Small Molecule-Based Fluorescent Organic Nanoassemblies with Strong Hydrogen Bonding Networks for Fine Tuning and Monitoring Drug Delivery in Cancer Cells

Article abstract of DOI:10.1002/smll.201802307

Bright supramolecular fluorescent organic nanoassemblies (FONs), based on strongly polar red-emissive benzothiadiazole fluorophores containing acidic units, are fabricated to serve as theranostic tools with large colloidal stability in the absence of a polymer or surfactant. High architectural cohesion is ensured by the multiple hydrogen-bonding networks, reinforced by the dipolar and hydrophobic interactions developed between the dyes. Such interactions are harnessed to ensure high payload encapsulation and efficient trapping of hydrophobic and hydrogen-bonding drugs like doxorubicin, as shown by steady state and time-resolved measurements. Fine tuning of the drug release in cancer cells is achieved by adjusting the structure and combination of the fluorophore acidic units. Notably delayed drug delivery is observed by confocal microscopy compared to the entrance of hydrosoluble doxorubicin, demonstrating the absence of undesirable burst release outside the cells by using FONs. Since FON-constituting fluorophores exhibit a large emission shift from red to green when dissociating in contact with the lipid cellular content, drug delivery could advantageously be followed by dual-color spectral detection, independently of the drug staining potentiality.

Full text of DOI:10.1002/smll.201802307

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Drug Delivery
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Small Molecule-Based Fluorescent Organic Nanoassemblies
with Strong Hydrogen Bonding Networks for Fine Tuning
and Monitoring Drug Delivery in Cancer Cells
Joanna Boucard, Camille Linot, Thibaut Blondy, Steven Nedellec, Philippe Hulin,
Christophe Blanquart,* Lénaïc Lartigue, and Eléna Ishow*
This dual imperative requires the combi-
Bright supramolecular fluorescent organic nanoassemblies (FONs), based on
strongly polar red-emissive benzothiadiazole fluorophores containing acidic
units, are fabricated to serve as theranostic tools with large colloidal stability
in the absence of a polymer or surfactant. High architectural cohesion is
ensured by the multiple hydrogen-bonding networks, reinforced by the dipolar
and hydrophobic interactions developed between the dyes. Such interactions
are harnessed to ensure high payload encapsulation and efficient trapping
of hydrophobic and hydrogen-bonding drugs like doxorubicin, as shown
by steady state and time-resolved measurements. Fine tuning of the drug
release in cancer cells is achieved by adjusting the structure and combination
of the fluorophore acidic units. Notably delayed drug delivery is observed by
confocal microscopy compared to the entrance of hydrosoluble doxorubicin,
demonstrating the absence of undesirable burst release outside the cells by
using FONs. Since FON-constituting fluorophores exhibit a large emission
shift from red to green when dissociating in contact with the lipid cellular
content, drug delivery could advantageously be followed by dual-color spec-
tral detection, independently of the drug staining potentiality.
nation of drugs with signaling units that
can now be smartly addressed by using
well-designed functional nanomaterials,
commonly referred as to nanometric drug
delivery systems (DDS).[1] NanoDDS actu-
ally offer the great advantage of preferen-
tial accumulation in cancerous tissues due
to passive enhanced and permeation reten-
tion effect,[2] while their nanometer size
allows for large surface-to-volume ratio
interactions, and simultaneous assem-
bling of various functional compounds
within a single object.[3] Using fluorescent
probes as drug tracers has appeared as
the most attractive approach for the very
high sensitivity of photodetectors and its
easy implementation to carry out manda-
tory pharmacokinetics and cellular inves-
tigations, prior to any small-animal and
clinical trials.[4] To reach this goal, most of
the engineered emissive nanoDDS incor-
porate the fluorescent labels and drugs
mixed together in liposomes,[5] polymeric vesicles,[6] micelles,[7]
or capsules,[8] and dendrimers[9] by taking advantage of electro-
static or van der Waals interactions between the active guests
and host matrices.[10] Since 60% of the active drugs display
low water solubility, their encapsulation requires carefully
engineered amphiphilic matrices to achieve good vectoriza-
tion and limit drug agglomeration in physiological media.[11]
This increases the level of synthetic complexity or compels to
tedious covalent binding between the matrix and the tracers
or drugs whose activation or release requires further adequate
stimuli.[12] In order to considerably simplify the amount of con-
stituents and avoid dye and drug leakage in water, self-assem-
blies of small hydrophobic molecules have recently appeared
as very attractive alternatives.[13] They are expected to dissociate
into individual components only in contact with the cellular
lipid constituents, which limits undesirable burst release in the
blood stream. Moreover, compared to polymeric matrices, they
offer the superior advantage of little local viscosity increase that
occurs by progressive erosion of polymers, impairing drug dif-
fusion into the diseased tissues.[14] Finally, they exhibit larger
drug payloads and minimized drug partition thanks to a higher
mixing entropy stemming from their small molecule-based
composition.[15] One particularly promising class of molecular
1. Introduction
Controlling and monitoring drug delivery constitute high med-
ical challenges to avoid harmful side effects and enhance the
efficiency of treatments. In this way, unprecedented levels of
therapeutic safety and personalized medicine will be achieved.
