Preparation and application of dopamine polyethyleneimine polyacrylic acid polymer film coating
The multi-layer polymer film coating formed by covalently self-assembly of dopamine, polyethyleneimine and polyacrylic acid solves the problem of the lack of antibacteriality of existing cannulation equipment, and effectively inhibits bacteria and fungi, reducing the risk of the disease. risk of infection.
Patent Information
- Application Number
- CN202411381941.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-30
- Publication Date
- 2025-02-21
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
Existing intubation equipment such as indwelling catheters and tracheal intubation catheters lack antibacterial properties, which leads to prone to infection in the long term. The existing anti-infection methods are cumbersome and may increase the probability of infection and the risk of drug-resistant strains.
A multi-layer stable polymer film coating is formed by covalently self-assemblying dopamine, polyethyleneimine and polyacrylic acid, which can effectively inhibit the formation of biological films of bacteria and fungi.
Effective inhibition of bacteria and fungi is achieved, reducing the colonization and growth of bacteria and fungi on the surface of the catheter and reducing the risk of infection.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure SMS_3
Abstract
Description
Technical field:
[0001] The invention relates to the technical field of materials and medicine, and more specifically to the preparation and application of a dopamine polyethyleneimine polyacrylic acid polymer film coating. Background technology:
[0002] At present, intubation is a commonly used method in clinical practice, including indwelling needles, indwelling urinary catheters, dialysis tubes, and endotracheal intubation. Whether it is an indwelling needle, indwelling urinary catheter, dialysis tube or endotracheal intubation catheter itself, it does not have antibacterial effects, and long-term use can easily cause infection. Indwelling urinary catheters are still the main cause of nosocomial urinary tract infections. 40% to 60% of critically ill patients have a central venous catheter indwelling requirement. After 24 hours of catheterization, the catheter is surrounded by a fibrin sheath, resulting in a large number of microorganisms breeding and gradually forming bacterial colonization. The anesthesia infection rate of general anesthesia with endotracheal intubation was 6.96%, which was much higher than the infection rate of other anesthesia methods. The infection rate in outpatient and emergency venous indwelling needle infusion patients was 6.84%. At present, in order to prevent the occurrence of infection, common methods include cumbersome care during the intubation process, prophylactic antibiotics, and regular replacement of new catheters, which not only bring great pain and economic burden to patients, but also increase the probability of infection and the risk of drug resistance of infected strains.
[0003] There is an urgent need for a material that can effectively inhibit the growth of bacteria and fungi, reduce the colonization and growth of bacteria and fungi on the surface of catheters, and reduce infections caused by catheters. The polymer film is formed by DA-PEI coprecipitation, and then the polymer film coating is formed by PAA-PEI covalent layer-by-layer self-assembly, which has the characteristics of low price and stable polymer film coating. It can be seen that DA-PEI-PAA polymer film coating is a polymer film coating technology that is urgently needed. Summary of the invention:
[0004] The object of the present invention is to provide a new coating method, wherein the new polymer film coating can inhibit the formation of bacterial and fungal biofilms, namely, the preparation and application of a dopamine polyethyleneimine polyacrylic acid polymer film coating.
[0005] We found that dopamine and polyethyleneimine can form a polymer film in pH 8.5 Tris buffer, and the polymer film can also form a multilayer stable polymer film through covalent layer-by-layer self-assembly of polyethyleneimine and polyacrylic acid. The multilayer stable polymer film coating can inhibit the formation of biofilms of bacteria and fungi.
[0006] Experiments have shown that the outermost layer of the dopamine polyethyleneimine polyacrylic acid polymer film coating is polyethyleneimine, which can effectively inhibit the formation of bacterial and fungal biofilms.
[0007] Example 1: Preparation process of dopamine polyethyleneimine polyacrylic acid coating
[0008] 1. Drug testing
[0009] Dopamine hydrochloride (DA-HCl): aladdin; purity: 98%; item number: D103111-25g; batch number: D2426148.
[0010] Polyethyleneimine (PEI, 70000Da): aladdin; purity: 50%; item number: E107080-100g; batch number: A2402230.
