Introduction
Many areas of our daily life need the control over the microbial contamination, as in food packaging and biomedical devices, to prevent severe threats for public health and safety [
1]. High volatility and leaching phenomena affect the traditional low molecular weight disinfectants; therefore antibacterial contact polymers are recently adopted in medical apparatuses, food packaging applications, and water pipe systems by virtue of their long-term stability and absence of toxic residues.
Macrocycle-based antibacterial materials display an effective activity against Gram-negative, Gram-positive, and drug-resistant bacteria [
2]. Their action is capable to complement traditional drugs, facilitating the development of modern medicine, and overcoming the antibiotic-resistance emergency. Calix[n]arenes, cyclic compounds with permanent porosity, are macrocycles with hydrophobic cavity interiors and the possibility to functionalize the upper and lower rims, resulting in customized formulations [
3]. Small molecules can be complexed within these macrocycles. More fascinating are the self-assembly properties of calixarenes that enable the production of functional nanomaterials, opening new opportunities for drug delivery of guest biomolecules [
4,
5]. Indeed, aggregated nanostructures can be obtained by manipulating calixarene units and exploited as carriers for bio relevant molecules, due to water solubility, low cytotoxicity, and good biocompatibility [
6]. Numerous pharmacological properties of calixarenes have also been described, including antibacterial, antifungal, antiviral [
7,
8] and anticancer activity [
9]. The introduction and spatial orientation of multiple cationic groups on the calix[4]arene scaffold has provided a variety of derivatives with effective antibacterial activity [
10].
Among the cationic calix[4]arene derivatives, an amphiphilic derivative (Chol-Calix), bearing choline groups and dodecyl aliphatic chains at the upper and lower rim of a calixarene platform, self-assembles in micellar nanoaggregates with demonstrated properties as gene [
11] and drug delivery system [
12,
13]. In the search for novel antibacterial agents, we previously demonstrated the potential of Chol-Calix as a nanocarrier for application in antimicrobial photodynamic and photo-induced therapy. The entrapment of a photosensitizer (porphyrin or phthalocyanine derivatives)[
14] and/or a nitric oxide photo-donor (
N-dodecyl-3-(trifluoromethyl)-4-nitrobenzenamine) [
15] in the Chol-Calix micelles resulted in a rapid and effective light-induced antibacterial activity against
Staphylococcus aureus and
Pseudomonas aeruginosa. Recently, we demonstrated that the micellar Chol-Calix is also a nanocontainer for conventional antibiotics (ofloxacin, tetracycline, and chloramphenicol) [
16] and that it possesses intrinsic antibacterial properties. Indeed, Chol-Calix showed MIC values in the range of 9.4–18.8 µg/mL and inhibited biofilm and mobility of
P. aeruginosa and
Escherichia coli strains [
17].
Polymeric Thin-Films represent a versatile approach for controlled and localized drug release [
18]. Embedding in Thin-Films could be a strategy to expand the range of applications of Chol-Calix, including the achievement of a novel material with antibacterial surface and medical devices enforced with antibacterial activity.
Swell-encapsulation or covalent immobilization are recognized methods to provide successful antimicrobial surfaces [
19]. However, solution blending of nanocarriers and selected polymers is low-cost and less time-consuming procedure with respect to polymer grafting that could require multiple surface modification steps. The preparation of gel membranes incorporating Ionic Liquids within a polymeric matrix was successfully carried out using Polyether block amides, commercially available as Pebax
® [
20,
21]. Pebax are plasticizer-free Thermoplastic Elastomers (TPE) [
22]. They are medically relevant polymers commonly adopted to fabricate catheters and medical tubings due to their good kink and chemical resistance. They have been explored for a range of biomedical applications [
23] as in the production of breathable films [
24], or antimicrobial surfaces incorporating photosensitizers [
25]. Being rubbery, Pebax can provide flexible films without requiring harmful plasticizers as typically done with polyvinyl chloride (PVC) which is widely used for medical devices [
26,
27].
In this work, we encapsulated Chol-Calix within a polymer matrix in order to produce novel flexible antibacterial materials. For immobilizing Chol-Calix into a Pebax
® copolymer, we adopted the solvent-casting method. A polymeric solution was obtained by using a solvent capable to dissolve both the polymer and the additive. Owing to the Chol-Calix solubility in alcohols, the hydrophobic Pebax
® 2533 grade that can be dissolved in different alcohols was selected [
28]. In the solvent solution, the polymer chains are extended, allowing the encapsulation of the complexes, while after the solvent evaporation the polymer entanglements trap the Chol-Calix.
The antibacterial activity of the prepared films was assessed against specimens of Gram negative (E. coli) and Gram positive (S. aureus) bacteria that represent clinically important strains with an active role in skin wound and nosocomial infections. Wettability, permeation to gases, thermal properties, and Chol-Calix release were evaluated on the filled films for complete characterization. Finally, mouse embryonic fibroblasts NIH/3T3 were exposed to Pebax® Chol-Calix blend films to exclude the potential toxicity of the Chol-Calix released.
