De-Hao D. Tsai

Determination of protein aggregation with differential mobility analysis: Application to IgG antibody†

Here we describe the use of electrospray differential mobility analysis (ES‐DMA), also known as gas‐phase electrophoretic mobility molecular analysis (GEMMA), as a method for measuring low‐order soluble aggregates of proteins in solution. We demonstrate proof of concept with IgG antibodies. In ES‐DMA, aqueous solutions of the antibody protein are electrosprayed and the various aerosolized species are separated according to their electrophoretic mobility using a differential mobility analyzer. In this way, complete size distributions of protein species present from 3 to 250 nm can be obtained with the current set up, including distinct peaks for IgG monomers to pentamers. The sizes of the IgG and IgG aggregates measured by DMA were found to be in good agreement with those calculated from simple models, which take the structural dimensions of IgG from protein crystallographic data. The dependence of IgG aggregation on the solution concentration and ionic strength was also examined, and the portion of aggregates containing chemically crosslinked antibodies was quantified. These results indicate that ES‐DMA holds potential as a measurement tool to study protein aggregation phenomena such as those associated with antibody reagent manufacturing and protein therapeutics. Biotechnol. Bioeng. 2008;101: 1214–1222.

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.

Quantification of ligand packing density on gold nanoparticles using ICP-OES

AbstractIn this study, a prototypical thiolated organic ligand, 3-mercaptopropionic acid (MPA), was conjugated on gold nanoparticles (AuNPs), and packing density was measured on an ensemble-averaged basis using inductively coupled plasma optical emission spectrometry. The effects of sample preparation, including centrifugation and digestion, as well as AuNP size and concentration, on recovery were investigated. For AuNPs with diameters of 5, 10, 30, 60, and 100xa0nm, calculated packing density is independent of size, averaging 7.8xa0nm−2 and ranging from 6.7 to 9.0xa0nm−2, and is comparable to reported values for MPA and similar short-chain ligands on AuNPs. These preliminary data provide fundamental information on the advantages and limitations of ICP-based analyses of conjugated AuNP systems. Moreover, they provide necessary information for the development of more broadly applicable methods for quantifying nanoparticle–ligand conjugates of critical importance to nanomedicine applications.n FigureInductively coupled plasma optical emission spectrometry was used to determine the packing density of S-containing ligands on gold nanoparticles based on the ratio of the measured S and Au mass fractions per square nanometer of surface area.

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.

Temperature-programmed electrospray-differential mobility analysis for characterization of ligated nanoparticles in complex media.

An electrospray-differential mobility analyzer (ES-DMA) was operated with an aerosol flow-mode, temperature-programmed approach to enhance its ability to characterize the particle size distributions (PSDs) of nanoscale particles (NPs) in the presence of adsorbed and free ligands. Titanium dioxide NPs (TiO2-NPs) stabilized by citric acid (CA) or bovine serum albumin (BSA) were utilized as representative systems. Transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry were used to provide visual information and elemental-based PSDs, respectively. Results show that the interference resulting from electrospray-dried nonvolatile salt residual nanoscale particles (S-NPs) could be effectively reduced using the thermal treatment process: PSDs were accurately measured at temperatures above 200 °C for CA-stabilized TiO2-NPs and above 400 °C for BSA-stabilized TiO2-NPs. Moreover, TEM confirmed the volumetric shrinkage of S-NPs due to thermal treatment and also showed that the primary structure of TiO2-NPs was relatively stable over the temperature range studied (i.e., below 700 °C). Conversely, the shape factor for TiO2-NPs decreased after treatment above 500 °C, possibly due to a change in the secondary (aggregate) structure. S-NPs from BSA-stabilized TiO2-NPs exhibited higher global activation energies toward induced volumetric shrinkage than those of CA-stabilized TiO2-NPs, suggesting that activation energy is dependent on ligand size. This prototype study demonstrates the efficacy of using ES-DMA coupled with thermal treatment for characterizing the physical state of NPs, even in a complex medium (e.g., containing plasma proteins) and in the presence of particle agglomerates induced by interaction with binding ligands.

Orthogonal Analysis of Functional Gold Nanoparticles for Biomedical Applications

We report a comprehensive strategy based on implementation of orthogonal measurement techniques to provide critical and verifiable material characteristics for functionalized gold nanoparticles (AuNPs) used in biomedical applications. Samples were analyzed before and after ≈50xa0months of cold storage (≈4xa0°C). Biomedical applications require long-term storage at cold temperatures, which could have an impact on AuNP therapeutics. Thiolated polyethylene glycol (SH-PEG)-conjugated AuNPs with different terminal groups (methyl-, carboxylic-, and amine-) were chosen as a model system due to their high relevancy in biomedical applications. Electrospray-differential mobility analysis, asymmetric-flow field flow fractionation, transmission electron microscopy, scanning electron microscopy, atomic force microscopy, inductively coupled plasma mass spectrometry, and small-angle X-ray scattering were employed to provide both complementary and orthogonal information on (1) particle size and size distribution, (2) particle concentrations, (3) molecular conjugation properties (i.e., conformation and surface packing density), and (4) colloidal stability. Results show that SH-PEGs were conjugated on the surface of AuNPs to form a brush-like polymer corona. The surface packing density of SH-PEG was ≈0.42xa0nm−2 for the methyl-PEG-SH AuNPs, ≈0.26xa0nm−2 for the amine-SH-PEG AuNPs, and ≈0.18xa0nm−2 for the carboxylic-PEG-SH AuNPs before cold storage, approximately 10xa0% of its theoretical maximum value. The conformation of surface-bound SH-PEGs was then estimated to be in an intermediate state between brush-like and random-coiled, based on the measured thicknesses in liquid and in dry states. By analyzing the change in particle size distribution and number concentration in suspension following cold storage, the long term colloidal stability of AuNPs was shown to be significantly improved via functionalization with SH-PEG, especially in the case of methyl-PEG-SH and carboxylic-PEG-SH (i.e., we estimate that >80xa0% of SH-PEG5K remained on the surface of AuNPs during storage). The work described here provides a generic strategy to track and analyze the material properties of functional AuNPs intended for biomedical applications, and highlights the importance of a multi-technique analysis. The effects of long term storage on the physical state of the particles, and on the stability of the ligand-AuNP conjugates, are employed to demonstrate the capacity of this approach to address critical issues relevant to clinical applications.