Curcumin-loaded polyvinyl butyral film with antibacterial activity

2.1 Materials

PVB (MW 40,000–70,000) and Cur (AR) were bought from Shanghai Macklin Biochemical Co., Ltd, China. Ethanol (AR) and sodium chloride (NaCl) (AR) were supplied by Beijing Chemical Works, China. Yeast extract (FMB Grade) was obtained from Shanghai Sangon Biotech Co., Ltd, China. Peptone (BR) was purchased from Beijing Aobox Biotechnology Co., Ltd, China. Agar powder (AR) was bought from Tianjin Guangfu Fine Chemical Research Institute, China. The species of bacteria were Staphylococcus aureus (S. aureus) (ATCC25923) and Escherichia coli (E. coli) (ATCC-25922), respectively.

2.2 Preparation of Cur-loaded PVB film

PVB films with Cur were prepared by a solution casting technique. Firstly, the PVB solution (6% w/v) was prepared by dissolving PVB in ethanol and the mixture was stirred using a magnetic stirrer hot plate (IKA, RT 15 S025, Germany) at 30°C with a rotor speed of 400 rpm. The transparent and homogeneous solution was obtained after being mixed for 90 min. Cur was subsequently added to the solution and continuously stirred for 30 min. The added content of Cur was 5% of the weight of PVB, which was determined through preexperiments of tensile strength and antibacterial activity. The mixtures were ultrasonically treated at an ultrasonic power of 200 W and a frequency of 40 kHz for 10 min. The stable and homogeneous solution was cast onto the polyfluortetraethylene plate and then dried at 37°C to obtain films. The prepared composite films were defined as PVB–Cur films (Figure 1). For comparison, the pure PVB films were prepared by the same method.

Figure 1

Schematic diagram of Cur-loaded PVB films.

2.3 FTIR analysis

The FTIR spectra of films were recorded on an iS50 FTIR spectrometer (ThermoFisher Nicolet, USA) with attenuated total reflection mode at room temperature, and the spectra of the films were scanned in the range of 500–4,000 cm⁻¹ with a resolution of 4 cm⁻¹.

2.4 Optical property

The ultraviolet and visible light barrier properties of PVB and PVB–Cur films were measured by using an ultraviolet-visible spectrophotometer (PerkinElmer, Lambda 25, USA). The film samples were cut into rectangular pieces (4 cm in length and 1 cm in width) and placed into the cuvette of spectrophotometer. The analyses were performed in triplicate. The transmittance of the film was measured in the wavelength range of 200–800 nm. Measurements were performed using air as a reference. The value of transparency is calculated, based on the transmittance of the film at 600 nm:

where T₆₀₀ is the percentage transmittance (%) at 600 nm and d is the film thickness (mm).

2.5 Thermogravimetric analysis (TGA)

The thermal stability and degradation characteristics of the films were carried out on a thermogravimetric analyzer (METTLER TOLEDO, TGA 2, Switzerland). The sample weights for the test were 2–4 mg. The samples were heated up from 50°C to 600°C at a heating rate of 10°C/min under nitrogen atmosphere with a nominal gas flow rate of 50 mL/min.

2.6 Film thickness

The thickness of the films was determined by using a spiral micrometer (Ningbo Shiji Bosi Tools Co., Ltd, China). Six thickness values were randomly measured at different locations on each film and their average thickness values were calculated.

2.7 Mechanical property

The mechanical properties of PVB and PVB–Cur films were determined by using a tensile tester (SHIMADZU, AGS-X, Japan) with a 100 N load cell at a tensile rate of 5 mm/min. The initial grip separation was set at 35 mm and all samples were cut into strips (size of 5 mm width × 60 mm height). All the determinations were performed at room temperature of 25°C and were made in triplicate. The values of tensile strength and elongation at break were directly determined from the stress–strain curves (32).

2.8 Contact angle measurement

The water contact angle of the films was determined using a contact angle measurement instrument (HARKE-SPCA, Beijing, China). The samples (20 mm × 60 mm) were fixed to the glass substrate and their wettability was evaluated by a sessile drop method. A droplet (10 μL) of deionized water was placed on the film surface using a micro-syringe. The side-view images of the water droplets were recorded. The contact angles were measured. All measurements were performed at room temperature. The contact angle was measured at five random sites and then the average angle was taken.

