Protease Inhibitor Cocktail Compound type Mean volume diamet
Mean volume diameter (nm)
Paclitaxel content in micelles (%)
Encapsulation eﬃciency (%)
AbsSample − AbsControl
AbsPurified water − AbsControl Cytotoxicity tests were performed using the WST-8 assay. Cell Count Reagent SF (Nacalai Tesque Inc.) was used as a reagent, and the assay was performed according to the manufacturer's instructions. B16 mel-anoma cells, which were cultured in DMEM containing 10% FBS, were used. The cell suspension was seeded in a 96-well plate at 5.0 × 103 cells. After culturing for 24 h, 10 μL of CrEL, drug-free micelles, PTX solution, or PTX-micelles were added and further cultured for 24 h. PTX solution was prepared by adding 2.5 mL of CrEL to 30 mg of PTX and bringing the total volume to 5.0 mL with absolute ethanol. Ten micro-liters of the reagent was added and color reaction was carried out for 1 h. Then, the absorbance of each well was measured using a microplate reader (ARVO X4, PerkinElmer Inc., Waltham, MA) at 450 nm to cal-culate cell viability.
2.6. Biodistribution study of PTX-encapsulated micelles in tumor-bearing mice
Mice (ddY, 4 weeks old, male) were housed in stainless-steel cages under standard environmental conditions (23 ± 1 °C, 55% ± 5% hu-midity and a 12/12 h light/dark cycle) and maintained with free access to water and a standard laboratory diet (carbohydrates 30%; proteins 22%; lipids 12%; vitamins 3%) ad libitum (Nihon Nosan Kogyo Co., Yokohama, Japan). B16 melanoma Protease Inhibitor Cocktail (1.0 × 106 cells in 50 μL of PBS) were subcutaneously injected into the footpad of the right hind limb of mice, and tumor-bearing mice were given after 2 weeks. Estimated tumor weights were calculated using the following formula:
Fig. 2. (a) Particle size distribution of PTX -encapsulated micelles prepared with compound 1 and compound 2 (n = 3, mean ± SD). (b) The intensity (peak height) ratios (I3/I1) of the third band (391 nm, I3) to the first band (373 nm, I1) of the pyrene fluorescence spectra analyzed as a function of compound concentration for calculation of the critical micelle concentrations (n = 3, mean ± SD).
Japan). Following centrifugation, the pellet was dispersed in cold PBS. Centrifugation and dispersing were repeated for a total of three cycles. The number of erythrocytes in the sample was counted using a counting chamber and diluted with PBS to be 2.0 × 10−8 cells/mL. Three mil-liliters of CrEL or drug-free micelles were added to the 2 mL of the di-luted sample and were incubated for 1 h at 37 °C. After centrifugation at 1500 rpm for 15 min, the release of hemoglobin was determined by photometric analysis of the supernatant at 540 nm. Complete hemolysis was achieved using purified water, yielding the 100% control value. Zero percent hemolysis was considered as PBS buﬀer-treated ery-throcyte solution (control) . Hemolysis (%) of each sample was calculated by the following equation. where L is the length (mm) and W is the width (mm) . The ex-periment started when the estimated tumor weight reached 80 mg. Then, 200 μL of PTX solution or PTX-micelles was injected into tumor-bearing mice via the tail vein under isoflurane anesthesia, and animals were maintained in metabolic cages. The injections were well tolerated and no adverse eﬀects were observed. After 24 h of sample adminis-tration, mice were euthanized via cervical dislocation under anesthesia and bled at the inferior vena cava. The tumor, brain, heart, lungs, liver, stomach, pancreas, spleen, and kidneys were immediately taken from the same individual. Tissue samples were homogenized in 0.7 mL of physiological saline. After addition of acetonitrile, samples were cen-trifuged at 4000 rpm for 20 min, and PTX concentration in the super-natant was determined using HPLC [42,43].
3. Results and discussion
3.1. Characterization of PTX -encapsulated micelles
Compounds in which 1-eicosanol or 1-octadecanol was introduced in the side chain formed micelles, whereas micelles were not confirmed in those in which 1-hexadecanol or 1-dodecanol was introduced. The formation and particle size of micelle-like assemblies are known to be controlled by the relative molecular weights of the hydrophobic and hydrophilic blocks . Therefore, it is assumed that the compound introduced with 1-hexadecanol or 1-dodecanol as a side chain was in-suﬃcient in molecular weight of the hydrophobic group and could not form micelles. The mean volume diameter, the mass percentage of PTX
Fig. 3. (a) Changes in mean diameters of PTX -encapsulated micelles prepared with compound 1 and compound 2 in PBS at 37 °C (n = 3, mean ± SD). (b) Transmission electron microscope image of PTX -encapsulated micelles pre-pared with compound 1.
contained in the prepared PTX-micelles (PTX content in micelles), and encapsulation eﬃciency of micelles prepared from the compound to which 1-eicosanol was introduced (compound 1) and the micelle pre-pared from the compound to which 1-octadecanol was introduced (compound 2) are summarized in Table 1. Fig. 2a shows the size dis-tribution of micelles. It was confirmed that PTX was encapsulated in both micelles. As shown in Fig. 2b, CMC of compound 1 and compound 2 were 5.0 mg/L and 13.2 mg/L, respectively. Generally, the higher proportions of hydrophobic segments in the amphiphilic copolymer exhibit lower CMC values. This suggests greater thermodynamic sta-bility . Fig. 3a shows time-dependent changes of the mean volume diameter of micelles in PBS. The stability of micelles prepared with compound 1 was shown. Although micelles prepared using compound 2 did not show aggregation, the mean volume diameter tended to in-crease. Therefore, it was suggested that compound 1 was more desir-able in preparation of PTX -encapsulated micelles. As shown in the TEM image, the micelles had a spherical shape (Fig. 3b).