Tae Joon Cho

Adsorption and conformation of serum albumin protein on gold nanoparticles investigated using dimensional measurements and in situ spectroscopic methods.

The adsorption and conformation of bovine serum albumin (BSA) on gold nanoparticles (AuNPs) were interrogated both qualitatively and quantitatively via complementary physicochemical characterization methods. Dynamic light scattering (DLS), asymmetric-flow field flow fractionation (AFFF), fluorescence spectrometry, and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy were combined to characterize BSA-AuNP conjugates under fluid conditions, while conjugates in the aerosol state were characterized by electrospray-differential mobility analysis (ES-DMA). The presence of unbound BSA molecules interferes with DLS analysis of the conjugates, particularly as the AuNP size decreases (i.e., below 30 nm in diameter). Under conditions where the γ value is high, where γ is defined as the ratio of scattering intensity by AuNPs to the scattering intensity by unbound BSA, DLS size results are consistent with results obtained after fractionation by AFFF. Additionally, the AuNP hydrodynamic size exhibits a greater proportional increase due to BSA conjugation at pH values below 2.5 compared with less acidic pH values (3.4-7.3), corresponding with the reversibly denatured (E or F form) conformation of BSA below pH 2.5. Over the pH range from 3.4 to 7.3, the hydrodynamic size of the conjugate is nearly constant, suggesting conformational stability over this range. Because of the difference in the measurement environment, a larger increase of AuNP size is observed following BSA conjugation when measured in the wet state (i.e., by DLS and AFFF) compared to the dry state (by ES-DMA). Molecular surface density for BSA is estimated based on ES-DMA and fluorescence measurements. Results from the two techniques are consistent and similar, but slightly higher for ES-DMA, with an average adsorbate density of 0.015 nm(-2). Moreover, from the change of particle size, we determine the extent of adsorption for BSA on AuNPs using DLS and ES-DMA at 21 °C, which show that increasing the concentration of BSA increases the measured change in AuNP size. Using ES-DMA, we observe that the BSA surface density reaches 90% of saturation at a solution phase concentration between 10 and 30 μmol/L, which is roughly consistent with fluorescence and ATR-FTIR results. The equilibrium binding constant for BSA on AuNPs is calculated by applying the Langmuir equation, with resulting values ranging from 0.51 × 10(6) to 1.65 × 10(6) L/mol, suggesting a strong affinity due to bonding between the single free exterior thiol on N-form BSA (associated with a cysteine residue) and the AuNP surface. Moreover, the adsorption interaction induces a conformational change in BSA secondary structure, resulting in less α-helix content and more open structures (β-sheet, random, or expanded).

Competitive Adsorption of Thiolated Polyethylene Glycol and Mercaptopropionic Acid on Gold Nanoparticles Measured by Physical Characterization Methods

Competitive adsorption kinetics between thiolated polyethylene glycol (SH-PEG) and mercaptopropionic acid (MPA) on gold nanoparticles (Au-NPs) were studied using a prototype physical characterization approach that combines dynamic light scattering (DLS) and electrospray differential mobility analysis (ES-DMA). The change in hydrodynamic particle size (intensity average) due to the formation of SH-PEG coatings on Au-NPs was measured by DLS in both two-component (Au-NP + MPA or Au-NP + SH-PEG) and three-component (Au-NP +MPA + SH-PEG) systems. ES-DMA was employed to quantify the surface coverage of SH-PEG and establish a correlation between surface coverage and the change in particle size measured by DLS. A change in the equilibrium binding constant for SH-PEG on Au-NPs at various concentrations of SH-PEG and MPA showed that the presence of MPA reduced the binding affinity of SH-PEG to the Au-NP surface. Kinetic studies showed that SH-PEG was desorbed from the Au-NP surface following a second-order desorption model after subsequently introducing MPA. The desorption rate constant of SH-PEG from the Au-NP surface by MPA displacement was strongly affected by the concentration of MPA and the excess SH-PEG in solution.

Hydrodynamic fractionation of finite size gold nanoparticle clusters.