[0011] Polyacrylic acid (PAA): Macklin: purity: 35%; item number: P865471-25ml; batch number: C16318338. Tris (hydroxymethylaminomethane): Solarbio.
[0012] 2. Materials
[0013] Frosted PVC
[0014] 3. Solution configuration
[0015] Tris-HCl buffer: Weigh 12.1 g of tris(hydroxymethyl)aminomethane, add deionized water to dissolve, make up to 1000 ml, adjust the pH to 8.5 with concentrated hydrochloric acid, and store at room temperature for later use.
[0016] Weigh DA-HCl and dissolve DA in pH 8.5 Tris-HCl buffer to make the final concentration 40 mg / mL; set aside.
[0017] Weigh PEI and dissolve it in pH 8.5 Tris-HCl buffer to make the final concentration 40 mg / mL; set aside.
[0018] Table 1 Preparation of different DA-PEI ratios
[0019]
[0020] 40 mg / mL DA and PEI stock solutions were used in the table.
[0021] Table 1 shows different preparations of DA-PEI. The DA-HCl mother solution and PEI mother solution were both 40 mg / mL, and the diluent was pH 8.5 Tris-HCl buffer.
[0022] 4. Instruments
[0023] Analytical balance (Nanjing Bonita Scientific Instrument Co., Ltd.); ZQZY-75CV incubator (Shanghai Zhichu Instrument Co., Ltd.).
[0024] 5. Preparation of polymer membrane formed by coprecipitation of dopamine and polyethyleneimine
[0025] PVC pretreatment: Cut the PVC sheet into 2cm×2cm squares, soak and clean it with acetone for 5 minutes, dry it at room temperature after cleaning, then clean it with anhydrous ethanol for 5 minutes, dry it at room temperature for later use.
[0026] The pretreated PVC sheet was immersed in the prepared DA-PEI solution. The deposition process was shaken at room temperature and the rotation speed was 60 rpm, and incubated until precipitation began to appear.
[0027] Table 2 Coating time of different groups
[0028]
[0029] Table 2 shows the coating formation time of different DA-PEI ratios. It can be seen that when the PEI concentration is higher than that of DA, the coating formation time is prolonged, and when the DA-PEI concentration increases, the coating formation time is also prolonged.
[0030] In summary, when the DA-PEI concentration increases, the coating formation time is prolonged; when the PEI concentration is higher than that of DA, the coating formation time is prolonged. The coating is most convenient when the DA concentration is 2mg / ml~6mg / ml and the PEI concentration is 2mg / ml~4mg / ml.
[0031] Example 2: Preparation process of polyethyleneimine and polyacrylic acid covalent layer-by-layer self-assembled polymer film 1. Reagent
[0032] Dopamine hydrochloride (DA-HCl): aladdin; purity: 98%; item number: D103111-25g; batch number: D2426148.
[0033] Polyethyleneimine (PEI, 70000Da): aladdin; purity: 50%; item number: E107080-100g; batch number: A2402230.
[0034] Polyacrylic acid (PAA): Macklin: purity: 35%; item number: P865471-25ml; batch number: C16318338. Tris (hydroxymethylaminomethane): Solarbio.
[0035] Sodium chloride: Runjie Chemical.
[0036] 2. Materials
[0037] In Example 1, coated PVC (2+2, 4+4 and 6+4) were prepared.
[0038] ATCC standard strain: Candida albicans SC5314 was donated by William A. Fonzi (Department of Microbiology and Immunology, Georgetown University, Washington, DC).
[0039] 3. Solution configuration
[0040] 0.5mol / LNaCl solution: weigh 29.25g of sodium chloride, add deionized water to dissolve, make up to 1000ml, and store at room temperature for later use.
[0041] Weigh PEI and dissolve it with 0.5 mol / L NaCl solution to a final concentration of 40 mg / mL; set aside. Weigh PAA and dissolve it with 0.5 mol / L NaCl solution to a final concentration of 40 mg / mL; set aside. Dilute the PEI solution to 2 mg / ml, 4 mg / ml, 6 mg / ml and 10 mg / ml with 0.5 mol / L NaCl solution.
