Alaaldin M. Alkilany

Toxicity and cellular uptake of gold nanoparticles: what we have learned so far?

Gold nanoparticles have attracted enormous scientific and technological interest due to their ease of synthesis, chemical stability, and unique optical properties. Proof-of-concept studies demonstrate their biomedical applications in chemical sensing, biological imaging, drug delivery, and cancer treatment. Knowledge about their potential toxicity and health impact is essential before these nanomaterials can be used in real clinical settings. Furthermore, the underlying interactions of these nanomaterials with physiological fluids is a key feature of understanding their biological impact, and these interactions can perhaps be exploited to mitigate unwanted toxic effects. In this Perspective we discuss recent results that address the toxicity of gold nanoparticles both in vitro and in vivo, and we provide some experimental recommendations for future research at the interface of nanotechnology and biological systems.

Cellular Uptake and Cytotoxicity of Gold Nanorods: Molecular Origin of Cytotoxicity and Surface Effects

Gold nanorods of different aspect ratios are prepared using the growth-directing surfactant, cetyltrimethylammonium bromide (CTAB), which forms a bilayer on the gold nanorod surface. Toxicological assays of CTAB-capped nanorod solutions with human colon carcinoma cells (HT-29) reveal that the apparent cytotoxicity is caused by free CTAB in solution. Overcoating the nanorods with polymers substantially reduces cytotoxicity. The number of nanorods taken up per cell, for the different surface coatings, is quantitated by inductively coupled plasma mass spectrometry on washed cells; the number of nanorods per cell varies from 50 to 2300, depending on the surface chemistry. Serum proteins from the biological media, most likely bovine serum albumin, adsorb to gold nanorods, leading to all nanorod samples bearing the same effective charge, regardless of the initial nanorod surface charge. The results suggest that physiochemical surface properties of nanomaterials change substantially after coming into contact with biological media. Such changes should be taken into consideration when examining the biological properties or environmental impact of nanoparticles.

Nanoparticle–Protein Interactions: A Thermodynamic and Kinetic Study of the Adsorption of Bovine Serum Albumin to Gold Nanoparticle Surfaces

Investigating the adsorption process of proteins on nanoparticle surfaces is essential to understand how to control the biological interactions of functionalized nanoparticles. In this work, a library of spherical and rod-shaped gold nanoparticles (GNPs) was used to evaluate the process of protein adsorption to their surfaces. The binding of a model protein (bovine serum albumin, BSA) to GNPs as a function of particle shape, size, and surface charge was investigated. Two independent comparative analytical methods were used to evaluate the adsorption process: steady-state fluorescence quenching titration and affinity capillary electrophoresis (ACE). Although under favorable electrostatic conditions kinetic analysis showed a faster adsorption of BSA to the surface of cationic GNPs, equilibrium binding constant determinations indicated that BSA has a comparable binding affinity to all of the GNPs tested, regardless of surface charge. BSA was even found to adsorb strongly to GNPs with a pegylated/neutral surface. However, these fluorescence titrations suffer from significant interference from the strong light absorption of the GNPs. The BSA-GNP equilibrium binding constants, as determined by the ACE method, were 10(5) times lower than values determined using spectroscopic titrations. While both analytical methods could be suitable to determine the binding constants for protein adsorption to NP surfaces, both methods have limitations that complicate the determination of protein-GNP binding constants. The optical properties of GNPs interfere with Ka determinations by static fluorescence quenching analysis. ACE, in contrast, suffers from material compatibility issues, as positively charged GNPs adhere to the walls of the capillary during analysis. Researchers seeking to determine equilibrium binding constants for protein-GNP interactions should therefore utilize as many orthogonal techniques as possible to study a protein-GNP system.

Exocytosis of nanoparticles from cells: Role in cellular retention and toxicity

Over the past decade, nanoparticles (NPs) have been increasingly developed in various biomedical applications such as cell tracking, biosensing, contrast imaging, targeted drug delivery, and tissue engineering. Their versatility in design and function has made them an attractive, alternative choice in many biological and biomedical applications. Cellular responses to NPs, their uptake, and adverse biological effects caused by NPs are rapidly-growing research niches. However, NP excretion and its underlying mechanisms and cell signaling pathways are yet elusive. In this review, we present an overview of how NPs are handled intracellularly and how they are excreted from cells following the uptake. We also discuss how exocytosis of nanomaterials impacts both the therapeutic delivery of nanoscale objects and their nanotoxicology.

