Control of Wnt/β-Catenin Signaling Pathway in Vivo via Light Responsive Capsules Alfredo Ambrosone,†,∥ Valentina Marchesano,†,∥ Susana Carregal-Romero,‡,§,∥ Daniela Intartaglia,†,∇ Wolfgang J. Parak,*,‡,§ and Claudia Tortiglione*,† †
Istituto di Scienze Applicate e Sistemi Intelligenti “E.Caianiello”, Consiglio Nazionale delle Ricerche, Pozzuoli 80078, Italy Fachbereich Physik, Philipps Universität Marburg, Marburg D-35032, Germany § CIC biomaGUNE, Donostia-San Sebastián 20009, Spain ‡
S Supporting Information *
ABSTRACT: The possibility to remotely manipulate intracellular pathways in single cells is among the current goals of biomedicine, demanding new strategies to control cell function and reprogramming cell fate upon external triggering. Optogenetics is one approach in this direction, allowing specific cell stimulation by external illumination. Here, we developed optical switchers of an ancient and highly conserved system controlling a variety of developmental and adult processes in all metazoans, from Hydra to mammals, the Wnt/β-catenin signaling pathway. An intracellular modulator of the Wnt pathway was enclosed into polyelectrolyte multilayer microcapsules engineered to include self-tracking (i.e., fluorescence labeling) and light mediated heating functionalities (i.e., plasmonic nanoparticles). Capsules were delivered in vivo to Hydra and NIR triggered drug release caused forced activation of the Wnt pathway. The possibility to remotely manipulate the Wnt pathway by optical switchers may be broadly translated to achieve spatiotemporal control of cell fate for new therapeutic strategies. KEYWORDS: controlled drug delivery, polyelectrolyte multilayer capsules, model organism
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throughout adulthood, controlling axial patterning during homeostasis and regeneration.8−10 Remarkably, forced activation of Wnt signaling by ALP results in the ectopic formation of tentacles or complete heads along the body column (Figure S1), and also induces the expression of genes of the Wnt pathway in the body column. We exploited the possibility of local delivery of ALP in Hydra by encapsulating it into optical switchers, i.e. light responsive polyelectrolyte multilayer capsules, which allow for drug shuttling into tissues and controlled release upon light illumination (Figure 1). While light triggered release of several compounds using this drug carrier concept has been shown in vitro,11 future application require in vivo demonstration. Simple animal models such as H. vulgaris, playing crucial role in fundamental discovery, may have high translational impact, presenting great advantages for feasibility and bioactivity testing of multifunctional nanoparticles.12−14 The body transparency and tissue like organization exposing the outer cell layer directly to the environmental medium, together with the fast functional readouts of ALP activity, make Hydra a unique system to screen
ignaling by the Wnt family of secreted glycolipoproteins via the transcriptional coactivator β-catenin (Wnt/β-cat signaling pathway) is one of the fundamental mechanisms that direct cell proliferation, cell polarity, and cell fate determination during embryonic development and tissue homeostasis.1,2 As expected, inappropriate activation of the Wnt pathway is implicated in a variety of human birth defects, cancers, and other diseases, and as consequence the modulation of this pathway at several levels might have profound therapeutic benefits both in regenerative medicine and anticancer strategies.3−5 Central to the well-documented canonical Wnt pathway are the effector β-catenin (β-cat), and a destruction complex that controls its stability.6 Figure 1 summarizes the molecular components of the pathway and the Wnt triggered switch from the OFF to ON state. Pharmacological compounds such as paullones can mimic the Wnt signaling and present the advantages to acting intracellularly, bypassing all the molecular processes downstream of Wnt ligand binding to membrane receptor. Among them, ALP acts by competing with adenosinetriphosphate (ATP) for binding to GSK3β.7 As this kinase controls β-cat phosphorylation for proteasome degradation, its inhibition causes nuclear accumulation of β-cat and constitutive Wnt signaling. In the small freshwater polyp Hydra vulgaris, the Wnt pathway is active © XXXX American Chemical Society
Received: December 12, 2015 Accepted: January 22, 2016
