Molecular imaging: current status and emerging strategies

Abstract

In vivo molecular imaging has a great potential to impact medicine by detecting diseases in early stages (screening), identifying extent of disease, selecting disease- and patient-specific treatment (personalized medicine), applying a directed or targeted therapy, and measuring molecular-specific effects of treatment. Current clinical molecular imaging approaches primarily use positron-emission tomography (PET) or single photon-emission computed tomography (SPECT)-based techniques. In ongoing preclinical research, novel molecular targets of different diseases are identified and, sophisticated and multifunctional contrast agents for imaging these molecular targets are developed along with new technologies and instrumentation for multi-modality molecular imaging. Contrast-enhanced molecular ultrasound (US) with molecularly-targeted contrast microbubbles is explored as a clinically translatable molecular imaging strategy for screening, diagnosing, and monitoring diseases at the molecular level. Optical imaging with fluorescent molecular probes and US imaging with molecularly-targeted microbubbles are attractive strategies as they provide real-time imaging, are relatively inexpensive, produce images with high spatial resolution, and do not involve exposure to ionizing irradiation. Raman spectroscopy/microscopy has emerged as a molecular optical imaging strategy for ultrasensitive detection of multiple biomolecules/biochemicals with both in vivo and ex vivo versatility. Photoacoustic imaging is a hybrid of optical and US techniques involving optically-excitable molecularly-targeted contrast agents and quantitative detection of resulting oscillatory contrast agent movement with US. Current preclinical findings and advances in instrumentation, such as endoscopes and microcatheters, suggest that these molecular imaging methods have numerous potential clinical applications and will be translated into clinical use in the near future.

Figure 1

Contrast agents used for molecular imaging are composed of at least 2 entities: one component such as an antibody, peptide, nucleic acid, or a small molecule for binding to the molecular target, and a label for readout by an imaging modality (see also Table 1). More sophisticated contrast agents can include multiple parts for targeting several molecules at once, as well as, several labels for multimodality imaging. Drugs can also be attached/encapsulated for targeted therapy.

Figure 2

Molecular contrast agent design and clinical use involve a series of steps similar to those used in drug development (Adapted from Willmann et al.). Preclinical steps (green-shaded boxes) involve target identification, validation, chemical labeling, in vitro cellular characterization, and in vivo animal testing. After many optimization steps (arrows between boxes 1, 2, and 3), the agent can be tested in humans after FDA applications for investigatory new drug (IND) or exploratory IND (eIND). Clinical testing (orange boxes) involves rigorous testing for agent properties (effectiveness, specificity, toxicity, off-target effects, kinetics, etc.) before potential approval by the FDA for routine clinical use, which is also heavily monitored for outcomes assessment.

Figure 3

Timeline representation of current and future utilization of molecular imaging in the clinic. Current approved molecular imaging techniques are mostly PET and SPECT, which are expected to continue for use in diagnosing advanced stage diseases. With a plethora of molecular targets identified preclinically for various diseases (e.g., cancer: Table 5) and advances in material engineering for nano- and micro-particle contrast agents, a large potential exists to expand clinical molecular imaging beyond nuclear medicine approaches. Molecular imaging techniques that do not expose patients to ionizing irradiation, such as MRI/MRS, optical, ultrasound and hybrid acoustic imaging approaches are ideal for early disease detection and screening. Hardware and software implementations for sensitive and quantitative detection of targeted contrast agents will also pave the way for targeted therapeutic delivery (tracking) and response monitoring. Optical, ultrasound, and hybrid acoustic imaging are expected to be forerunners in screening methodology since they can provide real-time, inexpensive, and high-resolution images.