J. Boucard, Dr. L. Lartigue, Prof. E. Ishow
CEISAM–UMR CNRS 6230
Université de Nantes
2 rue de la Houssinière, 44322 Nantes, France
E-mail: elena.ishow@univ-nantes.fr
Dr. C. Linot, T. Blondy, Dr. C. Blanquart
CRCINA INSERM
INSERM U1232
Université de Nantes, Université d’Angers
8 quai Moncousu, 44007 Nantes, France
E-mail: christophe.blanquart@inserm.fr
Dr. S. Nedellec, Dr. P. Hulin
INSERM Nantes UMS 016-UMS CNRS 3556
8 quai Moncousu, 44007 Nantes, France
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201802307.
DOI: 10.1002/smll.201802307
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nanoassemblies relies on fluorescent organic nanoparticles
(FONs) that have recently emerged for less than a decade and
are fabricated out of small π-conjugated fluorophores.[16] The
latter are merely self-assembled into bright FONs through a
straightforward solvent-shift nanoprecipitation process con-
sisting in the quick addition of a concentrated organic stock
solution into a large volume of water. Stable colloidal suspen-
sions are obtained without requiring additional surfactants,
so that FONs have proved remarkable for live cell imaging,[17]
near-infrared cell tracking,[18] metal cation sensors,[19] and more
recently super-resolution fluorescence microscopy upon photo-
switching.[20] They nowadays fiercely compete with quantum
dots in terms of emission versatility, brightness efficiency,
and biocompatibility thanks to the absence of heavy metals,
and their large amount of self-assembled dyes up to ≈104–105
depending on the size. Curiously, FONs have scarcely been
harnessed to entrap molecular hydrophobic drugs despite
their apparent structural match and the simplicity of the nano-
precipitation process. In this respect, only two examples have
been reported in the literature and involved for both of them
covalently linked fluorophores and dyes.[21] The first example
regarded tetraphenylethylene-based fluorophores bound to
doxorubicin,[22] a well-known hydrophobic anticancer drug,
whose ulterior photodecaging required blue light. In addition,
since full emission quenching was observed upon fluorescence
resonance energy transfer, FON internalization could not be
followed. Drug release was signaled through blue emission
recovery of the disconnected fluorophores, which unavoidably
yields large tissue autofluorescence due to the high excitation
energy. Similar limitations were also noticed with perylene
derivatives transformed as chlorambucil prodrugs that recov-
ered blue emission after bond breaking at low pH.[21a] These
rare examples show that the design of FONs, amenable to
develop strong association with hydrophobic and neutral phar-
maceuticals without resorting to covalent linkage, has not been
explored yet, and will represent a very attractive and versatile
approach to offer theranostic nanomedicines with minimized
amount of excipients or stabilizing agents. In addition, tar-
geting drug release upon FON dissociation, characterized by a
change of emission color in the red/green spectral region more
compatible with the first biological transparency window and
higher spectral detection sensitivity, will offer the great twofold
advantage of a monitoring independent on the drug electronic
nature and a large emission contrast. As numerous drugs con-
tain protic units (amino, carboxylic, or phosphonic), strong
networks of intermolecular hydrogen-bonds[23] are expected to
strongly enhance the cohesion between the dyes and the drugs
confined within the nanoassemblies, thereby contributing
to reinforce the already existing noncovalent van der Waals
interactions.[24] With this strategy in mind, we want to show
herein that smart FONs made of solvatochromic fluorophores,
endowed with acidic units (carboxylic, phosphonic), will repre-
sent a novel design to generate nanoDDS with dual capability
of high drug encapsulation and two-color drug release moni-
toring. To our knowledge, generating FONs displaying acidic
units to encapsulate hydrophobic and protic pharmaceuticals,
which requests subtle structural hydrophobic–hydrophilic bal-
ance to ensure FON formation, has surprisingly never been
explored so far. In order to demonstrate the applicability of this
Figure 1. A) Structures of p-BDZ, c-BDZ as FON units and doxorubicin.
