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Self-Assembly of a Triazolylferrocenyl Dendrimer in Water Yields

Nontraditional Intrinsic Green Fluorescent Vesosomes for
Nanotheranostic Applications
Gabriel Perli,∥ Qi Wang,∥ Carolyne B. Braga, Diego L. Bertuzzi, Liniquer A. Fontana, Marco C. P. Soares,
Jaime Ruiz, Jackson D. Megiatto Jr., Didier Astruc,* and Catia Ornelas*
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ABSTRACT: The promising field of nanomedicine stimulates a continuous search for multifunctional nanotheranostic systems for
imaging and drug delivery. Herein, we demonstrate that application of supramolecular chemistry’s concepts in dendritic assemblies
can enable the formation of advanced dendrimer-based nanotheranostic devices. A dendrimer bearing 81 triazolylferrocenyl terminal
groups adopts a more compact shell-like structure in polar solvents with the ferrocenyl peripheral groups backfolding toward the
hydrophobic dendrimer interior, while exposing the more polar triazole moieties as the dendritic shell. Akin to lipids, the compact
dendritic structure self-assembles into uniform nanovesicles that in turn self-assemble into larger vesosomes in water. The vesosomes
emit green nontraditional intrinsic fluorescence (NTIL), which is an emerging property as there are no classical fluorophores in the
dendritic macromolecular structure. This work confirms the hypothesis that the NTIL emission is greatly enhanced by rigidification
of the supramolecular assemblies containing heteroatomic subluminophores (HASLs) and by the presence of electron rich
functional groups on the periphery of dendrimers. This work is the first one detecting NTIL in ferrocenyl-terminated dendrimers.
Moreover, the vesosomes are stable in biological medium, are uptaken by cells, and show cytotoxic activity against cancer cells.
Accordingly, the self-organization of these dendrimers into tertiary structures promotes the emergence of new properties enabling
the same component, in this case, ferrocenyl group, to function as both antitumoral drug and fluorophore.

T he design of supramacromolecular assemblies1−3 func-

tioning as vesosomes is actively pursued in order to
optimize multifunctional theranostic systems involving fluo-
defined systems with higher levels of structural organization
that present emerging properties.20,21,32−35
In this context, herein we demonstrate that triazolylferro-
rescent imaging and drug delivery.4 Beyond a certain cenyl dendrimer 1 bearing 81 ferrocenyl termini (Figure 1a)
generation, dendrimers5−8 can be designed to function as self-assembles in aqueous medium into vesosomes with
nanozymes,9 to work as supramolecular reactors,10 and to spherical shape and low dispersity, which emit green
perform as building blocks for supramolecular assemblies of nontraditional intrinsic fluorescence upon excitation. More
biomedical interest.11 Indeed, their size, composition, branch- importantly, the vesosomes are stable and fluorescent in
ing degree, hydrophobicity, and hydrophilicity make dendritic biological medium, are uptaken by cells, and present
materials valuable synthons in self-assembly processes driven cytotoxicity against cancer cells. Therefore, the present work
by secondary interactions.12−15 Multifunctionality and multi- establishes that dendrimer molecular engineering in con-
valency in dendrimers enable the anchorage of fluorescence junction with self-assembly techniques allow the organization
probes, drugs, and targeting groups for applications as of multifunctional systems for nanomedicine, in which the
nanotheranostics platforms; however, supramolecular self- same component, in this case, ferrocenyl moieties, have a dual
assembly could simplify the procedures to obtain such complex role, i.e., acting as both anticancer drugs and fluorescent probe.
structures.16−19 Interestingly, this is an uncommon case of nonamphiphilic
A new photoluminescence phenomenon referred to as dendrimers forming such organized supramolecular structures
nontraditional intrinsic luminescence (NTIL) emerges when in aqueous medium. Moreover, this is the first report on the
dendrimers containing electron rich heteroatomic functional detection of NTIL on dendrimers with the electron-rich
groups (heteroatomic subluminophores, HASLs) form aggre- ferrocenyl periphery.36
gates or unique self-assemblies.20−24 It was recently suggested
that NTIL emission is greatly enhanced by rigidification of the
supramolecular assemblies containing HASLs and by the Received: May 29, 2021
presence of electron rich moieties on dendrimer’s periphery.20 Published: July 22, 2021
Combination of self-assembly, nanomaterials, and fluorescence
has enabled the construction of innovative supramolecular
constructs with interesting emerging properties.25−31 Accord-
ingly, dendrons and dendrimers can self-assemble into well-

