Table 1.
Variables in Box–Behnken design
| Factor Independent variables | Levels used | ||
|---|---|---|---|
| −1 | 0 | +1 | |
| Amount of oil (mL) (X1) | 5 | 10 | 15 |
| Smix (X2) | 1.0 | 1.5 | 2.0 |
| Homogenization Time (min) (X3) | 20 | 25 | 30 |
| Dependent variables | Constraints | ||
| Particle size (nm) (Y1) | Minimize | ||
| PDI (Y2) | Minimize | ||
| Entrapment Efficiency (%) (Y3) | Maximize | ||
Table 2.
Composition and observed responses in Box–Behnken design
| Batch | Drug (mg) | Independent Variables | Dependent variables | ||||
|---|---|---|---|---|---|---|---|
| A: Amount of Oil (ml) (X1) | Smix (X2) | Homogenization Time (min) (X3) | Particle Size (nm) | PDI | %EE | ||
| 1 | 100 | 5 | 1.5 | 30 | 269 | 0.141 | 79.81 |
| 2 | 100 | 10 | 1 | 30 | 224 | 0.106 | 96.01 |
| 3 | 100 | 5 | 1.5 | 20 | 278 | 0.133 | 71.12 |
| 4 | 100 | 10 | 1.5 | 25 | 229 | 0.112 | 85.89 |
| 5 | 100 | 5 | 2 | 25 | 257 | 0.148 | 71.89 |
| 6 | 100 | 5 | 1 | 25 | 271 | 0.141 | 72.45 |
| 7 | 100 | 15 | 1 | 25 | 202 | 0.117 | 84.31 |
| 8 | 100 | 15 | 1.5 | 20 | 203 | 0.105 | 86.93 |
| 9 | 100 | 15 | 2 | 25 | 197 | 0.116 | 89.18 |
| 10 | 100 | 10 | 1 | 20 | 219 | 0.119 | 91.43 |
| 11 | 100 | 10 | 2 | 30 | 218 | 0.121 | 92.87 |
| 12 | 100 | 10 | 1.5 | 25 | 215 | 0.112 | 82.16 |
| 13 | 100 | 15 | 1.5 | 30 | 198 | 0.109 | 84.57 |
| 14 | 100 | 10 | 2 | 20 | 211 | 0.104 | 90.79 |
| 15 | 100 | 10 | 1.5 | 25 | 222 | 0.115 | 87.11 |
| 16 | 100 | 10 | 1.5 | 25 | 217 | 0.111 | 84.94 |
| 17 | 100 | 10 | 1.5 | 25 | 212 | 0.11 | 86.91 |
Figure 1.
UV Spectrum of Flucytosine
Figure 2.
FTIR Spectrum A. Flucytosine (Pure drug), B. Flucytosine with Propylene Glycol and C. Flucytosine with Tween 80
Figure 3.
XRD of Flucytosine
Figure 4.
(A) Response 3D plots and Cube plots for the effect of amount of oil (X1), Smix(X2) and homogenization time (X3) on particle size, (B) Response 3D plots and Cube plots for the effect of amount of oil (X1), Smix(X2) and homogenization time (X3) on PDI, and (C) Response 3D plots and Cube plots for the effect of amount of oil (X1), Smix(X2) and homogenization time (X3) on % EE
Table 3.
Characterization Parameters of Flucytosine Nanoemulsions
| Formulation | Zeta Potential (mV) | pH | Drug Content |
|---|---|---|---|
| F1 | −14.2 ± 1.3 | 6.41 ± 0.04 | 91.2 ± 1.5 |
| F2 | −16.1 ± 1.4 | 6.46 ± 0.03 | 92.6 ± 1.4 |
| F3 | −17.8 ± 1.2 | 6.52 ± 0.02 | 93.8 ± 1.3 |
| F4 | −19.6 ± 1.3 | 6.58 ± 0.03 | 95.1 ± 1.2 |
| F5 | −21.4 ± 1.1 | 6.63 ± 0.02 | 96.4 ± 1.1 |
| F6 | −23.1 ± 1.4 | 6.69 ± 0.03 | 97.6 ± 1.0 |
| F7 | −27.6 ± 1.2 | 6.75 ± 0.02 | 98.5 ± 0.8 |
| F8 | −29.1 ± 1.3 | 6.82 ± 0.03 | 97.0 ± 0.7 |
| F9 | −31.5 ± 1.1 | 6.88 ± 0.02 | 96.3 ± 0.6 |
Figure 5.
