Supramolecular strategies to construct biocompatible and photoswitchable fluorescent assemblies

Article abstract of DOI:10.1021/ja107341f

We designed and synthesized an amphiphilic copolymer with pendant hydrophobic decyl and hydrophilic poly(ethylene glycol) chains along a common poly(methacrylate) backbone. This macromolecular construct captures hydrophobic boron dipyrromethene fluorophores and hydrophobic spiropyran photochromes and transfers mixtures of both components in aqueous environments. Within the resulting hydrophilic supramolecular assemblies, the spiropyran components retain their photochemical properties and switch reversibly to the corresponding merocyanine isomers upon ultraviolet illumination. Their photoinduced transformations activate intermolecular electron and energy transfer pathways, which culminate in the quenching of the boron dipyrromethene fluorescence. As a result, the emission intensity of these supramolecular constructs can be modulated in aqueous environments under optical control. Furthermore, the macromolecular envelope around the fluorescent and photochromic components can cross the membrane of Chinese hamster ovarian cells and transport its cargo unaffected into the cytosol. Indeed, the fluorescence of these supramolecular constructs can be modulated also intracellularly by operating the photochromic component with optical inputs. In addition, cytotoxicity tests demonstrate that these supramolecular assemblies and the illumination conditions required for their operation have essentially no influence on cell viability. Thus, supramolecular events can be invoked to construct fluorescent and photoswitchable systems from separate components, while imposing aqueous solubility and biocompatibility on the resulting assemblies. In principle, this simple protocol can evolve into a general strategy to deliver and operate intracellularly functional molecular components under optical control.

Full text of DOI:10.1021/ja107341f

ARTICLE
pubs.acs.org/JACS
Supramolecular Strategies To Construct Biocompatible and
Photoswitchable Fluorescent Assemblies
Ibrahim Yildiz,† Stefania Impellizzeri,† Erhan Deniz,† Bridgeen McCaughan,‡ John F. Callan,*,‡ and
Franc-isco M. Raymo*,†
†Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146-0431, United States
‡Department of Pharmacy and Pharmaceutical Sciences, School of Biomedical Sciences, University of Ulster, Coleraine BT52 1SA,
Northern Ireland, United Kingdom
S Supporting Information
b
ABSTRACT: We designed and synthesized an amphiphilic
copolymer with pendant hydrophobic decyl and hydrophilic
poly(ethylene glycol) chains along a common poly(methacrylate)
backbone. This macromolecular construct captures hydro-
phobic boron dipyrromethene fluorophores and hydropho-
bic spiropyran photochromes and transfers mixtures of both
components in aqueous environments. Within the resulting
hydrophilic supramolecular assemblies, the spiropyran com-
ponents retain their photochemical properties and switch
reversibly to the corresponding merocyanine isomers upon ultraviolet illumination. Their photoinduced transformations activate
intermolecular electron and energy transfer pathways, which culminate in the quenching of the boron dipyrromethene fluorescence.
As a result, the emission intensity of these supramolecular constructs can be modulated in aqueous environments under optical
control. Furthermore, the macromolecular envelope around the fluorescent and photochromic components can cross the
membrane of Chinese hamster ovarian cells and transport its cargo unaffected into the cytosol. Indeed, the fluorescence of these
supramolecular constructs can be modulated also intracellularly by operating the photochromic component with optical inputs. In
addition, cytotoxicity tests demonstrate that these supramolecular assemblies and the illumination conditions required for their
operation have essentially no influence on cell viability. Thus, supramolecular events can be invoked to construct fluorescent and
photoswitchable systems from separate components, while imposing aqueous solubility and biocompatibility on the resulting
assemblies. In principle, this simple protocol can evolve into a general strategy to deliver and operate intracellularly functional
molecular components under optical control.
’ INTRODUCTION
only one of the two states of the photochrome or the transfer of
energy from the excited fluorophore to only one of the two states
of the photochrome is generally responsible for fluorescence
modulation. The assembly of these fluorophore-photochrome
constructs, however, often requires tedious multistep proce-
dures, which considerably limit their synthetic accessibility. In
addition, most of these functional compounds are rather hydro-
phobic and can only be operated in organic solvents. In fact, it is
not entirely clear whether their photochemical and photophysi-
cal properties would at all survive the transition from organic to
aqueous environments necessary for the application of these
fluorescent photoswitches in biomedical research. Indeed, it is
becoming apparent that the ability of a photochromic element to
switch fluorescence, in combination with appropriate illumina-
tion protocols, can offer the opportunity to overcome diffraction
and permit the visualization of biological samples with resolution
at the nanometer level.19 In fact, fluorophore-photochrome
Photochromic molecules interconvert reversibly between
states with a distinct absorption signature across the ultraviolet
and visible regions of the electromagnetic spectrum.1-6 In most
instances, the interconvertible states also differ significantly in
their stereoelectronic properties. In fact, the structural and elec-
tronic changes, accompanying their photoinduced and reversible
transformations, can be engineered to control the emission of
complementary fluorophores.6-8 Specifically, fluorescent and
photochromic components can be integrated within a common
covalent skeleton, and the emission of the former can be modu-
lated by interconverting the latter under optical control. Accord-
ingly, numerous examples of fluorophore-photochrome dyads
have already been developed,9-13 and their operating principles
for fluorescence modulation have recently been extended to nano-
structured constructs14-18 and extensively reviewed.8 In most
instances, electron and energy transfer processes dominate the
excited-state dynamics of these multicomponent assemblies and
dictate their emission signature. In particular, the transfer of one
electron from/to the excited state of the fluorophore to/from
Received: August 20, 2010
Published: December 23, 2010
r
2010 American Chemical Society
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Figure 1. Photoinduced and reversible transformation of the spiropyr-
ans 1a and 2a into the merocyanines 1b and 2b.
constructs can become, in principle at least, invaluable analytical
tools for the super-resolution imaging of cells and tissues.20-32
However, it is first necessary to identify viable strategies to im-
pose biocompatibility on them, without complicating their
synthesis even further, and to operate effectively these functional
assemblies in water.
