Wellington Pham

In vivo imaging of siRNA delivery and silencing in tumors

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.

Human myeloperoxidase: A potential target for molecular MR imaging in atherosclerosis

Plaque rupture in atherosclerotic disease is the major cause of morbidity and correlates well with myeloperoxidase (MPO) secretion by activated neutrophils and macrophages in humans. We hypothesized that paramagnetic electron donor compounds that rapidly oxidize and polymerize in the presence of MPO could be designed to enable imaging of local MPO activity levels in arterial segments at risk. Several potential substrates for MPO were synthesized and tested. One lead compound consisting of a covalent conjugate of GdDOTA and serotonin (3‐(2‐aminoethyl)‐5‐hydroxyindole) was efficiently polymerized in the presence of human neutrophil MPO resulting in a 70–100% increase in proton relaxivity. As a result, we were able to demonstrate MPO activity in enzyme solutions and in a model tissue‐like system. These studies suggest that activatable paramagnetic MR imaging agents can be used to directly image MPO activity. Magn Reson Med 52:1021–1028, 2004.

In vivo imaging of tumor response to therapy using a dual-modality imaging strategy

In vivo assessment of the outcome of cancer therapy is hampered by the paucity of imaging probes that target tumors specifically and noninvasively. The importance of such probes increases with the continuous development of chemotherapeutics and the necessity to evaluate their effectiveness in a clinical setting. We have recently reported on a dual‐modality imaging probe specifically targeting the underglycosylated mucin‐1 tumor‐specific antigen (uMUC‐1), which is one of the early hallmarks of tumorigenesis in a wide variety of tumors. This probe consists of crosslinked superparamagnetic iron oxide nanoparticles (CLIO) for MR imaging, modified with Cy5.5 dye (for near infrared optical fluorescence imaging (NIRF)), and has peptides (EPPT), specifically recognizing uMUC‐1, attached to the nanoparticles dextran coat. In the present study, we demonstrated that this probe could not only detect orthotopically implanted preclinical models of adenocarcinomas but could also track tumor response to chemotherapy in vivo in real time. Considering the high cost associated with the development and testing of new cancer therapeutics and the need for accurate, noninvasive assessment of their effectiveness, we believe that the developed probe represents a valuable research tool relevant to clinical discovery.

Synthesis and Application of a Water-Soluble Near-Infrared Dye for Cancer Detection Using Optical Imaging

A novel water-soluble 2-[2-(2-chloro-3-{2-[3,3-dimethyl-5-sulfo-1-(4-sulfo-butyl)-3H-indol-2-yl]-vinyl}-cyclohex-2-enylidene)-ethylidene]-3,3-dimethyl-1-(4-sulfo-butyl)-2,3-dihydro-1H-indole-5-carboxylic acid (dye 2) was developed via an asymmetric approach. With an additional sulfonate group, the near-infrared feature of this dye exhibited a 2-fold increase in quantum yield compared to the previous generation. The current synthetic strategy provided a single carboxylic group as a handle for conjugation, thus allowing selectivity for bioconjugation. The stability of this dye was demonstrated by labeling peptides via solid-phase peptide chemistry. The in vivo optical imaging showed potential and broad applications of this dye in developing molecular-based beacons for cancer detection.

Crossing the blood–brain barrier: A potential application of myristoylated polyarginine for in vivo neuroimaging

As basic neurological research continues to reveal novel targets for therapy, the need to deliver therapeutic agents across the blood-brain barrier (BBB) becomes increasingly important. If developed, delivery modules would bring targeting molecules across the BBB to their respective active sites. In addition, it would be highly advantageous if the bioavailability of these delivered agents could be monitored over time using non-invasive imaging techniques. Here, we describe a versatile delivery module based on a myristoylated polyarginine backbone, which crosses the BBB. Incorporation of the fatty acid group was achieved using a Schotten-Bauman reaction with quantitative yield, and the peptide was further synthesized by conventional solid phase peptide synthesis (SPPS). We report for the first time the in vivo distribution of the delivery module over time into mouse brain using near-infrared (NIR) fluorescence imaging. The fluorescent cargo was detected in vivo from 24-48 h post IV injection and was further characterized in perfused brains. Immunohistochemical staining of excised brain showed that the delivery module primarily accumulated in neurons with occasional localization in astrocytes and endothelial cells. We conclude that this approach can be used for the delivery of imaging probes and potentially targeted therapeutics across the BBB.