We demonstrate a high-resolution in situ experimental method for performing simultaneous size classification and characterization of functional gold nanoparticle clusters (GNCs) based on asymmetric-flow field flow fractionation (AFFF). Field emission scanning electron microscopy, atomic force microscopy, multi-angle light scattering (MALS), and in situ ultraviolet-visible optical spectroscopy provide complementary data and imagery confirming the cluster state (e.g., dimer, trimer, tetramer), packing structure, and purity of fractionated populations. An orthogonal analysis of GNC size distributions is obtained using electrospray-differential mobility analysis (ES-DMA). We find a linear correlation between the normalized MALS intensity (measured during AFFF elution) and the corresponding number concentration (measured by ES-DMA), establishing the capacity for AFFF to quantify the absolute number concentration of GNCs. The results and corresponding methodology summarized here provide the proof of concept for general applications involving the formation, isolation, and in situ analysis of both functional and adventitious nanoparticle clusters of finite size.

Characterization of gold nanoparticles modified with single-stranded DNA using analytical ultracentrifugation and dynamic light scattering.

We report the characterization of gold nanoparticles modified with thiol-terminated single stranded DNA (ssDNA) using analytical ultracentrifugation. Dynamic light scattering was used to measure the diameter of bare and ssDNA modified gold nanoparticles to corroborate the predictions of our models. Sedimentation coefficients of nominally 10 and 20 nm diameter gold nanoparticles modified with thiol-terminated thymidine homo-oligonucleotides, 5-30 bases in length, were determined with analytical ultracentrifugation. The sedimentation coefficients of gold nanoparticles modified with ssDNA were found to decrease with increasing coverage of ssDNA and increasing length of ssDNA. The sedimentation coefficients of ssDNA modified gold particles were most closely predicted when the strands were modeled as fully extended chains (FEC). Apparent particle densities of bare gold nanoparticles calculated from measured sedimentation coefficients decreased significantly below that of bulk gold with decreasing size of nanoparticles. This finding suggests that hydration layer effects are an important factor in the sedimentation behavior for both bare and short ssDNA chain modified gold particles.

Highly Stable Positively Charged Dendron-Encapsulated Gold Nanoparticles

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.

Controlled Formation and Characterization of Dithiothreitol-Conjugated Gold Nanoparticle Clusters

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.

Surface Modification of Cisplatin-Complexed Gold Nanoparticles and Its Influence on Colloidal Stability, Drug Loading, and Drug Release

Cisplatin-complexed gold nanoparticles (PtII-AuNP) provide a promising strategy for chemo-radiation-based anticancer drugs. Effective design of such platforms necessitates reliable assessment of surface engineering on a quantitative basis and its influence on drug payload, stability, and release. In this paper, poly(ethylene glycol) (PEG)-stabilized PtII-AuNP was synthesized as a model antitumor drug platform, where PtII is attached via a carboxyl-terminated dendron ligand. Surface modification by PEG and its influence on drug loading, colloidal stability, and drug release were assessed. Complexation with PtII significantly degrades colloidal stability of the conjugate; however, PEGylation provides substantial improvement of stability in conjunction with an insignificant trade-off in drug loading capacity compared with the non-PEGylated control (<20% decrease in loading capacity). In this context, the effect of varying PEG concentration and molar mass was investigated. On a quantitative basis, the extent of PEGylation was characterized and its influence on dispersion stability and drug load was examined using electrospray differential mobility analysis (ES-DMA) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) and compared with attenuated total reflectance-FTIR. Using ES-DMA-ICP-MS, AuNP conjugates were size-classified based on their electrical mobility, while PtII loading was simultaneously quantified by determination of Pt mass. Colloidal stability was quantitatively evaluated in biologically relevant media. Finally, the pH-dependent PtII release performance was evaluated. We observed 9% and 16% PtII release at drug loadings of 0.5 and 1.9 PtII/nm2, respectively. The relative molar mass of PEG had no significant influence on PtII uptake or release performance, while PEGylation substantially improved the colloidal stability of the conjugate. Notably, the PtII release over 10 days (examined at 0.5 PtII/nm2 drug loading) remained constant for non-PEGylated, 1K-PEGylated, and 5K-PEGylated conjugates.