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

Journal name:
Nature Nanotechnology
Year published:
Published online


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


  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.


  1. Everhart, J. E. & Ruhl, C. E. Burden of digestive diseases in the United States part I: overall and upper gastrointestinal diseases. Gastroenterology 136, 376386 (2009).
  2. Dye, C. E., Gaffney, R. R., Dykes, T. M. & Moyer, M. T. Endoscopic and radiographic evaluation of the small bowel in 2012. Am. J. Med. 125, 1228.e11228.e12 (2012).
  3. Husebye, E. Gastrointestinal motility disorders and bacterial overgrowth. J. Intern. Med. 237, 419427 (1995).
  4. Soffer, E. E. Small bowel motility: ready for prime time? Curr. Gastroenterol. Rep. 2, 364369 (2000).
  5. Bassotti, G. et al. Gastrointestinal motility disorders in inflammatory bowel diseases. World J. Gastroenterol. 20, 3744 (2014).
  6. Lembo, A. & Camilleri, M. Chronic constipation. N. Engl. J. Med. 349, 13601368 (2003).
  7. Shafer, R. B., Prentiss, R. A. & Bond, J. H. Gastrointestinal transit in thyroid disease. Gastroenterology 86, 852855 (1984).
  8. Abrahamsson, H. Gastrointestinal motility disorders in patients with diabetes mellitus. J. Intern. Med. 237, 403409 (1995).
  9. Jost, W. H. Gastrointestinal motility problems in patients with Parkinson's disease. Drugs Aging 10, 249258 (1997).
  10. Kim, C., Favazza, C. & Wang, L. V. In vivo photoacoustic tomography of chemicals: high-resolution functional and molecular optical imaging at new depths. Chem. Rev. 110, 27562782 (2010).
  11. Ke, H., Erpelding, T. N., Jankovic, L., Liu, C. & Wang, L. V. Performance characterization of an integrated ultrasound, photoacoustic, and thermoacoustic imaging system. J. Biomed. Opt. 17, 056010 (2012).
  12. Ntziachristos, V., Ripoll, J., Wang, L. V. & Weissleder, R. Looking and listening to light: the evolution of whole-body photonic imaging. Nature Biotechnol. 23, 313320 (2005).
  13. Emelianov, S. Y., Li, P. C. & O'Donnell, M. Photoacoustics for molecular imaging and therapy. Phys. Today 62, 3439 (May, 2009).
  14. Stuart, S. et al. The smaller bowel: imaging the small bowel in paediatric Crohn's disease. Postgrad. Med. J. 87, 288297 (2011).
  15. Luke, G. P., Yeager, D. & Emelianov, S. Y. Biomedical applications of photoacoustic imaging with exogenous contrast agents. Ann. Biomed. Eng. 40, 422437 (2012).
  16. De la Zerda, A., Kim, J. W., Galanzha, E. I., Gambhir, S. S. & Zharov, V. P. Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics. Contrast Media Mol. Imaging 6, 346369 (2011).
  17. Zhang, W. et al. Synthesis and characterization of thermally responsive pluronic F127−chitosan nanocapsules for controlled release and intracellular delivery of small molecules. ACS Nano 4, 67476759 (2010).
  18. Tetko, I. V. & Tanchuk, V. Y. Application of associative neural networks for prediction of lipophilicity in ALOGPS 2.1 program. J. Chem. Inf. Comput. Sci. 42, 11361145 (2002).
  19. Lin, Y. & Alexandridis, P. Temperature-dependent adsorption of pluronic F127 block copolymers onto carbon black particles dispersed in aqueous media. J. Phys. Chem. B 106, 1083410844 (2002).
  20. Chandaroy, P., Sen, A., Alexandridis, P. & Hui, S. W. Utilizing temperature-sensitive association of pluronic F-127 with lipid bilayers to control liposome-cell adhesion. Biochim. Biophys. Acta Biomembr. 1559, 3242 (2002).
  21. Ahmed, F., Alexandridis, P. & Neelamegham, S. Synthesis and application of fluorescein-labeled pluronic block copolymers to the study of polymer–surface interactions. Langmuir 17, 537546 (2001).
  22. Phipps, J. S., Richardson, R. M., Cosgrove, T. & Eaglesham, A. Neutron reflection studies of copolymers at the hexane/water interface. Langmuir 9, 35303537 (1993).
  23. Mallidi, S. et al. Multiwavelength photoacoustic imaging and plasmon resonance coupling of gold nanoparticles for selective detection of cancer. Nano Lett. 9, 28252831 (2009).
  24. De la Zerda, A. et al. Family of enhanced photoacoustic imaging agents for high-sensitivity and multiplexing studies in living mice. ACS Nano 6, 46944701 (2012).
  25. Bayer, C. L., Nam, S. Y., Chen, Y. S. & Emelianov, S. Y. Photoacoustic signal amplification through plasmonic nanoparticle aggregation. J. Biomed. Opt. 18, 016001 (2013).
  26. Lovell, J. F. et al. Porphysome nanovesicles generated by porphyrin bilayers for use as multimodal biophotonic contrast agents. Nature Mater. 10, 324332 (2011).
  27. Jain, P. K., Lee, K. S., El-Sayed, I. H. & El-Sayed, M. A. Calculated absorption and scattering properties of gold nanoparticles of different size, shape, and composition:  applications in biological imaging and biomedicine. J. Phys. Chem. B 110, 72387248 (2006).
  28. Carino, G. P. & Mathiowitz, E. Oral insulin delivery. Adv. Drug Deliv. Rev. 35, 249257 (1999).
  29. Pu, K. et al. Semiconducting polymer nanoparticles as photoacoustic molecular imaging probes in living mice. Nature Nanotech. 9, 233239 (2014).
  30. Malmsten, M., Emoto, K. & Van Alstine, J. M. Effect of chain density on inhibition of protein adsorption by poly(ethylene glycol) based coatings. J. Colloid Interface Sci. 202, 507517 (1998).
  31. Kwon, S. & Sevick-Muraca, E. M. Non-invasive, dynamic imaging of murine intestinal motility. Neurogastroenterol. Motil. 23, 881e344 (2011).
  32. Gittes, G. K., Nelson, M. T., Debas, H. T. & Mulvihill, S. J. Improvement in survival of mice with proximal small bowel obstruction treated with octreotide. Am. J. Surg. 163, 231233 (1992).
  33. Ali, H. & van Lier, J. E. Metal complexes as photo- and radiosensitizers. Chem. Rev. 99, 23792450 (1999).
  34. Liu, T. W., MacDonald, T. D., Shi, J., Wilson, B. C. & Zheng, G. Intrinsically copper-64-labeled organic nanoparticles as radiotracers. Angew. Chem. Int. Ed. 51, 1312813131 (2012).

Download references

Author information


  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


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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary information (2,031 KB)

    Supplementary Information


  1. Supplementary Movie 1 (16,836 KB)

    Supplementary Movie 1

  2. Supplementary Movie 2 (1,384 KB)

    Supplementary Movie 2

Additional data