Molecular imaging probes targeting specific cancer markers have been long sought-after in applying tumor imaging for disease-specific detection and personalized therapeutics. However, the development of receptor-targeted imaging and its in vivo applications are particularly challenging, with current obstacles including: 1) the identification of cell surface biomarkers that are expressed sufficiently in tumor cells or tumor environments for sensitive tumor imaging; 2) the production of stable and high affinity targeting ligands in large amounts for in vivo studies; and 3) the development of safe and biodegradable contrast agents producing strong imaging signal or contrast.
The uPAR-targeted IO nanoparticle probe reported here provides an example that addresses these challenges and demonstrates the feasibility of in vivo
receptor-targeted tumor imaging. Results of our study showed that ATF-IO nanoparticles are capable of targeting uPAR-expressing tumor cells in vitro
and in vivo
, and enable receptor-targeted MR and optical imaging of the tumor in vivo
. We believe that the following characteristics of ATF-IO nanoparticles enabled uPAR-targeted imaging in vivo
. First, we used a tumor targeting ligand from a natural high affinity receptor binding domain of uPA. uPA is composed of three independently folded domain structures: growth factor domain (GFD), Kringle domain, and serine protease domain. uPA binds with high affinity to uPAR through the ATF of the GFD, with a Kd less than 1 nM (39
). Studies have shown that ATF (residues 1–135 aa) of uPA is a potent antagonist of uPA/uPAR binding since ATF lacks the serine protease domain of uPA, which negatively regulates its function by cleaving uPAR into a non-binding receptor (40
). Second, efficient internalization of the ligand/receptor complex may increase the concentration of the IO nanoparticles in tumor cells, which enhances the effect of uPAR-targeted tumor imaging (35
). Third, nanoparticles provide favorable pharmacokinetics by prolonging blood circulation time, allowing sufficient amounts of nanoparticles to reach the tumor. Furthermore, our ability to produce the recombinant protein in a large scale is essential for preclinical and eventually clinical studies. It should be mentioned that this study was done using mouse ATF peptides and the 4T1 mouse tumor model to investigate the feasibility of targeting uPAR. Although it has been shown that the interaction of uPA with its receptor has a species specificity (43
), we found that mouse ATF peptides bind efficiently to mouse tumor cells and also show cross reactivity with human uPAR-expressing tumor cells. A major advantage of using mouse ATF peptides to conduct studies in a mouse tumor model is that the targeting specificity and biodistribution in normal tissues of this imaging probe can be studied in greater detail. For future clinical application, it is desirable to use a human ATF peptide. We have produced recombinant human ATF peptides and have demonstrated target specificity in human breast cancer xenograft models in nude mice (unpublished results).
Extensive studies have shown that human breast cancer and tumor stromal cells have a much higher level of uPAR compared to normal breast tissues (44
). Differences between the level of uPAR present on the surface of normal and tumor cells suggest that an elevated uPAR in cancer may be sufficient for in vivo
uPAR-targeted tumor imaging. Several studies have shown that the highest level of uPAR expression is detected in the invasive edge of the tumor regions (23
), which are usually enriched in blood vessels, making this area particularly accessible for uPAR-targeted IO nanoparticles. Additionally, the ability of nanoparticles to leak cross tumor endothelium but not normal vasculatures by passive targeting may prevent the interaction of targeted nanoparticles with some cell types in normal tissues that express a low level of uPAR.
In this study, we prepared high quality and uniformly sized IO nanoparticles with a thin amphiphilic copolymer coating (estimated at ~2 nm). Compared with conventional dextran or PEG-coated nanoparticles used previously, amphiphilic copolymer-coated IO nanoparticles form a relatively small particle complex (~18 nm, in this study), which is desirable for in vivo
delivery of the imaging probe. Several previous studies used a commercially available superparamagnetic iron oxide (SPIO) nanoparticle, Feridex, which has a particle core size distribution of 20–30 nm or 80–150 nm in overall diameter with dextran coating (13
). We believe that size uniformity is essential for potential quantification using MRI signal of the amount of probe in vivo
Although other small molecule imaging agents may have better intratumoral distribution than nanoparticle-based imaging agents, these imaging agents are usually eliminated from the blood circulation in a relatively short time (less than 30 min), which makes it unlikely that sufficient levels of the targeted contrast agents can accumulate at the tumor site (2
). It has been shown that polymer-coated IO nanoparticles have over 8 hours of plasma retention time (11
). This longer circulation time could be an important factor enabling targeted IO nanoparticles to reach the tumor site and bind to tumor cells. ATF-IO nanoparticles were stable in vivo
and in intracellular environments for over 48 hrs during our imaging experiments. We observed that the intratumoral NIR signal increased over time and reached its highest level around 48 hrs after the administration of IO nanoparticles, suggesting that the long blood retention time may facilitate nanoparticle tumor targeting.
It has been reported by several other groups that large proportions of magnetic IO nanoparticles are taken up by the reticuloendothelial system in the liver and spleen, and then are subsequently metabolism or utilized for iron storage (12
). Our data showed that ATF-IO nanoparticles have reduced liver and spleen uptake compared with non-targeted IO nanoparticles. This suggests that conjugation of ATF-peptides to the IO nanoparticles attenuates their non-specific capture and retention in the liver and spleen, which commonly occurs after systemic delivery.
In conclusion, we have developed a uPAR-targeted molecular imaging nanoprobe that has a uniform-sized IO nanocrystal core, a thin amphiphilic copolymer coating, and a high affinity receptor binding domain of uPA conjugated with an NIR dye. This receptor-targeted nanoprobe selectively binds to and is internalized by tumor cells and can specifically accumulate in primary and metastatic tumors, facilitating in vivo
MR and optical imaging in a mouse mammary tumor model. Such uPAR-targeted imaging nanoparticles are promising probes for molecular MRI of breast cancer and several other cancer types, such as pancreatic, lung and brain, which express high levels of uPAR (46
). Given that chemotherapy drugs can be incorporated into the targeted nanoparticles (49
), it will be feasible to use uPAR-targeted IO nanoparticles for delivery of therapeutic agents into tumor cells and monitoring the response to therapy using MRI.
Breast cancer is the most common cancer in women with about 180,000 new cases and 40,000 deaths each year in the United States. Currently, breast magnetic resonance imaging (MRI) is used for early detection of breast cancer and is recommended as a routine screen for women at high risk of the disease. Although breast MRI using a non-targeted contrast agent has a relatively high sensitivity in detecting lesions, it lacks specificity in determining the pathological characteristics of the lesions. Recent studies show that patients receiving MRI have a higher rate of biopsy and mastectomy compared to those without this imaging procedure. MRI is a promising non-invasive imaging approach for preoperative staging of breast cancer and monitoring tumor response to therapy. Therefore, the development of biomarker-targeted MRI contrast probes that increase the specificity of breast cancer MRI should have a great impact on the detection and treatment of breast cancer.