[0042] The PAA solution was diluted with 0.5 mol / L NaCl solution to 2 mg / ml, 4 mg / ml, 6 mg / ml and 8 mg / ml respectively.
[0043] RPMI1640 culture medium: RPMI1640 (Gibco) 10g, NaHCO3 2.0g, morpholinepropanesulfonic acid (Sigma) 34.5g, add 900ml of triple-distilled water to dissolve, adjust the pH to 7.0 with 1N NaOH, make up to 1000ml, filter and sterilize, and store at 4°C.
[0044] Sandburg Dextrose Agar (SDA): 10 g peptone, 40 g glucose, 18 g agar, dissolve in 900 ml triple distilled water, adjust pH to 7.0, make up to 1000 ml, sterilize under high pressure and store at 4°C.
[0045] YEPD culture medium: 10g yeast extract, 20g peptone, 20g glucose, add 900ml triple-distilled water to dissolve and make up to 1000ml, sterilize under high pressure and store at 4℃.
[0046] 4. Instruments
[0047] Analytical balance (Nanjing Bonita Scientific Instrument Co., Ltd.); ZQZY-75CV incubator (Shanghai Zhichu Instrument Co., Ltd.); multifunctional microplate reader SpectraMax iD3 (Meigu Molecular Instrument (Shanghai) Co., Ltd.); biochemical incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.); HZ-9211KB air constant temperature oscillator (Taicang Science and Education Equipment Factory); ZQZY-78CV incubator (Shanghai Zhichu Instrument Co., Ltd.).
[0048] 5. Preparation of polymer membrane formed by coprecipitation of dopamine and polyethyleneimine
[0049] The coated PVC prepared in Example 1 was rinsed with deionized water for more than three times, and then immersed in deionized water for 12 hours of shaking cleaning. After cleaning, the coated PVC was placed at room temperature to dry for 12 hours.
[0050] The dried coated PVC was immersed in the PAA solution for 10 minutes and dried at room temperature for 12 hours.
[0051] The dried coated PVC was immersed in PEI solution for 10 minutes and dried at room temperature for 12 hours.
[0052] The dried coated PVC was immersed in the PAA solution for 10 minutes and dried at room temperature for 12 hours.
[0053] The dried coated PVC was immersed in the PEI solution for 10 minutes and dried at room temperature for 12 hours.
[0054] 6. Preparation of bacterial solution
[0055] Before the experiment, a small amount of Candida albicans was picked from the SDA medium stored at 4°C with an inoculation loop and inoculated into 1 ml of YEPD culture medium. The culture was shaken at 30°C and 200 rpm for 24 hours to activate the fungus in the late exponential growth phase. The bacterial solution was added to 1 ml of YEPD culture medium and activated again using the above method. After 16 hours, the bacterial solution was counted using a hemocytometer and the concentration of the bacterial solution was adjusted to 1×10 6 cells / ml.
[0056] 7. Biofilm preparation
[0057] Different polymer film-coated PVC materials were added to 6-well culture plates, 6 ml of bacterial solution was added to each well, and the culture plates were placed in a 37°C incubator for 72 hours.
[0058] 8. Judgment of the inhibition of biofilm formation by coatings with different ratios
[0059] After 72 hours, the PVC materials were taken out and placed in physiological saline. The surface of the PVC was blown with a 1ml pipette and then rinsed with physiological saline three times. The PVC materials were added to a new 6-well plate, and RPMI 1640 solution containing 0.5mg / ml of MTT was added to each well, 3ml, and cultured at 37℃ in the dark for 4 hours. The 1640 solution was taken out and discarded, and DMSO solution was added to the 6-well plate, 2ml per well, and oscillated at 50rpm in the dark for 10 minutes until the crystal violet was completely dissolved. 200ul was taken out and added to a 96-well plate, with 3 replicates per well, and the OD490 and OD570 were detected by an enzyme marker.
