GSK-3 inhibitor

Glycogen Synthase Kinase 3β Inhibitor Delivered by Chitosan
Nanocapsules Promotes Safe, Fast, and Efficient Activation of Wnt
Signaling In Vivo

Alfredo Ambrosone,* Laura De Matteis, Ines Serrano-Sevilla, Claudia Tortiglione, ́
and Jesus M. De La Fuente ́ *
Cite This: ACS Biomater. Sci. Eng. 2020, 6, 2893−2903 Read Online
ACCESS Metrics & More Article Recommendations *sı Supporting Information
ABSTRACT: The Wnt-β-catenin signaling is an evolutionarily
conserved pathway with a prominent role in different biological
processes such as stem cell renewal, cell proliferation, and
differentiation. Wnt signaling dysfunctions have been associated
with developmental and neurological diseases as well as formation
and progression of tumors. Nanomedicine may provide safe and
efficient drug delivery systems offering breakthrough innovation in
targeting Wnt signaling. The natural polymer chitosan represents
an excellent candidate for delivery platforms, showing interesting
biophysical properties such as high biocompatibility and
mucoadhesive properties. In this study, oily core chitosan nanocapsules were designed with the aim to deliver the Wnt signaling
agonist alsterpaullone in the model organism Hydra vulgaris. Chitosan nanocapsules show negligible impact on animal morphology,
without affecting the viability. Nile red-loaded nanocapsules reveal fast and efficient intracellular delivery of the fluorescent cargo.
Short incubations with alsterpaullone-loaded nanocapsules ensure a more effective activation of Wnt signaling with respect to the
same concentrations of the free drug. Altogether, these data provide evidence that chitosan nanocapsules may represent a very
promising strategy for future therapies targeting the diseases associated with canonical Wnt signaling.
KEYWORDS: drug delivery, chitosan nanocapsules, Wnt signaling, Hydra vulgaris
Nanomedicine is currently demanding smart nanosystems able
to ensure fast and safe delivery of bioactive molecules. In the past
decade, biomaterial engineering has provided a vast array of
nanocarriers with different shape, size, and composition.
Inorganic or organic nanoparticles made by polymeric matrices,
hydrogels, or nanocapsules (liposomes, emulsion-based, or
protein-based nanocapsules) undoubtedly represent the most
common nanodevices designed for biomedical purposes.1−3
Among this heterogeneous group of biomaterials, a great interest
is dedicated to nanomaterials based on natural polymers, which
may offer advantages in terms of environmental and human
Chitosan is a nontoxic naturally available polysaccharide,
obtained from crustacean shells, generally recognized as safe by
the American Food and Drug Administration for dietary use and
approved for wound-dressing products.5 Moreover, it has been
employed in different industrial sectors such as food,
pharmaceutics, cosmetics, and textiles.6,7 Over the last 20
years, a considerable amount of works has demonstrated that
chitosan-based nanostructures possess great colloidal stability,
efficient size control, and easy functionalization owing to the
presence of amino groups on their surface.8 Very interestingly,
the amino groups on chitosan structure are responsible also for
mucoadhesion, one of the most attractive properties from a
biological perspective.9 Indeed, chitosan-based nanocarriers
provide a fast and stable interaction with biological tissues,
increasing the residence time of drugs on the cell surface and
promoting the local release of biomolecules in manifold
applications.10−13 Notably, chitosan-based biomaterials were
shown to promote the transport of biomolecules between
adjacent cells by perturbing tight-junctions of epithelial
cells,14,15 which is of great interest for the delivery of
hydrophobic drugs that otherwise would be degraded during
the paracellular passage.16 Not least, consolidated evidence has
shown that chitosan nanocarriers are able to efficiently protect
the cargo and safely deliver it to cells in different physiological
and pathological contexts.17
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Altogether, these physicochemical properties make them
excellent nanocarriers in biomedical applications, especially for
targeted drug delivery.
In this work, we designed nanoreservoir carriers made of a
nanoemulsion core and a chitosan shell for the delivery of the
Wnt signaling agonist alsterpaullone (ALP) in the invertebrate
aquatic model Hydra vulgaris.
