Raffaella Rossin

Biodegradable dendritic positron-emitting nanoprobes for the noninvasive imaging of angiogenesis

A biodegradable positron-emitting dendritic nanoprobe targeted at αvβ3 integrin, a biological marker known to modulate angiogenesis, was developed for the noninvasive imaging of angiogenesis. The nanoprobe has a modular multivalent core-shell architecture consisting of a biodegradable heterobifunctional dendritic core chemoselectively functionalized with heterobifunctional polyethylene oxide (PEO) chains that form a protective shell, which imparts biological stealth and dictates the pharmacokinetics. Each of the 8 branches of the dendritic core was functionalized for labeling with radiohalogens. Placement of radioactive moieties at the core was designed to prevent in vivo dehalogenation, a potential problem for radiohalogens in imaging and therapy. Targeting peptides of cyclic arginine–glycine–aspartic acid (RGD) motifs were installed at the terminal ends of the PEO chains to enhance their accessibility to αvβ3 integrin receptors. This nanoscale design enabled a 50-fold enhancement of the binding affinity to αvβ3 integrin receptors with respect to the monovalent RGD peptide alone, from 10.40 nM to 0.18 nM IC50. Cell-based assays of the 125I-labeled dendritic nanoprobes using αvβ3-positive cells showed a 6-fold increase in αvβ3 receptor-mediated endocytosis of the targeted nanoprobe compared with the nontargeted nanoprobe, whereas αvβ3-negative cells showed no enhancement of cell uptake over time. In vivo biodistribution studies of 76Br-labeled dendritic nanoprobes showed excellent bioavailability for the targeted and nontargeted nanoprobes. In vivo studies in a murine hindlimb ischemia model for angiogenesis revealed high specific accumulation of 76Br-labeled dendritic nanoprobes targeted at αvβ3 integrins in angiogenic muscles, allowing highly selective imaging of this critically important process.

In Vivo Evaluation of 64Cu-Labeled Magnetic Nanoparticles as a Dual-Modality PET/MR Imaging Agent

A novel nanoparticle-based dual-modality positron emission tomograph/magnetic resonance imaging (PET/MRI) contrast agent was developed. The probe consisted of a superparamagnetic iron oxide (SPIO) core coated with PEGylated phospholipids. The chelator 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid (DOTA) was conjugated to PEG termini to allow labeling with positron-emitting (64)Cu. Radiolabeling with (64)Cu at high yield and high purity was readily achieved. The (64)Cu-SPIO probes produced strong MR and PET signals and were stable in mouse serum for 24 h at 37 degrees C. Biodistribution and in vivo PET/CT imaging studies of the probes showed a circulation half-life of 143 min and high initial blood retention with moderate liver uptake, making them an attractive contrast agent for disease studies.

Synthesis and Characterization of Core-Shell Star Copolymers for In Vivo PET Imaging Applications

The synthesis of core-shell star copolymers via living free radical polymerization provides a convenient route to three-dimensional nanostructures having a poly(ethylene glycol) outer shell, a hydrophilic inner shell bearing reactive functional groups, and a central hydrophobic core. By starting with well-defined linear diblock copolymers, the thickness of each layer, overall size/molecular weight, and the number of internal reactive functional groups can be controlled accurately, permitting detailed structure/performance information to be obtained. Functionalization of these polymeric nanoparticles with a DOTA-ligand capable of chelating radioactive (64)Cu nuclei enabled the biodistribution and in vivo positron emission tomography (PET) imaging of these materials to be studied and correlated directly to the initial structure. Results indicate that nanoparticles with increasing PEG shell thickness show increased blood circulation and low accumulation in excretory organs, suggesting application as in vivo carriers for imaging, targeting, and therapeutic groups.

Highly reactive trans-cyclooctene tags with improved stability for Diels-Alder chemistry in living systems.

