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.

Sizing up the Next Generation of Nanomedicines.

During the past two decades the nanomedicine field has experienced significant progress. To date, over sixty nanoparticle (NP) formulations have been approved in the US and EU while many others are in clinical or preclinical development, indicating a concerted effort to translate promising bench research to commercially viable pharmaceutical products. The use of NPs as novel drug delivery systems, for example, can improve drug safety and efficacy profiles and enable access to intracellular domains of diseased cells, thus paving the way to previously intractable biological targets. However, the measurement of their physicochemical properties presents substantial challenges relative to conventional injectable formulations. In this perspective, we focus exclusively on particle size, a core property and critical quality attribute of nanomedicines. We present an overview of relevant state-of-the-art technologies for particle sizing, highlighting the main parameters that can influence the selection of techniques suitable for a specific size range or material. We consider the increasing need, and associated challenge, to measure size in physiologically relevant media. We detail the importance of standards, key to validate any measurement, and the need for suitable reference materials for processes used to characterize novel and complex NPs. This perspective highlights issues critical to achieve compliance with regulatory guidelines and to support research and manufacturing quality control.

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.

Approaches for the quantitative analysis of oxidation state in cerium oxide nanomaterials.

Cerium oxide nanomaterials (nanoceria, CNMs) are receiving increased attention from the research community due to their unique chemical properties, most prominent of which is their ability to alternate between the Ce3+ and Ce4+ oxidation states. While many analytical techniques and methods have been employed to characterize the amounts of Ce3+ and Ce4+ present (Ce3+/Ce4+ ratio) within nanoceria materials, to-date no studies have used multiple complementary analytical tools (orthogonal analysis) with technique-independent oxidation state controls for quantitative determinations of the Ce3+/Ce4+ ratio. Here, we describe the development of analytical methods measuring the oxidation states of nanoceria analytes using technique-independent Ce3+ (CeAlO3:Ge) and Ce4+ (CeO2) control materials, with a particular focus on x-ray photoelectron spectroscopy (XPS) and electron energy loss spectroscopy (EELS) approaches. The developed methods were demonstrated in characterizing a suite of commercial nanoceria products, where the two techniques (XPS and EELS) were found to be in good agreement with respect to Ce3+/Ce4+ ratio. Potential sources of artifacts and discrepancies in the measurement results were also identified and discussed, alongside suggestions for interpreting oxidation state results using the different analytical techniques. The results should be applicable towards producing more consistent and reproducible oxidation state analyses of nanoceria materials.

Bridging communities in the field of nanomedicine.

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval. The workshop participants came up with recommendations highlighting methodological gaps that should be addressed in ongoing projects addressing the regulatory science of nanomedicines. In addition, individual opinions of experts relevant to progress of the regulatory science in the field of nanomedicine were summarised in the format of a survey.

Characterization of Size and Aggregation for Cellulose Nanocrystal Dispersions Separated by Asymmetrical-Flow Field-Flow Fractionation.

Cellulose nanocrystals (CNCs) derived from various types of cellulose biomass have significant potential for applications that take advantage of their availability from renewable natural resources and their high mechanical strength, biocompatibility and ease of modification. However, their high polydispersity and irregular rod-like shape present challenges for the quantitative dimensional determinations that are required for quality control of CNC production processes. Here we have fractionated a CNC certified reference material using a previously reported asymmetrical-flow field-flow fractionation (AF4) method and characterized selected fractions by atomic force microscopy (AFM) and transmission electron microscopy. This work was aimed at addressing discrepancies in length between fractionated and unfractionated CNC and obtaining less polydisperse samples with fewer aggregates to facilitate microscopy dimensional measurements. The results demonstrate that early fractions obtained from an analytical scale AF4 separation contain predominantly individual CNCs. The number of laterally aggregated "dimers" and clusters containing 3 or more particles increases with increasing fraction number. Size analysis of individual particles by AFM for the early fractions demonstrates that the measured CNC length increases with increasing fraction number, in good agreement with the rod length calculated from the AF4 multi-angle light scattering data. The ability to minimize aggregation and polydispersity for CNC samples has important implications for correlating data from different sizing methods.

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.

Separation and characterization of cellulose nanocrystals by multi-detector asymmetrical-flow field-flow fractionation.

