Nanobody-loaded nanobubbles targeting the G250 antigen with ultrasound/photoacoustic/fluorescence multimodal imaging capabilities for specifically enhanced imaging of RCC.

Clinicians have attempted to discover a noninvasive, easy-to-perform, and accurate method to distinguish benign and malignant renal masses. The targeted nanobubbles (NBs) we constructed that target the specific membrane antigen of renal cell carcinoma (RCC), G250, and contain indocyanine green (ICG) provide multimodal enhanced imaging capability in ultrasound/photoacoustic/fluorescence for RCC which may possibly solve this problem. In this study, we encapsulated ICG in the lipid shell of the NBs by mechanical oscillation, then anti-G250 nanobodies (AGN) were coupled to the surfaces by the biotin-streptavidin bridge method, and the nanobubble named AGN/ICG-NB was completely constructed. The average particle diameter of the prepared AGN/ICG-NBs was (427.2 ± 4.50) nm, and the zeta potential was (-13.33 ± 1.01) mV. Immunofluorescence and flow cytometry confirmed the specific binding capability of AGN/ICG-NBs to G250-positive cells. In vitro imaging experiments confirmed the multimodal imaging capability of AGN/ICG-NBs, and the in vivo imaging experiments demonstrated the specifically enhanced ability of AGN/ICG-NBs for ultrasound/photoacoustic/fluorescence imaging of human-derived RCC tumors. The biosafety of AGN/ICG-NB was verified by CCK-8 assay, organ H&E staining and blood biochemical indices. In conclusion, the targeted nanobubbles we prepared with ultrasound/photoacoustic/fluorescence multimodal imaging capabilities provide a potentially feasible approach to address the need for early diagnosis and differential diagnosis of renal masses.

Indocyanine Green-Loaded Nanobubbles Targeting Carbonic Anhydrase IX for Multimodal Imaging of Renal Cell Carcinoma.

Background and Purpose: The early diagnosis and differential diagnosis of renal cell carcinoma (RCC) has always been a clinical difficulty and a research focus. Carbonic anhydrase IX (CA IX) is highly expressed on the cell membrane of RCC but is not expressed in normal renal tissues. In this study, nanobubbles (NBs) targeting CA IX with ultrasound and photoacoustic multimodal imaging capabilities were prepared to explore a new method for the diagnosis and differential diagnosis of RCC. Methods: Indocyanine green (ICG)-loaded lipid NBs (ICG-NBs) were prepared by using the filming rehydration method, and anti-CA IX polypeptides (ACPs) were attached to their surfaces to prepare CA IX-targeted NBs (ACP/ICG-NBs). The particle size, zeta potential and ICG encapsulation efficiency of these nanobubbles were measured, and their specific targeting and binding abilities to RCC cells were determined. The in vitro and in vivo ultrasound, photoacoustic and fluorescence imaging characteristics of these nanobubbles were also assessed. Results: The particle size of the ACP/ICG-NBs was 475.9 nm in diameter, and their zeta potential was -2.65 mV. Laser confocal microscopy and flow cytometry both confirmed that ACP/ICG-NBs had specific binding activity and ideal affinity to CA IX-positive RCC cells (786-O) but not to CA IX-negative RCC cells (ACHN). The intensities of the in vitro ultrasound, photoacoustic and fluorescence imaging were positively correlated with the concentrations of ACP/ICG-NBs. In in vivo ultrasound and photoacoustic imaging experiments, ACP/ICG-NBs exhibited specific enhanced ultrasound and photoacoustic imaging effects in 786-O xenograft tumors. Conclusion: The ICG- and ACP-loaded targeted nanobubbles that we prepared had the capability of ultrasound, photoacoustic and fluorescence multimodal imaging and could specifically enhance the ultrasound and photoacoustic imaging of RCC xenograft tumors. This outcome has potential clinical application value for the diagnosis of RCC at the early stage and the differential diagnosis of benign and malignant kidney tumors.