Pre-Formulation StudiesFourier-Transform Infrared Spectroscopy (FTIR)

FTIR was used to study the interactions of fluconazole, cholesterol, span 60, physical mixture of fluconazole and cholesterol (PM: F/C), physical mixture of fluconazole and span 60 (PM: F/S), and physical mixture of fluconazole, cholesterol, and span 60 (PM: F/C/S). The results are shown in Fig. 1. Fluconazole shows six characteristic peaks at 1627 cm− 1 corresponding to the Triazole ring, 3120 cm− 1 corresponding to the -OH stretching, 1422 cm− 1corresponding to the -CH3 bending, and 1292 cm− 1corresponding to the bending vibration of the -CH2 group, 1089 cm− 1 corresponding to (C–OH bond) and 1107 cm− 1 corresponding to (C–F bond). The same results were obtained by Fatima et al., 2022 and Moraes et al., 2017 [28, 44]. Asymmetrical stretching of the cholesterol’s C-H bond was seen at 2941 cm-1, whereas the stretching -OH cholesterol was seen at 3359 cm− 1 as shown in Fig. 1. Farmoudeh et al., 2020 reported the same results of cholesterol [45]. Span 60 showed aliphatic C-H stretching at 2882 cm− 1, -CH3 group stretching at 1458 cm− 1, and O-H stretching at 3390 cm− 1. At 1744 cm− 1, the cyclic five-membered ring’s peak in span 60 was noted. The results were matched with Fatehi et al., 2020 and Miatmoko et al., 2021 [46, 47]. The characteristic peak of pure fluconazole has appeared in all physical mixtures with no change. In the three physical mixtures, FTIR showed all the peaks of cholesterol, and/or span 60 at their places confirming the absence of any chemical interactions between them.

Fig. 1

FTIR spectrum of fluconazole, cholesterol, span 60, physical mixture of fluconazole and cholesterol (PM: F/C), physical mixture of fluconazole and span 60 (PM: F/S), and physical mixture of fluconazole, cholesterol, and span 60 (PM: F/C/S)

Differential Scanning Calorimetry (DSC)

Figure 2 shows the DSC thermogram of Flu, Cholesterol, span 60, Flu/cholesterol physical mixture, Flu/ Span 60 physical mixture, and Flu/Cholesterol/span 60 physical mixture. Flu thermogram reveals endothermic peaks at 101, 146, 360, and 440 and another peak appeared at 246.75 °C. The first peak corresponds to crystal dehydration, and the temperature matches the boiling point of water. The second peak corresponds to the crystals melting point. Finally, the third and fourth peaks correspond to the degradation of fluconazole. The results are in good agreement with Akay et al., 2021 [48]. The melting points of cholesterol were observed at an endothermic peak at 149 ℃ and 208.1 °C due to degradation and abroad peak at 44.6 C, due to the loss of water molecules as reported by Abd-Elal et al., 2016 [49]. The same results were obtained by Yasam et al., 2016 when studying the DSC thermogram of cholesterol [50]. The main melting peaks of span 60 were observed at 64℃ which is matched with El-Ridy et al., 2018 [51]. The results of DSC of the physical mixtures demonstrated that Flu and the excipients melting peaks appeared in their places without undergoing shifting, and with no extra peaks appearing, showing an absence of interaction between Flu and excipients.

Fig. 2

DSC of fluconazole, cholesterol, span 60, physical mixture of fluconazole and cholesterol (PM: F/C), physical mixture of fluconazole and span 60 (PM: F/S), and physical mixture of fluconazole, cholesterol, and span 60 (PM: F/C/S)

X-Ray Diffraction Scanning (XRD)

The characteristic (XRD) patterns of fluconazole, Cholesterol, span 60, Physical mixture of fluconazole and Cholesterol (PM: F/C), Physical mixture of fluconazole and span 60 (PM: F/S), and Physical mixture of fluconazole, Cholesterol, and span 60 (PM: F/C/S) are compared and illustrated in Fig. 3. The diffraction pattern of fluconazole showed high-intensity crystallinity peaks at 16.584, 25.652, and 29.30 °2θ. The peak with 100% relative intensity was present at °2θ value of 20.03. These results are similar to that of Modha et al., 2010 [52]. On the other hand, the diffraction pattern of cholesterol showed an intense peak at 12.74, 14.14, 15.65, 16.85, 17.27, and 21.44 (100% relative intensity) and 23.89°2θ [53], while the diffraction pattern of span 60 showed a 100% relative intensity at 21.433 °2θ. When the diffractograms of the physical mixtures and fluconazole were compared, all the characteristic crystalline peaks for fluconazole could be observed with the same intensity revealing that no changes were made to the fluconazole.

