WO2024053896A1 - Antimicrobial polymer and antimicrobial polymer film containing same - Google Patents

Abstract

An antimicrobial polymer and an antimicrobial polymer film containing same are provided. The antimicrobial polymer bears a functional group that forms hydrogen bonds with bacterial cell membranes. The functional group can be located on at least one of the main chain and side chain of the antimicrobial polymer. The antimicrobial polymer film contains the antimicrobial polymer.

Description

Antibacterial polymer and antibacterial polymer film containing the same

The present invention relates to an antibacterial polymer and an antibacterial polymer film containing the same.

Due to the increase in resistant bacteria and stagnation in antibiotic development, there is an urgent need to develop effective antibiotics that can replace existing antibiotics. Antimicrobial peptides (AMPs), first discovered in the 1980s, are present in many organisms and act as an essential part of the innate immune system of various organisms, including humans. Antimicrobial peptides exhibit a broad spectrum of antibacterial activity, including against Gram-positive bacteria, Gram-negative bacteria, viruses and fungi. In addition, antibacterial peptides can effectively inhibit resistant bacteria due to a different mechanism of action induced by the cationic moiety of the antibacterial peptide, which promotes binding of the antibacterial peptide to the bacterial surface through electrostatic interactions. However, mass production of antibacterial peptides is difficult, including high costs.

The present invention provides a polymer with excellent antibacterial properties.

The present invention provides a polymer film with excellent antibacterial properties.

Other objects of the present invention will become clear from the following detailed description and accompanying drawings.

The antibacterial polymer according to one embodiment of the present invention includes a functional group that forms a hydrogen bond with the bacterial cell membrane.

The functional group may be located in at least one of the main chain and side chain of the antibacterial polymer.

The functional group may form a hydrogen bond with at least one of lipopolysaccharide and phospholipid of the bacterial cell membrane.

The functional group may include at least one of S and O. The functional group may include sulfoxide (SO).

The antibacterial polymer may include a citronellol-derived polymer. The functional group can link citronellyl analogs with methacrylate-based polymers.

The antibacterial polymer according to another embodiment of the present invention includes a sulfoxide (SO) functional group.

The sulfoxide functional group may be located in at least one of the main chain and side chain of the antibacterial polymer.

The sulfoxide functional group may form a hydrogen bond with at least one of lipopolysaccharide and phospholipid of the bacterial cell membrane.

Antibacterial polymer films according to embodiments of the present invention include the antibacterial polymer.

Polymers and polymer films according to embodiments of the present invention may have excellent antibacterial properties. The antibacterial polymer has a simple manufacturing method and is easy to mass produce.

Figure 1 shows the synthesis method of PCHM-#.

Figure 2 shows ¹ H NMR spectra of (a) PCHM-S, (b) PCHM-SO, (c) PCHM-SO ₂ , and (d) PCHM-O.

Figure 3 shows the synthesis method of PE_S_6,8 and PE_SO_6,8.

Figure 4 shows the ¹ H NMR spectra of PE_S_6,8 and PE_SO_6,8.

Figure 5 shows the FT-IR spectra of PE_S_6,8 and PE_SO_6,8.

Figure 6 shows the FT-IR spectrum of PCHM-#.

Figure 7 shows S 2p XPS spectra of PCHM-S, PCHM-SO, and PCHM-SO ₂ .

Figure 8 shows the antibacterial test results of PCHM-# against E. coli.

Figure 9 shows the bactericidal activity of PCHM-#.

Figure 10 shows a CLSM image of E. coli attached to the PCHM-# film.

Figure 11 shows the antibacterial test results of PE_S_6,8 and PE_SO_6,8 against E. coli.

Figure 12 shows the Raman spectrum of PCHM-# film in the range 1640 - 1800 cm ^-1 .

Figure 13 shows the Raman spectrum of PCHM-# film in the range of 1000 - 1200 cm ^-1 .

Figure 14 shows the DSC curve of the first heating scan of a mixture of PCHM-# and POPE (PCHM-#:POPE=2:1(w/w)).

Figure 15 shows the DSC curve of the second heating scan of a mixture of PCHM-# and POPE (PCHM-#:POPE=2:1(w/w)).

Hereinafter, the present invention will be described in detail through examples. The purpose, features, and advantages of the present invention will be easily understood through the following examples. The present invention is not limited to the embodiments described herein and may be embodied in other forms. The embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to enable the idea of the present invention to be sufficiently conveyed to those skilled in the art to which the present invention pertains. Accordingly, the present invention should not be limited by the following examples.

The antibacterial polymer according to one embodiment of the present invention includes a functional group that forms a hydrogen bond with the bacterial cell membrane.

The functional group may be located in at least one of the main chain and side chain of the antibacterial polymer.

The functional group may form a hydrogen bond with at least one of lipopolysaccharide and phospholipid of the bacterial cell membrane.

The functional group may include at least one of S and O. The functional group may include sulfoxide (SO).

The antibacterial polymer may include a citronellol-derived polymer. The functional group can link citronellyl analogs with methacrylate-based polymers.

The antibacterial polymer according to another embodiment of the present invention includes a sulfoxide (SO) functional group.

The sulfoxide functional group may be located in at least one of the main chain and side chain of the antibacterial polymer.

The sulfoxide functional group may form a hydrogen bond with at least one of lipopolysaccharide and phospholipid of the bacterial cell membrane.

The antibacterial polymer may include a citronellol-derived polymer. The functional group can link the citronellyl analog to the methacrylate-based polymer.

Antibacterial polymer films according to embodiments of the present invention include the antibacterial polymer.