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Figure 1. Schematic of Wnt/β-catenin signaling activation by optical switchers. In an adult polyp, the Wnt pathway is active only in a few cells of the hypostome, where it is involved in Hydra organizer formation and regeneration, while it is in the OFF state in the rest of body.8 At the molecular level in the absence of Wnt ligand, the destruction complex, which in vertebrates consists of the scaffold protein Axin, the tumor suppressor gene product APC, and the kinase GSK3β, phosphorylates β-catenin (orange hexagon) for ubiquitination and degradation (as triangles) by the proteasome. In contrast, when Wnt proteins reach signal-receiving cells, they bind to receptors of the Frizzled family and the coreceptor LRP, inhibiting the activity of the destruction complex and leading to the stabilization of β-catenin. Therefore, stabilized β-catenin accumulates in the cytoplasm and then enters into the nucleus where it interacts with members of the TCF/LEF transcription factor family, ensuring efficient transcriptional activation of downstream genes such as Wnt3, β-cat, dkk. Optical switchers designed in this work can induce a molecular switching ON the Wnt/β-cat signaling pathway by local delivery of intracellular modulator of this pathway, i.e. ALP (yellow ovals). On the left panel, a polyp treated by optical switchers (red circles) is NIR illuminated (red dots), determining local ALP release. On the right, key molecular events induced by GSK3β inhibition are shown together with the resulting polyp phenotype. As Wnt ligands are involved in Hydra organizer formation and maintenance of body axis polarity, forced activation of Wnt pathway induces appearance of ectopic tentacles along the body column, enabling fast functional readouts of optical switchers bioactivity.
Figure 2. Optical switchers uptake into Hydra tissue. (a) Schematic representation of polyelectrolyte multilayer capsules. The red color indicates the fluorescence property conferred by the outer layer (PAH-Dy547), in blue the PS-b-PAA micelles used to encapsulate hydrophobic ALP (yellow ovals) are shown, and in black, the gold nanoparticle aggregates conferring photothermal properties. Confocal images of nondegradable (PSS/PAH)4 AuNPs(PSS/PAH)2(PSS/PAH-Dy547) (PSS/PAH) capsules (b), and biodegradable (DEXS/ PARG)4(DEXS/PARG-AF488) (DEXS/PARG) capsules (c), both containing ALP, show homogeneous shape and size. In vivo dark field and fluorescence imaging of Hydra polyps incubated 3 h (d and e) and 24 h (f and g) with nondegradable capsule (5 × 106 caps mL−1, corresponding to 5 × 105 caps/animal) show efficient internalization into Hydra tissue. Dynamics of internalization of such microsized structures mirrors previous data obtained with fluorescent nanocrystals in Hydra indicating no adverse effects played by the size on the internalization mechanism.13,20 High degree of internalization was observed along the body column and on the tentacles (h and i), where single capsules are easily detected. Confocal images confirmed the presence of monodisperse capsules inside animal ectodermal cells (j). Actin filaments were counterstained with Alexa Fluor 488-phalloidin and nuclei with DAPI. Capsules appear as granular yellow structures. (k) Ultrastructural analysis by transmission electron microscopy shows a single capsule within the cytoplasm of a Hydra ectodermal cell. Scale bars: 10 μm in (b and c); 1 mm in (d and e); 500 μm in (f and g); 50 μm in (h−j); 5 μm in (k). B
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Figure 3. ALP delivery by biodegradable capsules switches ON the Wnt pathway in Hydra. (a) A scheme of the intracellular switch mediated by ALP. While in the OFF state of the Wnt pathway phosphorylated β-cat is degraded, in the ON state, by inhibiting the GSK3β kinase, stabilized β-cat is translocated into the nucleus where it drives the transcriptional activation of Wnt downstream genes. At the animal level, the forced activation of Wnt signaling drives cell fate toward specification of head structures, mainly tentacles, according to Wnt role in organizer and body axis formation. Polyps were incubated 24 h with (b) biodegradable (DEXS/PARG)6-ALP and (e) (DEXS/PARG)6-AuNP-ALP capsules (5 × 105 caps mL−1), singly inspected and imaged 48 h (c and f) and 72 h later (d and g). Animal morphologies mirroring those induced by free ALP were observed. The presence of AuNP aggregates within the capsule shell affects the degree of ALP release, as shown by a less sever phenotypical aberrations. Scale bars, 500 μm.
