Research ArticleHEALTH AND MEDICINE

A self-illuminating nanoparticle for inflammation imaging and cancer therapy

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Science Advances  09 Jan 2019:
Vol. 5, no. 1, eaat2953
DOI: 10.1126/sciadv.aat2953
  • Fig. 1 Engineering of a self-illuminating nanoparticle with luminescence, fluorescence, and intrinsic singlet oxygen generation capabilities.

    (A) Chemical structure and schematic of a designed amphiphile of a CLP conjugate. (B) Sketch of self-assembly of the CLP conjugate into a core-shell structured nanoparticle. Through BRET between the luminol and Ce6 units, luminescence originating from the energy donor luminol upon triggering by inflammatory mediators of ROS and MPO may excite the acceptor Ce6 to produce fluorescence and singlet oxygen (1O2). (C and D) UV-visible (C) and fluorescence (D) spectroscopic characterization of CLP. (E) Transmission electron microscopy (TEM) image (left) and size distribution profile in deionized water (right) of assembled CLP nanoparticles.

  • Fig. 2 In vitro characterization of BRET and luminescent properties of the CLP conjugate.

    (A) Luminescent spectra of a CLP conjugate, a Ce6-PEG conjugate, and luminol in the presence of 100 mM H2O2 and 100 mM ClO. (B) The luminescence spectrum of luminol in PBS and the absorption spectrum of Ce6 in DMSO. (C) Time-dependent luminescence of CLP (0.5 mg/ml) upon incubation with various concentrations of H2O2. (D and E) Luminescence at 1.0 mg/ml CLP (D) and time-resolved luminescent signals of CLP at 0.5 mg/ml (E) in the presence of 50 μM H2O2 with or without 15 mU MPO and 50 mM Cl. (F and G) Attenuation of the luminescence at 0.5 mg/ml CLP and in the presence of 50 μM H2O2 and 15 mU MPO by a ROS scavenger, Tempol (F), and an MPO inhibitor, 4-aminobenzoic hydrazide (4-ABAH) (G). (H) In vitro tissue penetration capacity of luminol and CLP. After a black 96-well plate containing CLP or luminol at the same dose of 0.5 mg/ml was covered with 3 mm of porcine muscle tissue, the penetrating luminescent signals were determined. (I) Time-resolved luminescence imaging of neutrophils. Immediately after 5.4 μM CLP (i.e., 25 μg/ml) was incubated with neutrophils (5 × 105 cells per well) with (PMA+) or without (PMA) stimulation with phorbol 12-myristate 13-acetate (PMA; 100 ng/ml ) for 1 hour, images were acquired at predetermined time points. (J) Intracellular and extracellular luminescent signals. The Blank group indicates neutrophils stimulated with PMA and without treatment with CLP nanoparticles. After PMA-stimulated neutrophils were incubated with 5.4 μM CLP for 0.5 hours, luminescence images were acquired and indicated as the Cells + medium group. Immediately after this imaging, cells and the culture medium containing PMA were separated, and images were separately collected for the Cells and Medium groups. For all luminescence imaging results (D, F, G, I, and J) measured with an IVIS Spectrum imaging system [exposure time = 5 min, focal length/stop (f/stop) = 1, binning = 8, field of view (FOV) = 12.8 cm], the left panels are luminescent images, while the right panels show quantitative data. Data in (D) and (F) to (J) are means ± SEM. (n = 3). One-way analysis of variance (ANOVA) was used for statistical analysis. **P < 0.01, ***P < 0.001; ns, no significance.

  • Fig. 3 In vivo imaging in mouse models of inflammation.

    (A) Luminescent imaging of mice with peritonitis immediately after intraperitoneal administration of different formulations. To induce peritonitis, a saline solution containing 1 mg of zymosan was intraperitoneally injected into each mouse at 6 hours before imaging. Mice in the saline group were treated with saline, while those in the CLP and luminol groups were each administered with 5 mg of CLP nanoparticles or free luminol at the same dose of the luminol unit, respectively. (B) Luminescence imaging of peritonitis in mice at 0.5 hours after intravenous injection of different probes. In the PeritonitisCLP+ group, healthy mice were treated with CLP nanoparticles. Peritonitis mice in the Peritonitis+Luminol+ and Peritonitis+CLP+ groups were administered with free luminol and CLP nanoparticles (5 mg in each mouse) at the same dose of the luminol unit, respectively. (C) Imaging of acute liver injury in mice at 24 hours after challenging via intraperitoneal injection of acetaminophen at 300 mg/kg. Mice in the saline group were intravenously injected with saline, while mice in the luminol and CLP groups were separately intravenously administered with free luminol and CLP nanoparticles (5 mg in each mouse) at the same dose of the luminol unit. After 0.5 hours, imaging was conducted. (D) In vivo luminescence imaging of mice with colitis induced by drinking water containing 3% (w/v) dextran sulfate sodium (DSS) for 7 days. (E) Ex vivo luminescent images of colonic tissues isolated immediately after in vivo imaging. In both cases, the control group represents healthy mice treated with CLP nanoparticles, while the model group denotes diseased mice administered with saline. In the luminol and CLP groups, mice with colitis were treated with free luminol and CLP nanoparticles (5 mg in each mouse) at the same dose of the luminol unit per mouse, respectively. At 15 min after different treatments via enema, luminescent images were acquired. Then, the mice were euthanized, and their colonic tissues were excised for ex vivo imaging. (F) Time-lapse in vivo luminescence imaging of mice with DSS-induced colitis after local administration of 5 mg of CLP nanoparticles or free luminol at the same dose of the luminol unit in each animal. In all images, the left panels show representative luminescent images, while the right panels illustrate quantified intensities. Data are means ± SEM (A to C and F, n = 4; D and E, n = 5). One-way ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 4 In vivo luminescence imaging of the development of colitis in mice.