TEM of Optimized Flucytosine Nanoemulsion (F7)
Figure 6.
In-vitro drug release profile of various batches of nanoemulsion formulations
Figure 7.
In-vitro drug release kinetics of Optimized Flucytosine nanoemulsion
Table 4.
Coefficient of determination (R2) values for different kinetic models for the optimized flucytosine nanoemulsion formulation (F7)
| Kinetic Model | Coefficient of determination (R2) |
|---|---|
| Zero Order | 0.8977 |
| Frist Order | 0.9978 |
| Higuchi Model | 0.9898 |
| Korsmeyer-Peppas | 0.9786 |
Table 5.
Antifungal Activity of Flucytosine Nanoemulsion by Zone of Inhibition
| Sample | Zone of Inhibition (mm) |
|---|---|
| Flucytosine Solution | 14.2 ± 0.8 |
| Flucytosine Nanoemulsion (F7) | 22.6 ± 1.1 |
| Blank Nanoemulsion | No zone |
| Standard Antifungal (Fluconazole) | 24.1 ± 0.9 |
Table 6.
Stability studies result of optimized formulation (F7)
| Storage Condition | Time (Months) | Appearance | Particle Size (nm) | PDI | Zeta Potential (mV) | pH | % EE | Drug Content (%) |
|---|---|---|---|---|---|---|---|---|
| Initial | 0 | Clear, homogeneous | 104.3 ± 1.9 | 0.15 ± 0.01 | −27.6 ± 1.2 | 6.75 ± 0.02 | 94.8 ± 1.1 | 98.5 ± 0.8 |
| 4 ± 2 °C | 1 | No change | 105.1 ± 2.1 | 0.16 ± 0.01 | −27.2 ± 1.3 | 6.74 ± 0.03 | 94.2 ± 1.2 | 98.1 ± 0.9 |
| 2 | No change | 106.4 ± 2.3 | 0.17 ± 0.02 | −26.9 ± 1.4 | 6.73 ± 0.03 | 93.6 ± 1.4 | 97.8 ± 1.0 | |
| 3 | No change | 107.2 ± 2.5 | 0.18 ± 0.02 | −26.5 ± 1.5 | 6.72 ± 0.04 | 92.9 ± 1.6 | 97.4 ± 1.1 | |
| 25 ± 2 °C / 60 ± 5% RH | 1 | No change | 106.3 ± 2.4 | 0.17 ± 0.01 | −26.8 ± 1.3 | 6.73 ± 0.03 | 93.8 ± 1.3 | 97.9 ± 0.9 |
| 2 | No change | 108.5 ± 2.6 | 0.18 ± 0.02 | −26.2 ± 1.4 | 6.71 ± 0.04 | 92.7 ± 1.5 | 97.2 ± 1.1 | |
| 3 | No change | 110.2 ± 2.9 | 0.19 ± 0.02 | −25.6 ± 1.6 | 6.69 ± 0.05 | 91.8 ± 1.7 | 96.6 ± 1.2 | |
| 40 ± 2 °C / 75 ± 5% RH | 1 | No change | 109.6 ± 2.8 | 0.19 ± 0.02 | −25.9 ± 1.4 | 6.70 ± 0.04 | 92.4 ± 1.6 | 96.9 ± 1.2 |
| 2 | Slight turbidity | 112.8 ± 3.1 | 0.21 ± 0.02 | −25.1 ± 1.6 | 6.68 ± 0.05 | 91.2 ± 1.8 | 96.1 ± 1.3 | |
| 3 | Slight turbidity | 116.5 ± 3.4 | 0.23 ± 0.03 | −24.3 ± 1.7 | 6.65 ± 0.06 | 89.7 ± 2.0 | 95.4 ± 1.5 |