Cryoplunge freezer

For cryogenic transmission electron microscopy, 4–5 μL of each formulation was applied to a 400-mesh lacy carbon copper grid. Specimens were then plunge-frozen with a Gatan Cryoplunge freezer. These specimens were imaged using a JEOL 3200FS transmission electron microscope operating at 300 keV at 4000× nominal magnification. All images were collected in vitreous ice using a total dose of ∼10 e⁻ Å⁻² and a nominal defocus range of 2.0–5.0 μm.
Small angle X-ray scattering (SAXS) studies were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) beamline at Argonne National Laboratory’s Advanced Photon Source (Argonne, IL, USA) with 10 keV (wavelength λ = 1.24 Å) collimated X-rays. SAXS was performed on undiluted 15 mg/mL polymer formulations, as described previously. Model fitting was performed using SASView and the built-in polymer micelle model.
Dynamic light scattering measurements (DLS) were performed on 15 μg/mL polymer formulations using a Nano 300 ZS zetasizer (Malvern Panalytical, Malvern, UK), using the number average distribution for calculation of the mean diameter and polydispersity of the formulations.

Specimens for cryoTEM were prepared by applying 4 μL of 1 mg/ml sample on a pretreated, holey carbon 400 mesh TEM grids and were plunge-frozen with a Gatan Cryoplunge freezer. Images were collected in vitreous ice using a JEOL 3200FSC transmission electron microscope operating at 300 keV at 4,000 x nominal magnification. A total dose of ~10 e⁻/Ų and a nominal defocus range of 2.0–5.0 μm were used. Micrographs were acquired using a Gatan 3.710 × 3,838 pixel K2 Summit direct electron detector operating in counting mode. Each micrograph was acquired as 20-frame movies during a 5 s exposure. After data acquisition, the individual frames of each micrograph were aligned using Digital Micrograph software (Gatan) to compensate for stage and beam-induced drift, and the aligned images were summed for further image processing.

PEG₄₄-PPS₂₀ block copolymer (from a minimum of 20 mg to a maximum of 40 mg) was dissolved in dichloromethane (0.5 mL) in the presence or absence of various amounts of CsA (from 4 to 6 mg) and was added dropwise to distilled water (1 mL). The mixture was stirred at room temperature and at open air until dichloromethane was completely removed by evaporation, at which point the aqueous phase contains nMIC or CsA-nMIC. When needed, the complete evaporation of the organic phase was achieved under vacuum. When the drug was present, any precipitate of unloaded drug was removed by fast centrifugation at 10,000 rpm for 5 minutes. The mean diameter and polydispersity of the obtained nMIC and CsA-nMIC were measured using a Malvem-Zetasizer Nano Range (Malvern, UK). The micellar morphology, in the presence or absence of CsA, was confirmed by cryo-transmission electron microscopy (cryo-TEM). For cryoTEM, 4–5 μL of each formulation was applied to a 400-mesh lacy carbon copper grid. Specimens were then plunge-frozen with a Gatan Cryo-plunge freezer. These specimens were imaged using a JEOL 3200FS transmission electron microscope operating at 300 keV on a CCD camera at 20000-50000× nominal magnification.