Non-invasive multimodal functional imaging of the intestine with frozen micellar naphthalocyanines

Journal name:
Nature Nanotechnology
Volume:
9,
Pages:
631–638
Year published:
DOI:
doi:10.1038/nnano.2014.130
Received
Accepted
Published online

Abstract

There is a need for safer and improved methods for non-invasive imaging of the gastrointestinal tract. Modalities based on X-ray radiation, magnetic resonance and ultrasound suffer from limitations with respect to safety, accessibility or lack of adequate contrast. Functional intestinal imaging of dynamic gut processes has not been practical using existing approaches. Here, we report the development of a family of nanoparticles that can withstand the harsh conditions of the stomach and intestine, avoid systemic absorption, and provide good optical contrast for photoacoustic imaging. The hydrophobicity of naphthalocyanine dyes was exploited to generate purified ∼20 nm frozen micelles, which we call nanonaps, with tunable and large near-infrared absorption values (>1,000). Unlike conventional chromophores, nanonaps exhibit non-shifting spectra at ultrahigh optical densities and, following oral administration in mice, passed safely through the gastrointestinal tract. Non-invasive, non-ionizing photoacoustic techniques were used to visualize nanonap intestinal distribution with low background and remarkable resolution, and enabled real-time intestinal functional imaging with ultrasound co-registration. Positron emission tomography following seamless nanonap radiolabelling allowed complementary whole-body imaging.

At a glance

Figures

  1. Spontaneous formation of non-exchangeable F127–naphthalocyanine frozen micelles.
    Figure 1: Spontaneous formation of non-exchangeable F127–naphthalocyanine frozen micelles.

    a, Retention of dyes of varying hydrophobicity added to an aqueous solution of 10% (wt/vol.) F127 and then dialysed against 20 mM cholate for 24 h. MB, methylene blue; QR, quinaldine red; 6G, rhodamine 6G; IR780, IR780 iodide. b, Chemical structure of napthalocyanines used. BNc: M = 2H; R1 = t-Bu; R2,R3 = H. VBNc: M = VO; R1 = t-Bu; R2,R3 = H. ZnBNc: M = Zn; R1 = t-Bu; R2,R3 = H. ONc: M = 2H; R1 = H; R2,R3 = O-(CH2)3CH3. Phthalocyanines contain single outer benzenes. BPc: M = 2H; R1 = t-Bu; R2,R3 = H. VBPc: M = VO; R1 = t-Bu; R2 = N(CH3)2; R3 = H.

  2. Temperature-mediated CMC switching to generate surfactant-free nanonaps.
    Figure 2: Temperature-mediated CMC switching to generate surfactant-free nanonaps.

    a, Generation of purified nanonaps. F127 PEO blocks are in blue, PPO blocks in black and Nc dyes in red. b, F127 retention as a function of centrifugal filtration washes at 4 °C (black) and 25 °C (red) (mean ± s.d. for n = 3). c, F127-solubilized dye retention as a function of centrifugal filtration washes at 4 °C for Nc (black) and MB (red) (mean ± s.d. for n = 3). d, Nanonap size distribution by dynamic light scattering in water. e, Negative-stained transmission electron micrograph of dried nanonaps. Scale bar, 50 nm. f, Equivalent absorbance from concentrated, reconstituted nanonaps (black) or liposomes (red; 1:19 molar ratio of Nc:lipid) following freeze-drying of nanoparticles formed with 2 mg ONc. Inset: Magnified liposomal absorbance.

  3. Multispectral nanonaps without peak wavelength shifting at ultrahigh optical densities.
    Figure 3: Multispectral nanonaps without peak wavelength shifting at ultrahigh optical densities.

    a, Normalized absorbance of nanonaps formed from BPc (blue), ZnBNc (dark green), BNc (light green) or ONc (bronze). b, Photograph of nanonaps in water. From left to right: BPc, ZnBNc, BNc and ONc. c, Absorption peak wavelength shift at high optical densities. Concentrated solutions were measured in a cuvette with a path length of 10 µm and compared with a 1,000-fold dilution in water. Indicated nanonaps are compared with indocyanine green (ICG) and MB. Mean ± s.d. for n = 3.

  4. Nanonaps pass safely through the intestine following oral administration.
    Figure 4: Nanonaps pass safely through the intestine following oral administration.

    a, Retention of ONc nanonaps dialysed in simulated gastric fluid (red) or simulated intestinal fluid (black) at 37 °C (mean ± s.d. for n = 3). b, Excretion of 100 ODs of ONc nanonaps in faeces (black) and urine (red) (mean ± s.d. for n = 3 mice). c, Excretion of 100 ODs of MB in faeces (black) and urine (red) (mean ± s.d. for n = 3 mice). d, Haematoxylin and eosin-stained intestine section of a control mouse (left) or a mouse 24 h after gavage of 100 ODs of ONc nanonaps (right). Villi and crypts were intact without influx of inflammatory cells. Scale bars, 100 µm.