    (A and B) Luminescent images (A) and quantitative analysis (B) of mice with colitis induced by drinking water containing 3% (w/v) DSS for different periods of time. For each time point, images were acquired at 15 min after enema administration of 5 mg of CLP nanoparticles in each mouse. (C to E) Changes in DAI (C) and the levels of MPO (D) and H2O2 (E) during colitis development. DAI is defined as the summation of the stool consistency index, fecal bleeding index, and weight loss index. Colonic tissues were excised from mice subjected to drinking water containing DSS at specific time points to quantify MPO and H2O2 levels. (F and G) Representative flow cytometric profiles (F) and quantitative data (G) of neutrophil counts. At different time points after mice were treated with drinking water containing DSS, they were euthanized, and cells in colonic tissues were isolated for analysis. (H) Correlation analyses of luminescent intensities with DAI, MPO, and H2O2 levels and neutrophil count. All data were fitted with a sigmoid growth function. (I and J) Microscopic images (I) and HAI scores (J) of H&E-stained sections of colonic tissues isolated from mice stimulated with DSS for different periods of time. Scale bars, 200 μm. Data in (B) to (E), (G), and (J) are means ± SEM (n = 4). One-way ANOVA was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001.

  • Fig. 5 In vitro and in vivo antitumor activity of CLP nanoparticles.

    (A and B) The effects of CLP dose (A) and H2O2 concentration (B) on the generation of 1O2, as quantified using DPA as a probe. In both cases, the images show the reduction in DPA absorbance at 355 nm using that at 0 hours as the baseline. For the dose effect, quantification was performed after CLP nanoparticles were incubated with 100 mM H2O2 for 6 hours. To examine the influence of the H2O2 concentration, absorbance was measured after 6 hours of incubation with 20 μM CLP nanoparticles. (C) Confocal microscopy images illustrating time-dependent endocytosis of CLP nanoparticles in A549 lung carcinoma cells. Images in the lower panel display quantitative analysis of fluorescent intensities corresponding to indicated yellow lines in single cells of the upper fluorescence images. After A549 cells were cultured with 10.8 μM CLP nanoparticles for predetermined time periods, nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI), while endolysosomes were labeled with Lyso. Scale bars, 20 μm. (D) The half-maximal inhibitory concentration (IC50) values of different cells [including RAW264.7 murine macrophages, B16F10 mouse melanoma cells, MOVAS (mouse vascular aortic smooth muscle cell), MCF-7 human breast cancer cells, and A549 cells] after incubation with various doses of CLP nanoparticles for 24 hours. (E) Quantification of intracellular ROS levels in different cells by flow cytometry. After cells were incubated with 10 μM DCF-DA for 20 min and washed with PBS, measurements were performed. The ROS level is proportional to the mean fluorescence intensity (MFI) of a fluorescent probe DCF-DA. (F) Correlation between IC50 values and ROS levels. (G) Changes in tumor volume during treatment by intratumoral administration of saline or CLP nanoparticles at 3.25 mg/kg of Ce6 every 3 days in nude mice bearing A549 xenografts. (H and I) Digital photos (H) and quantified tumor weight (I) of excised tumors at day 30 after different treatments. (J) DAPI and TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) double-labeling assay of cell apoptosis in paraffin sections. (K) Immunofluorescence analysis of proliferating cell nuclear antigen (PCNA) in tumor sections. (L) The ROS level in tumor tissues after treatment via intratumoral injection of saline or CLP nanoparticles. (M) Ex vivo fluorescence images (left) and quantitative data (right) of the distribution of CLP nanoparticles in A549 xenografts. At day 7 after the last intravenous injection of CLP nanoparticles at 5 or 25 mg/kg of Ce6, tumors were excised for imaging. (N) Tumor volume changes during treatment with intravenously administered saline or CLP nanoparticles at 5 or 25 mg/kg of Ce6. The control group was treated with saline. (O) DAPI/TUNEL double-labeling assay of apoptotic cells in tumor sections after 30 days of treatment with saline or CLP nanoparticles. Scale bars, 20 μm. Data are means ± SEM (A, B, D, E, and L to N, n = 4; G and I, n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. (Photo credit: Huijie An, Third Military Medical University).