[Example]

[Synthesis example of citronellyl bromide and citronellyl thiol]

Citronellyl bromide can be synthesized through the Appel reaction using carbon tetrabromide as a halogen source. A solution of citronellol (5 g, 32 mmol) and CBr ₄ (11.67 g, 35.20 mmol) in dry dichloromethane (135 mL) was added to a 250 mL one-neck round bottom flask equipped with a magnetic stir bar. PPh ₃ (10.07 g, 38.40 mmol) was added to the solution at 0° C. and the solution was stirred at room temperature under nitrogen atmosphere overnight. The reaction was terminated by adding water and the mixture was extracted with dichloromethane. The combined organic layer was dried with anhydrous magnesium sulfate and filtered. The obtained product was evaporated in a low pressure environment to obtain a colorless oil, which was purified by column chromatography (100% hexane). Citronellyl bromide (colorless oil) was obtained in 65% yield. ¹ H NMR of citronellyl bromide [400 MHz, CDCl ₃ , δ(ppm), TMS ref]: 5.09 (CH ₂ -CH-C), 3.41 (CH ₂ -Br) and 1.98 (CH-CH ₂ -CH ₂ ).

Thiourea (1.74 g, 22.81 mmol) was added to a solution of citronellyl bromide (5 g, 22.81 mmol) in ethanol (45 mL). This mixture was stirred under reflux conditions for 3 hours. The reaction was terminated by adding aqueous sodium hydroxide solution (10 wt%, 12.3 mL) and heated at 80°C for another 5 hours. After the reaction, the product was concentrated under reduced pressure, dissolved in chloroform, and transferred to a separatory funnel. After extraction with 0.1M HCl solution, the combined organic layer was dried over anhydrous magnesium sulfate and filtered. The product was evaporated under reduced pressure to give citronellyl thiol (colorless oil) in 89% yield. ¹ H NMR of citronellyl thiol [400 MHz, CDCl ₃ , δ(ppm), TMS ref]: 5.09 (CH ₂ -CH-C), 2.51 (CH ₂ -Br) and 1.97 (CH-CH ₂ -CH ₂ ).

[Synthesis example of poly(glycidyl methacrylate) (PGMA)]

PGMA (poly(glycidyl methacrylate)) uses 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as a chain transfer agent to form GMA. It can be synthesized by RAFT polymerization of (glycidyl methacrylate) GMA (10 g, 70.35 mmol), AIBN (16.42 mg, 0.1 mmol) and 4-cyano-4-(phenylcarbonothioylthio)phene. Carbonic acid (0.140 g, 0.5 mmol) was dissolved in 15 mL of dioxane and the solution was deoxygenated by three freeze-pump-thaw cycles. The solution was heated at 80°C for 16 hours in an oil bath under nitrogen conditions. After this, the product was precipitated in hexane three times to remove unreacted monomers. After filtering, it was vacuum dried at room temperature to obtain PGMA powder in 70% yield. ¹ H NMR of PGMA [400 MHz, CDCl ₃ , δ (ppm), TMS ref]: 4.29(O-CH ₂ -CH), 3.82(O-CH ₂ -CH), 3.24(CH ₂ -CH-CH ₂ -O), 2.85(COO-CH ₂ -CH-CH ₂ -O) , and 2.64(COO-CH ₂ -CH-CH ₂ -O).

[Synthesis example of poly(3-citronellylthio-2-hydroxypropyl methacrylate) (PCHM-S)]

PCHM-S (poly(3-citronellylthio-2-hydroxypropyl methacrylate)) can be synthesized by the epoxy-thiol reaction of PGMA using LiOH as a catalyst. Citronellyl thiol (2.43 g, 14.10 mmol) was added to a solution of PGMA (1.00 g, 7.05 mmol) in THF (25 mL). LiOH (33.77 mg, 1.41 mmol) dissolved in water (3.4 mL) was added to the solution at 0°C. Cooling was then removed and the reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was diluted with dichloromethane and washed with water. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The product was precipitated three times in hexane to remove unreacted citronellyl thiol. After filtration and vacuum drying at room temperature, PCHM-S powder was obtained in 80% yield. In PCHM-S, S represents the thioether moiety of the side chain. ^1H NMR of PCHM-S [400MHz, CDCl ₃ , δ(ppm), TMS ref]: 5.09(CH ₂ -CH-C), 4.30-3.85(COO-CH ₂ -CH), 2.75-2.48(CH ₂ -S-CH ₂ ) and 1.97(CH ₂ -CH-C).

[Synthesis example of poly(3-citronellylsulfinyl-2-hydroxypropyl methacrylate) (PCHM-SO)]

PCHM-SO (poly(3-citronellylsulfinyl-2-hydroxypropyl methacrylate)) can be formed by oxidizing PCHM-S. Hydrogen peroxide solution (0.36 g, 30 wt% solution in distilled water) and 10 mL of acetic acid were added to a 100 mL round bottom flask containing PCHM-S (1.00 g, 3.18 mmol) powder. The reaction mixture was stirred at 30°C overnight to obtain a homogeneous solution. The product was precipitated three times in diethyl ether, filtered, and vacuum dried at room temperature to obtain PCHM-SO powder in 90% yield. In PCHM-SO, SO represents the sulfoxide moiety of the side chain. 1H NMR of PCHM-SO [400MHz, CDCl ₃ , δ(ppm), TMS ref]: 5.08(CH ₂ -CH-C), 4.55-3.75(COO-CH ₂ -CH), 3.05-2.65(CH ₂ - SO-CH ₂ ) and 2.00(CH ₂ -CH-C).

