br Fig The effects of different studied parameters on
Fig. 8. The effects of different studied parameters on release efficiency of biotin-SUN-NLCs.
S. Taymouri et al.
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 Trichostatin A 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.
The desirability function was determined using Design Expert Software to achieve the optimized formulation. The optimum for-mulation was based on the set criteria of maximum EE, maximum zeta potential, minimum particle size, minimum PdI and RE8% in the range. Based on the modeling generated by Design Expert Software, the op-timized formulation suggested by desirability of 75% was D10O5L15S0.5 that was prepared using 6 mg SUN, 45 mg Chol, 9 mg labrafac and 6 mg B-SA conjugate when the surfactant level was almost 0.5% and the aqueous/organic phase ratio was 5. The optimized for-mulation exhibited a particle size of 125.50 nm, EE of 85.10%, zeta potential of 10.23 mV, drug release efficiency of about 62.85% during 8 h and PdI of 0.22. As it can be seen in Fig. 9, optimized biotin-SUN-NLCs were spherical with smooth surface. To evaluate the release ki-netics and mechanism of release from optimized biotin-NLCs, SUN re-lease data was studied by best curve fitting with different kinetic models such as Higuchi, Baker-lonsdale, first order, zero order and Korsmeyer-Peppas model. Considering the highest correlation coeffi-cient, SUN release kinetics from optimized biotin-NLCs followed Hi-guchi model (R2 = 0.9805). The slopes obtained from the Korsmeier-
Peppas model was found to 0.5783 indicating that the release was mainly controlled by diffusion coupled with erosion (anomalous dif-fusion mechanism). SUN loaded non-targeted optimized formulation (non-targeted D10O5L15S0.5) was also developed by replacing B-SA conjugates with Chol as described in section 2.3. The characteristics of non-targeted ones are summarized in Table 4. The release profile of the drug from non-targeted NLCs was shown in Fig. 7. The slightly faster release of drug from targeted D10O5L15S0.5 could be related to smaller particle size of these NPs compared to non-targeted ones. As particle sized decreased, the contacting surface area of NPs increased and the length of diffusion path decreased. As a result, the release rate drug increased .
3.8. Cell viability assay
The cellular toxicities of free SUN, SUN-NLCs and biotin-SUN-NLCs in A549 cells were studied by MTT assay which are known to express high level of biotin . As shown in Fig. 10, the cell suppression of all drug loaded NLCs and free SUN increased in dose dependent manner, in which biotin-SUN-NLCs had the highest cell cytotoxicity compared to SUN-NLCs and free SUN. The increased cytotoxicity of biotin-SUN-NLCs could be attributed to the fact that biotin-SUN-NLCs easily entered into the cells via receptor mediated endocytosis. From the results, SUN-NLCs also caused higher cytotoxicity compared to the free SUN at the same concentration. The results well correlated with previous studies which demonstrated higher cytotoxicity of lipid based NPs entrapping drug compared to free drugs [22,23]. This may be attributed to the efficient adherence of lipid NPs to the cell membrane, internalization inside the cell by endocytosis and enhance intracellular drug accumulation.