Water-soluble, deep-red fluorescent squaraine rotaxanes

Article abstract of DOI:10.1039/c2ob06783h

Eight fluorescent squaraine rotaxanes with deep-red absorption/emission wavelengths were prepared and assessed for chemical stability and suitability as water-soluble, fluorescent tracers. The most stable squaraine rotaxanes have four large stopper groups attached to the ends of the encapsulated squaraine, and two members of this structural class have promise as highly fluorescent tracers with rapid renal clearance and very low tissue uptake in living mice.

Full text of DOI:10.1039/c2ob06783h

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COMMUNICATION
Water-soluble, deep-red fluorescent squaraine rotaxanes†‡
Erin L. Cole,a Easwaran Arunkumar,b Shuzhang Xiao,a Bryan A. Smitha and Bradley D. Smith*a
Received 23rd October 2011, Accepted 22nd November 2011
DOI: 10.1039/c2ob06783h
macrocycle remains tightly held over the reactive squaraine dye.9
Eight fluorescent squaraine rotaxanes with deep-red absorp-
Here, we describe the synthesis and deep-red absorption/
emission properties of water-soluble squaraine rotaxanes, 1–8
(Scheme 1), and we assess chemical stabilities in conditions that
mimic physiological environments. We find that the size of the
four stopper groups appended to the ends of the encapsulated
squaraine dye is a crucial factor in determining long-term,
chemical stability in physiological solution. The most stable
squaraine rotaxanes have four large stopper groups and we have
identified two members of this structural class as non-targeted,
fluorescent tracers with bright, deep-red emissions and high suit-
ability for biological imaging.
The syntheses of squaraine rotaxanes 1–8 are described in the
Electronic Supplementary Information (ESI).‡ The rotaxanes
were prepared by conducting Leigh-type clipping reactions that
permanently trapped an organic soluble squaraine dye inside a
tetralactam macrocycle that was composed of two phenylene
side walls and two pyridine 2,6-carboxamide bridging units.4a In
most cases, copper catalyzed azide-alkyne cycloaddition chemi-
stry was used to attach appropriately protected polar groups to
the ends of the encapsulated squaraine dye, and subsequent
deprotection provided 1–8 with high water-solubility (each com-
pound readily dissolved to form a 20 μM aqueous solution).
Listed in Table 1 are the absorption/emission properties in water.
Each sample exhibited the expected narrow deep-red absorption
band in the region of 651–654 nm, fluorescence emission with
an approximate 20 nm Stokes shift, and fluorescence quantum
yields of 0.16–0.24.§ Chemical and photochemical stability was
measured by monitoring the squaraine absorbance maxima in
pure water and 10% FBS (fetal bovine serum). The two graphs
tion/emission wavelengths were prepared and assessed for
chemical stability and suitability as water-soluble, fluorescent
tracers. The most stable squaraine rotaxanes have four large
stopper groups attached to the ends of the encapsulated
squaraine, and two members of this structural class have
promise as highly fluorescent tracers with rapid renal clear-
ance and very low tissue uptake in living mice.