Figure 2. Structures of the BODIPY derivatives 3 and 4.
Recently, we designed and investigated a photoswitchable
fluorescent dyad consisting of a boron dipyrromethene (BODIPY)
fluorophore covalently connected to a spiropyran photochrome.13a
In acetonitrile, the ultraviolet illumination of this compound
promotes the conversion of the spiropyran component into the
corresponding merocyanine. This photoinduced transformation
facilitates the transfer of one electron or energy from the BODIPY
to the merocyanine component upon visible excitation of the
former with concomitant fluorescence quenching. The photo-
generated and nonfluorescent state reverts thermally to the
original fluorescent species and restores the initial emission
intensity. As a result, the fluorescence of this fluorophore-
photochrome dyad can be switched off and on for multiple
cycles simply by turning on and off, respectively, an ultraviolet
source, while illuminating the sample with visible radiations. To
impose hydrophilic character on this hydrophobic construct, we
appended covalently multiple copies of this dyad to a common
hydrophilic polymer backbone. The fluorescence of the resulting
macromolecular system could be modulated in water under
optical control, but only with slow switching speeds and poor
fatigue resistance. In addition, this hydrophilic assembly of fluo-
rescent and photochromic components required multiple and
tedious synthetic steps for its preparation. In search of strategies
to overcome these limitations, we envisaged the possibility of
invoking supramolecular events to assemble biocompatible
fluorescent switches with good photochemical performance in
water. Specifically, we relied on the established ability of amphi-
philic building blocks to form hydrophilic micellar aggregates
capable of encapsulating hydrophobic guests33-37 to construct
functional supramolecular assemblies of simple and separate
fluorescent and photochromic components. In this paper, we
report the preparation of these systems as well as the character-
ization of their spectroscopic properties, the investigation of their
ability to cross cell membranes, and the assessment of their
cytotoxicty.
Figure 3. Polymerization of the monomers 5 and 6 to generate the co-
polymer 7.
exergonic with an estimated free energy change of ca. -0.2 eV.13a
In addition, the formation of 1b results in the appearance of an
intense absorption band in the same region of wavelengths where
3 emits.13a The significant overlap between the absorption of one
and the emission of the other can promote the transfer of energy
from the excited fluorophore to the photogenerated state of the
photochrome. Thus, both electron and energy transfer processes
can contribute to quench the emission of the fluorophore with
the photochromic transformation. Fluorescent and photochro-
mic components, however, must be in close proximity for the
electron or energy transfer process to occur. In addition, the
modulation of fluorescence in biological media requires aqueous
compatibility, and 1a is not soluble in water. On the basis of these
considerations, we designed an amphiphilic polymer expected to
encapsulate both the fluorescent and photochromic components
in its interior and enforce a close separation between them, while
ensuring aqueous solubility of the overall supramolecular assem-
bly. In particular, we synthesized the methacrylate monomers 5
and 6 (Figure 3) with hydrophobic decyl and hydrophilic poly-
(ethylene glycol) chains, respectively, and reacted them, under
the assistance of azobisisobutyronitrile (AIBN), to generate the
amphiphilic copolymer 7.39 The 1H nuclear magnetic resonance
(NMR) spectrum of the resulting macromolecular construct
reveals the ratio between the hydrophobic and hydrophilic chains
appended to the main polymer backbone to be ca. 2.3. Gel per-
meation chromatography (GPC) indicates this particular co-
polymer to have a number average molecular weight (Mn) of 46 800
with a polydispersity index (PDI) of 1.73. Its hydrophilic
poly(ethylene glycol) chains ensure solubility in water, and its
’ RESULTS AND DISCUSSION
Design, Synthesis, and Spectroscopy. The spiropyran 1a
(Figure 1) switches to the merocyanine 1b upon ultraviolet
illumination in acetonitrile.38 This photoinduced transformation
shifts the reduction potential in the negative direction by 0.3 V
and turns the transfer of one electron from the excited BODIPY 3
(Figure 2) to the photochromic compound from endergonic to
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Figure 5. Absorption spectra of PBS dispersions (pH 7.4, 20 °C) of
polymer micelles containing 3 before (curve a) and after (curve b)
irradiation (365 nm, 0.4 mW cm-2, 5 min) or 4 before (curve c) and
after (curve d) irradiation. Emission spectrum (curve e, λex = 434 nm) of
a PBS dispersion of polymer micelles containing 4.
Figure 4. Absorption spectra of PBS dispersions (pH 7.4, 20 °C) of
polymer micelles containing 1a (curves a and b) or 2a (curves c and d)
recorded after equilibration in the dark for 12 h before (curves a and c)
and after (curves b and d) irradiation (365 nm, 0.4 mW cm-2, 5 min).
Emission spectrum (curve e, λex = 434 nm) of a PBS dispersion of
polymer micelles containing 2a after equilibration and irradiation.
indicative of the photoinduced degradation of the fluorophore
and suggests that, despite interactions with the amphiphilic
polymer, 3 is exposed to water (vide infra).
hydrophobic decyl chains encourage the formation of supramo-
lecular aggregates with an average hydrodynamic diameter of 18
nm, according to dynamic light scattering (DLS) measurements.