[Synthesis example of poly(3-citronellylsulfonyl-2-hydroxypropyl methacrylate)(PCHM-SO ₂ )]

PCHM-SO ₂ (poly(3-citronellylsulfonyl-2-hydroxypropyl methacrylate)) can be formed by oxidizing PCHM-S. PCHM-S (1.00 g, 3.18 mmol) was dissolved in 125 mL of DMF, and oxone (2.93 g, 9.54 mmol) dissolved in distilled water was added to the polymer solution. This solution was heated at 60°C for 24 hours in an oil bath under nitrogen conditions. The product was precipitated three times in distilled water, filtered, and vacuum dried at room temperature to obtain PCHM-SO ₂ powder in 85% yield. In PCHM-SO ₂ , SO ₂ represents the sulfone moiety of the side chain. ^1H NMR of PCHM-SO ₂ [400MHz, CDCl ₃ , δ(ppm), TMS ref]: 5.08(CH ₂ -CH-C), 4.60-3.85(COO-CH ₂ -CH), 3.40-3.00(CH ₂ -SO ₂ -CH ₂ ) and 1.99(CH ₂ -CH-C).

[Synthesis example of citronellyl glycidyl ether (CGE)]

CGE (citronellyl glycidyl ether) was synthesized to prepare a polymer containing an ether moiety instead of the thioether moiety of PCHM-S. Tetrabutylammonium bromide (0.52 g, 1.60 mmol) and sodium hydroxide solution (3.84 g, 95.99 mmol, 48% solution by weight in distilled water) were added to a solution of citronellol (5 g, 32.00 mmol) in 32 mL toluene. Epichlorohydrin (5.92 g, 63.99 mmol) was added to the solution while stirring at room temperature, and the reaction mixture was stirred at 50°C for 6 hours. After reaction, the product was diluted with dichloromethane and washed with water. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain citronellyl glycidyl ether (colorless oil) in 95% yield. ¹ H NMR of CGE [400 MHz, CDCl ₃ , δ(ppm), TMS ref]: 5.09(CH ₂ -CH-C), 3.69(O-CH ₂ -CH ₂ -CH), 3.53(CH ₂ -O- CH ₂ -CH ₂ -CH), 3.38(CH ₂ -O-CH ₂ -CH ₂ -CH), 3.14(O-CH ₂ -CH-CH ₂ -O), 2.80(O-CH ₂ -CH-CH ₂ -O-CH ₂ ) and 2.61(O-CH ₂ -CH-CH ₂ -O-CH ₂ ).

[Synthesis example of poly(methacrylic acid) (PMA)]

PMA (poly(methacrylic acid)) can be synthesized by RAFT polymerization of methacrylic acid using 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as a chain transfer agent. Methacrylic acid (5g, 58.10mmol), AIBN (22.72mg, 0.14mmol), and 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid (0.116g, 0.41mmol) were dissolved in 13mL of DMF and added to the solution. was deoxygenated through three freeze-pump-thaw cycles. The deoxygenated solution was heated at 80°C for 16 hours in an oil bath under nitrogen conditions. After the reaction, the product was precipitated in diethyl ether three times to remove unreacted monomers. After filtration, it was vacuum dried at room temperature to obtain PMA powder with a 75% yield. ¹ H NMR of PMA [400 MHz, DMSO, δ(ppm), TMS ref]: 12.36 (COOH), 1.85-1.60 (CH ₂ -C-CH ₃ ) and 1.07-0.87 (CH ₂ -C-CH ₃ ).

[Synthesis example of poly(3-citronellyloxy-2-hydroxypropyl methacrylate) (PCHM-O)]

CGE (4.93 g, 23.24 mmol) and tetrabutylammonium bromide (0.75 g, 2.32 mmol) were added to a solution of PMA (1 g, 11.62 mmol) in 57 mL of DMF. The reaction mixture was stirred at 100°C for 16 hours. The product was precipitated in ether three times to remove unreacted CGE, then dissolved in chloroform and dried under reduced pressure at room temperature to obtain PCHM-O as a sticky solid in 65% yield. In PCHM-O, O represents the ether moiety of the side chain. ^1H NMR of PCHM-O [400MHz, CDCl ₃ , δ(ppm), TMS ref]: 5.09(CH ₂ -CH-C), 4.15-3.85(COO-CH ₂ -CH), 3.65-3.30(CH ₂ -O-CH ₂ ) and 1.97(CH ₂ -CH-C).

[PCHM-#Film]

PCHM-# represents a PCHM polymer containing another functional group in the side chain, and # in PCHM-# represents a functional group in the side chain. For example, PCHM-S, PCHM-SO, PCHM-SO ₂ and PCHM-O represent PCHM polymers containing thioethers, sulfoxides, sulfones and ethers, respectively. A 1 wt% polymer solution in chloroform was coated on a glass substrate using a spin coating process (3,000 rpm, 30 seconds) and then dried in vacuum overnight. Using a polymer film prepared by spin coating, the bactericidal activity of the polymer film, bacterial attachment to the polymer surface, and surface characteristics were evaluated.

[Bactericidal activity of PCHM-# film]

The bactericidal activity of the PCHM-# film was investigated using a film adhesion test against E. coli (ATCC 700926). To prepare a bacterial suspension, E. coli cells were cultured in NB (Nutrient broth) solution at 37°C for 18 hours. Representative colonies were removed with a platinum loop, placed in 30 mL of nutrient solution, and cultured with shaking at 37°C for 18 hours. After washing twice with saline solution (0.9 wt% NaCl solution), it was resuspended in saline solution to generate 1×10 ⁵ CFU (colony forming unit)/mL. Bacterial cell concentration was estimated by measuring the absorbance of the cell dispersion at 600 nm and referring to a standard calibration curve. An optical density of 0.1 at 600 nm corresponds to approximately 10 ⁸ CFU/mL.

