br Independent variables levels br Dependent variables br
Independent variables levels
Lipid/drug ratio (w/w) 5 10 Particle size (nm)
Aqueous to organic phase volume ratio 5 10 Polydispersity index
Surfactant concentration (%) 0.5 1 Encapsulation efficiency
Release efficiency (%)
S. Taymouri et al.
Composition of different studied biotin modified NLCs loaded with SUN pro-duced by irregular full factorial design.
Formulations Lipid/ Aqueous to Oil content Surfactant
drug organic phase (% total concentration (%)
ratio volume ratio lipid)
D: Lipid/Drug ratio O: Aqueous/organic phase ratio L: Labrafac/lipid ratio S: Pluronic F127 (%).
The un-encapsulated drug in supernatant was determined by measuring the UV absorbance at 432 nm using a UV-VIS spectrophotometer. After calculating the quantity of free drug, the drug EE in the NPs were de-termined using following equations:
total amount of drug added
The in vitro release study was carried out using dialysis method. Briefly, the appropriate amount of drug-loaded NLCs were placed in a dialysis bag (molecular cut off 12 000 Da, Sigma, US.) which were sealed at both the ends and dialyzed against PBS (pH 7.4) containing 0.1% Tween 80 to provide sink condition. At predefined intervals, 1 mL of receiving buffer solution was withdrawn and replaced with equal volume of fresh PBS. Finally, the amount of SUN released was de-termined at 432 nm by a UV spectrophotometer. The parameter of RE8% was used to compare release profile and calculated by equation
Where y is the released percent at time t.
2.8. Cell viability assay
The cytotoxicity of free SUN, SUN-NLCs, and biotin-SUN-NLCs and blank NLCs was evaluated on A549 Nutlin-3 by MTT assay. The cells were seeded at a density of 5 × 103 cells/well in 96-well plates. After 24 h of incubation, they were incubated with different samples at the equiva-lent SUN concentrations varying from 0.5 to 4 μg/mL for 48 h. At the end of incubation time, MTT solution (20 μl, 5mg/ml) was added to each well, and the cells were incubated for another 4 h at 37 °C. Then, the medium was removed, and 150 μl of DMSO was added to each well to dissolve formazan crystals. The absorbance at 570 nm was measured using a microplate reader. Finally, the cell viability % was determined using equation (3). The cells treated with the same amount of PBS were taken as the negative control and the blank culture medium was used as the control. The statistical analysis of data was done using ANOVA by STATISTICA 18 (Statsoft1, Inc.) software. Journal of Drug Delivery Science and Technology 50 (2019) 237–247
Mean absorbance of sample
mean absorbance of blank
Mean absorbance of control
mean absorbance of blank
2.9. In vitro cellular uptake study
C6 as a fluorescent probe was loaded into the targeted and non-targeted NLCs and the cellular uptake was studied via fluorescence microscope (CETI, Belgium) and flow cytometry (BD FACSCalibur, US). The fluorescent NLCs were prepared in a same way as the NLCs loaded with SUN, except SUN was replaced with 1 mg of C6. For flow cyto-metry study, A549 cells were seeded in 12 well plates at the density of 2.5 × 105 per well and incubated overnight. After that, the cells were treated with 200 ng/ml of C6 -NLCs or biotin-C6-NLCs at 37 °C for 3 h. Subsequently, cells were washed three times with PBS, trypsinized, and resuspended in PBS. Then, resulting cell suspension was analyzed by flow cytometer.
For fluorescent microscopy study, A549 cells were seeded in 96 well plates at the density of 2 × 104 per well and incubated at 37 °C for 24 h. After 3 h incubation of cells with C6 -NLCs or biotin-C6-NLCs, cells were washed three times with PBS, and then the cell monolayer was imaged with fluorescent microscope.
3. Results and discussions
3.1. Synthesis and characterization of B-SA conjugate
The B-SA conjugate was successfully obtained in 71.8% yield. The structure of B-SA conjugate was confirmed by 1H NMR and FTIR spectra. Fig. 1 shows the 1H NMR spectra of biotin, stearylamine and B-SA conjugate in DMSO‑d6. The proton assignment of B-SA conjugate is as follows: σ (ppm): 0.9 (terminal methyl group of stearylamine), 1.2–1.7 (CH2, alkyl chain of stearylamine and HI, HJ and HK of biotin together), 2.1 (HH), 2.6 (HG’), 2.9 (HG), 3.1 (CH2 of stearylamine next to the amide group), 3.2 (HF), 4.2 (HE), 4.4 (HD), 6.4–6.6 (HB and HC), 7.9 (HA). Due to the amide bond formation bonding between the amine group of stearylamine and the carboxyl group of biotin, the HH (2.2 ppm) of biotin which is next to the carboxyl group, has appeared with a little up-field displacement (2.1 ppm). The CH2 group of stear-ylamine, next to the amide group, has appeared almost in down field (3.1 ppm) compared to the corresponding peak in stearylamine spec-trum (2.6 ppm). Also, the signal related to the carboxylic proton of biotin (Fig. 1 A) in 12 ppm, was completely disappeared after the conjugation with stearylamine (Fig. 1C). These spectral results are in-dicated that stearylamine and biotin are covalently bonded to each other. Fig. 2 shows the FTIR spectra of biotin, stearylamine, and B-SA conjugate. In the FTIR spectrum of B-SA conjugate, we see the char-acteristic absorption bands at 3306.36 cm−1 (N-H str.), 2922 and 2851.24 cm−1 (aliphatic C-H str.), 1699.94 cm-1 (C]O str. of amide group), 1644.02 cm−1 (C]O, biotin residue) along with the other specific bands for both of biotin and stearyl amine in the remaining area of the spectrum. As we can see in Fig. 2 A, the absorption band of COOH group in biotin at 1711 cm−1 was disappeared in the spectrum of product due to chemical bonding between the amine of stearyl amine and the carboxyl group of biotin. Furthermore, a newly absorption band around 1699.94 cm−1 (Fig. 2C) confirmed the amide bond between biotin and stearyl amine. We measured degree of biotin conjugation to stearylamine using the relative integral ratio of signals at 7.9 ppm for stearylamine and 6.6 ppm for Biotin. The integral intensities reveal the 1:1 conjugating ratio for this reaction.