Christian T. Farrar

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.

Influence of molecular parameters and increasing magnetic field strength on relaxivity of gadolinium- and manganese-based T1 contrast agents.

Simulations were performed to understand the relative contributions of molecular parameters to longitudinal (r(1)) and transverse (r(2)) relaxivity as a function of applied field, and to obtain theoretical relaxivity maxima over a range of fields to appreciate what relaxivities can be achieved experimentally. The field-dependent relaxivities of a panel of gadolinium and manganese complexes with different molecular parameters, water exchange rates, rotational correlation times, hydration state, etc. were measured to confirm that measured relaxivities were consistent with theory. The design tenets previously stressed for optimizing r(1) at low fields (very slow rotational motion; chelate immobilized by protein binding; optimized water exchange rate) do not apply at higher fields. At 1.5 T and higher fields, an intermediate rotational correlation time is desired (0.5-4 ns), while water exchange rate is not as critical to achieving a high r(1). For targeted applications it is recommended to tether a multimer of metal chelates to a protein-targeting group via a long flexible linker to decouple the slow motion of the protein from the water(s) bound to the metal ions. Per ion relaxivities of 80, 45, and 18 mM(-1) s(-1) at 1.5, 3 and 9.4 T, respectively, are feasible for Gd(3+) and Mn(2+) complexes.

Edema Control by Cediranib, a Vascular Endothelial Growth Factor Receptor–Targeted Kinase Inhibitor, Prolongs Survival Despite Persistent Brain Tumor Growth in Mice

PURPOSE Recent clinical trials of antivascular endothelial growth factor (VEGF) agents for glioblastoma showed promising progression-free and overall survival rates. However, available clinical imaging does not separate antitumor effects from antipermeability effects of these agents. Thus although anti-VEGF agents may decrease tumor contrast-enhancement, vascularity, and edema, the mechanisms leading to improved survival in patients remain incompletely understood. Our goal was to determine whether alleviation of edema by anti-VEGF agents alone could increase survival in mice. METHODS We treated mice bearing three different orthotopic models of glioblastoma with a VEGF-targeted kinase inhibitor, cediranib. Using intravital microscopy, molecular techniques, and magnetic resonance imaging (MRI), we measured survival, tumor growth, edema, vascular morphology and function, cancer cell apoptosis and proliferation, and circulating angiogenic biomarkers. RESULTS We show by intravital microscopy that cediranib significantly decreased tumor vessel permeability and diameter. Moreover, cediranib treatment induced normalization of perivascular cell coverage and thinning of the basement membrane, as mirrored by an increase in plasma collagen IV. These rapid changes in tumor vascular morphology and function led to edema alleviation -- as measured by MRI and by dry/wet weight measurement of water content -- but did not affect tumor growth. By immunohistochemistry, we found a transient decrease in macrophage infiltration and significant but minor changes in tumor cell proliferation and apoptosis. Systemically, cediranib increased plasma VEGF and placenta growth factor levels, and the number of circulating CXCR4(+)CD45(+) cells. However, by controlling edema, cediranib significantly increased survival of mice in the face of persistent tumor growth. CONCLUSION Anti-VEGF agents may be able to improve survival of patients with glioblastoma, even without inhibiting tumor growth.

Nanoparticles enhance brain delivery of blood-brain barrier-impermeable probes for in vivo optical and magnetic resonance imaging

Several imaging modalities are suitable for in vivo molecular neuroimaging, but the blood–brain barrier (BBB) limits their utility by preventing brain delivery of most targeted molecular probes. We prepared biodegradable nanocarrier systems made up of poly(n-butyl cyanoacrylate) dextran polymers coated with polysorbate 80 (PBCA nanoparticles) to deliver BBB-impermeable molecular imaging probes into the brain for targeted molecular neuroimaging. We demonstrate that PBCA nanoparticles allow in vivo targeting of BBB-impermeable contrast agents and staining reagents for electron microscopy, optical imaging (multiphoton), and whole brain magnetic resonance imaging (MRI), facilitating molecular studies ranging from individual synapses to the entire brain. PBCA nanoparticles can deliver BBB-impermeable targeted fluorophores of a wide range of sizes: from 500-Da targeted polar molecules to 150,000-Da tagged immunoglobulins into the brain of living mice. The utility of this approach is demonstrated by (i) development of a “Nissl stain” contrast agent for cellular imaging, (ii) visualization of amyloid plaques in vivo in a mouse model of Alzheimers disease using (traditionally) non–BBB-permeable reagents that detect plaques, and (iii) delivery of gadolinium-based contrast agents into the brain of mice for in vivo whole brain MRI. Four-dimensional real-time two-photon and MR imaging reveal that brain penetration of PBCA nanoparticles occurs rapidly with a time constant of ∼18 min. PBCA nanoparticles do not induce nonspecific BBB disruption, but collaborate with plasma apolipoprotein E to facilitate BBB crossing. Collectively, these findings highlight the potential of using biodegradable nanocarrier systems to deliver BBB-impermeable targeted molecular probes into the brain for diagnostic neuroimaging.