Squaraines are intensely colored and highly fluorescent dyes
with deep-red emission wavelengths.1 They have been studied
for many photonic applications but their utility for biological
imaging is limited by their susceptibility to chemical attack by
strong nucleophiles and hydrolytic decomposition in aqueous
solution.2 In 2005, we reported a method to permanently encap-
sulate squaraine dyes inside macrocyclic tetralactams and create
squaraine rotaxanes.3 This encapsulation process greatly enhances
dye stability and also circumvents the problem of fluorescence
quenching upon self-aggregation.4 We have subsequently shown
that squaraine rotaxanes can be employed as targeted fluorescent
molecular probes for cell microscopy and whole-body imaging of
disease models in small living animals.5 To date, most of the
squaraine rotaxane probes have been lipophilic or partially soluble
in water or related aqueous buffers. However, there is a need for
squaraine rotaxanes that are highly water-soluble. Applications for
these types of compounds would range from targeted bioconjugates
for molecular imaging to non-targeted fluorescent tracers for
various biomedical and analytical methods.6,7
The chemical conversion of hydrophobic fluorescent dyes into
water-soluble, non-aggregating compounds is a non-trivial task,
especially for near-infrared dyes because they have relatively
reactive chromophores and large hydrophobic surfaces.8 The
synthetic process has to introduce water-solubilizing groups into
the dye structure in a way that does not degrade the photophysi-
cal performance. In the case of squaraine rotaxanes, there is the
additional design constraint of ensuring that the protective
Table 1 Absorption/emission maxima in water
a
Compound
λabs (nm)
log ε
λem (nm)
Φf
1
2
3b
4
5
6
7
8
651
655
653
654
654
653
652
653
5.6
5.1
5.1
5.1
5.1
5.3
5.3
5.2
672
677
675
675
675
672
673
674
0.22
0.16
0.20
0.22
0.21
0.24
0.23
0.23
aDepartment of Chemistry and Biochemistry, University of Notre Dame,
Notre Dame, IN, 46556, USA. E-mail: smith.115@nd.edu
bMolecular Targeting Technologies Incorporated, 833 Lincoln Ave., Unit 9,
West Chester, PA, 19380, USA
†This article is part of the Organic & Biomolecular Chemistry 10th
Anniversary issue.
‡Electronic supplementary information (ESI) available: Synthetic
methods, spectral data, photophysical measurements, and protocol for
approved animal study. See DOI: 10.1039/c2ob06783h
a Fluorescence quantum yields (error limit 5%) were determined using
4,4′-[bis(N,N-dimethylamino)phenyl]squaraine dye as the standard (Φf =
0.70 in CHCl3). b Reported previously in ref. 5a.
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Scheme 1 Structures of squaraine rotaxanes and the trend for aqueous stability.
in Fig. 1 show stability curves for samples that were exposed to
laboratory lights (control samples stored in the dark gave the
same results). The data are consistent with the general stability
trend that is summarized in Scheme 1. That is, squaraine rotaxane
stability in water is related to the total number of large stopper
groups (i.e., groups larger than N-ethyl) attached to the two
terminal squaraine nitrogen atoms, in the approximate order of
four > three > two. The most stable squaraine rotaxanes in water
are 6–8, each with four large stopper groups, although 6 under-
goes some moderate decomposition in serum. The hydrolytic
susceptibility of rotaxanes 1–5, with only two or three large
stopper groups, suggests that the surrounding macrocycle has
enough translational mobility in aqueous solution to transiently
expose the reactive core of the encapsulated squaraine. This
macrocycle mobility is limited by the steric hindrance imposed
by the four large stopper groups in 7 and 8.¶ The excellent
photochemical stabilities of 7 and 8 were confirmed by irradiat-
ing aerated aqueous samples with a 45 W fluorescent bulb at a
distance of 30 cm for two hours and observing absorption losses
of only 4% and 1%, respectively. The high resistance to photo-
bleaching is not surprising since squaraine rotaxanes are weak
oxygen photosensitizers and relatively unreactive with singlet
oxygen.4a It is also worth noting that squaraine rotaxanes are
typically not phototoxic; for example, previous studies of bac-
terial cells that were stained with squaraine rotaxane 3 showed
that the cells remained viable after extended irradiation periods.5
The hydrolytic instabilities of 1–6 indicate that these structures
are not well-suited for further development into high stability,
fluorescent bioimaging probes. However, the excellent chemical
and photochemical stabilities of squaraine rotaxanes 7 and 8
prompted us to assess their potential as water-soluble, deep-red
fluorescent tracers. Fluorescent dyes that emit light in the wave-
length window of 650–900 nm are known to possess bioimaging
performance advantages such as maximum penetration of the
light through skin and tissue and inherently high contrast due to
minimized background autofluorescence.10 Nonetheless, deep-
red and near-infrared dyes are rarely employed as hydrophilic
fluorescent tracers, in part, because they self-quench in water at
concentrations above a few micromolar, which prohibits quanti-
tative fluorescence measurements.6,7,11 This drawback seems
especially prevalent with cyanine dyes, the most common class
of near-infrared dyes.12 Therefore, it is notable that squaraine
rotaxanes 7 and 8 exhibit excellent Beer–Lambert absorption/
emission behavior up to 14 μM, the highest concentration tested
(Fig. 2). As expected, the emission plots are slightly curved at
higher concentrations because the absorption/emission bands are
partially overlapping which produces an inner filter effect.13
Additional absorption/emission studies of 7 and 8 showed that the
brightness (molar absorptivity × fluorescence quantum yield) is
unchanged over the pH range of 6 to 10. Finally, equilibrium dialy-
sis experiments with mixtures of 7 or 8 with bovine serum albumin
indicated weak dye/albumin protein association (see ESI‡).