B) p-BDZ and c-BDZ in toluene (λexc = 365 nm) and as FONs in water
(λexc = 450 nm).
novel approach, we took as a hydrogen-bonding drug model
the archetypal amino-containing and anthracycline antican-
cerous doxorubicin, often used as liposomal formulation,[21b]
and studied its release in water and in cellulo by using confocal
microscopy as well as steady-state and time-resolved fluores-
cence measurements.
2. Results and Dicussion
2.1. Design and Structural Comparative Character
of Hydrogen-Bonding x-BDZ FONs
Two strongly polar and red-emissive fluorescent dyes, differing
by the strength of their acidic units, have been devised. They
incorporate a benzothiadiazole (BDZ) unit, known for its high
photostability and fluorescence efficiency (Figure 1),[25] and
imparting large charge transfer when combined to electron-
withdrawing groups.[26]
Their structure contains additional bulky groups to prevent
strong aggregation and emission quenching after self-assem-
bling as FONs.[27] No crystalline structure is indeed formed, as
revealed by thermal analyses using differential scanning calo-
rimetry showing glass transition temperatures Tg at 145 and
173 °C for p-BDZ and c-BDZ, respectively (Figure S1, Sup-
porting Information). Such amorphous behavior advantageously
limits segregation and expulsion risks in case of drug doping,
which can operate in solid lipid nanocarriers in the absence
of emulsifiers due to undesirable recrystallization at biological
temperatures.[28] We finally targeted the phophonic and carbox-
ylic acids as acidic units, since they are the most commonly
encountered organic acids, provide stable compounds, and dis-
play substantial distinct acidobasicity constants that can impact
the nanoDDS cohesion. After a six to eight-step synthetic pro-
cedure (Scheme S1 of the Supporting Information for synthetic
details), the phosphonic (p-BDZ) and carboxylic (c-BDZ) deriva-
tives were obtained as bright red powders that were readily
soluble in almost all solvents, except cyclohexane and alcoholic
solvents to a lesser extent. Both compounds p-BDZ and c-BDZ,
generically noted x-BDZ in the following, display significantly
higher Tg values (146 and 173 °C, respectively) than that of their
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Figure 2. A) Schematic description of the nanoprecipitation method to fabricate pristine FON and FON blends out of stock solutions in THF (50 µL)
added to a large amount of water (2.5 mL). B) TEM imaging of pristine and dox-doped x-BDZ FONs (scale bar: 50 nm). C) UV-vis absorption and
emission spectra (λexc = 465 nm) of p-BDZ and c-BDZ FONs, dox-doped p-BDZ and c-BDZ FONs and dox.HCl in water.
benzaldehyde precursor (Tg = 96 °C), devoid of protic units,
which already show the strong interactions developed between
the acidic units upon hydrogen-bonding.
2.2. Acidity Impact on Photophysical Properties of x-BDZ
Fluorophores and FONs
Nanoprecipitation in a large volume of water from a
1.45 mmol L−1 tetrahydrofuran (THF) solution yielded spher-
ical FONs that could clearly be characterized by transmis-
sion electron microcopy (TEM) without additional staining
(Figure 2A). The aromatic backbones thus efficiently counter-
balanced the protic character of the acidic units and ethylenoxy
chains, leading to water-insoluble nanomaterials.