The canonical Wnt/β catenin signaling is one of the most
important pathways in animal development. It regulates many
aspects of cell proliferation and differentiation and drives vital
developmental programs in metazoan. Aberrant regulation of
Wnt signaling caused by mutation of pathway components and/
or regulators results in developmental diseases and tumori￾genesis.18,19
Basically, canonical Wnt signaling switches on when Wnt
ligands bind to Frizzled receptors and coreceptors Lrp5/6. This
molecular event recruits Dishevelled (Dvl) to inhibit the
destruction complex where the glycogen synthase kinase 3β
(GSK-3β) phosphorylates and earmarks β-catenin for degrada￾tion. As a result, β-catenin is stabilized and translocates to the
nucleus, where it interacts with T-cell transcription factor/
lymphocyte enhancer factor (Tcf/Lef) promoting the tran￾scription of Wnt-target genes.20
Evolutionary speaking, the Wnt pathway arose early, and it is
well-conserved in metazoans, even in ancient multicellular
organisms. The freshwater polyp Hydra vulgaris is a prebilaterian
organism that arose approximately 650 million years ago, which
has been used as a model to trace back in animal evolution the
Wnt signaling transduction pathway. The Wnt/β catenin
pathway in Hydra controls axial patterning contributing to the
maintenance of the structure of the head, body column, and foot
in adult and regenerating polyps.21,22 Remarkably, pharmaco￾logical activation of this signaling by the GSK-3β inhibitor
alsterpaullone (ALP) promotes the stepwise formation of
ectopic tentacles or heads from the body column together
with the activation of Wnt-target genes.23,24
Herein, we report the synthesis and physicochemical
characterization of chitosan nanocapsules (CNCs) for in vivo
applications. In particular, we describe the release kinetics of
encapsulated fluorescent cargos in whole animals, pointing out
that chitosan nanocapsules ensure the fast delivery of the cargo
in the animal body. Finally, we provide evidence that ALP￾loaded chitosan nanocapsules trigger safe, fast, and robust
activation of Wnt signaling in H. vulgaris. Our data also show
that chitosan nanocapsules are excellent carriers of GSK-3β
inhibitors, encouraging translational research for therapeutic
2.1. Materials. Tween 20 and absolute EtOH were purchased from
Panreac Quimica S.L.U. Span 85 (sorbitanetrioleate), oleic acid, and ́
chitosan (medium molecular weight) and the fluorophore Nile red
were purchased from Sigma-Aldrich. Alsterpaullone was purchased
from Tebu-Bio. Water (double-processed tissue culture, endotoxin￾free) used in all nanocapsule synthesis was from Sigma-Aldrich.
2.2. Culture of Hydra vulgaris. Hydra vulgaris were cultured in
Hydra medium (1 mM calcium chloride, 0.1 mM sodium hydrogen
carbonate, pH 7) at 18 °C with a 12/12 h light/dark regime. Polyps
were fed on alternate days with freshly hatched Artemia salina nauplii.
Budless adult polyps were selected for in vivo tests. All tests were carried
out with 1 day-starved polyps. Hydra-conditioned medium was
prepared by incubating 20 polyps in 300 μL of Hydra medium for 24 h.
2.3. Chitosan Nanocapsule Synthesis. Chitosan nanocapsules
loaded with Nile red (NR-CNC) or alsterpaullone (ALP-CNC) were
prepared following the method already reported for the synthesis of
empty nanocapsules.15 The molecules were loaded into the oily core of
the nanocapsules by adding them to the organic phase during the
synthesis. Briefly, 0.1 mg of Nile red or 0.1 mg of ALP were mixed with
200 μL of absolute ethanol, and then they were incorporated into an
organic solution containing 8.6 mg of Span 85 and 40 mg of oleic acid in
4 mL of absolute ethanol. This organic phase was added dropwise to a
solution of 13.6 mg of Tween 20 dissolved in 8 mL of water. After 15
min under stirring, the nanoemulsion formed was stabilized by adding
0.5 mL of a 5 mg/mL chitosan solution in acetic acid 1% (v/v) and left
stirring again for 15 min. Then, the nanocapsules were added to 15 mL
of solution of 50 mM Na2SO4 under gentle stirring to obtain the final
polymeric hydrogel shell. The excess of Na2SO4 was removed by
ultracentrifugation (69673 × g, 30 min, 10 °C), nanocapsules were
washed with water until ethanol was removed and nanocapsules were
resuspended in 2 mL of water. The concentration of the nanocapsules
in water suspension was obtained by measuring the weight of a sample
after freeze-drying.
2.4. Determination of Encapsulation Efficiency and Drug
Loading in Alsterpaullone-Loaded Nanocapsules. Encapsulation
efficiency (EE) was intended as the percentage of encapsulated drug
over the amount added initially to the preparation of nanocapsules.
Drug loading (DL) is intended as the amount of encapsulated drug per
weight of carrier. To calculate the amount of encapsulated
alsterpaullone, 1.5 mg of nanocapsules were mixed with 900 μL of
methanol and sonicated for 30 min in order to obtain the complete
extraction of the encapsulated drug. The absorbance at 281 nm of the
solution containing the released drug was then measured using a Varian
Cary 50 UV/vis spectrophotometer after carrying out a calibration
curve of the compound in methanol.