Fig. 3

X-ray diffractograms of fluconazole, cholesterol, span 60, physical mixture of fluconazole and cholesterol (PM: F/C), physical mixture of fluconazole and span 60 (PM: F/S), and physical mixture of fluconazole, cholesterol, and span 60 (PM: F/C/S)

Experimental Design and Optimization of

The goal of the design expert optimization is to study the effect of experimental factors on the formulation’s responses. Table 2 shows the factors and the observed responses. The two factors’ interactions (2FI) model was the best fitting model for Y1, Y2, and Y3 responses because it has the highest R2 and the lowest predicted residual error sum of squares.

Table 2 Composition and the observed responses of the designThe Effect of Investigated Variables on ResponsesPS

The size of vesicles is an important consideration in the formulation and optimization of nanocarriers. Flu niosomes sizes ranged from 228.2 to 769.2 nm, as appeared in Table 2. Three FI model was significant at (p < 0.001), with a regression coefficient (R2) of 0.9998 according to ANOVA analysis indicating the goodness of fit. Also, there was an agreement (less than 0.2) between the predicted R2 (0.9990) and the adjusted R2 (0.9996). The equation that describes the relationship between particle size and formulation factors is:

$$\eqalign}\left( }_}}} \right)\,}\,}}\,}\,}}\,}\,}}\,}\,}} \cr & }\,}}\,}\,}}\,}\,}}\,}\,}} \cr}$$

(3)

Where A, B, and C correspond to X1, X2, and X3 respectively.

The prepared niosomes have a small particle size on the nanoscale with homogenous distribution according to the result of the polydispersity index (PDI). PDI was found to be lower than 0.7 for most formulations. The polydispersity index (PDI) has values less than 0.7, indicating a narrower size distribution, compared to the particles in a wide distribution which tend to aggregate [54]. Figure 4 and Eq. 3, reveal that PS increases significantly with the increased drug amount at (P < 0.001) and this result was in great agreement with the previous work done by El-Far et al., 2022 [55] which stated that drug interacts with the surfactant head groups, increases the charge and mutual repulsion of the surfactant bilayers and thus increases vesicle size. The particle sizes are influenced by the surfactant: cholesterol ratio as shown in Fig. 4; Table 2. Cholesterol is a necessary component in the formation of noise. It improves vesicle stability and plays an important role in bilayer packing [56]. Increasing the surfactant: cholesterol ratio to 2:1 reduced the mean diameter of the particles. This result agreed with the previous data, indicating that a decrease in cholesterol caused the vesicle size to decrease [57]. This could be interpreted by the high levels of cholesterol disrupting the niosomal membrane. Because cholesterol is amphiphilic, it intercalates within the bilayer structure of niosomes by orienting its polar head toward the water-soluble surface and aligning its aliphatic tail parallel to the hydrocarbon chains, forming large vesicles [58]. Teaima et al., 2020 showed that “cholesterol is a stiff, inverted cone-shaped molecule. When hydrated at a temperature above the gel/liquid transition temperature, it can be intercalated between the surfactant’s fluid hydrocarbon chains, increasing the size of the vesicle” [59]. The addition of CHO to a membrane increases the membrane bilayer rigidity and reduces water-soluble substance leakage through membranes. Surfactant monomers were closely packed with increasing curvature and decreasing size at low CHO concentrations. On the other hand, at high CHO concentration, and low nonionic surfactant content, the lipophilicity of the bilayer membrane increases (log P of 7.02). Also, it may increase the vesicle radius and create a thermodynamic stable form causing disturbance in the vesicular membrane [60]. Moreover, CHO can remove the vesicle’s phase transition temperature peak leading to stabilize the bilayer structure, thereby strengthening the bilayer structures and decreasing bilayer micro fluidity [61]. It was found that the increase in surfactant amount (span 60) and decrease in cholesterol amount resulted in a significant decrease in the particle size as shown in Fig. 4; Table 2. This may be due to the physical characteristic of span 60 as a solid surfactant with low HLB (HLB = 4.7), higher phase-transition temperature (53–57 °C), and surface free energy, which decreases with increasing lipophilicity [56, 62]. The produced niosomes had with larger mean vesicle diameter in the solvent injection method (B1) as compared to the thin film hydration method (B2). These findings were in great agreement with that of Kumar et al., 2013 who investigated the influence of various preparation techniques on the formulation of Diclofenac niosomes [63]. Also, the same findings were obtained by Mujeeb et al., 2017 [64].