To evaluate the bactericidal activity of the PCHM-# film, 0.1 mL of bacterial suspension was dropped on the surface of the PCHM-# film (2.5 cm × 2.5 cm) placed in a Petri dish, covered with an OHP film of the same size, and checked to see if it was in full contact. After 24 hours at 25°C, 0.9 mL of saline solution was poured into the Petri dish containing the sample. The adhered cells were separated from the film by shaking vigorously and the solution mixture was transferred to a microtube.

The resulting solution was serially diluted, and then 0.1 mL of each dilution was spread on agar plates. After incubation at 37°C for 18 hours, viable microbial flora were counted. Each test was repeated at least three times. Bactericidal activity was calculated as follows.

Bactericidal activity (%) = 100×(N ₀ - N _i )/N ₀

In the above formula, N ₀ represents the bacterial CFU of the blank and N _i represents the bacterial CFU of the test sample.

[Synthesis example of PE_S_6,8]

PE_S_6_8 represents an antibacterial polymer containing a thioether group in the main chain. 20 mL of 2.0 g (18.15 mmol) of 1,7-octadiene, 2.728 g (18.15 mmol) of 1,6-hexanedithiol, and 47.28 mg of DMPA (2,2-Dimethoxy-2-phenylacetophenone) It was dissolved in THF, placed in a round bottom flask, and then irradiated with UV light for 1 hour. After precipitating the product in methanol, unreacted substances were removed through filtering, and this process was repeated three times. The obtained powdered polymer was dried at room temperature under vacuum for one day.

[Synthesis example of PE_SO_6,8]

PE_SO_6,8 represents an antibacterial polymer containing a sulfoxide group in the main chain. PE_SO_6,8 can be prepared by oxidizing the thioether group of PE_S_6,8. 0.5 g (3.84 mmol) of the synthesized PE_S_6,8 polymer was added to a round bottom flask along with 0.131 g (3.84 mmol) of hydrogen peroxide and 5 mL of acetic acid, and then reacted with stirring at 30°C for 24 hours. The product was precipitated in diethyl ether and filtered, and this process was repeated three times. The obtained polymer was dried at room temperature under vacuum for one day.

[Confocal laser scanning microscopy (CLSM) image]

Images of live and dead bacteria attached to the polymer film were obtained using a confocal laser scanning microscope. SYTO9 is a green fluorescent nuclear and chromosome counterstain that can penetrate both living and dead cell membranes. PI is a red fluorescent dye that is membrane impermeable and is generally excluded from viable cells. Therefore, the SYTO9 and PI mixture is commonly used to distinguish between live and dead cells.

A polymer film (2.5 cm × 2.5 cm) coated on a glass substrate was placed in a 6-well plate and incubated with 5 mL of bacterial suspension (10 ⁸ CFU/mL). After 24 hours at 25°C without shaking, the glass substrate was washed twice with 1 mL of saline solution and stained for 20 minutes in the dark using SYTO9 (0.01mM) and PI (0.02mM). To prepare the SYTO9 and PI mixture used for bacterial staining, 1 μL of PI solution (20 mM in DMSO) and 2 μL of SYTO9 solution (5 mM) were added to 1 mL of distilled water and diluted to the appropriate concentration. The glass substrate was gently rinsed twice with saline solution and covered with a coverslip. Samples were characterized by confocal laser scanning microscopy (CLSM) using a 488 nm laser for SYTO9 and a 545 nm laser for PI.

[FITC-LPS binding analysis on polymer films]

To evaluate the LPS binding affinity of each polymer, a polymer film was prepared by drop casting a 1 wt% polymer solution in chloroform on a glass substrate (1 cm × 1 cm). 1 mL of FITC-LPS solution (0.1 mg/10 mL in saline solution) was added to each well of a 24-well plate, and a polymer film was placed in each well. After gently shaking for 24 hours at 25°C, avoiding light, 200 μL of the solution from each well was transferred from a transparent 24-well plate to a 96-well black plate. The adsorption of FITC-conjugated LPS by the polymer film was analyzed by exciting FITC-LPS at 485 nm and monitoring FITC emission at 535 nm using a multimode microplate reader.

[Feature Analysis]

¹ H NMR spectra were recorded on a spectrometer (400 MHz) at room temperature using CDCl ₃ and DMSO as solvents, with TMS as the reference. Molecular weight (Mn, Mw) and polydispersity index (PDI) were analyzed by gel permeation chromatography (GPC) using an Ultimate 3000 HPLC system. HPLC grade DMF was used as the eluent. The glass transition temperature of the polymer was investigated by differential scanning calorimetry (DSC) in a nitrogen atmosphere. Samples with a typical mass of 5 - 10 mg were encapsulated in sealed aluminum pans. The sample was first heated to 120°C and then quenched to -80°C and then a second heating scan was performed from -80°C to 120°C at a heating rate of 10°C/min.

To investigate the interaction between the polymer and phospholipid (POPE), 1 mg of POPE and 2 mg of the polymer were dissolved in chloroform, placed in an aluminum pan, and dried under reduced pressure at room temperature. The same procedure was performed to prepare DSC samples containing only POPE. The sample was cooled to -80°C and heated to 90°C at a heating rate of 10°C/min and then cooled to -80°C and subjected to a second heat scan from -80°C to 90°C at the same heating rate.

