A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals

Abstract

Stimuli-responsive nanomaterials are increasingly important in a variety of applications such as biosensing, molecular imaging, drug delivery and tissue engineering. For cancer detection, a paramount challenge still exists in the search for methods that can illuminate tumours universally regardless of their genotypes and phenotypes. Here we capitalized on the acidic, angiogenic tumour microenvironment to achieve the detection of tumour tissues in a wide variety of mouse cancer models. This was accomplished using ultra pH-sensitive fluorescent nanoprobes that have tunable, exponential fluorescence activation on encountering subtle, physiologically relevant pH transitions. These nanoprobes were silent in the circulation, and then strongly activated (>300-fold) in response to the neovasculature or to the low extracellular pH in tumours. Thus, we have established non-toxic, fluorescent nanoreporters that can nonlinearly amplify tumour microenvironmental signals, permitting the identification of tumour tissue independently of histological type or driver mutation, and detection of acute treatment responses much more rapidly than conventional imaging approaches.

Figures

Figure 1. Schematic of imaging tumor microenvironment by ultra-pH sensitive (UPS) nanoprobes

The UPS nanoprobes stay ‘OFF’ at pH 7.4 during blood circulation. After reaching tumors, the UPS nanoprobes are turned ON by acidic extracellular pHe (6.5–6.8) in the tumor milieu, or endocytic organelles (pHi, 5.0–6.0) in the tumor endothelial cells after receptor-mediated endocytosis.

Figure 2. Syntheses and characterization of UPS nanoprobes

a, Structural composition of two types of nanoprobes, UPSe and UPSi, with pH transitions at 6.9 and 6.2, respectively. The UPSe is specifically designed to activate in acidic tumor extracellular fluid (pHe = 6.5–6.8). The UPSi can be activated inside acidic endocytic organelles (e.g. pHi = 5.0–6.0). Cy5.5 is used as the NIR fluorophore in most of the animal studies. b, Normalized fluorescence intensity as a function of pH for UPSe and UPSi nanoprobes. At high pH (e.g. 7.4), both probes stay silent. At pH below their transitions (i.e. 6.9 and 6.2), the nanoprobes can be activated as a result of micelle dissociation. The blue dash-line simulates the pH response of a small molecular pH sensor with a pKa of 6.9 based on Henderson-Hasselbach equation. For UPS, the pH response (ΔpH10–90%) is extremely sharp (<0.25 pH unit between ON/OFF states) with >100-fold signal amplification. In contrast, small molecular pH sensors require 3 pH units for comparable signal change. c, Fluorescent images of UPSe-Cy5.5 nanoprobe solution in different pH buffers (λex/λem=675/710 nm). d, Transmission electron micrographs of UPSe nanoprobes at pH 7.4 and 6.7 (polymer concentration = 1 mg/mL, scale bar = 100 nm). e, UPSe nanoprobes remain stable in fresh mouse serum over 24 h at 37 °C.

Figure 3. UPSe nanoprobes can specifically image acidic tumor pHe

a, Aerobic glycolysis converts glucose to lactate in cancer cells. 2-DG and CHC are metabolic inhibitors for glucose uptake and lactic acid secretion, respectively. b, Effect of 2-DG or CHC on the rate of lactic acid secretion in A549 cells. c, Acidification of A549 cell culture medium in the presence of 2-DG or CHC after 6 h incubation. *P < 0.05, **P < 0.01, ***P < 0.001, compared with vehicle group. d, Overlaid fluorescent images of A549 tumor-bearing mice at 24 h postinjection of UPSe nanoprobes (10 mg/kg). In the control groups, 2-DG (250 mg/kg) or CHC (250 mg/kg) was injected 12 h before UPSe nanoprobe administration. Cy5.5 (red) and autofluorescence (green) are separately shown in the composite images. Yellow arrows indicate the tumor location. e, NIR fluorescence intensity ratio between tumor and normal tissues (T/N ratio) as a function of time after UPSe injection. Data are presented as mean ± s.d. (n = 4). f, Organ to blood ratios (see data in Table S5) 24 h post-injection of UPSe (n = 4). A549 tumor has 355-fold of signal amplification over blood by UPSe. **P < 0.01, compared with other organs. g, Hypoxia bands qualitatively correlate with activation pattern of UPSe in A549 tumor xenograft. Whole mount images of tumor slices stained for hypoxia (green). All images were obtained from the adjacent sections at ×200 magnification. Scale bar is 2 mm.

Figure 4. cRGD-UPSi nanoprobes can specifically image angiogenic tumor vasculature

a, Design of cRGDUPSi nanoprobe. b, Schematic of internalization and activation of cRGD-UPSi nanoprobes after αvβ3-mediated endocytosis in tumor endothelial cells. The nanoprobes are accumulated in the endosomes or lysosomes, where the acidic pH activates the nanoprobes. c, Superimposed fluorescent images of A549 tumor-bearing mice at 6 h post-injection of cRGD-UPSi or UPSi nanoprobe (10 mg/kg). In the competition group, a blocking dose of cRGD peptide (25 mg/kg) was injected 30 min before cRGD-UPSi administration. Cy5.5 (Red) and autofluorescence (Green) are separately shown in the composite images. d, T/N ratio after injection of nanoprobes as a function of time. Data are presented as mean ± s.d. (n = 4). e, Organ to blood ratios (see data in Table S7) 6 h post-injection of cRGD-UPSi nanoprobe (n = 4). A549 tumor has 628-fold of signal amplification over blood by cRGD-UPSi. **P < 0.01, compared with other organs. f, Representative images of ex vivo tumors, muscles, and blood at 6 h post-injection of nanoprobes. g, Plasma concentration versus time curves (n = 4) for and UPSi nanoprobes. h, Biodistribution profiles (n = 4) of cRGD-UPSi and UPSi nanoprobes 6 h after intravenous injection. i, Correlation of nanoprobe activation with tumor vasculature (anti-CD31). The co-localization between nanoprobe and tumor vasculature is indicated by the yellow color in the merged images (green: blood vessels; red: nanoprobes; blue: nuclei. Scale bar = 100 µm).

Figure 5. iUPS nanoprobes target both acidic pHe and tumor vasculature with broad tumor specificity

a, Intravital fluorescent images show complimentary pattern of spatial activation of cRGD-UPSi-RhoG (green) and UPSe-TMR (red) inside tumor vasculature and parenchyma, respectively. The dual nanoprobes were co-injected intravenously and the images were taken 6 h post-injection. Scale bar = 100 µm. b–c, iUPS nanoprobes show broad tumor imaging specificity and efficacy in 10 different tumor models of different cancer types (breast, prostate, head and neck, lung, brain, and pancreatic cancers) and organ sites. In 3LL lung cancer model (c), explanted lung was shown to illustrate the effective detection of small metastatic nodules (<1 mm). Scale bar = 2 mm. In each model, high T/N ratios were observed demonstrating the success of targeting tumor microenvironment signals as a universal strategy to achieve broad tumor specificity (see Fig. S20a–j).