Characterization
Functional groups of the prepared films were investigated by Attenuated Total Reflection (ATR) FTIR analysis (Spectrum One, Perkin Elmer). The spectra were recorded in the region from 4000 to 650 cm−1, with a resolution of 4 cm−1 (16 scans).
Thermal analysis
Calorimetric measurements of neat polymer and Chol-Calix blend films were conducted using a differential scanning calorimeter (DSC, TA Instruments Q100), equipped with a liquid sub ambient accessory. High purity standards (indium and cyclohexane) were used for calibration. All DSC runs were carried out at a rate of 10 °C/min from − 90 to 250 °C using nitrogen as purge gas. Sample weight was in the range 4–6 mg. Heating and cooling scans were performed after an initial equilibration to − 90 °C. To erase the previous thermal history, a first heating scan from − 90 °C to 250 °C was made. Then a cooling run (from 250 °C to − 90 °C) and a second heating one (from − 90 °C to 250 °C) were executed.
Thermogravimetric analysis (TGA) was carried out using a thermogravimetric apparatus (TGA, TA Instruments Q500) under a nitrogen atmosphere at 10 °C/min heating rate, from 40 to 600 °C. Dried samples of about 4–6 mg were put into a platinum pan. TGA data and their derivative (DTG) ones were recorded as a function of temperature.
Gas permeation tests
Permeation properties of the prepared films were measured at 25 °C. Circular samples with an effective area of 11.3 cm2 were used. Their thickness was measured using a digital micrometer (IP65, Mitutoyo), considering the average of multiple point measurements.
The testing apparatus is a constant-volume/variable-pressure device described elsewhere [
28]. Before each test, the films were thoroughly evacuated using a turbomolecular pump to remove eventually dissolved species (e.g., residual solvent, humidity, previously tested gases). The data, obtained measuring the increasing gas pressure signal in the permeate side
vs. time, were elaborated according to the time-lag method, evaluating the permeability (
P) as well as the diffusion coefficient (
D) of each gas through the membrane [
31] considering the “solution-diffusion” model that describes the transport in dense polymeric films [
32].
Scanning electron microscopy (SEM)
Samples for SEM analysis were prepared by sputter-coating with a thin film of gold. Gold sputtering thickness was about 5 nm. Sample images were acquired on a JEOL (JXA-8230 SuperProbe Electron Probe Microanalyzer) SEM operated at 20 kV.
Static water contact angle (WCA) measurements were determined using a DATAPHYSICS OCA 15EC apparatus and water as liquid. Specifically, a drop (2 μL) of deionized water was deposited on the sample surface and the measurement was made after its stabilization (~ 10 s). Three measurements on different areas of each sample were executed. WCA results are average values (± 1–3° standard deviation) of triplicate samples.
Release of Chol-Calix from the blend films
The release kinetics of Chol-Calix from the films (0.5, 1, 5 wt%) was determined by immersing a rectangular sample (5 mg, thickness 80 µm, size 7 × 10 mm) in 3 mL of PBS. The samples with and without Chol-Calix were kept at 37 °C; then, at specific interval times, the optical absorption at 210 nm was measured. By subtracting the absorption of the neat sample, the absorbances of the Pebax
®/Chol-Calix samples were converted to the amount of Chol-Calix released, based on a calibration curve. The absorption spectra were recorded on a Jasco V-770 spectrophotometer. The release data were fitted (coefficient of determination,
R2 = 0.999) by the following exponential function:
$$R\left( \% \right) = R\left( \% \right)_{{{\text{max}}}} \cdot \left( {1 - e^{ - kt} } \right)$$
where
R(%) is the release percentage at time
t,
R(%)
max is the maximum release percentage and
k the first order kinetic constant. Indeed, first-order equations well describe the dissolution of water-soluble drug from porous matrices [
33]. The release experiments were performed in triplicate.
To simulate the conditions of the antibacterial assays, the release of Chol-Calix from the Pebax® blend films was also investigated on larger samples (2 cm diameter, 32 mg, 80 µm) immersed in 1 mL of PBS for 24 h.
Antibacterial activity and biofilm biomass measurement
Test organisms used in this study were E. coli ATCC 10536 and S. aureus ATCC 6538. For the antibacterial tests, the cultures were grown overnight on LB and washed in phosphate buffered saline (PBS, pH 7.2) by centrifugation at 3500 rpm for 15 min and adjusted to a concentration of 5 × 105–1 × 106 CFU/mL, approximatively. The standardized cultures (1 mL) were dispensed into each well of a 12-well cell culture treated polystyrene microtiter plate. The different polymeric films (round samples, 2 cm in diameter, 32 mg, 80 µm), neat (control) or with Chol-Calix at various concentrations (0.5, 1, 5 wt%) were then placed in each well of the microtiter plate. After incubation at 37 °C for different time intervals (2, 4, 6, 8, 10 and 24 h), the planktonic phase was serially diluted in PBS, plated onto TSA and incubated for 24–48 h at 37 °C in order to evaluate the number of CFU/mL. All the determinations were performed in triplicate including the growth controls.