2.9 Antibacterial activity assay

The antibacterial activity of the films was evaluated by the colony-counting method. Colony forming unit (CFU) assay was performed on two bacteria of S. aureus (ATCC25923) and E. coli (ATCC25922) isolates to determine the antimicrobial activity of the PVB–Cur film. A suspension of 108 bacteria was incubated with 1.0 mL of Luria–Bertani (LB) liquid culture medium containing PVB–Cur film (5.0 mg mL⁻¹), and compared to the untreated control, PVB film (5.0 mg mL⁻¹), free Cur (5.0 mg mL⁻¹, turbid liquid) groups. Tubes were incubated at 37°C for 4 h using a tube rotator to ensure a uniform distribution. After incubation, suspensions were serially diluted in phosphate buffer solution, plated onto tryptic soy agar, and incubated for 24 h at 37°C under aerobic conditions. Individual colonies were counted and the number of CFUs was tabulated.

3.1 Chemical structure

The FTIR spectra of Cur (powders), PVB and PVB–Cur films are presented in Figure 2. In the spectrum of Cur, the peak at 3,501 cm⁻¹ corresponded to the phenolic –OH stretching vibration. The absorption peak at 1,626 cm⁻¹ was attributed to C–O stretching. The peaks at 1,601 and 1,505 cm⁻¹ corresponded to C–C stretching in the benzene ring (33). The bands at 1,274 cm⁻¹ corresponded to C–O of enol. Additionally, the bands in the region of 960–806 cm⁻¹ belonged to C–H out-of-plane bending and aromatic stretching. PVB showed an absorption band at 3,443 cm⁻¹ due to the –OH stretching, corresponding to the hydrophilic hydroxyl group in PVB. The peaks at 2,955 and 2,870 cm⁻¹ were attributed to the asymmetric and symmetric –CH₂ stretching, respectively. The peak presented at 1,737 cm⁻¹ was attributed to the carbonyl group double bond (C–O). The peak at 811 cm⁻¹ was related to the presence of acetal groups. The out-of-plane bending hydrogen bond to the carbonyl group was observed at 1,053 cm⁻¹ (34). The peak at 1,130 cm⁻¹ was related to the vibration of C–O–C, and the peak of 1,432 cm⁻¹ was due to –CH₂ bending. The characteristic peaks of Cur and PVB were presented in the spectrum of PVB–Cur films. It should be noted that in the spectrum of PVB–Cur film, the –OH stretching vibration of PVB showed a low-shift from 3,443 to 3,409 cm⁻¹. In addition, the C–O stretching of Cur shifted from 1,626 to 1,622 cm⁻¹; meanwhile, the C–C stretching in benzene ring at 1,601 and 1,505 cm⁻¹ was shifted to 1,586 and 1,512 cm⁻¹, respectively. These results demonstrated that the hydrogen bonding between PVB and Cur was generated in PVB–Cur films. In brief, PVB and Cur had been compounded together excellently.

Figure 2

FTIR spectra of Cur, PVB, and PVB–Cur film.

3.2 UV-vis analysis

Figure 3a shows the light transmittance of PVB and PVB–Cur films. The transmittance of the PVB film reduced at 600–200 nm. Moreover, the transmittance value of the PVB–Cur film almost reduced to 0% in the wavelength range of less than 500 nm. As we have known, Cur belongs to the class of phenolic compounds which is rich in unsaturated bonds, thus facilitating the absorption of ultraviolet radiation. Therefore, PVB–Cur film had superior UV-blocking properties than a PVB film. This film might effectively reduce the oxidative deterioration of food caused by light induction (35). Moreover, since UV light has a significant effect on the human wound, the PVB–Cur films with UV-blocking properties may be advantageous for inhibiting DNA rupture and dimerization of thymine molecules presented in the human wound. The transparency of the PVB–Cur film (2.94) and the PVB film (2.95) was almost equivalent at 600 nm, which indicated the good compatibility between Cur and PVB film matrix. Cur-loaded PVB films also had corresponding visualizations in Figure 3b and c.

Figure 3

Light transmittance characteristics of PVB and PVB–Cur films. (a) PVB film under visible light and (b) PVB–Cur film under visible light (c).

3.3 Thermal stability

The thermal stability of PVB and PVB–Cur films was evaluated by the TGA. The primary thermograms of the films are shown in Figure 4 and the thermal properties are listed in Table 1. From the thermogram, it could be found that the residues of Cur powder and PVB–Cur film at 600°C were 40% and 1%, respectively, and the pure PVB film was completely degraded. The residue of Cur should be attributed to the presence of the phenyl rings. The initial weight loss temperatures (T_onset) of Cur powder, PVB film, and PVB–Cur film were 255°C, 290°C, and 310°C, respectively. Their maximum decomposition rates (T_max) were recorded at 370°C, 376°C, and 396°C, respectively. The T_10% (T_10% is the temperature of degradation at 10 wt% loss) values of PVB film and PVB–Cur film were 327°C and 344°C, respectively. The PVB–Cur film showed higher T_10%, and T_onset and T_max values relative to the neat PVB film. This indicated that the degradation was shifted to a higher temperature, and the thermal stability of the composite film was improved by adding Cur to the PVB polymer matrix. The improved thermal stability observed for the PVB–Cur films might be attributed to the good dispersion of Cur in the polymer matrix. Meanwhile, these results indicated the potential of the PVB–Cur films applied at high temperatures.

