Aptamer-Conjugated Nanobubbles for Targeted Ultrasound Molecular Imaging

Abstract

Targeted ultrasound contrast agents can be prepared by some specific bioconjugation techniques. The biotin-avidin complex is an extremely useful noncovalent binding system, but the system might induce immunogenic side effects in human bodies. Previous proposed covalently conjugated systems suffered from low conjugation efficiency and complex procedures. In this study, we propose a covalently conjugated nanobubble coupling with nucleic acid ligands, aptamers, for providing a higher specific affinity for ultrasound targeting studies. The sgc8c aptamer was linked with nanobubbles through thiol-maleimide coupling chemistry for specific targeting to CCRF-CEM cells. Further improvements to reduce the required time and avoid the degradation of nanobubbles during conjugation procedures were also made. Several investigations were used to discuss the performance and consistency of the prepared nanobubbles, such as size distribution, conjugation efficiency analysis, and flow cytometry assay. Further, we applied our conjugated nanobubbles to ex vivo ultrasound targeted imaging and compared the resulting images with optical images. The results indicated the availability of aptamer-conjugated nanobubbles in targeted ultrasound imaging and the practicability of using a highly sensitive ultrasound system in noninvasive biological research.

Ultrasound imaging beyond the vasculature with new generation contrast agents

“…For example, Lanza et al used a 30MHz intravascular ultrasound system to detect thrombi with a targeted perfluorocarbon emulsion based contrast agent (250nm) 22 . Wang et al used a custom-made 40MHz transducer to image nanobubbles (485nm) targeting to CCRF-CEM cells in an ex vivo model 23 . Efforts have also been made to decrease the shell stiffness by incorporating surfactant in order to decrease the detection frequency for nanobubbles, as described in detail below 18 .…”

“…Although the mechanism is not fully understood, it has been postulated that the shell bending modulus resists increases in curvature below 1 μm, producing only a small number of nanobubbles 39 . Nanobubble formulations have thus been obtained by extraction of this nanometer subpopulation from microbubble solutions based on their buoyancy by centrifugation 23, 40-45 . Although this method produces a relatively small nanobubble yield, it has been demonstrated that these nanobubbles are capable of extravasating the leaky vasculature in tumors and improving signal intensity under diagnostic ultrasound 42-44 .…”

Functionalized nanomaterials: their use as contrast agents in bioimaging: mono- and multimodal approaches

“…The use of nanobubbles [29,[278][279][280][281][282][283][284][285][286] as US contrast agents is in constant progress. Even if named nanobubbles they are typically in the range 150 to 1000-nm in diameter.…”

“…Regardless of composition, surface functionalization of the nanomaterial is required to enable targeting and stealth for long circulation times with minimal nonspecific binding [239]. There is a plethora of entities that can be incorporated onto a NP's surface, with covalent bonding preferred over electrostatic interactions: DNA, RNA, [472] oligonucleotides (aptamers), [29,434,[473][474][475][476] peptides, [36,175,201,[477][478][479][480][481][482] proteins, [483][484][485][486][487] enzymes, [488][489][490][491] antibodies [492]. No matter what the surface moiety, its activity must not be altered once anchored to the NP surface ( Figure 13).…”

“…Clinical Imaging modalities generally include complementary techniques [14] such as: optical imaging, [15][16][17][18][19][20][21][22][23] magnetic resonance imaging (MRI), [24][25][26][27] computed tomography (CT), [28] ultrasound imaging (USI) [29][30][31][32][33] positron emission tomography (PET) [34][35][36][37] and single photon emission computed tomography (SPECT) [38][39][40]. Other techniques are also scrutinized, for example multi-photon plasmon resonance microscopy, [41] optical coherence tomography (OCT), [42] surface enhanced Raman spectroscopy (SERS), [15,[43][44][45][46][47] and diffuse optical spectroscopy [48].…”