A multifunctional targeting probe with dual-mode imaging and photothermal therapy used in vivo

Abstract

Background: Ag₂S has the characteristics of conventional quantum dot such as broad excitation spectrum, narrow emission spectrum, long fluorescence lifetime, strong anti-bleaching ability, and other optical properties. Moreover, since its fluorescence emission is located in the NIR-II region, has stronger penetrating ability for tissue. Ag₂S quantum dot has strong absorption during the visible and NIR regions, it has good photothermal and photoacoustic response under certain wavelength excitation.

Results: 200 nm aqueous probe Ag₂S@DSPE-PEG₂₀₀₀-FA (Ag₂S@DP-FA) with good dispersibility and stability was prepared by coating hydrophobic Ag₂S with the mixture of folic acid (FA) modified DSPE-PEG₂₀₀₀ (DP) and other polymers, it was found the probe had good fluorescent, photoacoustic and photothermal responses, and a low cell cytotoxicity at 50 μg/mL Ag concentration. Blood biochemical analysis, liver enzyme and tissue histopathological test showed that no significant influence was observed on blood and organs within 15 days after injection of the probe. In vivo and in vitro fluorescence and photoacoustic imaging of the probe further demonstrated that the Ag₂S@DP-FA probe had good active targeting ability for tumor. In vivo and in vitro photothermal therapy experiments confirmed that the probe also had good ability of killing tumor by photothermal.

Conclusions: Ag₂S@DP-FA was a safe, integrated diagnosis and treatment probe with multi-mode imaging, photothermal therapy and active targeting ability, which had a great application prospect in the early diagnosis and treatment of tumor.

Keywords: Ag2S; Cancer active targeting; Fluorescence imaging; Photoacoustic imaging; Photothermal therapy.

Fig. 1

White light (a) and fluorescence (b) image, TEM (c), absorption and emission spectra (d), EDS (e) of Ag₂S; the infrared spectrum of Ag₂S and DT (f)

Fig. 2

Absorption spectra of FA, DP-FA and DP-NH₂ (a), insert: Zeta potentials of DP-NH₂, DP-FA, Ag₂S@DP-FA and Ag₂S@DP (n = 5); dynamic light scattering results (b) (n = 5) and TEM (c) of Ag₂S@DP-FA; the change of particle size (d) and Zeta potential (e) of Ag₂S@DP-FA at different temperatures (n = 5); the PA response of the different concentrations of the probe (f) (n = 5), insert: PAI of probe

Fig. 3

The picture of temperature evolution of 2.5 mg/mL Ag₂S@DP-FA in different solvent under 1.8 W/cm² laser over time (a); 1.0 mg/mL Ag₂S@DP-FA in water under different laser power densities (b); different concentration Ag₂S@DP-FA in water under 1.8 W/cm² laser (c); d–f were the temperature evolution curves over time corresponding to (a, b, c), respectively (n = 5)

Fig. 4

MTT test of Ag₂S@DP-FA (a) (n = 5); white light results (b) and cell community data statistics (c) (n = 5) of colony formation assay method to detect the long-term toxicity of the probe

Fig. 5

White light and FI results of different cells incubated with different probes (a) and photoacoustic response results of different probe-labeled positive HeLa cells (b) (n = 5), *meant significant difference (p < 0.05), **meant great significant difference (p < 0.01)

Fig. 6

HeLa and A549 were incubated with positive probe (Ag₂S@DP-FA) or negative probe (Ag₂S@DP), and then treated with or without NIR laser irradiation. Cells were stained by calcein-AM. Laser power was 1.8 W/cm², and irradiation time was 5 min. White dotted line was the border of laser irradiation

Fig. 7

TEM of cells incubated with probes. a HeLa + PBS; b HeLa + Ag₂S@DP-FA; c HeLa + Ag₂S@DP; d A549 + PBS; e A549 + Ag₂S@DP-FA; f A549 + Ag₂S@DP. Inserts in b, c, e and f were the corresponding local magnification

Fig. 8

Blood sample analysis after injection (n = 5). a WBC; b RBC; c HGB; d PLT; e ALT; f AST

Fig. 9

The HE staining results of the main organ after injecting PBS and probe for 6 h, 1, 3, 7, 15 days shown in from top to bottom, from the left to right was heart, liver, spleen, lung, kidney, and intestine

Fig. 10

FI of the target tumor in the nude mice at 0 h, 5 min, 1, 2, 4, 8, 12, 24, 36 and 48 h (the tumor site in the dashed line cycle). a HeLa tumor-bearing nude mice + Ag₂S@DP-FA; b HeLa tumor-bearing nude mice + Ag₂S@DP; c A549 tumor-bearing nude mice + Ag₂S@DP-FA; d A549 tumor-bearing nude mice + Ag₂S@DP

Fig. 11

White and fluorescence imaging of heart (a), liver (b), spleen (c), lung (d), kidney (e), small intestine (f), and tumor (g) in HeLa tumor-bearing nude mice at 0 h, 5 min, 1, 2, 4, 8, 12, 24, 36 and 48 h after injection of Ag₂S@DP-FA

Fig. 12

In vivo PAI of the nude mouse tail intravenously injected with Ag₂S@DP-FA (a); the whole tumor after injecting 11 h under 744 nm laser excitation (b); the same part of tumor at different times under 744 nm laser excitation (c–j); the same part of tumor under 523 nm laser excitation (k)

Fig. 13

Infrared thermal images of HeLa tumor-bearing nude mouse tail intravenously injected with Ag₂S@DP-FA (a) or saline (b) on different times under 808 nm laser irradiation; the temperature of tumor evolution curves over time (c) (n = 5), **meant great significant difference (p < 0.01); representative photos of tumor-bearing nude mouse after treatments over time (d); H&E stain of tumor tissues from different treatment groups (e–h)

Fig. 14

Body weight curves (a), tumor growth curves (b) and survival curve (c) after different treatments (n = 5), **meant great significant difference (p < 0.01); H&E stained images of major organs from healthy nude mice, 1 and 40 days after Ag₂S@DP-FA and laser irradiation treatment mice, respectively (d)