Enhancing Membrane Permeability by Fatty Acylation of Oligoarginine Peptides

The improved bioavailability of drugs in the treatment of diseases produces a prolonged therapeutic effect and, as a consequence, reduced toxicity and cost. Most oligonucleotides, peptides, or proteins are poorly taken up by cells due to their insufficient association with the lipid bilayer of the plasma membrane. In many cases, therapeutic agents need to possess lipophilic properties in order to achieve the desired pharmacokinetic profile. Thus, the lipophilicity of the molecules assists in the penetration of cytoplasmic and intracellular membranes. Traditionally, the incorporation of homologous series of alkyl groups into a drug of interest produced increases in pharmacological effects. More recently, a short peptide (RKKRRQRRR), derived from HIV Tat-protein, as well as other arginine-containing peptides have attracted attention due to their high cellular uptake efficiency. These membrane-penetrating peptides have been applied as delivery vectors for various biological and medical applications. A systematic study of Tat-peptide indicated that the positively charged arginine residues are crucial to its membrane-penetrating ability, a property that has also been mimicked by a hepta-arginine peptide. It has thus been concluded that the cationic guanidine moiety on the arginine side chain provides the exceptional translocation properties that are not observed in peptides containing similar amino acids such as ornithine, lysine, histidine or citrulline. Although this membrane-translocalization phenomenon has been reported for more than a decade, the detailed mechanism still has to be determined. Here we described the systematic exploration of the changes in cell-localizing ability brought about by the modification of oligoarginine peptides with fatty acid analogues. We originally hypothesized that such modifications would lead to enhanced association with lipid membranes and, potentially, improved transmembrane delivery. The 7-mer oligoarginine Tat-peptide mimetic, originally reported by Wender et al. , was selected as the peptide template on which to study the effect of the length of the acyl chain. A series of fatty acid groups, including hexanoyl, octanoyl, decanoyl, lauroyl, myristoyl, and palmitoyl, were attached to the N terminus of the amidated peptide via a b-alanine spacer on solid support by using a modified Schotten±Baumann reaction. Thereafter, the lipopeptides were labeled with fluorescein isothiocyanate

NEAR-INFRARED DYES: Probe Development and Applications in Optical Molecular Imaging

The recent emergence of optical imaging has brought forth a unique challenge for chemists: development of new biocompatible dyes that fluoresce in the near-infrared (NIR) region for optimal use in biomedical applications. This review describes the synthesis of NIR dyes and the design of probes capable of noninvasively imaging molecular events in small animal models.

A near-infrared dye for multichannel imaging

A large Stokes shift dye, composed of water-solubility and near-infrared feature, was developed for multichannel imaging applications.

Open-Source Automated Parahydrogen Hyperpolarizer for Molecular Imaging Using 13C Metabolic Contrast Agents

An open-source hyperpolarizer producing 13C hyperpolarized contrast agents using parahydrogen induced polarization (PHIP) for biomedical and other applications is presented. This PHIP hyperpolarizer utilizes an Arduino microcontroller in conjunction with a readily modified graphical user interface written in the open-source processing software environment to completely control the PHIP hyperpolarization process including remotely triggering an NMR spectrometer for efficient production of payloads of hyperpolarized contrast agent and in situ quality assurance of the produced hyperpolarization. Key advantages of this hyperpolarizer include: (i) use of open-source software and hardware seamlessly allowing for replication and further improvement as well as readily customizable integration with other NMR spectrometers or MRI scanners (i.e., this is a multiplatform design), (ii) relatively low cost and robustness, and (iii) in situ detection capability and complete automation. The device performance is demonstrated by production of a dose (∼2–3 mL) of hyperpolarized 13C-succinate with %P13C ∼ 28% and 30 mM concentration and 13C-phospholactate at %P13C ∼ 15% and 25 mM concentration in aqueous medium. These contrast agents are used for ultrafast molecular imaging and spectroscopy at 4.7 and 0.0475 T. In particular, the conversion of hyperpolarized 13C-phospholactate to 13C-lactate in vivo is used here to demonstrate the feasibility of ultrafast multislice 13C MRI after tail vein injection of hyperpolarized 13C-phospholactate in mice.

Fluorescent magnetic hybrid nanoprobe for multimodal bioimaging

A fluorescent magnetic hybrid imaging nanoprobe (HINP) was fabricated by the conjugation of superparamagnetic Fe3O4 nanoparticles and visible light emitting (∼600 nm) fluorescent CdTe/CdS quantum dots (QDs). The assembly strategy used the covalent linking of the oxidized dextran shell of magnetic particles to the glutathione ligands of QDs. The synthesized HINP formed stable water-soluble colloidal dispersions. The structure and properties of the particles were characterized by transmission electron and atomic force microscopy, energy dispersive x-ray analysis and inductively coupled plasma optical emission spectroscopy, dynamic light scattering analysis, optical absorption and photoluminescence spectroscopy, and fluorescent imaging. The luminescence imaging region of the nanoprobe was extended to the near-infrared (NIR) (∼800 nm) by conjugation of the superparamagnetic nanoparticles with synthesized CdHgTe/CdS QDs. Cadmium, mercury based QDs in HINP can be easily replaced by novel water-soluble glutathione stabilized AgInS2/ZnS QDs to present a new class of cadmium-free multimodal imaging agents. The observed NIR photoluminescence of fluorescent magnetic nanocomposites supports their use for bioimaging. The developed HINP provides dual-imaging channels for simultaneous optical and magnetic resonance imaging.