Gatan 626

The vitrification of the samples was carried out in a homemade vitrification system. The chamber was held at 22 °C and the relative humidity at 80%. A 5 µL drop of the sample (1–2 mg·mL⁻¹) was deposited onto a lacey carbon film covered grid ((Ted Pella, Redding, CA, USA) rendered hydrophilic using an ELMO glow discharge unit (Cordouan Technologies, Bordeaux, France). The grid was automatically blotted to form a thin film which was plunged in liquid ethane held at −190 °C by liquid nitrogen. That way, a vitrified film was obtained, in which the native structure of the vesicles was preserved. The grid was mounted onto a cryo holder (Gatan 626, Pleasanton, CA, USA) and observed under low dose conditions in a Tecnai G2 microscope (FEI, Eindhoven, Netherland) at 200 kV. Images were acquired using an Eagle slow scan CCD camera (FEI).

Cryogenic Transmission Electron Microscopy (cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, OR, USA) equipped with field emission gun, operating at an acceleration voltage of 200 kV. Images were recorded on the Gatan Rio 16 CMOS 4k camera (Gatan Inc., Pleasanton, CA, USA) and processed with Gatan Microscopy Suite (GMS) software (Gatan Inc., Pleasanton, CA, USA). Specimen preparation was performed by vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using a Femto plasma cleaner (Diener Electronic, Ebhausen, Germany). Cryo-samples were prepared by applying a droplet (3 μL) of the suspension to the grid, blotting with filter paper and immediate freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, CA, USA) and analyzed in the TEM at −178 °C.

Cryogenic transmission electron microscopy (cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, OR, USA) equipped with a field emission gun, operating at an acceleration voltage of 200 kV. Images were recorded on the Gatan Rio 16 CMOS 4k camera (Gatan Inc., Pleasanton, CA, USA) and processed with Gatan Microscopy Suite (GMS) software (Gatan Inc., Pleasanton, CA, USA). The specimen preparation was done by vitrification of the aqueous solutions on grids with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using a Femto plasma cleaner (Diener Electronic, Ebhausen, Germany). Cryo-samples were prepared by applying a droplet (3 μL) of the suspension to the grid, blotting with filter paper, and immediately freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (Thermo Fisher Scientific, Waltham, MA, USA). After preparation, the vitrified specimens were kept under liquid nitrogen until they were inserted into a cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, CA, USA) and analyzed in the TEM at −178 °C.

The AAF solution with a protein concentration of 1 mg mL⁻¹ was placed in an ultrasonic chamber (Pol-Sonic, Poland) for 10 min at 40°. Next, the preparation of the AAF specimen consisted in vitrification of an aqueous suspension on the TEM grid with holey carbon film (Quantifoil R 2/2; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). Prior to use, the grids were activated for 15 s in oxygen plasma using the Femto plasma cleaner (Diener Electronic, Ebhausen, Germany). The samples of AAF were vitrified by applying a droplet (3 μL) of the suspension to the grid, blotting with filter paper, and immediate freezing in liquid ethane using a fully automated blotting device Vitrobot Mark IV (FEI Company, Hillsboro, Oregon, USA). After preparation, the vitrified specimens were kept in liquid nitrogen until they were inserted into the Cryo-TEM-holder Gatan 626 (Gatan Inc., Pleasanton, USA) providing a sufficiently low temperature (− 178 °C) during the transfer of the samples to the microscope and during the TEM analyses^{25 (link)}. Cryogenic Transmission Electron Microscopy (Cryo-TEM) images were obtained using a Tecnai F20 X TWIN microscope (FEI Company, Hillsboro, Oregon, USA) equipped with a field emission gun (FEG) operating at the acceleration voltage of 200 kV. Images were recorded with an Eagle 4k HS camera (FEI Company, USA) and processed with TIA software (FEI Company, USA).

Cryo-TEM imaging was performed using an FEI (Thermo Fisher Scientific) Talos 200C high-resolution TEM (Center for Electron Microscopy of Soft Matter, Wolfson Department of Chemical Engineering, Technion). The specimens were prepared at 25°C and 100% relative humidity under a controlled environment vitrification system. Drops of diluted liposomes were placed in a carbon-coated perforated polymer film, mounted on a 200-mesh TEM grid, and manipulated using tweezers. Then, 10¹⁰ particles/ml GNP-TF-liposomes or GNP with unconjugated untargeted liposomes were used for the imaging measurement; for the BBB in-vitro crossing evaluation, imaging was performed on a 24-h medium sample with TF-liposomes without a dilution step. After thinning with a filter paper-covered metal strip, the drop was immersed in liquid ethane at its freezing point (-183°C). Under controlled conditions, the grid was transferred into a Gatan 626 (Gatan, Pleasanton, CA) cryo-holder and imaged at -175°C. Digital images were captured using a highly sensitive FEI Falcon III direct-imaging camera. A volta phase plate was used to enhance image contrast.

Cryogenic transmission electron microscopy (cryo-TEM) images were acquired with a JEOL JEM microscope (JEOL JEM 2011, Tokyo, Japan) operating at 200 kV under low-dose conditions. First, 10µL of the sample was deposited onto the holey carbon grid and, immediately after, vitrified by rapid immersion in liquid ethane. The vitrified sample was mounted on a cryo-transfer system and introduced into the microscope (Gatan 626, Gatan, Pleasanton, CA, USA). Images were recorded on a CCD camera (Gatan Ultrascan US1000, Gatan, Pleasanton, CA, USA).