Zetasizer 3000 hs particle size analyzer

The pcDNA3.1-hMDA-7 plasmid (a generous gift from Professor Jicheng Yang of the Department of Molecular Biology, Suzhou University) was constructed by inserting the hMDA-7 DNA fragment from the Puc19- hMDA-7 plasmid into pcDNA3.1 through Kpn I and Xba I restriction sites. The recombinant plasmid was transformed into DH5α competent cells by the CaCl₂ method.
A total of 0.1 mg pcDNA3.1-hMDA-7 plasmid DNA dissolved in Tris-EDTA buffer was mixed with 3 ml of 2% BSA (Sijiqing Company, China), and water was added to a total mixture volume of 5 mL. Absolute ethyl alcohol (6.5 mL) was gradually added as well as 40% glutaraldehyde, and incubated at room temperature overnight. After centrifugation, the supernatant was discarded and the precipitate dissolved in water after ethanol evaporation. The solution was submitted to high speed centrifugation on an ultracentrifuge from BECKMAN (USA), and BSA nanoparticles obtained in the supernatant were stored at -20°C. Blank BSA nanoparticles (BSA-NP) were prepared as described above but without the hMDA-7 plasmid DNA. BSA-NP morphology and size were examined on a JEM-1200EX transmission electron microscope (HITACH, Japan). Zeta-potential was measured on a Zetasizer 3000HS Particle size analyzer (Malvern Instruments, UK).

Transmission electron microscopy (TEM) images were obtained in a JEOL JEM-2100F electron microscope, at an acceleration voltage of 120 kV. Samples for TEM analysis were prepared by adding a single drop (2 µL) of the aqueous solution (ca. 0.1 mg/mL in milliQ water) of particles onto a copper grid coated with a carbon film (Electron Microscopy Sciences). Further details on the preparation of grids can be found in the SI, Section A4.
UV-Vis spectra were measured in an Agilent 8453 UV-Vis diode-array spectrophotometer. Zeta potential measurements were performed in a Malvern Zetasizer 3000 HS particle size analyzer (Malvern Instruments, UK). Fluorescence measurements were carried out with a PerkinElmer LS55 Fluorescence Spectrometer. ICP-MS measurements were performed on a Thermo iCAP Q ICP-MS (Thermo Fisher Scientific GmbH, Bremen, Germany). A ASX560 autosampler was coupled to the ICP-MS (CETAC Tech, Omaha, NE, USA). In order to digest the different materials for ICP experiments, 100 µL of CaF₂ particles aqueous solution was digested overnight in 10 mL HNO₃ (65%, Scharlau) at 70 ^oC. Full beads were digested overnight in aqua regia. Spheroids were digested in aqua regia as well, but they required sonication before and after a 24h digestion time.