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  • br Materials and methods br SUN base was provided

    2020-08-18


    2. Materials and methods
    SUN base was provided by Parsian Pharmaceutical Co (Iran). Pluronic F127 (PF127), coumarin 6 (C6), biotin, cholesterol (Chol), stearylamine, anhydrous dimethyl sulfoxide (DMSO), 4- N-hydro-xysuccinimide (NHS), dicyclohexylcarbodiimide (DCC), and dialysis bag (molecular cut off 12 000 Da) were purchased from Sigma (US). Labrafac was obtained from BASF (Ludwigshafen, Germany). For cell culture study, A549 cell line was provided from Iranian Biological Research Center (Iran). 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylte-trazolium bromide (MTT) were from Sigma Company (USA). Trypsin, fetal bovine serum (FBS), phosphate buffer saline (PBS), Dulbecco's Modified Eagle Medium (DMEM), penicillin, and streptomycin were purchased from Gibco Laboratories (USA).
    2.2. Synthesis of biotin-stearylamine (B-SA) conjugate
    B-SA conjugate was synthesized via the reaction of the activated carboxyl group of biotin with the amine group of stearylamine in the presence of DCC and NHS. For this coupling, biotin (700 mg, 2.8 mmol) was dissolved in the anhydrous DMSO. Then, DCC (693 mg, 3.4 mmol) and NHS (387 mg, 3.4 mmol) were added, and the mixture was stirred at room temperature under nitrogen U-0126 for 8 h. The resulting white solid byproduct, dicyclohexylurea, was removed via centrifuga-tion. Then, the supernatant was added dropwise through a syringe to the solution of stearylamine (772 mg, 2.3 mmol) in the anhydrous DMSO. The reaction mixture was stirred for further 24 h at room tem-perature in the same condition as previous. Finally, the mixture was lyophilized to get the product. The chemical structure of B-SA conjugate was approved using 1H NMR (Bruker, Biospin, AC-400, Mannheim, Germany) and FTIR (WQS-510/520, Raileigh, China) spectra.
    2.3. Preparation NLCs
    NLCs containing SUN were prepared by the emulsion-solvent  Journal of Drug Delivery Science and Technology 50 (2019) 237–247
    diffusion and evaporation method as previously described [9]. The total amount of lipid was employed were 60 mg of which 10% was B-SA conjugate and depending on the formulation code, labrafac was con-stituted 15% or 30% and Chol made up the remaining 75% or 60% of the lipid. For biotin-SUN-NLCs preparation, the desired amount of Chol, labrafac, B-SA conjugate along with 6 mg or 12 mg of SUN were dis-solved into mixed organic solvent of ethanol and acetone (1:1, v/v). Meanwhile, PF127 in concentration 0.5 or 1% w/v was dissolved in 30 mL of distilled water. The two phases were heated separately to the same temperature (at 60 °C). Then, the organic solution was added dropwise using an injection needle into hot aqueous solution of PF127 and stirred with a magnetic stirrer for 5 min at 800 rpm. After the coarse emulsion is formed, the mixture was further sonicated using a probe sonicator (bandelin, Germany) for 2 min at 50 W. In the final step, the obtained nanoemulsion (O/W) was cooled down at room temperature and mixed on magnetic stirrer for 3 h to permit solvent evaporation. To optimize conditions of the technical procedure, irre-gular full factorial design was employed for preparation of biotin-SUN-NLCs. Four different factors each at 2 levels including lipid/drug ratio (w/w), aqueous/organic phase ratio (v/v), liquid lipid to total lipid ratio (w/w) and surfactant concentration were studied (Table 1). Table 2 illustrates the twelve formulations were investigated. The evaluated response were particle size, polydispersity index (PdI), zeta potential, EE %, release efficiency % during 8 h (RE8%). The experi-mental factors and factor levels were chosen on the basis of the result of various initial trials. For the statistical data analysis and determine the contribution effect of each factor, Design Expert software (version 10, US) was used. Analysis of variance (ANOVA) was performed to con-clude the significance of the factor and their interaction.
    2.4. Characterization of biotin-SUN-NLCs
    The mean particle size, PdI, and zeta potential of developed biotin-SUN-NLCs were determined with size/zeta potential analyzer (ZEN 3600 Malvern, U.K) at 25 °C after 5 fold dilution with distilled water. All test performed triplicate.
    2.5. Morphology of biotin-SUN-NLCs
    Scanning electron microscope (SEM) images were utilized for ex-amining the morphology of NLCs. For this purpose, sample were ap-plied on a metal stubs and then coated with thin layer of gold under argon gas atmosphere. Afterward the samples were analyzed with an upper detector.
    2.6. Encapsulation efficiency
    For determination of EE of SUN in the NLCs, centrifuge technique was used. 0.5 mL of each biotin-SUN-NLCs formulation was transferred into Amicon microcentrifugation tubes (cutoff 10 000 Da, Ireland) and centrifuged (Microcentrifuge Sigma 30 k, UK) at 14 000 rpm for 10 min.
    Table 1
    Different factors and their levels investigated by irregular full factorial design in production of biotin modified NLCs loaded with SUN.