Probing the Mechanism of LAL-32, a Gold Nanoparticle-Based Antibiotic Discovered through Small Molecule Variable Ligand Display.

The unrelenting rise of antimicrobial-resistant bacteria has necessitated the search for novel antibiotic solutions. Herein we describe further mechanistic studies on a 2.0-nm-diameter gold nanoparticle-based antibiotic (designated LAL-32). This antibiotic exhibits bactericidal activity against the Gram-negative bacterium Escherichia coli at 1.0 µM, a concentration significantly lower than several clinically available antibiotics (such as ampicillin and gentamicin), and acute treatment with LAL-32 does not give rise to spontaneous resistant mutants. LAL-32 treatment inhibits cellular division, daughter cell separation, and twin-arginine translocation (Tat) pathway dependent shuttling of proteins to the periplasm. Furthermore, we have found that the cedA gene imparts increased resistance to LAL-32, and shown that an E. coli cedA transposon mutant exhibits increased susceptibility to LAL-32. Taken together, these studies further implicate cell division pathways as the target for this nanoparticle-based antibiotic and demonstrate that there may be inherently higher barriers for resistance evolution against nanoscale antibiotics in comparison to their small molecule counterparts.

Gold nanoparticles to improve HIV drug delivery.

BACKGROUND: Antiretroviral therapy (ART) has improved lifespan and quality of life of patients infected with the HIV-1. However, ART has several potential limitations, including the development of drug resistance and suboptimal penetration to selected anatomic compartments. Improving the delivery of antiretroviral molecules could overcome several of the limitations of current ART. RESULTS & CONCLUSION: Two to ten nanometer diameter inorganic gold crystals serve as a base scaffold to combine molecules with an array of properties in its surface. We show entry into different cell types, antiviral activity of an HIV integrase inhibitor conjugated in a gold nanoparticle and penetration into the brain in vivo without toxicity. Herein, gold nanoparticles prove to be a promising tool to use in HIV therapy.

Thiol-modified gold nanoparticles for the inhibition of Mycobacterium smegmatis.

Antimicrobial drug discovery has slowed considerably over the last few decades. One major cause for concern is the lack of innovative approaches to treat infections caused by mycobacteria such as TB. Herein we demonstrate that our Small Molecule Variable Ligand Display (SMLVD) method for nanoparticle antibiotic discovery can be expanded around a ligand feed ratio parameter space to identify gold nanoparticle conjugates that are potent inhibitors of mycobacteria growth, with our most potent inhibitor able to reduce growth by five orders of magnitude at 8 µM.

Nanoscale structure-activity relationships, mode of action, and biocompatibility of gold nanoparticle antibiotics.

The emergence of resistance to multiple antimicrobial agents by pathogenic bacteria has become a significant global public health threat. Multi-drug-resistant (MDR) Gram-negative bacteria have become particularly problematic, as no new classes of small-molecule antibiotics for Gram-negative bacteria have emerged in over two decades. We have developed a combinatorial screening process for identifying mixed ligand monolayer/gold nanoparticle conjugates (2.4 nm diameter) with antibiotic activity. The method previously led to the discovery of several conjugates with potent activity against the Gram-negative bacterium Escherichia coli. Here we show that these conjugates are also active against MDR E. coli and MDR Klebsiella pneumoniae. Moreover, we have shown that resistance to these nanoparticles develops significantly more slowly than to a commercial small-molecule drug. These results, combined with their relatively low toxicity to mammalian cells and biocompatibility in vivo, suggest that gold nanoparticles may be viable new candidates for the treatment of MDR Gram-negative bacterial infections.

Methods for isolating RNA sequences capable of binding to or mediating the formation of inorganic materials.

The ability of oligonucleotides to mediate the formation of inorganic materials is now well established. RNA and DNA are proving to be capable of mediating the formation of inorganic nanoparticles with sequence-specific control over nanoparticle size, shape, and even atomic-level crystallinity. Here we describe methods for isolating specific RNA sequences from large random sequence libraries that either bind to precut inorganic crystal wafers with high affinity or influence inorganic crystal growth.

Gastrointestinal bioavailability of 2.0 nm diameter gold nanoparticles.