Fig. 4

3D surface response plots showing the impact of the independent variables on (A)PS(Y1), (B)ZP(Y2), and (C)EE% (Y3)

ZP

The developed 3FI model for the preparing method factor (Y2) was significant at (p < 0.0006), with a regression coefficient (R2) of 0.9473 according to ANOVA analysis showing the model is well fit. The predicted R2 (0.7246) agrees with the adjusted R2 (0.8946) (less than 0.2). The adequate precision was 13.94 suggesting that the signal was adequate. The equation that describes the relationship between ZP and formulation factors is:

$$\eqalign}\left( }_}}} \right)\,}\,}}\,}\,}}\,}\,}}\,}\,}} \cr & }\,}}\,}\,}}\,}\,}}\,}\,}} \cr}$$

(4)

Where A, B, and C correspond to X1, X2, and X3 respectively. Values were between − 18.1 and − 60.2 mV in all cases. All the formulations presented negative values of zeta potential due to hydroxyl ions adsorption on the surface of the vesicle by non-ionic surfactants and to the effect of CHO, which produces a negative charge on the vesicle surface [55]. The higher electrostatic repulsion results in higher negative values providing more stability [65]. The thin film hydration method (B2) provided more stable niosomes than that of the injection method (B1). These findings were in great agreement with Mujeeb et al., 2017 [64].

EE%

The EE% of fluconazole niosomes ranged from 51.3 to 75%, as demonstrated in Table 2. Three FI model was significant at (p < 0.0005), with a regression coefficient R2 of 0.9496 according to ANOVA analysis showing that the model is well fit. Also, the predicted R2 (0.7369 agrees with the adjusted R2 (0.8994), and the difference is less than 0.2. The equation that shows the relationship between EE% and formulation factors is:

$$\eqalign}\,\left( }_}}} \right)\,}\,}}\,}\,}}\,}\,}}\,}\,}} \cr & }\,}}\,}\,}}\,}\,}}\,}\,}} \cr}$$

(5)

Where A, B, and C correspond to X1, X2, and X3 respectively.

As represented in Fig. 4, increasing the drug ratio resulted in a non-significant increase in the EE% of fluconazole, with p values = 0.5529. A higher surfactant ratio led to a significantly higher EE%, which may be related to span 60 has phase transition temperature (53 °C) which resulted in a decrease in the fluidity and breakage of the bilayer. Also, span 60 has a HLB value of (4.7) with a long C17 chain), which increases its hydrophobic character to be able to hold the hydrophilic drugs inside its core [66]. Previous research has shown that when CHO is used above a certain optimum concentration, it may decrease hydrophilic drugs EE%, the reason may be because of a disruption in the bilayer physical organizational structure, leading to leakage [66].

Selection of the Optimized Flu-Nio

The desirability function was based on the conditions for attaining minimum PS (Y1), maximum ZP (Y2), and maximum EE% (Y3). The Flu-Nio optimized formula was developed by using a fluconazole amount of 20 mg (X1), solvent injection as a method of preparation (X2), and a Surfactant: cholesterol ratio of 2:1 (X3). The optimized formula is predicted to have PS (Y1) of 257.1 nm, ZP (Y2) of -46.35 mv as shown in Fig. 5, and EE% (Y3) of 71.5% achieving 0.747 the highest desirability. In addition, it was evaluated and compared to the observed values. The percentage error was 1.021, 2.52, and 4.67 for PS, ZP, and EE% respectively. Also, the chosen formula showed a PDI of 0.11 ± 0.09 indicating the homogeneity of the chosen formula.

Transmission electron Microscopy (TEM)

The selected formula morphology is shown in Fig. 5. Flu-Nio-laden contact lenses have a smooth well-defined sphere shape with a relatively uniform size distribution and a definite wall with an aqueous core. Flu-Nio-laden contact lens size obtained from the TEM examination agreed with the PS analysis and it showed a uniform size distribution.

