Preparation and characterization of the micelles

p(NIPAAM-co-PEGMEA)-b-PCL micelles (empty) were prepared as follows. The polymer (5 mg) was dissolved in an appropriate amount of acetone and slowly added drop-wise to 5 mL phosphate buffer saline (PBS). The residual acetone was eliminated by evaporation under ambient temperature overnight, and the micelles self-assembled.

The IR-780/17-AAG–loaded micelle was prepared using the oil-in-water emulsion solvent evaporation method.22 Briefly, IR-780, 17-AAG, and the polymer were dissolved in 1.5 mL acetone and then slowly added drop-wise to PBS. The ratio of the drug (IR-780 or 17-AAG) to the polymer was 1:6. The residual acetone was eliminated by evaporation under ambient temperature overnight, and the drug-encapsulated micelles self-assembled. The solution was then filtered through a 0.45 mm polyether-sulfone membrane filter (EMD Millipore, Billerica, MA, USA) to remove free drugs and polymer aggregates (Figure S1). The fraction of IR-780–loaded micelles was determined by fluorescence with an excitation of 690 nm and emission of 780 nm. IR-780–loaded micelle solution was added to DMSO (final micelle solution 10%, v/v), and fluorescence was detected using a spectrofluorometer (SpectraMax Gemini XS; Molecular Devices, Sunnyvale, CA, USA) or enzyme-linked immunosorbent assay (ELISA) microplate reader (SpectraMax M2 Multi-mode Microplate Reader, Molecular Devices). In addition, the fraction of 17-AAG–loaded micelles was determined by measuring the absorbance at 332 nm. Using free IR-780 or 17-AAG in DMSO under the same conditions as standards, the drug encapsulation efficiency (EE) and drug loading content (DL) were calculated as follows:

The particle mean diameters, polydispersity indexes (PDIs), and zeta potentials of the micelles were measured by dynamic light scattering (DLS) at 25°C with a scattering angle of 90° using a Zetasizer Nano-ZS90 (Malvern Instruments, Malvern, UK).

The morphology of the micelles was observed by transmission electron microscopy (H-7500; Hitachi, Tokyo, Japan). The samples were prepared by adding a drop of micelle solution onto 200-mesh carbon-coated copper grids followed by negative staining with 1% phosphotungstic acid at pH 7.0. Excess liquid on the copper grids was removed with filter papers and dried in a desiccator until examination.