  5. Non-invasive anatomical and functional PA imaging of the intestine using nanonaps.
    Figure 5: Non-invasive anatomical and functional PA imaging of the intestine using nanonaps.

    a, PA MIP of nanonaps following gavage of 100 ODs of ZnBNc nanonaps using a single-transducer PA system. Red arrows show nanonap transit. b, Depth-encoded PA MIP of the intestine visualizing ZnBNc nanonaps. c, Real-time multimodal mouse intestinal transverse plane with PA signal (colour) and simultaneous US (grey) acquisition following gavage of 100 ODs of ONc nanonaps. d, Nanonap movement in the intestine. Black arrow shows inflow and white arrow shows outflow. e, Intestinal region-of-interest analysis. First-derivative zero crossings provide the time of maximal nanonap inflow (black triangles) and outflow (grey triangles) points. f, Rate of contractile motion from the region, plotted over time. g, Co-registered US for anatomical mapping of nanonaps. The bladder (B) and kidneys (K) are located with US (grey), and the nanonap PA signal is shown in colour. h, US (grey)/PA(colour) MIPs of transverse slices, showing ONc nanonap intestinal transit over time. The MIP was used to orient the PA signal within a single slice of interest (lower left). Outflow quantification over time of nanonaps in area A (red) shown with reference to two others that maintained steady nanonap content in areas B (blue) and C (grey). The fluctuations in A are due to contractile inflow and outflow of nanonaps. i, US/PA detection of intestinal obstruction. Mice were subjected to duodenal ligations or sham surgery. 100 ODs of ONc nanonaps were administered and mice were imaged 1 h later. The top images show a transverse slice 2.4 cm above the bladder, showing the swollen stomach in the obstructed mice. The bottom images show US/PA MIPs. An unobstructed flow of nanonaps is clear in the sham group. The dashed line indicates the approximate surgical incision site and the image width corresponds to 2.4 cm. Representative images for n = 3 per group. All scale bars, 5 mm.

  6. Seamless nanonap labelling with 64Cu for whole-body PET imaging of the GI tract.
    Figure 6: Seamless nanonap labelling with 64Cu for whole-body PET imaging of the GI tract.

    a, Nanonap labelling using 64Cu. F127 PEO blocks are shown in blue, PPO blocks in black, Nc dyes in red and 64Cu is shown as the radioactive yellow circle. b, Retention stability of 64Cu chelation in radiolabelled nanonaps in simulated gastric fluid (red), simulated intestinal fluid (blue) and water (black) incubated at 37 °C (mean ± s.d. for n = 3). c, Faecal clearance of ONc nanonaps and chelated 64Cu in mice 24 h after gavage of 100 ODs of ONc nanonaps. 64Cu was assessed using gamma counting and nanonaps using absorption. (Mean ± s.d. for n = 3–4 mice.) d, Biodistribution of 64Cu and nanonaps 24 h after gavage. No data (ND) were obtained for some samples because they were not measured. (Mean ± s.d. for n = 3–4 mice.) e, Representative PET imaging of nanonaps. 100 ODs of 64Cu-labelled ONc nanonaps were gavaged and mice were imaged at the indicated time points. Scale bars, 1 cm. f, Representative 0.8-mm-thick coronal slices through the mouse, 3 h after gavage.

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Author information

Affiliations

  1. Department of Biomedical Engineering, University at Buffalo, State University of New York, Buffalo, New York 14260, USA

    • Yumiao Zhang,
    • Mansik Jeon,
    • Jumin Geng,
    • Chulhong Kim &
    • Jonathan F. Lovell
  2. Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, New York 14260, USA

    • Yumiao Zhang,
    • Paschalis Alexandridis &
    • Jonathan F. Lovell
  3. Department of Creative IT Engineering, POSTECH, Pohang, Korea

    • Mansik Jeon &
    • Chulhong Kim
  4. Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA

    • Laurie J. Rich &
    • Mukund Seshadri
  5. Department of Radiology and Department of Medical Physics, University of Wisconsin, Madison, Wisconsin 53705, USA

    • Hao Hong,
    • Yin Zhang,
    • Sixiang Shi,
    • Todd E. Barnhart &
    • Weibo Cai
  6. Farncombe Family Digestive Health Research Institute, Department of Medicine, McMaster University, Hamilton, Ontario L8N 3Z5, Canada

    • Jan D. Huizinga

Contributions

Yu.Z. and J.F.L. conceived the project. Yu.Z, M.J., L.J.R., H.H. and J.G. were responsible for most data collection. Yu.Z., P.A. and J.F.L. planned experiments and interpreted the data related to nanonap formulation. H.H., Yi.Z., S.S., T.E.B. and W.C. planned experiments and interpreted the data related to nanonap radiolabelling. Yu.Z., M.J., L.J.R., J.D.H., M.S., C.K. and J.F.L. planned experiments and interpreted the data related to photoacoustic imaging. Yu.Z., J.G. and J.F.L. planned toxicity studies and interpreted the data. Yu.Z., M.J., H.H., J.D.H., W.C., C.K. and J.F.L. wrote the manuscript.

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The authors declare no competing financial interests.

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