© 2021 American Chemical Society https://doi.org/10.1021/jacs.1c05551

12948 J. Am. Chem. Soc. 2021, 143, 12948−12954
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Figure 1. (a) Chemical structure of dendrimer 1; inset shows the triazolylferrocenyl termini. (b) Absorption spectra of 1 normalized to the
ferrocenyl band in CHCl3 (blue) and DMSO (red). (c) Hydrodynamic diameter of the self-assemblies formed by 1 in water obtained by DLS:
nonfiltered solution (red) and filtered solution (blue). (d) Size distribution plot of the nanoassembly formed by 1 in aqueous medium (filtered
solution) obtained by NTA. (e) Representative NTA video frame of the nanoparticles in solution. (f−i) SEM images of the self-assembled
microparticles formed by 1.

Dendrimer 137 was prepared from the classic organometallic groups (Δδ = 0.03 ppm) were not significantly affected by the
strategy developed by Astruc’s group to afford the nonaallyl change in solvent polarity. Such findings suggest the
dendritic core, while the 81 branches were built following the 1 hypothesized compact structure of 1 in DMSO, which is
→ 3 connectivity using a sequence of hydrosilylation, further corroborated by UV−vis. The absorption spectra of 1
Williamson etherification, nucleophilic substitution, and in CHCl3 shows the characteristic ferrocenyl and triazole
“click” reaction as described in our previous works.37−40 absorptions at λmax = 440 and 270 nm, respectively.
The dendritic structure of 1 is hydrophobic and flexible, Congruently, there are no significant electronic interactions
which is in sharp contrast with the amphiphilic dendrimers and among the ferrocenyl termini and triazole rings in 1 (Figure
dendrons commonly used in dendrimer self-assembly S2), which thereby must adopt the expected extended
processes. We reasoned that dendrimer 1 would adopt a macromolecular conformation in CHCl3. However, the
more compact structure in polar organic solvents with the absorption spectrum of 1 in DMSO shows a significant
nonpolar ferrocenyl moieties backfolding toward the hydro- broadening of the ferrocene absorption (Figure 1b). This
phobic dendritic interior, while exposing the more polar broadening informs considerable electronic interactions among
triazole groups as the dendritic shell to the polar solvent. We the ferrocenyl groups, which in turn confirms that they are
investigated that hypothesis by 1H NMR and UV−vis packed close together in DMSO. Conversely, the triazole band
absorption spectroscopies,41 using chloroform and dimethyl at λmax = 270 nm remains virtually unchanged, hence informing
sulfoxide (DMSO) as the good and poor solvents for 1, that they are well-solvated in DMSO as they are in CHCl3.
respectively. Thus, this preliminary spectroscopic investigation strongly
The 1H NMR spectrum of 1 in DMSO-d6 (Figure S1) suggests that solvophobic forces drives the folding of
reveals that the resonance assigned to the triazole proton was dendrimer 1 into a more compact structure with the ferrocenyl
deshielded (Δδ = 0.63 ppm) when compared to the same moieties buried inside the dendritic interior and the triazole
signal in CDCl3. Such deshielding is most likely due to the groups exposed to the solvent.
formation of hydrogen bonds between the triazole rings and The disposition of the ferrocene and triazole moieties in the
DMSO solvent molecules, thereby confirming that the former dendritic shell structure of 1 in DMSO led us to question
are exposed to the latter. On the other hand, the signals whether similar solvophobic forces assisted by hydrogen
attributed to the −SiCH2− moieties (Δδ = 0.12 ppm) as well bonding could drive the self-assembly of 1 into organized
as those assigned to the cyclopentadienyl rings of ferrocenyl superstructures in water. Therefore, 36 μL of an orange DMSO
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dendrimer solution (2.8 mg/mL, 69 μM of 1, 5.6 mM of Fc)