One of the challenges of pretargeted radioimmunotherapy, which centers on the capture of a radiolabeled probe by a preinjected tumor-bound antibody, is the potential immunogenicity of biological capturing systems. A bioorthogonal chemical approach may circumvent this drawback, but effective in vivo chemistry in mice, larger animals, and eventually humans, requires very high reagent reactivity, sufficient stability, and retained selectivity. We report here that the reactivity of the fastest bioorthogonal reaction, the inverse-electron-demand-Diels-Alder cycloaddition between a tetrazine probe and a trans-cyclooctene-tagged antibody, can be increased 10-fold (k2 = 2.7 × 10(5) M(-1) s(-1)) via the trans-cyclooctene, approaching the speed of biological interactions, while also increasing its stability. This was enabled by the finding that the trans-cyclooctene tag is probably deactivated through isomerization to the unreactive cis-cyclooctene isomer by interactions with copper-containing proteins, and that increasing the steric hindrance on the tag can impede this process. Next, we found that the higher reactivity of axial vs equatorial linked TCO can be augmented by the choice of linker. The new, stabilized, and more reactive tag allowed for improved tumor-to-nontumor ratios in pretargeted tumor-bearing mice.

Click to Release: Instantaneous Doxorubicin Elimination upon Tetrazine Ligation†

Eliminated without a trace: The fastest click reaction, the highly selective inverse-electron-demand Diels-Alder reaction, has been modified to enable selective bioorthogonal release. Thus, the click reaction of a tetrazine with a drug-bound trans-cyclooctene caused the instantaneous release of the drug and CO2 (see scheme). One possible application is the chemically triggered release, and thereby activation, of a drug from a tumor-bound antibody-drug conjugate.

In Vivo Imaging of 64Cu-Labeled Polymer Nanoparticles Targeted to the Lung Endothelium

Nanoparticles (NPs) targeting the intercellular adhesion molecule 1 (ICAM-1) hold promise as a mean of delivering therapeutics to the pulmonary endothelium in patients with acute and chronic respiratory diseases. As these new materials become available, strategies are needed to understand their behavior in vivo. We have evaluated the use of 64Cu and PET to noninvasively image the lung uptake and distribution of NPs coated with an anti-ICAM antibody. Methods: Model fluorescent NPs were coated with a mixture of an anti-ICAM antibody (or nonspecific IgG) and 64Cu-DOTA-IgG (where DOTA is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Biodistribution and small-animal PET and CT studies were performed in healthy mice and in mice pretreated with lipopolysaccharides (LPSs). Metabolism studies were also performed to evaluate the stability of 64Cu-labeled NPs in lungs in vivo. Results: The lungs of mice administered anti-ICAM NPs labeled with 64Cu were clearly imaged by small-animal PET 1, 4, and 24 h after administration. Both biodistribution and small-animal imaging showed a 3- to 4-fold higher uptake in the lungs of mice injected with ICAM-targeted NPs relative to that of the control group. Lung uptake was further enhanced by pretreating the mice with LPS, presumably because of ICAM-1 upregulation. However, an approximately 2-fold decrease in lung signal was observed in each experimental group over 24 h. Metabolism studies in lung tissues harvested from mice injected with 64Cu-labeled anti-ICAM NPs showed considerable release of a small 64Cu-radiometabolite from the NPs beginning as early as 1 h after injection. A decrease in lung fluorescence was also observed, most likely reflecting partial release of NPs from the lungs in vivo. Conclusion: The use of small-animal PET to track 64Cu-labeled nanostructures in vivo shows potential as a strategy for the preclinical screening of new NP drug delivery agents targeting the lung endothelium and other tissues. Future design optimization to prolong the stability of the radiolabel in vivo will further improve this promising approach.

Facile, efficient approach to accomplish tunable chemistries and variable biodistributions for shell cross-linked nanoparticles.

The in vivo behavior of shell cross-linked knedel-like (SCK) nanoparticles is shown to be tunable via a straightforward and versatile process that advances SCKs as attractive nanoscale carriers in the field of nanomedicine. Tuning of the pharmacokinetics was accomplished by grafting varied numbers of methoxy-terminated poly(ethylene glycol) (mPEG) chains to the amphiphilic block copolymer precursors, together with chelators for the radioactive tracer and therapeutic agent (64)Cu, followed by self-assembly into block copolymer micelles and chemical cross-linking throughout the shell regions. (64)Cu-radiolabeling was then performed to evaluate the SCKs in vivo by means of biodistribution experiments and positron emission tomography (PET). It was found that the blood retention of PEGylated SCKs could be tuned, depending on the mPEG grafting density and the nanoparticle surface properties. A semiquantitative model of the density of mPEG surface coverage as a function of in vivo behavior was applied to enhance the understanding of this system.

