Journal of Fungi

Article Novel Colloidal Nanocarrier of Cetylpyridinium Chloride: Activities on Candida Species and Cytotoxic Potential on Murine Fibroblasts

Heitor Ceolin Araujo 1, Laís Salomão Arias 1, Anne Caroline Morais Caldeirão 2, Lanay Caroline de Freitas Assumpção 3, Marcela Grigoletto Morceli 3, Francisco Nunes de Souza Neto 1, Emerson Rodrigues de Camargo 4, Sandra Helena Penha Oliveira 5, Juliano Pelim Pessan 1 and Douglas Roberto Monteiro 2,3,*

1 Department of Preventive and Restorative , School of Dentistry, Araçatuba, São Paulo State University (UNESP), Araçatuba SP 16015-050, Brazil; [email protected] (H.C.A.); [email protected] (L.S.A.); [email protected] (F.N.d.S.N.); [email protected] (J.P.P.) 2 Graduate Program in Dentistry (GPD—Master’s Degree), University of Western São Paulo (UNOESTE), Presidente Prudente SP 19050-920, Brazil; [email protected] 3 School of Dentistry, Presidente Prudente, University of Western São Paulo (UNOESTE), Presidente Prudente SP 19050-920, Brazil; [email protected] (L.C.d.F.A.); [email protected] (M.G.M.) 4 Department of Chemistry, Federal University of São Carlos (UFSCar), São Carlos SP 13565-905, Brazil; [email protected] 5 Department of Basic Sciences, School of Dentistry, São Paulo State University (UNESP), Araçatuba SP 16015-050, Brazil; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +55-18-3229-1000

 Received: 4 September 2020; Accepted: 9 October 2020; Published: 12 October 2020 

Abstract: Nanocarriers have been used as alternative tools to overcome the resistance of Candida species to conventional treatments. This study prepared a nanocarrier of cetylpyridinium chloride (CPC) using oxide nanoparticles (IONPs) conjugated with chitosan (CS), and assessed its antifungal and cytotoxic effects. CPC was immobilized on CS-coated IONPs, and the nanocarrier was physico-chemically characterized. Antifungal effects were determined on planktonic cells of Candida albicans and Candida glabrata (by minimum inhibitory concentration (MIC) assays) and on single- and dual-species biofilms of these strains (by quantification of cultivable cells, total biomass and metabolic activity). Murine fibroblasts were exposed to different concentrations of the nanocarrier, and the cytotoxic effect was evaluated by MTT reduction assay. Characterization methods confirmed the presence of a nanocarrier smaller than 313 nm. IONPs-CS-CPC and free CPC showed the same MIC 1 1 values (0.78 µg mL− ). CPC-containing nanocarrier at 78 µg mL− significantly reduced the number of cultivable cells for all biofilms, surpassing the effect promoted by free CPC. For total biomass, metabolic activity, and cytotoxic effects, the nanocarrier and free CPC produced statistically similar outcomes. In conclusion, the IONPs-CS-CPC nanocarrier was more effective than CPC in reducing the cultivable cells of Candida biofilms without increasing the cytotoxic effects of CPC, and may be a useful tool for the treatment of oral fungal .

Keywords: antifungal agents; biofilms; Candida albicans; Candida glabrata; cetylpyridinium chloride; chitosan; iron oxide nanoparticles

1. Introduction Candida albicans is a commensal microorganism that may be isolated from oral, vaginal and gastrointestinal microbiomes in most healthy individuals [1]. In cases of unbalanced immune system,

J. Fungi 2020, 6, 218; doi:10.3390/jof6040218 www.mdpi.com/journal/jof J. Fungi 2020, 6, 218 2 of 14 however, this fungus may act opportunistically forming biofilms and leading to oral or systemic infections associated with high rates of morbidity and mortality [2]. In oral fungal infections such as denture stomatitis and oropharyngeal , Candida glabrata is the second or third most prevalent Candida species, and exerts interactions of competition [3], antagonism [4] or indifference [5] with C. albicans, which may modulate the virulence factors of these species. A cooperative behavior may also be observed, since C. glabrata adheres to hyphae of C. albicans in order to facilitate its invasion into deeper tissues of the host [6,7]. Cetylpyridinium chloride (CPC) is a quaternary ammonium compound used in mouth-rinses and topical formulations, presenting antifungal activity [8–10]. Its effects have been shown to be superior to [9], also presenting high efficacy in reducing C. albicans adhesion to the oral mucosa [11]. However, the low substantivity of CPC [12] can be considered as a drawback of therapies involving this agent due to the need for frequent exposure, which may result in antimicrobial resistance by some Candida strains [13,14]. Furthermore, CPC at high concentrations produce significant reductions in the viability of human gingival fibroblasts, regardless of the treatment period (24–72 h) [15]. As for murine fibroblasts and human keratinocytes, even at low concentrations, CPC was cytotoxic after 15 and 60 min of exposure [16]. In response to this scenario, antimicrobial agents have been redesigned to make them more effective against microorganisms and less toxic to human cells. In the field of nanotechnology, nanoparticles of silver [17,18], gold [19], zinc oxide [20] and chitosan [21], applied alone or in combination with other compounds, have shown interesting results in managing Candida species. Iron oxide nanoparticles (IONPs) have also been used in several biomedical applications (e.g., drug delivery, tissue engineering, contrast agents) due to their nano-sized and magnetic properties [22,23]. Literature data show that IONPs-based nanocarriers were able to improve the physico-chemical properties of the aggregated drugs and to combat yeasts, and spores [24–29]. Moreover, in order to avoid the aggregation of IONPs and to increase their biocompatibility, coatings with natural polymers such as chitosan (CS) have been adopted in the nanocarrier engineering, forming associations known as core-shell [30,31]. Faced with the aforementioned CPC limitations, the objectives of the present study were to develop and characterize a novel nanocarrier of CPC based on a core-shell association of IONPs and CS, as well as to assess its antifungal and cytotoxic effects using Candida species and murine fibroblasts, respectively. The null hypothesis was that the antifungal and cytotoxic effects of the nanocarrier would not differ from those found for the CPC alone.

2. Materials and Methods

2.1. Experimental Design For the current study, the IONPs-CS-CPC nanocarrier was prepared by loading CPC on CS-coated IONPs. Characterization tests using transmission electron (TEM), dynamic light scattering (DLS), X-ray powder diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA) were performed in order to determine the shape and size of the particles, the type of crystalline structure, the main functional chemical groups, and the thermal behavior of the nanocarrier. Then the minimum inhibitory concentrations (MICs) of IONPs-CS-CPC against planktonic cells of C. albicans and C. glabrata were determined. In addition, the antifungal effects 1 of the CPC-containing nanocarrier at 15.6, 39 and 78 µg mL− on single- and dual-species biofilms of C. albicans and C. glabrata were evaluated by quantification of cultivable cells, biofilm biomass and metabolic activity. The cytotoxic potential of the nanocarrier on murine fibroblasts was investigated by MTT assay.

2.2. Assembly and Characterization of the IONPs-CS-CPC Nanocarrier The assembly of the IONPs-CS-CPC nanocarrier involved two distinct stages: (i) coating IONPs with CS in a simple mixing process, as previously detailed [24]; (ii) incorporation and solubilization J. Fungi 2020, 6, 218 3 of 14

1 of CPC (1000 µg) (Sigma-Aldrich, St. Louis, MO, USA) in 700 µg mL− of CS-coated IONPs under magnetic stirring for 1 h. In order to characterize the colloidal nanocarrier, TEM, DLS, XRD, FTIR and TGA were performed following protocols previously described [24].

2.3. Candida Strains and Growth Conditions Two reference strains were evaluated, both from American Type Culture Collection (ATCC): C. albicans (ATCC 10231) and C. glabrata (ATCC 90030). Stock cultures of each strain at 80 C were − ◦ reactivated on Sabouraud Dextrose Agar (SDA; Difco, Le Pont de Claix, France) and, subsequently, suspended in Sabouraud Dextrose Broth (Difco) under aerobic conditions at 37 ◦C. After incubation for 7 1 20–22 h, the inoculum of each strain was adjusted in artificial saliva [32] to 10 cells mL− , according to the protocol described by Monteiro et al. [33].

2.4. Antifungal Effect of the IONPs-CS-CPC Nanocarrier The antifungal effect of the nanocarrier was evaluated on C. albicans and C. glabrata in their planktonic states or when forming biofilms. For planktonic susceptibility, the standard broth microdilution method in 96-well plates (kasvi, São José dos Pinhais, PR, Brazil) was used to find the 1 minimum inhibitory concentration (MIC). Briefly, CPC-containing nanocarrier at 1000 µg mL− was diluted in geometric progression in the Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich) 1 culture medium, resulting in concentrations ranging from 200 to 0.39 µg mL− . Next, 100 µL of each dilution was mixed with an equal volume of the inoculum of each Candida species at 2.5–3.0 103 colony-forming units (CFUs) mL 1 in RPMI 1640. MIC values (100% inhibition of visible × − fungal growth) were determined after 48 h of incubation at 37 ◦C. MICs of CPC, CS and IONPs alone were also determined. The analysis of antifungal synergy among the nanocarrier compounds was obtained by calculating the fractional inhibitory concentration index (FICI) in checkerboard assays, according to the criteria reported in detail by Wei and Bobek [34]. For biofilm susceptibility testing, 96-well microtiter plates containing 200 µL of the inoculum 7 1 (10 cells mL− in artificial saliva) of each Candida species in single culture were incubated for 48 h at 37 C. For dual-species biofilms, 100 µL of the inoculum of each strain at 2 107 cells mL 1 (both at ◦ × − final concentration of 1 107 cells mL 1) were mixed in 96-well plates and then incubated during 48 h × − to form biofilms. For both single- and dual-species cultures, biofilm refreshment was performed after the first 24 h of incubation. After biofilm formation period (48-h), artificial saliva was removed, and the resulting biofilms were washed with phosphate buffered saline (0.1 M; pH 7.0) to remove non-adherent cells. Biofilm-containing wells were then filled with 200 µL of the colloidal nanocarrier diluted in artificial saliva to attain CPC concentrations of 15.6 (IONPs-CS-CPC15.6), 39 (IONPs-CS-CPC39) and 1 78 µg mL− (IONPs-CS-CPC78). These concentrations denote values 20-, 50- and 100-fold times 1 1 the nanocarrier MIC values. Treatments with CPC (78 µg mL− ), CS (54.6 µg mL− ) and IONPs 1 (54.6 µg mL− ) alone were also performed. As a negative control (NC), biofilms were treated with pure artificial saliva. After 24 h of treatment at 37 ◦C with the different compounds, the number of CFUs was counted using SDA and CHROMagar Candida (Difco), respectively for single- and dual-species biofilms. Total biomass and metabolic activity quantifications were also performed using the well-established colorimetric methods of crystal violet and XTT reduction, respectively, which were detailed in previous studies [35,36]. Quantitative results were represented according to the 2 2 well area (log10 CFU cm− and absorbance cm− ). All microbiological assays (MIC and biofilms) were conducted on three different occasions, each in triplicate.

2.5. Cytotoxic Effect of the IONPs-CS-CPC Nanocarrier Mouse L929 fibroblast cells from ATCC were employed for cytotoxicity testing. These cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated under ideal conditions of humidity and temperature, as reported J. Fungi 2020, 6, x FOR PEER REVIEW 4 of 14

2.5. Cytotoxic Effect of the IONPs-CS-CPC Nanocarrier Mouse L929 fibroblast cells from ATCC were employed for cytotoxicity testing. These cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Invitrogen Life Technologies, Carlsbad, CA, USA) and incubated under ideal conditions of humidity and temperature, as reported J. Fungi 2020, 6, 218 4 of 14 by Takamiya et al. [37]. Briefly, after reaching 90–100% confluence, fibroblast cells were seeded in 24- well plates at 1 × 105 cells well−1 and incubated for 24 h. Subsequently, the cell monolayer was exposed to differentby Takamiya concentrations et al. [37]. Briefly,(0.24–500 after µg reaching mL−1) of 90–100%pure CPC confluence, and IONPs-CS-C fibroblastPC cells during were 24 seeded or 48 h. in In 5 1 order24-well to assess plates the at 1cytotoxicity10 cells wellafter− eachand incubatedexposure period, for 24 h. MTT Subsequently, assay was the performed, cell monolayer as detailed was × 1 elsewhereexposed [38]. to di Thefferent results concentrations were expressed (0.24–500 as %µ gcell mL viability− ) of pure in relation CPC and to IONPs-CS-CPC the NC group during(fibroblasts 24 exposedor 48 h.to Inpure order DMEM). to assess the cytotoxicity after each exposure period, MTT assay was performed, as detailed elsewhere [38]. The results were expressed as % cell viability in relation to the NC group 2.6.(fibroblasts Statistical Analysis exposed to pure DMEM).

2.6.All Statistical biofilm data Analysis showed normal distribution (Shapiro-Wilk test) and were statistically examined by 1-way ANOVA followed by Fisher LSD’s test. In turn, cytotoxicity data showed normal and non- All biofilm data showed normal distribution (Shapiro-Wilk test) and were statistically examined by normal1-way distribution, ANOVA followed respectively by Fisher for LSD’s pure test. CPC In (res turn,ults cytotoxicity transformed data into showed square normal root) and and non-normal IONPs-CS- CPCdistribution, and were submitted respectively to for 2-way pure ANOVA CPC (results followed transformed by Tukey’s into squaretest. SigmaPlot root) and software IONPs-CS-CPC (version 12.0;and Systat were Software submitted Inc., to 2-waySan Jose, ANOVA USA) followed was used by in Tukey’s data analysis, test. SigmaPlot adopting software a significance (version level 12.0; of 5%.Systat Software Inc., San Jose, CA, USA) was used in data analysis, adopting a significance level of 5%.

3. Results3. Results

3.1.3.1. Characterization Characterization of ofthe the IONPs-CS-CPC IONPs-CS-CPC Nanocarrier Nanocarrier TheThe characterizations characterizations of of the the colloidal colloidal suspension suspension of of pure IONPs andand thethe IONPs-CS IONPs-CS compound compound have been previously reported [24]. According to Figure1a, a predominantly spherical shape was have been previously reported [24]. According to Figure 1a, a predominantly spherical shape was found for IONPs and CPC in the nanocarrier, which showed a diameter lower than 50 nm. It was also found for IONPs and CPC in the nanocarrier, which showed a diameter lower than 50 nm. It was also possible to visualize the IONP core coated with CS, as well as CPC particles attached to CS, forming possible to visualize the IONP core coated with CS, as well as CPC particles attached to CS, forming a core-shell nanocarrier (Figure1a,b). By using DLS, however, the average hydrodynamic diameter of a core-shell nanocarrier (Figure 1a,b). By using DLS, however, the average hydrodynamic diameter the nanocarrier was 313 46 nm (Figure1c). Chlorine (Cl; Figure1e), iron (Fe; Figure1f) and oxygen ± of the(O; nanocarrier Figure1g) atoms was were313 ± identified 46 nm (Figure by energy-dispersive 1c). Chlorine (Cl; spectroscopy Figure 1e), (EDS) iron (Fe; in the Figure nanocarrier 1f) and image oxygen (O;represented Figure 1g) inatoms Figure were1d, confirming identifiedthe by presenceenergy-di ofspersive CPC, IONPs spectroscopy and CS in the(EDS) sample. in the nanocarrier image represented in Figure 1d, confirming the presence of CPC, IONPs and CS in the sample.

FigureFigure 1. 1. CharacterizationCharacterization of of the ironthe oxideiron nanoparticles-chitosan-cetylpyridiniumoxide nanoparticles-chitosan-cetylpyridinium chloride nanocarrier. chloride nanocarrier.Transmission Transmission electron microscopy electron images microscopy (a,b,d) and images size distribution (a,b,d) and histogram size distribution obtained by histogram dynamic obtainedlight scattering by dynamic (c) for light the nanocarrier.scattering (c Image) for the “b” nanocarrier. represents an Image increase “b” in therepresents area highlighted an increase with in the the areared highlighted arrow in image with “a”, the where red itarrow is possible in image to clearly “a”, observewhere theit is cetylpyridinium possible to clearly chloride observe particles the adhered to the core of iron oxide nanoparticles coated with chitosan. The orange area in image (d) was analyzed by X-ray energy-dispersive spectroscopy and allowed the identification of chlorine (e), iron (f) and oxygen (g) atoms. J. Fungi 2020, 6, x FOR PEER REVIEW 5 of 14 J. Fungi 2020, 6, x FOR PEER REVIEW 5 of 14

cetylpyridinium chloridecetylpyridinium particles adheredchloride toparticles the core adhered of iron tooxide the corenanoparticles of iron oxide coated nanoparticles with coated with chitosan. The orange area in image (d) was analyzed by X-ray energy-dispersive spectroscopy and chitosan. TheJ. Fungiorange2020 area, 6, 218in image (d) was analyzed by X-ray energy-dispersive spectroscopy and 5 of 14 allowed the identificationallowed of thechlorine identification (e), iron (off) andchlorine oxygen (e), (irong) atoms. (f) and oxygen (g) atoms.

The XRD resultsThe Thefound XRD XRD resultsfor resultsIONPs-CS-CPC found found for IONPs-CS-CPC for were IONPs-CS-CPC similar were to similar thosewere to seensimilar those for seen toa forthosechlorhexidine a chlorhexidine seen for a chlorhexidine nanocarrier nanocarrier developeddevelopednanocarrier by byour ourdeveloped group group [24]. [24by]. Inour In ge general, groupneral, the[24]. positions positionsIn general, andand the widths widths positions observed observed and for widthsfor di ffractograms observed offor diffractograms ofIONPs, diffractogramsIONPs, IONPs-CS IONPs-CS of and IONPs, and IONPs-CS-CPC IONPs-CS IONPs-CS-CPC and were were IONPs-CS equivalent, equivalent,-CPC demonstrating demonstratingwere equivalent, that that demonstrating the the assembly ofthat the the assembly of thenanocarrier nanocarrierassembly didof did notthe not structurallynanocarrier structurally affdidect affect thenot IONPs.structurally the IONPs. In addition, affect In addition, ditheff ractogramsIONPs. diffractograms In revealedaddition, the diffractograms presence of revealed the presencenanoparticlesrevealed of nanoparticles the with presence spinel-like wi ofth nanoparticles spinel-like crystalline crystalline structurewith spinel-like structure [24]. crystalline [24]. structure [24]. Absorption peaksAbsorptionAbsorption representing peaks peaks the representing mainrepresenting function the theal main groups main functional function of IONPsal groups groupsand CS of of IONPs[24] IONPs were and andalso CS CS [24 [24]] were were also also detected in thedetected FTIRdetected spectrum in thein FTIRthe of spectrumFTIR the spectrumIONPs- of theCS-CPC IONPs-CS-CPCof the nanocarrierIONPs- nanocarrierCS-CPC (Figure nanocarrier (Figure 2a). 2Furthermore,a). Furthermore,(Figure 2a). asymmetricFurthermore, 3 asymmetric and symmetrical stretching vibrations33 of C–H (ʋCsp –H) were represented by bands asymmetric andand symmetrical symmetrical stre stretchingtching vibrations vibrations of of C–H C–H (ʋCsp –H)–H) were were represented byby bands bands around 2910 and −1 around1 −29101 and 2850 cm , respectively, which are typical of the CPC methylene chain (Figure 2a) around 2910 and2850 2850 cm cm− , respectively,, respectively, which which are are typical typical of of the the CPC CPC methylene methylene chain chain (Figure (Figure2a) 2a) [ 39 ]. According to

[39]. According TGA,to [39].TGA, theAccording the total total decomposition decompositionto TGA, the oftotal theof decompositionthe CPC CPC present present in of the inthe nanocarrierthe CPC nanocarrier present occurred in occurred the atnanocarrier temperatures at occurred up to at temperatures up to 400 °C (Figure 2b). Finally, as the final mass loss of the nanocarrier at 800 °C was temperatures up400 to ◦400C (Figure °C (Figure2b). Finally,2b). Finally, as the as final the massfinal mass loss of loss the of nanocarrier the nanocarrier at 800 at◦ C800 was °C similarwas to that found similar to that foundforsimilar IONPs for IONPsto and that IONPs-CS andfound IONPs-CS for [24 IONPs], it can[24], and be it assumed IONPs-CScan be assumed that [24], the it bindingthat can thebe ofassumedbinding CPC to of IONPs-CSthat CPC the to binding was successful. of CPC to IONPs-CS was successfulIONPs-CS. was successful.

Figure 2. Characterization of the iron oxide nanoparticles-chitosan-cetylpyridinium chloride nanocarrier. Figure 2. Characterization of the iron oxide nanoparticles-chitosan-cetylpyridinium chloride Figure 2. CharacterizationFourier-transform of the infrared iron oxide spectra nanoparticles-chitosan-cetylpyridinium (a) and thermogravimetric curve (b) obtained chloride for the nanocarrier. nanocarrier. Fourier-transformnanocarrier. infrared Fourier-transform spectra (a) andinfrared thermogravimetric spectra (a) and curve thermogravimetric (b) obtained for curve the (b) obtained for the 3.2. Antifungal E ect of the IONPs-CS-CPC Nanocarrier nanocarrier. nanocarrier.ff For the two strains evaluated, CPC, either free or forming the nanocarrier, showed the same MIC 1 values (0.78 µg mL − ). Thus, the effect of the interaction among components of the nanocarrier on planktonic cells was classified as indifferent (Table1).

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3.2. Antifungal Effect of the IONPs-CS-CPC Nanocarrier For the two strains evaluated, CPC, either free or forming the nanocarrier, showed the same MIC J. Fungi 2020, 6, 218 6 of 14 values (0.78 µg mL−1). Thus, the effect of the interaction among components of the nanocarrier on planktonic cells was classified as indifferent (Table 1). 1 Table 1. Minimum inhibitory concentration (MIC, µg mL− ) values of free cetylpyridinium chloride Table(CPC) 1. Minimum or forming inhibitory the nanocarrier concentration in conjunction (MIC, with µg ironmL− oxide1) values nanoparticles of free cetylpyridinium (IONPs) and chitosan chloride (CPC)(CS), or against formingCandida the nanocarrierstrains. in conjunction with iron oxide nanoparticles (IONPs) and chitosan

(CS), against Candida strains. Forming the Nanocarrier Species Free CPC FICI Classification FormingIONPs the CS Nanocarrier CPC Species Free CPC FICI Classification C. albicans ATCC 10231 0.78IONPs 0.54 0.54CS CPC 0.78 >1.00 Indifference C. albicans ATCC 10231 0.78 0.54 0.54 0.78 >1.00 Indifference C. glabrata ATCC 90030 0.78 0.54 0.54 0.78 >1.00 Indifference C. glabrata ATCC 90030 0.78 0.54 0.54 0.78 >1.00 Indifference Note: Free iron oxide nanoparticles (IONPs) and CS resulted in minimum inhibitory concentration (MIC) values 1 Note:higher Free than iron 140 oxideµg mL nanoparticles− , as previously (IONPs) reported and [28 ].CS FICI resulted - fractional in minimum inhibitory inhibitory concentration concentration indices for (MIC)the nanocarrier.values higher than 140 µg mL−1, as previously reported [28]. FICI - fractional inhibitory concentration indices for the nanocarrier. Concerning the effect on single-species biofilms, all tested compounds significantly reduced Concerning the effect on single-species biofilms, all tested compounds significantly reduced the the number of CFUs compared to NCs, except for treatment with IONPs alone (Figure3a,b). number of CFUs compared to NCs, except for treatment with IONPs alone (Figure 3a,b). IONPs-CS- IONPs-CS-CPC78 was the most effective treatment, statistically differing from the other groups CPC78 was the most effective treatment, statistically differing from the other groups and achieving and achieving reductions of 5.51- and 4.18-log10 compared to controls, respectively for C. albicans reductionsand C. glabrata of 5.51-(Figure and 4.18-log3a,b). As10 forcompared dual-species to controls, biofilms, respectively IONPs-CS-CPC78 for C. alsoalbicans di ff eredand C. from glabrata the (Figureother 3a,b). groups As and for promoteddual-species the highestbiofilms, decreases IONPs-CS-CPC78 in CFUs compared also differed to NC, from with the values other of groups 4.71- and and promoted the highest decreases in CFUs compared to NC, with values of 4.71- and 3.67-log10, 3.67-log10, respectively for C. albicans and C. glabrata (Figure3c). respectively for C. albicans and C. glabrata (Figure 3c).

FigureFigure 3. 3.QuantificationQuantification of of biofilm biofilm cultivable cultivable cells. cells. Mean Mean values ofof thethe logarithmlogarithm of of colony-forming colony-forming 2 unitsunits per per cm cm2 forfor single single biofilms biofilms of of CandidaCandida albicans albicans ATCCATCC 10231 10231 (a) Candida glabrataglabrataATCC ATCC 90090 90090 (b (b) ) µ 1 µ 1 andand dual-species dual-species biofilms biofilms (c ()c )treated treated with with 54.6 54.6 µgg mL−−1 ironiron oxide nanoparticlesnanoparticles (IONPs), (IONPs), 54.6 54.6g µg mL mL− −1 chitosan (CS), 78 µg mL 1 cetylpiridinium chloride (CPC) and nanocarrier containing CPC at chitosan (CS), 78 µg mL−1 cetylpiridinium− chloride (CPC) and nanocarrier containing CPC at 15.6 15.6 (IONPs-CS-CPC15.6), 39 (IONPs-CS-CPC39) and 78 µg mL 1 (IONPs-CS-CPC78). Negative control (IONPs-CS-CPC15.6), 39 (IONPs-CS-CPC39) and 78 µg mL−1− (IONPs-CS-CPC78). Negative control (NC) indicates biofilms treated with pure artificial saliva (without drugs). Error bars represent standard (NC) indicates biofilms treated with pure artificial saliva (without drugs). Error bars represent deviations of the means. Different lower case letters indicate significant differences among the groups standard deviations of the means. Different lower case letters indicate significant differences among by one-way ANOVA and Fisher LSD’s test (p < 0.05). the groups by one-way ANOVA and Fisher LSD’s test (p < 0.05).

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TotalTotal biomass biomass results results showed showed that that CPC, CPC, IONPs- IONPs-CS-CPC39CS-CPC39 and and IONPs-CS-CPC78 IONPs-CS-CPC78 did did not not diff erdiffer fromfrom each each other, other, and and resulted resulted in inthe the highest highest reductions reductions comparedcompared to to NCs, NCs, both both for for single single biofilm biofilm of of C. albicansC. albicans (Figure(Figure 4a)4 a)and and dual-species dual-species biofilm biofilm (Figure4 c).4c). These These significant significant reductions reductions ranged ranged from from 42 to42 55%. to 55%. For Forsingle single biofilm biofilm of ofC.C. glabrata glabrata, ,however, however, nono significantsignificant reduction reduction in biomassin biomass was was found found after afterexposure exposure to the to thedifferent different compounds compounds (Figure (Figure4 4b).b).

FigureFigure 4. Quantification 4. Quantification of oftotal total biofilm biofilm biomass biomass by crystalcrystalviolet violet staining. staining. Mean Mean absorbance absorbance values values 2 per cmper2 for cm singlefor single biofilms biofilms of Candida of Candida albicans albicans ATCCATCC 10231 10231 (a), (a Candida), Candida glabrata glabrata ATCCATCC 90030 90030 ( (bb),), and and dual-species biofilms (c) treated with 54.6 µg mL 1 iron oxide nanoparticles (IONPs), 54.6 µg mL 1 dual-species biofilms (c) treated with 54.6 µg mL−1 −iron oxide nanoparticles (IONPs), 54.6 µg− mL−1 chitosan (CS), 78 µg mL 1 cetylpyridinium chloride (CPC) and nanocarrier containing CPC at −1 − chitosan (CS), 78 µg mL cetylpyridinium chloride (CPC) and1 nanocarrier containing CPC at 15.6 15.6 (IONPs-CS-CPC15.6), 39 (IONPs-CS-CPC39) and 78 µg mL− (IONPs-CS-CPC78). Negative control −1 (IONPs-CS-CPC15.6),(NC) indicates biofilms 39 (IONPs-CS-CPC39) treated with pure artificial and saliva 78 µg (without mL (IONPs-CS-CPC78). drugs). Error bars represent Negative standard control (NC)deviations indicates of biofilms the means. treated Different with lower pure case artifici lettersal indicate saliva significant (without di drugs).fferences Error among bars the groupsrepresent standardby one-way deviations ANOVA of the and means. Fisher LSD’sDifferent test (lowerp < 0.05). case letters indicate significant differences among the groups by one-way ANOVA and Fisher LSD’s test (p < 0.05). For both single biofilms of C. albicans (Figure5a) and C. glabrata (Figure5b), CPC, IONPs-CS-CPC39 Forand both IONPs-CS-CPC78 single biofilms significantly of C. albicans reduced (Figure the 5a) metabolic and C. activity glabrata compared (Figure 5b), to NCs. CPC, ForIONPs-CS- the CPC39dual-species and IONPs-CS-CPC78 biofilm, a similar significan trendtly was reduced observed, the except metabolic that CPCactivity and compared IONPs-CS-CPC78 to NCs. ledFor the to the highest reductions (Figure5c). dual-species biofilm, a similar trend was observed, except that CPC and IONPs-CS-CPC78 led to the highest reductions (Figure 5c).

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FigureFigure 5. 5.QuantificationQuantification of of biofilm biofilm metabolicactivity activity by by XTT XTT reduction reduction assay. assay. Mean Mean absorbance absorbance 2 valuesvalues per per cm cm2 forfor single single biofilms biofilms of ofCandida Candida albicans albicansATCC ATCC 10231 10231 (a), Candida (a), Candida glabrata glabrataATCC 90030 ATCC (b), 90030 µ 1 µ 1 (b),and and dual-species dual-species biofilms biofilms (c) treated (c) treated with 54.6with g54.6 mL −µgiron mL oxide−1 iron nanoparticles oxide nanoparticles (IONPs), 54.6 (IONPs),g mL− 54.6 µg µ 1 chitosan−1 (CS), 78 g mL− cetylpyridinium−1 chloride (CPC) and nanocarrier containing CPC at mL chitosan (CS), 78 µg mL cetylpyridinium chloride (CPC)1 and nanocarrier containing CPC at 15.6 (IONPs-CS-CPC15.6), 39 (IONPs-CS-CPC39) and 78 µg mL− (IONPs-CS-CPC78). Negative control 15.6 (IONPs-CS-CPC15.6), 39 (IONPs-CS-CPC39) and 78 µg mL−1 (IONPs-CS-CPC78). Negative (NC) indicates biofilms treated with pure artificial saliva (without drugs). Error bars represent standard controldeviations (NC) indicates of the means. biofilms Different treated lower with case pure letters arti indicateficial saliva significant (without differences drugs). among Error the bars groups represent standardby one-way deviations ANOVA of andthe Fishermeans. LSD’s Different test (p lower< 0.05). case letters indicate significant differences among the groups by one-way ANOVA and Fisher LSD’s test (p < 0.05). 3.3. Cytotoxic Effect of the IONPs-CS-CPC Nanocarrier

1 3.3. CytotoxicCPC was Effect not of cytotoxic the IONPs-CS-CPC at concentrations Nanocarrier equal to or lower than 1.95 µg mL− , either in the free state or forming the nanocarrier, regardless of the exposure period evaluated (Figure6a,b). However, −1 CPC was not cytotoxic at concentrations equal to or lower1 than 1.95 µg mL , either in the free for free CPC, concentrations equal to or higher than 3.9 µg mL− promoted significant reductions in stateL929 or forming cell viability, the nanocarrier, ranging from regardless 74.8–89.9% andof the 78.1–90.5%, exposure respectively period evaluated for 24 and (Figure 48 h of6a,b). exposure However, for free CPC, concentrations equal to or higher than 3.9 µg mL−1 promoted significant reductions1 in (Figure6a). In turn, the CPC-containing nanocarrier at the same concentration (3.9 µg mL− ) led to L929significant cell viability, reductions ranging in cell from viability 74.8–89.9% of 51.5% and and 78.1–90.5%, 83.7%, respectively respectively for 24 for and 24 48and h of48 exposureh of exposure 1 −1 (Figure(Figure 6a).6b). In The turn, nanocarrier the CPC- atcontaining CPC concentrations nanocarrier> 7.8 atµ gthe mL same− further concentration induced to (3.9 higher µg levels mL of) led to significanttoxicity (fibroblastreductions viability in cell< viability11.4%; Figure of 51.5%6b). and 83.7%, respectively for 24 and 48 h of exposure (Figure Comparisons6b). The nanocarrier between 24at andCPC 48 concentrations h of exposure for > 7.8 each µg concentration mL−1 further revealed induced that to freehigher CPC levels at of 1 toxicity3.9–250 (fibroblastµg mL− wasviability significantly < 11.4%; more Figure cytotoxic 6b). after 48 h of exposure (Figure6a). For the nanocarrier, 1 CPCComparisons only at 3.9 and between 7.8 µg 24 mL and− induced 48 h of to exposure more cytotoxicity for each after concentration 48 h of exposure revealed (Figure that6b). free CPC at 3.9–250 µg mL−1 was significantly more cytotoxic after 48 h of exposure (Figure 6a). For the nanocarrier, CPC only at 3.9 and 7.8 µg mL−1 induced to more cytotoxicity after 48 h of exposure (Figure 6b).

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Figure 6. Cytotoxic potential evaluated by MTT reduction assay. Cell viability percentages of Figure 6. Cytotoxic potential evaluated by MTT reduction assay. Cell viability percentages of L929 L929 murine fibroblasts after exposure for 24 or 48 h to different concentrations (0.24–500 µg mL 1) murine fibroblasts after exposure for 24 or 48 h to different concentrations (0.24–500 µg mL−1) −of of cetylpyridinium chloride (a) and iron oxide nanoparticles-chitosan-cetylpyridinium chloride cetylpyridinium chloride (a) and iron oxide nanoparticles-chitosan-cetylpyridinium chloride nanocarrier (b). Negative control (NC) represents murine fibroblasts exposed to pure culture medium nanocarrier (b). Negative control (NC) represents murine fibroblasts exposed to pure culture medium (without drugs). Error bars represent standard deviations of the means. Different uppercase and (without drugs). Error bars represent standard deviations of the means. Different uppercase and lowercase letters indicate significant differences among the different concentrations of the compounds, lowercase letters indicate significant differences among the different concentrations of the respectively for 24 and 48 h of exposure. *Indicates significant difference between 24 and 48 h of compounds, respectively for 24 and 48 h of exposure. *Indicates significant difference between 24 and exposure, within each concentration tested (two-way ANOVA followed by Tukey’s test; p < 0.05). 48 h of exposure, within each concentration tested (two-way ANOVA followed by Tukey’s test; p < 4. Discussion0.05).

4. DiscussionIn the present study, the IONPs-CS-CPC nanocarrier was significantly more effective in reducing the number of CFUs in biofilms, despite promoting similar effects to those of CPC on planktonic cells and murineIn the fibroblasts.present study, Thus, the the IONPs-CS-CPC study’s null hypothesis nanocarrier was partiallywas significantly rejected. more effective in reducingRegarding the number the assembly of CFUs of the in nanocarrier,biofilms, despit the characterizatione promoting similar results effect showeds to that those CS successfullyof CPC on planktoniccoated the IONPs,cells and while murine all the fibroblasts. added CPC Thus, remained the study’s anchored null hypothesis on the core-shell was partially structure rejected. (IONPs-CS) (FiguresRegarding1 and2). IONPsthe assembly display advantagesof the nanocarrier, that allow the their characterization use in drug delivery results systems, showed such that as theCS successfullylargest ratio ofcoated surface the area IONPs, to volume while due all tothe their added nanometric CPC remained size. In addition,anchored these on the nanoparticles core-shell structurehave grafted (IONPs-CS) ligands and(Figures portions 1 and of selective2). IONPs binding display sites advantages that favor that the allow binding their of bothuse in natural drug deliverybiomolecules systems, and such other as compounds the largest ratio (organic of surfac or inorganic)e area to usedvolume as shelldue to [40 their]. In nanometric turn, CS has size. been In widelyaddition, employed these nanoparticles as shell for have IONPs, grafted as it ligands prevents and the portions agglomeration of selective of nanoparticles binding sites (i.e., that actingfavor theas a binding stabilizing of both agent), natural and limitsbiomolecules nonspecific andcell other uptake, compounds in addition (organic to enabling or inorganic) the immobilization used as shell [40].of di ffInerent turn, drugs CS has [41 been]. For widely the IONPs-CS-CPC employed as nanocarrier,shell for IONPs, a strong as it bondprevents between the agglomeration hydrogen of the of nanoparticles (i.e., acting as a stabilizing agent), and limits nonspecific cell uptake, in addition to primary amino group (-NH2) of CS and oxygen of IONPs is the likely mechanism responsible for the enablingconjugation the ofimmobilization IONPs with CS of [42different]. It is alsodrugs believed [41]. For that the CPC IONPs-CS-CPC forms a hydrogen nanocarrier, interaction a strong with bondthe amino between group hydrogen of CS, which of the is primary protonated amino in angroup acidic (-NH medium2) of CS (Figure and oxygen7). Interestingly, of IONPs is despite the likely the mechanismfact that a carrier responsible system for of the nanometric conjugation dimensions of IONPs was with formed, CS [42]. larger It is also sizes believed were observed that CPC by forms DLS acompared hydrogen to interaction TEM. Such with diff theerences amino may group be explainedof CS, which by is the protonated methodological in an acidic principles medium that (Figure make 7).up Interestingly, each technique. despite For TEM,the fact the that electron a carrier beam system crosses of nanometric through an dimensions ultrafine and was dry formed, sample larger [43], whilesizes inwere the indirectobserved DLS by technique DLS compared the particle to sizeTEM. is obtainedSuch differences through the may frequency be explained of movement by the in methodologicalan aqueous medium, principles which that may make generate up each clusters technique. of particles For TEM, [44]. the electron beam crosses through an ultrafine and dry sample [43], while in the indirect DLS technique the particle size is obtained through the frequency of movement in an aqueous medium, which may generate clusters of particles [44].

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Figure 7. Schematic representation of the possible chemical bonds/interactions among the compounds Figure 7. Schematic representation of the possible chemical bonds/interactions among the compounds that form the iron oxide nanoparticles-chitosan-cetylpyridinium chloride nanocarrier. A bond between that form the iron oxide nanoparticles-chitosan-cetylpyridinium chloride nanocarrier. A bond oxygen from iron oxide nanoparticles (IONPs) and hydrogen from amine group (-NH2) of chitosan (CS) is responsiblebetween oxygen for coating from IONPs iron oxide with CS.nanoparticles In turn, cetylpyridinium (IONPs) and hydrogen chloride (shown from amine in red) group establishes (-NH2) of a hydrogenchitosan interaction (CS) is responsible with the aminefor coating group IONPs of CS. with CS. In turn, cetylpyridinium chloride (shown in red) establishes a hydrogen interaction with the amine group of CS. Although previous studies have shown that carriers based on IONPs improve the effect of some drugsAlthough on bacterial previous and studies fungal have planktonic shown cellsthat carrie [28,45rs,46 based], the on present IONPs study improve found the equal effect MICof some valuesdrugs for on CPC bacterial alone and or conjugated fungal planktonic to the IONPs-CS cells [28,45,46], complex the present (Table1 ).study This found demonstrates equal MIC that values thefor additional CPC alone antimicrobial or conjugated effects to promoted the IONPs-CS by the nanocarrierscomplex (Table may 1). be associatedThis demonstrates with the typethat the of immobilizedadditional antimicrobial drug and microorganism effects promoted tested. by Inthe addition, nanocarriers FICI may values be classifiedassociated the with interaction the type of amongimmobilized the compounds drug and present microorganism in the IONPs-CS-CPC tested. In nanocarrier addition, FICI as indi valuesfferent classified (Table1), suggestingthe interaction thatamong its eff ectthe oncompoundsCandida planktonicpresent in the cells IONPs-CS-CPC is primarily dependentnanocarrier onas indifferent the presence (Table ofCPC 1), suggesting itself. CPCthat is formedits effect by on a Candida hydrophilic planktonic region, cells which is primarily interacts withdependent microbial on the cell presence surfaces, of including CPC itself. the CPC cytoplasmicis formed membrane by a hydrophilic [47]. As recentlyregion, which reported, interacts the interaction with microbial between cell the surfaces, negative including charge of the thecytoplasmic microorganism membrane with the [47]. pyridinium As recently ion reported, (positively the charged)interaction may between trigger the changes negative in charge the of the membranemicroorganism [48]. Furthermore, with the atpyridinium low concentrations, ion (positiv CPCely impairs charged) cell osmoregulationmay trigger changes and homeostasis, in the lipid whilemembrane at high concentrations [48]. Furthermore, it promotes at low leakage concentrations, of cytoplasmic CPC content impairs due tocell the osmoregulation disintegration of and the cellhomeostasis, membrane while [48]. at high concentrations it promotes leakage of cytoplasmic content due to the disintegrationFor biofilm analyzes, of the cell the membrane IONPs-CS-CPC78 [48]. nanocarrier was more effective in reducing the number of CFUs ofFor single- biofilm and analyzes, dual-species the biofilmsIONPs-CS-CPC78 than free CPC nano (Figurecarrier3 ).was For more CPC alone,effective an einffect reducing limited the to thenumber middle of andCFUs outer of single- layers and of biofilms dual-species has been biofil reportedms than [49free], probablyCPC (Figure due 3). to For electrostatic CPC alone, or an hydrophobiceffect limited interactions to the middle between and extracellular outer layers matrix of bi andofilms CPC, has which been hinders reported drug [49], penetration probably [48due]. to Consequently,electrostatic it or seems hydrophobic rational interactions that the favorable between results extracellular found matrix in the and present CPC, study which are hinders related drug to thepenetration greater ease [48]. of Consequently, the nanostructure it seems in rational penetrating that intothe favorable the deeper results layers found of Candida in the presentbiofilms, study whichare allowedrelated to the the conjugated greater ease CPC of tothe act nanostruct in broaderure and in deeperpenetrating areas into of biofilms the deeper compared layers of to Candida free CPC.biofilms, In fact, previouswhich allowed studies the by conjugated EDS mapping CPC have to act shown in broader the presence and deeper of Fe areas atoms of (characteristic biofilms compared of IONPs)to free well CPC. distributed In fact, on previousCandida biofilmsstudies andby EDS on polystyrene mapping have surfaces, shown proving the thepresence effectiveness of Fe atoms of IONPs(characteristic as drug carriers of IONPs) [24,28 well]. The distributed results also on revealed Candida that biofilms IONPs and alone on polyst did notyrene lead surfaces, to significant proving reductionsthe effectiveness in CFUs, of which IONPs could as drug be expected carriers [24,28]. based on The previous results also findings revealed showing that IONPs that IONPs alone candid not eitherlead stimulate to significant or inhibit reductions microbial in CFUs, growth which depending could be on expected the strain based and on concentration previous findings tested showing [50]. that IONPs can either stimulate or inhibit microbial growth depending on the strain and concentration tested [50]. CS, on the other hand, was able to decrease this parameter for single

J. Fungi 2020, 6, 218 11 of 14

CS, on the other hand, was able to decrease this parameter for single biofilms, which could be associated with its ability to impair cell membrane permeability, resulting in release of cytoplasmic content and death of the microorganisms [51]. Therefore, the effects of the nanocarrier on CFUs may be attributed to a combined action of CS and CPC, with greater emphasis on the latter. In general, total biomass and metabolic activity results should also be considered as positive, 1 since the nanocarriers at 39 and 78 µg mL− showed similar effects to those found for pure CPC at 1 78 µg mL− (Figures4 and5). Another aspect of clinical relevance refers to the mucoadhesive property of CS [52], which might solve the issue of low substantivity observed for CPC [12]. The greater substantivity of CPC in the nanocarrier would reduce the number of applications of the product per day (which may enhance patient’s adherence to the treatment), as well as the emergence and spread of microbial resistance. As for the analysis in murine fibroblasts, both free CPC and the nanocarrier significantly reduced 1 cell viability at concentrations equal to or higher than 3.9 µg mL− , regardless of the contact period (24 or 48 h) (Figure6). Considering that the presence of IONPs and CS did not increase the cytotoxic potential of the nanocarrier compared to free CPC, it is possible to assume that the effect on murine fibroblasts was essentially related to CPC. The simultaneous analysis of cytotoxicity results and MIC values evidences that both free CPC or CPC conjugated to the nanocarrier were not cytotoxic at the 1 effective concentration against Candida planktonic cells (0.78 µg mL− ). However, at the concentrations 1 tested on biofilms (15.6, 39 and 78 µg mL− ) both compounds led to significant reductions in L929 cell viability (~90%). Müller et al. [53] also observed that mouthwashes containing CPC had antimicrobial properties but were highly toxic to gingival fibroblasts. In addition, this compound is able to induce apoptosis in pulmonary epithelial cells [54], and to inhibit mitochondrial oxygen consumption and ATP synthesis for different cell cultures [55]. On the other hand, CPC may prevent osteoclastic formation in mouse bone marrow cells, positively contributing to the prevention of bone loss without leading to cytotoxic effects, depending on the concentration assessed [56]. Based on the above, the issues related to CPC’s cytotoxicity (either alone or forming nanocarriers) point to the need for the study of alternatives to reduce possible side effects. Finally, it is worth emphasizing that the oral cavity is formed by different cell types in addition to fibroblasts, and that Candida species coexist with several microorganisms. Consequently, future studies evaluating the performance of the IONPs-CS-CPC nanocarrier on microcosms of oral fungal infections, as well as on reconstituted oral tissues with multiple layers, may provide additional data on the carrier’s actual benefits. Such analyzes, along with the evaluation of the molecular mechanisms of action of the nanocarrier, may help in the development of effective and safe strategies to manage .

5. Conclusions Summarizing, a nanocarrier of CPC was effectively formed by immobilizing this drug on CS-coated IONPs. The IONPs-CS-CPC nanocarrier was shown to be more effective than pure CPC in reducing the cultivable cells of Candida biofilms without increasing the cytotoxic effects of CPC. These results suggest that this new nanocarrier may be a useful tool for the treatment of oral fungal infections.

Author Contributions: Conceptualization, D.R.M.; Formal analysis, H.C.A., S.H.P.O., J.P.P. and D.R.M.; Funding acquisition, D.R.M.; Investigation, H.C.A., L.S.A., A.C.M.C., L.C.d.F.A., M.G.M., F.N.d.S.N., E.R.d.C. and D.R.M.; Project administration, D.R.M.; Writing—original draft, H.C.A.; Writing—review & editing, L.S.A., A.C.M.C., F.N.d.S.N., E.R.d.C., S.H.P.O., J.P.P. and D.R.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil; Grant number 404721/2016-8), São Paulo Research Foundation (FAPESP, Grant number 2017/24416-2), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Finance Code 001). Acknowledgments: The authors thank nChemi Engenharia de Materiais (São Carlos, São Paulo, Brazil) for supplying the colloidal suspension of iron oxide nanoparticles used in this study. J. Fungi 2020, 6, 218 12 of 14

Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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