br As indicated in Fig EE was mostly influenced
As indicated in Fig. 3, EE was mostly influenced by lipid/drug weight ratio. Increment of drug content significantly increased EE (Fig. 5a) possibly due to good entrapment of drug in the lipids . Surfactant content and aqueous/organic phase volume ratio and
Fig. 4. Effects of different studied parameters on particle size of biotin-SUN-NLCs.
Fig. 5. The effects of different studied parameters on encapsulation efficiency of biotin-SUN-NLCs.
Fig. 6. The effects of different studied parameters on zeta potential of biotin-SUN-NLCs.
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Fig. 7. SUN release profiles from each studied formulation of biotin-SUN-NLCs.
interaction between of each two pairs of factors were also significantly affecting EE. EE decreased with increasing surfactant concentration (Fig. 5c) probably due to increase in the solubility of SUN in the aqu-eous phase owing to the solubilization effect of the emulsifier . Increasing aqueous/organic phase ratio also decreased the EE (Fig. 5d). This could be attributed to rapid diffusion of the organic solvent mo-lecules in water and subsequently fast partitioning of the drug mole-cules into the aqueous phase before the droplet hardening occurred . This finding was in accordance with the previous study of Var-shosaz et al.  who showed a decrease in valproic 1,2-Dioleoyl-sn-glycero-3-PC EE with in-creasing aqueous/organic phase ratio.
Zeta potential is the electric charge at the surface of particle and considered as a key factor to evaluate the physical stability of colloidal suspensions or emulsions. Particle aggregation is less likely to occur for charged particles due to electric repulsion. It is known that zeta
potential between ± 5 and 15 mV are in the region of limited floccu-lation. Steric hindrance provided by sterically stabilizing surfactants such as non-ionic surfactants also impact on particle stability. When the surfactants are employed, even lower zeta potential value is sufficient to ensure good colloidal stability . As depicted in Table 3, the zeta potential values of biotin-SUN-NLCs were positive and varied from 0.96 to 17.33. The positive charges on particles originated from projection of amino groups of B-SA conjugates on the outer surface of the NLCs ex-posed to the external phase. The following equation shows the effect of each studied factor on zeta potential.
Analysis of zeta potential data revealed that the lipid/drug weight ratio is the most effective (p < 0.05) variable on zeta potential of the NPs (Fig. 3). DOE result indicated drug content, liquid lipid to total lipid ratio, surfactant concentration in interaction with aqueous/or-ganic phase volume ratio or drug content, aqueous/organic phase vo-lume ratio in interaction with drug content had significant effect on the zeta potential of NLCs. As shown in Fig. 6a, zeta potential value de-creased when drug content in NLC was increased. As described earlier, increasing drug content increased the particle size of biotin-SUN-NLCs. The bigger particle size owned the lower charge density which might be the result of smaller surface-to-volume area of larger particles . The zeta potential was also strongly influenced by the liquid lipid to total lipid ratio. It is evident from Fig. 6b, zeta potential increased with in-creasing liquid lipid to total lipid ratio. This could be related to change in the crystalline structure and crystalline re-orientation of the lipid. Given that different sides of a crystal can possess a different charge density, variation in the crystalline structure and crystalline re-or-ientation of the lipid may be led to change charges on the particle surface .
Fig. 8. The effects of different studied parameters on release efficiency of biotin-SUN-NLCs.
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Fig. 7 shows the drug release profiles for each studied formulation. To compare release profiles, RE8% was calculated. Based on in-vestigated results, the range of RE8% was 28.84–64.47%. Fig. 3 shows the most effective parameter on RE8% was interaction of liquid lipid/ total lipid ratio and aqueous/organic phase volume ratio. Except the interaction of surfactant concentration and liquid lipid/total lipid ratio that did not have significant effect on the RE8%, other main variables and their interaction had significant effect on this parameter. The fol-lowing equation shows the effect of each studied factor on RE8%.
As can be seen in Fig. 8a,b&c, RE8% decreased when drug content, labrafac/lipid ratio as well as organic/aqueous phase ratio increased (P < 0.05). These changes also increased particle size. Larger particles showed slower rate of drug release owning to decrease surface area of NPs and increase length of the diffusion path that the drug had to travel to reach dissolution medium. Taymouri and co-workers  also found that reduction in particle size of nanomicelles increased release rate of docetaxel. It can be seen from Fig. 8d, increasing in PF127 concentra-tion in the external aqueous phase caused an increase RE8% owing to the solubilization effect of the emulsifier.