sized and characterized using the same techniques abovedescribed, i.e, UV-spectroscopy, optical and electron microscopy, zeta potential measurement (Figure 2c; Figure S4). In case of the light responsive PSS/PAH capsule release of ALP only is initiated upon photothermal heating, ALP is released from biodegradable capsules spontaneously upon enzymatic degradation.19 Therefore, four types of capsules were prepared to study and compare different drug carriers having different release mechanisms: biodegradable and nondegradable capsules with and without AuNPs in their shell. The specific architecture, together with the shell composition, name, size, and zeta potential of the four different polyelectrolyte capsules used in this study are indicated in Tables S1 and S2. For these microcapsules first, their internalization into Hydra tissues was studied. In vivo imaging shows efficient capsule uptake into polyp tissues, from tentacles to the foot region, shown as uniform fluorescent labeling 3 h post incubation, and patterned as granular spotted fluorescence 24 h post incubation (Figure 2d−g; Figures S6−S8). Regardless of the nature of the capsules, confocal images confirmed the presence of individual capsules both within the tentacles (Figure 2h,i) and in the body tissue ectoderm (Figure 2j), facing the outer medium and representing the portal of entry of medium suspended particles (as the animal mouth is tightly closed in absence of prey),20 while TEM analysis showed a cytoplasmic localization within lysosomes (Figure 2k). Second, toxicity assays based on the developmental potential of the polyp (see Figure S9) previously developed to test different nanomaterials in Hydra ensured the absence of potential adverse effects of the polymers out of which the capsules are composed,21−25 in our experimental conditions. Dose−response assays showed normal morphology
bioactivity of such light responsive microdevices to manipulate the Wnt pathway, and suggest its use for functional testing of other intracellular pathways, at whole animal level, reducing vertebrate experimentation and avoiding ethical issues.
RESULTS AND DISCUSSION As universal carrier, light responsive capsules were synthesized using layer-by-layer self-assembly of poly(styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) polyelectrolytes.15 To achieve efficient encapsulation of small hydrophobic molecules such as ALP, CaCO3 microparticles predoped with polystyrene-block-poly(acrylic acid) (PS-b-PAA) micelles were employed,16 enabling ALP loading after core extraction. Gold nanoparticle (AuNP) agglomerates embedded into the layers were used to mediate capsule wall opening through plasmon assisted photothermal heating,11,17,18 while fluorescently labeled PAH polymers on the outer layer allowed for insertion of self-tracking properties on the capsule shell. Light responsive capsules presenting the general structure (PSS/ PAH)2AuNPs(PSS/PAH)2(PSS/PAH-Dy547) (PSS/PAH) were synthesized and characterized by UV-spectroscopy (Figures S2 and S3), confocal and electron microscopy (Figure S4), zeta potential and size measurement (Table S1). Capsule loading by ALP was confirmed by UV−vis spectroscopy of the capsule solution before and after encapsulation (Figure S5). A scheme of the capsule structure and chemical composition is shown in Figure 2a,b and Figure S3. As control, also biodegradable capsules based on dextran sulfate (DEXS) and polyarginine (PARG) presenting similar structure (DEXS/ PARG)4(DESX/PARG-AF488) (DESX/PARG) were syntheC
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Figure 4. NIR triggered activation of Wnt signaling pathway. (a) Scheme of the Wnt signaling pathway activation by ALP. Following NIR irradiation of (PSS/PAH)6 -AuNP-ALP capsules internalized into Hydra cells, the Wnt molecular cascade is switched ON. (b and c) Quantitative real time PCR was used to profile Hydra β-catenin and Hydra Wnt3 mRNA levels in NIR illuminated animals, using the whole animal approach (Figure S17a). Twenty-five animals were homogenated at the indicated time post irradiation and processed for qRT-PCR analysis. Data represent the mean ± SD of three technical repeats from two biological replicates. (d) Schematic representation of the NIR laser used to irradiate living polyps. A 100 mW laser (830 nm CW diode) was coupled to an upright microscope (63× objective) leading to a focused light spot of about 3 μm2 in the image plane. Animals were incubated with 5 × 105 caps mL−1 and after 24 h irradiated one by one. Single nondegradable (PSS/PAH)6-AuNP-ALP fluorescent capsules were illuminated one by one with a laser intensity of 3.8 mW μm−2 for a few seconds as shown by fluorescence and phase contrast images, respectively, before (e), during (f), and after irradiation (g). Spherical structures detected by contrast phase imaging are nematocytes, i.e., the stinging cells present on Hydra ectoderm, employed by the animal for prey capture. At 72 h after irradiation, no effects were detectable in (h) untreated animals, irradiated; (i) polyps treated with (PSS/PAH)6ALP, not irradiated; (j) polyps treated with light responsive (PSS/PAH)6-AuNP-ALP, not irradiated. Typical ALP morphologies were induced in (k) polyps treated with (PSS/PAH)6-AuNP-ALP and irradiated. The dynamic of tentacle emergence is shown at 48 h (m) and 72 h (n) post irradiation. Scale bars: 20 μm in (e), 20 μm in (f and g); 500 μm in (h−n).