The comparison of this value to the hydrodynamic diameters of
polystyrene standards of known molecular weight40 suggests that
an average of four amphiphilic copolymer units assemble into a
single micellar construct. Similarly, transmission electron micros-
copy (TEM) images (Figure S1a, Supporting Information) of
the resulting assemblies show globular particles with an average
diameter of 17 nm.
The spiropyran 1a is essentially insoluble in water at ambient
temperature, but dissolves in phosphate-buffered saline (PBS)
with a pH of 7.4 in the presence of 7. Indeed, the corresponding
absorption spectrum (curve a in Figure 4) reveals the character-
istic band of 1a at 340 nm. The low absorbance of this band,
however, indicates that 7 can transfer only micromolar amounts
of 1a in aqueous environments. Nonetheless, the photochromic
components entrapped within the polymer micelles retain their
photochemical character, and the band of 1b appears at 538 nm
upon ultraviolet irradiation (curve b in Figure 4).
To facilitate the entrapment of guests within 7 and prevent
their exposure to water, we designed and synthesized the spiro-
pyran 2a (Figures 1 and S2, Supporting Information) and the
BODIPY 4 (Figure 2). Both compounds bear a hydrophobic
decyl chain, which is expected to bury the photochromic and
fluorescent components further into the hydrophobic interior of
the supramolecular construct. Indeed, the absorption spectrum
(curve c in Figure 4) of a PBS dispersion of 2a and 7 reveals an
intense absorption at 340 nm for the spiropyran guests trapped
within the micellar hosts. In addition, a band for 2b appears at
544 nm after ultraviolet irradiation (curve d in Figure 4), while
that of 1b is positioned at 538 nm under otherwise identical con-
ditions. The elongation in wavelength demonstrates that the
environment around 2b is more hydrophobic than that sur-
rounding 1b. In fact, the absorption wavelength of merocyanines
is known to increase with a decrease in solvent polarity.41 Further-
more, the spectroscopic signature of 2a and 2b within the
polymer micelles resembles that recorded in acetonitrile (curves a
and b inFigure S3, SupportingInformation). Inbothinstances, the
ratio between the two isomers is ca. 70:30 at the photostationary
state, albeit the absorption band of the photogenerated species is
positioned at 562 nm in acetonitrile.
The BODIPY 3 is also sparingly soluble in water, but readily
dissolves in PBS in the presence of 7. Consistently, the absorp-
tion spectrum (curve a in Figure 5) of a PBS dispersion of 3
and 7 shows the characteristic band of the BODIPY fluorophore
at 522 nm. Upon ultraviolet irradiation under conditions nor-
mally required to operate spiropyrans, however, the absorbance
of 3 decreases significantly (curve b in Figure 5). This trend is
The merocyanine 2b reverts spontaneously back to 2a within
the polymer micelles, and concomitantly, the absorbance at 544 nm
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absorption at 523 nm and, once again, does not change with
ultraviolet irradiation (curves a and b in Figure S4, Supporting
Information). Instead, the BODIPY absorbance decreases sig-
nificantly in a mixture of acetonitrile and water (2:1, v/v) under
identical irradiation conditions (curves c and d in Figure S4).
Thus, the amphiphilic copolymer 7 is particularly effective in
maintaining either 2a or 4 within a relatively hydrophobic envi-
ronment and limiting their exposure to water. On the contrary,
conventional phospholipid micelles cannot ensure the same level
of protection to their hydrophobic guests, at least in the case of 4.
Indeed, the absorption spectra (Figure S5, Supporting Infor-
mation) of 4, entrapped within micelles prepared from the am-
monium salt of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol-
amine N-(methoxy(polyethylene glycol)-2000),42 reveal a significant
decrease in absorbance with ultraviolet irradiation. Nonetheless,
the behavior of 2a under these conditions (Figure S6, Supporting
Information) is similar to that observed in the presence of 7. In
both instances, 2a switches to 2b under ultraviolet irradiation
with the appearance of a band essentially at the same wavelength
and the photogenerated isomer reverts back to the original one
with similar kinetics.
The emission spectrum (curve e in Figure 5) of a PBS dis-
persion of 4 and 7 shows the characteristic BODIPY fluorescence
at 544 nm with a quantum yield of 0.44. In acetonitrile, the
emission of 4 is centered at a similar wavelength (cf. 539 nm), but
the quantum yield is 0.81. The depressive effect of the polymer
micelles on the fluorescence quantum yield is, presumably, a
result of their ability to bring multiple dyes in close proximity
within their hydrophobic interior and encourage self-quenching.
In any case, the emission band of 4 within the supramolecular
constructs is in the same region of wavelengths where 2b absorbs
under the same experimental conditions. Thus, the entrapment
of 4 and 2b within the same macromolecular host is expected to
result in the transfer of energy from the former to the latter upon
excitation. In addition, electron transfer from the excited
BODIPY to the photogenerated isomer of the photochromic
system is exergonic13a and, if the two components are sufficiently
close within their supramolecular container, can also occur as an
alternative to energy transfer. It follows that the photoinduced
and reversible interconversion of 2a and 2b within a polymer
micelle also containing 4 is expected to modulate the emission
intensity of the BODIPY fluorophores. Indeed, the absorption
and emission spectra (curves a and c in Figure 7) of a PBS dis-
persion of 2a, 4, and 7 show the typical absorbance and fluo-
rescence of the BODIPY components at 528 and 542 nm,
respectively. Upon ultraviolet irradiation, 2a switches to 2b,
and the absorption band of the merocyanine isomer appears in
the corresponding spectrum (curve b in Figure 7). The absor-
bance of this band indicates the ratio between 2a and 2b to be,
once again, ca. 70:30 at the photostationary state. This transfor-
mation activates the expected electron and energy transfer path-
ways and causes a significant decrease in the emission intensity at
542 nm (curve d in Figure 7) with a quenching efficiency of 0.8.