  • Fig. 6 In vitro mechanistic studies of the antitumor activity of CLP nanoparticles in A549 cells.

    (A) Confocal microscopy images showing the mitochondrial localization of CLP nanoparticles. Images in the lower panel display quantitative analysis of the fluorescent intensities in the single cells as highlighted by the yellow lines in the upper fluorescence images. After cells were incubated with 10.8 μM CLP nanoparticles for different periods of time, mitochondria were stained with MitoTracker Green FM (MitoTracker). Scale bar, 20 μm. (B and C) The effects of the CLP nanoparticle dose (B) and incubation time (C) on mitochondrial membrane potential as analyzed by flow cytometry using a fluorescent probe tetramethylrhodamine ethyl ester (TMRE). For the dose-response experiments, cells were incubated with different doses of CLP nanoparticles for 12 hours, while they were cultured with 64.8 μM CLP to examine the time effect. FCCP (carbonyl cyanide p-trifluoromethoxy phenylhydrazone) was used as a positive control that can effectively depolarize the mitochondrial membrane potential. (D and E) Representative Western blot bands (D) and quantitative analysis (E) of total and cleaved caspase-3 in A549 cells after treatment with different doses of CLP nanoparticles for 24 hours. (F) Correlation between the percentage of apoptotic cells and the level of cleaved caspase-3. (G and H) Western blot analysis of total and cleaved caspase-8 (G) and caspase-9 (H) in A549 cells after 24 hours of incubation with CLP nanoparticles. (I) Schematic illustration of the antitumor mechanisms of CLP nanoparticles. Data are means ± SEM (n = 3). *P < 0.05, ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/1/eaat2953/DC1

    Supplementary Materials and Methods

    Fig. S1. Synthesis and characterization of a CLP conjugate.

    Fig. S2. Characterization of the CLP conjugate and its nanoparticles.

    Fig. S3. Luminescence properties of the CLP conjugate under different conditions.

    Fig. S4. Intracellular uptake of CLP nanoparticles and in vitro luminescence imaging in neutrophils.

    Fig. S5. In vivo imaging in different mouse models of inflammation.

    Fig. S6. The retention of CLP and luminol in the colon after enema administration in colitis mice.

    Fig. S7. In vitro antitumor activity of CLP nanoparticles.

    Fig. S8. Tumor therapy after intratumoral administration of CLP nanoparticles in mice bearing A549 xenografts.

    Fig. S9. In vivo pharmacokinetic, tissue distribution, and elimination profiles of CLP after intravenous administration.

    Fig. S10. Tissue distribution of CLP nanoparticles after intravenous administration in mice bearing A549 xenografts.

    Fig. S11. Tumor therapy after intravenous administration of CLP nanoparticles in mice bearing A549 xenografts.

    Fig. S12. Comparison of in vivo efficacy and safety of CLP nanoparticles with CDDP.

    Fig. S13. Safety study after intratumoral administration of CLP nanoparticles in A549 xenograft–bearing mice.

    Fig. S14. Safety profiles of CLP nanoparticles in A549 xenograft–bearing mice during and after 30 days of intravenous injection.

    Fig. S15. Acute toxicity evaluation of CLP nanoparticles after a single intravenous injection in healthy mice.

    Fig. S16. Mechanistic studies of in vitro antitumor activity of CLP nanoparticles in A549 cells.

  • Supplementary Materials

    This PDF file includes:

    • Supplementary Materials and Methods
    • Fig. S1. Synthesis and characterization of a CLP conjugate.
    • Fig. S2. Characterization of the CLP conjugate and its nanoparticles.
    • Fig. S3. Luminescence properties of the CLP conjugate under different conditions.
    • Fig. S4. Intracellular uptake of CLP nanoparticles and in vitro luminescence imaging in neutrophils.
    • Fig. S5. In vivo imaging in different mouse models of inflammation.
    • Fig. S6. The retention of CLP and luminol in the colon after enema administration in colitis mice.
    • Fig. S7. In vitro antitumor activity of CLP nanoparticles.
    • Fig. S8. Tumor therapy after intratumoral administration of CLP nanoparticles in mice bearing A549 xenografts.
    • Fig. S9. In vivo pharmacokinetic, tissue distribution, and elimination profiles of CLP after intravenous administration.
    • Fig. S10. Tissue distribution of CLP nanoparticles after intravenous administration in mice bearing A549 xenografts.
    • Fig. S11. Tumor therapy after intravenous administration of CLP nanoparticles in mice bearing A549 xenografts.
    • Fig. S12. Comparison of in vivo efficacy and safety of CLP nanoparticles with CDDP.
    • Fig. S13. Safety study after intratumoral administration of CLP nanoparticles in A549 xenograft–bearing mice.
    • Fig. S14. Safety profiles of CLP nanoparticles in A549 xenograft–bearing mice during and after 30 days of intravenous injection.
    • Fig. S15. Acute toxicity evaluation of CLP nanoparticles after a single intravenous injection in healthy mice.
    • Fig. S16. Mechanistic studies of in vitro antitumor activity of CLP nanoparticles in A549 cells.

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