Cellulose nanocrystals (CNCs) are renewable, naturally derived polymeric nanomaterials receiving substantial attention for a wide range of potential applications. The recent availability of high quality reference materials will facilitate the development and validation of measurement methods needed to advance the scientific and commercial use of CNCs. In the present study, we demonstrate an optimized method to fractionate CNCs with narrow size dispersion based on asymmetrical-flow field-flow fractionation (AF4) coupled with on-line multi-angle light scattering (MALS), dynamic light scattering (DLS), and differential refractometry (dRI). A stable suspension of CNC (Certified Reference Material CNCD-1, National Research Council-Canada) in deionized water was prepared using a dispersion method provided by NRC and adopted from a protocol originally developed at the National Institute of Standards and Technology. The as-prepared material was initially characterized in batch mode to validate the NRC dispersion method. AF4 was then optimized for channel and cross flow, mobile phase composition, and injection volume, among other parameters. Additionally, suspensions containing (1.25-10) mg mL-1 CNC were injected directly into the dRI detector (off-line), yielding a dn/dc value of 0.148 ± 0.003 mL g-1. dRI was then used as an on-line mass sensitive detector to quantify recovery. Results show that maximum recovery (≈ 99%) was achieved under optimized conditions. The weight-averaged molar mass (Mw) was estimated at roughly 107 Da from a partial Zimm analysis. The optical radius of gyration, Rg, and the hydrodynamic radius, Rh, were measured during elution. The shape factor (Rg/Rh) ranged from 1.5 to 1.9 for the fractionated material, supporting an elongated or rod-like structure. To our knowledge, this is the first time that both the morphology and molar mass of CNCs have been directly measured for the full distribution of species. Finally, we developed and demonstrated a semi-preparatory fractionation method to separate CNCs at the milligram scale for off-line research and analysis.

Agglomeration of Escherichia coli with Positively Charged Nanoparticles Can Lead to Artifacts in a Standard Caenorhabditis elegans Toxicity Assay.

The increased use and incorporation of engineered nanoparticles (ENPs) in consumer products requires a robust assessment of their potential environmental implications. However, a lack of standardized methods for nanotoxicity testing has yielded results that are sometimes contradictory. Standard ecotoxicity assays may work appropriately for some ENPs with minimal modification but produce artifactual results for others. Therefore, understanding the robustness of assays for a range of ENPs is critical. In this study, we evaluated the performance of a standard Caenorhabditis elegans ( C. elegans) toxicity assay containing an Escherichia coli ( E. coli) food supply with silicon, polystyrene, and gold ENPs with different charged coatings and sizes. Of all the ENPs tested, only those with a positively charged coating caused growth inhibition. However, the positively charged ENPs were observed to heteroagglomerate with E. coli cells, suggesting that the ENPs impacted the ability of nematodes to feed, leading to a false positive toxic effect on C. elegans growth and reproduction. When the ENPs were tested in two alternate C. elegans assays that did not contain E. coli, we found greatly reduced toxicity of ENPs. This study illustrates a key unexpected artifact that may occur during nanotoxicity assays.

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.

Overcoming challenges in single particle inductively coupled plasma mass spectrometry measurement of silver nanoparticles.

Single particle ICP-MS has evolved rapidly as a quantitative method for determining nanoparticle size and number concentration at environmentally relevant exposure levels. Central to the application of spICP-MS is a commonly used, but not rigorously validated, calibration approach based on the measured transport efficiency and the response of ionic standards. In this work, we present a comprehensive and systematic study of the accuracy, precision and robustness of spICP-MS using the rigorously characterized reference material (RM) 8017 (Polyvinylpyrrolidone Coated Nominal 75 nm Silver Nanoparticles), recently issued by the National Institute of Standards and Technology (NIST). We report for the first time, statistically significant differences in frequency-based and size-based measures of transport efficiency with NIST RM 8013 Gold Nanoparticles and demonstrate that the size-based measure of transport efficiency is more robust and yields accurate results for the silver nanoparticle RM relative to TEM-based reference values. This finding is significant, because the frequency-based method is more widely applied. Furthermore, we demonstrate that the use of acidified ionic standards improves measurement of ICP-MS Ag response, but does not degrade the accuracy of the results for AgNP suspensions in water or various other diluents. Approaches for controlling AgNP dissolution were investigated and are shown to effectively improve particle stability in dilute suspensions required for spICP-MS analysis, while minimally affecting the measured intensity and allowing for more robust analysis. This study is an important and necessary advancement toward full validation and adoption of spICP-MS by the broader research community. Graphical abstract Measurement challenges in spICP-MS analysis.

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.

In situ monitoring, separation, and characterization of gold nanorod transformation during seed-mediated synthesis.

The control of gold nanorod (GNR) solution-based syntheses has been hindered in part by the inability to examine and control the conversion of precursor seed populations to anisotropic materials, which have resulted in low yields of desired products and limited their commercial viability. The advantages offered by tandem separation and characterization methods utilizing asymmetric-flow field flow fractionation (A4F) are principally achieved as a result of their non-disruptive nature (minimizing artefacts), fast throughput, and in-situ analysis. With hyphenated A4F methods, resolved populations of seeds and secondary products, up to long aspect ratio rods, have been achieved and exemplify progress towards elucidating mechanistic aspects of formation and thus rational design. While there have been previously reported studies on A4F separation of GNRs, to our knowledge, this is the first published investigation of in situ GNR growth, separation, and characterization based on A4F, where its utilization in this capacity goes beyond traditional separation analysis. By using hydroquinone as the reducing agent, the conversion of the initial seed population to a distribution of products, including the GNRs, could be monitored in real time using A4F hyphenated with a diode array detector. Transmission electron microscopy confirms that the number of peaks observed during fractionation corresponds with size and shape dispersity. This proof-of-principle study introduces A4F as a technique that establishes a foundation for future mechanistic studies on the growth of GNRs from gold seeds, including conversion of the seed population to initial products, a topic highly relevant to advancing progress in nanomanufacturing.

