No abstract

Nanoparticle surfaces functionalized with proteins or other biomolecules provide a mechanism for interfacing the unique properties of nanomaterials with biological samples. In most of these studies, the biomolecule is conjugated to a gold nanoparticles (AuNP) surface through the thiol group of native or introduced cysteine residues. Here we demonstrate the direct attachment of a hexa-histidine tagged (His(6)) peptide to a 1.5 nm AuNP. Binding occurs via a specific interaction between the Ne of the His imidazole, forming a 1:1 stoichiometric complex. Given the widespread use of histidine tags in producing recombinant proteins, this approach promises to expand the applications of AuNP in biological applications.

A new advance in cell transfection protocol using a bimodal nanoparticle agent to selectively manipulate protein expression levels within mammalian cells is demonstrated. The nanoparticle based transfection approach functions by controlled release of gene regulatory elements from a 6 nm AuNP (gold nanoparticle) surface. The endosomal release of the regulatory elements from the nanoparticle surface results in endogenous protein knockdown simultaneously with exogenous protein expression for the first 48 h. The use of fluorescent proteins as the endogenous and exogenous signals for protein expression enables the efficiency of codelivery of siRNA (small interfering RNA) for GFP (green fluorescent protein) knockdown and a dsRed-express linearized plasmid for induction to be optically analyzed in CRL-2794, a human kidney cell line expressing an unstable green fluorescent protein. Delivery of the bimodal nanoparticle in cationic liposomes results in 20% GFP knockdown within 24 h of delivery and continues exhibiting knockdown for up to 48 h for the bimodal agent. Simultaneous dsRed expression is observed to initiate within the same time frame with expression levels reaching 34% after 25 days although cells have divided approximately 20 times, implying daughter cell transfection has occurred. Fluorescence cell sorting results in a stable colony, as demonstrated by Western blot analysis. The simultaneous delivery of siRNA and linearized plasmid DNA on the surface of a single nanocrystal provides a unique method for definitive genetic control within a single cell and leads to a very efficient cell transfection protocol.

Bacterial surface peptide display has gained popularity as a method of affinity reagent generation for a wide variety of applications ranging from drug discovery to pathogen detection. In order to isolate the bacterial clones that express peptides with high affinities to the target molecule, multiple rounds of manual magnetic activated cell sorting (MACS) followed by multiple rounds of fluorescence activated cell sorting (FACS) are conventionally used. Although such manual methods are effective, alternative means of library screening which improve the reproducibility, reduce the cost, reduce cross contamination, and minimize exposure to hazardous target materials are highly desired for practical application. Toward this end, we report the first semi-automated system demonstrating the potential for screening bacterially displayed peptides using disposable microfluidic cartridges. The Micro-Magnetic Separation platform (MMS) is capable of screening a bacterial library containing 3×1010 members in 15 minutes and requires minimal operator training. Using this system, we report the isolation of twenty-four distinct peptide ligands that bind to the protective antigen (PA) of Bacilus anthracis in three rounds of selection. A consensus motif WXCFTC was found using the MMS and was also found in one of the PA binders isolated by the conventional MACS/FACS approach. We compared MMS and MACS rare cell recovery over cell populations ranging from 0.1% to 0.0000001% and found that both magnetic sorting methods could recover cells down to 0.0000001% initial cell population, with the MMS having overall lower standard deviation of cell recovery. We believe the MMS system offers a compelling approach towards highly efficient, semi-automated screening of molecular libraries that is at least equal to manual magnetic sorting methods and produced, for the first time, 15-mer peptide binders to PA protein that exhibit better affinity and specificity than peptides isolated using conventional MACS/FACS.

Multimodal, biocompatible contrast agents for high magnetic field applications represent a new class of nanomaterials with significant potential for tracking of fluorescence and MR in vitro and vivo. Optimized for high-field MR applicationsincluding biomedical imaging at 21.1 T, the highest magnetic field available for MRI-these nanoparticles capitalize on the improved performance of chelated With the commissioning of stronger magnets, high-field MRI has increased in both clinical application (1,2) and animal research (3). New magnets with strengths above 14.1 T, culminating in the 21.1-T (900-MHz) system (4), create not only new opportunities for biomedical research but also many challenges, particularly with respect to optimized exogenous contrast agents (CA). Nanomaterials offer a tantalizing opportunity to tailor new agents for high-field MRI while also incorporating multimodal contrast and increasing or targeting payload delivery (5,6). This report describes the fabrication and characterization of a novel bimodal nanoparticle CA for use as an intracellular probe at high magnetic fields. The configuration of this high-field CA incorporates a fluorescent quantum dot (QD) as a stable, bimodal platform for the delivery of intracellular MR contrast. In particular, fluorescent Indium Phosphide/Zinc Sulfide (InP/ ZnS) QD nanoparticles (7) have been functionalized with a Dy 3þ lanthanide ion to yield T 1 , T 2 , T 2 *, and optical contrast. These modifications have been accomplished by attaching either a simple (CAAKA) or cell penetrating peptide (CAAKATat) to the QD; in turn, this peptide is bound to a Dy 3þ ion chelated with 1,4,7,10-tetraazacyclododecane-N,N 0 ,N 00 ,N 000 -tetra acetic acid (DOTA). Intracellular nanoparticles are evaluated in an in vitro tissue model at 21.1 T (900 MHz), the highest field currently available for MRI, and through confocal microscopy to analyze their contrast enhancement and intracellular localization. The focus of most clinical CAs has been the utilization of iron oxide or gadolinium compounds (8-14), motivated by the high sensitivity of these agents and wellknown chemistry involved in their fabrication. However, these particles may not be as beneficial at higher fields (>7 T). Most conventional iron oxides are known to saturate at field strengths less than 1 T, while gadolinium chelates also prove optimal at fields below 1 T (15,16). For example, even at clinical field strengths (1.5-3.0 T), the effectiveness of Gd is limited and drastically decreased-by as much as one-third with respect to its T 1 relaxivity-as field strength increases. Other paramagnetic lanthanides may offer improved performance at higher fields. In fact, for some cases, like dysprosium, relaxivity is expected to increase.Dysprosium has been used extensively in MRI as a chemical shift reagent (17). Recently, ionic dysprosium (Dy 3þ ) has received attention as a potential candidate for high-field applications because the magnetism of Dy complexes is impacted largely by Curie relaxation, which manifests as a l...