br Preparation of PTX encapsulated micelles
2.2. Preparation of PTX-encapsulated micelles
PTX-encapsulated micelles (PTX-micelles) using four kinds of alkyl di(MePEG-lactate) phosphates with diﬀerent side chains were prepared. Fifty milligrams of compounds and 2.5 mg of PTX were dissolved in 10 mL of chloroform. A thin film was formed by evaporation, and then chloroform was removed under reduced pressure. After adding phos-phate buﬀered saline (PBS) to the sample, PTX-encapsulated micelles were obtained using a bath sonicator (FU-9H, Tokyo Glass Kikai Co., Ltd., Tokyo, Japan). Sonication was carried out for 5 min. The mean volume diameter and size distribution of the prepared PTX-micelles were measured using a particle size analyzer (ELSZ-2, Otsuka Electronics Co., Ltd., Osaka, Japan). PTX contents in the micelles were measured using high-performance liquid chromatography (HPLC; SIL-20 A prominence, SPD-20 A prominence, LC-20AD prominence, CTO-10ASvp, DGU-20A3 prominence, Shimadzu Corp., Kyoto, Japan) at 227 nm with an ODS column (STR ODS-M, size: 4.6 mm–150 mm, Shinwa Chemical Industries Ltd., Kyoto, Japan). The mobile phase consisted of a solution of phosphate buﬀer solution (0.01 M, pH 6.8) Colloids and Surfaces B: Biointerfaces 177 (2019) 356–361
and acetonitrile with a volume ratio of 2:3. HPLC measurements were carried out at 40 °C (flow rate: 1.0 mL/min), and 50 μL of sample so-lution were applied. All HPLC measurements were carried out under the same conditions. Encapsulation eﬃciency was expressed as a percen-tage of the total amount of PTX initially used .
The critical micelle concentrations (CMC) of the compounds were determined by fluorescent probe techniques using pyrene as a hydro-phobic fluorescent probe. Pyrene solution in acetone (1.2 × 10−4 M) was added into a container, and acetone was removed under N2 at-mosphere. Then, 10 mL of a diﬀerent concentration aqueous CCK8 solution ranging from 2.5 × 10−4 to 0.5 mg/mL was added into the containers, and all the solutions were equilibrated for 24 h prior to measurements. The final pyrene concentration in the compound solu-tion was 6 × 10-7 M. The fluorescence spectra were recorded on a fluorescence spectrophotometer (RF-5300, Shimadzu Corp.). The emission spectra were recorded from 350 to 600 nm with an excitation wavelength of 340 nm, and bandwidths were 2.5 nm. From the pyrene emission spectra, the intensity (peak height) ratios (I3/I1) of the third band (391 nm, I3) to the first band (373 nm, I1) were analyzed as a function of compound concentration, and a CMC value was calculated [35,36]. The stability of the PTX-encapsulated micelles was in-vestigated. The micellar solution was added to PBS and shaken at 100 rpm at 37 °C. Then, particle size was measured after 0.5, 1, 2, 4, 8, 12, and 24 h. Morphological observation of the micelles was carried out using a transmission electron microscope (TEM; H-7650, Hitachi High-Technologies Co., Ltd., Tokyo, Japan). Samples were negatively stained using a 0.2% (w/v) phosphotungstic acid solution, and they were stored overnight in a desiccator to completely remove the solvent .
2.3. In vitro release study of PTX from micelles
To confirm the influence of pH on drug release behavior, PTX re-lease from micelles was studied by using the dialysis membrane method . Two milliliters of solution containing PTX-micelles was placed in a dialysis tubing cellulose membrane (molecular weight cut-oﬀ: 12,400; Sigma-Aldrich, St. Louis, MO, USA) and added to 98 mL of PBS (pH 7.4) or citrate buﬀer solution (pH 5.0, containing 0.5% [w/v] polysorbate 80). Sample solutions were shaken at 100 rpm at 37 °C. After 1, 2, 4, 6, 8, 12, and 24 h, the external solution was exchanged. The amounts of PTX in external solution were quantified using HPLC. In addition, 0.01 mg/mL of PTX solution as the control group was also studied in the same manner. To increase the solubility of PTX, dimethyl sulfoxide was added to the solution to a concentration of 0.1%.
2.4. In vitro study of compound degradation
2.5. In vitro hemolysis and cytotoxicity tests of micelles
Male Sprague-Dawley rats (retired) were purchased from Japan SLC Inc. (Tokyo Japan). All animal care was conducted under the Guidelines for Animal Experimentation of Tokyo University of Science, which is based on the Guidelines for Animal Experimentation of the Japanese Association for Laboratory Animal Science. Rats were killed by iso-flurane. Blood was then collected by cardiac puncture and centrifuged at 1500 rpm for 15 min at 4 °C (Model 2410, Kubota Corp., Tokyo,
Properties of paclitaxel encapsulated micelles (n = 3, mean ± S.D.).