Tumor-Targeted Multimodal Optical Imaging with Versatile Cadmium-Free Quantum Dots

Abstract

The rapid development of fluorescence imaging technologies requires concurrent improvements in the performance of fluorescent probes. Quantum dots have been extensively used as an imaging probe in various research areas because of their inherent advantages based on unique optical and electronic properties. However, their clinical translation has been limited by the potential toxicity especially from cadmium. Here, a versatile bioimaging probe is developed by using highly luminescent cadmium-free CuInSe₂/ZnS core/shell quantum dots conjugated with CGKRK (Cys-Gly-Lys-Arg-Lys) tumor-targeting peptides. This probe exhibits excellent photostability, reasonably long circulation time, minimal toxicity, and strong tumor-specific homing property. The most important feature of this probe is that it shows distinctive versatility in tumor-targeted multimodal imaging including near-infrared, time-gated, and two-photon imaging in different tumor models. In a glioblastoma mouse model, the targeted probe clearly denotes tumor boundaries and positively labels a population of diffusely infiltrating tumor cells, suggesting its utility in precise tumor detection during surgery. This work lays a foundation for potential clinical translation of the probe.

Figure 1

Preparation, characterization, and surface modification of QDs. a) Scheme for PEGylation and peptide conjugation of the QDs. b) Absorbance and PL spectra of CISe/ZnS QDs in chloroform (black) and QD—NH₂ in PBS (blue). Emission peaks are at 692 nm (black) and 709 nm (blue), respectively. The insets are images of CISe/ZnS QDs before (1,3) and after phase transfer (2,4), taken under ambient light (1,2) or under 365 nm UV light (3,4). Each vial contains two solvent layers: at the top is PBS and at the bottom is chloroform. c) TEM image of QD—NH₂ in PBS. d,e) Hydrodynamic diameter and Zeta potential of the QD samples. f) Absorbance spectra of QD-PEG (blue), QD-PEG-P-FAM (fluorescein) (red), and free FAM (green) in PBS. FAM-CGKRK was used here instead of unlabeled CGKRK in order to verify the conjugation of the peptide onto the QDs. After extensive washing to remove nonconjugated peptide, significant FAM peak at around 495 nm could be detected, validating the conjugation method.

Figure 2

In vitro cell labeling with QDs. a–c) Time- and concentration-dependent QD binding to MCF10CA1a human breast cancer cells quantified with a,b) a fluorescence plate reader and c) a flow cytometer. d) One-photon confocal images of MCF10CA1a cells treated with QD-PEG (25 × 10⁻⁶ M) or QD-PEG-P (25 × 10⁻⁶ M) for 4 h. F-actin staining outlines the cells. Scale bars: 20 μm.

Figure 3

Circulation time, distribution, and blood chemistry of QDs in vivo. a) Plasma concentrations of QD-PEG and QD-PEG-P at different time points after intravenous injection, n = 9 mice per group. b,c) Quantification of the QD signal per area in each tissue based on the ex vivo imaging results (see Figure S6a,b, Supporting Information) of b) QD-PEG or c) QD-PEG-P treated mice, n = 3 mice per group. H, heart; Li, liver; Sp, spleen; Lu, lung; K, kidney; B, brain. d) Blood chemistry was evaluated 24 h after the injection of PBS (control), QD-PEG or QD-PEG-P, n = 4–6 mice per group. Differences between QD-PEG or QD-PEG-P versus PBS group were not significant with a criterion of P < 0.05 for significance. ALB, albumin (g L⁻¹); ALP, alkaline phosphatase (units L⁻¹); ALT, alanine transaminase (units L⁻¹); AMY, amylase (units L⁻¹); TBIL, total bilirubin (μM); BUN, blood urea nitrogen (mM); CA, calcium (mM); PHOS, phosphorus (mM); CRE, creatinine (μM); GLU, glucose (mM); Na⁺, sodium (mM); K⁺, potassium (mM); TP, total protein (g L⁻¹); GLOB, globulin (g L⁻¹).

Figure 4

Breast tumor targeting with QD-PEG-P. a) Intravital whole body fluorescent imaging (ventral view) of MCF10CA1a breast-tumor-bearing nude mice at 28 h after intravenous injection of PBS, QD-PEG, or QD-PEG-P. Images were taken under 700 nm channel of Li Cor Pearl Impulse small animal imaging system. White arrows show the position of MCF10CA1a tumors (circled in black). b) Ex vivo CW and TG imaging of the tissues harvested from the mice in (a) after the in vivo imaging. H, heart; Li, liver; Sp, spleen; Lu, lung; K, kidney; T, tumor; B, brain. c) Quantitative analysis of indium in the tumors by ICP-OES, n = 3 in each group. **P < 0.01. d) Representative confocal microscopy images of QD distribution in sections from the tumors in panel (b). Scale bars: 50 μm.

Figure 5

Brain tumor targeting with QD-PEG-P. a) Intravital whole body fluorescent imaging (dorsal view) of the brain tumor-bearing mice injected with PBS, QD-PEG, or QD-PEG-P. Tumor mice with shaved head were imaged at 3.5 h postinjection using the 700 nm channel of Li Cor Pearl Impulse small animal imaging system. b) Ex vivo fluorescent imaging of brains excised from the mice imaged in (a). The upper panel image was obtained with Xenogen IVIS200 imaging system, and the lower panel one was with a Li Cor Odyssey CLx infrared imaging system. c) Quantification of the 700 nm channel signals from tumors, n = 3 in each group. *P < 0.05. d–f) Representative confocal microscopy images of QD distribution in sections from the brains in panel (b). The sections were immunostained with either d,e) NG2 antibody (green) or f) endothelial cell marker CD31 antibody (green). e) Magnified images corresponding to the square areas in the QD-PEG-P group in (d). White dashed line in (f) indicates the tumor boundary in brain, where normal brain is above the line. Scale bars: d) 40 μm, e) 5 μm, f) 50 μm.