was added to 1.964 mL of deionized water in a 15 mL Falcon
tube, and the mixture was vortexed vigorously for 5 min and
left to rest overnight at room temperature (Figure S3). The
resulting solution (1.24 μM of 1) was colorless with no visual
formation of precipitates.
Dynamic light scattering (DLS) analysis (Figure 1c) reveals
that 1 formed two discrete larger structures: a nanoassembly in
the 100 nm size range and a larger structure in the micrometer
size range. The aqueous solution containing the self-assembled
dendrimers was filtered through a 0.45 μm syringe filter and
analyzed by DLS and nanoparticle tracking analysis (NTA).
The filtered solutions clearly showed the presence of
nanoparticles of around 100 nm with low dispersity (90 ±
44 nm by DLS and 111 ± 33 nm by NTA; Figures 1d,e). In
order to unveil the time frame for the self-assembly process,
the aqueous solution was analyzed by DLS at 15 min, 2 h, 4 h,
8 h, and 10 h after addition of the dendrimer. These data show
that the dendrimers start to self-assemble upon rapidly forming
nanoparticles and microparticles (as early as we can measure
by DLS, i.e., < 15 min; Figures S4−S8).
Characterization by scanning electron microscopy (SEM) in
the dry state reveals that 1 self-assembles into symmetrical and
low dispersity microspheres with an average diameter of 2.5
μm (Figures 1f−i). In the SEM images, smaller nanoparticles
with an average diameter of 100 nm are visible in the
background; a finding that is in good agreement with the DLS
measurements. Furthermore, a close inspection of the
magnified SEM image reveals nanoparticles on the surface of
the microparticles. Such observation suggests that dendrimer 1
preorganizes into nanoparticles, which in turn self-assemble
into the larger micrometer spheres. Due to the proposed
compact shell-like macromolecular conformation adopted by
dendrimer 1 in DMSO, in which the dendritic hydrophobic Figure 2. EF-TEM images of the suprastructures formed by the
interior packed with the backfolded ferrocenyl groups is dendrimer 1. (a) Dendrimer 1, nanoparticles, and vesosome at 0 eV
surrounded by the more polar peripheral triazole groups, we energy loss. (b) Dendrimer 1, nanoparticles, and vesosome at 125 eV
postulate that 1 functions as a lipid that self-assembles into energy loss. (c) Zoom on (a) to show the individual dendrimers. (d)
nanovesicle secondary structures that in turn self-organize into Self-assembly of dendrimer 1 to form a nanoparticle (0 eV energy
multivesicular vesicle tertiary structures (vesosomes). loss). (e) Vesosomes formed by the self-assembly of nanoparticles at 0
The hierarchical organization of the dendrimers into eV. (f) Vesosomes formed by the self-assembly of nanoparticles at 100
nanoparticles and then into vesosomes is further confirmed eV energy loss.
by energy-filtered transmission electron microscopy (EF-
TEM) that provides information about the internal morphol- (FBS) under the same conditions as in deionized water.
ogy of thick particles and aggregates with spatial resolution.42 Microscopy analysis of the resulting mixtures reveal that
Figure 2a shows the primary, secondary, and tertiary structures formation of the vesosomes is not affected by the presence of
formed by dendrimer 1, namely, (1) monodisperse individual all compounds present in the cell culture medium, which
dendrimers with 12 nm (zoom in on the Figure 2c), (2) includes enzymes, proteins, amino acids, antibiotics, lipids,
nanoparticles, and (3) vesosomes. Figure 2b was acquired at hormones, vitamins, and salts, thereby confirming the high
125 eV energy loss and it shows that the internal morphology selectivity of the hierarchical self-assembly process of
of the vesosomes is composed of nanoparticles (for complete dendrimer 1.
sequences of EF-TEM images see Figures S9 and S10). Figure To determine the chemical composition of the vesosomes,
2d suggests that the individual dendrimers self-assemble into the samples were submitted to energy-dispersive X-ray
nanoparticles, whereas Figures 2e and 2f show vesosomes spectroscopy (EDS) elemental analyses. The EDS spectrum
emerging from the self-assembly of nanoparticles. Remarkably, (Figure 3) confirms the high purity of the vesosomes by
the nanovesicles and vesosomes are stable in water for at least showing the typical peaks for all the elements in the dendritic
two months. structure of 1.
DLS data and SEM images of the nanovesicles and The EDS analysis informs no contamination of the
vesosomes afforded from dendrimer solutions at varying vesosomes by residual copper ions, which is a common serious
concentrations (from 1 to 50 μM) display no changes in the drawback in the synthesis of other macromolecules through
average size nor in the morphology of the assemblies. “click” reactions.43 However, the EDS investigation also reveals
Therefore, the self-assembly is independent of the dendrimer the presence of alkaline ions Na, K, and Ca in the vesosomes as
concentration. We also carried out the self-assembly process of trace impurities. The sulfur signal in the EDS spectrum
1 in cell culture medium containing 10% of fetal bovine serum indicates the presence of DMSO solvent molecules. SEM/EDS
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Figure 3. Energy-dispersive X-ray spectroscopy elemental analyses