Concomitantly, the characteristic fluorescence of the photogen-
erated merocyanines38 develops at 638 nm. Indeed, this addi-
tional band resembles that observed for 2b (curve e in Figure 4)
within the polymer micelles in the absence of 4, under otherwise
identical conditions. Nonetheless, the presence of 4 translates
into a significant enhancement in the fluorescence of 2b. In fact, a
comparison of the integrated emission intensities, recorded with
and without 4, suggests that the fractional contribution of direct
excitation to the total emission intensity of 2b is only 0.3 and that
Figure 6. Absorbance evolution at 545 nm of a PBS dispersion (pH 7.4,
20 °C) of polymer micelles containing 2a recorded after equilibration in
the dark for 12 h and either with ultraviolet irradiation (curve a, 365 nm,
0.4 mW cm-2, 5 min) or with alternating irradiation steps (curve b) at
ultraviolet and visible (562 nm, 0.3 mW cm-2, 5 min) wavelengths.
(curve a in Figure 6) decays over the course of several minutes.
Curve fitting of the corresponding absorbance profile indicates
the reisomerization rate constant to be 4ꢀ10-4 s-1. This value
is approximately 1 order of magnitude smaller than that (cf. 2 ꢀ
10-3 s-1) measured in acetonitrile (curve c in Figure S3, Sup-
porting Information) and comparable to that determined for
similar merocyanines in dimethylformamide,41 suggesting, once
again, the presence of a relatively hydrophobic environment
around the photochromic switch within the polymer micelles.
Similarly, 2b also reverts back to 2a upon visible illumination, and
in fact, the photochromic compounds entrapped within the
polymeric assembly can be switched back and forth between
their two states by alternating ultraviolet and visible irradiation
steps (curve b in Figure 6).
In analogy to the photochrome 2a, the fluorophore 4 also
tends to localize within the hydrophobic interior of the polymeric
assembly. The corresponding absorption spectrum (curve c in
Figure 5) shows the BODIPY absorption at 528 nm and does not
change after ultraviolet illumination (curve d in Figure 5), indi-
cating that the macromolecular envelope protects the fluorophore
from the aqueous environment. Indeed, control experiments in
the absence of the polymer host demonstrate that the exposure
of 4 to water encourages photobleaching. Specifically, the
absorption spectrum of 4 in acetonitrile shows the BODIPY
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Figure 7. Absorption spectra of a PBS dispersion (pH 7.4, 20 °C) of
polymer micelles containing 2a and 4 recorded after equilibration in the
dark for 12 h before (curve a) and after (curve b) irradiation (365 nm, 0.4
mW cm-2, 5 min). Emission spectra (λex = 434 nm) of the same dis-
persion after equilibration and before (curve c) and after (curve d) irra-
diation. Evolution of the emission intensity at 545 nm of the same
dispersion after equilibration and with alternating irradiation steps
(curve e) at ultraviolet and visible (562 nm, 0.3 mW cm-2, 5 min)
wavelengths.
Figure 8. Confocal fluorescence images (λex = 514 nm, λem = 535-635
nm, scale bar = 50 μm) of CHO cells recorded before (a) and after (b)
incubation with a PBS dispersion (10%, v/v) of 2a, 4, and 7 for 24 h.
steps (curve e in Figure 7). Interestingly, a plot of the absorbance
of 2b against the number of switching steps is essentially identical
to that recorded in the absence of the BODIPY component
(curve b in Figure 6) and shows a decrease of ca. 8% after four
switching cycles. Instead, the emission intensity of the BODIPY
component drops by ca. 40% after the same number of switching
cycles. This behavior suggests that the fluorescent component is
more susceptible to photodegradation than the photochromic
one under these experimental conditions.
Intracellular Fluorescence Modulation and Cytotoxicity
Assays. The ability of the amphiphilic copolymer 7 to transport
2a and 4 across cell membranes was assessed by confocal micros-
copy using Chinese hamster ovarian (CHO) cells. Indeed,
polymers with pendant poly(ethylene glycol) chains are known
to enter cells and transport intracellularly even relatively large
inorganic nanoparticles.43,44 Although the exact mechanism re-
sponsible for intracellular accumulation is not clear at this stage,
endocytosis has previously been shown to result in the localiza-
tion of similarly sized nanostructured assemblies in endosomes.43
In our experiments, CHO cells were imaged before and after
(parts a and b, respectively, of Figure 8) incubation with a PBS
dispersion of 2a, 4, and 7 for 24 h. The resulting images reveal
fluorescence within the cells only after their incubation with the
polymer micelles. In particular, the fluorescent supramolecular
assemblies cross the cell membrane and accumulate preferentially
in the cytosol with limited localization in the nucleus. Further-
more, the punctuate intracellular distribution of the fluorescent
constructs suggests their accumulation in restricted cytosolic
compartments, such as endosomes/vesicles. In addition, a stack
of images (Figure S8, Supporting Information), recorded along
the expected transfer of energy from the excited BODIPY
components to the photogenerated isomers of the photochromic
switches is predominantly responsible for the detected merocya-
nine fluorescence. Thus, these observations demonstrate that the
copolymer 7 is able to wrap around the fluorescent and photo-
chromic components and maintain them in sufficiently close
proximity to permit fluorescent modulation. Interestingly, DLS
measurements reveal that the hydrodynamic diameter of the
polymer micelles grows from 18 nm (vide supra) to 37 nm in the
presence of 2a and 4 with an increase of the average number of
polymer chains per micelle from 4 to 19. Consistently, TEM
images (Figure S1b, Supporting Information) also show an
increase in the average diameter of the polymer micelles from
17 nm (vide supra) to 35 nm in the presence 2a and 4. Thus, the
hydrophobic guests affect the ability of the macromolecular host
to assemble into supramolecular constructs and control the
physical dimensions of the resulting aggregates.
