Assessment of Charged AuNPs: From Synthesis to Innate Immune Recognition

Abstract

Gold nanoparticle (AuNP) physicochemical characteristics, mainly size and charge, modulate their biodistribution, cytotoxicity, and immunorecognition as reported fromin vitroandin vivostudies. While data fromin vitrostudies could be biased by several factors including activation of cells upon isolation and lack of sera proteins in the microenvironment of primary generated cell lines,in vivostudies are costly and time-consuming and require ethics consideration. In this study, we developed a simple and novelin vivo-like method to test for NP immunorecognition from freshly withdrawn human blood samples. AuNPs with a size range of 30 ± 5 nm coated with cationic poly(L-lysine) (PLL) dendrigraft and slightly negative poly(vinyl alcohol) (PVA) were synthesized in water. PLL-capped AuNPs were further coated with poly(ethylene glycol) (PEG) to obtain nearly neutrally charged PEG-AuNPs. Physicochemical properties were determined using zeta potential measurements, UV-Vis spectroscopy, dynamic light scattering (DLS), and scanning electron microscopy (SEM). Gel electrophoretic separation, zeta potential, and DLS were also used to characterize our NPs after human blood plasma treatment. PLL-AuNPs showed similar variation in charge and binding affinity to plasma proteins in comparison with PVA-AuNPs. However, PLL-AuNPs.protein complexes revealed a drastic change in size compared to the other tested particles. Results obtained from the neutrophil function test and pyridine formazan extraction revealed the highest activation level of neutrophils (~70%) by 50 μg/mL of PLL-AuNPs compared to a null induction by PEG- and PVA-AuNPs. This observation was further verified by flow cytometry analysis of polymorphonuclear cell size variation in the presence of coated AuNPs. Overall, ourin vivo-like method, to test for NP immunorecognition, proved to be reliable and effective. Finally, our data supports the use of PEG-AuNPs as promising vehicles for drug delivery, as they exhibit minimal protein adsorption affinity and insignificant charge and size variation once introduced in whole blood.

Human blood biocompatibility and immunogenicity of scFvD2B PEGylated gold nanoparticles

“…AuNPs-PEG were generated through drop-by-drop additions of 240 μl of SH-PEG5000 (111 μM) over 3 min onto 5 ml of AuNPs-citrate under stirring, then left stirring overnight [34]. UV-Vis and DLS analysis were performed to confirm the successful PEGylation of AuNPs-citrate.…”

“…Blood of human origin was intravenously withdrawn from three different individuals in anti-coagulative lithium heparin tubes then centrifuged at 1000 g for 5 min to separate the serum. The serum was collected separately and AuNPs-citrate, AuNPs-scFvD2B, AuNPs-PEG and AuNPs-scFvD2B-PEG (50 μg ml −1 ) were incubated in at a 1:1 ratio at 37 °C for 15 min Then, the AuNPs-protein complexes were pelleted down by centrifugation (Thermo scientific, heraeus fresco 21 centrifuge) at 11 000 rpm for 10 min and washed using dH 2 O for 3 times, then resuspended in dH 2 O for further characterization [34].…”

“…Based on the route of AuNPs-scFvD2B administration, the first contact with body components could be either with white blood cells (WBCs), namely neutrophils, if intradermally; the other route, intravenously, leads to AuNPs-scFvD2B interaction with various blood components including, WBCs, red blood cells (RBCs), plasma and complement proteins [32]. Immunogenicity assessment can be done using neutrophil function test (NFT) [33,34], since neutrophils are the most abundant leukocytes and the first immune cells to be recruited to the site of infection with professional phagocytic abilities [35]. Their activity is enhanced through ROS generation [36] and NETosis [37].…”

“…For example, PEGylated AuNPs delivering recombinant tumor necrosis factor alpha showed reduced systemic toxicity and omitted any protein-specific antibodies formation [50]. Also, PEG-AuNPs were described as safe drug delivery vehicle of reduced neutrophil activation once incubated in whole blood [34]. Moreover, PEG was successfully proved to stop the opsonization process [51] and increase the blood circulation and half-life of NPs such as in liposomes [52], poly(lactic-coglycolic acid) (PLGA) NPs [53], and Hollow gold nanospheres (HAuNS) [54].…”

Application of dissolvable Mg/Al layered double hydroxides as an adsorbent for the dispersive solid phase extraction of gold nanoparticles prior to their determination by atomic absorption spectrometry

“…This was attributed to the surface charge of citrate-capped AuNPs as compared to other coatings such as PVP, PVA and cysteine. Although all AuNPs that were examined in this work have a negative charge [36][37][38] citrate-capped AuNPs have lower zeta potential. In addition, citrate is a small molecule as compared to large molecules such as PVP and PVA where steric repulsion forces are signicant.…”