Targeted Nanoprobes Reveal Early Time Point Kinetics In Vivo by Time-Resolved MRI

Abstract

This commentary highlights the findings by Kessinger et al. (Theranostics 2011; 1: 263-275) that dynamic T₂*-weighted magnetic resonance imaging (MRI) of cyclic RGD peptide-encoded superparamagnetic polymeric micelle (SPPM) nanoparticles allows quantitative analysis of tumor integrin αvβ3 expression, which can exclude the effect of blood volume and extravascular signal components and thus provide less biased tumor contrast and receptor specificity of probes.

Targeted nanoprobes have made the field of molecular imaging an exciting arena with capabilities of delivering contrast agents to the desired site in vivo ^1-3. Targeted nanoprobes have been employed in a variety of imaging modalities such as positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI) and optical imaging ^4-6. Among the imaging modalities, MRI is a powerful medical diagnostic imaging technique for soft tissue imaging ⁷. Magnetic nanoprobes have been around for a while in the field of MRI due to their exceptional spatial and temporal contrast enhancement ^7-8. Iron oxide nanoparticle (IONP) is one of the most adopted nanoprobes for T₂ weighted MRI studies. Several IONP formulas have been approved for clinical application, for example, Ferumoxide (Endorem^®-Europe, Feridex^® in the USA and Japan) and Ferucarbotran (Resovist^®-Europe and Japan) ^9-13. Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles have shown potential in the imaging of molecular biomarkers such as integrins ^{12, 14-15}. Integrins are heterodimeric transmembrane glycoproteins, a family of adhesion molecules which have been shown to play a major role in angiogenesis and tumor metastasis ¹⁶. The α_vβ₃ integrin is significantly over-expressed on activated endothelial cells during angiogenesis but not on dormant endothelial cells. A salient feature of α_vβ₃ integrin is that it binds to extracellular matrix proteins via the three amino acid sequence arginine-glycine-aspartic acid (RGD). It is indicated that inhibition of integrin α_vβ₃ activity by cyclic RGD peptides, monoclonal antibodies and peptidomimetics may trigger endothelial cell apoptosis and result in inhibition of angiogenesis ^17-18.

Data related to early events of binding and cellular uptake of targeted nanoprobes in cancer cells will reveal important insights on the critical factors that affect nanoprobe accumulation in vivo. In developing new anti-angiogenic agents and for response evaluation, early time point kinetic studies are very important, where targeted nanoprobes can specifically bind to the cell surface receptors on the tumor endothelium from circulating blood. Gadolinium based T₁ based MRI contrast probes have been used for studying kinetic parameters using DCE-MRI ^19-20. Low molecular weight probes such as Gd-DTPA worked well in tumor perfusion studies and pharmacokinetic parameters were measured ^21-22. However, these probes are non-specific and require millimolar concentrations for detection, indicating lack of sensitivity. Developing sensitive and target specific nanoprobes for DCE-MRI would provide more reliable kinetic data.

In this volume of Theranostics Kessinger et al. ²³ examined for the first time early time point kinetic studies of the binding events of targeted nanoprobes in vivo using time-resolved MRI. In this work, ultra-sensitive superparamagnetic polymeric micelle (SPPM) nanoprobes were employed to quantify the targeting kinetics of c(RGDfK) (cRGD)-encoded SPPM to angiogenic endothelium in subcutaneous lung, breast and glioblastoma xenograft models in mice using T₂*-weighted HTR-MRI. A cluster of SPIO nanoparticles loaded into the core-shell of the polymeric micelle was used in the three different animal models. The nanoprobes were shown to increase the transverse relaxivity of the nanoprobes to over 400 Fe mM^-1 s^-1 at 7T, a 4-fold higher relaxivity compared to Feridex. T₂*-weighted HTR-MRI with 1.3 s temporal resolution over 30 min shed light on the early phase of the targeting kinetics of α_vβ₃-specific SPIO nanoprobes in different tumor xenografts models. SPIO nanoprobes conjugated with α_vβ₃-specific cyclic RGDfK (cRGD) peptide and non-targeting cyclic RADfK (cRAD) as a control, were evaluated for difference in MR signal intensity (SI) over time. Comparison of the targeted and non-targeted SPPM data allowed for the subtraction of signal intensity contributions from blood concentration, clearance and enhanced permeability and retention (EPR) effect. Accumulation rates (K_a) in the initial phase (<5 min) were found to be similar across all the three tumor models. However, a sustained accumulation of targeted cRGD-SPPM over 30 min was observed.

DCE-MRI is usually applied for characterization of abnormalities of perfusion, endothelial and epithelial permeability with T₁ weighted contrast imaging ²⁴. In this study, a new opportunity for characterizing the tumor specific targeting kinetics of nanoparticles by T₂ weighted contrast imaging is reported and the initial results of α_vβ₃ integrin receptor density quantification are achieved. Since the signal intensity curves of each region of interest (ROI) can be derived from T₂ images, kinetic modeling analysis is able to estimate the pharmacokinetics of cRGD-SPPM contrast agent which reflects the tumor integrin level. Considering the non-specific binding of cRGD-SPPM via the leakage of contrast agent from capillary to the extravascular parenchymal space, they also studied the cRAD-SPPM as a non-targeting control. By subtracting the mean value of cRAD from cRGD curves, a compartment which only represents the specific cRGD concentration is achieved and relates to the specific accumulation rate independently.

As is the case with the two- or three-compartment kinetic modeling widely used in PET dynamic studies, one-compartment modeling from which non-targeting contrast agent is subtracted is also a direct method for receptor density study. The one-compartment model is easier to fit and more efficient for quantification of parameters; however, it is premised on accurate subtraction. For PET quantitative analysis of receptor density, the optimized model is comparable to the three compartment model for binding potential measurement.

In conclusion, this paper describes early time point kinetic binding studies of targeted nanoprobes that offer new opportunities in quantitative characterization of the targeting kinetics of cancer-specific nanoparticles to their intended biological targets in an intact animal. The data provide less biased tumor contrast and receptor specificity of probes and can exclude the effect of blood volume and extra-vascular signal components.