2.5. Characterization of Alsterpaullone-Loaded and Fluo￾rescently Labeled Nanocapsules. Dynamic light scattering (DLS)
analysis was carried out to obtain hydrodynamic diameter of
nanocapsules using a Brookhaven 90Plus DLS instrument, using the
photo-correlation spectroscopy (PCS) technique. All measurements
were carried out in water at the concentration of 0.05 mg/mL of
nanocapsules at 25 °C.
Electrophoretic mobility (zeta potential) of nanocapsules was
determined by measuring the surface potential of a 0.01 mg/mL
nanoparticle suspension in 1 mM KCl at 25 °C with a Plus Particle Size
Analyzer (Brookhaven Instruments Corporation). In order to detect
potential leaking of encapsulated cargo, Nile red-loaded nanocapsules
were mixed with Hydra culture medium, freshly prepared or previously
incubated with animals (Hydra conditioned medium), and water for
comparison at a concentration of 0.75 mg/mL nanocapsules.
Samples were analyzed after 5 min and 24 h by separating
nanocapsules by filtration and extracting the retained drug with 600
μL of methanol. The concentration of Nile red in methanol was
measured by spectrophotometric analysis at 555 nm, and the retained
drug was expressed as percentage over the initially encapsulated
2.6. Toxicological Assessment. Toxicological studies were
performed to assess the animal response and to optimize the CNC
concentration for safe biomolecule delivery. Different concentrations of
empty CNCs (from 0.3 to 1.0 mg/mL) were added to living Hydra in
order to assess the progressive effects on the morphology and
physiology of individual polyps. Toxicological tests were carried out
in plastic multiwells where groups of 25 polyps were incubated with
CNCs up to 24 h. Three independent experiments were carried out.
Animal morphological changes were recorded by microscopic
examination of each polyp and scored by using the Wilby’s evaluation
score system,25 adapted for nanotoxicology as previously described.26
Nonparametric Mann−Whitney U-test was used to compare medians
of treated and untreated polyps.
2.7. Uptake of Nanocapsules in Hydra. For in vivo fluorescence
imaging, inverted microscope (Axiovert 100, Ziess) equipped with a
digital color camera (Olympus, DP70) and fluorescence filters set (BP
450-490/FT 510/LP515) was used. Images were acquired and
analyzed by the software system Cell F (Olympus). In order to
investigate cell uptake of fluorescent nanocapsules, treated polyps were
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ACS Biomater. Sci. Eng. 2020, 6, 2893−2903
macerated into a suspension of fixed single cells, according to standard
procedures.27 Macerates were stained with 4′,6-diamidine-2′-phenyl￾indole dihydrochloride (DAPI) 2 min and washed for 3 × 10 min in
phosphate saline buffer (PBS). Slides were observed by phase-contrast
and fluorescence microscopy.
2.8. Scanning Electron Microscopy. Scanning electron micros￾copy (SEM) was used to analyze the early interactions of chitosan
nanocapsules in Hydra. Treated polyps were exposed to CNCs,
vigorously rinsed by pipetting in Hydra medium in order to remove
excess of CNCs, relaxed with 2% (w/v) urethane in Hydra medium and
fixed for 1 h in 2% (w/v) glutaraldehyde in Hydra medium. Samples
were then washed (3 × 10 min) with Hydra medium and postfixed for
45 min with 1% (w/v) buffered OsO4. After a series of additional
washes, animals were dehydrated in a graded ethanol series (30−50−
70−90−100%) (v/v). Environmental scanning electron microscopy
(ESEM) micrographs of polyps were collected using a Quanta FE6-250
(FEI Company) field emission ESEM for high-resolution imaging
working at low vacuum mode.
Cryogenic transmission electron microscopy (cryo-TEM) was used
to characterize empty nanocapsules. Samples were vitrified in an FEI
Vitrobot: a 4 μL drop of an aqueous suspension of the material was
placed on a TEM Quantifoil carbon grid, the excess of water was blotted
away at the Vitrobot with filter paper, and the grid was freeze-plunged in
liquid ethane. Samples were then transferred under liquid nitrogen
atmosphere to a Gatan TEM cryo-holder equipped with a liquid
nitrogen reservoir. Samples were handled and observed at T = 100 K.
Cryo-TEM images were obtained using a Tecnai T20 (Thermofisher
Scientific, formerly FEI) at a working voltage of 200 kV and coupled
with a Veleta CCD Camera.
2.9. Assessment of Wnt Signaling Activation in Hydra
vulgaris. Groups of 20 Hydra polyps were treated with alsterpaul￾lone-loaded CNCs and free ALP for 5 and 15 min. Following extensive
washes (3 × 10 min) to rinse out the excess of nanocapsules and drugs,
treated animals were cultured in fresh medium and inspected daily for 1
week to check ectopic tentacle emergence and body swelling. Three
independent experiments were carried out.
