Synthetic silica fibers of different length, diameter and shape: synthesis and interaction with rat (NR8383) and human (THP-1) macrophages in vitro, including chemotaxis and gene expression profile.

BACKGROUND: Inhalation of biopersistent fibers like asbestos can cause strong chronic inflammatory effects, often resulting in fibrosis or even cancer. The interplay between fiber shape, fiber size and the resulting biological effects is still poorly understood due to the lack of reference materials. RESULTS: We investigated how length, diameter, aspect ratio, and shape of synthetic silica fibers influence inflammatory effects at doses up to 250 µg cm-2. Silica nanofibers were prepared with different diameter and shape. Straight (length ca. 6 to 8 µm, thickness ca. 0.25 to 0.35 µm, aspect ratio ca. 17:1 to 32:1) and curly fibers (length ca. 9 µm, thickness ca. 0.13 µm, radius of curvature ca. 0.5 µm, aspect ratio ca. 70:1) were dispersed in water with no apparent change in the fiber shape during up to 28 days. Upon immersion in aqueous saline (DPBS), the fibers released about 5 wt% silica after 7 days irrespectively of their shape. The uptake of the fibers by macrophages (human THP-1 and rat NR8383) was studied by scanning electron microscopy and confocal laser scanning microscopy. Some fibers were completely taken up whereas others were only partially internalized, leading to visual damage of the cell wall. The biological effects were assessed by determining cell toxicity, particle-induced chemotaxis, and the induction of gene expression of inflammatory mediators. CONCLUSIONS: Straight fibers were only slightly cytotoxic and caused weak cell migration, regardless of their thickness, while the curly fibers were more toxic and caused significantly stronger chemotaxis. Curly fibers also had the strongest effect on the expression of cytokines and chemokines. This may be due to the different aspect ratio or its twisted shape.

Ultrastructure and Surface Composition of Glutathione-Terminated Ultrasmall Silver, Gold, Platinum, and Alloyed Silver-Platinum Nanoparticles (2 nm).

Alloyed ultrasmall silver-platinum nanoparticles (molar ratio Ag:Pt = 50:50) were prepared and compared to pure silver, platinum, and gold nanoparticles, all with a metallic core diameter of 2 nm. They were surface-stabilized by a layer of glutathione (GSH). A comprehensive characterization by high-resolution transmission electron microscopy (HRTEM), electron diffraction (ED), X-ray diffraction (XRD), small-angle X-ray scattering (SAXS), differential centrifugal sedimentation (DCS), and UV spectroscopy showed their size both in the dry and in the water-dispersed state (hydrodynamic diameter). Solution NMR spectroscopy (1H, 13C, COSY, HSQC, HMBC, and DOSY) showed the nature of the glutathione shell including the number of GSH ligands on each nanoparticle (about 200 with a molecular footprint of 0.063 nm2 each). It furthermore showed that there are at least two different positions for the GSH ligand on the gold nanoparticle surface. Platinum strongly reduced the resolution of the NMR spectra compared to silver and gold, also in the alloyed nanoparticles. X-ray photoelectron spectroscopy (XPS) showed that silver, platinum, and silver-platinum particles were at least partially oxidized to Ag(+I) and Pt(+II), whereas the gold nanoparticles showed no sign of oxidation. Platinum and gold nanoparticles were well crystalline but twinned (fcc lattice) despite the small particle size. Silver was crystalline in electron diffraction but not in X-ray diffraction. Alloyed silver-platinum nanoparticles were almost fully amorphous by both methods, indicating a considerable internal disorder.

Water-Based Synthesis of Ultrasmall Nanoparticles of Platinum Group Metal Oxides (1.8 nm).

Ultrasmall nanoparticles of platinum group metal oxides (core diameter of about 1.8 nm) were prepared by alkaline hydrolysis of metal precursors in the presence of NaBH4 and by colloidal stabilization with tripeptide glutathione. We obtained water-dispersed nanoparticles of Rh2O3, PdO, RuO2, IrO2, Os/OsO2, and Pt/PtO. Their size was probed using high-resolution transmission electron microscopy, differential centrifugal sedimentation, small-angle X-ray scattering, and diffusion-ordered 1H NMR spectroscopy (1H DOSY). Their oxidation state was clearly determined using X-ray photoelectron spectroscopy, X-ray powder diffraction, and electron diffraction. The chemical composition of the nanoparticles, that is, the ratio of the metal oxide core and glutathione capping agent, was quantitatively determined by a combination of these methods.