As observed in the absence of 4, 2b spontaneously reverts back
to 2a with first-order kinetics on a time scale of minutes. As a
result, its absorbance in the visible region decays (Figure S7,
Supporting Information) and the initial emission intensity of 4 is
restored. Similarly, the photogenerated species 2b switches back
to 2a also under visible irradiation, and in fact, the emission inten-
sity of its fluorescent partner 4 can be modulated for multiple
switching cycles by alternating ultraviolet and visible irradiation
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cells incubated with a PBS dispersion of only 4 and 7, under
otherwise identical conditions, does not change significantly with
ultraviolet illumination. Thus, the presence of the photochromic
component within the cells is essential to switch fluorescence
with optical inputs.
The cytotoxicity of the polymer micelles, containing 2a and 4,
as well as of the irradiation conditions required to switch intra-
cellularly the photochromic component was assessed with the
Trypan Blue assay.45 This particular organic dye stains exclu-
sively dead/dying cells and offers the opportunity to determine
the fraction of living cells (viability). Specifically, the viability was
determined for cells either incubated with increasing amounts
(0-25%, v/v) of a PBS dispersion of 2a, 4, and 7 (Figure S12a,
Supporting Information) or illuminated for increasing irradiation
times (0-1200 s) at ultraviolet wavelengths (Figure S12b). In
both instances, the cell viability remains essentially unchanged.
Thus, both the polymer micelles and the illumination conditions
required for their operations do not have any significant toxicity
on the CHO cells.
Figure 9. Emission intensity of CHO cells measured with a plate reader
(λex = 434 nm, λem = 550 nm) after incubation with a PBS dispersion
(10%, v/v) of 2a, 4, and 7 for 24 h before irradiation, immediately after
irradiation (365 nm, 0.4 mW cm-2, 1 min), and 10, 20, and 30 min after
irradiation, reported relative to that determined for identical cells incu-
bated without 2a under otherwise identical conditions.
’ CONCLUSIONS
An amphiphilic copolymer with hydrophobic decyl chains and
hydrophilic poly(ethylene glycol) tails along a common poly-
(methacrylate) backbone captures mixtures of BODIPY fluo-
rophores and spiropyran photochromes and transfers them in
aqueous phase. Hydrophobic decyl tails, however, must be at-
tached to the fluorescent and photochromic guests to bury them
within the hydrophobic interior of the macromolecular envelope
to prevent their exposure to water and preserve their photoche-
mical and photophysical properties. Within the resulting supra-
molecular constructs, the photoinduced and reversible transfor-
mation of the spiropyrans into the corresponding merocyanines
activates electron and energy transfer pathways and quenches the
BODIPY fluorescence. As a result, the emission intensity of these
systems can be modulated under optical control by switching the
photochromic components between their two states. Further-
more, the amphiphilic supramolecular container can cross cell
membranes, transport its cargo into the cytosol, and permit the
intracellular modulation of fluorescence. In addition, both the
supramolecular systems and the irradiation conditions required
for their operation are not cytotoxic. Thus, this simple supra-
molecular strategy to assemble photoswitchable fluorescent
constructs from separate fluorophores and photochromes and
impose biocompatibility on them can evolve into a valuable
protocol for the intracellular delivery and operation of functional
molecular components. In particular, the cellular internalization
of photoswitchable fluorophores can offer the opportunity to
visualize subcellular components with subdiffraction resolution
under the influence of patterned or sequential multiphoton
illumination. Hence, our protocol for the assembly of biocom-
patible fluorescent probes with photoswitchable character can
have profound implications in super-resolution imaging and,
ultimately, facilitate the experimental implementation of this
collection of promising analytical techniques for the investigation
of biological specimens at the nanoscale.
the optic axis, reveals consistent fluorescence evolution in the vertical
direction, indicating that the fluorophores remain trapped inside the
polymer micelles, rather than associating with the cell membrane.
The profile of the emission intensity measured along lines
drawn across cells (curves a-f in Figure S9, Supporting Infor-
mation) incubated with a PBS dispersion of 2a, 4, and 7 for 24 h
shows the predominant localization of the fluorescent constructs
at the periphery of the cells, rather than in their central region.
Upon ultraviolet illumination, 2a switches to 2b and the fluo-
rescence of 4 decreases (curves g-l in Figure S9). A similar
analysis for cells incubated only with 4 and 7, under otherwise
identical conditions, shows instead negligible changes in fluo-
rescence with ultraviolet irradiation (Figure S10, Supporting
Information). In fact, a comparison of the average emission
intensity, integrated along the drawn lines, shows a decrease of
ca. 33% upon ultraviolet irradiation only in the presence of 2a
within the polymer micelles (Figure S11, Supporting In-
formation). Thus, the ability of the photochromic component
to switch the emission of its fluorescent partner under optical
control, observed in aqueous solutions (vide supra), can be
reproduced even within living cells. These observations indicate
that the copolymer 7 can transport the fluorescent and photo-
chromic components 2a and 4 inside cells and maintain them in
close proximity within the intracellular environment to permit
fluorescence modulation.
