J. Manuel Perez

Magnetic relaxation switches capable of sensing molecular interactions

Highly sensitive, efficient, and high-throughput biosensors are required for genomic and proteomic data acquisition in complex biological samples and potentially for in vivo applications. To facilitate these studies, we have developed biocompatible magnetic nanosensors that act as magnetic relaxation switches (MRS) to detect molecular interactions in the reversible self-assembly of disperse magnetic particles into stable nanoassemblies. Using four different types of molecular interactions (DNA–DNA, protein–protein, protein–small molecule, and enzyme reactions) as model systems, we show that the MRS technology can be used to detect these interactions with high efficiency and sensitivity using magnetic relaxation measurements including magnetic resonance imaging (MRI). Furthermore, the magnetic changes are detectable in turbid media and in whole-cell lysates without protein purification. The developed magnetic nanosensors can be used in a variety of biological applications such as in homogenous assays, as reagents in miniaturized microfluidic systems, as affinity ligands for rapid and high-throughput magnetic readouts of arrays, as probes for magnetic force microscopy, and potentially for in vivo imaging.

Oxidase activity of polymeric coated cerium oxide nanoparticles

Inorganic enzyme? Ceria nanoparticles exhibit unique oxidase-like activity at acidic pH values. These redox catalysts can be used in immunoassays (ELISA) when modified with targeting ligands (see picture; light blue and yellow structures are nanoparticles with attached ligands). This modification allows both for binding and for detection by the catalytic oxidation of sensitive colorimetric dyes (e.g. TMB).

Drug/Dye‐Loaded, Multifunctional Iron Oxide Nanoparticles for Combined Targeted Cancer Therapy and Dual Optical/Magnetic Resonance Imaging

A biocompatible, multimodal, and theranostic functional iron oxide nanoparticle is synthesized using a novel water-based method and exerts excellent properties for targeted cancer therapy, and optical and magnetic resonance imaging. For the first time, a facile, modified solvent diffusion method is used for the co-encapsulation of both an anticancer drug and near-infrared dyes. The resulting folate-derivatized theranostics nanoparticles could allow for targeted optical/magnetic resonance imaging and targeted killing of folate-expressing cancer cells.

Use of magnetic nanoparticles as nanosensors to probe for molecular interactions.

Biocompatible magnetic nanosensors have been designed to detect molecular interactions in biological media. Upon target binding, these nanosensors cause changes in the spin–spin relaxation times of neighboring water molecules, which can be detected by magnetic resonance (NMR/MRI) techniques. These magnetic nanosensors have been designed to detect specific mRNA, proteins, enzymatic activity, and pathogens (e.g., virus) with sensitivity in the low femtomole range (0.5–30 fmol).

Synthesis of Biocompatible Dextran‐Coated Nanoceria with pH‐Dependent Antioxidant Properties

Recent reports indicate that cerium oxide nanoparticles (nanoceria) are potent free-radical scavengers with neuroprotective, radioprotective, and anti-inflammatory properties. Nanoceria also have the unique property of being regenerative or autocatalytic. These results point to the possibility of engineering nanoceria with selective antioxidant properties that promote cell survival under conditions of oxidative stress. However, most of these studies have been done with nanoparticles with poor water solubility or synthesized by procedures involving toxic solvents, therefore hindering their potential clinical use. Herein, we report a facile synthesis of monodisperse, water-soluble, and highly crystalline dextran-coated nanoceria (DNC). The improved water solubility of DNC allowed for studies that show a unique pHdependent antioxidant activity that could have important applications in the design of improved therapeutics and in tailoring its antioxidant properties. Our synthetic procedure involves the alkaline-based precipitation of cerium oxide (Ce2O3:CeO2) from a solution containing cerium salt and dextran. Briefly, an aqueous solution of cerium nitrate and dextran was added to an ammonia solution under continuous stirring. Upon formation of the cerium oxide nanocrystals, molecules of dextran coat the nanoparticles’ surface, preventing further growth and resulting in dextran-coated nanoceria (DNC). The DNC preparation is stable in phosphate-buffered saline (PBS) at concentrations of 40mM or higher for months. DNC demonstrates good water stability even after several heating cycles (70–80 8C) with no sedimentation upon centrifugation at 8000 rpm for 30min. These characteristics make our waterbased synthetic method advantageous over organic-solventbased preparations, which are prone to aggregation when suspended in aqueous media.

Iron oxide nanoparticles: Hidden talent

A catalytic property widely used for laboratory tests and the treatment of waste water has been discovered in iron oxide nanoparticles and could lead to many applications in medical diagnostics.

Role of nanoparticle valency in the nondestructive magnetic-relaxation-mediated detection and magnetic isolation of cells in complex media.

Nanoparticle-based diagnostics typically involve the conjugation of targeting ligands to the nanoparticle to create a sensitive and specific nanosensor that can bind and detect the presence of a target, such as a bacterium, cancer cell, protein, or DNA sequence. Studies that address the effect of multivalency on the binding and detection pattern of these nanosensors, particularly on magnetic relaxation nanosensors that sense the presence of a target in a dose-dependent manner by changes in the water relaxation times (DeltaT2), are scarce. Herein, we study the effect of multivalency on the detection profile of cancer cells and bacteria in complex media, such as blood and milk. In these studies, we conjugated folic acid at two different densities (low-folate and high-folate) on polyacrylic-acid-coated iron oxide nanoparticles and studied the interaction of these magnetic nanosensors with cancer cells expressing the folate receptor. Results showed that the multivalent high-folate magnetic relaxation nanosensor performed better than its low folate counterpart, achieving single cancer cell detection in blood samples within 15 min. Similar results were also observed when a high molecular weight anti-folate antibody (MW 150 kDa) was used instead of the low molecular weight folic acid ligand (MW 441.4 kDa), although better results in terms of sensitivity, dynamic range, and speed of detection were obtained when the folate ligand was used. Studies using bacteria in milk suspensions corroborated the results observed with cancer cells. Taken together, these studies demonstrate that nanoparticle multivalency plays a key role in the interaction of the nanoparticle with the cellular target and modulate the behavior and sensitivity of the assay. Furthermore, as detection with magnetic relaxation nanosensors is a nondestructive technique, magnetic isolation and further characterization of the cancer cells is possible.

