Parallel Multiparameter Study of PEI-Functionalized Gold Nanoparticle Synthesis for Biomedical Applications: Part 2. Elucidating the Role of Surface Chemistry and Polymer Structure in Performance.

Elucidating the polyethyleneimine (PEI) chemistry to predictively and reproducibly synthesize gold nanoparticle (AuNP)-PEI conjugates with desired properties has been elusive despite evaluation in numerous studies and reported enhanced properties. The lack of reproducible methods to control the core size and stability has led to contradictory results for performance and safety; thus, advancement of the conjugate platform for commercial use has likely been hindered. Recently, we reported a robust, reproducible method for synthesizing PEI-functionalized AuNPs (Au-PEIs), providing an opportunity to investigate structure-function relationships and to further investigate synthesis parameters affecting performance, where only materials stable in biological media are candidates for use. The properties of Au-PEIs prepared by the optimized reduction of HAuCl4 using four different structural variants of PEI changed significantly with the PEI molar mass and backbone form (branched or linear). In the present study using our previously reported synthesis procedure, comprehensive analysis of properties such as size distribution, surface plasmon resonance (SPR), morphological state, surface functionality, and the shelf life has been systematically evaluated to elucidate the role of surface chemistry and reactive groups involved in conjugation, as a function of conjugate size and morphology. Being important for commercial adoption, the chemistry was related to the observed colloidal stability of the product in relevant media, including exposure to physiological variables such as salt, pH, proteins, and thermal changes. Overall, this work advances progress toward smart design of engineered nanoscale drug delivery systems and devices by providing unreported details of contributions affecting formation, stability, and fate.

Fast quantification of nanorod geometry by DMA-spICP-MS.

A fast, quantitative method for determining the dimensions of nanorods (i.e., length and diameter) is described, based on hyphenation of differential mobility analysis (DMA) with single particle inductively coupled plasma mass spectrometry (spICP-MS). Seven gold nanorod samples with different dimensions (diameters 11.8 nm to 38.2 nm, aspect ratios 1.8 to 6.9) were used to validate the method. We demonstrate that DMA-spICP-MS can (1) achieve quantification of both length and diameter comparable with TEM analysis, (2) make statistically meaningful measurements in minutes at low concentrations (<108 mL-1) and (3) separate nanorods from spheres and quantify the geometry of each population. A robustness analysis of this method was performed to evaluate potential biases in this approach.

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.

An assessment of retention behavior for gold nanorods in asymmetrical flow field-flow fractionation.

Applications of asymmetrical flow field-flow fractionation (AF4) continue to expand rapidly in the fields of nanotechnology and biotechnology. In particular, AF4 has proven valuable for the separation and analysis of particles, biomolecular species (e.g., proteins, bacteria) and polymers (natural and synthetic), ranging in size from a few nanometers to several micrometers. The separation of non-spheroidal structures (e.g., rods, tubes, etc.) with primary dimensions in the nanometer regime, is a particularly challenging application deserving of greater study and consideration. The goal of the present study was to advance current understanding of the mechanism of separation of rod-like nano-objects in the AF4 channel. To achieve this, we have systematically investigated a series of commercially available cetyltrimethylammonium bromide stabilized gold nanorods (AuNRs), with aspect ratios from 1.7 to 10. Results show clearly that the retention time is principally dependent on the translational diffusion coefficient of the AuNRs. Equations used to calculate translational and rotational diffusion coefficients (cylinder and prolate ellipsoid models) yield similarly good fits to experimental data. Well characterized gold nanorods (length and diameter by transmission electron microscopy) can be used as calibrants for AF4 measurements allowing one to determine the aspect ratio of nanorod samples based on their retention times. Graphical abstract ᅟ.

Electrospray-Differential Mobility Hyphenated with Single Particle Inductively Coupled Plasma Mass Spectrometry for Characterization of Nanoparticles and Their Aggregates.

The novel hyphenation of electrospray-differential mobility analysis with single particle inductively coupled plasma mass spectrometry (ES-DMA-spICPMS) was demonstrated with the capacity for real-time size, mass, and concentration measurement of nanoparticles (NPs) on a particle-to-particle basis. In this proof-of-concept study, the feasibility of this technique was validated through both concentration and mass calibration using NIST gold NP reference materials. A detection limit of 10(5) NPs mL(-1) was determined under current experimental conditions, which is about 4 orders of magnitude lower in comparison to that of a traditional ES-DMA setup using a condensation particle counter as detector. Furthermore, independent and simultaneous quantification of both size and mass of NPs provides information regarding NP aggregation states. Two demonstrative applications include gold NP mixtures with a broad size range (30-100 nm), and aggregated gold NPs with a primary size of 40 nm. Finally, this technique was shown to be potentially useful for real-world samples with high ionic background due to its ability to remove dissolved ions yielding a cleaner background. Overall, we demonstrate the capacity of this new hyphenated technique for (1) clearly resolving NP populations from a mixture containing a broad size range; (2) accurately measuring a linear relationship, which should inherently exist between mobility size and one-third power of ICPMS mass for spherical NPs; (3) quantifying the early stage propagation of NP aggregation with well-characterized oligomers; and (4) differentiating aggregated NPs and nonaggregated states based on the "apparent density" derived from both DMA size and spICPMS mass.

Absolute quantification method for protein concentration.

A fast and accurate assay to determine the absolute concentration of proteins is described based on direct measurement of droplet entrapped oligomer formation in electrospray. Here we demonstrate the approach using electrospray differential mobility analysis (ES-DMA), which can distinguish monomers and dimers from higher order oligomers. A key feature of the method is that it allows determination of the absolute number concentration of proteins eliminating the need for protein-specific calibration. The method was demonstrated by measuring the concentration of a NIST Standard Reference Material 927e (bovine serum albumin), a high-purity immunoglobulin G 1κ, and a formulated Rituximab. The method may be applied to any electrospray source, regardless of diagnostic tool (e.g., MS or ion-mobility, etc.), provided the electrospray is operated in a droplet-fission mode.