(EDS) spectrum of the vesosomes formed by self-assembly of 1 in
water.

elemental mapping profiles of the vesosomes confirms the high

homogeneity of the samples with carbon and oxygen evenly
distributed in the large microparticles as well as in the
nanoparticles, whereas the sulfur, calcium, and potassium are
only detected in the large vesosomes (Figure 4).
These data suggest that solvophobic effects are the main
driving force for the self-assembly of the nanovesicles, which
self-organize into the vesosomes also driven by the solvophobic
forces but assisted by formation of hydrogen bonds with
solvent molecules.
These findings on the size and morphology of the vesosomes
prompted us to investigate eventual emerging properties of our
dendritic supramolecular microparticles. We have found that
the vesosomes emit green fluorescence light (λ = 525 nm)
when excited at 469 nm and visualized with Cytation 5 cell
imaging multimode reader equipped with a green fluorescent
protein (GFP) filter (Figures 5a,b). The fluorescence emission
of the vesosomes were also detected by high-resolution laser-
scanning confocal microscopy (LSCM) upon excitation at 406 Figure 4. SEM/EDS elemental mapping profiles of the vesosomes.
nm. The LSCM images clearly show the green fluorescence
emission of the vesosomes at λmax = 600 nm (Figures 5c,d and
S11). Since the DMSO solution of the dendrimer 1 is not Moreover, some vesosomes were able to penetrate the MCF-7
fluorescent due to the lack of classical fluorophores, which in cells remaining fluorescent in the intracellular medium. The
conjunction with the high purity of the vesosomes determined images also reveal that the cells containing the dendrimer-
by EDS44 led us to conclude that the observed green based vesosomes changed their morphology to round-shaped
fluorescence occurs via aggregate-induced emission mecha- cells suggesting that the vesosomes are able to induce cell
nisms, which are a result of the close packing of the ferrocenyl death (Figure S12).
groups within the dendritic structure and the consequent The cytotoxicity of the vesosomes was evaluated against
structural rigidification upon formation of the vesosomes that MCF-7 cells using the cell counting kit-8 (CCK-8) assay.
discourage nonemissive decay pathways. MCF-7 cells were incubated for 24 and 48 h in the presence of
Capitalizing on the emergent green fluorescence emission of the vesosomes at different concentrations (from 0.10 and
the vesosomes and the well-known antitumoral properties of 150.0 μM of ferrocenyl groups; from 1.2 nM to 1.8 μM of
ferrocenyl derivatives,45,46 we have carried out a preliminary dendrimer 1). The highest concentration tested resulted in
study on the in vitro properties of the dendritic vesosomes 48% cell viability after 24 h corresponding to an IC50 of 168
upon incubation with MCF-7 cells (breast cancer cells), using μM of Fc groups or 2.1 μM of dendrimer (Figure S13).
imaging with Cytation 5 imaging with a GFP filter (λex = 469 Incubation for 48 h resulted in 38% cell viability corresponding
nm; emission detector λ = 525 nm).47 Figure S12 shows to an IC50 of 124 μM of Fc groups or 1.5 μM of dendrimer
merged images of bright field and fluorescent images of the (Figure S14). These IC50 values are in the same range of values
MCF-7 cells incubated for 24 h with the vesosomes formed by obtained for known ferrocenyl derivatives tested against breast
dendrimer 1 in cell culture medium containing FBS. These cancer cells.45 Therefore, the cytotoxicity of the vesosomes is
images demonstrate that the vesosomes are stable and present most likely caused by the known mechanism for ferrocenyl
green fluorescence in cell culture medium containing FBS. derivatives that in physiological medium generate reactive
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■
pubs.acs.org/JACS Communication