Fig. 5

(A and B) Zeta potential and particle size of fluconazole niosmal optimized formula (C)TEM of fluconazole niosmal optimized formula

Cytotoxicity Test

The cytotoxicity of the optimized formula Flu- Nio, Flu solution, and control were performed to demonstrate their effect on Mouse endothelial cells. The results revealed that Flu- Nio, Flu solution at a concentration of 10 µg/ml, and the control have a cell viability % of 99.22, 99.67, and 100% respectively, with no statistically significant difference detected. These results indicate that Flu- Nio, Flu solution at a concentration of 10 µg/ml had no harmful impact on normal Mouse endothelial cells as shown in Fig. 6. According to cytotoxicity test results, Flu-Nio is biocompatible and has low cytotoxicity. Fluconazole contact lenses showed non-significant variation in the loaded Flu concentration after soaking at both 4 and 7 days at p < 0.001.

Fig. 6

Cytotoxicity profile of the optimized Flu-Nio, Flu solution, (at a concentration of 10 µg/ml) and control on C-166: Mouse Endothelial Cell after 24 h exposure to the Flu-Nio. Cytotoxicity test was done using sulforhodamine B (SRB) (0.4% w/v) and is represented as the cell viability percentage after applying the treatment

Fluconazole Quantification in the Contact Lenses

The concentration of Flu up taken in the contact lenses was assessed at different time intervals (at 4 days and 7 days) to show the maximum drug loaded. The results showed each contact lens contained 3.02 ± 0.23 and 3.11 ± .081for both 4 and 7 days respectively.

Light Transmission

The free contact lenses were clear and transparent; their light transmission measured at 600 nm showed a high transmission of 95.65 ± 0.55%. The incorporation of Flu-Nio did lower the transmission to 90.743 ± 0.47% with no significant difference at (p > 0.05) with the free contact lenses because the nanosized particles diffract and scatter the incident lights [6]. The use of contact lenses with that amount of Flu-Nio would not affect the vision of the wearer which is essential [67].

In vitro Release of Fluconazole from (Flu-Nio) Laden Contact Lenses

In-vitro cumulative release of fluconazole from the optimized (Flu-Nio) laden contact lenses was compared with free drug solution and the results are shown in Fig. 7. The fluconazole from Flu- Nio-laden contact lenses was released and showed an initial burst release in the first hour (27.2% at 1 h), then it showed a prolonged release up to 48–72 h with a cumulative release of 79.62%. In contrast, the release of fluconazole from the control solution showed a high burst release in the first hour (46.4% at 1 h) and a 100% cumulative drug release after three hours. In addition, Flu-Nio is an efficient fluconazole carrier. The initial phase release of fluconazole from Flu-Nio-laden contact lenses could be attributed to free fluconazole penetration and drug desorption from the niosome surface. Free drug solution exhibited a significant (P < 0.05) higher and faster release than that from optimized Flu-Nio-laden contact lenses which may be attributed to the cholesterol of noisome that decreases the leakage of encapsulating fluconazole by reducing the niosomal membrane fluidity [68]. The results suggest that niosomes could be used to extend the release of fluconazole. According to the higher correlation coefficient, the drug release from the optimized Flu-Nio-laden contact lenses is best fitted to Baker and Lonsdale equation, indicating that fluconazole release from the vesicles could be attributed to the diffusion mechanism [69].

Fig. 7

Percentage cumulative release of fluconazole from Flu-Nio-laden contact lenses and fluconazole solution in simulated tear fluid (pH 6) at 37ºC (n = 3, mean ± standard deviation)

Antimicrobial Activity StudyAntifungal Susceptibility by Disc Diffusion Method

Figure 8 shows the zone of inhibition of Flu-Nio-laden contact lenses (contact lens impregnated with Flu-Nio), Flu laden contact lenses (contact lens impregnated with Flu solution) and, Flu-Nio disc (sterile filter paper disc impregnated with Flu-Nio) and Flu disc (sterile filter paper disc impregnated with Flu solution) on C. albicans (ATCC® 10231). Flu-Nio-laden contact lenses and Flu-Nio disc increased the diameter of the zone of inhibition and showed an increase in antifungal activity. Ciolino et al., 2011 [70] suggested that econazole-releasing contact lenses made of econazole-PLGA film encased within a pHEMA hydrogel preserved their fungicidal effects for three weeks. In addition, the formulation provides an alternate treatment for fungal keratitis and serves as a platform for ocular medication delivery. When designing a contact lens for antifungal ocular drug delivery, Phan and colleagues demonstrated that some aspects should be considered, such as (a) increasing the drug amounts to be loaded or released from the contact lenses; (b) the drug release rate from the contact lenses; and (c) consistent release with known concentration. The qualities of the lens material, the properties of the target drug, and the interactions between the polymer and the drug on the lens all have a substantial impact on the drug’s uptake and release [71]. Previous research suggested that medications with a higher water solubility, such as fluconazole, might partition more quickly into aqueous systems. Furthermore, this causes fast drug release from the contact lenses, as seen with several ophthalmic medicines.