Targeting the Surface of the Protein 14-3-3 by Ultrasmall (1.5†nm) Gold Nanoparticles Carrying the Specific Peptide CRaf.

The surface of ultrasmall gold nanoparticles with an average diameter of 1.55 nm was conjugated with a 14-3-3 protein-binding peptide derived from CRaf. Each particle carries 18 CRaf peptides, leading to an overall stoichiometry of Au(115)Craf(18). The binding to the protein 14-3-3 was probed by isothermal titration calorimetry (ITC) and fluorescence polarization spectroscopy (FP). The dissociation constant (KD ) was measured as 5.0 µM by ITC and 0.9 µM by FP, which was close to the affinity of dissolved CRaf to 14-3-3σ. In contrast to dissolved CRaf, which alone did not enter HeLa cells, CRAF-conjugated gold nanoparticles were well taken up by HeLa cells, opening the opportunity to target the protein inside a cell.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.

Controlling the Surface Functionalization of Ultrasmall Gold Nanoparticles by Sequence-Defined Macromolecules.

Ultrasmall gold nanoparticles (diameter about 2 nm) were surface-functionalized with cysteine-carrying precision macromolecules. These consisted of sequence-defined oligo(amidoamine)s (OAAs) with either two or six cysteine molecules for binding to the gold surface and either with or without a PEG chain (3400 Da). They were characterized by 1 H NMR spectroscopy, 1 H NMR diffusion-ordered spectroscopy (DOSY), small-angle X-ray scattering (SAXS), and high-resolution transmission electron microscopy. The number of precision macromolecules per nanoparticle was determined after fluorescent labeling by UV spectroscopy and also by quantitative 1 H NMR spectroscopy. Each nanoparticle carried between 40 and 100 OAA ligands, depending on the number of cysteine units per OAA. The footprint of each ligand was about 0.074 nm2 per cysteine molecule. OAAs are well suited to stabilize ultrasmall gold nanoparticles by selective surface conjugation and can be used to selectively cover their surface. The presence of the PEG chain considerably increased the hydrodynamic diameter of both dissolved macromolecules and macromolecule-conjugated gold nanoparticles.

Pathways for Oral and Rectal Delivery of Gold Nanoparticles (1.7 nm) and Gold Nanoclusters into the Colon: Enteric-Coated Capsules and Suppositories.

Two ways to deliver ultrasmall gold nanoparticles and gold-bovine serum albumin (BSA) nanoclusters to the colon were developed. First, oral administration is possible by incorporation into gelatin capsules that were coated with an enteric polymer. These permit the transfer across the stomach whose acidic environment damages many drugs. The enteric coating dissolves due to the neutral pH of the colon and releases the capsule's cargo. Second, rectal administration is possible by incorporation into hard-fat suppositories that melt in the colon and then release the nanocarriers. The feasibility of the two concepts was demonstrated by in-vitro release studies and cell culture studies that showed the easy redispersibility after dissolution of the respective transport system. This clears a pathway for therapeutic applications of drug-loaded nanoparticles to address colon diseases, such as chronic inflammation and cancer.

Enhanced dissolution of silver nanoparticles in a physical mixture with platinum nanoparticles based on the sacrificial anode effect.

A strategy to reduce implant-related infections is the inhibition of the initial bacterial implant colonization by biomaterials containing silver (Ag). The antimicrobial efficacy of such biomaterials can be increased by surface enhancement (nanosilver) or by creating a sacrificial anode system for Ag. Such a system will lead to an electrochemically driven enhanced Ag ion release due to the presence of a more noble metal. Here we combined the enlarged surface of nanoparticles (NP) with a possible sacrificial anode effect for Ag induced by the presence of the electrochemically more noble platinum (Pt) in physical mixtures of Ag NP and Pt NP dispersions. These Ag NP/Pt NP mixtures were compared to the same amounts of pure Ag NP in terms of cell biological responses, i.e. the antimicrobial activity against Staphylococcus aureus and Escherichia coli as well as the viability of human mesenchymal stem cells (hMSC). In addition, Ag NP was analyzed by ultraviolet-visible (UV-vis) spectroscopy, cyclic voltammetry, and atomic absorption spectroscopy. It was found that the dissolution rate of Ag NP was enhanced in the presence of Pt NP within the physical mixture compared to a dispersion of pure Ag NP. Dissolution experiments revealed a fourfold increased Ag ion release from physical mixtures due to enhanced electrochemical activity, which resulted in a significantly increased toxicity towards both bacteria and hMSC. Thus, our results provide evidence for an underlying sacrificial anode mechanism induced by the presence of Pt NP within physical mixtures with Ag NP. Such physical mixtures have a high potential for various applications, for example as antimicrobial implant coatings in the biomedicine or as bactericidal systems for water and surface purification in the technical area.