Interestingly, their TEM diameters dTEM were found system-
atically larger for c-BDZ (44 3 nm) than for p-BDZ (24 2 nm)
(Figure 2B). Such differences have been ascribed to the higher
Significant differences regarding the photophysical properties
were actually noted between p-BDZ and c-BDZ. Both com-
pounds display similar absorption spectra with a low-energy
band centered at 454 nm in THF solution, and 466 nm as FON
(Figure S3, Supporting Information) as a result from charge
transfer from the triphenylamino to the benzothiadiazole
moiety. By contrast, a noticeable bathochromic shift of the emis-
sion band, peaking at 664 and 661 nm for p-BDZ and c-BDZ,
respectively, was observed in THF solution. This shift reveals
to be exacerbated in FONs with an emission maximum at 638
and 625 nm for p-BDZ and c-BDZ, respectively (Figure 2C;
Table S1, Supporting Information). We ascribed this discrep-
first acidity for PO3H2 (pKa1 = 1.1–2.3)[29] than for CO2H
(pKa = 3.2–4.2)[29a] units. Since FONs are here formed after a
nucleation-aggregation process given the respective amounts
of solvents used, the lower the solute concentration, the larger
the FON final size. Hence, ionized or strong hydrogen-bonding
dyes tend to generate smaller FONs, as noticed for p-BDZ or
already reported urea dyes.[30]



ancy to the stronger acidic character of PO3H2 acid units as

compared to CO2H, consequently inducing stronger hydrogen
bonding interactions. This impact of hydrogen bonding on the
radiative excited state is particularly true when considering the
fluorescence quantum yield Φf which notably drops from THF
solution to aqueous FON suspensions, namely from 25 to 2.2%
for p-BDZ and 27 to 11% for c-BDZ, respectively (Table S1, Sup-
porting Information). This considerable decrease was found
to partly result from radiationless deactivation through strong
In the prospect of biological studies, colloidal stability was
eventually investigated in saline media and culture media
((Roswell Park Memorial Institute medium (RPMI) supple-
mented with 8% serum). Since dynamic light scattering meas-
urements could not properly be carried out with p-BDZ FONs
given their small size and scattering effects, UV–vis absorp-
tion and emission measurements, as well as epifluorescence
microscopy were privileged. They revealed no detectable micro-
metric aggregation of FONs that would have caused significant
light scattering and hypochromic change in the intensity signal,
making FONs fully suitable for further in cellulo experiments
(Figure S2, Supporting Information).

vibrational coupling with water O H oscillators. Indeed,
replacing H2O with D2O caused a 50 and 37% increase in
the emission quantum yield Φf for p-BDZ and c-BDZ FONs,
respectively, confirmed by a similar increase in the intensity
averaged lifetimes of the excited states (33 and 22%, respec-
tively) (Figure S4 and Table S2, Supporting Information). We
can thus conclude on strong cohesion of a water shell around
the protic FONs and suspect that similar results would apply to
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Table 1. Structural and photophysical properties of dox-free x-FON and
dox-doped dox/x-FON nanossemblies in water. All syntheses have been
repeated three times.
solution to remove nonassociated compounds, especially doxo-
rubicin, could reasonably be ruled out. Interestingly, nanopre-
cipitation of x-BDZ FONs in a solution containing dox.HCl also
showed quite a large fluorescence quenching of FONs com-
pared to aqueous solution, though to a lesser extent than that
observed for doped dox/x-BDZ FONs (84 and 65% for dox/p-
BDZ and dox/c-BDZ FONs, respectively) (Figures S6 and S7,
and Table S4, Supporting Information). These results indicate
that the encapsulation strategy starting from neutral com-
pounds is superior to the approach based on electrostatic inter-
actions between charged water-soluble molecules, as recently
reported.[32] They also indirectly prove the higher hydrogen-
bonding interactions of phosphonic acid units with dox, since
p-BDZ underwent larger quenching effects in the presence of
dox. Finally, from the above-reported photophysical measure-
ments and the millisecond process of nanoprecipitation,[33]
composition of the nanoblends can reasonably be assumed as
mirroring the composition of the stock organic mixtures.
FON
d
TEM (σ) in nm
ζ (mV) λmaxabs (nm) λmaxem (nm) Φf (× 10−2
)
p-BDZ
466, 335
473, 336
466, 334
472, 337
464, 333
472, 334
497
638
613
625
616
630
613
595
2.2a)
0.2b)
11.4a)
2.3b)
4.5a)
0.5b)
4.2
24 2 (0.20 0.03) −26
33 1 (0.23 0.01) −23
44 3 (0.27 0.01) −30
29 1 (0.17 0.01) −20
1
2
2
1
2
1
dox/p-BDZ
c-BDZ
dox/c-BDZ
cp-BDZ
34 (0.21 0.01)
38 (0.19 0.01)
–
−30
−24
dox/cp-BDZ
dox.HCl
–
a)Measured from coumarin 540 A; b)One can notice a 81, 58, and 61% drop of the
intensity-averaged lifetimes 〈τs〉 of dox-free p-BDZ, c-BDZ, and cp-BDZ FONs when
the latter are doped with doxorubicin upon nanoprecipitation.