region enhanced tentacle formation in this area (Figure S12j− l), suggesting a possible local activation of Wnt pathway as consequence of high ALP doses released in this region. We noticed that the presence of AuNPs in the polymer shell of the biodegradable capsules decreased the rate of ALP release (Figures S13 and S14), lowering the percentage of polyps presenting ALP-like phenotype to 74% (Table S3). This decrease could be associated with a lower degree of degradation due to the presence of AuNPs as already observed in similar capsules.19 When the concentration was decreased 20-fold (from 5 × 105 to 2.5 × 104 caps mL−1), the efficiency of the process dropped down to 33% and 16.7%, respectively, for (DEXS/PARG)6-ALP and (DEXS/PARG)6-AuNP-ALP, confirming a drug dependent effect. These data demonstrate that a sufficient amount of ALP in active form is retained upon
of polyps treated with empty biodegradable and nondegradable capsules (up to 1 × 108 caps mL−1, corresponding to around 1 × 107 capsules per Hydra) and normal regenerative capabilities (Figures S10 and S11). Finally, drug encapsulation and release were tested. Animals treated with biodegradable (DEX/ PARG)6-ALP capsules (Figure 3) presented ectopic tentacles along the animal column (90% of treated polyps), perfectly mirroring the ALP induced activation of Wnt signaling.10 The emerging tentacles covered body regions increasing proportionally to capsule concentration and incubation time (Figure S12b−i) as also observed in animals treated with increasing dose of free ALP (Figure S1h,k). These data demonstrate the efficiency of the drug encapsulation methodology and the occurrence of polyelectrolyte shell degradation in vivo. Of interest, slightly higher capsule binding and uptake into the foot D
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or even clinics in the near future.3 In the study described above, we have demonstrated that Wnt signaling can be turned ON in vivo by using optical switchers. Thus, the therapeutic potential of Wnt optical switchers may find widespread applications in all those pathologies where Wnt signaling is aberrantly regulated, from regenerative medicine to anticancer strategies. Several physical stimulations are being developed to trigger controlled drug release. Delivery mechanism based on magnetothermal heating of capsules with magnetic NPs in their shells upon exposure to alternating magnetic fields seems feasible,29 which would allow for application of this technique deep inside tissue. For instance, magnetic nanoparticles have been used, which upon local magnetothermal heating via alternating magnetic fields can trigger heat-sensitive proteins,30,31 or upon local oscillation via periodic magnetic fields can control mechanically sensitive cells.32 However, also NIR light offers some unique advantages over other triggers because it can be spatially selective (e.g., with a narrow beam), because lasers or light-emitting diodes can be readily manufactured for in vivo point-of-care use and are amenable to clinical translation because tissue is most transparent in that regime. Optogenetic approaches work in this direction, but rely on transgenic expression of light-sensitive proteins to achieve selective neuronal stimulation,33,34 and are time- and cost-consuming. In the case of light-mediated heating, the switch is integrated in the delivery system. While typically light-mediated release is carried out for molecules that are bound to the surface of a carrier particle, in the present approach the molecules to be released are encapsulated in the cavity of a carrier particle, which allows for higher quantities of released molecules. Obviously there are also pitfalls associated with photothermal heating. While temperature increases of only a few °C are required for the opening to the capsules,35 overheating upon applying too high laser intensities can damage cells.36 In addition, also some encapsulated molecules close to the capsule walls may be degenerated/deactivated by the heat, whereas those located in the center of the cavity would not be affected. While the release of biologically active molecules has been demonstrated,18 quantitative release of a controlled number of active molecules so far has not been demonstrated with this system and is also technically challenging. Potentially lightmediated release also allows for multiplexed stimulation. Instead of using agglomerated AuNPs resulting on a very broad absorption band, also dispersed AuNPs of different shape could be used, which have distinct plasmon resonance peaks37 and thus could be excited individually. Light-mediated opening with dispersed instead of agglomerated AuNPs has been demonstrated.38 Thus, although at an early stage of development, the spatially and temporally controlled optical switch system has the potential to be a useful tool for the manipulation of cell function at the illuminated region. Optical switchers may function as universal tools to remotely control, cell-by-cell, cellular and molecular processes minimizing potentially harmful side effects of pharmacological treatments.