[0060] The above experiments were repeated for 3 samples, and the coating inhibition rate = [1-(Polymer film coated PVC sample OD490-Polymer film coated PVC sample OD570) / (Blank PVC sample OD490-Blank PVC sample OD570)] × 100%
[0061] Table 3: Inhibition rate of fungal biofilm by polymer film coatings with different ratios
[0062]
[0063] Table 3 shows the inhibitory effect of polymer film coatings of different proportions on the biofilm formation of Candida albicans SC5314. It can be seen that polymer film coatings of different proportions have an inhibitory effect on the biofilm formation of Candida albicans SC5314, among which the 2+2 (4+6+8+10) preparation method has the best inhibitory effect.
[0064] In summary, polymer film coatings in different proportions have inhibitory effects on the biofilm formation of Candida albicans SC5314, among which the polymer film coating prepared by the DA+PEI (PAA+PEI+PAA+PEI) [2+2(4+6+8+10)] multilayer assembly method has the best inhibitory effect on the biofilm formation of Candida albicans SC5314.
[0065] Example 3: Effect of polymer films in inhibiting fungal film formation
[0066] 1. Drug testing
[0067] Dopamine hydrochloride (DA-HCl): aladdin; purity: 98%; item number: D103111-25g; batch number: D2426148.
[0068] Polyethyleneimine (PEI, 70000Da): aladdin; purity: 50%; item number: E107080-100g; batch number: A2402230.
[0069] Polyacrylic acid (PAA): Macklin: purity: 35%; item number: P865471-25ml; batch number: C16318338. Tris (hydroxymethylaminomethane): Solarbio.
[0070] Dimethyl sulfoxide (DMSO): China National Pharmaceutical Group Shanghai Chemical Reagent Company.
[0071] 2. Strains
[0072] ATCC standard strain: Candida albicans SC5314 was donated by William A. Fonzi (Department of Microbiology and Immunology, Georgetown University, Washington, DC). Clinical strains: drug-resistant Candida albicans (100, 103); Cryptococcus neoformans; Candida auris. They were provided by the Fungi Laboratory of Changhai Hospital and can also be purchased from the Institute of Materia Medica, Chinese Academy of Sciences. They were collected from clinical samples of different departments of Changhai Hospital and identified by morphology and biochemistry.
[0073] All experimental strains were activated by streaking on Sandburg Dextrose Agar (SDA). After Candida albicans was cultured at 30°C for 48 h, single clones were picked out and streaked again for activation. The single clones obtained for the second time were placed on SDA slants, cultured using the above method, and stored at 4°C for later use.
[0074] 3. Materials
[0075] In Example 2, [2+2(4+6+8+10)]DA-PEI-PAA polymer film-coated PVC was prepared.
[0076] 4. Culture medium
[0077] RPMI 1640 culture medium: RPMI1640 (Gibco) 10g, NaHCO3 2.0g, morpholinepropanesulfonic acid (Sigma) 34.5g, add 900ml of triple distilled water to dissolve, adjust pH to 7.0 with 1N NaOH, make up to 1000ml, filter and sterilize, and store at 4°C.
[0078] Sandburg Dextrose Agar (SDA): 10 g peptone, 40 g glucose, 18 g agar, dissolve in 900 ml triple distilled water, adjust pH to 7.0, make up to 1000 ml, sterilize under high pressure and store at 4°C.
[0079] YEPD culture medium: 10g yeast extract, 20g peptone, 20g glucose, add 900ml triple-distilled water to dissolve and make up to 1000ml, sterilize under high pressure and store at 4℃.
[0080] 5. Instruments
[0081] Analytical balance (Nanjing Bonita Scientific Instrument Co., Ltd.); multifunctional microplate reader SpectraMax iD3 (MeiGu Molecular Instrument (Shanghai) Co., Ltd.); biochemical incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.); HZ-9211KB air constant temperature oscillator (Taicang Science and Education Equipment Factory); ZQZY-78CV incubator (Shanghai Zhichu Instrument Co., Ltd.).
[0082] 6. Preparation of bacterial solution
[0083] Before the experiment, a small amount of Candida albicans was picked from the SDA medium stored at 4°C with an inoculation loop and inoculated into 1 ml of YEPD culture medium. The culture was shaken at 30°C and 200 rpm for 24 hours to activate the fungus in the late exponential growth phase. The bacterial solution was added to 1 ml of YEPD culture medium and activated again using the above method. After 16 hours, the bacterial solution was counted using a hemocytometer and the concentration of the bacterial solution was adjusted to 1×10 6 cells / ml.