3.1. Synthesis and Characterization of Alsterpaullone￾Loaded Nanocapsules. Alsterpaullone-loaded nanocapsules
were obtained and characterized according to the schematiza￾tion reported in Figure 1. Alsterpaullone belongs to the
paullones,28 a family of benzazepinones with promising
antitumor properties, especially for group 3 medulloblasto￾mas.29 Owing to the well-known specific inhibitory effect on
Hydra GSK-3β, but not on other cyclin-dependent kinases,23
alsterpaullone has been extensively employed to activate the
Wnt pathway in Hydra. The chemical structure and mode of
action of ALP are displayed in Figure S1.
Herein, alsterpaullone was successfully loaded into chitosan￾based nanocapsules (ALP-CNC) using a nanoemulsion method
to produce the lipophilic oily core of the nanocapsules where the
Figure 1. Synthesis and physicochemical characterization of chitosan nanocapsules. (A) Schematic representation of chitosan nanocapsule preparation
including reagents, synthetic procedures, and final products obtained in the present work. (B) Representative Cryo-TEM image of chitosan
nanocapsules. Arrows indicate CNC with different sizes. (C) DLS measurements of Nile red-loaded (red box) and ALP-loaded CNCs (green box).
(D) Zeta potential of nanocapsules.
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drug could be hosted. Thanks to its hydrophobic nature and
solubility in organic solvents, alsterpaullone was dissolved in the
organic phase of the mixture prepared for the synthesis of
nanoemulsion cores.
Then, the unstable nanoemulsion structure was stabilized
producing a chitosan hydrogel shell. Chitosan was chosen in this
work because of the advantages that this natural polysaccharide
presents in terms of biosafety and favorable interactions with
living organisms, tissues, and cells.6 The mucoadhesive proper￾ties of chitosan derive from the presence of amino groups on its
structure that are protonated at acidic and slightly acidic pH
(below 6−6.5). As a result, they are prone to interact with
negatively charged biological membranes and mucosae provid￾ing a useful tool for drug delivery.30
According to the same procedure, we also synthesized
nanocapsules loaded with the fluorophore Nile red (NR￾CNC), in order to obtain fluorescently labeled CNCs for
tracking the dynamics of cargo release in Hydra (Figure 1 A).
Nile red has been widely employed for intracellular lipid
staining,31,32 and in our experimental setup it represents an
excellent dye to track hydrophobic payload release from
nanostructures with an oily core.
The amount of Nile red (Figure S2) or alsterpaullone loaded
into the chitosan nanocapsules was measured spectrophoto￾metrically after the extraction of the drug from the final
suspension of loaded nanocapsules. With this aim, the cargo
release from CNC lipophilic core was forced using methanol and
the solubilized molecules were separated from the capsules. The
absorbance of the free molecule was then measured, and the
encapsulation efficiency (EE, percentage of encapsulated drug
over the amount initially added) and drug loading (DL, amount
of encapsulated drug per weight of carrier) were calculated. As
for alsterpaullone, the encapsulation efficiency was 73%, while
the DL was 1.55 μg/mgCNC. The encapsulation efficiency was
high and comparable to that achieved to date for the
encapsulation of other liposoluble drugs such as capsaicin.33,34
Size and zeta potential of ALP-CNC and NR-CNC were
determined. CNC hydrodynamic diameters together with their
percentage distribution are reported in Figure 1C. Both
nanocapsules presented a prevailing population corresponding
to a hydrodynamic diameter between 80 and 120 nm, although
NR-CNCs also contained some aggregates of 450 nm
(corresponding to approximately 15% of the total amount of
nanocapsules). Nanomaterial size is a critical parameter for
effective active molecule administration through biological
tissues. Therefore, it appears important to underline that our
synthetic procedure allows us to obtain an enriched fraction of
oily core-chitosan nanocarriers with a size comparable to those
currently proposed for biomedical applications.35,36
The positive zeta potential (Figure 1D) in both cases indicates
that most amino groups were exposed on the surface and
protonated. Differences that were very slight and not relevant for
biological interaction studies were observed comparing ALP￾and NR-CNCs. In light of this, herein we used fluorescent NR￾CNCs to explore in vivo kinetics of uptake and cargo release of
chitosan nanocapsules using H. vulgaris polyps, while ALP￾loaded CNCs were exploited for biological functional assays.
