Recent advances in molecular, multimodal and theranostic ultrasound imaging

Abstract

Ultrasound (US) imaging is an exquisite tool for the non-invasive and real-time diagnosis of many different diseases. In this context, US contrast agents can improve lesion delineation, characterization and therapy response evaluation. US contrast agents are usually micrometer-sized gas bubbles, stabilized with soft or hard shells. By conjugating antibodies to the microbubble (MB) surface, and by incorporating diagnostic agents, drugs or nucleic acids into or onto the MB shell, molecular, multimodal and theranostic MBs can be generated. We here summarize recent advances in molecular, multimodal and theranostic US imaging, and introduce concepts how such advanced MB can be generated, applied and imaged. Examples are given for their use to image and treat oncological, cardiovascular and neurological diseases. Furthermore, we discuss for which therapeutic entities incorporation into (or conjugation to) MB is meaningful, and how US-mediated MB destruction can increase their extravasation, penetration, internalization and efficacy.

Keywords: Angiogenesis; Blood–brain barrier; Cardiovascular; Cavitation; Drug delivery; Molecular imaging; Nanomedicine; Sonography; Theranostics; Tumor.

Figure 1

Examples for the use of CEUS in clinical liver imaging in comparison with MRI. A+B: In B-mode US and in T₂-weighted MRI, a benign liver tumor (fibronodular hyperplasia, FNH) can be delineated. C: Twenty minutes after the administration of the hepatocyte-specific MR contrast agent Gd-EOB-DTPA, the lesion and the surrounding tissue provide comparable contrast enhancement, which is typical for a FNH. D-F: In contrast-specific US mode, one can depict the rapid centrifugal (“spoke-wheel”) enhancement of the lesions. In the late phase (G; i.e. 180 s after injection), the lesion provides no wash-out pattern. Knowledge on the microbubble (MB) kinetics in the late phase enables the exclusion a malignant tumor. The enhancement pattern in the arterial phase further supports the diagnosis of a FNH.

Figure 2

3D molecular ultrasound imaging with SPAQ, showing the principle of SPAQ imaging (A). Stepwise movement of the US-transducer leads to MB destruction in the overlapping slice. Smaller step sizes yield less saturated and therefore better quantifiable images. (B) Shows a 3D-reconstructed SPAQ image of VEGFR2-targeted MB binding in an experimental breast cancer xenograft. White arrows outline the tumor. Yellow arrows show artifacts due to breathing of the mouse. The MB-destruction events are displayed as red dots (see e.g. blue arrowheads). The higher VEGFR2 expression at the angiogenic tumor margin compared with the tumor center is clearly demonstrated.

Figure 3

BR55 highly sensitively depicts very early breast cancer lesions. Representative SPAQ images of one slice in a 4 mm³ (A) and 34 mm³ (B) MCF-7 tumor (tumor marked by yellow arrowheads; white arrows show representative signals of destructed BR55 MB (red overlay)). Quantitative analysis of 3D SPAQ imaging demonstrates the highest binding of BR55 in 4 mm³ small tumours and a significantly reduced binding in larger tumors (C), whereas the relative blood volume (rBV) is constant (E). US data were confirmed by immunohistochemistry (D, F). *p<0.05; **p<0.01 (adapted with permission from [82])

Figure 4

Multimodal US contrast agents. USPIO-loaded PBCA MB as contrast agents for MRI / US showing TEM images of PBCA MB with increasing USPIO (a–e) concentrations (A), phantom MR imaging showing signal enhancement in MRI, which increases with MB destruction (B), and signal enhancement observed in vivo in both US and MRI upon the i.v. injection of USPIO-loaded MB in tumor bearing mice (C; adapted with permission from [87]). D-F: Rhodamine-b loaded PBCA MB for use in optical and US imaging. Comparison between fluorescent and non-fluorescent MB for the evaluation of targeted-MB binding to cells in vitro (D). Two-photon microscopic validation and evaluation of ICAM-1 targeted MB binding to activated (upper panel) vs non-activated (bottom panel) HUVEC (E). Panel F shows the application of fluorescent MB for validation of in vivo molecular US studies. I-III represents sections of the tumor during molecular imaging, while IV shows the evaluation of bound MB by destruction replenishment analysis. By subsequent fluorescence microscopy of tumor cryosections, the attachment of fluorescent MB (red) to FITC-lectin-stained (green) tumor vasculature (V) could be validated (adapted with permission from [101]). Biodistribution analysis of ¹¹¹In-labelled PBCA-MB over time by gamma counting (G–H) and US (I). Both modalities showed a high amount of MB accumulated in the liver compared to kidney and tumor. Unlike gamma counting, where both signals from MB and shell fragments are registered and quantified over time, upon SPAQ imaging, all MB are destroyed and cannot be monitored longitudinally by US. Radioactively labeled MB therefore provide a more reliable means for quantitatively monitoring the biodistribution of MB and their shell fragment in vivo over time as compared to US (adapted with permission from [107]).

Figure 5

Mechanisms of sonoporation. Acoustic micro-streaming under stable cavitation (1). (2) MB compression leading to invagination and membrane opening. (3) MB expansion leading to membrane extension (push-force) and opening. (4) MB destruction releasing acoustic shock-waves and jet-streams that permeabilize the membranes. (Adapted with permission from [134])

Figure 6

Drug delivery to tumors and across the BBB using (model)-drug loaded MB. A-B: The entrapment of drug molecules in the MB shell can either be achieved during MB synthesis or upon post-loading. C-D: By fluorescence microscopy, the encapsulation of in the MB shell was validated. E-F: Upon US-mediated destruction of VEGFR2-targeted model drug-loaded MB in tumors, the accumulation of rhodamin-b and coumarin-6 in and around tumor blood vessels for animals treated with MB plus US (panels II and III; vs. without US, panel I) exemplified effective model drug delivery upon US guidance and triggering (adapted with permission from [102]). G: US- plus MB-mediated drug delivery across the BBB was studied using lipid-shelled MB. The extravasation of evans blue dye was used as verification of BBB-opening induced by DOX-SPIO-MB with US in normal brain tissue and C6 tumors (delineated by yellow line) H-J: H & E stained images of tumor-bearing brain after applying DOX-SPIO-MB and US validated the absence of brain hemorrhage. Region of interest for further analysis were selected from the tumor (T1, T2), the tumor-tissue boundary (B1, B2), and normal brain tissue (N1, N2). Panel I demonstrates increases in doxorubicin accumulation in brain tissue in the DOX-SPIO-MB + US group compared to the DOX group. K: SPIO accumulation in US-treated brain tissues also increased upon the combined application of MB plus US (adapted with permission from [94]).