Early-stage imaging of nanocarrier-enhanced chemotherapy response in living subjects by scalable photoacoustic microscopy

Abstract

Conventional evaluation methods of chemotherapeutic efficacy such as tissue biopsy and anatomical measurement are either invasive with potential complications or dilatory to capture the rapid pathological changes. Here, a sensitive and resolution-scalable photoacoustic microscopy (PAM) with theranostic nanoformulation was developed to noninvasively monitor the therapy response in a timely manner. Ultrasmall graphene oxide nanosheets were designed as both drug-loading vehicle and photoacoustic signal amplifier to the tumor. With the signal enhancement by the injected contrast agents, the subtle microvascular changes of the chemotherapy response in tumor were advantagely revealed by our PAM system, which was much earlier than the morphological measurement by standard imaging techniques. High tumor uptake of the enhanced nanodrug with Cy5.5 labeling was validated by fluorescence imaging. At different observation scales, PAM offered unprecedented sensitivity of optical absorption and high spatial resolution over optical imaging. Our studies demonstrate the PAM system with synergistic theranostic strategy to be a multiplexing platform for tumor diagnosis, drug delivery, and chemotherapy response monitoring at a very early stage and in an effective way.

Keywords: chemotherapy response; early prediction; graphene oxide; nanocarrier; photoacoustic microscopy; scalable imaging; signal amplification.

Figure 1

(a) Scheme of the PAM system used for in vivo animal imaging. (b) MIP image of three pieces of horse hair obtained by the PAM system. Scale bar = 1 mm. (c) Image profile along the red dashed line from part b.

Figure 2

(a) UV–vis–NIR of GO, GO-Cy5.5, Dox, and GO-Cy5.5-Dox, respectively. (b) Fluorescence spectra of GO-Cy5.5-Dox excited at 480 nm (black) and 680 nm (red), respectively. (c) MIP image of tumor supply vessels before injection of GO-Cy5.5-Dox. (d) MIP image of tumor supply vessels 6 h after injection of GO-Cy5.5-Dox. (e) MIP image of tumor supply vessels before injection of PBS. (f) MIP image of tumor supply vessels 6 h after injection of PBS. Scale bar = 1 mm.

Figure 3

(a) Blood circulation of GO-Cy5.5-Dox in mice by tail vein injection. Blood samples of 5 μL were collected at indicated time points into Eppendorf tubes for fluorescence imaging. (b) Fluorescence signal curve quantified from part a (n = 4/group). (c) In vitro drug release measurement of GO-Cy5.5-Dox in various pH conditions.

Figure 4

(a) Fluorescence imaging of GO-Cy5.5-DOX distribution in vivo in H1975-tumor-bearing mice. (b) Fluorescence imaging of major organs and tumor collected 48 h after drug injection. (c) Fluorescence signal of liver and tumor in mice at different time points by measuring the Cy5.5 fluorescence signal (n = 5/group).

Figure 5

(a) Representative MIP images of a region-of-interest in tumor receiving GO treatment, Dox treatment, and GO-Dox treatment, respectively, at different time points. Scale bar = 1 mm. (b) Quantitative PA signals of blood vessels inhibited by the treatments (n = 6/group). *p < 0.01. (c) CD31 immunohistochemical histology of the tumor section 6 days after treatment with Dox and GO-Dox chemotherapy. Scale bar = 20 μm.

Figure 6

(a) Photoacoustic imaging of the tumor receiving chemotherapy by the conventional PA tomography system (VisualSonics). Scale bar = 1 mm. (b) Photoacoustic imaging of the tumor receiving chemotherapy by the PAM system. Scale bar = 1 mm. (c) PA signal change on the tumor measured by the conventional PA system and home-built PAM system (n = 6/group). *p < 0.01. (d) PA signal, total length, and junction number of the vasculature from PAM images (n = 6/group).

Figure 7

(a) Relative tumor volume of untreated and Dox- and GO-Dox-treated mice. (b) Body weight curves of the mice in the untreated and Dox- and GO-Dox-treated groups. (c) HE staining (10-fold magnification) of the major organs 1 week after PBS or GO-Dox (Dox concentration at 8 mg/kg) injection.