DOI: 10.1002/cbic.201200350
Selective Multicolour Imaging of Zebrafish Muscle Fibres by Using
Fluorescent Organic Nanoparticles
E. Ramanjaneya Reddy,[a] Rakesh Kumar Banote,[b, c] Kiranam Chatti,[b, c] Pushkar Kulkarni,[b, c] and
Marina S. Rajadurai*[a]
The design and preparation of fluorescent organic nanoparti-
cles (FONs) from organic molecules is one of the rapidly
emerging fields of organic-based nanoscience, because of the
broad range of potential applications in optoelectronic nano-
devices, (bio-)chemical sensing, drug delivery and monitoring
systems, diagnostics, immunofluorescent labelling and in vitro/
in vivo imaging.[1–5] Particularly for fluorescent imaging studies,
fluorescent proteins, organic dyes and probes have been
widely used in all areas of biology and preclinical research for
the visualisation of cellular and subcellular biological process-
es.[6] Among fluorescence-based imaging agents, organic mole-
cules are of interest, because of their diversity, easy synthesis,
good colour tunabilty and high quantum yield (Ff), although
poor photostability is an issue.[5b] On the other hand, FONs are
very attractive candidates for fluorescent imaging, as they offer
definite advantages over bulky organic chromophores, such as
easy cellular uptake,[7] size-dependent fluorescent properties[8]
and longer fluorescence lifetime.[2c]
fish an ideal research model for studying early development,
understanding the pathogenesis of human disease and provid-
ing systems for developing and testing new therapies.
Though there are a number of reports available on zebrafish
tissues fluorescent imaging, including green fluorescent pro-
tein expression, quenched fluorescent protein (EnzChek) and
phospholipid (PED6), antibody labelling and staining with fluo-
rescent small molecules,[12–15] to the best of our knowledge
there is no report of whole-body imaging with FONs. FONs
based on purine scaffolds seem particularly promising, as sub-
stituted purines, pyrimidines and their tautomers are the most
widely distributed nitrogen-containing heterocycles in nature
and have high biological activity. We envisioned that the use
of nontoxic FONs self-assembled from biologically active mole-
cules and carrying chromophoric tags with high quantum yield
and broad emission could be useful for cell/tissue/organism
multicolour imaging, and for biological process visualisation in
zebrafish embryos.
Although the nature of the organic fluorophores does not
allow them to generate fluorescent signals at significant tissue
depths (>1–2 cm), this limitation can be overcome by using
either fluorescence imaging with near-infrared (NIR) light[9,10] or
by taking advantage of small animals with transparent tissues,
for example frog (Xenopus) or zebrafish (Danio rerio) embryos.
Zebrafish are particularly attractive for fundamental and pre-
clinical research because of an exceptional combination of
experimental and genetic advantages, such as the significant
similarity between zebrafish and human genomes, high fe-
cundity, external fertilization, rapid embryo development and
optical transparency of embryos. An additional advantage is
the straightforward generation of a zebrafish mutant for mod-
elling a particular human disease; for example, human muscu-
lar dystrophies show very similar cellular pathology to that of
zebrafish muscle degeneration.[11] These qualities make zebra-
Herein, for the first time, we report the syntheses of a novel
pyrene-based blue chromophore linked to biologically active
guanine analogues, PPy and GPy, and their self-assembly in
DMSO/H2O to form solution-stable, blue emissive, biocompat-
ible, nontoxic FONs. We also report their photophysical proper-
ties and the results of scanning electron microscopy (SEM),
atomic force microscopy (AFM) and confocal fluorescence mi-
croscopy (CFM) studies. Finally, we demonstrate the use of
FONs as an excellent technique for in vivo whole-body fluores-
cent imaging of zebrafish, and in particular for the selective
imaging of muscle fibres.
The target guanosine analogues, 6-chloro-7-(12-(4-(pyren-1-
yl)-1H-1,2,3-triazol-1-yl)dodecyl)-7H-purin-2-amine (PPy) and 2-
amino-7-(12-(4-(pyren-1-yl)-1H-1,2,3-triazol-1-yl)dodecyl)-1H-
purin-6(7H)-one (GPy) were synthesised from commercially
available 6-chloro-7H-purin-2-amine in four steps (Scheme 1).
N-alkylation of 6-chloro-7H-purin-2-amine with 3 equiv of 1,12-
dibromododecane in the presence of anhydrous potassium
carbonate gave N9-alkylated purine 1 in 53% yield. Such low
yield can be explained by the fact that alkylation of purines is
not regiospecific, and product mixtures will contain significant
amounts of N7-substituted product and unutilised material.[16]
Reaction of 1 with sodium azide formed the azide precursor 2
in good yield. Subsequent 1,3-dipolar Huisgen cycloaddition
reaction of 2 with 1-ethynylpyrene[17] under “click reaction”
conditions with CuSO4·5H2O/sodium ascorbate in water/DMF
[a] E. R. Reddy,+ Dr. M. S. Rajadurai
Organic and Medicinal Chemistry, Institute of Life Sciences
University of Hyderabad Campus
Gachibowli, Hyderabad, 500046 (India)
[b] R. K. Banote,+ Dr. K. Chatti, Dr. P. Kulkarni
Zephase Therapeutics, Institute of Life Sciences
University of Hyderabad Campus
Gachibowli, Hyderabad, 500046 (India)
[c] R. K. Banote,+ Dr. K. Chatti, Dr. P. Kulkarni
Biology Department, Institute of Life Sciences
University of Hyderabad Campus
1
(2:1) gave purine clicked with pyrene PPy. H NMR spectrosco-
py confirmed the formation of a five-membered triazole ring
by exhibiting a CtriazoleÀH proton at 8.23 ppm and disappear-
ance of the terminal acetylene proton at 3.63 ppm. Subse-
quently, treatment of PPy with 0.1n HCl under reflux condition
Gachibowli, Hyderabad, 500046 (India)
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201200350.
ChemBioChem 0000, 00, 1 – 6
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