Nanophox

The zeta potential of the subtilosome-based siRNA nanoparticles was measured with the Zetasizer Nano ZS instrument (Malvern Instrument Limited, Worcestershire, UK). Electron microscopy of the subtilosome-siRNA nanoparticles was conducted to characterize the nanoparticles’ size, surface morphology, and shape. For DLS, the lyophilized preparation of the subtilosome-siRNA nanoparticles was suspended in distilled water (2 mg/mL), and a single drop was analyzed with the particle-size analyzer, NANOPHOX (Sympatec GmbH, Clausthal-Zefferfeld, Germany).

The diameter and size distribution of the aqueous NP dispersions were measured by cryo-TEM. Briefly, a small droplet was placed on a perforated lacey carbon film supported on a TEM copper grid. The drop was blotted with a piece of filter paper, resulting in formation of a thin film of 100–300 nm. The specimen was subsequently plunged into a reservoir of liquid ethane cooled by liquid nitrogen to ensure vitrification (rapid freezing) and prevent ice crystal formation. The vitrified specimen was transferred under liquid nitrogen and mounted on a cryogenic sample holder cooled to −170 °C. All samples were observed under low-dose conditions. Vitrified samples were examined using a FEI T12 G2 Cryo-TEM (Hillsboro, Oregon, USA) operating at 120 kV and equipped with a Gatan 626 cryo-holder system. The mean diameter was determined by measuring at least 200 particles using the image analysis software AnalySIS Auto version 3.2 (Soft Imaging System GmbH, Münster, Germany). In addition, the hydrodynamic diameter and size distribution of the aqueous NP dispersions were measured at room temperature with a particle DLS analyzer (Nanophox, Sympatec GmbH, Clausthal-Zellerfeld, Germany).

Dynamic light scattering using photon cross-correlation spectroscopy (PCCS) (NanoPhox, Sympatec, Germany) was used to determine the hydrodynamic size of the NPs (10 μg/mL) in cell medium. In brief, 1 mL dispersions of Ni and NiO (10 μg/mL) were prepared in cuvettes and analyzed directly after dispersion (0 h), after 2 and 24 h. Triplicate samples were analyzed to identify the size distribution pattern and data is presented based on single sample analysis measured three times each. Calibration was made using standard latex samples (20 ± 2 nm) and blank samples were analyzed prior to all measurements [28 (link)].

The mean diameter and particle distribution of these GKRK-APO carriers were measured by dynamic light scattering (Nanophox, Sympatec GmbH, Germany). Morphology of the VCR-loaded GKRK-APO was characterized via an atomic force microscopy (AFM) (NanoWizarc, JPK Ltd., Berlin, Germany) and transmission electron microscopy (TEM) (HITACHI, H-7650, Tokyo, Japan), respectively. The stability of VCR-loaded GKRK-APO in 10% FBS was evaluated using a Turbiscan Lab^® Expert (Formulaction, L'Union, France). The analysis of stability was carried out by the software of the instrument, as a variation of back-scattering (ΔBS) profiles.
The VCR encapsulation efficiency (EE) of VCR-loaded GKRK-APO and VCR-loaded APO was calculated using the following equation:
$\mathrm{E}\mathrm{E}\mathrm{\%}=(W_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{ }\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}-W_{f\mathrm{r}\mathrm{e}\mathrm{e}\mathrm{ }\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}})/W_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}\mathrm{ }\mathrm{d}\mathrm{r}\mathrm{u}\mathrm{g}}\times 100\%$
where, W_{total drug} and W_{free drug} represent the total drug in APO and the amount of free drug in the ultrafiltrate, respectively.

Protein particles were analyzed by dynamic light scattering (DLS) (NanoPhox, Sympatec, Clausthal-Zellerfeld, Germany) and measured directly as suspension in their ACN:water mixture with the following settings: 25°C, refraction 1.35, viscosity 0.53 mPas. The viscosity settings were verified by measuring known 100 nm polystyrene particles (43302, Sigma) in the same ACN:water mixture. The z-average was used to report the average particle size. Samples were diluted if the observed kilocounts per second (KCPS) exceeded 500. Further data analysis was performed using Python 3.0.

The particle size analysis was performed on Nanophox (Sympatec, Germany).