Functionalization of agents for specific targeting to molecular sites is an important tool for molecular specific imaging. Along with the synthesis of these nanoprobes, the chemistry of bioconjugation has also contributed significantly to the development of targeted probes that can be conjugated to different biomarkers, proteins, peptides, or oligonucleotides. Development of functionalized nanoprobes for in vivo targeting is extremely challenging and involves multidisciplinary skills. A number of factors determine the clinical usefulness of the nanoparticles. Their physical and functional biokinetic properties, clearance profiles, biodistribution, biocompatibility, and long-term toxicity need to be carefully considered for their potential use in in vivo applications.
Magnetomotive OCT of single cells dispersed in a 3-D gel. Cells containing magnetite are clearly identified.
|Oldenburg AL, Gunther JR, Boppart SA. Imaging magnetically labeled cells with magnetomotive optical coherence tomography. Optics Letters, 30:747-749, 2005.||PubMed
In vivo magnetomotive OCT of magnetic nanoparticles in a Xenopus (African frog) tadpole model.
|Oldenburg AL, Toublan FJ, Suslick KS, Wei A, Boppart SA. Magnetomotive contrast for in vivo optical coherence tomography. Optics Express, 13:6597-6614, 2005.||n/a|
Plasmon-resonant gold nanorods exhibit specific longitudinal (L) and transverse (T) resonances and can function as highly absorbing and scattering contrast agents.
|Oldenburg AL, Hansen MN, Zweifel DA, Wei A, Boppart SA. Plasmon-resonant gold nanorods as low backscattering albedo contrast agents for optical coherence tomography. Opt. Express 14(15):6724-6738, 2006.||n/a|
In this study, we demonstrate the use of MM-OCT for quantitative measurement of magnetic iron-oxide nanoparticle transport and concentration in ex vivo muscle, lung and liver tissues. The effect of temperature on the dynamics of these nanoparticles is also analyzed. We observe that the rate of transport of nanoparticles in tissues is directly related to the elasticity of tissues, and describe how the origin of the MM-OCT signal is associated with nanoparticle binding. These results improve our understanding of how iron-oxide nanoparticles behave dynamically in biological tissues, which has direct implications for medical and biological applications of targeted nanoparticles for contrast enhancement and therapy.
The paper reports on the method's use in imaging mammary tumor tissue but Boppart said that the technology has many potential applications.
Boppart said the technique uses magnetic forces to move nanoparticles bound to proteins, cells, tissue; phase-sensitive OCT then measures changes in light scattering from the movement. The method's uniqueness comes from the ability to target the nanoparticles to a specific site, such as a tumor, where their multifunctionality can then be utilized.
The nanoparticles can provide information on contrast (especially valuable when it comes, for example, to distinguishing between healthy cells and cancerous cells) due to the fact that the MNPs are magnetic while the rest of the tissue is not. Another use is to record biomechanical tissue measurements (such as cell elasticity and viscosity, important factors in disease detection). It works by using a magnetic field to make the nanoparticles vibrate, thereby providing signals from the tissue through the rate and frequency of the movements.
Another use enabled by the technology is therapeutic. By turning up the frequency of the magnetic field, the nanoparticles move so quickly they begin to heat up, providing a possible treatment tool.
"So now we have a platform where we can target these to a tumor, find them with contrast, measure the mechanical properties, and treat it right there," Boppart said.
The magnetic nanoparticles in this project were antibody functionalized to target the human epidermal growth factor receptor 2 (HER2 neu) protein, a receptor which is over expressed in breast cancer. The fabrication of the antibody-directed functionalized nanoparticles was also rather unique for biomedicine: conjugating the antibody to the nanoparticle in a specific way to provide the highest target specificity to the receptor.
