In the present study, we demonstrated that our previously developed nanoparticle platform can be functionalized with RGD to serve as a contrast agent that allows the detection of ongoing angiogenesis and the distinction between angiogenesis intensities of different tumor models by two complementary and noninvasive imaging modalities, i.e., MRI and NIRF. The combination of both modalities provided spatiotemporal information about the accumulation and fate of the probe. Analysis of the MR images revealed a significant difference in distribution of the RGD-targeted nanoemulsions in the fast growing and highly vascularized human colorectal LS174T model compared to the EW7 model, characterized by slow growth (50% of LS174T), highly elevated vascular mimicry in the tumor rim [32
], and lower MVD [31
]. In the case of the EW7 model with a lower angiogenesis level and vascular mimicry, the majority of the RGD nanoparticle uptake occurred nonspecifically and might explain the relatively homogeneous nanoparticle distribution pattern, which was similar to the LS174T control group injected with the untargeted nanoparticles. In this way, the EW7 RGD group served as a control for both, i.e., the targeted as well as untargeted LS174T groups. On the contrary, in the LS174T RGD nanoparticle tumors, the high expression of the αv
integrin, predominantly observed at the periphery of the tumor, caused a shift away from the homogenous accumulation pattern toward a pattern corresponding prevalently to documented expression of the integrin [36
]. Because αv
integrin expressed at endothelial cells is directly accessible from the circulation, targeting of RGD-functionalized nanoparticles is faster than the passive accumulation owing to the EPR effect. Therefore, the first one of these two competing processes dominates the second in the case of a high receptor expression at the tumor vasculature [37,38
Whereas MRI served to show the accumulation pattern within the tumors 4 hours after injection, NIRF imaging provided time-resolved information about the fate of the particles on the whole tumor level for a period of 24 hours. The statistically significant difference in accumulation kinetics between the three investigated groups for the time points up to 6 hours was given as follows: LS174T RGD > LS174T control > EW7 RGD.
Histologic examination served to corroborate the accumulation of nanoparticles in the tumor tissue. Perls staining, used to visualize iron oxide deposits in tissue, demonstrated the co-localization of the targeted particles with the vessel walls, whereas untargeted nanoemulsions were found extravasated and diffusely spread throughout the tissue. CLSM of rhodamine B-labeled nanoparticles corroborated the Perls staining results. The biodistribution, assessed by measuring the Cy7 fluorescence counts of the whole organs and the tumors after excision, revealed a high-dose percentage of the targeted contrast agent in the LS174T tumors (normalized to the liver: 35%), a highly desirable property of a contrast agent.
The MVD of the two used tumor models was assessed using CD31 staining and revealed LS174T tumors to have a much higher angiogenesis level than the EW7 counterpart. It is important to stress that such an assessment of MVD alone does not provide information about the proliferating fraction of endothelial cells within a tumor at a given time point. However, in most mouse models—unlike human tumors—high MVD is associated to intense ongoing angiogenesis [39
]. Usually, the degree of MVD increases with tumor types that have higher rates of nutrient or oxygen consumption compared to others with a lower level metabolic requirement. Whereas MVD has often been shown to be a prognostic indicator in many tumor studies, its measurement for monitoring of antiangiogenic therapy has not been demonstrated to be reliable. A decrease in MVD following antiangiogenic therapy is certainly a confirmation of its efficacy, but an unchanged MVD is not necessarily a proof for its inefficacy [10
]. In cases of equal tumor cell and endothelial cell dropout, no changes in MVD are detectable, as, e.g., shown in a case of multiple myeloma treatment with thalidomide, where not all tumor regressions were associated with an MVD decrease [40
In light of this, because our nanoparticles were shown to be able to distinguish between different levels of angiogenesis in two distinct tumor models by directly targeting the αv
receptor as visualized by NIRF and MRI, it is imaginable that they could be not only used as a noninvasive contrast agent for angiogenic phenotyping but also to reliably monitor response to antiangiogenic therapy, like we have shown with paramagnetic liposomes [41
]. The efficacy of the latter then would be expressed as a change in nanoparticle kinetics monitored by NIRF imaging and/or differences in T2
* signal loss pattern using MRI after injection over time. Moreover, we found in a very recent parallel study that our nanoparticle platform could be modified by implementation of cholesterol to form a stable nanocarrier with a PEG content that could be judiciously varied in a range of 5 to 50 mol%. Lower PEG contents proved to even highly increase its targeting capabilities to the αv
receptors of newly forming vessels [37
]. An improved modification of our nanoemulsion of this sort might result in a much higher sensitivity for detecting changes in neoangiogenesis during the course of treatment. Furthermore, by using this modified version of our nanoemulsion formulation, it could be convincingly shown that RGD-targeted nanoparticles began to accumulate as early as 10 to 30 minutes after i.v. injection and gave a clear binding pattern 2 hours after administration. In contrast, untargeted control particles showed almost no accumulation within the first 30 minutes and a very heterogeneous pattern after 2 to 4 hours. Only 8 hours after injection, all the particles extravasated into the tumor tissue. These data corroborate our finding that RGD-targeted nanoparticles show a higher accumulation compared to the untargeted control within the first hours after i.v. administration, as presented in the 24-hour kinetics herein. Another recent study using RGD-targeted, superparamagnetic polymeric micelle nanoprobes, combined with T2
*-weighted time-resolved MRI, demonstrated an increased accumulation of the probe over the control in subcutaneous tumor animal models during the first 30 minutes after i.v. injection, showing an onset already within the first 5 minutes [38
In a recently published study with a smaller, 50-nm version of the RGD nanoemulsion presented here, which also had hydrophobic glucocorticoids incorporated, we achieved significant tumor growth inhibition, demonstrating the versatility of this nanocarrier and its use for theranostics [42
In conclusion, the RGD-conjugated nanoparticle contrast agent presented in this study can be used to noninvasively investigate differences in angiogenic activity in tumors and for angiogenesis phenotyping of tumors. Its biodegradability, flexibility, and capability of encapsulating hydrophobic materials/drugs make this platform suitable for theranostics and the tailored antiangiogenesis combination therapy with highly potent but water-insoluble cytotoxic agents.