Fluorescent nanoprobes dedicated to in vivo imaging: from preclinical validations to clinical translation

Abstract

With the fast development, in the last ten years, of a large choice of set-ups dedicated to routine in vivo measurements in rodents, fluorescence imaging techniques are becoming essential tools in preclinical studies. Human clinical uses for diagnostic and image-guided surgery are also emerging. In comparison to low-molecular weight organic dyes, the use of fluorescent nanoprobes can improve both the signal sensitivity (better in vivo optical properties) and the fluorescence biodistribution (passive "nano" uptake in tumours for instance). A wide range of fluorescent nanoprobes have been designed and tested in preclinical studies for the last few years. They will be reviewed and discussed considering the obstacles that need to be overcome for their potential everyday use in clinics. The conjugation of fluorescence imaging with the benefits of nanotechnology should open the way to new medical applications in the near future.

Figure 1

Definition of the optical window for in vivo imaging. Reprinted from Kobayashi, H.; Ogawa, M.; Alford, R.; Choyke, P.L.; Urano, Y. New strategies for fluorescent probe design in medical diagnostic imaging. Chem. Rev. 2010, 110, 2620–2640 [20]. Copyright (2010) American Chemical Society.

Figure 2

Sentinel lymph node mapping in gastric cancer surgery using fluorescence imaging. Fluorescence (a,b) and video (c,d) images showing fine lymphatic vessels (a,c) and the succession of lymph nodes (b,d). Reproduced from Miyashiro, I.; Miyoshi, N.; Hiratsuka, M.; Kishi, K.; Yamada, T.; Ohue, M.; Ohigashi, H.; Yano, M.; Ishikawa, O.; Imaoka, S. Detection of sentinel node in gastric cancer surgery by indocyanine green fluorescence imaging: comparison with infrared imaging. Ann. Surg. Oncol. 2008, 15, 1640–1643 [8], with kind permission from Springer Science+business Media.

Figure 3

Near infrared fluorescent dyes that have been used in clinical trials until now.

Figure 4

Basic chemical structures of (a) fluoresceins, (b) rhodamines, (c) bodipys, (d) indocyanines, (e) porphyrines and (f) phthalocyanines.

Figure 5

Passive and active tumour targeting of nanoparticles.

Figure 6

Schematic structures of fluorescent nanoprobes for in vivo imaging. Inorganic nanoprobes are quantum dots (a) or dye-loaded silica, calcium phosphate, gold or oxide nanoparticles, for which the organic dye can be included in the inorganic matrix (b), or grafted on the nanoparticle surface (c). Organic nanoprobes can be divided in two main families: dye-loaded polymer-based and dye-loaded lipid-based nanoparticles. In each family, different architectures can be found: polymer- or lipid- core particles (polymer nanospheres (d), proteins (e), lipid nanoparticles (f), lipoproteins (g)), self-assembled constructions (polymer (h) or lipid (i) micelles), nanocapsules (polymersomes (j) or liposomes (k)). The fluorescent organic dye can be either included in the hydrophobic core or shell of the structure, or grafted on the nanoparticle surface (hydrophilic organic dye).

Figure 7

Different architectures of “polymer-core nanoparticles”.

Figure 8

Design of activatable probes for protease activity imaging. (a) Architecture of the probe, based on linear poly(lysine) with peptide-fluorophore moieties on pending amino groups. In this example, the peptide sequence can be cleaved by cathepsin D (CaD). (b) In vivo fluorescence images obtained 24 h after i.v. injection of the reporter probe in Nude mice implanted with a CaD+ (red arrow) and CaD− (blue arrow) tumours. Adaptedfrom Tung, C.-H.; Mahmood, U.; Bredow, S.; Weissleder, R. In vivo imaging of proteolytic enzyme activity using a novel molecular reporter. Cancer Res. 2000, 60, 4953–4958 [116] by permission from the American Association for Cancer Research.

Figure 9

NIR-emissive polymersomes. In aqueous solution, amphiphilic poly(ethylene oxide)/poly(butadiene) polymers (PEO₃₀-PBD₄₆) self-assemble into polymer vesicles (polymersomes) with the hydrophobic PBD tails oriented end-to-end to form a bilayer membrane, in which (porphynato)zinc(II) oligomers can be encapsulated. Adapted from Duncan, T.V.; Ghoroghchian, P.; Rubtsov, I.V.; Hammer, D.; Therien, M. Ultrafast excited-state dynamics of nanoscale near-infrared emissive polymersomes. J. Am. Chem. Soc. 2008, 130, 9773–9784 [132]. Copyright (2008) American Chemical Society.

Figure 10

Dye-loaded lipid nanoparticles (“lipidots™”) for in vivo imaging. (a) Structure of the particles. (b) Photograph of 50 nm diameter lipidots loaded with different organic dyes, covering the visible and near-infrared range. (c) Lymph node imaging 4 hours after sub-dermal injection of ICG-lipidots in the right paw. (d) Specific uptake of cRGD-functionalized lipidots in comparison to non-functionalized nanoparticles in HEKβ3 xenografted tumours in Nude mouse. This tumour model is known for its poor EPR effect and over-expression of α_vβ₃ integrins, for which the cRGD ligand presents a strong affinity (right images: microscopy photographs of HEKβ3 cells (nuclei stained in blue) after 24 h incubation in the presence of DiD-loaded lipidots (in red)). Adapted from [24,143].