To further support the ability of the photochromic component
to modulate the emission of its fluorescent partner intracellularly,
the fluorescence of CHO cells (Figure 9), incubated with a PBS
dispersion of 2a, 4, and 7 in a well plate for 24 h, was measured
with a plate reader relative to that of identical cells incubated
without 2a, under otherwise identical conditions. After ultravio-
let irradiation for only 30 s, the detected emission intensity
decreases to ca. 40% of the original value (Figure 9), in agree-
ment with the expected photoinduced interconversion of 2a into
2b with concomitant suppression of the fluorescence of 4. Upon
storage of the sample in ambient light, the photogenerated state
2b of the photochromic system gradually reverts back to the
original isomer 2a, and the fluorescence of 4 is restored over the
course of several minutes. On the contrary, the fluorescence of
’ EXPERIMENTAL PROCEDURES
Materials and Methods. CH2Cl2 and MeCN were distilled over
CaH2. THF was distilled over Na/benzophenone. H2O (18.2 MΩ cm)
was purified with a Barnstead International NANOpure DIamond Ana-
lytical system. Methacrylic acid was vacuum distilled. AIBN was crystallized
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twice from methanol. All other chemicals were used as received from
commercial sources. Compounds 1a and 3 were prepared following
literature procedures.13a,38 All reactions were monitored by thin-layer
chromatography, using aluminum sheets coated with silica (60, F254).
Fast atom bombardment mass spectrometry (FABMS) spectra were
recorded with a VG Mass Lab Trio-2 spectrometer in a 3-nitrobenzyl
alcohol matrix. NMR spectra were recorded with Bruker Avance 300 and
400 spectrometers. GPC was performed with a Phenomenex Phenogel
5-μm MXM column (7.8 ꢀ 300 mm) operated with a Varian ProStar
system, coupled to a ProStar 330 photodiode array detector, in THF
at a flow rate of 1.0 mL min-1. Monodisperse polystyrene standards
(2700-200000) were employed to determine the Mn and PDI of the
polymers from the GPC traces. DLS experiments were performed in
quartz cells (3 ꢀ 3 mm) with a Coulter N4 Plus apparatus, operating at a
wavelength of 632.8 nm (10 mW) with an orthogonal geometry. The
samples were dissolved in H2O (3 mL), filtered through Pall Corp.
syringe filters (0.1 μm) five times, and stored. The concentration was
adjusted to ensure scattering intensities in the range from 5ꢀ104 to 1ꢀ
106 counts/s. The nanoparticle size was calculated by averaging the
values of five runs of 300 s in unimodal size mode. TEM images were
recorded with an FEI Tecnai 12 microscope on copper grids (200 mesh)
using a uranyl acetate stain. Absorption spectra were recorded with a
Varian Cary 100 Bio spectrometer, using quartz cells with a path length
of 0.5 cm. Emission spectra were recorded with a Varian Cary Eclipse
spectrometer in aerated solutions. Fluorescence quantum yields were
determined with fluorescein and rhodamine B standards, according to a
literature procedure.46 The samples were irradiated at 365 nm (0.4 mW
cm-2) with a Mineralight UVGL-25 lamp and at 562 nm (0.3 mW
cm-2) with a Spectral Energy LH 150/1 light source. The output power
at both wavelengths was determined with a Newport 1815-C power meter.
Synthesis of 8. A solution of 2,3,3-trimethylindolenine (1 g, 6 mmol)
and 1-bromodecane (4.2 g, 19 mmol) in MeCN (15 mL) was heated
under reflux and Ar for 12 h. After the solution was cooled to ambient
temperature, the solvent was distilled off under reduced pressure. The
residue was washed with Et2O (3 ꢀ 30 mL) and dried under reduced
pressure to afford 8 (1.2 g, 50%) as a red waxy solid. FABMS: m/z = 300
distilled off under reduced pressure. The residue was purified by column
chromatography [SiO2:CHCl3/MeOH (98:2, v/v)] to afford 4 (63 mg,
47%) as a red solid. FABMS: m/z = 563 [M þ H]þ. 1H NMR (CDCl3):
δ = 0.89 (3H, t, 13 Hz), 0.98 (6H, t, 15 Hz), 1.13-1.41 (14H, m), 1.25
(6H, s), 1.65-1.68 (2H, m), 2.27-2.32 (4H, m), 2.54 (6H, s), 3.49
(2H, m), 6.41 (1H, br s), 7.37 (2H, d, 8 Hz), 7.90 (2H, d, 8 Hz). 13C
NMR (CDCl3): δ = 12.3, 12.9, 14.5, 14.9, 17.5, 23.1, 27.5, 29.7, 29.8,
29.9, 30.1, 32.3, 40.7, 128, 129.1, 130.8, 133.4, 135.5, 138.6, 139.2, 139.5,
154.6, 167.
