Perfluoropolyether (PFPE) Intermediate Molds for High-Resolution Thermal Nanoimprint Lithography

Masciullo, Cecilia; Sonato, Agnese; Romanato, Filippo; Cecchini, Marco

doi:10.3390/nano8080609

Cited by 13 publications

(6 citation statements)

References 25 publications

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“…The topography with GRs (lines of micron ridges and grooves) was fabricated by means of electron-beam lithography (EBL) and reactive ion etching (RIE) starting from a commercial p-doped silicon wafer (SYLTRONIX, France), as previously reported [45, 46]. Perfluoropolyether (PFPE) resin (FLUOROLINK® MD 700, Solvay Specialty Polymers, Bollate, Italy) was mixed with 3% w/w photoinitiator Darocur 1173 (C10H12O2, 405655 Sigma Aldrich), poured on top of the GR initial silicon mold, and crosslinked by UV-light (365 nm, 25 mW cm −2 ) as reported in [47]. Cyclic olefin copolymer (COC) foils (thickness of 140 μm, Microfluidic ChipShop GmbH, Jena, Germany) were imprinted using an Obducat Nanoimprint 24 system (Obducat, Lund, Sweden) using the PFPE molds.…”

Section: Methodsmentioning

confidence: 99%

The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons

et al. 2019

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BackgroundAlthough neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway.MethodsHere, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance.ResultsWe found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber–binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype.ConclusionsWe identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.

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Section: Methodsmentioning

confidence: 99%

The role of ubiquitin ligase E3A in polarized contact guidance and rescue strategies in UBE3A-deficient hippocampal neurons

et al. 2019

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“…Given our previous results 40 , the T1 geometry is here considered as the gold standard guidance topography for our neuronal cell model. NG patterns were replicated from initial molds into biocompatible and transparent Cyclic Olefin Copolymer (COC) thermoplastic polymer foils by exploiting perfluoropolyether (PFPE) intermediate molds, as described in Materials and Methods and in 54 . Briefly, PFPE intermediate molds were obtained by pouring a PFPE dispersion on top of each initial mold, and then crosslinking with UV light (Fig.…”

Section: Resultsmentioning

confidence: 99%

Neuronal contact guidance and YAP signaling on ultra-small nanogratings

Tonazzini

Masciullo

Savi

et al. 2020

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contact interaction of neuronal cells with extracellular nanometric features can be exploited to investigate and modulate cellular responses. By exploiting nanogratings (nGs) with linewidth from 500 nm down to 100 nm, we here study neurite contact guidance along ultra-small directional topographies. the impact of nG lateral dimension on the neuronal morphotype, neurite alignment, focal adhesion (fA) development and YAp activation is investigated in nerve growth factor (nGf)differentiating PC12 cells and in primary hippocampal neurons, by confocal and live-cell total internal reflection fluorescence (TIRF) microscopy, and at molecular level. We demonstrate that loss of neurite guidance occurs in NGs with periodicity below 400 nm and correlates with a loss of FA lateral constriction and spatial organization. We found that YAP intracellular localization is modulated by the presence of nGs, but it is not sensitive to their periodicity. nocodazole, a drug that can increase cell contractility, is finally tested for rescuing neurite alignment showing mild ameliorative effects. Our results provide new indications for a rational design of biocompatible scaffolds for enhancing nerveregeneration processes.With its chemical and physical complexity, the extracellular environment can drive cell morphogenesis, migration and differentiation 1-4 , In particular, the contact interaction of cells with extracellular nanometric features has a primary role in regulating many physiological 5-12 and pathological processes 13 in vivo, and can be exploited to modulate cell responses in vitro [14][15][16][17] . Cells gather morphological information about the Extracellular matrix (ECM) through integrin-mediated adhesion clusters, called the focal adhesions (FAs), which act as topographical sensors. FAs can integrate multiple nanotopographical details into specific biomolecular instructions via the cytoskeletal signaling, which lastly regulates cell contractility, morphology and migration 9,18,19 , Artificial scaffolds with controlled micro/nano-topographies are definitively the apparatus of choice to better understand the response mechanisms of cells to external stimuli. They indeed allow to select and finely tune specific topographical aspects and selectively study their interaction with living system in vitro. The scientific literature reports many examples of nanostructures that can, for example, direct stem cell fate 20-22 , neuronal and glial adhesion 23-25 , differentiation 26 , polarization, and neurite orientation 14,18 , Our previous studies have demonstrated that plastic nanogratings (NGs) (i.e. alternating lines of sub-micron grooves and ridges, in the range between 500 and 2000 nm in linewidth) can promote neurite alignment and bipolarity of PC12 neuronal cells upon administration of nerve grow factor (NGF), simply by the contact guidance mechanism 27,28 , In these studies, adhesion on the ridges imposes a geometrical and directional constraint to FAs that results in neuronal polarization via the ROCK-mediated pathway 14,29-31 ...

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“…PDMS compliance has been recognized to affect the imprint quality. 26 Despite this limitation, the three volume distributions obtained with three different imprints greatly overlap with each other. We also note that the volume standard deviation is much lower than the 10% limit necessary for proper quantification.…”

Section: Discussionmentioning

confidence: 99%