Figure 4

Raman Spectroscopy/Microscopy techniques are an emerging molecular imaging tool for potential translation to clinical applications with endoscopes or catheters. Left to right: Nanoparticles with specific Raman signatures (a), including quantum dots, metal (gold (Au) or silver (Ag)) nanoparticles which can be coated for several different spectra (e.g., SERS nanoparticles such as silica coated gold nanoparticles), and carbon nanotubes/fullerenes, can be used for attaching specific moieties to bind to molecular targets (as in Figure 1 where the label is the nanoparticle). The nanoparticles (when excited with an optical laser) produce specific Raman spectra (b), which can be used to generate in vivo imaging signal (c), ex vivo imaging signal (d), and for ex vivo biosensing assays (e). Image in (c) represents in vivo imaging of SERS nanoparticles (S421 and S441) injected subcutaneously in nude mice, and the spectra obtained for S421, S441, and an equal mix of both S421 and S441. Reprinted with permission from Keren et al. Image in (d) represents ex vivo Raman imaging of excised subcutaneous tumor xenografts that were pre-injected (in mouse prior to euthanization) with either plain, non-targeted single-walled carbon nanotubes (Plain SWNT) or SWNT’s conjugated to RGD peptide (binds to α_Vβ₃ integrin); note that RGD-targeted SWNTs show high Raman signal compared to non-targeted, plain SWNTs. SWNT structure and Raman image were reprinted with permission from de la Zerda et al. Image in (e) represents a schematic for using Raman Spectroscopy for analyzing blood samples with nanoparticles (a) targeted to specific biomarkers (DNA, RNA, or proteins); this technique can be advantageous for ultrasensitive, rapid screening for early detection of cancer. Not shown: Raman imaging can also be used for resolution microscopy (e.g., CARS).

Figure 5

Contrast-enhanced ultrasound imaging with molecularly-targeted microbubbles (MBs; molecular ultrasound imaging) is moving rapidly towards clinical translation. Left to right: Proof-of-principle preclinical studies include testing targeted MBs constructed by conjugating an antibody (blue “Y” molecule) to a MB using strept(avidin)-biotin (green line-red circle) binding chemistry (a) for endothelial cell target specificity and validation. Then, MBs using peptides (purple lines) with high binding affinity for the human targets can be constructed with direct conjugation to lipid MB shell (b), and after further rounds of preclinical testing (see Figure 2), peptide-conjugated MBs can progress to clinical testing. Functional ultrasound imaging methods including perfusion analysis with assessment of first-pass time-intensity curves (c) and maximum intensity persistence (MIP) (d) can provide levels of tissue vascularity. Molecular ultrasound imaging with KDR-targeted MBs can be used to measure KDR expression levels (e; for more details on calculating the attached MB signal, refer to Willmann et al.). (f) A clinical ultrasound system was used to acquire B-mode images (right side) and contrast images (see Deshpande et al. in this issue for a more detailed review of contrast imaging) of breast cancer in a transgenic mouse model.

Figure 6

Photoacoustic (PA), thermoacoustic, and magnetoacoustic imaging techniques are emerging as a highly sensitive and versatile tool for molecular imaging. Left to right: Contrast agents for use with hybrid acoustic imaging methods are optically-absorbing nanoparticles, such as small molecule dyes, nanoparticles made of silica, gold, or cobalt, and carbon nanotubes/fullerenes, for use with photoacoustic imaging; and, magnetic nanoparticles for use with magnetoacoustic imaging. Energy is applied to the nanoparticles in the form of near-infrared light, radiofrequency, microwaves, or magnetic field; this energy causes the contrast agent nanoparticles (a) to oscillate and produce sound waves for measurement with ultrasound. PA imaging devices have been developed for imaging at the microscale (b), and macroscale (c) levels. In vivo and in vitro micromolecular analyses can be performed with PA imaging devices such as PA microscopy, PA flow cytometry, and PA spectrophotometry (b). Image represents an in vivo PA spectrophotometry system used to image surrounding vascularity and sentinal lymph node with gold nanorods (activated with 807 nm laser) in male rats (Reprinted with permission from Song et al.). Macromolecular analyses can be performed with PA tomography for in vivo imaging applications (c). Images (reprinted with permission from de la Zerda et al.) represent the device setup of a PA imaging system for small animals; this system was used to generate the adjacent, lower right image of subcutaneous glioma tumor xenografts in nude mice. Plain (non-targeted)- or RGD- (targeting α_Vβ₃ integrin) single-walled carbon nanotubes (a) were injected in the mouse tail vein, and PA signal (green) was imaged with B-mode US imaging (grey).