3.2. Kinetics of Chitosan Nanocapsule Uptake and
Cargo Delivery In Vivo. Living in uncontaminated freshwater,
Hydra species are extremely sensitive to environmental
pollutions such as organic toxicants,37 heavy metals,38,39 and
industrial effluents.40 In recent years, Hydra vulgaris has been
fruitfully introduced as a living model for nanoecotoxicology,
allowing the exploration of nano-bio interactions at tissue,
cellular, and molecular levels.41,42 For instance, we previously
provided evidence that exposure to Cd-based nanocrystals
produces immediate tissue damage together with severe changes
of gene expression,43,44 while silica nanoparticles, either SiO2 or
hybrid diatomite, affect minimally the animal morphology with
poor impact on the transcriptome landscape.45,46
In this work, CNC were initially tested for biosafety
assessment by measuring the toxicological impact on Hydra.
With this aim, living polyps were incubated with different doses
of empty CNC (from 0.3 mg/mL to 1 mg/mL) for 30 min and
24 h. Animal phenotypes were monitored by stereomicroscopic
inspections during CNC exposure. A numerical score system
was assigned to quantify progressive morphological damages of
polyps, ranging from 10 (healthy animal) to 0 (animal
disintegrated). A detailed description of morphological changes
and scores is reported in the Supporting Information (Table S1).
The toxicological evaluation reported in Figure 2 indicates that
concentrations up to 0.3 mg/mL for 24 h did not affect
significantly animal phenotype. Morphological alterations were
reported at 0.3 mg/mL, although their low grade (ranging from
score 9 to 6), means they are considered reversible and, in the
absence of diffuse cell damage, do not really impact animal
survival, even after 24 h of incubation. Conversely, animals
treated with 1 mg/mL CNCs presented mild/severe phenotyp￾ical damages especially after 24 h, grouped as scores 4 and 5,
indicating that prolonged exposure to very high concentration of
CNCs may trigger toxic effects in invertebrate models. As a
consequence, we selected doses of 0.3 and 0.6 mg/mL and short
incubation (from 5 to 30 min) for further experiments on CNCs
uptake in Hydra tissue.
The Hydra body consists of two epithelial cell layers, namely
the ectoderm (outer cell layer) and the endoderm (inner cell
layer), separated by a mesoglea extracellular matrix called the
mesoglea;47 this simple body architecture and tissue trans￾parency facilitate the study of nanoparticle uptake and dynamics
with respect to more complex animal models. Indeed, our
previous studies showed that Hydra tissues allow easy
localization and tracking of diverse nanostructures over time
by means of fluorescence and transmission electron micros￾copy.48−50
Figure 2. Toxicity analysis of Hydra polyps exposed to different
concentrations of CNC for 30 min or 24 h. Morphological scores are
reported as median ± median absolute deviation of three independent
experiments. Asterisks denote significant difference between untreated
and treated polyps (*, P < 0.05; **, P < 0.01) according to a posthoc
Mann−Whitney U test.
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In order to assess nanomaterial interactions with Hydra, NR￾CNCs treated polyps were accurately rinsed with fresh medium
to remove the excess of NR-CNC and immediately imaged by
fluorescence microscopy. Impressively, we noticed that after
short incubation of 5 min with NR-CNCs, animals showed a
diffuse fluorescent staining from tentacles to the foot region,
indicating that, shortly after the administration, the CNCs are
able to deliver the Nile red cargo to the outer cell layer of Hydra
polyp (Figure 3A,D). Moreover, additional images in Figure 3
Figure 3. Uptake of Nile red-loaded nanocapsules (NR-CNC) in Hydra vulgaris. Groups of 10 polyps were exposed to 0.3 and 0.6 mg/mL NR-CNC,
then accurately rinsed and imaged after 5, 15, and 30 min of incubation with NR-CNCs by fluorescence microscopy. Polyps show diffuse fluorescent
staining indicating an efficient cargo delivery to the Hydra outer epithelial layer. The highest degree of NR delivery was observed after 30 min. Scale
bars: 100 μm in panels A−C; 50 μm in panels D−F.
Figure 4. Intracellular delivery of nanocapsules in Hydra. Polyps incubated with 0.3 mg/mL NR-CNCs for 5 and 15 min were macerated in single cells
and imaged by bright field (BF) (A, D, and G) and fluorescence microscopy for Nile red (B, E, and H) and DAPI (C, F, and I) signal detection. Arrows
indicate large intracellular vacuoles representing compartmentalization of exogenous materials. Scale bars in A, D and G: 50 μm.