Unexpected Changes in Functionality and Surface Coverage for Au Nanoparticle PEI Conjugates: Implications for Stability and Efficacy in Biological Systems.

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.

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 ≈50 months of cold storage (≈4 °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 ≈0.42 nm(-2) for the methyl-PEG-SH AuNPs, ≈0.26 nm(-2) for the amine-SH-PEG AuNPs, and ≈0.18 nm(-2) for the carboxylic-PEG-SH AuNPs before cold storage, approximately 10 % 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 >80 % 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.

Dissolution, agglomerate morphology, and stability limits of protein-coated silver nanoparticles.

Little is understood regarding the impact that molecular coatings have on nanoparticle dissolution kinetics and agglomerate formation in a dilute nanoparticle dispersion. Dissolution and agglomeration processes compete in removing isolated nanoparticles from the dispersion, making quantitative time-dependent measurements of the mechanisms of nanoparticle loss particularly challenging. In this article, we present in situ ultra-small-angle X-ray scattering (USAXS) results, simultaneously quantifying dissolution, agglomeration, and stability limits of silver nanoparticles (AgNPs) coated with bovine serum albumin (BSA) protein. When the BSA corona is disrupted, we find that the loss of silver from the nanoparticle core is well matched by a second-order kinetic rate reaction, arising from the oxidative dissolution of silver. Dissolution and agglomeration are quantified, and morphological transitions throughout the process are qualified. By probing the BSA-AgNP suspension around its stability limits, we provide insight into the destabilization mechanism by which individual particles rapidly dissolve as a whole rather than undergo slow dissolution from the aqueous interface inward, once the BSA layer is breached. Because USAXS rapidly measures over the entire nanometer to micrometer size range during the dissolution process, many insights are also gained into the stabilization of NPs by protein and its ability to protect the labile metal core from the solution environment by prohibiting the diffusion of reactive species. This approach can be extended to a wide variety of coating molecules and reactive metal nanoparticle systems to carefully survey their stability limits, revealing the likely mechanisms of coating breakdown and ensuing reactions.

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.

PEGylated gold nanorod separation based on aspect ratio: characterization by asymmetric-flow field flow fractionation with UV-Vis detection.

The development of highly efficient asymmetric-flow field flow fractionation (A4F) methodology for biocompatible PEGylated gold nanorods (GNR) without the need for surfactants in the mobile phase is presented. We report on the potential of A4F for rapid separation by evaluating the efficiency of functionalized surface coverage in terms of fractionation, retention time (t R ) shifts, and population analysis. By optimizing the fractionation conditions, we observed that the mechanism of separation for PEGylated GNRs by A4F is the same as that for CTAB stabilized GNRs (i.e., according to their AR) which confirms that the elution mechanism is not dependent on the surface charge of the analytes and/or the membrane. In addition, we demonstrated that A4F can distinguish different surface coverage populations of PEGylated GNRs. The data established that a change in Mw of the functional group and/or surface orientation can be detected and fractionated by A4F. The findings in this study provide the foundation for a complete separation and physicochemical analysis of GNRs and their surface coatings, which can provide accurate and reproducible characterization critical to advancing biomedical research.

Stability-Enhanced Cisplatin Gold Nanoparticles As Therapeutic Anticancer Agents.

Using dendron chemistry, we developed stability enhanced, carboxylate surface modified (negatively charged dendron) AuNPs (Au-NCD). Since the carboxylate surface of Au-NCD is optimal for complexation with cisplatin (Pt) moieties, we further synthesized Pt loaded Au-NCD (Au-NCD/Pt) to serve as potential therapeutic anticancer agents. The size distribution, zeta potential and surface plasmon resonance of both Au-NCDs and Au-NCD/Pt were characterized via dynamic light scattering, scanning transmission electron microscopy and ultraviolet-visible spectrophotometry. Surface chemistry, Pt uptake, and Pt release were evaluated using inductively coupled plasma-mass spectrometry and X-ray photoelectron spectroscopy. Colloidal stability in physiological media over a wide pH range (1 to 13) and shelf-life stability (up to 6 months) were also assessed. Finally, the cytotoxicity of both Au-NCD and Au-NCD/Pt to Chinese hamster ovary cells (CHO K1; as a normal cell line) and to human lung epithelial cells (A549; as a cancer cell line) were evaluated. The results of these physicochemical and functional cytotoxicity studies with Au-NCD/Pt demonstrated that the particles exhibited superlative colloidal stability, cisplatin uptake and in vitro anticancer activity despite low amounts of Pt release from the conjugate.