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Cationic polyethylenimine conjugated gold nanoparticles (AuNP-PEI) are a widely studied vector for drug delivery and an effective probe for interrogating NP-cell interactions. However, an inconsistent body of literature currently exists regarding the reproducibility of physicochemical properties, colloidal stability, and efficacy for these species. To address this gap, we systematically examined the preparation, stability, and formation mechanism of PEI conjugates produced from citrate-capped AuNPs. We considered the dependence on relative molar mass, Mr, backbone conformation, and material source. The conjugation mechanism of Au-PEI was probed using attenuated total reflectance FTIR and X-ray photoelectron spectroscopy, revealing distinct fates for citrate when interacting with different PEI species. The differences in residual citrate, PEI properties, and sample preparation resulted in distinct products with differentiated stability. Overall, branched PEI (25 kDa) conjugates exhibited the greatest colloidal stability in all media tested. By contrast, linear PEI (25 kDa) induced agglomeration. Colloidal stability of the products was also observed to correlate with displaced citrate, which supports a glaring knowledge gap that has emerged regarding the role of this commonly used carboxylate species as a "place holder" for conjugation with ligands of broad functionalities. We observed an unexpected and previously unreported conversion of amine functional groups to quaternary ammonium species for 10 kDa branched conjugates. Results suggest that the AuNP surface catalyzes this conversion. The product is known to manifest distinct processes and uptake in biological systems compared to amines and may lead to unintentional toxicological consequences or decreased efficacy as delivery vectors. Overall, comprehensive physicochemical characterization (tandem spectroscopy methods combined with physical measurements) of the conjugation process provides a methodology for elucidating the contributing factors of colloidal stability and chemical functionality that likely influence the previously reported variations in conjugate properties and biological response models.

We report the development of a novel cationic dendron (TAG1-PCD) and a positively charged gold nanoparticle-dendron conjugate (PCD-AuNP). TAG1-PCD was designed by considering the reactivity, hydrophilicity, and cationic nature that is required to yield a stable gold conjugate in aqueous media. The PCD-AuNPs, nominally 10 nm in size, were synthesized by reduction of chloroauric acid in the presence of TAG1-PCD. The physicochemical properties of PCD-AuNPs were characterized by dynamic light scattering, transmission electron microscopy, UV-vis absorbance, and X-ray photoelectron spectroscopy for investigation of size distribution, shape uniformity, surface plasmon resonance bands, and Au-dendron bonding. Asymmetric-flow field flow fractionation was employed to confirm the in situ size, purity, and surface properties of the PCD-AuNPs. Additionally, the stability of PCD-AuNPs was systematically evaluated with respect to shelf life determination, stability in biological media and a wide range of pH values, chemical resistance against cyanide, redispersibility from lyophilized state, and stability at temperatures relevant to biological systems. Dose dependent cell viability was evaluated in vitro using the human lung epithelial cell line A549 and a monkey kidney Vero cell line. Observations from in vitro studies are discussed. Overall, the investigation confirmed the successful development of stable PCD-AuNPs with excellent stability in biologically relevant test media containing proteins and electrolytes, and with a shelf life exceeding 6 months. The excellent aqueous stability and apparent lack of toxicity for this conjugate enhances its potential use as a test material for investigating interactions between positively charged NPs and biocellular and biomolecular systems, or as a vehicle for drug delivery.

We report a systematic study of the controlled formation of discrete-sized gold nanoparticle clusters (GNCs) by interaction with the reducing agent dithiothreitol (DTT). Asymmetric-flow field flow fractionation and electrospray differential mobility analysis were employed complementarily to determine the particle size distributions of DTT-conjugated GNCs (DTT-GNCs). Transmission electron microscopy was used to provide visualization of DTT-GNCs at different states of aggregation. Surface packing density of DTT and the corresponding molecular conformation on the Au surface were characterized by inductively coupled plasma mass spectrometry and X-ray photoelectron spectroscopy. Results show that DTT increases the aggregation rate of gold nanoparticles (AuNPs) up to ≈100 times. A mixed conformation (i.e., combining vertically aligned, horizontally aligned, and cross-linking modes) exists for DTT on the Au surface for all conditions examined. The primary size of AuNPs, concentration of DTT, and the starting concentration of AuNPs influence the degree of aggregation for DTT-GNCs, indicating that the collision frequency, energy barrier, and surface density of DTT are the key factors that control the aggregation rate. DTT-GNCs exhibit improved structural stability compared to the citrate-stabilized GNCs (i.e., unconjugated) following reaction with thiolated polyethylene glycol (SH-PEG), indicating that cross-linking and surface protection by DTT suppresses disaggregation normally induced by the steric repulsion of SH-PEG. This work describes a prototype methodology to form ligand-conjugated GNCs with high-quality and well-controlled material properties.