The low affinity of squaraine rotaxanes 7 or 8 for albumin
protein suggested that they may be useful as non-targeted,
control tracers in our ongoing project to develop targeted squar-
aine rotaxane probes for in vivo molecular imaging. Therefore, a
mouse biodistribution study was conducted to determine the
extent of non-selective tissue and organ retention of 7 and 8. All
animal procedures were approved by the University of Notre
Dame Institutional Animal Care and Use Committee. As a
benchmark, we also included the near-infrared fluorophore, Indo-
cyanine Green (ICG), a clinically approved dye that is com-
monly employed as a non-targeted tracer for in vivo imaging
studies.14 Three cohorts of mice were dosed intravenously with
an aqueous solution of one of the three dyes (40 pmol g−1) and
each animal was sacrificed two hours later. The skin overlaying
5770 | Org. Biomol. Chem., 2012, 10, 5769–5773
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and a sensitive CCD camera. Shown in Fig. 3 are representative
brightfield image and fluorescence intensity maps for mice
treated with each dye. The fluorescence images indicate relative
biodistribution of the tracers, and in the case of 7 and 8 the
tracers are primarily in the urine remaining in the bladder, with
very little uptake by the other organs. In comparison, there was
very little ICG in the bladder but a very intense signal in the
intestines. This was expected since ICG is known to be cleared
from the bloodstream by the biliary system and subsequently
excreted into the intestines.14
Fig. 1 Change in squaraine rotaxane absorption (5 μM) at wavelength
maxima in pure water (top) and 10% FBS in water (bottom), T = 25° C.
Samples were exposed to laboratory lights throughout the experiment.
Note that the fluorescence spectra of 7 and 8 were also unchanged over
time indicating no longitudinal change in fluorescence quantum yield.
Fig. 3 Fluorescence intensity maps (ex: 630 10 nm, em: 700 20 nm)
and brightfield images (50 mm field of view) of the exposed abdominopelvic
cavities of representative mice treated intravenously with 40 pmol g−1 of
7 (A,D), 8 (B,E), or ICG (C,F) and sacrificed at two hours post-injection
of fluorophore. Abbreviations: bladder (Bl), intestines (In), liver (Li).
Note that the physical area of the bladder signal in A is smaller than B
but the pixel intensity is higher. The fluorescence intensity scale for each
image is in arbitrary units.
The biodistributions of 7 and 8 were further compared by
ex vivo fluorescence imaging of the excised organs. Region of
interest analysis of the pixel intensity maps in Fig. 4 allowed
calculation of mean pixel intensity for each excised tissue. The
squaraine fluorophores in 7 and 8 have essentially the same
deep-red absorption/emission wavelengths and the same bright-
ness, so the mean pixel intensity values reflect relative levels of
dye in each tissue sample. The sum of mean pixel intensity
values for 7 in all the excised tissues is 9.3 times greater than the
analogous value for 8. We conclude that non-selective retention
of water-soluble, squaraine rotaxane 7 by the tissues and organs
of living animals is quite low, and that the tissue retention of
Fig. 2 Absorption and emission intensities at different concentrations
in phosphate buffered saline (sodium phosphate 10 mM, NaCl 150 mM,
pH 7.4): (a) 7 em (red open squares); (b) 8 em (red open circles); (c) 7 abs
(blue filled diamonds); (d) 8 abs (blue filled circles). Linear fits of the
two absorption plots have R2 values >0.99; the curved lines for the
fluorescence plots are simply to guide the eye.