The use of gold nanoparticles as imaging agents and therapeutic delivery systems is growing rapidly. However, a significant limitation of gold nanoparticles currently is their low absorption efficiencies in the gastrointestinal (GI) tract following oral administration. In an attempt to identify ligands that facilitate gold nanoparticle absorption in the GI tract, we have studied the oral bioavailability of 2.0 nm diameter gold nanoparticles modified with the small molecules p-mercaptobenzoic acid and glutathione, and polyethylene glycols (PEG) of different lengths and charge (neutral and anionic). We show that GI absorption of gold nanoparticles modified with the small molecules tested was undetectable. However, the absorption of PEGs depended upon PEG length, with the shortest PEG studied yielding gold nanoparticle absorptions that are orders-of-magnitude larger than observed previously. As the oral route is the most convenient one for administering drugs and diagnostic reagents, these results suggest that short-chain PEGs may be useful in the design of gold nanoparticles for the diagnosis and treatment of disease.

In vivo toxicity, biodistribution, and clearance of glutathione-coated gold nanoparticles.

Gold nanoparticles are emerging as promising materials from which to construct nanoscale therapeutics and therapeutic delivery systems. However, animal studies have shown that gold nanoparticles modified with certain thiol monolayers such as tiopronin can cause renal complications and morbidity. Although these effects may be eliminated by coadsorbing small amounts of polyethylene glycol (PEG) onto the nanoparticle surface, PEG can also lower cellular internalization efficiency and binding interactions with protein disease targets, significantly reducing the potential for using gold nanoparticles as therapeutics. Using ICP-MS analysis of blood, urine, and several organs, we show in this article that glutathione-coated gold nanoparticles (1.2 nm ± 0.9 nm) cause no morbidity at any concentration up to and including 60 µM and target primary organs although providing gradual dissipation and clearance over time. This study suggests that glutathione may be an attractive alternative to PEG in the design of gold nanoparticle therapeutics. FROM THE CLINICAL EDITOR: This study describes the utility and toxicity of glutathione coated gold nanoparticles in comparison to PEGylated counterparts that are commonly used to increase "Stealth" properties and lower cytotoxicity. Too much PEG on the NPs can lead to lower cellular internalization efficiency and less efficient binding interactions with protein disease targets, significantly reducing the potential for using gold nanoparticles as therapeutics.

Identification of antibiotics using small molecule variable ligand display on gold nanoparticles.

Here we describe the use of simple 1-pot thiol exchange reactions to generate a library of mixed ligand-coated gold nanoparticles that was screened for antibiotic activity. A library of 120 nanoparticle conjugates was assembled and antibiotic activity toward E. coli was determined and found to depend upon the combination of thiols assembled onto the nanoparticles. The most active conjugate displayed 99.9% growth inhibition at 0.5 µM.

Unusual reactivity of a silver mineralizing peptide.

The ability of peptides selected via phage display to mediate the formation of inorganic nanoparticles is now well established. The atomic-level interactions between the selected peptides and the metal ion precursors are in most instances, however, largely obscure. We identified a new peptide sequence that is capable of mediating the formation of Ag nanoparticles. Surprisingly, nanoparticle formation requires the presence of peptide, HEPES buffer, and light; the absence of any one of these compromises nanoparticle formation. Electrochemical experiments revealed that the peptide binds Ag+ in a 3 Ag+:1 peptide ratio and significantly alters the Ag+ reduction potential. Alanine replacement studies yielded insight into the sequence-function relationships of Ag nanoparticle formation, including the Ag+ coordination sites and the residues necessary for Ag synthesis. In addition, the peptide was found to function when immobilized onto surfaces, and the specific immobilizing concentration could be adjusted to yield either spherical Ag nanoparticles or high aspect ratio nanowires. These studies further illustrate the range of interesting new solid-state chemistries possible using biomolecules.

Inhibition of HIV fusion with multivalent gold nanoparticles.

The design and synthesis of a multivalent gold nanoparticle therapeutic is presented. SDC-1721, a fragment of the potent HIV inhibitor TAK-779, was synthesized and conjugated to 2.0 nm diameter gold nanoparticles. Free SDC-1721 had no inhibitory effect on HIV infection; however, the (SDC-1721)-gold nanoparticle conjugates displayed activity comparable to that of TAK-779. This result suggests that multivalent presentation of small molecules on gold nanoparticle surfaces can convert inactive drugs into potent therapeutics.

Scanning probe-based fabrication of 3D nanostructures via affinity templates, functional RNA, and meniscus-mediated surface remodeling.