Nanoscopic Porous Iridium/Iridium Dioxide Superstructures (15†nm): Synthesis and Thermal Conversion by In†Situ Transmission Electron Microscopy.

Porous particle superstructures of about 15 nm diameter, consisting of ultrasmall nanoparticles of iridium and iridium dioxide, are prepared through the reduction of sodium hexachloridoiridate(+IV) with sodium citrate/sodium borohydride in water. The water-dispersible porous particles contain about 20 wt % poly(N-vinylpyrrolidone) (PVP), which was added for colloidal stabilization. High-resolution transmission electron microscopy confirms the presence of both iridium and iridium dioxide primary particles (1-2 nm) in each porous superstructure. The internal porosity (≈58 vol%) is demonstrated by electron tomography. In situ transmission electron microscopy up to 1000 °C under oxygen, nitrogen, argon/hydrogen (all at 1 bar), and vacuum shows that the porous particles undergo sintering and subsequent compaction upon heating, a process that starts at around 250 °C and is completed at around 800 °C. Finally, well-crystalline iridium dioxide is obtained under all four environments. The catalytic activity of the as-prepared porous superstructures in electrochemical water splitting (oxygen evolution reaction; OER) is reduced considerably upon heating owing to sintering of the pores and loss of internal surface area.

Click Chemistry on the Surface of Ultrasmall Gold Nanoparticles (2 nm) for Covalent Ligand Attachment Followed by NMR Spectroscopy.

Ultrasmall gold nanoparticles (core diameter 2 nm) were surface-conjugated with azide groups by attaching the azide-functionalized tripeptide lysine(N3)-cysteine-asparagine with ∼117 molecules on each nanoparticle. A covalent surface modification with alkyne-containing molecules was then possible by copper-catalyzed click chemistry. The successful clicking to the nanoparticle surface was demonstrated with 13C-labeled propargyl alcohol. All steps of the nanoparticle surface conjugation were verified by extensive NMR spectroscopy on dispersed nanoparticles. The particle diameter and the dispersion state were assessed by high-resolution transmission electron microscopy (HRTEM), differential centrifugal sedimentation (DCS), and 1H-DOSY NMR spectroscopy. The clicking of fluorescein (FAM-alkyne) gave strongly fluorescing ultrasmall nanoparticles that were traced inside eukaryotic cells. The uptake of these nanoparticles after 24 h by HeLa cells was very efficient and showed that the nanoparticles even penetrated the nuclear membrane to a very high degree (in contrast to dissolved FAM-alkyne alone that did not enter the cell). About 8 fluorescein molecules were clicked to each nanoparticle.

Solution NMR Spectroscopy with Isotope-Labeled Cysteine (13C and 15N) Reveals the Surface Structure of l-Cysteine-Coated Ultrasmall Gold Nanoparticles (1.8 nm).

Ultrasmall gold nanoparticles with a diameter of 1.8 nm were synthesized by reduction of tetrachloroauric acid with sodium borohydride in the presence of l-cysteine, with natural isotope abundance as well as 13C-labeled and 15N-labeled. The particle diameter was determined by high-resolution transmission electron microscopy and differential centrifugal sedimentation. X-ray photoelectron spectroscopy confirmed the presence of metallic gold with only a few percent of oxidized Au(+I) species. The surface structure and the coordination environment of the cysteine ligands on the ultrasmall gold nanoparticles were studied by a variety of homo- and heteronuclear NMR spectroscopic techniques including 1H-13C-heteronuclear single-quantum coherence and 13C-13C-INADEQUATE. Further information on the binding situation (including the absence of residual or detached l-cysteine in the solution) and on the nanoparticle diameter (indicating the well-dispersed state) was obtained by diffusion-ordered spectroscopy (1H-, 13C-, and 1H-13C-DOSY). Three coordination environments of l-cysteine on the gold surface were identified that were ascribed to different crystallographic sites, supported by geometric considerations of the nanoparticle ultrastructure. The particle size data and the NMR-spectroscopic analysis gave a particle composition of about Au174(cysteine)67.