AUTHOR INFORMATION
Corresponding Authors
Didier Astruc − Univ. Bordeaux, ISM, UMR CNRS 5255,
33405 Talence Cedex, France; orcid.org/0000-0001-
6446-8751; Email: [email protected]
Catia Ornelas − Institute of Chemistry, Rua Josue de Castro,
Cidade Universitaria Zeferino Vaz, University of Campinas,
13083-970 Campinas, SP, Brazil; orcid.org/0000-0001-
8020-9776; Email: [email protected]
Authors
Gabriel Perli − Institute of Chemistry, Rua Josue de Castro,
Cidade Universitaria Zeferino Vaz, University of Campinas,
13083-970 Campinas, SP, Brazil; Present
Address: Université de Lyon, CNRS UMR 5233,
Ingénierie des Matériaux Polymères, INSA Lyon, F-69621
Villeurbanne, France; orcid.org/0000-0003-2588-5100
Qi Wang − Univ. Bordeaux, ISM, UMR CNRS 5255, 33405
Figure 5. (a,b) Merged images of bright field and fluorescent images Talence Cedex, France; Present Address: Institutes of
of the vesosomes afforded from the self-assembly of dendrimer 1 in Physical Science and Information Technology, Anhui
water captured with Cytation 5 (GFP filter, excitation wavelength: University, Hefei, 230601, PR China.
469 nm; emission detector wavelength: 525 nm). (c,d) Laser- Carolyne B. Braga − Institute of Chemistry, Rua Josue de
scanning confocal microscopy (LSCM) images of the vesosomes Castro, Cidade Universitaria Zeferino Vaz, University of
upon excitation at 406 nm and monitoring at 600 nm. Campinas, 13083-970 Campinas, SP, Brazil; orcid.org/
0000-0003-1800-386X
oxygen species (ROS) that can damage DNA and other Diego L. Bertuzzi − Institute of Chemistry, Rua Josue de
biomolecules causing cell death. These preliminary data Castro, Cidade Universitaria Zeferino Vaz, University of
suggest that the self-assembly of the triazolylferrocenyl Campinas, 13083-970 Campinas, SP, Brazil; orcid.org/
dendrimers produce fluorescent vesosomes able to perform 0000-0002-0638-0791
the dual-role of imaging and cytotoxic agent for cancer cells. Liniquer A. Fontana − Institute of Chemistry, Rua Josue de
In conclusion, herein we demonstrated that a hydrophobic Castro, Cidade Universitaria Zeferino Vaz, University of
triazolylferrocenyl dendrimer self-assembles into uniform Campinas, 13083-970 Campinas, SP, Brazil
nanovesicles and vesosomes in a hierarchical process in Marco C. P. Soares − Laboratory of Photonic Materials and
water. In aqueous medium these dendrimers behave as soft Devices, Rua Mendeleyev 200, Cidade Universitaria Zeferino
nanoelements that self-assemble into nanocompounds (nano- Vaz, School of Mechanical Engineering, University of
vesicles), that in turn self-assemble into microcompounds Campinas, 13083-860 Campinas, SP, Brazil; orcid.org/
(vesosomes).48 These new supramolecular entities present 0000-0002-5219-5124
green NTIL fluorescence, which is an emerging property as the Jaime Ruiz − Univ. Bordeaux, ISM, UMR CNRS 5255,
dendrimer has no fluorophores in its structure. This work 33405 Talence Cedex, France
confirms the hypothesis that the NTIL emission is greatly Jackson D. Megiatto Jr. − Institute of Chemistry, Rua Josue de
enhanced by rigidification of the supramolecular assemblies Castro, Cidade Universitaria Zeferino Vaz, University of
containing HASLs and by the presence of electron rich groups Campinas, 13083-970 Campinas, SP, Brazil; orcid.org/
on the periphery of dendrimers. We have also demonstrated 0000-0002-1258-4678
that the peripheral triazolylferrocenyl groups can act as both
fluorophores and cytotoxicity inducing agents enabling the Complete contact information is available at:
development of innovative theranostic systems. Therefore, the https://pubs.acs.org/10.1021/jacs.1c05551
present work paves the way to use supramolecular techniques
to assemble dendrimers into large and well-defined super- Author Contributions
∥
structures in a simple one-pot strategy. The variety of GP and QW contributed equally to this work and should be
biological active moieties available to decorate dendrimers as considered as co-first authors.
well as the possibility to use other dendrimer generations in Notes
the self-assembly process render the present technique a The authors declare no competing financial interest.
powerful methodology for the design of nanotheranostic
systems. ■ ACKNOWLEDGMENTS