Thermogravimetric analysis (TGA) was performed in a nitrogen (N ₂ ) atmosphere. The sample was heated to 100°C, isothermal for 10 minutes, and then heated to 700°C at a heating rate of 10°C. FT-IR spectra were recorded in the absorption mode of the spectrophotometer with a resolution of 8 cm ^-1 in the vibration frequency range of 600 to 4000 cm ^-1 . Raman spectra were recorded on a Raman spectrometer using a 532 nm laser.

To prepare a polymer film containing POPE, 1 mg of POPE and 2 mg of the polymer were dissolved in chloroform, drop-casted on a glass substrate (1 cm × 1 cm), and dried under reduced pressure at room temperature for one day. The absorbance of the bacterial suspension at 600 nm was measured with a UV-Visible spectrometer at room temperature. Surface morphology and topology were investigated by atomic force microscopy (AFM). The surface composition of the polymer film was analyzed using X-ray photoelectron spectroscopy (XPS) using Mg/Al (1486.69 eV) as a radiation source. After the survey scan was performed, high-resolution scans in the range of 0-1500 eV at an angle of 30° were performed in the C 1s, O 1s, N 1s and S 2p regions.

The contact angle between diiodo-methane and water on the polymer surface was measured with a droplet shape analyzer connected to a computer running droplet shape analysis software. The contact angle for each sample was measured more than five times on independently prepared polymer films, and the average value was used as data. The Owens-Wendt-Rabel-Kaelble (OWRK) method was used to calculate the surface energy of the polymer film. The zeta potential of PCHM-# films was measured by electrophoretic light scattering spectrophotometry.

Figure 1 shows the synthesis method of PCHM-#, and Figure 2 shows the ¹ H NMR spectra of (a) PCHM-S, (b) PCHM-SO, (c) PCHM-SO ₂ , and (d) PCHM-O. indicates. In PCHM-#, # refers to the functional group linking the citronellyl analogue to the 2-hydroxypropyl methacrylate-based polymer, and S, SO, SO ₂ and O refer to thioether, sulfoxide, sulfone and ether, respectively. do.

Referring to Figures 1 and 2, the epoxy of PGMA and the thiol of citronellyl thiol were synthesized by raft polymerization of GMA using 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as a chain transfer agent. It is synthesized through liver reaction. Citronellyl thiol is synthesized by the reaction of thiourea with citronellyl bromide prepared by the Appel reaction of citronellol using carbon tetrabromide as a halide source. The chemical structures of nellyl bromide and citronellyl thiol were confirmed by ¹ H NMR.

The degree of substitution of citronellyl thiol is 100% as confirmed by ¹ H NMR, and the proton peak of the epoxy moiety of PGMA disappears after the epoxy-thiol reaction. The thioether moiety of PCHM-S is oxidized to sulfoxide and sulfone using hydrogen peroxide and oxone as oxidizing agents, respectively, and oxone is used to prevent oxidation of the double bond in the citronellyl analogue.

The proton peak of the carbon attached to sulfur in PCHM-S, PCHM-SO and PCHM-SO ₂ gradually shifts to the lower field with the increase in the oxidation number of sulfur and the number of electron-withdrawing oxygen, which leads to the corresponding functional group. indicates successful oxidation.

PCHM-O is synthesized by the reaction of the epoxy group of citronellyl glycidyl ether (CGE) and the carboxyl group of PMA using TBAB as a catalyst, and PMA is synthesized by methacrylic acid using the same chain transfer agent used in the synthesis of PGMA. It is synthesized by raft polymerization.

CGE is synthesized through the reaction of citronellol and epichlorohydrin in the presence of TBAB, a phase transfer catalyst, and its chemical structure can be confirmed by ¹ H NMR. The proton peak from the carboxylic acid of PMA disappears after reaction with CGE, indicating that all carboxylic acid functional groups of PMA reacted with CGE.

Figure 3 shows the synthesis method of PE_S_6,8 and PE_SO_6,8, Figure 4 shows ^{the 1} H NMR spectra of PE_S_6,8 and PE_SO_6,8, and Figure 5 shows the FT-IR spectrum of PE_S_6,8 and PE_SO_6,8. indicates.

Referring to Figures 3 to 5, PE_S_6_8 uses DMPA (2,2-Dimethoxy-2-phenylacetophenone) as a catalyst to react 1,7-octadiene and 1,6-hexanedithiol. It is synthesized by PE_SO_6,8 is manufactured by placing the synthesized PE_S_6,8 polymer in a round bottom flask with hydrogen peroxide and acetic acid and then reacting.

The structures of PE_S_6,8 and PE_SO_6,8 were confirmed by ¹ H NMR. After oxidation of the thioether group, the peak corresponding to the hydrogen of the methylene group attached to sulfur moved to the lower field, confirming that oxidation had progressed. In addition, in the FT-IR spectrum of PE_SO_6,8, a peak at 1098 cm ^-1 corresponding to sulfoxide was observed, confirming that oxidation had proceeded well.

Figure 6 shows the FT-IR spectrum of PCHM-#, and Figure 7 shows the S 2p XPS spectrum of PCHM-S, PCHM-SO, and PCHM-SO ₂ .

Referring to Figures 6 and 7, the characteristic peaks corresponding to the functional groups in the side chain of PCHM-# show the successful synthesis of the polymer. In the FT-IR spectrum of PCHM-#, a characteristic hydroxyl peak in the range of 3100 - 3300 cm ^-1 appears due to the ring-opening reaction between the epoxy moiety of PGMA and CGE. The hydroxyl peak of PCHM-SO appears at the lowest wave number compared to other polymers because the polar sulfoxide group and hydroxyl group of the side chain can form hydrogen bonds. The characteristic peak assigned to the epoxy moiety of PGMA at 904 cm ^-1 disappears after the thiol-epoxy reaction, showing that all epoxy moieties were reacted with citronellyl thiol, consistent with the ¹ H NMR spectrum.

