Here, we report on an ultrasmall, high-affinity, and efficiently cleared silica nanoparticle probe, which has been recently approved for first-in-human clinical trials and which successfully overcomes a number of the limitations of other particle platforms. This multimodal platform has advanced to the point of clinical translatability. Its applications include real-time, intraoperative detection and imaging of nodal metastases, differential tumor burden, and lymphatic drainage patterns in melanoma. Although several investigators have synthesized radiolabeled fluorescent particle probes (25
), our multimodal agent has been radiolabeled with the long-lived positron-emitter iodine-124, and, thus, we believe it can provide unique longer-term pharmacokinetic clearance and targeting information over the course of days. The complementary nature of this platform, coupled with its small size (~7-nm i.d.), may facilitate clinical assessments by enabling the seamless integration of imaging data acquired at different spatial, temporal, and sensitivity scales, potentially providing new insights into fundamental molecular processes governing tumor biology.
The results of this study underscore the clear-cut advantages offered by PET. Using this quantitative and highly sensitive imaging tool, we were able to noninvasively extract an accurate, reproducible, and comprehensive body of data for the targeted probe: (a) molecular information, including receptor expression levels, binding affinity, and specificity; (b) in vivo distribution and targeting kinetics; (c) clearance and dehalogenation profiles; (d) blood/tissue residence times and bioavailability; and (e) radiation dosimetry.
Our in vitro results show receptor-binding specificity of the ~7-nm–targeted particle probe to M21 cells and HUVECs. Similar findings have been reported with receptor-binding assays using the same cell types but with the monovalent form of the peptide (36
). Importantly, the multivalency enhancement of the cRGDY-bound particle probe, along with the extended blood and tumor T1/2
values, are key properties associated with the particle platform that are not found with the monovalent form of the peptide.
The integrin-binding peptide, cRGD, a well-established integrin-binding molecular marker, was selected to elucidate the biological and kinetic properties of the peptide-bound particle and additionally enable the biological performance of our multimodal platform to be benchmarked against other cRGD agents (i.e., peptide tracers). However, another more tumor-specific, high-affinity ligand could be investigated as the targeting moiety in future studies. The biological properties of particle-based systems are generally quite different from those of simple (i.e., small monovalent) targeting peptides, and the choice of the proper targeting agent (i.e., molecular versus particle-based probes) in the clinical setting will rest, in part, on the application of interest, the tumor type/composition, and standard-of-care considerations.
The relatively long blood T1/2
for the 124
I-PEG-dot tracer may be a consequence of the chemically neutral PEG-coated surface, rendering the probe biologically inert and significantly less susceptible to phagocytosis by the RES. However, recognition of the 124
I-cRGDY-PEG-dot tracer by target integrins and/or more active macrophage activity may have led to reductions in the T1/2
value. These values, however, are substantially longer than published blood T1/2
values of existing cRGDY peptide tracers (~13 minutes) (5
), potentially leading to increased probe bioavailability, facilitating tumor targeting, and yielding higher tumor uptake over longer time periods. Moreover, the tumor T1/2
for the 124
I-cRGDY-PEG-dot was found to be 13-times greater than that for blood, compared with only a 5-fold difference for the 124
I-PEG-dot, suggesting substantially greater target-tissue localization of the former than the latter. Such mechanistic interpretations of the in vivo data can be exploited clinically to refine diagnostic, treatment-planning, and treatment-monitoring protocols.
The greater accumulation in and slower clearance from M21 tumors, relative to that of surrounding normal structures, allows discrimination of specific tumor uptake mechanisms from nonspecific mechanisms (i.e., tissue perfusion, leakage) in normal tissues. However, a small component of the M21 tumor uptake can presumably be attributed to vascular permeability alterations (i.e., enhanced permeability and retention effects) (41
), largely reflected in the observed %ID/g increases for the control tracer (124
I-PEG-dots, Figure C) at earlier p.i. time points. At 1-hour p.i., no significant %ID/g increases were seen in the M21 tumors over the controls. This observation may represent the effects of differential perfusion in the first hour, with tumor accumulation and retention primarily seen at later p.i. times (i.e., 24 hours). Further, in comparison with those of the clinically approved peptide tracer, 18
F-galacto RGD (42
), nearly 2-fold greater maximum uptake values were found in M21 tumors for the targeted dots at 2-hours p.i. (data not shown), while additionally offering advantages of multivalent binding and extended circulation times.
The advantages of the combined optical-PET probe highlight the versatility of the platform for in vivo applications. This is particularly true in small-animal models, in which the ability to assess anatomic structures having sizes at or well below the resolution limit of the PET scanner (i.e., the so-called partial-volume effect) may undermine detection and quantitation of activity in lesions. In these cases, assessment of metastatic disease in small local/regional nodes, important clinically for melanoma staging and treatment, may not be adequately resolved by PET imaging, given that the size of the nodes we typically observed was on the order of the PET spatial resolution (1–2 mm). By exploiting the significantly improved photophysical features of encapsulated dyes, such as Cy5, as well as the enhanced detection sensitivity and contrast achievable at longer emission wavelengths, detailed information pertaining to the localization of superficial nodes, lymphatic function, and clearance can be acquired using deep-red/NIR fluorescence imaging strategies.
Larger-animal spontaneous melanoma models may more accurately reflect human disease and enable improved simulation of surgical procedures used in humans (i.e., SLN mapping). Locally injected 124
I-cRGDY-PEG-dot tracer and dynamic PET imaging enabled superior detection sensitivity and discrimination of metastatic tumor burden within hypermetabolic neck nodes compared with the PET imaging agent, 18
F-FDG is an indicator of glucose metabolism, accumulating within metabolically active tumors that use this substrate. Traditionally used to stage clinical melanoma, it failed to accurately stage nodal disease in this representative miniswine. A number of well-known limitations are associated with whole-body 18
F-FDG PET (44
), particularly when imaging patients with early-stage melanoma, including a low mean sensitivity of 17.3% (0%–40%) for detecting SLNs and an inability to detect micrometastases less than about 1-cm i.d. In the present study, the 124
I-cRGDY-PEG-dot tracer was able to discriminate at least order of magnitude differences in metastatic tumor burden (i.e., 10- to 20-times greater) between the bilateral neck nodes, as determined by high-power microscopy, while 18
F-FDG PET could not. Although metastatic disease was detected within the SLN, tumor was missed within the smaller (1.0 × 0.6 × 1.0 cm3
) contralateral node.
Thus, for metastatic disease assessment, the use of dynamic PET imaging as a depth-sensitive, volumetric imaging tool, in conjunction with 124I-cRGDY-PEG-dots, will offer distinct advantages in large-animal models in the intraoperative setting. Superior detection sensitivity, metabolic nodal status, and enhanced penetration for the mapping of deep-seated nodal and tissue activities may complement extended real-time fluorescence imaging assessments of metastatic tumor burden in the future. The use of PET will be critical for confirming the findings of depth-insensitive optical imaging tools, particularly in anatomic regions associated with unpredictable patterns of metastatic disease spread.