Photoacoustic imaging of tumor targeting with riboflavin-functionalized theranostic nanocarriers

Abstract

Photoacoustic imaging is an emerging method in the molecular imaging field, providing high spatiotemporal resolution and sufficient imaging depths for many clinical applications. Therefore, the aim of this study was to use photoacoustic imaging as a tool to evaluate a riboflavin (RF)-based targeted nanoplatform. RF is internalized by the cells through a specific pathway, and its derivatives were recently shown as promising tumor-targeting vectors for the drug delivery systems. Here, the RF amphiphile synthesized from a PEGylated phospholipid was successfully inserted into a long-circulating liposome formulation labeled with the clinically approved photoacoustic contrast agent - indocyanine green (ICG). The obtained liposomes had a diameter of 124 nm (polydispersity index =0.17) and had a negative zeta potential of -26 mV. Studies in biological phantoms indicated a stable and concentration-dependent photoacoustic signal (Vevo^® LAZR) of the ICG-containing RF-functionalized liposomes. In A431 cells, a high uptake of RF-functionalized liposomes was found and could be blocked competitively. First, studies in mice revealed ~3 times higher photoacoustic signal in subcutaneous A431 tumor xenografts (P<0.05) after injection of RF-functionalized liposomes compared to control particles. In this context, the application of a spectral unmixing protocol confirmed the initial quantitative data and improved the localization of liposomes in the tumor. In conclusion, the synthesized RF amphiphile leads to efficient liposomal tumor targeting and can be favorably detected by photoacoustic imaging with a perspective of theranostic applications.

Keywords: active targeting; indocyanine green; long-circulating liposomes; photoacoustic imaging; riboflavin.

Figure 1

Scheme of control and targeted ICG liposomes (top left); fluorescence spectra of control and targeted liposomes (top right); and major characteristics of prepared formulations (bottom). Abbreviations: ICG, indocyanine green; PDI, polydispersity index; DPPC, dipalmitoyl phosphocholine; DSPE-PEG, distearoyl phrosphoethanolamine-polyethylene glycol; DSPE-PEG-RF, distearoyl phrosphoethanolamine-polyethylene glycol-riboflavin.

Figure 2

Fluorescence microscopy images of control and targeted liposomes’ uptake in A431 cells (left) and uptake quantification (right). Notes: RhB (red) and cell nuclei marked with DAPI (blue); results are presented as mean from three individual experiments with n=10 images for each condition. *** P<0.001. Abbreviations: RhB, rhodamine B; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

(A) PA spectra of ICG liposomes at various concentrations, colors corresponding to ROIs from (B). (B) Overlay of US (grey) and PA (red) images from ICG liposomes at various concentrations. (C) Depth dependence of IPA signal from ICG liposomes in chicken breast phantom. Abbreviations: PA, photoacoustic; ICG, indocyanine green; ROI, region of interest; US, ultrasound; Avr, average; IPA, PA average intensity.

Figure 4

(A) Examples of two-dimensional tumor slices at different time points after injection of targeted or control liposomes. (B) Normalized PS% signal intensity in three-dimensional tumor volume after liposome injection. Notes: US signal (black and white) overlapped with PA signal (red). Arrows indicate signal accumulation in the tumor. Data are presented as mean ± standard deviation from n=5 mice/group. *P=0.05. Abbreviations: US, ultrasound; PA, photoacoustic; h, hours; min, minutes.

Figure 5

(A) Merge of US (black and white) and PA unmixed images of 2D tumor slices 4 hours after injection of control or targeted liposomes. (B) Quantification of unmixed IPA from 2D tumor slices. Notes: Green – ICG signal from liposomes; red – OxyHemo; and blue – deOxyHemo. Data are presented as mean ± standard deviation from n=2 mice/group. Abbreviations: US, ultrasound; PA, photoacoustic; 2D, two-dimensional; ICG, indocyanine green; IPA, PA average intensity; h, hours; min, minutes.