[0084] 7. Biofilm preparation
[0085] Different groups of PVC materials were added to 6-well culture plates, 6 ml of bacterial solution was added to each well, and the culture plates were placed in a 37°C incubator for 72 hours.
[0086] 8. Biofilm determination
[0087] After 72 hours, the PVC materials of different groups were taken out and placed in physiological saline. The surface of the materials was blown with a 1ml pipette and then rinsed with physiological saline for 3 times. The PVC materials were added to a new 6-well plate, and RPMI 1640 solution containing 0.5mg / ml MTT was added to each well, 3ml, and cultured at 37℃ in the dark for 4 hours. The 1640 solution was taken out and discarded, and DMSO solution was added to the 6-well plate, 2ml per well, and oscillated at 50rpm in the dark for 10 minutes until the crystal violet was completely dissolved. 200ul was taken out and added to a 96-well plate, with 3 replicates per well, and the OD490 and OD570 were detected by an enzyme marker.
[0088] The above experiments were repeated for 3 samples, and the coating inhibition rate = [1-(coated PVC sample OD490-coated PVC sample OD570) / (blank PVC sample OD490-blank PVC sample OD570)] × 100%
[0089] Table 4: Inhibition rate of coating on different fungal biofilms
[0090]
[0091]
[0092] Table 4 shows the effect of polymer film coating on biofilms of different fungi. It can be concluded that the polymer film coating has an inhibitory effect on the biofilm formation of different fungi, including sensitive Candida albicans strains Y0109 and SC5314, resistant Candida albicans strains 100 and 103, Candida auris, and Cryptococcus neoformans.
[0093] In summary, the polymer film coating has an inhibitory effect on the biofilm formation of different fungi, including the biofilm formation of sensitive Candida albicans strains Y0109 and SC5314, resistant Candida albicans strains 100 and 103, Candida auris, and Cryptococcus neoformans.
[0094] Example 4: Application of polymer film in inhibiting bacterial capsule formation
[0095] 1. Drug testing
[0096] Dopamine hydrochloride (DA-HCl): aladdin; purity: 98%; item number: D103111-25g; batch number: D2426148.
[0097] Polyethyleneimine (PEI, 70000Da): aladdin; purity: 50%; item number: E107080-100g; batch number: A2402230.
[0098] Polyacrylic acid (PAA): Macklin: purity: 35%; item number: P865471-25ml; batch number: C16318338. Tris (hydroxymethylaminomethane): Solarbio.
[0099] Dimethyl sulfoxide (DMSO): China National Pharmaceutical Group Shanghai Chemical Reagent Company.
[0100] 2. Strains
[0101] Standard strains: Pseudomonas aeruginosa (PA): ATCC 15442; purchased from China Food and Drug Administration. Escherichia coli (E.coli): CMCC44102. Staphylococcus aureus (SA): No. CMCC26003, purchased from Institute of Materia Medica, Chinese Academy of Sciences.
[0102] 3. Materials
[0103] In Example 2, [2+2(4+6+8+10)]DA-PEI-PAA polymer film PVC was prepared.
[0104] 4. Culture medium
[0105] Eluent: Dissolve 1 ml Tween-80, 10 g peptone and 8.5 g sodium chloride in 900 ml deionized water, adjust the pH to 7.0 ± 0.2, make up to 1000 ml, sterilize at 121 °C for 15 min, cool to room temperature, and store at 4 °C for later use.
[0106] Dilution solution (tryptone saline solution TPS): dissolve 10g tryptone and 8.5g sodium chloride in 900ml deionized water, adjust the pH to 7.0, make up to 1000ml, sterilize at 121℃ for 15min, cool to room temperature, and store at 4℃ for later use.