encapsulation followed by release, to physiologically activate Wnt signaling. For analysis of nondegradable capsules, we first investigated drug leaking by performing UV−vis spectroscopy analysis of the capsule medium, showing not detectable levels of ALP within the experimental error, and thus negligible leaking as functionally proved by the morphological analysis of polyps exposed to increasing capsule dose (Figures S15 and S16). For in vivo laser irradiation, treated polyps were immobilized on a glass slide under round coverslips and illuminated one by one, using different equipment for whole animal and localized NIR excitation (Figure S17). When irradiating the whole polyp, ALP abnormalities (of entity comparable to those induced by low ALP doses, Figure S1h) were induced 48 h post irradiation (p.i.) on 40% of treated polyp (Figure S18). The activation of the Wnt signaling was confirmed at molecular level by assessing the expression levels of known Wnt/β-cat target genes, such as β-cat and Wnt3 through quantitative real time RT-PCR. Expression profiles (Figure 4b,c) show strong accumulation of both gene transcripts in irradiated animals, demonstrating the light triggered activation of the molecular machinery controlled by β-catenin transcription factor. By exciting single capsules with a light spot of ≈3.8 mW/μm2 intensity (12 mW laser power in the focus of the objective, ≈3 μm2 laser spot area), we finally restricted the ALP release to a small tissue area on the body column (Figure 4d). Short irradiation times (less than 3 s) were used to induce fluorescence guided capsule opening (at least 5 capsules/animal, Figure 4e−g; Figure S19). Figure 4k shows that 48 h p.i. the polyps clearly exhibited the morphology of forced Wnt pathway activation, presenting isolated tentacles emerging throughout the body. These structures appeared further elongated at 72 h, as expected (Figure 4l−n; Figure S20). The absence of phenotype in any other experimental condition tested (untreated-irradiated animals, treated-not irradiated animals, Figure 4h−j) indicates a specific light triggered release. The efficiency of the process was high (80%), showing the controlled delivery reliable and reproducible, thus confirming the possibility to manipulate an intracellular pathway by optical switchers (Table S4). To date, ALP has been administered to Hydra by soaking at micromolar range; thus, the effect of local delivery within cells presenting different positional information (i.e., morphogen gradients) is completely unknown. It is indeed possible, due to the systemic propagation of Wnt signaling over long distances, that a local release of Wnt agonist induces same effect as administration to the whole animal. Here, by focusing the NIR laser to a few micrometers, we illuminated single capsules located in the body column resulting in ALP delivered to restricted regions. However, we cannot exclude the simultaneous rupture of some other adjacent capsules not directly in the center of the focus light spot; thus, the observed phenotype may results from Wnt pathway activation on a wider area than the actual spot size. The ongoing development of a dedicated equipment for single cell irradiation on a whole animal (i.e., microchip hosting immobilized polyps) may allow in the near future to deeply investigate the effect of optical switcher on single cells.
