The reference strain C. albicans SC 5314 was used in this study. After being streaked on a Sabouraud dextrose agar (SDA, Liofichelm) plate, from a frozen stock solution, this strain was grown for 24 h at 37°C. For each assay, some colonies were collected from the SDA plates and grown overnight in batches of Sabouraud dextrose broth (SDB, Liofichelm) at 37°C and 120 rpm. Cells were then harvested by centrifugation (3,000 g, 10 min, 4°C) and washed twice with phosphate buffered saline (PBS, pH = 7.5). Cellular density was then adjusted using a Neubauer counting chamber.

Amphotericin deoxycholate (AmBdeox) was purchased from Sigma (Sigma-Aldrich, USA) and Liposomal Amphotericin B (LAmB) was supplied by Gilead^® (Foster City, CA, USA). Aliquots of AmBdeox were prepared using DMSO according to the indications of the manufacturer while the aliquots of LAmB were prepared in 10 mM bicine buffer.

PDMS (kit Sylgard 184, Dow Corning, USA) was prepared as recommended by the manufacturer's instructions. In summary, base and curing agents provided in the kit were mixed in 10:1 (w/w), casted in a petri dish and kept at room temperature for 48 h. Afterwards, PDMS was cut into circle pieces with 0.9 cm diameter and a thickness of ~0.3 cm. Before utilization, surfaces were subjected to an ultrasonic cleaning treatment to remove impurities and grease, in a commercial detergent (Sonasol, Henkel Ibérica, Portugal) for 5 min followed by methanol for 20 min and finally rinsed with distilled water. Cleaned coupons were sterilized at 121°C for 15 min, in autoclave.

Liposomal amphotericin B immobilization was performed using a two-step mussel-inspired coating strategy previously reported (Lee et al., 2007) and it is schematically presented in Figure 1. The first step comprises the formation of a pDA layer which was accomplished by immersing the coupons in 7 mL of a freshly prepared solution of dopamine (Sigma, 2 mg/mL) in 10 mM bicine buffer (Sigma, pH 8.5) for 18 h at room temperature under agitation (70 rpm). Surfaces were then rinsed with ultrapure water. For LAmB immobilization, pDA coated coupons were immersed on a solution of LAmB prepared at different concentrations (0.5, 1, and 2 mg/mL) in bicine buffer, and they were incubated overnight at room temperature, under agitation (70 rpm). Functionalized surfaces were washed with ultrapure water and dried prior utilization.

The contact-killing properties of PDMS functionalized with LAmB were evaluated as previously reported (Alves and Pereira, 2016). Briefly, a yeast suspension was adjusted in SDB to a final concentration of approximately 10⁶ colony-forming units (CFU) CFU/mL, and 20 μL of this solution was added on top of PDMS coupons placed in a petri dish. The plate was afterwards sealed and incubated at 37°C under static conditions for 24 h. After contact, coupons were placed on a SDA plate, with the face exposed to the yeast suspension, in contact with the agar, and they were incubated for additional 24 h at 37°C. Yeast growth was evaluated for the conditions tested and tabulated as “+” for growth and “–” for no visible growth.

To evaluate the possible leaching of LAmB from the PDMS modified surfaces, an analytical method previously reported was adapted (Asri et al., 2014). Surfaces were placed on top of an agar plate previously streaked with C. albicans adjusted to a concentration of approximately 10⁸ CFU/mL. Plates were then incubated for 72 h at 37°C and the presence or absence of an inhibition zone was evaluated. The inhibition zone as an indication of LAmB leaching from the surfaces. Three independent assays with three replicates for each condition were performed.

The performance of modified surfaces against yeast adhesion was evaluated by preparing a suspension with approximately 10⁸ CFU/mL in artificial urine (Brooks and Keevil, 1997) to better mimic the environment of CAUTI. PDMS coupons, before and after modifications were placed on 48-well microtiter plates (Orange Scientific, USA), which were covered with 300 μL of yeast suspension and plates were incubated for 4 h, at 37°C and 120 rpm. Samples were then washed with saline solution (0.9% NaCl), stained with a live/dead stain (BacLight Bacterial Viability Kit, Invitrogen) and observed in a fluorescent inverted microscope (Olympus, BX51). The staining solution is composed by propidium iodide and the SYTO 9, two nucleic acid staining agents, which allow the differentiation between viable and non-viable cells, evaluating membrane's integrity. Propidium iodide is a red-fluorescent nuclei acid staining agent which only penetrates damaged cell membranes while SYTO 9 is a green-fluorescent nucleic acid staining agent, which can penetrate cells both with intact and damaged membranes. At least three images per coupon were collected and at least three coupons were inspected per assay.