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.

High separation efficiency of gold nanomaterials of different aspect ratio and size using capillary transient isotachophoresis.

Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP), were compared for the detection and separation of spherical gold nanoparticles (AuNPs) and gold nanorods (AuNRs). The development of ctITP using two different leading ions is described. Overall, when compared to traditional capillary zone electrophoresis (CZE), ctITP resulted in improved peak shape and peak efficiency. Specifically, the number of theoretical plates for AuNR samples increased by a factor of 2-2.5 depending on the choice of leading ion. Further, using ctITP two AuNRs differing by aspect ratio were baseline resolved, whereas the same AuNRs could not be separated using CZE or other techniques like single particle inductively coupled plasma mass spectrometry (spICP-MS) and asymmetric flow field-flow fractionation (AF4). The results of this study demonstrate that ctITP is an efficient on-line technique for the improved detection and separation of gold nanomaterials in CE.

Parallel multi-parameter study of PEI-functionalized gold nanoparticle synthesis for bio-medical applications: part 1-a critical assessment of methodology, properties, and stability.

Cationic polyethyleneimine (PEI)-conjugated gold nanoparticles (AuNPs) that are chemically and physically stable under physiological conditions are an ideal candidate for certain bio-medical applications, in particular DNA transfection. However, the issue remains in reproducibly generating uniform stable species, which can cause the inadequate characterization of the resulting product under relevant conditions and timepoints. The principal objective of the present study was to develop an optimized and reproducible synthetic route for preparing stable PEI-conjugated AuNPs (Au-PEIs). To achieve this objective, a parallel multi-parametric approach involving a total of 96 reaction studies evaluated the importance of 6 key factors: PEI molar mass, PEI structure, molar ratio of PEI/Au, concentration of reaction mixtures, reaction temperature, and reaction time. Application of optimized conditions exhibited narrow size distributions with characteristic surface plasmon resonance absorption and positive surface charge. The optimized Au-PEI product generated by this study exhibits exceptional stability under a physiological isotonic medium (phosphate-buffered saline) over 48 h and shelf-life in ambient condition without any significant change or sedimentation for at least 6 months. Furthermore, the optimized Au-PEI product was highly reproducible. Contributions from individual factors were elucidated using a broad and orthogonal characterization suite examining size and size distribution, optical absorbance, morphological transformation (agglomeration/aggregation), surface functionalities, and stability. Overall, this comprehensive multi-parametric investigation, supported by thorough characterization and rigorous testing, provides a robust foundation for the nanomedicine research community to better synthesize nanomaterials for biomedical use.

Ultraviolet photo-oxidation of polyvinylpyrrolidone (PVP) coatings on gold nanoparticles.

Polymeric coatings are commonly applied to impart functionality and colloidal stability to engineered nanoparticles. In natural environments, transformations of the coating can modify the particle transport behavior, but the mechanisms and outcomes of these transformations have not yet been thoroughly evaluated. This study investigates the photo-transformations of polyvinylpyrrolidone (PVP) coatings on gold nanoparticles (AuNPs) under ultraviolet (UV) irradiation, representing light exposure in surface waters or other sunlit environments, and the impact on the AuNP colloidal stability. Multiple orthogonal characterization methods were applied to interrogate UV-induced transformations and their consequences. Rapid oxidation of the PVP coating occurred upon UV exposure. The transformed PVP largely persisted on the AuNP surface, albeit in a collapsed polymer layer around the AuNP surface. This transformation resulted in drastically diminished colloidal stability of the AuNPs, consistent with loss of steric stabilization. While the residual coating modified the interaction of the AuNPs with calcium counterions, it did not prevent subsequent stabilization by humic acid. This study demonstrates the importance of both chemical and physical coating transformations on nanoparticles, and hence the need for orthogonal and complementary characterization methods to fully characterize the coating transformations. Finally, the specific transformations of the PVP-coated AuNPs investigated here are discussed more broadly with respect to generalizability to other polymer-coated NPs and the implications for their fate in sunlit or other reactive environments.

Time-dependent density functional theory study of the excited-state dihydrogen bonding: clusters of 2-pyridone with diethylmethylsilane and triethylgermanium.

Density functional theory (DFT) was carried out to identify the existence of intermolecular dihydrogen bonds of the 2-pyridone (2PY)-diethylmethylsilane (DEMS) and 2PY-triethylgermanium (TEGH) clusters in the ground state. The H···H distances of both clusters are shorter than the sum of their van der Waals radii. Thus, intermolecular dihydrogen bonds N-H•••H-Si and N-H•••H-Ge exist in the 2PY-DEMS and 2PY-TEGH clusters, respectively. Based on the ground-state conformations, intermolecular dihydrogen bonds N-H•••H-Si and N-H•••H-Ge in the electronically excited state of the 2PY-DEMS and 2PY-TEGH clusters were also investigated using time-dependent density functional theory (TDDFT). Electronic transition of the 2PY-DEMS cluster resembles that of the 2PY-TEGH cluster. Their S(1) state is a locally excited (LE) state centered on 2PY moiety. The H•••H distances of the 2PY-DEMS and 2PY-TEGH clusters both stretch in the S(1) state compared to those in the ground state. Upon electronic excitation, intermolecular dihydrogen bonding N-H•••H-Si and N-H•••H-Ge can weaken with decreasing dihydrogen bonding energies.