Targeting placental growth factor/neuropilin 1 pathway inhibits growth and spread of medulloblastoma

Medulloblastoma is the most common pediatric malignant brain tumor. Although current therapies improve survival, these regimens are highly toxic and are associated with significant morbidity. Here, we report that placental growth factor (PlGF) is expressed in the majority of medulloblastomas, independent of their subtype. Moreover, high expression of PlGF receptor neuropilin 1 (Nrp1) correlates with poor overall survival in patients. We demonstrate that PlGF and Nrp1 are required for the growth and spread of medulloblastoma: PlGF/Nrp1 blockade results in direct antitumor effects in vivo, resulting in medulloblastoma regression, decreased metastasis, and increased mouse survival. We reveal that PlGF is produced in the cerebellar stroma via tumor-derived Sonic hedgehog (Shh) and show that PlGF acts through Nrp1-and not vascular endothelial growth factor receptor 1-to promote tumor cell survival. This critical tumor-stroma interaction-mediated by Shh, PlGF, and Nrp1 across medulloblastoma subtypes-supports the development of therapies targeting PlGF/Nrp1 pathway.

Vessel architectural imaging identifies cancer patient responders to anti-angiogenic therapy

Measurement of vessel caliber by magnetic resonance imaging (MRI) is a valuable technique for in vivo monitoring of hemodynamic status and vascular development, especially in the brain. Here, we introduce a new paradigm in MRI termed vessel architectural imaging (VAI) that exploits an overlooked temporal shift in the magnetic resonance signal, forming the basis for vessel caliber estimation, and show how this phenomenon can reveal new information on vessel type and function not assessed by any other noninvasive imaging technique. We also show how this biomarker can provide new biological insights into the treatment of patients with cancer. As an example, we demonstrate using VAI that anti-angiogenic therapy can improve microcirculation and oxygen saturation and reduce vessel calibers in patients with recurrent glioblastomas and, more crucially, that patients with these responses have prolonged survival. Thus, VAI has the potential to identify patients who would benefit from therapies.

Correction for artifacts induced by B0 and B1 field inhomogeneities in pH-sensitive chemical exchange saturation transfer (CEST) imaging

Chemical exchange saturation transfer (CEST) imaging provides an indirect detection mechanism that allows quantification of certain labile groups unobservable using conventional MRI. Recently, amide proton transfer (APT) imaging, a variant form of CEST imaging, has been shown capable of detecting lactic acidosis during acute ischemia, providing information complementary to that of perfusion and diffusion MRI. However, CEST contrast is usually small, and therefore, it is important to optimize experimental conditions for reliable and quantitative CEST imaging. In particular, CEST imaging is sensitive to B0 and B1 field, while on the other hand; field inhomogeneities persist despite recent advances in magnet technologies, especially for in vivo imaging at high fields. Consequently, correction algorithms that can compensate for field inhomogeneity‐induced measurement errors in CEST imaging might be very useful. In this study, the dependence of CEST contrast on field distribution was solved and a correction algorithm was developed to compensate for field inhomogeneity‐induced CEST imaging artifacts. In addition, the proposed algorithm was verified with both numerical simulation and experimental measurements, and showed nearly complete correction of CEST imaging measurement errors caused by moderate field inhomogeneity. Magn Reson Med, 2007.

Mechanism of dynamic nuclear polarization in high magnetic fields

Solid-state NMR signal enhancements of about two orders of magnitude (100–400) have been observed in dynamic nuclear polarization (DNP) experiments performed at high magnetic field (5 T) and low temperature (10 K) using the nitroxide radical 4-amino TEMPO as the source of electron polarization. Since the breadth of the 4-amino TEMPO EPR spectrum is large compared to the nuclear Larmor frequency, it has been assumed that thermal mixing (TM) is the dominate mechanism by which polarization is transferred from electron to nuclear spins. However, theoretical explanations of TM generally assume a homogeneously broadened EPR line and, since the 4-amino TEMPO line at 5 T is inhomogeneously broadened, they do not explain the observed DNP enhancements. Accordingly, we have developed a treatment of DNP that explicitly uses electron–electron cross-relaxation to mediate electron–nuclear polarization transfer. The process proceeds via spin flip–flops between pairs of electronic spin packets whose Zeeman temperatures di...

Magneto-fluorescent core-shell supernanoparticles

Magneto-fluorescent particles have been recognized as an emerging class of materials that exhibit great potential in advanced applications. However, synthesizing such magneto-fluorescent nanomaterials that simultaneously exhibit uniform and tunable sizes, high magnetic content loading, maximized fluorophore coverage at the surface, and a versatile surface functionality has proven challenging. Here we report a simple approach for co-assembling magnetic nanoparticles with fluorescent quantum dots to form colloidal magneto-fluorescent supernanoparticles. Importantly, these supernanoparticles exhibit a superstructure consisting of a close packed magnetic nanoparticle “core” which is fully surrounded by a “shell” of fluorescent quantum dots. A thin layer of silica-coating provides high colloidal stability and biocompatiblity and a versatile surface functionality. We demonstrate that after surface pegylation, these silica-coated magneto-fluorescent supernanoparticles can be magnetically manipulated inside living cells while being optically tracked. Moreover, our silica-coated magneto-fluorescent supernanoparticles can also serve as an in vivo multi-photon and magnetic resonance dual-modal imaging probe.

Dynamic nuclear polarization at 9T using a novel 250GHz gyrotron microwave source.

In the 1990s we initiated development of high frequency gyrotron microwave sources with the goal of performing dynamic nuclear polarization at magnetic fields (∼5-23 T) used in contemporary NMR experiments. This article describes the motivation for these efforts and the developments that led to the operation of a gyrotron source for DNP operating at 250 GHz. We also mention results obtained with this instrument that would have been otherwise impossible absent the increased sensitivity. Finally, we describe recent efforts that have extended DNP to 460 GHz and 700 MHz (1)H frequencies.