br Optimization br The desirability function was determined
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 Kainic acid 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.
The IC50 values of free SUN, SUN-NLCs, and biotin-SUN-NLCs in A549 cells were 3.14, 2.17, and 1.66 μg/mL, respectively, which ex-hibited better cellular cytotoxicity of biotin-SUN-NLCs in A549 cells. A similar result was also reported for biotin-decorated pluronic® P123/ F127 mixed micelles used for niclosomide delivery compared to the non-targeted micelles and free niclosomide .
In addition to biotin-SUN-NLCs and SUN-NLCs, the cytotoxicity of drug free targeted and non-targeted NLCs, in the same concentration as used for drug loaded NLCs was also investigated. As it can be seen in Fig. 10, at low concentrations, the drug free non-targeted NLCs showed negligible toxicity towards A549 cells, but at higher concentrations, a little toxicity was seen with cell viability of near 70%. Drug free tar-geted NLCs showed higher cytotoxicity than non-targeted ones due to projection of positively charged B-SA conjugates on the surface of the NLCs . In agreement with our study, the study conducted by Var-shosaz et al.  also showed that positively charged blank NLCs had cytotoxic effects on K562 cells. This phenomenon may be also involved
Fig. 9. SEM images of optimized biotin-SUN-NLCs.
Physical properties of SUN loaded targeted NLCs and non-targeted ones.
in higher toxicity of biotin-SUN-NLCs in comparison with SUN-NLCs and free SUN. To compensate impaired safety of these nanocarriers, increment of drug loading % or biotinylation of other fat soluble che-micals such as cholesterol is needed.
3.9. Cellular uptake assay
For investigating the cellular uptake of developed NLCs, both biotin-NLCs and NLCs were loaded with C6. C6 is an efficient compound commonly used as a fluorescent probe for cellular uptake investigation due to its unique properties such as high fluorescent intensity and small leakage rate from NPs formulation . Flow cytometry demonstrated enhanced uptake of biotin-C6-NLCs by A549 cell compared with C6-NLCs. Based on the geometric means of each histogram in Fig. 11, A549 cells treated with biotin-C6-NLCs exhibited higher fluorescence intensity than C6-NLCs, indicating that biotin decoration on particle surface could considerably facilitate the uptake of NLCs by A549 cells via receptor mediated endocytosis. Fluorescent microscope further