Dendrimer-Based Responsive MRI Contrast Agents (G1–G4) for Biosensor Imaging of Redundant Deviation in Shifts (BIRDS)

Biosensor imaging of redundant deviation in shifts (BIRDS) is a molecular imaging platform for magnetic resonance that utilizes unique properties of low molecular weight paramagnetic monomers by detecting hyperfine-shifted nonexchangeable protons and transforming the chemical shift information to reflect its microenvironment (e.g., via temperature, pH, etc.). To optimize translational biosensing potential of BIRDS we examined if this detection scheme observed with monomers can be extended onto dendrimers, which are versatile and biocompatible macromolecules with modifiable surface for molecular imaging and drug delivery. Here we report on feasibility of paramagnetic dendrimers for BIRDS. The results show that BIRDS is resilient with paramagnetic dendrimers up to the fourth generation (i.e., G1–G4), where the model dendrimer and chelate were based on poly­(amido amine) (PAMAM) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA^4–) complexed with thulium ion (Tm³⁺). Temperature sensitivities of two prominent signals of G<i>n</i>-PAMAM-(TmDOTA^–)_<i>x</i> (where <i>n</i> = 1–4, <i>x</i> = 6–39) were comparable to that of prominent signals in TmDOTA^–. Transverse relaxation times of the coalesced nonexchangeable protons on Gn-PAMAM-(TmDOTA^–)_<i>x</i> were relatively short to provide signal-to-noise ratio that was comparable to or better than that of TmDOTA^–. A fluorescent dye, rhodamine, was conjugated to a G2-PAMAM-(TmDOTA)₁₂ to create a dual-modality nanosized contrast agent. BIRDS properties of the dendrimer were unaltered with rhodamine conjugation. Purposely designed paramagnetic dendrimers for BIRDS in conjunction with novel macromolecular surface modification for functional ligands/drugs could potentially be used for biologically compatible theranostic sensors

pH-Dependent Cellular Internalization of Paramagnetic Nanoparticle

A hallmark of the tumor microenvironment in malignant tumor is extracellular acidosis, which can be exploited for targeted delivery of drugs and imaging agents. A pH sensitive paramagnetic nanoaparticle (NP) is developed by incorporating GdDOTA-4AmP MRI contrast agent and pHLIP (pH Low Insertion Peptide) into the surface of a G5–PAMAM dendrimer. pHLIP showed pH-selective insertion and folding into cell membranes, but only in acidic conditions. We demonstrated that pHLIP-conjugated Gd₄₄-G5 paramagnetic nanoparticle binds and fuses with cellular membrane at low pH, but not at normal physiological pH, and that it promotes cellular uptake. Intracellular trafficking of NPs showed endosomal/lysosomal path ways

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGF, HIF-1α, SDF-1 and vessel morphology.

<p>Expression of vascular endothelial growth factor (VEGF) (dark brown colored) at different parts of the PTK787 treated and vehicle treated tumors. There were no differences observed in the expression of VEGF on immunohistochemistry at different parts of the tumors treated with either PTK787 or vehicle. However, delineation of vessels using FITC tagged tomato lectin indicated higher number of dilated vessels at the tumor periphery in rats that received PTK787 treatment. These dilated vessels may be indicative of increased permeability, fPV and signal intensity changes in PTK787 treated tumors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g001" target="_blank"><b>Figures 1</b></a><b> and </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008727#pone-0008727-g002" target="_blank"><b>2</b></a>). Increased permeability may also be due to increased VEGF expression. HIF-1α expression was mostly seen in the central part of the vehicle treated tumors (arrows), however, SDF-1 expressions were observed both in PTK787 and vehicle treated tumors (arrows).</p

SPECT analysis of <i>in vivo</i> accumulation of Tc-99m-HYNIC-VEGF-c.

<p>VEGF-c (which targets both VEGFR2 and VEGFR3) was tagged with HYNIC chelators and then labeled with Tc-99m-pertechnetate (Tc-99m) and injected intravenously in PTK787 and vehicle treated rats. One hour after injection, SPECT images were obtained using dedicated animal scanner. All PTK787 treated rats showed increased accumulation of Tc-99m-HYNIC-VEGF-c in the tumors (lower panel, arrows) compared to that of vehicle treated tumors (upper panel, arrows).</p

Western blot images and densitometry analysis of western blots.