Moreover, after 24 h-incubation the biofilm formed on the polymeric films with Chol-Calix at 5 wt% was evaluated by biomass measurement. The polymeric films were washed twice with PBS, dried, stained for 1 min with 0.1% safranin, and then washed with water as previously reported [
34]. The stained biofilms were suspended in 30% acetic acid aqueous solution and the mean optical density at 492 nm (OD
492) was measured using a spectrophotometer EIA reader (Bio-Rad Model 2550, Richmond, CA, USA). The reduction percentage of biofilm was calculated using the following equation:
$${\text{Biofilm Reduction }}\left( \% \right) = 100 - \frac{{{\text{OD}}_{492} \;{\text{Pebax}}^{\textregistered } / {\text{CholCalix}}}}{{{\text{OD}}_{492} \;{\text{neat Pebax}}^{\textregistered } }} \times 100$$
The films were sterilized under a UV lamp (256 nm) for 5 min before each assay.
Cell viability
Cell culture
The mouse embryonic fibroblasts NIH/3T3, purchased from ATCC cells bank (CRL-1658), were maintained in DMEM-F12 (Gibco, Thermofisher) supplemented with 10% heat-inactivated (HI) fetal bovine serum (Gibco, Thermofisher), 100 mg/mL penicillin, and streptomycin (Gibco, Thermofisher), and 2 mM l-glutamine at 37 °C, 5% CO2. NIH/3T3 cells for the performed experiments were used at passage 7–9. One day before experiments, 25 × 103 cells were plated on 96 multi-well plates in DMEM-F12 with 5% fetal calf serum (FCS).
Before treatments, cells were washed with phosphate-buffered saline (PBS) and the medium was replaced with fresh DMEM F-12 with 5% FCS.
Cytotoxicity test
To test the potential cytotoxicity of the Pebax®/Chol-Calix blend films, we studied the effect of the Chol-Calix released from differently loaded films according to the MTT assay. N = 9 round samples (3 mm in diameter, 3 mg in weight) of neat Pebax® and its films containing 0.5, 1, and 5% of Chol-Calix, were firstly kept for 5 min under UV lamp to sterilize them, thus avoiding cellular contamination. Then they were incubated in 100 μL of 5% FBS DMEM/ F12 for 4 h at 37 °C in separated wells of a 96well-microplate, the fibroblasts were washed with PBS and the medium was replaced with those containing the released compounds (100 μL). After 48 h of treatments, we added 20 μL of MTT (0.5 mg/mL) to each well for 2 h at 37 °C, then the medium was discarded and the water-insoluble formazan crystals were dissolved in DMSO. The formazan production due to the conversion of MTT by living cells, was evaluated in a microplate reader (Varioskan® Flash Spectral Scanning Multimode Readers, Thermo Scientific, Waltham, MA, USA) by reading the absorbance at 570 nm.
Conclusions
Chol-Calix, a nanoconstruct obtained by the self-assembly of multiple units of an amphiphilic calix[4]arene derivative bearing choline moieties and dodecyl chains, was synthesized and incorporated in an elastomeric polyether block amide. Flexible free-standing films based on Pebax®2533 loaded with the Chol-Calix nanoconstructs at 0.5, 1 and 5 wt% were prepared by solution casting, utilizing a non-toxic solvent (ethanol), without plasticizers.
The thermal stability of the copolymer matrix is preserved in the blends, while the crystallinity of the copolymer blocks is increased. The presence of the additive can be observed on the film surface, according to its concentration in the matrix. The wettability of the films increases upon the Chol-Calix loading in the blend membranes. Low content of Chol-Calix slightly reduces the gas permeability through the films, whereas an increase in this parameter is observed as Chol-Calix amount rises, due to the additive aggregation tendency.
Low leaching of Chol-Calix from the film surface was confirmed through release tests carried out in PBS medium. The blend films displayed antimicrobial activity within 10 h against Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria that represent clinically important strains. In addition, the Chol-Calix was capable to interfere with the biofilm formation since the films loaded with the higher concentration of Chol-Calix demonstrated a one-third reduction in biofilm formation on their surface. Furthermore, the blend films did not exert cytotoxicity on mouse embryonic fibroblasts NIH/3T3.
The obtained results suggest the Pebax2533/Chol-Calix combination as a promising approach for the development of novel flexible antibacterial Thin-films that could be upgraded by loading antibacterial drugs in the Chol-Calix nanocarrier.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.