Successful oxidation of PCHM-S results in the appearance of the characteristic peak of sulfoxide at 996 cm ^-1 in the FT-IR spectrum of PCHM-SO, and the appearance of characteristic peaks of sulfone at 1123 and 1274 cm ^-1 in the FT-IR spectrum of PCHM-SO ₂ It is clearly confirmed that

In the FT-IR spectrum of PCHM-O, a peak at 1113 cm ^-1 assigned to the ether group appears, and after CGE substitution, the characteristic peak of the carbonyl group of PMA shifts from 1699 to 1728 cm ^-1 , resulting in the successful synthesis of PCHM-O with 100% substitution degree. represents.

The oxidation state of sulfur in PCHM-S, PCHM-SO and PCHM-SO ₂ was analyzed by the XPS spectrum of S 2p, and the sulfur peak shifts to higher binding energy with increasing sulfur oxidation number. The molecular weight and polydispersity of PCHM-# were investigated by GPC analysis using DMF as eluent due to the low solubility of PCHM-SO in THF. The molecular weight of sulfur-containing polymers increases with increasing sulfur oxidation number, because the addition of oxygen increases the molecular weight of the repeating unit and the solubility of the polymer in DMF. The molecular weight (Mn) of PCHM-# is in the range of 19,000 - 32,000, which is not much different and is sufficient to produce a physically stable film.

Figure 8 shows the antibacterial test results of PCHM-# against E. coli, Figure 9 shows the bactericidal activity of PCHM-#, and Figure 10 shows a CLSM image of E. coli attached to the PCHM-# film.

Referring to Figures 8 to 10, the bactericidal activity of the PCHM-# film prepared by spin coating was evaluated against E. coli using glass washed with ethanol as a negative control. Among the PCHM-# films, the PCHM-SO film showed the most effective bactericidal activity of 99.99%, showing no bacterial flora on the agar plate.

While there were a few bacterial flora on the agar plates of PCHM-SO ₂ and PCHM-O, many bacterial flora were found on the agar plates of PCHM-S, and the bactericidal activities of PCHM-SO ₂ , PCHM-O and PCHM-S were 88.19, respectively. , 84.25, 15.34%. PCHM-S shows relatively low bactericidal activity despite the presence of citronellyl analogs in the side chain, indicating that the functional groups in the side chain have a significant impact on the bactericidal activity of the polymer.

Bacterial adhesion to the polymer film was measured by observing fluorescence microscopy images of E. coli stained with a dye mixture of SYTO9 and PI to distinguish between live and dead bacteria. The number of attached live and dead bacteria was calculated by the image program, and CLSM images of PCHM-SO, PCHM-SO ₂ and PCHM-O showed few or no live bacteria stained by SYTO9 with green fluorescence. Many live bacteria were observed on the PCHM-S film, which is consistent with the bactericidal activity test results.

The total number of bacteria attached to PCHM-S, PCHM-SO, PCHM-SO2, and PCHM-O is 141, 39, 31, and 122, respectively, and the number of bacteria attached to PCHM-S and PCHM-O films is 141, 39, 31, and 122, respectively. and about 4 times that of the PCHM-SO ₂ film. In other words, the interaction between the polymer film and bacteria is highly dependent on the functional groups of the polymer side chain, resulting in different bactericidal activity and bacterial adhesion.

Figure 11 shows the antibacterial test results of PE_S_6,8 and PE_SO_6,8 against E. coli. The antibacterial test of PE_S_6,8 and PE_SO_6,8 was performed in the same manner as the antibacterial test of PCHM-#.

Referring to Figure 11, PE_SO_6,8 showed over 95% antibacterial activity against E. coli and appeared to have superior antibacterial activity to PE_S_6,8, confirming that the antibacterial activity of the PE_SO_6,8 polymer was due to the sulfoxide group located in the main chain.

Because inhibition of bacterial adhesion is important to reduce the likelihood of harmful bacterial infection, many studies have been conducted to elucidate the relationship between surface properties and bacterial adhesion, especially for biomedical devices. To understand bacterial attachment on PCHM-# films, the surface properties of PCHM-# films were analyzed using surface roughness, zeta potential and surface energy, which are shown in Table 1.

[Table 1]

Referring to Table 1, all polymer films had smooth surfaces with small Ra values in the range of 1–3 nm, so surface roughness was excluded as a factor affecting bacterial adhesion. The outermost component of the E. coli outer membrane is lipopolysaccharide (LPS), which contains a hydroxyl group in the repeating sugar group and a phosphate group in the inner core. These hydroxyl and phosphate groups are of E. coli. This can explain the overall negative zeta potential. Therefore, the attachment of bacteria to negatively charged surfaces can be greatly inhibited by electrostatic repulsion. All PCHM-# films exhibit negative zeta potential due to the presence of highly electronegative oxygen in the carbonyl moiety of the polymer backbone.

The zeta potentials of PCHM-S, PCHM-SO, PCHM-SO ₂ , and PCHM-O are -31.28, -44.81, -41.85, and -17.85 mV, respectively. In general, as the dipole moment of the functional group increases, the absolute value of the zeta potential decreases. increases. This is due to the negatively charged oxygen being attached to sulfur by a coordinating single bond with both shared electrons coming from sulfur. Therefore, PCHM-SO and PCHM-SO ₂ films exhibit a larger negative zeta potential than other polymer films, so the electrostatic repulsion between these polymer films and bacteria is large, effectively inhibiting bacterial attachment.