RGD targeted protein microspheres
Air-filled microbubbles and perfluorocarbon-filled protein microspheres that are commercially available as ultrasound contrast agents have been reported in the past as scattering contrast agents in OCT. Protein microspheres with different cores can be fabricated using high frequency ultrasound. A wide variety of materials like gases, lipids, and water can be selected for the core region of these protein-shell microspheres [Figure]. The encapsulation process is mediated through sonication using high frequency ultrasound. The protein shell is crosslinked through the reduction of disulphide bonds by the radicals formed during the sonication process. The sizes of microspheres can be controlled from hundreds of nanometers to a few tens of microns by the addition of surfactants to change the surface tension of the protein mixture.
Novel molecularly-targeted contrast agents are being developed which enhance the diagnostic imaging capabilities of OCT and other non-fluorescent or non-bioluminescent imaging techniques. These include scattering microspheres as contrast and drug-delivery agents, magnetomotive nanoparticles, plasmon-resonant nanoparticles, and absorbing near-infrared dyes. In addition, there are a large number of optical properties and characteristics from contrast agents that can be leveraged to enable optical molecular imaging of cells and tissues.
Metaphorical diagram of a contrast agent showing the wide range of substrates, optical properties, and applications.
|Boppart SA, Oldenburg AL, Xu C, Marks DL. Optical probes and techniques for molecular contrast enhancement in coherence imaging. Article and Cover Figure. J Biomedical Optics, 10:041208, 2005.||PubMed Abstract|
TEM of gold microspheres.
(a) OCT image of an in vivo mouse liver before contrast agents were introduced. (b) OCT image of an in vivo mouse liver after injection of gold microspheres into the mouse tail vein. Liver sinusoids are now apparent.
|Lee TM, Toublan FJ, Sitafalwalla S, Oldenburg AL, Suslick KS, Boppart SA. Engineered microsphere contrast agents for optical coherence tomography. Optics Letters, 28(17): 1546-1548, Sept 2003.||PubMed
Tumor cell targeting. RGD-peptide conjugated microspheres containing fluorescent Nile Red dye are shown targeting to HT29 tumor cells.
|Toublan FJJ, Boppart SA, Suslick KS. Tumor targeting by surface modified microspheres. J. Am. Chem. Soc., 128:3472-3473, 2006.||PubMed Abstract|
Ex vivo and In vivo Disease Targeting Using Protein Microspheres
These protein microspheres are versatile and can be modified for a variety of different imaging modalities, including MM-OCT, positron emission tomography (PET), and Cerenkov luminescence imaging, which is a type of radioluminescence imaging. They have been functionalized with RGD peptides for both ex vivo and in vivo targeting of atherosclerotic plaques.
Conceptual schematic (A) and TEM image (B) of the magnetomotive microspheres.
Ex vivo targeting of atherosclerotic plaques in rabbit aorta. (A) Representative photographs of aorta specimens. (B) H&E and OCT images of aortas from different experimental groups.
|Kim J, Ahmad A, Li J, Marjanovic M, Chaney EJ, Suslick KS, Boppart SA. Intravascular magnetomotive optical coherence tomography of targeted early-stage atherosclerotic changes in ex vivo hyperlipidemic rabbit aortas. J. Biophotonics 9: 109-116, 2016.|
Conceptual schematic (a) and SEM image (b) of 64Cu-labeled quantum dot (QD)-microspheres.
In vivo targeting of cyclic RGD-functionalized microspheres and PET-CT (a), Cerenkov luminescence-excited fluorescence (b), and quantum dot fluorescence (c) imaging of microsphere binding on the aortic arch. Evidence of fibrous plaque and thickening of the aorta are identified in histology sections (f). Scale bars: (a) 1 cm; (b-c) 5 mm, (f-g) 25 µm.
|Li J, Dobrucki LW, Marjanovic M, Chaney EJ, Suslick KS, Boppart SA. Enhancement and wavelength-shifted emission of Cerenkov luminescence using multifunctional microspheres. Phys. Med. Biol. 60, 727-739, 2015.|