METHODS Synthesis and Encapsulation of Cargo in Polyelectrolyte Microcapsules. Biodegradable and nondegradable microcapsules were prepared by layer-by-layer (LbL) coating of a sacrificial template, consisting of spherical calcium carbonate (CaCO3) particles loaded with block copolymer micelles. Dextran sulfate (DEX) and poly-Larginine (PARG) polyelectrolytes were used for shell assembly of degradable, while poly(sodium 4-styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) were employed for nondegradable
CONCLUSION The study of Wnt signaling in human diseases,4 in stem cell biology,26 and regenerative medicine5,27,28 holds promises for translational medicine. Both cancer and osteoporosis will likely see Wnt-signaling-based therapeutics moving into clinical trials E
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ACS Nano capsule synthesis. Capsules were fluorescently labeled by using green or red fluorophores covalently linked to the positive polyelectrolytes PARG (with AF488) or PAH (with Dy547). To provide light-responsiveness to capsules, AuNP agglomerates were deposited between the polyelectrolyte layers through electrostatic interactions. After the core removal, capsules were extensively washed and incubated for 1 h with a DMSO solution containing 0.5 mg mL−1 alsterpaullone. Samples were washed once with ethanol and twice with Milli-Q water. The size of the capsules was 7.8 ± 1.1 μm. Synthesis, functionalization and characterization of microcapsules are extensively described in Supporting Information. Animal Handling. H. vulgaris (strain Zürich, originally obtained by P. Tardent) were cultured at 18 °C on a 12 h light/dark cycle, in Hydra medium (1 mM CaCl2, 0.1 mM NaHCO3, pH 7.0), fed on alternate days with Artemia nauplii. Polyps from homogeneous populations, three-weeks-old and without buds, were selected for the experiments. For capsule treatments, groups of 24 h starved animals were collected in plastic multiwells and incubated with different capsule concentrations for 24 h. Following extensive washes, animals were inspected by stereo, optical, confocal and transmission electron microscopy to monitor capsule uptake, localization and possible cytotoxic effects. An extensive description of microscopic investigations and toxicity methods are reported in the Supporting Information. In Vivo Animal NIR Irradiation. Hydra polyps were NIR irradiated using different optical settings in order to open the capsules in the whole body or, alternatively, in a confined animal region. For both purposes, a 830 nm laser diode was employed to trigger the alsterpaullone release. In the case of whole animal irradiation, polyps were anesthetized and placed on a microscope slide. The laser beam was directly oriented and focused on a 1.4 mm2 area of the animal body. Animals were individually irradiated for 3 min. For local capsule opening, the laser was coupled to an upright microscope. A 63× magnification objective was employed on ≈3 μm2 area. Capsules were singly opened by fluorescence-guided NIR irradiation. Each capsule was exposed to short laser pulses (approximately 3−5 s). After NIR illumination, Hydra polyps were collected in plastic multiwells and cultured as described above. Further details concerning animal irradiation methods are provided in the Supporting Information. Detection of Wnt Signaling Pathway Activation. Upon whole animal or local NIR irradiation, Hydra polyps were examined at different time points by a stereomicroscope in order to detect ectopic tentacle formation. To get molecular evidence of Wnt signaling activation, the expression levels of Wnt target genes were investigated. To this aim, total RNA were extracted from Hydra polyps by using TRIzol (Life Technologies). Total RNA was retro-transcribed by High Capacity cDNA Reverse Transcription Kit (Applied Biosystem). mRNA expression levels of Hydra β-catenin and Wnt3 were profiled by Real-Time Quantitative Reverse Transcription PCR (qRT-PCR).