Static contact angle and surface energy were also measured to understand bacterial attachment on polymer surfaces (Table 1). Since the bactericidal activity and bacterial attachment were investigated when the polymer film was in full contact with the bacterial suspension, the static contact angle and surface energy were measured using the polymer film immersed in a saline solution at 25°C for 24 hours. When a polymer film is immersed in water at a certain temperature, the hydrophilic moiety of the polymer tends to be located at the interface between the polymer film and water. Additionally, when the temperature is close to the glass transition temperature of the polymer, water molecules act as a plasticizer and the polymer is in an amorphous state at that temperature, so the polymer can be easily assembled into a thermodynamically stable structure. Therefore, the water contact angle value of the polymer film becomes smaller than that of the polymer film cast after being immersed in saline solution for 24 hours.

PCHM-S and PCHM-O have low glass transition temperatures of 34.5℃ and 15.2℃, and are close to 25℃, so the water contact angle value of PCHM-S and PCHM-O decreases after immersion compared to PCHM-SO and PCHM-SO ₂ is bigger. For cast films, the water contact angle values of PCHM-SO and PCHM-SO ₂ are smaller than those of PCHM-S and PCHM-O due to the polar functional groups in the side chains.

Comparing the water contact angle values of PCHM-SO and PCHM-SO ₂ before and after immersion in saline solution, the amount of water absorbed by the polymer film is the largest in the PCHM-SO film, so the decrease in water contact angle value after immersion of PCHM-SO is observed for PCHM-SO. It is greater than the decrease in -SO ₂ . The polar sulfoxide group of PCHM-SO can form strong hydrogen bonds with water, causing the polymer to absorb a large amount of water, which results in the FT-IR spectrum of PCHM-SO having hydroxyl peaks at lower wavenumbers compared to other polymers. You can also check this.

E. coli exhibits a low water contact angle of 30.7° and a high surface energy of 67.1 mN/m, indicating that the surface of E. coli is hydrophilic due to the polar phosphate and hydroxyl groups of the outer membrane. Bacterial adhesion to a polymer surface increases as the difference in surface energy between the polymer surface and bacterial cells decreases, so in the case of PCHM-#, bacterial adhesion is suppressed on polymer surfaces with low surface energy. Therefore, bacterial adhesion is effectively inhibited on PCHM-SO and PCHM-SO2 films, which have relatively low surface energy values and larger negative zeta potential compared to other polymer films.

Since antibacterial polymers destroy the integrity of the outer membrane of bacteria, leading to their death, the interaction between the polymer and the outer membrane of bacteria is one of the important factors that determines the antibacterial properties of polymers. Since the outermost component of the E. coli outer membrane is LPS containing phosphate and hydroxyl moieties, the LPS binding affinity to the polymer film is measured by measuring the fluorescence of FITC-conjugated LPS using a microplate reader. It was measured and analyzed. 1 mL of FITC-LPS solution (0.1 mg/10 mL in saline solution) was added to each well of a 24-well plate, and PCHM-# film was placed in each well and mixed gently for 24 hours at 25°C. After mixing, the fluorescence intensity between the FITC-LPS solution treated with the polymer film and the solution treated with clean glass was compared to measure the amount of LPS bound to the polymer film, which is shown in Table 2. Table 2 shows the fluorescence intensity of the FITC-LPS solution at 535 nm and the amount of LPS adsorbed on the PCHM-# film.

[Table 2]

Referring to Table 2, the fluorescence intensity of solutions treated with PCHM-SO films is significantly lower than those treated with other polymers, which is due to the ability of the sulfoxide moiety to form strong hydrogen bonds between PCHM-SO and LPS. indicates effective interaction. The fluorescence intensity of solutions treated with polymer films of PCHM-SO ₂ and PCHM-O is lower than that of solutions treated with clean glass and higher than that of solutions treated with PCHM-SO films, as LPS has a lower fluorescence intensity than that of solutions treated with PCHM-SO. It indicates that it is bound to the polymer films of PCHM-SO ₂ and PCHM-O through relatively weak hydrogen bonds.

Because the ether moiety has a smaller hydrogen bond radius than the thioether moiety, the ether moiety forms a stronger hydrogen bond complex than the thioether moiety. Therefore, the interaction between PCHM-S film and LPS is the weakest among all polymer films, as evidenced by the similar fluorescence intensity between solutions treated with PCHM-S and those treated with pristine glass. LPS binding affinity varies greatly depending on the side chain functional group of the polymer, and the higher the hydrogen bond strength of the side chain functional group, the greater the amount of bound LPS.

Figure 12 shows the Raman spectrum of the PCHM-# film in the range of 1640 - 1800 cm ^-1 , and Figure 13 shows the Raman spectrum of the PCHM-# film in the range of 1000 - 1200 cm ^-1 . In Figures 12 and 13, the solid line corresponds to the PCHM-# film containing no POPE, and the dotted line corresponds to the PCHM-# film containing POPE (POPE mixed PCHM-S film). The PCHM-# film without POPE is manufactured by drop casting a 1 wt% polymer solution in chloroform, and the POPE mixed PCHM-# film is made by drop casting of POPE and polymer (POPE: polymer = 1:2 (w/w)) in chloroform. It is prepared by drop casting the solution mixture.

Referring to Figures 12 and 13, the characteristic peak assigned to the carbonyl moiety of POPE appears at 1740 cm ^-1 , which is a higher wave number compared to all POPE blended polymer films except the POPE blended PCHM-S film, which is This indicates that the interactions between polymers other than PCHM-S and PCHM-S are stronger than those between POPE molecules.