■
*
ASSOCIATED CONTENT
sı Supporting Information
This communication is dedicated to Professor René Dabard
(University of Rennes 1) at the occasion of his 90th birthday.
The authors are grateful to Fundaçaõ de Amparo à Pesquisa do
The Supporting Information is available free of charge at Estado de São Paulo (FAPESP) for funding support (#2018/
https://pubs.acs.org/doi/10.1021/jacs.1c05551. 02093-0 and 2021/00555-9 for CO, #2013/22160-0 for JDMJ;
Detailed experimental procedures, NMR spectra, #2017/06146-8 for CBB, #307635/2018-0 for LAF). CO and
absorption spectra, EF-TEM images, cytation images JDMJ thank Conselho Nacional de Desenvolvimento Cientí-
of cellular uptake experiments, CCK-8 cytotoxicity data fico e Tecnológico (CNPq) for the research fellowship
(PDF) (#307403/2018-1 for CO and #307635/2018-0 for JDMJ).
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This study was financed in part by the Coordenaçaõ de C.; Klein, M. L.; Percec, V.; Good, M. C. Direct Visualization of
Aperfeiçoamento de Pessoal de Nível Superior, Brasil Vesicle Disassembly and Reassembly Using Photocleavable Den-
(CAPES), Finance Code 001. The authors are also thankful drimers Elucidates Cargo Release Mechanisms. ACS Nano 2020, 14,
for the access to equipment and assistance provided by the 7398−7411.
(16) Lee, C. C.; MacKay, J. A.; Fréchet, J. M. J.; Szoka, F. C.
National Institute of Science and Technology on Photonics
Designing Dendrimers for Biological Applications. Nat. Biotechnol.
Applied to Cell Biology (INFABIC) at the State University of 2005, 23, 1517−1526.
Campinas (FAPESP #08/57906-3). The authors thank Dr. (17) Ornelas, C.; Pennell, R.; Liebes, L. F.; Weck, M. Construction
Douglas Soares da Silva and INCT/Inomat (FAPESP #2014/ of a Well-Defined Multifunctional Dendrimer For Theranostics. Org.
50906-9 and CNPq/MCTIC #465452/2014-0) for electron Lett. 2011, 13, 976−979.
microscopy support. Financial support from the Chinese (18) Caminade, A.-M.; Turrin, C.-O. Dendrimers for Drug Delivery.
Scientific Council (CSC, grant to QW), the FP7 European J. Mater. Chem. B 2014, 2, 4055−4066.
Network “Nanosolution” (JR and DA), the CNRS and the (19) Mignani, S.; El Kazzouli, S.; Bousmina, M.; Majoral, J.-P.
University of Bordeaux is gratefully acknowledged. Expand Classical Drug Administration Ways By Emerging Routes

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Using Dendrimer Drug Delivery Systems: A Concise Overview. Adv.
Drug Delivery Rev. 2013, 65, 1316−1330.
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