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also show that exposure time up to 30 min increases the staining
The delivery was extremely rapid in comparison to many
other nanomaterials investigated in Hydra so far. In our
experience, except for CdSe/CdS rod-shaped nanocrystals,50
the internalization of exogenous nanomaterials typically
necessitates longer incubations (>2 h) to be sharply detected
by fluorescence microscopy in Hydra tissues.48,51,52
It is worth noting that the internalization pattern of CNC
appeared to be unusual with respect to those currently observed
in Hydra. In fact, our previous studies demonstrate that
exogenous nanomaterials (gold nanoparticles, cadmium-based
QDs, polymeric microcapsules, and others) accumulate as small
or large aggregates throughout the whole animal body, which
normally result in a punctuated staining throughout the animal
body. This typical pattern is determined in Hydra by
pynocitosis-mediated uptake followed by lysosome and
autophagosome compartmentalization of nanomaterials.49
In order to assess intracellular accumulation of the fluorescent
cargo, we macerated the body of treated polyps, obtaining
suspensions of single cells with intact morphology. Hydra cells
were fixed, DAPI-stained, and observed by fluorescence
microscopy. Nile red dye is detectable as large cytoplasmic
foci after incubations for 5 and 15 min (Figure 4E,H),
confirming that CNCs allow very fast intracellular deliver of
the cargo. Nile red-positive intracellular vacuoles are also
appreciable in bright field images (Figure 4D,G), likely
representing lipid accumulation coming from the oily core.
Regularly, cells from untreated polyps do not present such
structures (Figure 4 A). Moreover, cells from treated polyps
present an evident fluorescent background, suggesting that Nile
red passively diffuses through cell membranes and reaches the
In light of this evidence, nanocapsule-mediated cargo delivery
may occur through distinct mechanisms, including cell uptake of
intact nanomaterial, payload diffusion at the epithelial surface
upon capsule opening, or through the combination of both
To gain more insight regarding these mechanisms, we
investigated deeper about the dynamics of cargo delivery.
First, we checked whether the animal staining could be provoked
by undesired fluorophore leaking from intact nanocapsules. To
this aim, freshly prepared NR-CNCs were filtered and the eluate
was inoculated in the animal medium and kept for 24 h in the
presence of Hydra polyps. No fluorescent staining was detected
in Hydra tissues by fluorescence microscopy inspections (Figure
Figure 5. Nanocapsules−Hydra interaction at the animal surface by scanning electron microscopy. SEM shows nanocapsule adhesion to the Hydra
ectodermal layer after exposures of 5 min (D−F) and 15 min (G−I). The number of CNCs attached at the animal surface increases according to the
exposure time. Magnifications increase from left to the right side. Scale bars are embedded in the photograms: 100 μm (A and D); 40 μm (B); 10 μm
(C, F, and H); 30 μm in (E and G); 5 μm (I).
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S3B). In addition, we proved that more than 95% Nile red was
retained in CNCs, kept for 24 h in water or even in the presence
of Hydra-conditioned medium (Figure S4A). Finally, we also
confirmed the absence of Nile red in the culture media recovered
after 5 min of incubation with Hydra polyps (Figure S4B).
These results substantiate that the cargo uptake by Hydra
polyps is due to a fast local release of Nile red occurring at
epithelial cell membranes rather than an unintentional leaking
from intact nanocapsules. Interestingly, these data also establish
that NR-CNCs prevent cargo leakage after the synthesis and
encourages their use as efficient nanocontainers for drug delivery
in vivo.
To image closer the interactions between the Hydra outer
layer and nanocapsules, scanning electron microscopy analysis
of animals treated with 0.3 mg/mL NR-CNCs was performed.
Polyps were incubated 5 and 15 min with NR-CNCs, extensively
washed, fixed, and inspected individually. SEM images in Figure
5D−F show a significant number of nanocapsules attached to
the Hydra ectodermal layer upon 5 min of incubation.
A high number of nanocapsules was detected also after 15 min
of exposure, confirming that CNC adhesion to Hydra body
occurs very early after CNC exposure. As mentioned above,
electrostatic interaction between positively charged CNCs and
outer cell envelops, including membranes and glycocalyx, may
drive these fast outcomes. Regardless of the composition,
driving interactions between positively charged nanomaterials
and cell surfaces have been widely described in cell cultures and
other animal models.53−57
Swelling of polymers, drug diffusion through the polymeric
matrix, and polymeric erosion or degradation and a combination
of them have been described as possible mechanisms of drug
release from chitosan nanomaterials.5,58,59
By examining all our data together, in the absence of overt
fluorescence leaking, we exclude the occurrence of spontaneous
polymer swelling in the Hydra medium. On the other hand, we
reported that CNCs massively approach Hydra epithelium
where their structure may be rapidly perturbed or degraded
before delivering the cargo. Thereafter, Nile red and the
hydrophobic core may easily diffuse through cell membranes
and be compartmentalized in the cytoplasm.