Synthesis of 5. A solution of DCC (2.3 g, 11 mmol) in CH2Cl2 (20
mL) was added dropwise, over the course of 20 min, to a solution of
1-decanol (1.5 g, 10 mmol), DMAP (232 mg, 2 mmol), and methacrylic
acid (816 mg, 10 mmol) in CH2Cl2 (60 mL) maintained at 0 °C under
Ar. The reaction mixture was allowed to warm to ambient temperature
and was stirred for 24 h under these conditions. The resulting precipitate
was filtered off, and the solvent was distilled off under reduced pressure.
The residue was purified by column chromatography [SiO2:hexane/
EtOAc (2:1, v/v)] to afford 5 (1.5 g, 70%) as a colorless oil. FABMS:
1
m/z=228 [M þ H]þ. H NMR (CDCl3): δ = 0.90 (3H, t, 13 Hz),
1.20-1.36 (14H, m), 1.63-1.70 (2H, m), 1.95 (3H, s), 4.15 (2H, t, 13
Hz), 5.54 (1H, s), 6.09 (1H, s). 13C NMR (CDCl3): δ = 14.5, 18.7, 23.1,
26.4, 29, 29.6, 29.7, 29.9, 32.3, 65.2, 125.5, 136.9, 167.9.
Synthesis of 6. A solution of DCC (1.2 g, 3.6 mmol) in CH2Cl2
(20 mL) was added dropwise, over the course of 20 min, to a solution of
poly(ethylene glycol) monomethyl ether (Mn = 2000, 10 g, 5 mmol),
DMAP (244 mg, 2 mmol), and methacrylic acid (860 mg, 10 mmol) in
CH2Cl2 (80 mL) maintained at 0 °C under Ar. The reaction mixture was
allowed to warm to ambient temperature and was stirred for 24 h under
these conditions. The resulting precipitate was filtered off, and the
solvent was distilled off under reduced pressure. The residue was
purified by column chromatography [SiO2:CHCl3/MeOH (19:1,
1
v/v)] to afford 6 (6 g, 60%) as a white solid. H NMR (CDCl3): δ =
1.95 (3H, s), 3.38 (3H, s), 3.54-3.88 (180H, m), 4.30 (2H, t, 5.32,
10 Hz), 5.57 (1H, s), 6.13 (1H, s).
Synthesis of 7. A solution of 5 (73 mg, 0.3 mmol), 6 (1 g, 0.5 mmol),
and AIBN (3 mg, 0.03 mmol) in degassed THF (8 mL) was heated for
72 h at 75 °C under Ar in a sealed vial. After the solution was cooled to
ambient temperature, the reaction mixture was transferred to a centri-
fuge tube and diluted with THF to a total volume of 10 mL. Hexane was
added in portions of 1 mL, and the tube was shaken vigorously after each
addition until the formation of a precipitate was clearly observed. After
centrifugation, the oily layer at the bottom of the tube was separated
from the supernatant and dissolved in THF (10 mL). Hexane was added
in portions of 1 mL, and the tube was shaken vigorously after each
addition until the formation of a precipitate was clearly observed. After
centrifugation, the oily residue was separated from the supernatant and
dried under reduced pressure to give 7 (0.8 g) as a white solid. GPC:
Mn = 46 800, PDI = 1.73. 1H NMR (CDCl3): δ = 0.70-0.89 (3H, br s),
1.14-1.28 (8H, br s), 1.61-1.68 (2H, m), 1.95-2.07 (3H, br s), 3.38
(2H, s), 3.54-3.90 (135H, m), 4.25-4.30 (3H, br s).
1
[M - Br]þ. H NMR (CDCl3): δ = 0.82 (3H, t, 13 Hz), 1.19-1.32
(12H, m), 1.41 (2H, m), 1.61 (6H, s), 1.89 (2H, t, 15 Hz), 2.87 (3H, s),
4.68 (2H, t, 15 Hz), 7.44-7.47 (1H, m), 7.51-7.54 (2H, m), 7.63 (1H,
m). 13C NMR (CDCl3): δ = 14.4, 15.1, 22.9, 23.1, 23.5, 27.2, 28.4, 29.4,
29.5, 29.7, 32.2, 50, 54.5, 115.7, 123.1, 129.6, 130.4, 141.3, 142.1, 196.3.
Synthesis of 2a. A solutionof2-hydroxy-5-nitrobenzaldehyde(0.38 g,
2 mmol), 8 (0.86 g, 2 mmol), and piperidine (0.19 g, 2.3 mmol) in EtOH
(10 mL) was heated under reflux for 3 h. The reaction mixture was
allowed to cool to ambient temperature, and the solvent was distilled off
under reduced pressure. The residue was purified by column chroma-
tography [SiO2:hexane/EtOAc (19:1, v/v)] to afford 2a (0.35 g, 35%) as
a light-orange solid. FABMS: m/z = 449 [M þ H]þ. 1H NMR (CDCl3):
δ = 0.89 (3H, t, 14 Hz), 1.14(3H, s), 1.19-1.34 (16H, m), 1.29 (3H, s),
3.14 (2H, m), 5.85 (1H, d, 8 Hz),6.58 (1H,d, 8 Hz), 6.75 (1H, d, 8 Hz),
6.89 (2H, m), 7.10 (1H, d, 8 Hz), 7.20 (1H, m), 8.01 (2H, m). 13C NMR
(CDCl3): δ = 14.5, 20.3, 23.1, 26.4, 27.7, 29.4, 29.7, 29.8, 29.9, 30, 32.3,
44.2, 53.1, 107.2, 115.9, 118.9, 119.6, 122, 122.5, 123.1, 126.3, 128.1,
128.3, 136.3, 141.3, 147.6, 160.1.