Bimetallic silver-platinum nanoparticles with combined osteo-promotive and antimicrobial activity.

Bimetallic alloyed silver-platinum nanoparticles (AgPt NP) with different metal composition from Ag10Pt90 to Ag90Pt10 in steps of 20 mol% were synthesized. The biological effects of AgPt NP, including cellular uptake, cell viability, osteogenic differentiation and osteoclastogenesis as well as the antimicrobial activity towards Staphylococcus aureus and Escherichia coli were analyzed in comparison to pure Ag NP and pure Pt NP. The uptake of NP into human mesenchymal stem cells was confirmed by cross-sectional focused-ion beam preparation and observation by scanning and transmission electron microscopy in combination with energy-dispersive x-ray analysis. Lower cytotoxicity and antimicrobial activity were observed for AgPt NP compared to pure Ag NP. Thus, an enhanced Ag ion release due to a possible sacrificial anode effect was not achieved. Nevertheless, a Ag content of at least 50 mol% was sufficient to induce bactericidal effects against both Staphylococcus aureus and Escherichia coli. In addition, a Pt-related (≥50 mol% Pt) osteo-promotive activity on human mesenchymal stem cells was observed by enhanced cell calcification and alkaline phosphatase activity. In contrast, the osteoclastogenesis of rat primary precursor osteoclasts was inhibited. In summary, these results demonstrate a combinatory osteo-promotive and antimicrobial activity of bimetallic Ag50Pt50 NP.

Deciphering the Surface Composition and the Internal Structure of Alloyed Silver-Gold Nanoparticles.

Spherical bimetallic AgAu nanoparticles in the molar ratios 30:70, 50:50, and 70:30 with diameters of 30 to 40 nm were analyzed together with pure silver and gold nanoparticles of the same size. Dynamic light scattering (DLS) and differential centrifugal sedimentation (DCS) were used for size determination. Cyclic voltammetry (CV) was used to determine the nanoalloy composition, together with atomic absorption spectroscopy (AAS), energy-dispersive X-ray spectroscopy (EDX) and ultraviolet-visible (UV/Vis) spectroscopy. Underpotential deposition (UPD) of lead (Pb) on the particle surface gave information about its spatial elemental distribution and surface area. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were applied to study the shape and the size of the nanoparticles. X-ray powder diffraction gave the crystallite size and the microstrain. The particles form a solid solution (alloy) with an enrichment of silver on the nanoparticle surface, including some silver-rich patches. UPD indicated that the surface only consists of silver atoms.

Comparison of different methods to study effects of silver nanoparticles on the pro- and antioxidant status of human keratinocytes and fibroblasts.

In times of antibiotic-resistant bacteria new strategies to avoid the septic-inducing threat of dangerous microorganisms are needed. Silver ions (Ag+) in the forms of silver nitrate or silver sulfadiazine have been used as antimicrobial agents for years. A step further was the development of micro and silver particles (AgNP). In contrast to other Ag+ ion sources, AgNP allow a sustained release of Ag+ ions, due to their high surface to volume ratio. However, AgNP are also toxic to eukaryotic cells and the mechanisms of cytotoxicity have not yet been fully elucidated. In this study, the impact of different AgNP preparations on a human keratinocyte cell line was investigated. The intracellular radical formation was confirmed by the 2',7'-dichlorodihydrofluorescein di-acetate (H2DCF-DA) assay on two cell types (HaCaT cells and normal human dermal fibroblasts) as well as by electron paramagnetic resonance (EPR) spectroscopy, which showed comparable results. EPR spectroscopy was performed for the first time for 24h in experiments using keratinocytes. Drastic changes in the mitochondrial activity were induced in cells incubated with AgNP containing high concentrations of Ag+ ions. It was also possible to show that the quantitative uptake of AgNP was dependent on the AgNP concentration. In addition, the effects of AgNP on the GSH/GSSG system were elucidated. The results showed a batch- and concentration-dependent decrease of the total glutathione concentration which correlated well with the decrease of cell viability. Furthermore, the results suggest a direct reaction of GSH molecules with Ag+ ions. In conclusion, this study proves the efficacy of the H2DCF-DA assay and the EPR spectroscopy. The investigations show that AgNP formulations containing high amounts of released Ag+ ions induce radicals in human keratinocytes and deplete them of their natural anti-oxidative molecules. On the contrary, nanoparticles prepared and stored under argon did not induce significant adverse effects, suggesting that slowing down the release of Ag+ may help to reduce AgNP-related side effects without affecting the antibacterial impact.