any kind of polar FONs, since local organization of water mole-
cules has recently been evidenced by nonlinear hyper-Rayleigh
measurements.[31]
In order to test dox release using the nanoblends, we used
fluorescence imaging, since doxorubicin stains the nuclei in red
upon intercalation into DNA to inhibit cell replication. Meso
cancer cells, found in malignant mesothelioma, a very severe
cancer caused by asbestos inhalation, were here chosen since
nanomedicine nowadays represents great promise as therapeutic
alternative to molecular drugs displaying very poor patient out-
come.[34] Control experiments were carried out using dox.HCl as
well as x-BDZ FONs for which no significant cell mortality was
detected for concentrations up to 1 µmol L−1 (Figure 3A).
2.3. Doxorubicin Encapsulation and Delivery Monitoring
in Cancer Cells
We next introduced doxorubicin to form theranostic mole-
cular nanoblends. The fluorescent dye and anticancerous drug
were mixed in a 1:1 ratio in THF and added in water under
fast stirring as above described. This ratio could be regarded
as the optimal one, as it ensures high drug loading, efficient
FON formation, and dox encapsulation, and finally easier com-
parison with nonassociated components to understand the
behavior of the resulting doped nanoassemblies. The resulting
nanoassemblies showed by TEM similar morphologies as those
of pristine BDZ FONs, proving that dox does not impede the
nanoprecipitation process (Figure 2B). It is worth noting that
the negative charge of the surface potential ζ measured by zeta-
metry decreased from −26 to −23 mV for p-BDZ and −30 to
−20 mV for c-BDZ FONs upon dox doping (Table 1).
This evolution tends to prove strong association between
x-BDZ and dox in its neutral form, confirmed by a change in the
nanoparticle size (Table 1). Photophysical measurements also
allowed us to rule out segregation and formation of nanopar-
ticles made exclusively of dox and x-BDZ during precipitation.
Indeed, both doped nanoassemblies dox/p-BDZ and dox/c-BDZ
underwent dramatic emission quenching as evidenced by the
considerable decrease in the emission intensity (up to 91 and
80% respectively) (Figure 2C) and fluorescence intensity decay
(Table 1; Figure S5 and Table S3, Supporting Information). In
this respect, time-resolved fluorescence measurements are par-
ticularly instructive, since the time components inferred from
decay modeling of the dox-doped dox/x-BDZ nanoassembly
emission did not contain the lifetime of free doxorubicin, found
at 1.00 ns (Table S3, Supporting Information). The large change
undergone in the emission decay of doped nanoassemblies
compared to those of the separated constituents thus indicates
full association of the molecular drug and dyes into nanoassem-
blies. At this stage, the necessity to purify the dox-doped FON
Confocal fluorescence microscopy done after various times
of incubation (t = 24, 48, 72 h) revealed small bright red-
orange spots inside the cytoplasm for both dox/x-BDZ and
x-BDZ FONs, which is typical of FON cell uptake by endocy-
tosis. The image purposely shows false colors to better evi-
dence FON evolution (Figure 3C). White and red colors were
assigned to emission in the green and red channels, respec-
tively. At time 24 h, x-BDZ FONs appeared pinkish as a result
of the overlay of their emission signals in both green and red
channels. At times 48 and 72 h, one can notice a very inter-
esting feature for c-BDZ FONs showing a signal mostly white,
while such evolution took longer for p-BDZ FONs owing to
the larger cohesion imparted by the intermolecular hydrogen
bonds between the phosphonc acid units. This observation
indicates that FONs structurally evolved due to the modi-
fication of the fluorophore surroundings. Previous studies
involving strongly solvatochromic fluorophores constituting
FONs let us suggest that FONs dissociate in contact with the
phospholipids of the endosome membrane instead of the cyto-
plasmic membrane.[35] In the latter case, FONs would have
dissolved and stained the cytoplasmic membrane, which is
actually not observed and contradicts the dot-like spreading of
FONs inside the cells after uptake. Hence, the separated fluo-
rophores underwent apolar surroundings where they emit at
lower wavelengths. A similar assumption could be applied to
dox/x-BDZ FONs, delivering their dox content upon progres-
sive dissociation. Indeed, red staining of the nuclei by doxoru-
bicin was found for all conditions, albeit notably delayed for
dox/x-BDZ FONs blends, with a maximum reached after 72 h
against 48 h for dox.HCl (Figure 3B). The green-grayish color
that could be detected for cells treated by dox.HCl, known for
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