Nanoparticles for Molecular Imaging

Over the past decade there has been an explosion in the number of nanotechnology-based agents that have been applied to biological and medical applications. It is generally believed that these agents will revolutionize how medicine is practiced. One particularly promising direction that has garnered a great deal of interest is molecular imaging.

The development of nanotechnology-based imaging probes offers to substantially improve the specificity and sensitivity of diagnostic imaging by allowing for the non-invasive and quantitative detection of specific biomolecules in living subjects.

In general, molecular imaging probes consist of a nanoparticle that has been functionalized with a targeting agent. The targeting agent is typically selected to recognize a disease biomarker located on the cell surface;^1-4 however, probes have also been developed that strictly bind healthy tissue, thus leaving malignancies within target tissues unlabeled.^5-7 In either case, the nanoparticles serve to enhance the contrast between malignant and benign tissue.

Interest in the use of nanoparticles stems from their ability to provide improved contrast compared with more traditional contrast agents and the ability to control their pharmacokinetics through variations of their size, surface properties, and shape.⁸ The strong contrast enhancing capabilities of nanoparticles can typically be attributed to atomic constraints that occur at the nanometer size-scale and/or the cumulative effect that results from packing many contrast agents into nanometer-sized particles.

For example, when iron oxide particles are synthesized at the nanometer-size scale they exhibit "superparamagnetic" properties because they can exist as single-domain crystals. In contrast, larger iron oxide particles generally consist of multiple magnetic domains that are aligned in the short range, but at longer distances the domains are anti-aligned and thus exhibit a reduced net magnetic effect per iron ion.

As a result, on a per iron ion basis, nano-sized iron oxide particles are generally able to generate more contrast on magnetic resonance images than larger micron-sized nanoparticles. Of course, the total iron content cannot be ignored. Since, micron-sized iron oxide particles are composed of significantly more iron ions than nanoparticles, they exhibit much stronger MR contrast on a per particle basis. This has allowed single micron-sized particles to be imaged via MR.⁹

To date, most nanoparticles that have been developed for magnetic resonance imaging applications have been characterized in terms of their relaxivity per ion (e.g. Fe, Gd, etc). Although this is certainly of great value, it can be argued that for molecular imaging applications it is even more important to calculate relaxivity on a per particle basis. For example, if a tumor cell has ten receptors on its surface, the binding of ten micron-sized particles of iron oxide would certainly provide more contrast than ten nanoparticles, even though the smaller nanoparticles would likely have a higher relaxivity per iron ion than the micron-sized particle.

This argument is, of course, not limited to iron oxide particles. A recent comparison that we made between Gd-labeled dendrimers and Gd-labeled dendrimer nanoclusters (DNCs) also highlights the importance of calculating the relaxivity per nanoparticle.¹ In this study, Gd-labeled DNCs were formed by simply cross-linking Gd-labeled dendrimers into a higher-order structure, with a mean hydrodynamic diameter of ~150nm. While both the dendrimers and DNCs exhibited a similar relaxivity per Gd ion, the DNCs possessed >1,000-times more Gd per particle. As a result of this higher payload, the tumor-targeted Gd-labeled DNCs provided a dramatic improvement in contrast compared with Gd-labeled dendrimers, in tumor-bearing mice.

Aside from the contrast-enhancing capabilities of molecular imaging agents, it is also of critical importance to characterize the pharmacokinetics of new nanoparticle formulations. Particle size, shape, and charge are all known to be major driving forces responsible for dictating the blood half-life and biodistribution.

In general, nanoparticles at the length scale of ~10-100nm have generally exhibited longer circulations times and improved tissue penetration than micron-sized particles. These pharmacokinetic properties can lead to improved targeting and in many cases can be used to overcome the lower contrast-enhancing capabilities of smaller particles - hence the growing interest in using nanoparticles as opposed to micron-sized particle for molecular imaging applications.

In applications where long circulation times and additional contrast is not necessary, there has even been a movement to make molecular imaging probes that are <5.5nm in diameter to encourage renal filtration.¹⁰ This would allow for more rapid imaging, since unbound nanoparticles would be cleared much faster, and reduced toxicity for the same reasons.

