4.1. Nanoparticle Generation and Characterization

The polymeric nanoparticles were prepared by the oil-in-water emulsion–solvent evaporation method. Briefly, PLGA (Acid terminated, lactide: glycilide—50:50, M_W 24.000–38.000, Sigma-Aldrich) and fluorescent dye (Nile red, Sigma-Aldrich, St. Louis, MO, USA) were dissolved in Dichloromethane (Bio-Lab Ltd., Jerusalem, Israel) and transferred dropwise to a 1% polyvinyl alcohol (PVA, 87–90% hydrolyzed, M_W 30.000–70.000, Sigma-Aldrich, St. Louis, MO, USA) solution. The solution was then emulsified in ice water by a probe sonicator (Vibra-Cell™ Ultrasonic, Sonics & Materials, Inc., Newtown, Connecticut, USA) followed by solvent evaporation.

BSA, GPVI-Fc and GPVI-CD39 were conjugated to the nanoparticles via NHS-EDC chemistry. EDC-NHS coupling is widely used for protein coupling [23,24] and has been implemented previously for PLGA NP-GPVI [20], whereas measurements we performed on non-covalent absorption showed minimal coupling to the surface. Average coating densities on the NPs were estimated by measuring the remaining protein after bioconjugation, performed via a standard Bradford assay - Pierce™ Coomassie Plus (TS-23236, Thermo Scientific, Waltham, MA, USA) [24,25]. Briefly, to estimate the number of protein molecules per particle, first, the unbound protein concentration was measured in the supernatant using a standard Bradford assay. It was then subtracted from the initial concentration to evaluate the amount of protein that remained on the particles. By dividing the amount of protein by the estimated number of particles, the number of protein molecules per particle was evaluated.

PLGA particle size distribution was determined by dynamic light scattering (Zetasizer Nano-ZS, Malvern Instruments, Malvern, UK) at 25 °C with a scattering angle of 173°. Particle surface charge (Zeta-potential) was measured using laser Doppler micro-electrophoresis (Zetasizer Nano-ZS, Malvern Instruments, Malvern, UK). For the final preparation stage, the particles were reacted in a blocking buffer (50 mM Tris, pH 8.0, 0.5% (w/v) casein) for 2 h at room temperature. The particles were washed again and preserved in the blocking buffer at 4 °C. The nanoparticles were suspended in Dulbecco’s phosphate buffered saline (PBS) (Sigma-Aldrich, St. Louis, MO, USA) at a stock concentration of 5 mg/mL and stored at −20 °C.

Primary morphological assessment of the nanoparticles was achieved through negative staining, ultra-thin sectioning, electron microscopy (Libra 120, Zeiss, Jena, Germany) and confocal laser scanning microscopy (LSM 800, Zeiss, Jena, Germany). Quantitative analysis of morphology parameters was executed by atomic force measurements (MFP3D-BIO Asylum Research, Santa Barbara, CA, USA) on NP-BSAs. Cantilever D (k = 5 pN/nm) by Brucker AFM Samples (MLCT) with a scanning rate of 0.1 Hz was employed to image nanoparticles on collagen-coated cover slips. Data analysis was performed with the open-source SPM software Gwyddion (http://gwyddion.net/; accessed on 2 March 2021).