Polymer Micelles. A solution of 7 (2.5 mg mL-1, 100 μL) in
CHCl3 was added to a solution of 1a (0.1 mg mL-1, 100 μL), 2a (0.1 mg
mL-1, 40 μL), 3 (0.1 mg mL-1, 12 μL), or 4 (0.1 mg mL-1, 30 μL) in
CHCl3. Alternatively, a solution of 7 (2.5 mg mL-1, 200 μL) in CHCl3
was mixed with solutions of 2a (0.1 mg mL-1, 20 μL) and 4 (0.1 mg
mL-1, 100 μL) in CHCl3. Each mixture was heated at 40 °C in an open
vial. After the evaporation of the solvent, the residue was purged with air
and dispersed in PBS (1 mL, pH 7.4). After vigorous shaking, the disper-
sion was filtered, and the filtrate was used for the spectroscopic and
imaging experiments without further purification.
Synthesis of 4. A solution of N,N0-dicyclohexylcarbodiimide (DCC;
58 mg, 0.3 mmol) in CH2Cl2 (5 mL) was added dropwise over the course
of 10 min to a solution of 2 (100 mg, 0.2 mmol), N-hydroxysuccinimide
(33 mg, 0.3 mmol), and 4-(dimethylamino)pyridine (DMAP; 3 mg, 0.02
mmol) in CH2Cl2 (15 mL) maintained at 0 °C under Ar. The reaction
mixture was allowed to warm to ambient temperature and was stirred
for 15 h under these conditions. The precipitate was filtered off, and
n-decylamine (45 mg, 0.3 mmol) was added dropwise to the filtrate over
the course of 10 min. The resulting solution was stirred at ambient
temperature for 12 h. The precipitate was filtered off, and the solvent was
Phospholipid Micelles. A solution of 1,2-dipalmitoyl-sn-glycero-
3-phosphoethanolamine N-(methoxy(polyethylene glycol)-2000) am-
monium salt (20 mg mL-1, 100 μL) in CHCl3 was added to a solution of
2a (1.2 mg mL-1, 33 μL) or 4(0.6 mg mL-1, 33 μL) in CHCl3 maintained
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Journal of the American Chemical Society
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(2) Brown, G. H., Ed. Photochromism; Wiley: New York, 1971.
at ambient temperature in an open vial. After the evaporation of the
solvent, the residue was dried under reduced pressure, dispersed in H2O
(1.2 mL), heated to 80 °C for 30 s, and passed through a syringe filter
(0.2 μm). Aliquots of the filtrate (100 μL) were diluted with H2O (300 μL)
and used for the spectroscopic experiments without further purification.
Intracellular Fluorescence Modulation. CHO cells were cul-
tured in Hams-F12 essential media, supplemented with fetal bovine
serum (10%, v/v), penicillin (200 U mL-1), streptomycin (200 μg
mL-1), and glutamine (2 mM), and incubated overnight at 37 °C in O2/
CO2/air (20:5:75, v/v/v). The cultured cells were incubated further
with PBS dispersions (10%, v/v) of 4 and 7 with and without 2a for 24 h
on glass coverslips. After being washed three times with PBS (1 mL), the
coverslips were imaged with an inverted Leica SP5 confocal/multi-
photon microscope. The samples were excited at 514 nm, and the
emission was recorded from 535 to 635 nm. Alternatively, the coverslips
were transferred in well plates, and their fluorescence was measured with
a Flex station luminescent plate reader. The samples were excited at 434
nm, and the emission was measured at 550 nm from the bottom of the
plate with the automatic emission cut-off switched off. Ultraviolet
irradiation was performed with a UVP UVGL-58 lamp, operating at
365 nm (0.4 mW cm-2).
Cytotoxicity Assays. CHO cells were seeded in well plates at a
density of 5 ꢀ 104 cells mL-1, incubated overnight at 37 °C in O2/CO2/
air (20:5:75, v/v/v), and spiked with increasing volumes (0-25%, v/v)
of a stock PBS dispersion of 2a (0.02 mg mL-1), 4 (0.01 mg mL-1), and
7 (0.25 mg mL-1). The cells were incubated for a further 24 h, harvested
by trypsinization, and resuspended in Hams-F12 media and Trypan Blue
(0.4%, v/v). After incubation at ambient temperature for 1 min, the cells
were counted with an Invitrogen Countess hemocytometer, and their
viability was determined. Ultraviolet irradiation was performed with a
UVP UVGL-58 lamp, operating at 365 nm (0.4 mW cm-2).
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’ ASSOCIATED CONTENT
S
Supporting Information. TEM images of polymer mi-
b
celles without and with 2a and 4 in their interior, synthesis of 2a,
absorption spectra of 2a in MeCN, absorption spectra of 4 in
MeCN and MeCN/H2O, absorption spectra of 4 in phospho-
lipid micelles, absorption spectra and absorbance evolution after
irradiation of 2a in phospholipid micelles, absorbance evolution
after irradiation of 2a and 4 in polymer micelles, vertical stack of
fluorescence images of CHO cells incubated with 2a, 4, and 7,
profiles of the emission intensity measured across CHO cells
incubated with 2a, 4, and 7, profiles of the emission intensity
measured across CHO cells incubated with 4 and 7, relative
emission intensity measured across CHO cells incubated with
and without 2a, and cytotoxicity assays. This material is available
free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
J.Callan@ulster.ac.uk; fraymo@miami.edu
’ ACKNOWLEDGMENT
We thank the National Science Foundation (CAREER Award
CHE-0237578 and CHE-0749840) and the University of Miami
for financial support.
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