Figure 4

Thermal stability of PVB, Cur, and PVB–Cur films.

3.4 Mechanical property

Figure 5 shows the tensile stress–strain curves of the PVB and PVB–Cur films. The tensile strength of pure PVB film and PVB–Cur film was 6 and 7.5 MPa, and the elongation at break was 207% and 222%, respectively. The results demonstrated that the incorporation of Cur did not impair the mechanical properties of the films. The good mechanical properties of PVB–Cur composite films could be explained by a good interfacial interaction between Cur and PVB (20). It can be observed in Figure 5 that both PVB and PVB–Cur films exhibited yield behavior during tensile testing. This indicated that the film was not brittle, but an elastic plastic material. The stress–strain curves of PVB–Cur films exhibited elastic deformation regions with high modulus at the initial stage. The yield points were observed at approximately 3.5% strain. Subsequently, the strain-hardening region appeared in the stress–strain curve. At this stage, the enhancement in stress was accompanied by further elongation of the film. The excellent mechanical properties made the resulting films suitable candidates for food packaging (36).

Figure 5

Strain–stress curves of PVB and PVB–Cur composite films.

3.5 Wettability property

The wettability of the film surfaces was investigated by measuring the water contact angle. Generally, when the angle between the water droplet and the surface of the film is less than 90° the surface of the film is judged to be hydrophilic, whereas the surface exceeding 90° is hydrophobic. Figure 6 shows the contact angle of the PVB film with and without Cur. Contact angle values at different times (0, 30, 60, 90, 120 s, respectively) were measured to analyze the time dependence. The starting contact angle of the PVB–Cur film was slightly lower than that of the PVB film. The starting contact angles of the PVB film and the PVB–Cur film were 78.1 ± 0.5° and 75.8 ± 0.6°, respectively. Therefore, both the PVB film and the PVB–Cur film were hydrophilic. Typically, the surface of the wound dressing with water contact angle of 60° to 80° could facilitate the adhesion and proliferation of cells at the wound (37). In addition, the contact angles of the PVB film and the PVB–Cur film were not constant, and there was a slight decrease of about 3° during the contact time of 0–60 s. After 60 s, the contact angle basically reached equilibrium. The incorporation of Cur slightly enhanced the hydrophilicity of the film, and the contact angle of the composite film could be maintained within the range that was favorable for wound healing.

Figure 6

(a) Contact angle pictures and (b and c) contact angle values of water droplets on the surface of PVB and PVB–Cur films at different times.

3.6 Antibacterial activity

Bacterial proliferation is a key factor in causing food spoilage and wound infections. Therefore, the antibacterial property is very important for the application of film materials. Since S. aureus and E. coli are two common infectious species of bacteria in the natural environment, the activities of PVB–Cur film against these species were evaluated in vitro. The CFU quantification method was used to evaluate the antibacterial behavior of the materials. As shown in Figure 7, the PVB film did not exhibit noticeable antibacterial activity, compared to the Control group (p > 0.05). PVB–Cur film and Cur groups both possessed the ability to inhibit the growth of S. aureus and E. coli bacteria. The quantitative result of the colony count of S. aureus in the PVB–Cur film was 0.61 ± 0.17 × 10⁸, which was not different from that of the Cur group (0.45 ± 0.09 × 10⁸), but significantly lower than those of the Control group and the PVB film group (***p < 0.001, one-way ANOVA with statistical software SPSS 14.0 (SPSS Inc., Chicago, USA)). The quantitative result of the colony count of E. coli in the PVB–Cur film was 1.65 ± 0.22 × 10⁸, which was also significantly lower than those of the Control group and the PVB film group (***p < 0.001). Therefore, the PVB–Cur film has robust antibacterial properties against both S. aureus and E. coli.

Figure 7

Antibacterial assays of S. aureus and E. coli. CFU photographs of S. aureus (a) and quantitative results (c). CFU photographs of E. coli. (b) and quantitative results (d).

So far, the antibacterial mechanism of Cur has not been fully understood. Some studies had shown that the antibacterial effect of Cur was related to FtsZ, which was a cytoskeletal protein that plays an important role in the process of prokaryotic cell division. Here, Cur inhibited the growth of E. coli mainly by inhibiting the assembled FtsZ. In addition, Cur could damage bacterial membranes and improve their permeability. Cur is believed to have antibacterial properties by anchoring the cell wall of the bacterial cell, causing it to rupture and then infiltrating into the cell, destroying the structure of the organelle (38,39,40).