Developing generic platforms to organize discrete molecular elements and nanostructures into deterministic patterns on surfaces is one of the central challenges in the field of nanotechnology. Here we review three applications of the atomic force microscope (AFM) that address this challenge. In the first, we use two-step nanografting to create patterns of self-assembled monolayers (SAMs) to drive the organization of virus particles that have been either genetically or chemically modified to bind to the SAMs. Virus-SAM chemistries are described that provide irreversible and reversible binding, respectively. In the second, we use similar SAM patterns as affinity templates that have been designed to covalently bind oligonucleotides engineered to bind to the SAMs and selected for their ability to mediate the subsequent growth of metallic nanocrystals. In the final application, the liquid meniscus that condenses at the AFM tip-substrate contact is used as a physical tool to both modulate the surface topography of a water soluble substrate and guide the hierarchical assembly of Au nanoparticles into nanowires. All three approaches can be generalized to meet the requirements of a wide variety of materials systems and thereby provide a potential route toward development of a generic platform for molecular and materials organization.

Cellular uptake of gold nanoparticles passivated with BSA-SV40 large T antigen conjugates.

Internalization and subcellular localization in HeLa cells of gold nanoparticles modified with the SV40 large T antigen were quantified using inductively coupled plasma optical emission spectroscopy (ICP-OES). Internalization was monitored as a function of incubation time, temperature, nanoparticle diameter, and large T surface coverage. Increasing the amount of large T peptides per gold nanoparticle complex, by either increasing the coverage at constant nanoparticle diameter or by increasing the nanoparticle diameter at constant large T coverage, resulted in more cellular internalization. In addition, nuclear fractionation was performed to quantify nuclear localization of these complexes as a function of large T coverage. In contrast to our prior qualitative investigations of nuclear localization by video-enhanced color differential interference contrast microscopy (VEC-DIC), ICP-OES was able to detect nanoparticles inside fractionated cell nuclei. Although increasing the large T coverage was found to afford higher cell internalization and nuclear targeting, quantitative evaluation of cytotoxicity revealed that higher large T coverages also resulted in greater cytotoxicity. The ICP-OES and nuclear fractionation techniques reported here are valuable tools that can add important quantitative information to optical and electron imaging methods such as VEC-DIC and transmission electron microscopy regarding the fate of nanoparticles in cells.

Synthesis, stability, and cellular internalization of gold nanoparticles containing mixed peptide-poly(ethylene glycol) monolayers.

Gold nanoparticles have shown great promise as therapeutics, therapeutic delivery vectors, and intracellular imaging agents. For many biomedical applications, selective cell and nuclear targeting are desirable, and these remain a significant practical challenge in the use of nanoparticles in vivo. This challenge is being addressed by the incorporation of cell-targeting peptides or antibodies onto the nanoparticle surface, modifications that frequently compromise nanoparticle stability in high ionic strength biological media. We describe herein the assembly of poly(ethylene glycol) (PEG) and mixed peptide/PEG monolayers on gold nanoparticle surfaces. The stability of the resulting bioconjugates in high ionic strength media was characterized as a function of nanoparticle size, PEG length, and monolayer composition. In total, three different thiol-modified PEGs (average molecular weight (MW), 900, 1500, and 5000 g mol-1), four particle diameters (10, 20, 30, and 60 nm), and two cell-targeting peptides were explored. We found that nanoparticle stability increased with increasing PEG length, decreasing nanoparticle diameter, and increasing PEG mole fraction. The order of assembly also played a role in nanoparticle stability. Mixed monolayers prepared via the sequential addition of PEG followed by peptide were more stable than particles prepared via simultaneous co-adsorption. Finally, the ability of nanoparticles modified with mixed PEG/RME (RME = receptor-mediated endocytosis) peptide monolayers to target the cytoplasm of HeLa cells was quantified using inductively coupled plasma optical emission spectrometry (ICP-OES). Although it was anticipated that the MW 5000 g mol-1 PEG would sterically block peptides from access to the cell membrane compared to the MW 900 PEG, nanoparticles modified with mixed peptide/PEG 5000 monolayers were internalized as efficiently as nanoparticles containing mixed peptide/PEG 900 monolayers. These studies can provide useful cues in the assembly of stable peptide/gold nanoparticle bioconjugates capable of being internalized into cells.

Selection of biomolecules capable of mediating the formation of nanocrystals.