In addition to the physical-chemical properties of the nanoparticle itself, the targeting agent also plays an instrumental role in the utility of nanoparticle-based contrast agents. For the most part, targeting agents that have been evaluated for molecular imaging applications have mirrored those used for targeted therapeutics (e.g. folic acid, transferrin, anti-HER2/neu antibodies, etc.).

For cancer imaging, these agents have shown a great deal of promise when used to assess the availability of therapeutic targets and monitor the efficacy of treatment; however, tumor cell receptors are unlikely to be adopted for diagnostic imaging due to the lack of any single receptor that is highly up-regulated across most tumors.

For diagnostic imaging, a biomarker that is universally present would have to be identified for clinical utility. Borrowing from FDG-PET imaging, one option that is being explored involves taking advantage of the increased metabolic rate of cancer cells and the resultant acidic microenvironment. Accordingly, various agents are being developed that specifically bind to cells that exist in subphysiologic pH.¹¹ Since, an acidic microenvironment is common to most tumors, it is expected that tumor pH could serve as a more universal target. Ligands that target angiogeneisis or hypoxia could also potentially be utilized to expand the versatility of targeted molecular imaging probes. Of course, when biomarkers with increased universality are selected, it often comes at the cost of reduced specificity - a criticism that has often plagued FDG-PET.

In conclusion, nanoparticles have shown great promise as molecular imaging probes. However, as the number of nanoparticle formulations continues to expand it will be increasingly important to establish proper indices by which they can be compared. It will also be important to develop creative targeting strategies that can be used to identify disease with high sensitivity and high predictive value.

References

1. Cheng Z, Thorek DL, Tsourkas A. Gadolinium-conjugated dendrimer nanoclusters as a tumor-targeted T1 magnetic resonance imaging contrast agent. Angew Chem Int Ed Engl. 2010;49(2):346-50.
2. Thorek DL, Chen AK, Czupryna J, Tsourkas A. Superparamagnetic iron oxide nanoparticle probes for molecular imaging. Ann Biomed Eng. 2006 Jan;34(1):23-38.
3. Tsourkas A, Shinde-Patil VR, Kelly KA, Patel P, Wolley A, Allport JR, Weissleder R. In vivo imaging of activated endothelium using an anti-VCAM-1 magnetooptical probe. Bioconjug Chem. 2005 May-Jun;16(3):576-81.
4. Zhang CY, Lu J, Tsourkas A. Iron chelator-based amplification strategy for improved targeting of transferrin receptor with SPIO. Cancer Biol Ther. 2008 Jun;7(6):889-95.
5. Montet X, Weissleder R, Josephson L. Imaging pancreatic cancer with a peptide-nanoparticle conjugate targeted to normal pancreas. Bioconjug Chem. 2006 Jul-Aug;17(4):905-11.
6. Reimer P, Weissleder R, Shen T, Knoefel WT, Brady TJ. Pancreatic receptors: initial feasibility studies with a targeted contrast agent for MR imaging. Radiology. 1994 Nov;193(2):527-31.
7. Tanimoto A, Kuribayashi S. Hepatocyte-targeted MR contrast agents: contrast enhanced detection of liver cancer in diffusely damaged liver. Magn Reson Med Sci. 2005;4(2):53-60.
8. Moghimi SM, Hamad I. Factors Controlling Pharmacokinetics of Intravenously Injected Nanoparticulate Systems. In: de Villiers MM, Aramwit P, Kwon GS, editors. Nanotechnology in Drug Delivery. New York: Springer; 2009. p. 267-82.
9. Shapiro EM, Skrtic S, Sharer K, Hill JM, Dunbar CE, Koretsky AP. MRI detection of single particles for cellular imaging. Proc Natl Acad Sci U S A. 2004 Jul 27;101(30):10901-6.
10. Choi HS, Liu W, Misra P, Tanaka E, Zimmer JP, Itty Ipe B, Bawendi MG, Frangioni JV. Renal clearance of quantum dots. Nat Biotechnol. 2007 Oct;25(10):1165-70.
11. Reshetnyak YK, Andreev OA, Lehnert U, Engelman DM. Translocation of molecules into cells by pH-dependent insertion of a transmembrane helix. Proc Natl Acad Sci U S A. 2006 Apr 25;103(17):6460-5.