Present Addresses ⊥
Instituto de Ciencias de Materiales de Aragón (ICMA), Universidad de Zaragoza-CSIC, 50009 Zaragoza, Spain. ∇ Telethon Institute of Genetics & Medicine, Via Campi Flegrei, 34, 80078 Pozzuoli, Italy. Author Contributions ∥
A.A., V.M., and S.C.-R. contributed equally to this work. S.C.-R. designed and performed all capsules synthesis and characterization; S.C.-R, A.A, V.M. designed the experiments, carried out animal treatments, imaging and irradiation experiments. D.I. performed toxicological evaluations. All authors analyzed the data. W.J.P. and C.T. conceived the experiments and wrote the paper, with contributions from all authors. Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS We thank Dr. A. Tino (CNR-Istituto di Scienze Applicate e Sistemi Intelligenti “E. Caianiello”) for fruitful discussions; Giuseppe Marino, for technical assistance with Hydra culturing; Stazione Zoologica Anton Dohrn (Napoli) for access to TEM facilities. This project was supported by the German Research Society (DFG project PA 794/12-1 to W.J.P.), NanoSciEranet (project NANOTRUCK to C.T.). REFERENCES (1) van Amerongen, R.; Nusse, R. Towards an Integrated View of Wnt signaling in Development. Development 2009, 136, 3205−3214. (2) Holstein, T. W. The Evolution of the Wnt Pathway. Cold Spring Harbor Perspect. Biol. 2012, 4, a007922. (3) Whyte, J.; Smith, A.; Helms, J. Wnt Signaling and Injury Repair. Cold Spring Harbor Perspect. Biol. 2012, 4, a008078. (4) Anastas, J. N.; Moon, R. T. WNT Signalling Pathways as Therapeutic Targets in Cancer. Nat. Rev. Cancer 2013, 13, 11−26. (5) Abo, A.; Clevers, H. Modulating WNT Receptor Turnover for Tissue Repair. Nat. Biotechnol. 2012, 30, 835−836. (6) MacDonald, B.; Tamai, K.; He, X. Wnt/beta-Catenin Signaling: Components, Mechanisms, and Diseases. Dev. Cell 2009, 17, 9−26. (7) Leost, M.; Schultz, C.; Link, A.; Wu, Y.; Biernat, J.; Mandelkow, E.; Bibb, J.; Snyder, G.; Greengard, P.; Zaharevitz, D.; Gussio, R.; Senderowicz, A.; Sausville, E.; Kunick, C.; Meijer, L. Paullones are Potent Inhibitors of Glycogen Synthase Kinase-3beta and CyclinDependent kinase 5/p25. Eur. J. Biochem. 2000, 267, 5983−5994. (8) Hobmayer, B.; Rentzsch, F.; Kuhn, K.; Happel, C. M.; von Laue, C. C.; Snyder, P.; Rothbacher, U.; Holstein, T. W. WNT Signalling Molecules Act In Axis Formation in the Diploblastic Metazoan. Nature 2000, 407, 186−9. (9) Lengfeld, T.; Watanabe, H.; Simakov, O.; Lindgens, D.; Gee, L.; Law, L.; Schmidt, H. A.; Ozbek, S.; Bode, H.; Holstein, T. W. Multiple Wnts Are Involved in Hydra Organizer Formation and Regeneration. Dev. Biol. 2009, 330, 186−99. (10) Broun, M.; Gee, L.; Reinhardt, B.; Bode, H. R. Formation of the Head Organizer in Hydra Involves the Canonical Wnt Pathway. Development 2005, 132, 2907−16. (11) Ochs, M.; Carregal-Romero, S.; Rejman, J.; Braeckmans, K.; De Smedt, S. C.; Parak, W. J. Light-Addressable Capsules as Caged Compound Matrix for Controlled Triggering of Cytosolic Reactions. Angew. Chem., Int. Ed. 2013, 52, 695−699. (12) Conde, J.; Ambrosone, A.; Sanz, V.; Hernandez, Y.; Marchesano, V.; Tian, F.; Child, H.; Berry, C. C.; Ibarra, M. R.; Baptista, P. V.; Tortiglione, C.; de la Fuente, J. M. Design of Multifunctional Gold Nanoparticles for In Vitro and In Vivo Gene Silencing. ACS Nano 2012, 6, 8316−8324. (13) Marchesano, V.; Hernandez, Y.; Salvenmoser, W.; Ambrosone, A.; Tino, A.; Hobmayer, B.; de la Fuente, J. M.; Tortiglione, C.
ASSOCIATED CONTENT S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.5b07817. Additional materials and methods (capsule synthesis and characterization, in vivo fluorescence imaging and dynamic of capsule internalization, toxicological analysis; molecular analysis methods); Supporting Figures S1− S20; Supporting Tables S1−S3; additional references (PDF)
AUTHOR INFORMATION Corresponding Authors
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DOI: 10.1021/acsnano.5b07817 ACS Nano XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsnano.5b07817 ACS Nano XXXX, XXX, XXX−XXX