the abdominopelvic cavity was resected and optical images were
taken of the intact organs using an in vivo imaging station that
was equipped with appropriate illumination/emission filter sets
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Scheme 2 Molecular model of squaraine rotaxane 8 at physiological
pH showing electrostatic surface with positive centers colored blue and
negative centers red. The longest diagonal length of the extended
molecule is ∼4.6 nm.
body molecular imaging studies of living animals. We expect
that deep-red fluorescent tracers 7 and 8 will be broadly useful in
other types of biomedical, analytical, and biotechnology
applications.7
Fig. 4 Comparative fluorescence intensity maps (ex: 630 10 nm, em:
700 20 nm) of excised tissue and organs of mice treated intravenously
with 40 pmol g−1 of 7 (A) or 8 (B) and sacrificed at two hours post-injection
of fluorophore. Abbreviations: liver (Lvr), kidney (Kdn), intenstines (Int),
spleen (Spl), lung (Lng), heart (Hrt), muscle (Msc). Fluorescence
intensity scale in arbitrary units.
Acknowledgements
This work that was supported by NIH grants R43-EB009266
and T32GM075762. The mouse imaging protocol was approved
by the Notre Dame Institutional Committee for Animal Care and
the molecular model in Scheme 2 was created by the Notre
Dame Center for Research Computing.
tracer 8 is even lower. The rapid excretion of 8 through the
kidneys is especially notable, and quite obvious to the naked eye
since the mouse urine turns blue within a few minutes after intra-
venous dosage. Recently, other research groups have observed
high renal clearance of dyes and small nanoparticles (hydro-
dynamic radius ≤ 5 nm) with structures having a mixed and geo-
metrically balanced distribution of positive and negative
charges.6,15 The structure of squaraine rotaxane 8 seems to agree
with this trend but there is one significant difference. Choi and co-
workers reported that the dyes must have a mixture of permanent
charges such as quaternary ammoniums and sulfonates, and not
weakly charged groups like carboxylates and protonated ammo-
niums.6 We believe that the dendritic and polyionic structure of 8
(primarily a mixture of eight anionic carboxylates and four cationic
ammoniums at physiological pH, see Scheme 2), is an additional
structural factor that promotes rapid renal clearance because it
favors extensive hydration of the molecular periphery, screening of
electrostatic charge, and avoidance of biological surfaces.
In summary, the long-term hydrolytic instability of water-
soluble squaraine rotaxanes 1–5 is attributed to the relatively
small steric size of the N-ethyl stopper group that is attached to
one or both ends of the encapsulated squaraine (Scheme 1). The
most stable squaraine rotaxanes in water are 6–8, each with four
large stopper groups, although 6 undergoes some moderate
decomposition in serum. As deep-red fluorescent tracers, squar-
aine rotaxane 7, and especially 8, exhibit an attractive collection
of properties including very high chemical and photochemical
stability, low phototoxicity, resistance to self-quenching, high
and pH-independent brightness, and negligible uptake by mam-
malian tissue. In the near future, we will publish reports showing
the utility of 8 as a non-targeted, fluorescent tracer for whole-
Notes and references
§The ESI‡ contains photophysical properties for rotaxanes 1–8 in phos-
phate buffered saline (PBS, sodium phosphate 10 mM, NaCl 150 mM,
pH 7.4) and 10% FBS (fetal bovine serum), and they are nearly identical
to the PBS data Table 1. Also provided in the ESI‡ are photophysical
properties of the precursor squaraine dyes, and inspection of this data
shows that encapsulation inside the macrocyclic tetralactam induces a
7–10 nm red-shift in squaraine absorption/emission maxima and a slight
increase in fluorescence quantum yield.
¶Even at 37 °C, compound 8 exhibited extremely high stability in 10%
FBS.
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