The potential of the modified surfaces to impair biofilm formation, was investigated by enumerating the number of yeast cells adhered to the surfaces. A yeast suspension was first prepared from an overnight culture and adjusted to a final concentration of approximately 10⁸ CFU/mL in artificial urine. The coupons were placed in a 48-well microtiter plates and 300 μL of this suspension was added to each well. Plates were incubated at 37°C, under agitation (120 rpm) and cells were allowed to attach to the surfaces for 1 h. Coupons were then washed twice with saline solution and placed in new wells of a microtiter plate and 300 μL of artificial urine were then added to each well. Plates were then incubated for more 23 h under the same conditions. After this period, coupons were washed again with saline solution to remove free-floating yeast, transferred to new wells and 300 μL of PBS were finally added to each well and the surfaces were scraped. Solutions resulting from the scraping were collected, vortexed to disrupt possible cell aggregates. Serial 10-fold dilutions were performed and plated into SDA plates which were incubated overnight at 37°C in an aerobic incubator prior enumeration. To infer about pDA role on LAmB immobilization, the sample PMDS-LAmB was also evaluated in this assay. This one was prepared by simple immersion of PDMS coupons on a solution of LAmB (2 mg/mL) using the same conditions of pDA-LAmB (overnight at 70 rpm and room temperature).

Cytotoxicity evaluation was performed according to the ISO 10993-5:2006, using fibroblast cells 3T3 (CCL 163) obtained from ATCC, commonly used for biomaterial surface biocompatibility studies (Lee et al., 2004a; Zhu et al., 2006). Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% of fetal bovine serum and 1% penicillin/streptomycin was used to culture these cells at 37°C, 5% CO₂. Once achieved the confluence, trypsin was used to detach cells and a cell suspension was prepared with 10⁵ cells/mL, which was then added to each well of a 48-well microtiter plate, in which the PDMS surfaces were previously inserted. Plates were incubated at 37°C, 5% CO2 for 48 h and after that period of time, cytotoxicity was evaluated by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) inner salt reduction assay as a measure of cellular metabolic viability. For that, medium was removed and a solution containing 100 μL of MTS (Promega CellTiter 96^® AQueous Non-Radioactive Cell Proliferation Assay) per each 1 mL of DMEM without phenol red was added to each well. After 1 h of incubation in the dark, at 37°C, 5% CO2, the absorbance of the resulting solution was red at 490 nm and results were expressed as percentage of viable cells using the metabolic activity of cells grown on pDA-coated PDMS as controls.

Results were presented as mean ± standard deviation (SD). Statistical analysis was performed by Kolmogorov-Smirnov normality test using Graph Pad Prism 7.0 for Mackintosh. Afterwards, parametric tests (one-way Anova followed by Tukey's test) or non-parametric (Kruskal-Wallis test) were implemented, depending on whether the samples were from normally distributed populations or not, respectively.

For LAmB immobilization on PDMS surfaces, a two-step bio-inspired approach was applied (Figure 1). The first step comprised the deposition of an adhesive pDA “primer” coating on PDMS surfaces from a mildly basic solution of dopamine. The thickness of this layer formed in similar conditions has been previously measured by ellipsometry and it was of approximately 50 nm (Lee et al., 2007; Zhou et al., 2014). This parameter should not be significantly changed after LAmB immobilization as it has been demonstrated for other compounds, such as antimicrobial peptides (Cui et al., 2014). The presence of quinone functional groups in pDA coating allows further covalent immobilization of nucleophilic biomolecules via Michael addition and/or Schiff base reactions (Liebscher et al., 2013). Free Amphotericin B contains primary amines, so it was hypothesized that if some AmB was released from the liposomes during functionalization of pDA-coated surface, this one could be grafted onto the surfaces through Michael addition and/or Schiff base reactions. As far as liposomes as concerned, since they do not present any amine or thiol groups in their composition, it was intended to promote its adsorption, taking advantage of the increased roughness provided by pDA-based coatings previously reported (Alves et al., 2016). It has been shown that low roughness promotes liposome rupture, but high roughness induces adsorption of whole liposomes (Duarte et al., 2015). Different concentrations of LAmB were tested in this second step. Since it was intended to provide PDMS with the ability to contact-kill fungal cells but without active AmB release, to avoid cytotoxic issues, optimization parameters in this phase included the following factors: contact-killing and leaching activities (Table 1).

Results showed that no contact-killing was observed for bare PDMS, before or after pDA coating (Supplementary Figure 1). Further functionalization with LAmB yielded the surfaces with fungal contact-killing activity only for the higher concentration tested (2 mg/mL), as evidenced by the absence of fungal growth in these modified coupons. Regarding the release of LAmB, for all the concentrations tested, no inhibition zone was found which was an evidence of no release from the surfaces or from the liposomes. If some AmB was released from the liposomes during immobilization, these results suggest that it was covalently bounded trough their amine groups via Michael addition and/or Schiff base reactions. Additionally, for comparison purposes, free AmBdeox was also immobilized, using the higher concentration of LAmB tested, and it was possible to observe an inhibition zone surrounding the functionalized coupons (Supplementary Figure 2), an evidence of AmB leaching, which highlight the role of liposomes on protecting AmB also when they are immobilized.

The overall results from these optimization tests allowed to determine the best concentration for the immobilization of LAmB to provide the surfaces of PDMS with antifungal activity but with no LAmB release from the surfaces (2 mg/mL). This coating strategy was further evaluated.