[0107] TSA medium: Weigh 40g of TSA medium, dissolve in 1000mL of deionized water, sterilize at 121℃ for 15min, cool to about 80℃, pour into a 90mm plate, cool to room temperature, and store at 4℃ for later use. TSB culture medium: Weigh 30g of TSB medium, dissolve in 1000mL of deionized water, sterilize at 121℃ for 15min, cool to room temperature, and store at 4℃ for later use.
[0108] 5. Instruments
[0109] Analytical balance (Nanjing Bonita Scientific Instrument Co., Ltd.); biochemical incubator (Shanghai Yiheng Scientific Instrument Co., Ltd.); HZ-9211KB air constant temperature oscillator (Taicang Science and Education Equipment Factory); ZQZY-78CV incubator (Shanghai Zhichu Instrument Co., Ltd.); microscope (Shanghai Optical Instrument Factory).
[0110] 6. Preparation of bacterial solution
[0111] Before the experiment, use a pipette to take a small amount of bacterial liquid from the cryopreserved tube stored at -80℃, inoculate it into 1ml TSB culture medium, and culture it at 30℃ and 200rpm for 24h. Take 100ul of activated bacterial liquid and inoculate it into 1ml TSB culture medium, and culture it at 30℃ and 200rpm for 24h. Adjust the concentration of bacterial liquid and prepare bacterial suspension so that the concentration of bacterial liquid is 1×103~5×103 cells / mL for use.
[0112] 7. Biofilm preparation
[0113] Different groups of PVC materials were added to 6-well culture plates, 6 ml of bacterial solution was added to each well, and the culture plates were placed in a 37°C incubator, shaken at 50 rpm for 4 hours, and then statically cultured for 20 hours. The cultured PVC materials were placed in new 6-well culture plates, 6 ml of culture solution was added to each well, and the above culture steps were repeated for a total of 5 days.
[0114] 8. Biofilm determination
[0115] After 5 days, the PVC materials were taken out and placed in physiological saline. The surface of the materials was blown with a 1 ml pipette and then rinsed with physiological saline for 3 times. The PVC materials were placed in a test tube containing 10 mL of eluent and cleaned in 40 kHz ultrasound for 5 minutes. Then, they were diluted at different times and 100 μl of the diluted bacterial solution was smeared on a TSA plate. The bacteria were counted after 24 hours of incubation at 37°C.
[0116] The above experiments were repeated for 3 samples, and the coating inhibition rate = (1-the number of bacteria in the coated PVC sample / the number of bacteria in the blank PVC sample) × 100%
[0117] Table 5: Inhibition rate of coating on different bacterial biofilms
[0118]
[0119] Table 5 shows the effect of polymer film coating on biofilms of different bacteria. It can be concluded that the polymer film coating has an inhibitory effect on the biofilm formation of different bacteria including PA, SA and E. coli.
[0120] In summary, the polymer film coating has an inhibitory effect on the biofilm formation of different bacteria, including the biofilm formation of PA, SA and E. coli.
Claims
1. A dopamine polyethyleneimine polyacrylic acid polymer film coating preparation method characterized by: The following steps are involved: (1) coprecipitating dopamine and polyethyleneimine to form a polymer film; (2) based on step (1), covalently self-assembling polyethyleneimine and polyacrylic acid to form a polymer film.
2. The preparation method according to claim 1, characterized in that: Step (1) The concentration of dopamine and polyethyleneimine is 2-10 mg / ml, and the solvent is tris(hydroxymethyl)aminomethane (Tris) at pH 8.5; the optimal feed ratio of dopamine to polyethyleneimine is 1:
1. The polyethyleneimine and polyacrylic acid solvent in step (2) is 0.5 mol / L sodium chloride, and the concentration is 2-10 mg / ml.
3. The polymer film coating prepared according to the method of claim 1 or 2 can inhibit bacterial film formation.
4. Use of the polymer film coating according to claim 3 in inhibiting fungal pellicle formation, characterized in that: The fungi are sensitive Candida albicans Y0109, SC5314; resistant Candida albicans 103, 100; Cryptococcus neoformans; and Candida auris.
5. Use of the polymer film according to claim 3 in inhibiting bacterial capsule formation, characterized in that: The bacteria are Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli.