Essential Oil-Based Design and Development of Novel Anti-Candida Azoles Formulation

Candida is the most common fungal class, causing both superficial and invasive diseases in humans. Although Candida albicans is the most common cause of fungal infections in humans, C. auris is a new emergent serious pathogen causing complications similar to those of C. albicans. Both C. albicans and C. auris are associated with high mortality rates, mainly because of their multidrug-resistance patterns against most available antifungal drugs. Although several compounds were designed against C. albicans, very few or none were tested on C. auris. Therefore, it is urgent to develop novel effective antifungal drugs that can accommodate not only C. albicans, but also other Candida spp., particularly newly emergent one, including C. auris. Inspired by the significant broad-spectrum antifungal activities of the essential oil cuminaldehyde and the reported wide incorporation of azoles in the antifungal drugs, a series of compounds (UoST1-11) was designed and developed. The new compounds were designed to overcome the toxicity of the aldehyde group of cuminaldehyde and benefit from the antifungal selectivity of azoles. The new developed UoST compounds showed significant anti-Candida activities against both Candida species. The best candidate compound, UoST5, was further formulated into polymeric nanoparticles (NPs). The new formula, UoST5-NPs, showed similar activities to the nanoparticles-free drug, while providing only 25% release after 24 h, maintainng prolonged activity up to 48 h and affording no toxicity. In conclusion, new azole formulations with significantly enhanced activities against C. albicans and C. auris, while maintaining prolonged action and no toxicities at lower concentrations, were developed.


Introduction
Candida species are the most common cause of fungal infections in humans. Although Candida can cause treatable superficial infections [1], they can also generate serious health problems, particularly to immunocompromised patients [2,3]. C. auris is a newly-emerging Candida species [4,5]. Both C. albicans and C. auris are considered as serious global health threats mainly because of their multidrug-resistance patterns associated with high mortality rates that can reach 60% [6][7][8].  Structural modification of cuminaldehyde to examine the importance of isoproyl group a series of tert-butyl group was synthezied Replacing the aldehyde group with 1,2,4-triazole ring with reported antifungal activity to decrease toxicity and improve phsiochemical properties The introduction of 3-trifluoro-methylbenzylsulfanyl moeity to enhance the activity Structural modification of cuminaldehyde to examine the importance of isoproyl group a series of tert-butyl group was synthezied Replacing the aldehyde group with 1,2,4-triazole ring with reported antifungal activity to decrease toxicity and improve phsiochemical properties The introduction of 3-trifluoro-methylbenzylsulfanyl moeity to enhance the activity Initially, cuminaldehyde (1) was oxidized to cumnic acid (2) by iodine, NaOH and TBHP as catalyst in moderate yield. The acids 2, 3 were then converted to acid chlorides 4, 5 using oxalyl chloride, DCM and DMF under nitrogen at 0 • C. The acid hydrazide derivatives 6, 7 were obtained in high yields by refluxing the acid chlorides with hydrazine hydrate [18]. The hydrazides derivatives 6, 7 were treated with substituted phenyl isothiocyanates 8a-k for the synthesis of the corresponding thiosemicarbazides R (1-11), which were subsequently converted to triazole thiols S (1-11) using 2N NaOH. The target compounds UoST1-11 were formed in moderate yields (~70%) by nucleophilic substitution reactions between triazole thiols S (1-11) and 3-trifluorobenzyl chloride.

Selective Inhibition Activities of UoST Compounds Against Candida Spp.
The anti-Candida activities of the developed compounds UoST1-11 were tested against C. albicans and C. auris. The results obtained indicated that compounds UoST5, 7, 8 and 11 caused significant variable inhibition activities on the growth of both C. albicans ( Figure 2A) and C. auris ( Figure 2B) at different concentrations (2,5,15,30,50 and 100 µg/mL) when compared to negative control. Compound UoST5 showed persistent and significant promising activities on both Candida spp. when compared to other compounds (two-way ANOVA, P < 0.0001, Figure 2) and with the lowest MIC 50 (2 µg/mL) on C. auris when compared to amphotericin B, employed as positive control (Table 1). On the other hand, compound UoST7 showed selective inhibition activities on C. auris but not C. albicans ( Figure 2). Initially, cuminaldehyde (1) was oxidized to cumnic acid (2) by iodine, NaOH and TBHP as catalyst in moderate yield. The acids 2, 3 were then converted to acid chlorides 4, 5 using oxalyl chloride, DCM and DMF under nitrogen at 0 °C. The acid hydrazide derivatives 6, 7 were obtained in high yields by refluxing the acid chlorides with hydrazine hydrate [18]. The hydrazides derivatives 6,7 were treated with substituted phenyl isothiocyanates 8a-k for the synthesis of the corresponding thiosemicarbazides R (1-11), which were subsequently converted to triazole thiols S (1-11) using 2N NaOH. The target compounds UoST1-11 were formed in moderate yields (~70%) by nucleophilic substitution reactions between triazole thiols S (1-11) and 3-trifluorobenzyl chloride.

Selective Inhibition Activities of UoST Compounds Against Candida Spp.
The anti-Candida activities of the developed compounds UoST1-11 were tested against C. albicans and C. auris. The results obtained indicated that compounds UoST5, 7, 8 and 11 caused significant variable inhibition activities on the growth of both C. albicans ( Figure 2A) and C. auris ( Figure 2B) at different concentrations (2,5,15,30,50 and 100 µg∕mL) when compared to negative control. Compound UoST5 showed persistent and significant promising activities on both Candida spp. when compared to other compounds (two-way ANOVA, P < 0.0001, Figure 2) and with the lowest MIC50 (2 µg/mL) on C. auris when compared to amphotericin B, employed as positive control (Table 1). On the other hand, compound UoST7 showed selective inhibition activities on C. auris but not C. albicans ( Figure 2).  (2,5,15,30,50, 100 µg/mL). (B) C. auris growth inhibition by UoST5, 7, 8 and 11 compounds at different concentrations (2,5,15,30,50, 100 µg/mL). Candida at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 °C for 24 h prior to measurement at 600 nm using plate reader. Cultures without compounds were employed as negative control. The data was analyzed using two-way ANOVA and statistical significance was calculated with Bonferroni's multiple comparisons test and significance level indicated by asterisks (* P < 0.05; ** P < 0.01: *** P < 0.001). The data display the mean ± standard error (SEM) of three replicas.   (2,5,15,30,50, 100 µg/mL). Candida at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 • C for 24 h prior to measurement at 600 nm using plate reader. Cultures without compounds were employed as negative control. The data was analyzed using two-way ANOVA and statistical significance was calculated with Bonferroni's multiple comparisons test and significance level indicated by asterisks (* P < 0.05; ** P < 0.01: *** P < 0.001). The data display the mean ± standard error (SEM) of three replicas.

Formulation and Characterization of UoST5-Loaded NPs
Compound UoST5 was formulated into PLGA NPs using the emulsion/evaporation method. The size and shape of the developed NPs were analyzed by a Zeta analyzer and scanning electron microscope (SEM), respectively. The particle size and size distribution were evaluated by a Zetasizer and found to be 199.5 ± 0.035 nm with narrow size distribution (0.098 ± 0.016) ( Figure 3A). Additionally, the particle surface charge (Zeta potential) was −1.58 ± 0.25 mV ( Figure 3B). The surface morphology and topography of UoST5-loaded NPs showed well-defined spherical shaped NPs with smooth surfaces without any cracks ( Figure 3C). The amount of drug entrapped was calculated directly by dissolving the NPs in DCM. The drug release was measured by UV/VIS spectrophotometer and the entrapment efficiency was calculated to be 67.2%.
Compound UoST5 was formulated into PLGA NPs using the emulsion/evaporation method. The size and shape of the developed NPs were analyzed by a Zeta analyzer and scanning electron microscope (SEM), respectively. The particle size and size distribution were evaluated by a Zetasizer and found to be 199.5 ± 0.035 nm with narrow size distribution (0.098 ± 0.016) ( Figure 3A). Additionally, the particle surface charge (Zeta potential) was −1.58 ± 0.25 mV ( Figure 3B). The surface morphology and topography of UoST5-loaded NPs showed well-defined spherical shaped NPs with smooth surfaces without any cracks ( Figure 3C). The amount of drug entrapped was calculated directly by dissolving the NPs in DCM. The drug release was measured by UV/VIS spectrophotometer and the entrapment efficiency was calculated to be 67.2%.
The release of UoST5 from PLGA NPs was determined within 10 days by incubating certain amount of UoST5-NPs in a release media at 37 °C followed by measurement at different time intervals. The release pattern was biphasic with an initial burst release of 19.79% after 4 h ( Figure 3D). This was followed by a gradual release as the cumulative release percentage reached 25.039% after 24 h and 92.9% after 6 days and stayed at the same percentage for another 4 days ( Figure 3D).

NP Formulation Enhanced and Prolonged the Anti-Candida Activities of UoST5
The bioactivity of UoST5-loaded NPs on C. auris was compared to the free drug (suspended in water or dissolved in DMSO). The results showed that UoST5-NPs (suspended in water) achieved significantly higher activity than when the compound was suspended in water but lower than when dissolved in DMSO (two-way ANOVA, P < 0.0001, Figure 4). The anti-Candida activities of UoST5-NPs were comparable to the compound in DMSO (Figure 4), while afforded no toxicity to mammalian cells at six times the MIC50 concentrations (Data not shown). Drug-free NPs, employed as control, showed no effects on Candida or mammalian cells. The release of UoST5 from PLGA NPs was determined within 10 days by incubating certain amount of UoST5-NPs in a release media at 37 • C followed by measurement at different time intervals. The release pattern was biphasic with an initial burst release of 19.79% after 4 h ( Figure 3D). This was followed by a gradual release as the cumulative release percentage reached 25.039% after 24 h and 92.9% after 6 days and stayed at the same percentage for another 4 days ( Figure 3D).

NP Formulation Enhanced and Prolonged the Anti-Candida Activities of UoST5
The bioactivity of UoST5-loaded NPs on C. auris was compared to the free drug (suspended in water or dissolved in DMSO). The results showed that UoST5-NPs (suspended in water) achieved significantly higher activity than when the compound was suspended in water but lower than when dissolved in DMSO (two-way ANOVA, P < 0.0001, Figure 4). The anti-Candida activities of UoST5-NPs were comparable to the compound in DMSO (Figure 4), while afforded no toxicity to mammalian cells at six times the MIC 50 concentrations (Data not shown). Drug-free NPs, employed as control, showed no effects on Candida or mammalian cells. Anti-Candida activity of UoST5-Loaded NPs compared to free synthesized compound. The anti-Candida auris activity of UoST5-NPs in water was compared to UoST5 in water and DMSO at different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/mL). C. auris at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 °C for 24 h prior to measurement at 600 nm using a plate reader. Cultures without compounds were employed as negative control. The data display the mean of the percentage of the survival rate of mammalian cells ± standard error of the mean. A P-value < 0.05 was considered as significant. The level of significance was indicated by asterisks (** P < 0.01: *** P < 0.001) auris. Candida growth inhibition was measured at different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/mL). Candida at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 °C for 24 h prior to measurement at 600 nm using plate reader. Cultures without compounds were employed as negative control in order to measure percent inhibition. The data was analyzed using two-way ANOVA and statistical significance was calculated with Bonferroni's multiple comparisons test and significance level indicated by asterisks (* P < 0.05; ** P < 0.01: *** P < 0.001). The data display the mean ± standard error (SEM) of three replicates. The anti-Candida auris activity of UoST5-NPs in water was compared to UoST5 in water and DMSO at different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/mL). C. auris at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 • C for 24 h prior to measurement at 600 nm using a plate reader. Cultures without compounds were employed as negative control. The data display the mean of the percentage of the survival rate of mammalian cells ± standard error of the mean. A P-value < 0.05 was considered as significant. The level of significance was indicated by asterisks (** P < 0.01: *** P < 0.001) The anti-Candida activities of UoST5-loaded NPs were compared to NPs-free drug dissolved in DMSO at concentrations range 3.12-100 µg/mL within 48 h ( Figure 5). Both the free drug and NPs formulations exhibited significant growth inhibition activity on both C. albicans and C. auris with different magnitudes depending on the drug concentrations and time of incubation. In the case of C. albicans, the inhibition effect of UoST5-NPs after 24 h incubation was 75.46% at 100 µg/mL which is significantly lower than the effect of free drug (two-way ANOVA, P < 0.0001, Figure 5A). The inhibition was further enhanced after 48 h to reach 91.8% similarity to the effect of free drug ( Figure 5B). At 3.12 µg/mL, the inhibition effect was 30.58%, and 39.3% when compared to free drug (40% and 19%, two-way ANOVA, P < 0.0001) after 24 h and 48 h, respectively. A similar pattern was obtained in the case of C. auris ( Figure 5C,D). UoST5-NPs at 100 and 3.12µg/ml after 24 h showed 74.34% and 30.96% inhibition activity, respectively. Unlike the effect on C. albicans, the inhibition effect of UoST5-NPs was slightly reduced after 48 h to reach 60.2% and 15.96% at 100 and 3.12 µg/mL, respectively ( Figure 5C,D). Comparing the anti-Candida activities of UoST5-loaded NPs to free UoST5 drug at the same concentration indicated that UoST5-loaded NPs showed lower inhibition activity at 24 h and higher inhibition activity at 48 h ( Figure 5), although the released amount of the drug from the NPs was only 25%; thus providing a significant increase in activity. Collectively, we have developed novel triazole derivatives effective against both C. albicans and C. auris and its formulation in NPs enhanced and prolonged the activities of the drug. different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/mL). C. auris at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 °C for 24 h prior to measurement at 600 nm using a plate reader. Cultures without compounds were employed as negative control. The data display the mean of the percentage of the survival rate of mammalian cells ± standard error of the mean. A P-value < 0.05 was considered as significant. The level of significance was indicated by asterisks (** P < 0.01: *** P < 0.001) auris. Candida growth inhibition was measured at different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/mL). Candida at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 °C for 24 h prior to measurement at 600 nm using plate reader. Cultures without compounds were employed as negative control in order to measure percent inhibition. The data was analyzed using two-way ANOVA and statistical significance was calculated with Bonferroni's multiple comparisons test and significance level indicated by asterisks (* P < 0.05; ** P < 0.01: *** P < 0.001). The data display the mean ± standard error (SEM) of three replicates. auris. Candida growth inhibition was measured at different concentrations (3.125, 6.25, 12.5, 25, 50 and 100 µg/mL). Candida at 10 4 CFU/mL was grown in 96 well microplate containing YPD broth. The plates were incubated at 37 • C for 24 h prior to measurement at 600 nm using plate reader. Cultures without compounds were employed as negative control in order to measure percent inhibition. The data was analyzed using two-way ANOVA and statistical significance was calculated with Bonferroni's multiple comparisons test and significance level indicated by asterisks (* P < 0.05; ** P < 0.01: *** P < 0.001).
The data display the mean ± standard error (SEM) of three replicates.

Discussion
Candida spp. are responsible for 75-88% of all fungal infections in the US, causing an extra annual health cost of $1.7 billion [19]. The general resistance of Candida spp. to most available antifungal drugs [20] and the emergence of multidrug resistant C. auris [21] make urgent the need to develop novel effective anti-Candida drugs. On the other hand, most compounds synthesized nowadays are developed against C. albicans, while very few are tested against C. auris. Thus, we proposed to develop novel azoles derivatives with effective antifungal activities against Candida spp. including in particular C. auris. Azole derivatives show great potential activities against Candida infections. Chandrika et al. developed 12 novel azole derivatives that showed significant antifungal activities against different types of Candida spp., but none of them was C. auris [22]. Similarly, Shrestha et al., successfully developed some novel alkylated-azoles that showed great activities against 13 clinically fungal isolates, but C. auris was not one of them [23]. Here, we have developed a series of novel S-alkylated azole derivatives (compounds UoST1-11) by cyclization reactions of thiosemicarbazide intermediates R(1-11) followed by S-alkylation in alkaline media.
Generally, 1,2,4-triazole-3-thiones have attracted considerable attention because of their broad biological activities [24]. Some of our developed compounds (compounds UoST5, 7, 8 and 11) showed variable promising activities against both C. albicans and C. auris. The variability of the activity is attributed to the variation in the substitution on the newly developed structures including the isopropyl or tert-butyl substituents at the C-4 position of the phenyl ring (R) and the substituted phenyl attached to the N-4 position of the triazole ring (R1) carrying 3-Cl, 4-Cl, 4-CH 3 , 4-OCH 3 , 4-F, 3-Br or unsubstituted phenyl. Looking at the chemical structures of the most active compounds UoST5, 7, 8 and 11 that showed significant anti-Candida activity, it can be declared that 3-Br (UoST5), 4-OCH 3 (UoST8) substitution on R1 with an isopropyl substituent on R, and the tert-butyl substituent on R with 4-F on R1 (UoST11) are active against both Candida spp., while the substitution with 4-CH 3 on R1 with tert-butyl on R (UoST7) was significantly selective against C. auris.
Compared to the positive control, UoST5 was selected as candidate for further development mainly because of its significant anti-Candida activities against both Candida spp., its low MIC 50 value and its safety on mammalian cells. Like 90% of the molecules in the discovery pipeline, UoST5 showed limited aqueous solubility that consequently leads to poor bioavailability [25]. In view of the foregoing, we have enhanced the bioavailability of UoST5 by encapsulation into PLGA-NPs. The single emulsion solvent evaporation method was employed for UoST5-NPs fabrication [26]. The aforementioned fabrication method of hydrophobic drugs affords simplicity with the ability to scale up [26][27][28]. The obtained drug entrapment efficiency of 67.2% is similar to other reports using the same method [14,29]. The relatively higher entrapment efficiency reported here is one of the main advantages of encapsulating hydrophobic drugs into PLGA-NPs. This can be attributed to the hydrophobic nature of the biodegradable polymer (PLGA) employed [15,26]. Furthermore, a relatively uniform particle distribution was obtained as the polydispersity index value was 0.098. The relatively low particle charge obtained was also favorable since it increases the contact and interaction of the NPs with the Candida outer membrane. Generally, Candida carry negative surface charge; thus it was essential to develop NPs that carry either neutral or low negative charge to avoid any repulsion forces that may prevent the attachment of NPs to the Candida surface [30,31]. The release of UoST5 from the NPs followed the common biphasic pattern with an initial burst release followed by sustained release of the drug over 6 days. The initial burst release of the drug is mainly related to the fraction of the drug located and adsorbed on the surface of the NPs that has been reported and described elsewhere [32,33]. The sustained release is relevant to the amount of the drug encapsulated and the degradation rate of PLGA employed.
The bioactivity of UoST5-NPs compared to the free UoST5 can be explained by the UoST5-NPs' release pattern. Although 25% of the entrapped drug was released from the NPs after 24 h, the inhibition effects on C. albicans and C. auris were 62.8-91.7% of the inhibition effect obtained by the free drug (100% in direct contact with the Candida cells). Interestingly, UoST5 release after 48 h reached up to 32.95%, while exerting similar or higher activities on Candida cells when compared to 100% free drug. Initially, this confirms that the method and reagents used to encapsulate UoST5 did not modify or degrade the UoST5 molecule as the drug conserved its bioactivity. Additionally, as the PLGA-NPs free of UoST5 has no inhibitory effect on Candida, the enhancement of the antifungal activity of UoST-NPs can be related to the ability of the NPs to adhere and attach to the outer surface of Candida cells. Therefore, it provides direct contact and reduces the loss of the drug. The ability of the NPs to adhere and even infiltrate to fungal cells was previously studied and explained [14,34]. A model represents the synthesis of NPs, attachment to Candida cells and delivery of UoST5 is described in Figure 6. Essential oils including cuminaldehyde are known for their promising antimicrobial activities [35], while inducing toxic effects on human cells [11]. Therefore, extraction and purification of essential oil followed by few synthetic steps in order to optimize its selective antimicrobial activities and provide no toxicity will be of great value for both human and market uses.
the NPs to adhere and even infiltrate to fungal cells was previously studied and explained [14,34]. A model represents the synthesis of NPs, attachment to Candida cells and delivery of UoST5 is described in Figure 6. Essential oils including cuminaldehyde are known for their promising antimicrobial activities [35], while inducing toxic effects on human cells [11]. Therefore, extraction and purification of essential oil followed by few synthetic steps in order to optimize its selective antimicrobial activities and provide no toxicity will be of great value for both human and market uses.

General Information
Reactions were monitored by thin-layer chromatography (TLC) using pre-coated silica gel plates (Kieselgel 60 F254, BDH, Taufkirchen, Germany), and spots were visualized under UV light (254 nm). Melting points (mp) were determined using a Gallenkamp melting point apparatus (London, UK). 1 H-NMR and 13 C-NMR spectra were recorded at 500/125 MHz on a Bruker spectrometer (Karlsruhe, Germany). Chemical shifts were expressed in parts per million (ppm) relative to tetramethylsilane, and coupling constant (J) values were represented in Hertz (Hz) and the signals were designated as follows: s, singlet; d, doublet; t, triplet; m, multiplet. Mass spectroscopic data were obtained using electrospray ionization (ESI) mass spectroscopy (Bruker Daltonics mass spectrometer, Bremen, Germany).

General Procedure for the Synthesis of 4-Substituted Benzoyl Chlorides 4, 5
Oxalyl chloride (12 mmol) was added dropwise to a mixture of the appropriate 4-substituted benzoic acid 2, 3 (10 mmol), in anhydrous DCM (30 mL) and anhydrous DMF (10 µL) under nitrogen at 0 • C. The reaction mixture was stirred at room temperature for sn additional 3 h until the reaction was completed. The reaction mixture was evaporated under vacuum and the oily residue was used without further purification for the acid hydrazide preparation.

General Procedure for the Synthesis of 4-Substituted Benzoic Acid Hydrazides 6, 7
The synthesis of 4-substituted benzoic acid hydrazides was performed as previously described [18]. To a solution of ethyl 4-substituted benzoyl chloride 4, 5 (10 mmol) in ethanol (10 mL), excess hydrazine monohydrate (5 mL) was added. The reaction mixture was refluxed for 24 h, and left to cool to room temperature. The formed precipitate was collected by filtration, washed with water followed by cold ethanol to remove excess hydrazine and left to dry to give the corresponding acid hydrazide. The 4-substituted benzoyl-N-substituted phenyl thiosemicarbazide R derivatives were synthesized as previously described [18]. To a solution of 4-substituted benzoic acid hydrazide 6, 7 (10 mmol) in ethanol (30 mL) a solution of substituted phenyl isothiocyanate 8a-k (10 mmol) in ethanol (20 mL) was added with continuous stirring. The reaction mixture was refluxed for 3 h. After being cooled to room temperature, the precipitate formed was collected by filtration, washed with cold ethanol (30 mL) to give the corresponding thiosemicarbazide R(1-10), Yield 79%.

Entrapment Efficiency
For the appropriate determination of the entrapment efficiency of the drug (UoST5), an efficient assay was developed based on the UV/VIS spectrophotometery absorbance of the drug. The maximum absorbance (ň max ) of UoST5 was determined by UV/VIS spectrophotometer (SYNERGY H1, Biotek Ltd, Winoosk, VT, USA) and the selected ň max (350 nm) was validated based on the selectivity, linearity and quantification limit. A calibration curve was developed for UoST5 in DCM (Supplementary Figure  S1). The prepared NPs were mixed with DCM in order to completely dissolve the entrapped drug. The amount of entrapped drug was measured at 350 nm and the percentage of entrapment efficiency was calculated using the following formula (Equation (1)): Entrapment efficiency % = (Amount of drug entrapped/Total amount of drug used) × 100 (1)

Topology and Particle Size Measurements
The morphological characteristics of the UoST5-loaded NPs were evaluated using a scanning electron microscope (JSM-633OF; JEOL Ltd, Tokyo, Japan). NPs samples were suspended in distilled water and a drop of the solution was placed on a clean slide cover and left to dry under vacuum. The dried sample was mounted on carbon tape and sputter-coated with gold. The gold-coated samples were scanned and photomicrographs were taken at an acceleration voltage of 3 kV. The effective diameter, the size distribution, and zeta-potential of the developed NPs were measured by a Zetasizer (Malvern, Cambridge, UK). NPs were diluted with distilled water and the effective diameter and polydispersity index were determined using the Dynamic Light Scattering (DLS) mode at 25 • C. The Laser Doppler Velocimetry (LDV) mode was employed to measure the zeta potential (mV).

In-Vitro Drug Release Study
Certain weights of NPs equivalent to 2.1 mg of UoST5 were suspended in dialysis membrane (MWCO = 12 kDa). The membranes were then immersed in 10 mL release media consisting of 0.9% NaCl: isopropanol (85:15) followed by incubation in water bath at 37 • C with shaking at 50 rpm. Samples were taken at different time intervals and the concentrations of UoST5 were determined. The cumulative percentage release (%) was calculated according to the following formula (Equation (2)): The cumulative percentage release (%) = [Volume of sample withdrawn (ml)/Bath volume (v)] × P (t − 1) + Pt (2) where Pt = percentage release at time (t) and P (t − 1) = percentage release previous to t.

Cell Toxicity Assay Using MTT Staining
The cell viability was performed using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay as described before [38]. Briefly, 96-well micro-plates were seeded with normal fibroblast cells (F180) (4000 cells per well) for 24 h. The best candidates UoST5, 7, 8, 11 at 3 µg/mL and UoST5-NPs at 3, 6.25, 12.5 µg/mL were suspended in DMEM media and filter sterilized prior to application on seeded cells. The color change was measured using a Multiskan Go instrument (Thermo Fisher Scientific, Japan) at 570 nm. Each experiment was repeated six times. Cell viability was calculated, using the following formula (Equation (3) where % of cell death was calculated by subtracting the living cells from the total number of cells [39].

Statistical Analysis
The data was collected and graphed using GraphPad Prism (5.04, GraphPad Inc., La Jolla, CA, USA). The data were analyzed by two-way analysis of variance (ANOVA) using Bonferroni's Multiple Comparison Test. A P-value < 0.05 was considered as significant.

Conclusions
In conclusion, we have developed some novel azole derivatives by replacing the toxic aldehyde group of cuminaldehyde, a natural essential oil, with 1,2,4-triazole rings and the introduction of a 3-trifluoromethylbenzylsulfanyl moiety to selectively enhance the antifungal activity and reduce the toxicity. The anti-Candida activity of the best candidate UoST5 was enhanced and prolonged by encapsulation in polymeric NPs. The formulated drug showed significant anti-Candida activities against C. albicans and C. auris, known multidrug-resistant human pathogens. The superior and prolonged activities were proposed to be due to the nature of the polymer employed since it can afford favorable adherence characteristics to Candida cells and hence can provide direct contact while establishing continuous release.

Conflicts of Interest:
The authors declare there is no competing interest.