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MR molecular imaging and fluorescence microscopy for identification of activated tumor endothelium using a bimodal lipidic nanoparticle

Willem J. M. Mulder^*,1, Gustav J. Strijkers, Jo W. Habets^*, Egbert J. W. Bleeker^*, Daisy W.J. van der Schaft^#, Gert Storm, Gerben A. Koning^,¶, Arjan W. Griffioen^# and Klaas Nicolay^*

^* Biomedical NMR, Department of Biomedical Engineering, Eindhoven University of Technology, MB Eindhoven, The Netherlands;
^# Angiogenesis Laboratory, Research Institute for Growth and Development, Department of Pathology/Internal Medicine, Maastricht University & University Hospital, AZ Maastricht, The Netherlands;
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht, The Netherlands; and
^¶ Department of Radiation, Radioisotopes, and Reactors, Faculty of Applied Sciences, Delft University of Technology, JB Delft, The Netherlands

¹Correspondence: Biomedical NMR, Dept of Biomedical Engineering, Eindhoven University of Technology, P.O. Box 513, 5600 MB Eindhoven, The Netherlands. E-mail: w.j.m.mulder{at}tue.nl

SPECIFIC AIMS

The first aim of the present study was to assess whether MR molecular imaging of angiogenesis is possible using vß3-specific bimodal lipidic nanoparticles (LNP) in tumor-bearing mice. The second aim was to unravel the mechanism and site of accumulation of the lipidic nanoparticles in the tumor.

PRINCIPAL FINDINGS

1. Paramagnetic and fluorescent RGD lipidic nanoparticles target proliferating EC in vitro
We have described the association of paramagnetic and fluorescent RGD lipidic nanoparticles with proliferating human EC (HUVEC). Liposomes conjugated with the vß3-specific RGD peptide or nonspecific RAD peptide were assessed for their ability to associate to proliferating EC. After incubation, cells were washed and assessed for liposome association with confocal laser scanning microscopy (CLSM) and MRI. HUVEC incubated with RGD liposomes showed a strong fluorescence signal that originated from an intracellular location. RAD liposomes either did not or only marginally associated to the HUVEC. Quantitative MRI of a pellet of 10⁶ packed HUVEC revealed a significant reduction in the T₁ relaxation time of cells incubated with RGD liposomes vs. cells incubated with RAD liposomes and cells not incubated with lipidic nanoparticles.

2. RGD lipidic nanoparticles accumulate in the rim of the tumor
A distinct difference in the distribution pattern of contrast-enhanced MRI voxels was observed between mice injected with RGD liposomes or RAD liposomes. The tumors of mice that received RGD liposomes revealed a signal intensity increase mainly found at the rim of the tumor (Fig. 1 A–C), whereas tumors of mice that received RAD liposomes showed signal intensity increase throughout the whole tumor (Fig. 1D-F ).

View larger version (79K): [in this window] [in a new window]   Figure 1. In vivo localization of paramagnetic liposomes in the tumor. A–C) MR images of slices through a tumor of an animal 35 min after it was injected with paramagnetic RGD liposomes. A) A slice through the periphery of the tumor showed a large amount of imaging voxels with significant signal enhancement. B) In plane the signal enhancement was also mainly found at the edge of the tumor. C) A slice through the middle of the tumor showed a low fraction of imaging voxels with signal enhancement, predominantly in the rim of the tumor. D–F) MR images of slices through the tumor of an animal 35 min after it was injected with paramagnetic RAD liposomes. Signal enhancement was observed throughout the tumor at all slice positions. G–I) Slices through the tumor of an animal that was first injected with nonparamagnetic RGD liposomes to block the vß3-integrin sites, and 35 min thereafter injected with paramagnetic RGD liposomes. Only a small number of voxels showed contrast enhancement. The color indicates the % of signal enhancement according to the pseudo-color scale on the right.

3. Competitive blocking led to a decrease of contrast enhanced voxels
The tumors of mice first injected with nonparamagnetic RGD liposomes to block the vß3-integrin sites, followed after 35 min by the injection of paramagnetic RGD liposomes, showed a 4-fold decrease in the percentage of tumor voxels with significantly increased signal intensity in the T₁-weighted images. This assay demonstrated that little of the RGD liposomes extravasated into the tumor tissue and that the major part of the measured contrast enhancement is related to specific interaction of the RGD liposomes.

4. Fluorescence microscopy revealed that RGD-LNP were associated with blood vessels
Fluorescence microscopy revealed a distinct difference in the localization and distribution pattern of RGD or RAD liposomes. In the case of RGD liposomes, rhodamine fluorescence was organized in circular and longitudinal patterns (Fig. 2 A,B); these were invariably associated with blood vessels, suggesting a specific association with vß3 expressed at the angiogenic endothelium. In contrast, after RAD liposome administration, a diffuse pattern of fluorescence was found around blood vessels within the tumor tissue (Fig. 2C ), indicative of extravasation of the liposomes (Fig. 2D ). In the competitive assay, both the fluorescein-labeled nonparamagnetic RGD liposomes (Fig. 2E ) and the rhodamine-labeled paramagnetic liposomes (Fig. 2F ) targeted the vessel wall.