Characteristic peaks of the phosphate group of POPE appear at 1064, 1096, and 1128 cm ^-1 , which correspond to the vibrational modes of -PO- in the head group, PO ₂ - and -PO- in the tail group. These three characteristic peaks are clearly distinguished in the Raman spectra of the POPE blended film with PCHM-S, whereas these peaks are indistinguishable in the Raman spectra of the POPE blended film with PCHM-SO and PCHM-SO ₂ . The interaction between the phosphate group of POPE and the polymer is stronger for PCHM-SO, PCHM-SO ₂ , and PCHM-O than for PCHM-S.

As a result of investigating the interaction between POPE and PCHM-# by comparing the positions and shapes of the characteristic functional group peaks of POPE and the polymer using Raman spectra, it was found that the interaction between POPE and PCHM-S was the weakest compared to other polymers.

Figure 14 shows the DSC curve of the first heating scan of the mixture of PCHM-# and POPE (PCHM-#:POPE=2:1 (w/w)), and Figure 15 shows the DSC curve of the mixture of PCHM-# and POPE (PCHM-#:POPE=2:1(w/w)). #:POPE=2:1(w/w)) shows the DSC curve of the second heating scan.

Phospholipids have diverse phase behaviors through self-assembly resulting from polar head groups and hydrophobic tails, and phase transitions in phospholipids can be recognized through DSC analysis. The phase transition of phospholipids depends on internal factors such as the composition and structure of the phospholipids and external factors such as temperature, pH, water content, and the presence of solutes. The presence of other molecules, such as solutes, can inhibit POPE self-assembly by inhibiting interactions between POPE molecules.

The phase behavior of POPE, a mixture of POPE and polymer, was determined by adding a POPE solution or a polymer mixed POPE solution in chloroform (POPE: polymer = 1:2 (w/w)) to a DSC pan and vacuum drying the sample at room temperature. was investigated using DSC analysis.

Referring to Figure 14, the first heating scan showed that there were two phase transitions. The first transition at a lower temperature of 10.18 °C corresponds to the chain melting transition of hydrophobic alkyl chains from gel to liquid crystal, followed by a lower enthalpy transition at a higher temperature of 53.01 °C, corresponding to the transition from a bilayer structure to an inverted hexagonal structure. . In the first heating scan, the melting peaks of PCHM-S, PCHM-SO, PCHM-SO ₂ and PCHM-O appear around 5 to 8°C, which is lower than the first melting temperature of POPE, and the alkyl chains of POPE are PCHM-S and PCHM. Less tightly packed in the presence of -SO ₂ and PCHM-O. In the case of PCHM-SO, the melting peak appears at 47.09°C, which is similar to the second transition temperature of POPE. Since the strong interaction between PCHM-SO and POPE prevents gel formation of POPE, the first melting peak occurs in the presence of PCHM-SO. The enthalpy change appears to be the smallest.

Referring to Figure 15, in the second heating scan, the first chain melting transition is dominant in POPE and the melting peak of polymer blend POPE appears at a lower temperature than that of POPE. The decrease in melting temperature after polymer incorporation is much larger for PCHM-SO compared to other polymers, indicating the strongest interaction between POPE and PCHM-SO due to the sulfoxide bonds that can form strong hydrogen bonds. Since the melting peak of the polymer-mixed POPE is due to the phase transition of POPE, the change in phase transition enthalpy of the polymer-mixed POPE was recalculated by correcting the weight of POPE to the total weight. In the case of PCHM-SO, PCHM-SO ₂ , and PCHM-O, the transition enthalpy is smaller than that of POPE, indicating that the interaction between POPE and these polymers inhibits chain packing between POPE molecules.

Because the interaction between POPE and PCHM-S is weaker than that of other polymers, the transition enthalpy of POPE mixed with PCHM-S is almost similar to that of POPE. When a polymer is present, the phase behavior of POPE is greatly influenced by the functional groups on the side chains of the polymer, and when functional groups are present on the polymer, forming strong hydrogen bonds with POPE effectively disrupts the chain packing between POPE molecules.

So far, we have looked at specific embodiments of the present invention. A person skilled in the art to which the present invention pertains will understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative rather than a restrictive perspective. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the equivalent scope should be construed as being included in the present invention.

Polymers and polymer films according to embodiments of the present invention may have excellent antibacterial properties. The antibacterial polymer has a simple manufacturing method and is easy to mass produce.

Claims (11)

1. An antibacterial polymer containing functional groups that hydrogen bond with bacterial cell membranes.
2. According to claim 1,

   An antibacterial polymer, characterized in that the functional group is located on at least one of the main chain and the side chain of the antibacterial polymer.
3. According to claim 1,

   An antibacterial polymer characterized in that the functional group forms a hydrogen bond with at least one of lipopolysaccharide and phospholipid of the bacterial cell membrane.
4. According to claim 1,

   An antibacterial polymer, characterized in that the functional group includes at least one of S and O.
5. According to claim 3,

   An antibacterial polymer characterized in that the functional group includes sulfoxide (SO).
6. According to claim 1,

   The antibacterial polymer is an antibacterial polymer, characterized in that it contains a citronellol-derived polymer.
7. According to claim 6,

   An antibacterial polymer characterized in that the functional group connects a citronellyl analog to a methacrylate-based polymer.
8. Antibacterial polymer containing a sulfoxide (SO) functional group.
9. According to claim 8,

   An antibacterial polymer, characterized in that the sulfoxide functional group is located in at least one of the main chain and the side chain of the antibacterial polymer.
10. According to claim 8,

    An antibacterial polymer characterized in that the sulfoxide functional group forms a hydrogen bond with at least one of lipopolysaccharide and phospholipid of the bacterial cell membrane.
11. An antibacterial polymer film comprising the antibacterial polymer of any one of claims 1 to 10.

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