Biopolymers in the biosphere are well known to mediate the formation of a wide array of inorganic materials, such as bone, shells, lenses, and magnetic particles to name a few. Recently, in vitro experiments with biopolymers such as peptides, RNA, and DNA have shown that templating by these macromolecules can yield a variety of materials under mild reaction conditions. The primary sequence of the biopolymer can be viewed as a proteomic or genomic signature for the templating of an inorganic material from defined metal precursors and reaction conditions. Together with the rapid advances in inorganic particle synthesis by other combinatorial methods, these bioinspired in vitro materials experiments may provide additional insights into possible inorganic materials yet to be discovered and subsequently synthesized by conventional methods. Some of the concepts important to understanding the crystallization phenomena occurring during biopolymer mediation are discussed. A simple kinetic model is provided in the context of known biopolymer-mediated inorganic crystallizations.

Mimicking the biological activity of diazobenzo[b]fluorene natural products with electronically tuned diazofluorene analogs.

Under appropriate electronic modulation, simple diazofluorene analogs recapitulate the DNA cleavage activity of kinamycin D under thiol-based reducing conditions. Achieving DNA cleavage under these reducing conditions is key to anticancer activity, as the most active compound, 1-methoxydiazofluorene, inhibits the proliferation of HeLa cells.

RNA-mediated synthesis of palladium nanoparticles on Au surfaces.

RNA catalysts for the shape-controlled synthesis of Pd particles from the precursor complex trisdibenzylideneacetone dipalladium ([Pd2(DBA)3] were recently discovered in our laboratory (J. Am. Chem. Soc. 2005, 127, 17814-17818). In the work described here, RNA codes for hexagonal Pd platelets and Pd cubes were covalently immobilized on gold surfaces and evaluated for their activity toward particle synthesis. When coupled to gold via oligoethylene glycol linkers, both RNA sequences were able to catalyze the formation of Pd particles with the same shape control previously observed in solution. For low surface coverages, the average distance between RNA molecules on the surface was estimated at ca. 300 nm, yet large (e.g., dimensions of hundreds of nanometers) Pd hexagons and cubes still formed. This surprising result suggests that a single RNA molecule may be sufficient for nucleating and controlling the shapes of these particles. Finally, the use of surface-bound RNA as a tool for directing the orthogonal synthesis of materials on surfaces was demonstrated. Patterning the RNA code for Pd hexagons next to the code for Pd cubes, followed by incubation in a solution containing [Pd2(DBA)3], resulted in the spontaneous formation of spatially distinct spots of hexagonal and cubic particles.

Gold and silica-coated gold nanoparticles as thermographic labels for DNA detection.

The infrared emissivity of Au and silica-coated Au nanoparticles (Au NPs) deposited on indium tin oxide substrates was investigated. NPs were irradiated with laser light at a frequency close to the Au plasmon resonance band, and the blackbody radiation emitted as a result was monitored with an IR camera equipped with an InAs array detector. The differences in temperature before and after laser irradiation were recorded (T-jumps) and were found to be directly proportional to the number of particles present on the slide and to the laser power used in the experiment. Coating Au NPs with silica increased the measured T-jumps 2-5 times, depending on the thickness of the silica shell. This was in agreement with the observation that silica has a much higher IR emissivity than Au. Both Au and silica-coated Au NPs were then tested as labels for thermographic DNA detection. Target DNA concentrations as low as 100 pM were recorded when Au NPs were used as labels and as low as 10 pM when silica-coated Au NPs were used.

RNA-mediated control of metal nanoparticle shape.

RNA sequences previously isolated by in vitro selection were further characterized for their ability to control palladium particle growth. Five pyridyl-modified RNA sequences (Pdases) representing each of the different evolved families were found to form hexagonal plates with a high degree of shape specificity. However, a sixth nonrelated pyridyl-modified RNA sequence was found to form exclusively cubic particles under identical conditions. Replacing pyridyl-modified RNA with native RNA resulted in a complete loss of RNA function. Removing the 3'-fixed sequence region from the Pdase had little effect on particle growth; however, further truncations into the variable region resulted in a significant loss of activity and particle shape control. These Pdases were selected using the organometallic precursor complex tris(dibenzylideneacetone) dipalladium(0) ([Pd2(DBA)3]). Changing the metal center and ligand of the group VIII organometallic precursor complex revealed a strong dependence of particle growth and shape on the DBA ligands. Changing the metal center from Pd to Pt while retaining the DBA ligands gave predominantly hexagonal Pt, but with a decrease in shape control. Taken together, the results of this study suggest that the full-length Pdases contain active sites capable of highly specific molecular recognition of organometallic complexes as particle formation reagents.