<p>(<b>A</b>) Expression of different angiogenic factors (left panel) in the vehicle- and PTK787 treated tumors from representative cases at the peripheral (P), central part of the tumors (C) and contralateral brains (B). Note the increased expression of VEGF, SDF-1 and HIF-1α at the peripheral part of PTK787 treated tumors. Right panel shows the densitometry analysis of the blot (normalized to β-actin and contralateral brain). The analysis also confirmed the finding of the blot. Note the patterns of VEGF, SDF-1 and HIF-1α in PTK787 treated tumors which are different from that of vehicle treated tumors. (<b>B</b>) Densitometry analysis of VEGFR2, VEGFR3 and EGFR blot (normalized to β-actin and contralateral brain). Expression of VEGFR2, VEGFR3 and EGFR showed higher normalized values at the peripheral part of the PTK787 treated tumors compared to that of central part and the expression patterns are different in vehicle treated tumors. Please note that PTK787 treated tumors showed lower normalized values of VEGFR2 and EGFR both at the periphery and central parts of the tumors compared to that of corresponding contralateral brain; whereas vehicle treated tumors did not show any changes in the normalized values of VEGFR2 and EGFR compared to that of corresponding contralateral brain. Data are expressed as mean ± SEM, n = 3.</p

Immunohistochemistry of PTK787 and vehicle treated tumor showing expression of VEGFR2, VEGFR3 and EGFR.

<p>Immunohistochemistry confirmed the findings of SPECT studies, where PTK787 treated tumors showed increased expression of VEGFR2 and VEFGR3 at the peripheral parts of the tumors, especially around the vessels (arrows) compared to that of vehicle treated tumors (right column). Both PTK787 (lower panel, left column) and vehicle treated (lower panel, right column) showed expression of EGFR in the tumors. Lower panel, middle column show no brown cells in negative control slide.</p

Spectroscopic Characterization of the 3+ and 2+ Oxidation States of Europium in a Macrocyclic Tetraglycinate Complex

The 3+ and 2+ oxidation states of europium have drastically different magnetic and spectroscopic properties. Electrochemical measurements are often used to probe Eu^III/II oxidation state changes, but a full suite of spectroscopic characterization is necessary to demonstrate conversion between these two oxidation states in solution. Here, we report the facile conversion of an europium­(III) tetraglycinate complex into its Eu^II analogue. We present electrochemical, luminescence, electron paramagnetic resonance, UV–visible, and NMR spectroscopic data demonstrating complete reversibility from the reduction and oxidation of the 3+ and 2+ oxidation states, respectively. The Eu^II-containing analogue has kinetic stability within the range of clinically approved Gd^III-containing complexes using an acid-catalyzed dissociation experiment. Additionally, we demonstrate that the 3+ and 2+ oxidation states provide redox-responsive behavior through chemical-exchange saturation transfer or proton relaxation, respectively. These results will be applicable to a wide range of redox-responsive contrast agents and Eu-containing complexes

MRI relaxation parameters in tumors.

<p>Analyses of R2* values normalized to contralateral normal hemisphere (an indirect indicator of iron positive cell accumulation ) showed significantly higher (p<0.05) accumulation of iron positive cells in animals that received previously cryopreserved, FePro labeled CB AC133+EPCs that were <i>in vitro</i> expanded for 10–15 and 25–30 days. Bars: means ± SD. * p<0.05 frozen day 10–15 versus fresh day 25–30; <b>^§</b> p<0.05 frozed day 25–30 versus fresh day 10–15 and fresh day 25–30.</p

CB AC133+ EPCs expression of CD31, vWF and KDR and DiI-Ac-LDL uptake in differentiated progenitors – effect of FePro labeling and cryopreservation.

<p>Expression of CD31, VEGFR2 and vWF in differentiated cells that were prior to differentiating (at days 25–30 of the primary culture) labeled with FePro and cryopreserved for few weeks (D, E, F and G). Control cells were induced to differentiate at days 25–30 of the primary culture without previous FePro labeling and cryopreservation (A, B and C). Positive signals for CD31, VEGFR2 and vWF were visualized with a FITC conjugated secondary antibody (green). Nuclei were visualized with DAPI (blue). VEGFR2 positive (middle panels B and E) cells also exhibited the uptake of DiI-Ac-LDL (red). Representative photomicrographs (40×) of differentiated cells. Scale bar = 100 µm.</p

MRI detection of FePro labeled long-term cultured frozen and fresh CB AC133+ EPCs in glioma.

<p>At days 10–15 (A, B) and 25–30 (E, F) of the primary culture, cells were labeled with FePro and cryopreserved for few weeks. On the day of IV administration, the cells were thawed, incubated for 1–2 hours in stem cell media, washed and IV injected. A control group of rats received freshly prepared FePro labeled cells at 10–15 (C, D) and 25–30 (G, H) days of cultures. Seven days after cell administration multi-echo gradient-echo MRI were obtained using a 7 Tesla small animal MRI system. All animals receiving either frozen or fresh FePro labeled cells exhibited low signal intensity areas around tumors (arrows). Corresponding DAB enhanced Prussian blue stained sections showed iron positive cells at the tumor margins. Both frozen and fresh FePro labeled cells migrated and accumulated in tumor sites.</p