Cation Exchange on the Surface of Gold Nanorods with a Polymerizable Surfactant: Polymerization, Stability, and Toxicity Evaluation

Gold nanorods were synthesized using a seed-mediated wet chemical approach with a quaternary ammonium surfactant, cetyltrimethylammonium bromide (CTAB), that forms a bilayer on the surface of the nanorods. The CTAB molecules in the bilayer were exchanged with a similar polymerizable analog, 11-(acryloyloxy) undecyltrimethyl ammonium bromide (p-CTAB). Mass spectrometric analysis of the degree of exchange of CTAB for p-CTAB, after gold digestion, gave 77 +/- 3 and 23 +/- 1% for p-CTAB and CTAB, respectively. On-rod polymerization with a cationic free-radical initiator was confirmed by FTIR analysis and did not induce aggregation as judged by ultraviolet-visible spectroscopy, transmission electron microscopy, and dynamic light scattering measurements after polymerization. In contrast to the nanorods before polymerization, the nanorods with a polymerized bilayer showed improved stability against dialysis as well as enhanced biocompatibility as measured using a viability assay on cultured human cells. Our results indicate that (1) CTAB molecules on the surface of the gold nanorods are exchangeable with similar surfactants that have a positively charged headgroup and (2) surfactant polymerization on the surface of the gold nanorods enhances both the stability and biocompatibility of these nanomaterials, probably by decreasing the degree of surfactant desorption from the surface.

Gold nanorods as nanoadmicelles: 1-naphthol partitioning into a nanorod-bound surfactant bilayer

As-prepared gold nanorods, stable in aqueous solution, bear a bilayer of the cationic surfactant cetyltrimethylammonium bromide (CTAB). This bilayer provides a approximately 3 nm thick hydrophobic layer that could be used to sequester hydrophobic organic molecules from aqueous solution. We have investigated the uptake of 1-naphthol as a model hydrophobic compound by CTAB-coated gold nanorods using both ultraviolet-visible spectroscopy and gas chromatography with flame ionization detection. We find the adsorption isotherm of 1-naphthol partitioning into the CTAB bilayer on gold nanorods fits the Langmuir model. The maximum number of bound 1-naphthol molecules is 14.6 +/- 2.2 x 10(3) molecules per gold nanorod, with an equilibrium binding constant of 1.97 +/- 0.79 x 10(4) M(-1) at room temperature.

Protein corona: Opportunities and challenges

In contact with biological fluids diverse type of biomolecules (e.g., proteins) adsorb onto nanoparticles forming protein corona. Surface properties of the coated nanoparticles, in terms of type and amount of associated proteins, dictate their interactions with biological systems and thus biological fate, therapeutic efficiency and toxicity. In this perspective, we will focus on the recent advances and pitfalls in the protein corona field.

Toxicity and Cellular Uptake of Gold Nanorods in Vascular Endothelium and Smooth Muscles of Isolated Rat Blood Vessel: Importance of Surface Modification

Gold nanorods (GNRs) have promising applications in drug delivery and cancer treatment and are generally administered via direct injection into the circulation. Thus it is necessary to evaluate their potential adverse effects on blood vessels. Herein, GNRs with various surface modifications are used to evaluate the toxicity and cellular uptake of GNRs into vascular endothelial and smooth muscle cells of isolated rat aortic rings. Surfactant-capped GNRs are synthesized and either coated with polyelectrolyte (PE) to prepare PE-GNRs, or modified with thiolated polyethylene glycol (PEG) to prepare PEG-GNRs. Using toxicity assays, small-vessel myography, fluorescence microscopy, and electron microscopy, it is shown that therapeutic concentrations of PE-GNRs but not PEG-GNRs are toxic to the vascular endothelium, which leads to an impaired relaxation function of aortic rings. However, no toxicity to smooth muscles is observed. Moreover, electron microscopy analysis confirms the cellular uptake of PE-GNRs but not PEG-GNRs into the endothelium of exposed aortic rings. The difference in toxicity and cellular uptake of PE-GNRs versus PEG-GNRs is explained and linked to free surfactant molecules and protein adsorption, respectively. The results indicate that toxicity and cellular uptake in the vascular endothelium in blood vessels are potential adverse effects of systemically administered GNR solutions, which can be prevented by appropriate surface functionalization.

Clickable polyglycerol hyperbranched polymers and their application to gold nanoparticles and acid-labile nanocarriers

A one-step, large-scale preparation of alkyne-containing hyper-branched polyglycerols (HPG) is reported. The HPGs undergo click reactions to organic azides allowing a range of applications.

Polyelectrolyte coating provides a facile route to suspend gold nanorods in polar organic solvents and hydrophobic polymers.

The widely used and versatile polyelectrolyte layer-by-layer (LbL) nanoparticle coating strategy allows for gold nanorods to be transferred from aqueous media into a broad range of polar organic solvents without aggregation. The uniform dispersity and stability of the nanorods in organic solvents allows for uniform incorporation of nanorods into a variety of hydrophobic polymers.