Peculiarities in thermal evolution of precipitated amorphous calcium phosphates with an initial Ca/P ratio of 1:1.

Thermal evolution of amorphous calcium phosphate (ACP) powder from a fast nitrate synthesis with a Ca/P ratio of 1:1 were studied in the range of 20-980 °C. The powder consisted of amorphous dicalcium phosphate anhydrate (CaHPO4) after heating to 200 °C. CaHPO4 gradually condensed to amorphous calcium pyrophosphate Ca2P2O7 (CPP) between 200 to 620 °C. Amorphous CPP crystallized at 620-740 °C to a metastable polymorph α'-CPP of the high-temperature phase α-CPP and ß-CPP. The α'-CPP/ ß-CPP phase ratio reached a maximum at 800 °C (60 wt% α'-CPP/40 wt% ß-CPP), and α'-CPP gradually transformed to ß-CPP at a higher temperature. Some ß-TCP occurred at 900 °C, so that a three-phasic mixture was obtained in the powder heated to 980 °C. The occurrence of metastable α'-CPP is attributed to Ostwald's step rule, and a mechanism for ß-TCP formation is proposed. The advantages of prospective biomaterials from these powders are discussed.

The Molecular Footprint of Peptides on the Surface of Ultrasmall Gold Nanoparticles (2 nm) Is Governed by Steric Demand.

Ultrasmall gold nanoparticles were functionalized with peptides of two to seven amino acids that contained one cysteine molecule as anchor via a thiol-gold bond and a number of alanine residues as nonbinding amino acid. The cysteine was located either in the center of the molecule or at the end (C-terminus). For comparison, gold nanoparticles were also functionalized with cysteine alone. The particles were characterized by UV spectroscopy, differential centrifugal sedimentation (DCS), high-resolution transmission electron microscopy (HRTEM), and small-angle X-ray scattering (SAXS). This confirmed the uniform metal core (2 nm diameter). The hydrodynamic diameter was probed by 1H-DOSY NMR spectroscopy and showed an increase in thickness of the hydrated peptide layer with increasing peptide size (up to 1.4 nm for heptapeptides; 0.20 nm per amino acid in the peptide). 1H NMR spectroscopy of water-dispersed nanoparticles showed the integrity of the peptides and the effect of the metal core on the peptide. Notably, the NMR signals were very broad near the metal surface and became increasingly narrow in a distance. In particular, the methyl groups of alanine can be used as probe for the resolution of the NMR spectra. The number of peptide ligands on each nanoparticle was determined using quantitative 1H NMR spectroscopy. It decreased with increasing peptide length from about 100 for a dipeptide to about 12 for a heptapeptide, resulting in an increase of the molecular footprint from about 0.1 to 1.1 nm2.

Automated analysis of transmission electron micrographs of metallic nanoparticles by machine learning.

Metallic nanoparticles were analysed with respect to size and shape by a machine learning approach. This involved a separation of particles from the background (segmentation), a separation of overlapping particles, and the identification of individual particles. An algorithm to separate overlapping particles, based on ultimate erosion of convex shapes (UECS), was implemented. Finally, particle properties like size, circularity, equivalent diameter, and Feret diameter were computed for each particle of the whole particle population. Thus, particle size distributions can be easily created based on the various parameters. However, strongly overlapping particles are difficult and sometimes impossible to separate because of an a priori unknown shape of a particle that is partially lying in the shadow of another particle. The program is able to extract information from a sequence of images of the same sample, thereby increasing the number of analysed nanoparticles to several thousands. The machine learning approach is well-suited to identify particles at only limited particle-to-background contrast as is demonstrated for ultrasmall gold nanoparticles (2 nm).

Cell-Biological Response and Sub-Toxic Inflammatory Effects of Titanium Dioxide Particles with Defined Polymorphic Phase, Size, and Shape.

