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  • br Materials http www glpbio com simage GC Veratridine png

    2022-09-16


    2. Materials and methods
    PTX was obtained from the Department of Chemical Engineering in Chosun University. (2-Hydroxypropyl)-β-cyclodextrin (HP-β-CD) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kolliphor® (poly-oxyethylene (160) polyoxylpropylene (30) glycol [P188®] and poly-oxyethylene (196) polyoxylpropylene (67) glycol [P407®]), poly-vinylpyrrolidone [Kollidon® 30, K30®], Kolliphor® (polyglycol mono-and di-esters of 12-hydroxystearic Bafilomycin A1 (lipophilic part) and ~30% free polyethylene glycol (hydrophilic part) [HS-15®]), TPGS, polyvinyl ca-prolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus®), and polyethylene glycol (PEG) 6000 were obtained from BASF (Ludwigshafen, Germany). Vinylpyrrolidone to vinyl acetate (60/ 40 ratio) (PVP/VA S-630) was purchased from ISP Technologies Inc. (Wayne, NJ, USA). Aerosil® 200 was obtained from Evonik (Essen, Germany). Coumarin6 (Cou-6) and 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma–Aldrich. MCF-7, BT-474, RAW 264.7, and Madin-Darby Canine Kidney (MDCK) cells were purchased from the Korean Cell Line Bank (KCLB) (Seoul, Korea). Fetal bovine serum (FBS), antibiotics, Roswell 
    Park Memorial Institute (RPMI) 1640 medium, and Dulbecco's modified Eagle medium (DMEM) were purchased from Hyclone (Logan, UT, USA). Acetonitrile was purchased from Burdick & Jackson (Honeywell, NJ, USA).
    2.2. Screening of solubilizers
    Solubilizers were screened to improve solubility of PTX because a solubilizer usually enhances drug solubility via forming micelles. The PTX solubility test was performed in polymeric solution (5%, w/v) [31]. PTX (10 mg) was added to 15 mL tubes containing 5 mL distilled water (DW) and 5 mL polymeric solutions of P188, P407, HS-15, TPGS, Soluplus®, HP-β-CD, PEG6000, PVP/VA S630, or K30. The nine samples were shaken at 200 rpm at 37 ± 0 .5 °C for 24 h in an incubator (LSI-3016A, Daihan Lab Tech Co., Ltd., Namyangju, Korea). The insoluble PTX was removed by centrifugation (10,000 ×g, 10 min, MICRO-12 microcentrifuge; Hanil Scientific Industrial Co., Ltd. Korea). The su-pernatants were evaluated by high-performance liquid chromatography (HPLC) (n = 3) (as indicated in Section 2.6).
    2.3. Preparation of PTX formulations
    To evaluate the effect of the copolymer system, SD formulations were prepared with the ratio of TPGS: polymers = 3:1, as shown in Table 1 (SD1–SD9). The preparation method was the same as described directly above. Physical mixtures (PM) were prepared using the above method, without solvent.
    PTX-loaded micelles were prepared by a modified method pre-viously reported [29]. Briefly, Soluplus® and TPGS (4:1), and PTX (15 mg) were dissolved in methylene chloride (2 mL), as shown in Table 2. The organic solvent was completely evaporated in an oven at 40 °C for 4 h. The films were hydrated in DW (6 mL) and sonicated at 240 W for 1 min with a probe-type sonicator (KFS-300 N, Ultrasonic Processor, Korea Process Technology, Seoul, Korea) in an ice bath (4 °C). The hydrated solutions were stirred at 500 rpm overnight. Free PTX was removed by filtration with a 0.45-μm filter (Acrodisc® Supor® Membrane, Pall Life Sciences, Ann Arbor, MI, USA).
    Table 2
    Composition of micelles formulations (mg, 1 batch).
    2.4. Apparent solubility of SD formulations
    2.5. Particle size and surface charge of micelles
    The particle size of micelles was determined using a Zeta-sizer Nano-90 (Malvern Instruments Ltd., Malvern, UK), and the surface charge was determined by measuring the Zeta-potential (Zetasizer Nano-Z, Malvern Instruments Ltd. Malvern, UK). The particle size and surface charge of each sample were measured in triplicate, and average values were calculated.
    2.6. Drug content, drug loading, and encapsulation efficiency
    Drug content, drug loading, and encapsulation efficiency were de-termined using an HPLC method. PTX content in SD formulations (equivalent to 1 mg of PTX) was dissolved in acetonitrile (20 mL) and sonicated in bath-sonication for 30 min. PTX content in micelles (100 μL of 6 mL) was dissolved in acetonitrile (5 mL) and sonicated in bath-sonication for 30 min. The insoluble PTX and excipients in SD and micelles were removed by centrifugation (10,000 ×g, 10 min).