Active degradation of chitosan polymer could be mediated by
specific Hydra enzymes on the body surface. Overall, chitosan
has been shown to be degraded by enzymes such as chitinases
and chitosanases,60 as well as other enzymes such as
chitobiases61 and lysozime.62 Hydra normally feed on small
crustaceans like water fleas (Daphnia magna) or Artermia salina
nauplii (under laboratory conditions), both possessing a chitin
skeleton.63,64 Actually, the presence of chitinase activity has been
reported to be confined to the endoderm of the body column in
65 Interestingly, our data reported in Figure S3A
demonstrate that the Hydra-conditioned medium does not
induce release of the encapsulated dye, thus suggesting that
capsule degradation may occur by enzymatic activity at the
animal cell surface and not by secreted enzymes. Therefore, even
though we did not investigate further this aspect of cargo relief
mechanisms, Nile red discharge may occur through nanocapsule
degradation/erosion mediated by chitinases or other enzymatic
activities available at the epithelial cell layer, including
membranes, glycocalyx, and associated bacteria (see Figure S5).
3.3. Wnt Signaling Activation Mediated by a Very Fast
Release of Alsterpaullone from Chitosan Nanocapsules.
GSK-3β inhibitor alsterpaullone promotes nuclear translocation
of β-catenin and transcriptional activation of Wnt targeted genes
in Hydra.
23 As a consequence, canonical Wnt signaling
activation produces the well-known phenotype characterized
by a robust body swelling and the gradual emergence of ectopic
heads and tentacles, which normally takes 3−5 days after
alsterpaullone treatment. Wnt signaling components and
alsterpaullone agonist activity are schematized in Figure S2.
Generally, alsterpaullone is provided directly to the animal
Figure 6. Fast and effective delivery of alsterpaullone mediated by chitosan nanocapsules. (A) Animals were incubated for 30 min with 0.3 and 0.6 mg/
mL ALP-CNC with a concentration of approximatively 0.5 and 1.0 μg/mL ALP, respectively. As comparison, Hydra polyps were treated with the same
concentrations of free alsterpaullone. Data represent the percentage of polyps showing ALP-dependent phenotype, namely, the emergence of ectopic
tentacles and body swelling. Asterisks denote significant difference between ALP-CNC-treated and free ALP-treated polyps (*, P < 0.05; ***, P <
0.001) according to Student’s test. (B and C) Representative phenotypes observed after incubation with 0.5 and 1.0 μg/mL encapsulated
alsterpaullone, respectively.
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medium for 24−48 h, in a variable range of concentrations
spanning from 0.2 to 5 μM.22,24 Timing and severity of
phenotypic manifestation may vary significantly according to
dosage, animal stage, feeding regime, and chemical stability of
the drug, making it sometimes difficult to compare alsterpaul￾lone pharmacological activity when experiments are carried out
in different laboratories.
Herein, we exploited the rapid kinetics of CNC-mediated
cargo release to obtain smart nanocarriers for fast and effective
delivery of alsterpaullone in living animals. To this aim, groups of
15 Hydra polyps were incubated with 0.3 and 0.6 mg/mL ALP￾CNCs containing approximatively 0.5 and 1.0 μg/mL ALP,
respectively, and with equivalent doses of free alsterpaullone,
known to be active in Wnt signaling activation. A schematization
of the method is reported in Figure S6.
The fast kinetics of drug release was assessed by incubating
the polyps for just 30 min, washing, and then monitoring for the
alsterpaullone-specific phenotype (ectopic tentacle formation)
up to 6 days. Interestingly, 4 days after treatment, more than
10% of animals treated with 0.3 mg/mL ALP-CNC showed
ectopic tentacles (Figure 6A), while the higher dose (0.6 mg/
mL ALP-CNCs) induced the ALP phenotype in about 80% of
polyps, more than 4 times the efficiency of the equivalent
concentration (1 μg/mL) of free ALP (Figure 6A). These data
suggest that higher concentration of ALP-CNCs trigger more
robust activation of Wnt signaling (Figure 6B,C) as indicated by
the greater number of ectopic tentacles and faster tentacle
All together, these data suggest that CNCs deliver in a very
short time a sufficient amount of ALP to trigger Wnt signaling
activation and subsequent specific phenotype which normally
appears after 72−96 h upon the pharmacological stimulation.
Therefore, on the basis of our experimental evidence, the
alsterpaullone released from the nanocapsules can enrich the
cytoplasm, exerts its function of GSK-3β inhibitor, and activates
the Wnt signaling within the expected time observed in Hydra.
Nanotechnology applied to the Wnt signaling manipulation is
still in its infancy, and currently available techniques target
almost exclusively cell culture models. Polymersomes loaded
with the small molecule Wnt agonist, 6-bromoindirubin-3′-
oxime (BIO), were able to activate Wnt signaling promoting the
osteogenic differentiation in human primary bone marrow
stromal cells.66 Polyethylene glycol-polyethylenimine-chlorin e6
(PEG-PEI-Ce6) nanoparticles were demonstrated to inhibit the
growth of oral squamous carcinoma cells by combining the
delivery of Wnt-1 small interfering RNAs and photodynamic
More interestingly, liposomal vesicles loaded with purified
mouse Wnt3a were able to stimulate the proliferation of skeletal
progenitor cells and accelerated their differentiation into
osteoblasts of mice.68 Fascinatingly, we recently developed
optical switchers for remote activation of Wnt signaling in vivo.