Fig. 8

Shows the zone of inhibition of Flu-Nio laden contact lenses, Flu-laden contact lenses, Flu-Nio disc, and Flu disc on C. albicans (ATCC® 10231), the growth inhibition was measured in mm after overnight incubation at 37 °C. Each experiment was done in triplicates, and an average data set is shown

Determination of the Fungal Adhesion

Figure 9 shows the adherence of C. albicans on Flu-Nio-laden contact lenses compared with Flu-laden contact lenses, and the control lenses. The number of viable cells that could be cultivated after being released from the surface of contact lenses is used for measuring adhesion. Adhesion was reduced for both Flu-Nio and Flu-laden contact lenses. The 1 × 103 cells/ml that were initially added to the control lenses had increased to 8.3 ± 0.74 log CFU/lens on the lenses after the culture. Figure 9 shows that there was almost a 2 log-fold reduction of cell adherence to Flu-Nio-laden contact lenses compared with only a 1 log-fold reduction of cell adherence to Flu-laden contact lenses.

Fig. 9

Shows the effect on Candida albicans adhered to Flu-Nio-laden CL and Flu-laden CL, compared to that attached to control lenses. Adhesion, measured as the number of culturable viable cells after release from the lens surface, was reduced for both treated contact lenses compared to control lenses. The viability of adhered cells is represented as a median with an interquartile range after overnight incubation at 37 °C. Each experiment was done in three different cultures, and an average data set was taken. Statistical analysis was done using the student’s t-test

Statistical analysis using student’s t-test showed that there was a significant difference between the adhesion of C. albicans on Flu-Nio laden contact lenses and Flu-laden contact lenses compared with the control lenses (P < 0.01) and showed that Flu-Nio laden contact lenses had significantly lower fungal adherence than Flu laden contact lenses. This reduction in the fungal adhesion may be attributed to the small-sized Flu noisome which slowly diffuses Flu and acts by interacting with lanosterol 14-α demethylase, a cytochrome P-450 enzyme that converts lanosterol to ergosterol (essential for the fungal cell membrane) when its synthesis is suppressed, cells become more permeable and their contents pass out [33, 72, 73].

Determination of the Remaining C. Albicans in the Solution

The number of C. albicans cells in the PBS (pH) around the lenses throughout incubation was also measured (Fig. 10). The count of CFU per 1 mL of PBS after 24 h incubation surrounding the control lens was 8.5 ± 0.72 log CFU/lens. Flu-Nio-laden contact lenses showed a 2 log-fold reduction of cell number compared with Flu-laden contact lenses that showed only a 1 log-fold reduction of cell growth. These results indicate that during incubation, the drug was released into the fluid around the lenses and was effective against C. albicans. Statistical analysis using student’s t-test showed that there was a significant difference between the remaining viable cells in the surrounding solution of Flu-Nio-laden contact lenses and Flu-laden contact lenses in comparison with control lenses (P < 0.0001) and showed that Flu-Nio laden contact lenses significantly reduced the microbial viable count than Flu laden contact lenses (P < 0.001). This could be attributed to the low diffusion of the Flu from the Flu-laden contact lens through the media. On the contrary, niosomes had resulted in better diffusion of the Flu from Flu-Nio-laden contact lenses, with a reduction in the fungal adhesion. The reason may be attributed to the small, lipophilic nature, and abundance of non-ionic surfactants [74]. Previous studies conducted by Willcox and co-workers found that the bacterial viability and adhesion for the tested strains was reduced by > 5 log reduction in solution or on the lens surface using silver (20 ppm) nanoparticles lenses [42].

Fig. 10

Shows the effect on C. albicans remaining viable cells in the solutions surrounding Flu-Nio-laden CL and Flu-laden CL, compared to that surrounding the control lenses. The viability of adhered cells is represented as a median with an interquartile range after overnight incubation at 37 °C. Each experiment was done in three different cultures, and an average data set was taken. Statistical analysis was done using a student’s t-test where statistical significance is represented by ***P < 0.001 and **** P < 0.0001