Surface morphology after each step of functionalization was inspected by SEM analysis (Figure 2A). It was possible to observe that bare PDMS exhibits a smooth surface as compared to further modified surfaces. After pDA coating, surfaces revealed some aggregates resulting from self-polymerization of dopamine previously described (Alves et al., 2016). Further functionalization with LAmB did not introduce significant changes on this morphology. Since dopamine agglomerates are in the same range of liposomes loaded with AmB, it was not possible to discriminate them. For further surface morphology characterization, samples were also evaluated by AFM and from the images obtained, it was possible to determine the average roughness of surfaces (Figure 2B). Results corroborated that pDA coating increased the surface roughness, which may be explained by the presence of polydopamine agglomerates and it is in agreement with other studies (Xu et al., 2017). The additional layer of LAmB decreased the surface roughness as compared to pDA coating. Chemical analyses were also conducted using EDS and results are presented in Table 2. Results showed that PDMS before and after pDA coating displayed a similar chemical signature, which may be attributed to the sampling depth achieved by the EDS, which is higher than 50 nm (the maximal thickness of pDA coatings). Therefore, it is expected that PDMS is the major contributor to the chemical signature detected by EDS analysis. On the other hand, pDA-coated PDMS further functionalization with LAmB, resulted in chemical changes, namely an increase in carbon (C) content and decrease on silicon (Si) content, which is an evidence of LAmB successful coating.

The hydrophobicity parameters of surfaces were also determined using van Oss approach (Van Oss and Giese, 1995). Contact angles, surface tension parameters and free energy of interaction are summarized in Table 3. Results showed that bare PDMS presented a water contact angle higher than 65° and a negative value of free energy of interaction (ΔGiwiTOT), which reveals the hydrophobic features of this material (Vogler, 1998). Application of a pDA coating, introduced hydrophilic features to the modified surfaces, as evidenced by the positive value of free energy of interaction and a lower water contact angle of 77.8°. This surface modification is attributed to dopamine polymerization that introduces new hydrophilic functional groups, especially the catechol and amine groups, on the PDMS surface. Further functionalization with LAmB increased the value of free energy of interaction which was attributed to the successful decoration with liposomes on the pDA-coated PDMS surfaces.

After confirming that LAmB retained its activity after immobilization, its antifungal performance was further evaluated. To inspect the ability of modified surfaces to interfere with C. albicans adhesion and/or viability, fungal cells were allowed to adhere for 4 h and the remaining cells on the PDMS surfaces were imaged with fluorescence microscopy (Figure 3). It has been established that the first 6 h are the critical time for preventing pathogens adhesion to the surfaces of biomaterial implants (Poelstra et al., 2002). It was possible to observe that fungal cells were able to adhere to both unmodified PMDS and pDA-coated surfaces without compromising their viability. Further immobilization with LAmB, on the other hand, provided PDMS surfaces with the ability to prevent fungal adhesion and promote cellular membrane disintegration, as evidenced by the lower number of attached cells and their red fluorescence color. The decreased number of adhered cells found on these surfaces is an evidence of their anti-adhesive features which may be attributed to the increased hydrophilicity and lower roughness, as compared to pDA coating alone (Figure 2 and Table 3). Contact-killing features observed may be attributed to AmB released from the liposomes during immobilization and subsequently grafted onto pDA coating via Michael addition and/or Schiff base reactions as well as to the immobilized liposomes that, upon contact with fungi will allow AmB release to target these cells membrane (Walker et al., 2018).

The antifungal activity of LAmB was also investigated for a longer period of time: fungal colonization was allowed to proceed for 1 h, being afterwards exposed to urine for additional 23 h, to better mimic the physiological conditions. The number of fungal cells adhered to surfaces was enumerated by CFU counting (Figure 4). Results showed that PDMS with and without pDA coating favored the attachment of C. albicans as demonstrated for a shorter period of time. LAmB immobilized was able to impair fungal attachment as evidenced by an approximately 3 Log CFU reduction. To better understand the role of pDA layer on LAmB functionalization, physical adsorption of LAmB was allowed to proceed onto PDMS surfaces, and their antifungal properties were evaluated. Results showed that simple adsorption of LAmB did not impart the surfaces of PDMS with antifungal features, as C. albicans was able to adhere to these surfaces at the same extent as it did to PDMS and pDA controls. Such results are an evidence of pDA role for a stable functionalization of PDMS with LAmB.

An important advantage associated to the use of LAmB, is its reduced toxicity provided by their incorporation within the liposomes (Stone et al., 2016). Toxicity of LAmB, after functionalization, on mammalian cells was then evaluated. Results presented in Figure 5 showed that surfaces functionalized with LAmB did not compromise 3T3 fibroblast metabolic activity, as compared to pDA coating, which is an evidence of no toxicity. This ladder evidence also suggests no release of active AmB, which may be attributed to its entrapment on liposomes or its covalently attachment to the surface.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.