Basically, light-responsive polyelectrolyte multilayer capsules
were successfully employed to shuttle and release on demand
alsterpaullone in Hydra tissue upon near-infrared light
illumination. We further proved that microscope-guided NIR
irradiation allowed the opening of single capsules with a precise
control of Wnt signaling local activation.52,69 In addition to this
outstanding evidence, herein we proved that chitosan nano￾capsules ensure the shortest effective delivery of alsterpaullone
in Hydra, holding promise for advanced pharmacological
approaches to the control of the canonical Wnt signaling
pathway in vivo.
Advanced nanomaterials for a viable drug delivery strategy
should provide protection of active biomolecules from
degradation; favorable interactions with cells and tissues to
ensure local release; and, possibly, improved pharmacokinetic
profile of the loaded drug. In recent years, chitosan-based
nanomaterials have been proposed as nanocarriers for
biomolecule delivery in nanomedicine. Herein we report the
synthesis and physicochemical characterization of alsterpaul￾lone-loaded chitosan nanocapsules, as well as the in vivo release
kinetics and functionality of the encapsulated drug in Hydra
vulgaris, a small invertebrate introduced to bridge the gap
between testing advanced nanomaterials in cell culture and
clinical models.
Our data indicate that CNCs represent safe nanoreservoir
carriers, which protects the small and hydrophobic drug
alsterpaullone from undesired leaking or degradation, preserving
cargo bioactivity and enhancing its stability and solubility.
Because of the high loading capacity, chitosan nanocapsules
could allow the simultaneous encapsulation of different cargos,
such as hydrophobic molecules and markers, combining in a
single device multiple functionalities in nanomedicine.
Finally, mucoadhesive properties of chitosan ensure an
efficient delivery of alsterpaullone and robust activation of
Wnt signaling in a very short time, showing advantages with
respect to free drug administered directly to the animal medium.
The fast and efficient delivery of the GSK3β inhibitor by
chitosan nanocapsules allows reducing the exposure time of the
drug, thus lowering drug toxicity associated with prolonged and
repeated administrations. Importantly, the encapsulation of the
Wnt signaling agonist alsterpaullone in chitosan nanocapsules
paves the way to improved pharmacokinetics of this small drug
and to target Wnt signaling and associated pathological
*sı Supporting Information
The Supporting Information is available free of charge at
Additional information on CNC toxicity, Wnt signaling
pathway components, and a possible model of cargo
delivery (PDF)
Corresponding Authors
Alfredo Ambrosone − Instituto de Ciencia de Materiales de
Aragon-CSIC/Universidad de Zaragoza and CIBER in ́
Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),
Zaragoza, Spain;;
Email: [email protected]
Jesús M. De La Fuente − Instituto de Ciencia de Materiales de
Aragon-CSIC/Universidad de Zaragoza and CIBER in ́
Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),
Zaragoza, Spain; CIBER-BBN, Instituto de Salud Carlos III,
Madrid, Spain;;
Email: [email protected]
Laura De Matteis − CIBER-BBN, Instituto de Salud Carlos III,
Madrid, Spain; Instituto de Nanociencia de Aragon (INA), ́
Universidad de Zaragoza, 50018 Zaragoza, Spain
ACS Biomaterials Science & Engineering Article

ACS Biomater. Sci. Eng. 2020, 6, 2893−2903
Ines Serrano-Sevilla ́ − Instituto de Ciencia de Materiales de
Aragon-CSIC/Universidad de GSK-3 inhibitor Zaragoza and CIBER in ́
Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN),
Zaragoza, Spain; CIBER-BBN, Instituto de Salud Carlos III,
Madrid, Spain
Claudia Tortiglione − Istituto di scienze applicate e sistemi
intelligenti “E. Caianiello”, Consiglio Nazionale delle Ricerche,
Pozzuoli, Italy;
Complete contact information is available at:
The authors declare no competing financial interest.
We thank Carlos Cuestas for the technical support in SEM
analysis. The authors acknowledge financial support from
Spanish MINECO (project BIO2017-84246-C2-1-R) and
DGA and Fondos Feder (Bionanosurf E15_17R). A.A. thanks
European Union for financial support with a Marie Curie
Fellowship (H2020-MSCA-IF-2014-657566). I.S.S. acknowl￾edges Ministerio de Economıa y Competitividad del Gobierno ́
de España for her FPI grant (BES-2015-071304).
CNC, chitosan nanocapsules; ALP, alsterpaullone; NR, Nile red
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