Dextran-Coated Gold Nanoparticles for the Assessment of Antimicrobial Susceptibility

Bacteria rapidly evolve mechanisms to become resistant to antibiotics. Therefore, identifying an effective antibiotic or antibacterial agent and administering it at concentrations that will successfully prevent bacterial growth (antimicrobial susceptibility) is critical for health care decision making and vital for the battle against multi-drug-resistant bacteria. Currently, the determination of antimicrobial susceptibility requires at least 24 h. Herein, we describe a nanoparticle-based antimicrobial susceptibility assay based on the concanavalin A-induced clustering of dextran-coated gold nanoparticles, which sense the presence of available complex carbohydrates in bacterial suspension. Under conditions of bacterial growth inhibition, addition of concanavalin A results in the formation of extensive dextran gold nanoassemblies, which are facilitated by the presence of free carbohydrates in solution and result in large shifts in the surface plasmon band of the nanoparticles. Meanwhile, at conditions of increased bacterial growth, a decrease in the amount of free carbohydrates in solution will occur due to an increased carbohydrate uptake by the proliferating bacteria. This will result in a decrease in the size of the gold nanoparticle clusters and an increase in the number of nanoparticles that bind to bacterial surface carbohydrates, causing lower shifts in the plasmonic band. The gold nanoparticle-based assessment of antimicrobial susceptibility yields results within 3 h and can be used for the high-throughput screening of samples during epidemics and identification of potential antimicrobial agents to expedite clinical decision-making in point-of-care diagnostics.

Cytochrome c Encapsulating Theranostic Nanoparticles: A Novel Bifunctional System for Targeted Delivery of Therapeutic Membrane-Impermeable Proteins to Tumors and Imaging of Cancer Therapy

The effective administration of therapeutic proteins has received increased attention for the treatment of various diseases. Encapsulation of these proteins in various matrices, as a method of protein structure and function preservation, is a widely used approach that results in maintenance of the proteins function. However, targeted delivery and tracking of encapsulated therapeutic proteins to the affected cells is still a challenge. In an effort to advance the targeted delivery of a functional apoptosis-initiating protein (cytochrome c) to cancer cells, we formulated theranostic polymeric nanoparticles for the simultaneous encapsulation of cytochrome c and a near-infrared dye to folate-expressing cancer cells. The polymeric nanoparticles were prepared using a novel water-soluble hyperbranched polyhydroxyl polymer that allows for dual encapsulation of a hydrophilic protein and an amphiphilic fluorescent dye. Our protein therapeutic cargo is the endogenous protein cytochrome c, which upon cytoplasmic release, initiates an apoptotic response leading to programmed cell death. Results indicate that encapsulation of cytochrome c within the nanoparticles cavities preserved the proteins enzymatic activity. The potential therapeutic property of these nanoparticles was demonstrated by the induction of apoptosis upon intracellular delivery. Furthermore, targeted delivery of cytochrome c to folate-receptor-positive cancer cells was achieved via conjugation of folic acid to the nanoparticles surface, whereas the nanoparticles theranostic properties were conferred via the coencapsulation of cytochrome c and a fluorescent dye. Considering that these theranostic nanoparticles can carry an endogenous cellular apoptotic initiator (cytochrome c) and a fluorescent tag (ICG) commonly used in the clinic, their use and potential translation into the clinic is anticipated, facilitating the monitoring of tumor regression.

Gadolinium-Encapsulating Iron Oxide Nanoprobe as Activatable NMR/MRI Contrast Agent

Herein we report a novel gadolinium-encapsulating iron oxide nanoparticle-based activatable NMR/MRI nanoprobe. In our design, Gd-DTPA is encapsulated within the poly(acrylic acid) (PAA) polymer coating of a superparamagnetic iron oxide nanoparticle (IO-PAA), yielding a composite magnetic nanoprobe (IO-PAA-Gd-DTPA) with quenched longitudinal spin-lattice magnetic relaxation (T(1)). Upon release of the Gd-DTPA complex from the nanoprobes polymeric coating in acidic media, an increase in the T(1) relaxation rate (1/T(1)) of the composite magnetic nanoprobe was observed, indicating a dequenching of the nanoprobe with a corresponding increase in the T(1)-weighted MRI signal. When a folate-conjugated nanoprobe was incubated in HeLa cells, a cancer cell line overexpressing folate receptors, an increase in the 1/T(1) signal was observed. This result suggests that, upon receptor-mediated internalization, the composite magnetic nanoprobe degraded within the cells lysosome acidic (pH 5.0) environment, resulting in an intracellular release of Gd-DTPA complex with subsequent T(1) activation. In addition, when an anticancer drug (Taxol) was coencapsulated with the Gd-DTPA within the folate receptor targeting composite magnetic nanoprobe, the T(1) activation of the probe coincided with the rate of drug release and corresponding cytotoxic effect in cell culture studies. Taken together, these results suggest that our activatable T(1) nanoagent could be of great importance for the detection of acidic tumors and assessment of drug targeting and release by MRI.