Diels–Alder Reaction for Tumor Pretargeting: In Vivo Chemistry Can Boost Tumor Radiation Dose Compared with Directly Labeled Antibody

Current pretargeting systems use noncovalent biologic interactions, which are prone to immunogenicity. We previously developed a novel approach based on the bioorthogonal reaction between a radiolabeled tetrazine and an antibody-conjugated trans-cyclooctene (TCO). However, the tumor-to-blood ratio was low due to reaction with freely circulating antibody-TCO. Methods: Here we developed 2 tetrazine-functionalized clearing agents that enable rapid reaction with and removal of a TCO-tagged antibody (CC49) from blood. Next, we incorporated this approach into an optimized pretargeting protocol in LS174T-bearing mice. Then we compared the pretargeted 177Lu-labeled tetrazine with 177Lu-labeled CC49. The biodistribution data were used for mouse and human dosimetry calculations. Results: The use of a clearing agent led to a doubling of the tetrazine tumor uptake and a 125-fold improvement of the tumor-to-blood ratio at 3 h after tetrazine injection. Mouse dosimetry suggested that this should allow for an 8-fold higher tumor dose than is possible with nonpretargeted radioimmunotherapy. Also, humans treated with CC49-TCO–pretargeted 177Lu-tetrazine would receive a dose to nontarget tissues 1 to 2 orders of magnitude lower than with directly labeled CC49. Conclusion: The in vivo performance of chemical pretargeting falls within the range of results obtained for the clinically validated pretargeting approaches in mice, with the advantage of potentially allowing for fractionated radiotherapy as a result of a lower likelihood of immunogenicity. These findings demonstrate that biologic pretargeting concepts can be translated to rapid bioorthogonal chemical approaches with retained potential.

Small-animal PET of tumor angiogenesis using a (76)Br-labeled human recombinant antibody fragment to the ED-B domain of fibronectin.

The aim of this study was to image the extra domain B (ED-B) of fibronectin, an angiogenesis-related target, in solid tumors using small-animal PET. Toward this aim, an ED-B fibronectin-binding human antibody derivative (L19-SIP) was labeled with 76Br via an enzymatic approach. Biodistribution and imaging studies were performed in human teratoma–bearing mice for up to 48 h after injection. Methods: L19-SIP was labeled with 76Br using bromoperoxidase/H2O2. The stability of the labeled antibody was tested both in vitro and in vivo. Biodistribution and small-animal imaging studies (PET and CT) were performed in F9-bearing 129/sv mice (n = 3 or 4). Results: The enzymatic radiobromination approach afforded the labeled antibody in high yield (>55%) under mild reaction conditions. 76Br-L19-SIP stability in mouse serum proved to be similar to that of the 125I-labeled analog (>80% of intact material at 48 h after injection). Fast and specific in vivo targeting was obtained in tumors and other organs expressing ED-B fibronectin (i.e., ovaries and uterus). However, slow renal clearance and persistent activity predominately in blood and stomach suggests partial 76Br-L19-SIP debromination in vivo. This debromination was confirmed in a metabolism study in normal mice. The F9 tumors were clearly imaged by small-animal PET at each considered time point, starting at 5 h up to 48 h after injection. Conclusion: 76Br-L19-SIP specifically accumulated at the target site, enabling detailed small-animal PET of tumor neovasculature. Therefore, targeting the angiogenesis-associated expression of ED-B fibronectin can be a valuable tool for tumor detection using molecular imaging with PET.

Triggered Drug Release from an Antibody–Drug Conjugate Using Fast “Click-to-Release” Chemistry in Mice

The use of a bioorthogonal reaction for the selective cleavage of tumor-bound antibody-drug conjugates (ADCs) would represent a powerful new tool for ADC therapy, as it would not rely on the currently used intracellular biological activation mechanisms, thereby expanding the scope to noninternalizing cancer targets. Here we report that the recently developed inverse-electron-demand Diels-Alder pyridazine elimination reaction can provoke rapid and self-immolative release of doxorubicin from an ADC in vitro and in tumor-bearing mice.