CNPs were successfully formed by using ionic gelation technique using sodium tripolyphosphate (TPP) which act as cross-linking agent. Figure 4.2.1 summarizes that the particles size and PDI values of CNPs with the addition of TPP volume (0-300 ?L). These result showed that the particle size of CNPs decreased with increasing TPP, which indicates an increase in cross-linking interaction between positive charge amino groups of chitosan and negative charge of TPP forming nanoparticles 4. Based on the result, the smallest nanoparticles size formed under 100 nm required an at least 150 ?L of TPP, producing CNPs of size 100.5±2.94 nm, 80.22±7.08 nm, 61.45±0.53 nm and 89.79±3.37 nm with PDI values of 0.374, 0.259, 0.226 and 0.200, respectively. Therefore, the smallest particles size of nanoparticles was formed at a volume of 250 ?L of TPP which is 61.45±0.53 nm. The PDI decreased with increasing amount of TPP volume to form a uniform and homogeneous nanoparticles. At lower levels of TPP (<100?L), there is a lower number of linkage between chitosan and TPP to make interaction causing the nanoparticles to agglomerate. Figure 4.2.1 Particles size and polydispersity index (PDI) of empty CNPs with subsequent additions of TPP volume 4.2.2 Formation of CNPs encapsulated with L-ascorbic acid Figure 4.2.2 shows the L-ascorbic acid-loaded into CNPs. These trends show that the CNPs size was directly dependent on the concentration of drug loading. The particle size of the CNPs encapsulated with L-ascorbic acid was the smallest (87.28±3.13) with PDI 0.266 when the concentration of L-ascorbic acid is less concentrated (160 ?M) whereas the particle size was maximized (100.39±2.92 nm) with PDI value 0.333 by increasing the concentration of the drug. However, if the concentration of the drug is too concentrated (310 ?M) the particle size becomes smaller which was 99.33±1.88 nm and its PDI was 0.277 due to the not enough cross-linking of polyanion TPP to hold all L-ascorbic acid particles in chitosan nanoparticles. Generally, encapsulation of the drug causes an increase in CNP size. Therefore, at 235 ?M of L-ascorbic acid, the particle size of CNPs was showed the best size of encapsulation of drug as compared to CNPs without the drug. Figure 4.2.2 Particle size and polydispersity index (PDI) of encapsulated CNPs with increasing concentration of L-ascorbic acid 4.2.3 Formation of CNPs encapsulated with thymoquinone Figure 4.2.3 Particle size and polydispersity index (PDI) of encapsulated CNPs with increasing concentration of thymoquinone DLS data above showed that 100 ?M of thymoquinone producing CNPs of size 100.57±19.69 nm with PDI value of 0.406. Although its PDI value was the highest among the PDI values of other concentrations of thymoquinone, its particles size showed the best size of encapsulation of drug as compared to the empty CNPs. 4.2.4 Formation of CNPs encapsulated with L-ascorbic acid and thymoquinoneTable 4.1 Formulation of L-ascorbic acid and thymoquinone encapsulated CNPsSampleConcentration of L-ascorbic Acid (?M)Concentration of thymoquinone (?M)C1160100C2235100C3310100C4160150C5235150C6310150 Figure 4.2.4 Particle size and polydispersity index (PDI) of encapsulation CNPs with dual drug Figure 4.2.4 showed that L-ascorbic acid and thymoquinone was encapsulated in CNPs. When the dual drug was encapsulated in CNPs, the particle size of CNPs become larger. The sample C1, C3, C4 and C5 showed the smaller particle size of CNPs which was 120.57±9.71 nm, 143.70±2.42 nm, 141.53±7.81 nm and 156.63±12.25 nm with PDI values of 0.238, 0.267, 0.207and 0.235, respectively. However, the best particle size of CNPs for the dual drug is at C2, producing 173.73±9.81 nm with PDI value of 0.210 because the particle size of CNPs was doubled as compared with encapsulation of single drug. Its also have lower PDI value which indicates more monodisperse CNPs with higher particle stability. Thus, it is proven that both drugs are successfully encapsulated in CNPs. 4.3 Entrapment Efficiency 4.3.1 Entrapment efficiency of CNPs encapsulated with single drug. The encapsulated efficiency was detected by measuring the absorbance of total drug and absorbance of free drug in CNPs by varying L-ascorbic acid and thymoquinone concentration as depicted in Figure 4.3.1 (a), Figure 4.3.1 (b) and Figure 4.3.2. The L-ascorbic acid absorbance was taken using UV spectrophotometer at 244 nm while absorbance of thymoquinone was at 254 nm. Figure 4.3.1 (a) Percentage encapsulation of L-ascorbic acid in CNPs at 244 nm Figure 4.3.1 (b) Percentage encapsulation of thymoquinone in CNPs at 254 nm For Figure 4.3.1 (a), the graph showed that 160 ?M of L-ascorbic acid had highest entrapment efficiency which was 69.3±1.8 %. The result showed that as the concentration of the L-ascorbic acid increases, the entrapment efficiency will decrease, which can be explained by the competition between CS and L-ascorbic acid to bind to TPP. However, when compared to the particle size of CNPs, 160 ?M of L-ascorbic acid showed smallest particles size. Therefore, 235 ?M showed the best EE of 64.2±2.8% with a diameter size of 100.39±2.92 nm. For Figure 4.3.1 (b), the obtained result outlined that EE was significantly affected by the drug concentration. The graph showed that 150 ?M of thymoquinone had a greater encapsulated efficiency which was 82.9±1.6%. But, 150 ?M of thymoquinone had smallest particles size. Thus, 100 ?M had the better EE of 72.5±1.3% with a particles size of 100.57±19.69 nm. The calculation of EE for single drug is shown in Appendix I. 4.3.2 Entrapment efficiency of CNPs encapsulated with dual drug. Figure 4.3.2 (a) Percentage encapsulation of L-ascorbic acid in CNPs for dual drug system at wavelength of 244 nm Based on the Figure 4.3.2 (a), the graph showed that C1, C2, and C3 had the highest entrapment efficiency for the dual drug which was 47.9±5.7 %, 31.8±2.8 %, and 73.5±2.4 %, respectively. However, the particles size of CNPs of C3 and C1 was smaller which was 143.70±2.42 nm and 120.57±9.71 nm, respectively. Therefore, C2 showed the best-loaded efficiency of a dual drug in CNPs where it had a particle size of 173.73±9.81 nm and EE of 31.8±2.8%. Figure 4.3.2 (b) Percentage encapsulation of thymoquinone (100 ?M) in CNPs for dual drug system at 254 nm For Figure 4.2.2 (b), the graph showed that C1 and C3 had the highest entrapment efficiency for the dual drug which was 89.4±2.9% and 66.2±2.1%, respectively. Although, C2 had lower EE (62.3±0.9%) it had greater particles size with proof that the both drugs were successfully encapsulated in CNPs. The percentage encapsulation of dual drug is smaller than a single drug because there might have a greater competition for both drugs and CS to bind with TPP.
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