures that the combined action of triclosan and chlorhexidine can generate a more excellent antibacterial effect on bacteria, and can significantly inhibit bacterial reproduction.
Test example 3
The biocompatibility of the sample was measured by co-culturing the uncoated silicone rubber, the silicone rubbers obtained in examples 2 to 4, and the cells for 1 day and 3 days, and the results are shown in fig. 9, in which the left graph of fig. 9 is the absorbance (OD) results of co-culturing for 1 day; FIG. 9 shows the results of absorbance (OD) at 3 days of co-culture.
As can be seen from fig. 9, the coated silicone rubber has good biocompatibility as compared to the silicone rubber without the coating.
The embodiments of the present invention have been described in detail, but the present invention is not limited to the embodiments, and various changes can be made without departing from the gist of the present invention within the knowledge of those skilled in the art. Furthermore, the embodiments of the present invention and the features of the embodiments may be combined with each other without conflict.
Claims (6)
1. An antimicrobial coating, characterized by: the modified chitosan microsphere comprises a modified chitosan coating and polylactic acid-glycolic acid copolymer microspheres, wherein the polylactic acid-glycolic acid copolymer microspheres are loaded on the surface of the modified chitosan coating;
the modified chitosan coating is an acylation modified chitosan coating;
the reagent adopted by the acylation modification is dihydroxyphenyl propionic acid;
the diameter of the polylactic acid-glycolic acid copolymer microspheres is 30-50 mu m;
the antibacterial coating further comprises at least one drug, and the drug is loaded on the polylactic acid-glycolic acid copolymer microspheres;
the drug loading rate of the polylactic acid-glycolic acid copolymer microspheres accounts for 10-13% of the mass of the polylactic acid-glycolic acid copolymer microspheres;
the medicine is triclosan and/or chlorhexidine.
2. The antimicrobial coating of claim 1, wherein: the mass ratio of the modified chitosan coating to the polylactic acid-glycolic acid copolymer microspheres is (35 to 55): 1.
3. a method for preparing an antibacterial coating according to any one of claims 1 to 2, characterized in that: the method comprises the following steps: adding the polylactic acid-glycolic acid copolymer microspheres into the modified chitosan solution, and stirring to obtain the polylactic acid-glycolic acid copolymer microspheres.
4. A silicone rubber characterized by: the antibacterial coating comprises dopamine-modified silicone rubber and the antibacterial coating of any one of claims 1 to 2, and is coated on the surface of the dopamine-modified silicone rubber.
5. The silicone rubber according to claim 4, wherein: the dopamine modified silicone rubber is prepared by soaking silicone rubber in a dopamine hydrochloride solution.
6. A method for preparing the silicone rubber of claim 4 or 5, comprising the steps of:
s1: soaking the silicone rubber in a dopamine hydrochloride solution to prepare dopamine modified silicone rubber;
s2: applying the antibacterial coating of any one of claims 1-2 to the surface of the dopamine-modified silicone rubber.
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