Six types of titanium dioxide particles with defined size, shape, and crystal structure (polymorphic form) were prepared: nanorods (70 × 25 nm2), rutile sub-microrods (190 × 40 nm2), rutile microspheres (620 nm), anatase nanospheres (100 nm), anatase microspheres (510 nm), and amorphous titania microspheres (620 nm). All particles were characterized by scanning electron microscopy, X-ray powder diffraction, dynamic light scattering, infrared spectroscopy, and UV spectroscopy. The sub-toxic cell-biological response to these particles by NR8383 macrophages was assessed. All particle types were taken up well by the cells. The cytotoxicity and the induction of reactive oxygen species (ROS) were negligible for all particles up to a dose of 100 µg mL-1, except for rutile microspheres which had a very rough surface in contrast to anatase and amorphous titania microspheres. The particle-induced cell migration assay (PICMA; based on chemotaxis) of all titanium dioxide particles was comparable to the effect of control silica nanoparticles (50 nm, uncoated, agglomerated) but did not show a trend with respect to particle size, shape, or crystal structure. The coating with carboxymethylcellulose (CMC) had no significant biological effect. However, the rough surface of rutile microspheres clearly induced pro-inflammatory cell reactions that were not predictable by the primary particle size alone.

Incorporation of Fluoride into Human Teeth after Immersion in Fluoride-Containing Solutions.

Toothpastes and mouth rinses contain fluoride as a protective agent against caries. The aim of this study was to determine the degree of fluoride-uptake by human tooth mineral during immersion into fluoride-containing aqueous solutions as different pH. Human teeth were immersed in fluoride-containing solutions to assess the extent of fluoride incorporation into tooth enamel. A total of 16 extracted teeth from 11 patients were immersed at 37 °C for one minute into aqueous fluoride solutions (potassium fluoride; KF) containing either 250 ppm or 18,998 ppm fluoride (1-molar). Fluoride was dissolved either in pure water (neutral pH) or in a citrate buffer (pH 4.6 to 4.7). The elemental surface composition of each tooth was studied by energy-dispersive X-ray spectroscopy in combination with scanning electron microscopy and X-ray powder diffraction. The as-received teeth contained 0.17 ± 0.16 wt% fluoride on average. There was no significant increase in the fluoride content after immersion in 250 ppm fluoride solution at neutral or acidic pH values. In contrast, a treatment with a 1-molar fluoride solution led to significantly increased fluoride concentrations by 0.68 wt% in water and 9.06 wt% at pH 4.7. Although such fluoride concentrations are far above those used in mouth rinses or toothpastes, this indicates that fluoride can indeed enter the tooth surface, especially at a low pH where a dynamic dissolution-reprecipitation process may occur. However, precipitations of calcium fluoride (globuli) were detected in no cases.

Silencing of proinflammatory NF-κB and inhibition of herpes simplex virus (HSV) replication by ultrasmall gold nanoparticles (2 nm) conjugated with small-interfering RNA.

Azide-terminated ultrasmall gold nanoparticles (2 nm gold core) were covalently functionalized with alkyne-terminated small-interfering siRNA duplexes by copper-catalyzed azide-alkyne cycloaddition (CuAAC; click chemistry). The nanoparticle core was visualized by transmission electron microscopy. The number of attached siRNA molecules per nanoparticle was determined by a combination of atomic absorption spectroscopy (AAS; for gold) and UV-Vis spectroscopy (for siRNA). Each nanoparticle carried between 6 and 10 siRNA duplex molecules which corresponds to a weight ratio of siRNA to gold of about 2.2 : 1. Different kinds of siRNA were conjugated to the nanoparticles, depending on the gene to be silenced. In general, the nanoparticles were readily taken up by cells and highly efficient in gene silencing, in contrast to free siRNA. This was demonstrated in HeLa-eGFP cells (silencing of eGFP) and in LPS-stimulated macrophages (silencing of NF-κB). Furthermore, we demonstrated that nanoparticles carrying antiviral siRNA potently inhibited the replication of Herpes simplex virus 2 (HSV-2) in vitro. This highlights the strong potential of siRNA-functionalized ultrasmall gold nanoparticles in a broad spectrum of applications, including gene silencing and treatment of viral infections, combined with a minimal dose of gold.