View larger version (94K): [in this window] [in a new window]   Figure 2. Histology of dissected tumors. Fluorescence microscopy of DAPI colored 10 µm sections from dissected tumors revealed a distinct difference between tumors of mice injected with RGD liposomes (A, B) or RAD liposomes (C). In RGD liposomes, circular (A) and longitudinal distribution patterns (B) of the rhodamine fluorescence were observed and these were associated with blood vessels. A diffuse pattern of fluorescence within the tumor tissue was found for RAD liposomes (C). A slice through the middle of the tumor (D) (also depicted in panel A) showed no rhodamine fluorescence. Fluorescence microscopy of a section from a tumor of a mouse injected with nonparamagnetic fluorescein RGD liposomes, followed by paramagnetic rhodamine RGD liposomes recorded with a red filter (E) and a green filter (F).

5. CD-31 costaining revealed the exclusive association of RGD-LNP with tumor endothelium
The difference between RGD and RAD liposome localization was further validated by immunohistochemical detection of blood vessels using the endothelial cell-specific CD31 antibody conjugated to FITC. The red fluorescence from liposomal rhodamine colocalized with the CD31-FITC staining and originated from the vessel lumen and wall. No RGD liposomes were outside the vessel wall or lumen. In contrast, RAD liposomes were extravasated into the tumor tissue, outside of the vessels.

6. The multi-modality imaging approach gave insight in the exact mechanism of accumulation in the tumor
Many pathological processes are associated with the enhanced expression of cell surface receptors at the diseased endothelium, often accompanied by an increase in the permeability of the endothelial barrier. Accumulation of the contrast agent at the pathological site may be caused by 1) the desired specific association with the targeted cell surface receptor, but also by 2) extravasation of the contrast agent into the extravascular compartment or 3) binding to cell surface receptors of nonendothelial cells after extravasation of the contrast agent. The MRI contrast enhancement therefore needs to be critically validated, since accumulation caused by 2) and 3) will strongly increase the background signal and may lead to misinterpretation. This is especially true for vß3-integrin in tumors, as the angiogenic endothelium has an increased permeability and the integrin is expressed at tumor cells. Large proteins conjugated with multiple RGD peptides and RGD-4C-phages attach exclusively to endothelial cells and do not reach the tumor cells. In the present study we used liposomes with a mean size of 150 nm conjugated with 700 cyclic RGD peptides each. We incorporated a fluorescent label into the liposomal MRI contrast agent to determine its fate at microscopic resolution and discern the mechanism of accumulation. As a control, nonspecific RAD liposomes were used. The difference in contrast enhancement between RGD-LNP and RAD-LNP at anatomical level was established with MRI, and the fluorescence pattern was established with fluorescence microscopy of slices of dissected tumors.

CONCLUSIONS AND SIGNIFICANCE

The current paper demonstrates a multimodality imaging approach for detection of the vß3-integrin to identify the activated endothelium in vitro on HUVEC and in vivo in tumor-bearing mice by using RGD-conjugated LNP. LNP specifically targeted angiogenically activated tumor EC and that accumulation of the LNP can be detected by MRI in vivo and by fluorescence microscopy ex vivo.

In vitro studies demonstrate that RGD-LNP associate massively with proliferating HUVEC whereas nonspecific RAD-LNP only marginally associate with HUVEC. RGD-LNP were internalized by the cells and localized to a perinuclear compartment.

In the in vivo tumor mouse model, distinct differences in the accumulation pattern of the specific LNP and the nonspecific LNP were found. The RGD-LNP were detected mainly at the rim of the tumor whereas the RAD liposomes were found diffuse throughout. This distribution pattern of the RGD-LNPs closely correlates with the position of angiogenic blood vessels in the tumor, which are found mainly at the rim. The specificity of the RGD-LNP was further validated with a competitive blocking assay of the vß3-integrin sites with a nonparamagnetic RGD-LNP before injection with paramagnetic RGD-LNP. This resulted in a 4-fold lower percentage of imaging voxels in the tumor that showed contrast enhancement.

For validation of the MRI findings and to gain insight into the exact mechanism of accumulation of the LNP, fluorescence microscopy of the excised tumor was performed. Colloidal particles like liposomes or micelles accumulate in tumor tissue due to the leakiness of the endothelial cell layer, often referred to as the enhanced permeability and retention effect and used for drug targeting. The ex vivo fluorescence microscopic validation was of key importance in establishing the site and mechanism of accumulation and interpreting the MRI findings. The circular and longitudinal fluorescence distribution patterns of the rhodamine from RGD liposomes demonstrated the association with tumor blood vessels. Endothelial cell-specific costaining with a CD31 antibody revealed that the RGD-LNP were associated exclusively with tumor blood vessels, indicative of a specific association with vß3-expressing angiogenic endothelium. In contrast, a diffuse pattern of fluorescence was found around blood vessels within the tumor tissue in case of RAD liposomes. The endothelial cell costaining further established that these liposomes were present in the extravascular compartment.

The current results indicate that MRI in combination with the RGD-conjugated LNP is a powerful tool for investigating angiogenesis at the molecular level and shows promise for the noninvasive monitoring of the effect of anti-angiogenesis therapy and improved diagnosis.

View larger version (38K): [in this window] [in a new window]   Figure 3. Schematic representation of the study. Tumor-bearing mice were injected with either vß3-specific RGD-LNP or nonspecific RAD-LNP. In vivo MRI revealed the location of contrast enhancement, while ex vivo fluorescence microscopy yielded critical insight in the mechanism of accumulation of both LNPs.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4145fje;

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