Silvia Cavalli

Reversible Holographic Patterns on Azopolymers for Guiding Cell Adhesion and Orientation

Topography of material surfaces is known to influence cell behavior at different levels: from adhesion up to differentiation. Different micro- and nanopatterning techniques have been employed to create patterned surfaces to investigate various aspects of cell behavior, most notably cellular mechanotransduction. Nevertheless, conventional techniques, once implemented on a specific substrate, fail in allowing dynamic changes of the topographic features. Here we investigated the response of NIH-3T3 cells to reversible topographic signals encoded on light-responsive azopolymer films. Switchable patterns were fabricated by means of a well-established holographic setup. Surface relief gratings were realized with Lloyds mirror system and erased with circularly polarized or incoherent light. Cell cytoskeleton organization and focal adhesion assembly proved to be very sensitive to the underlying topographic signal. Thereafter, pattern reversibility was tested in air and wet environment by using temperature or light as a trigger. Additionally, pattern modification was dynamically performed on substrates with living cells. This study paves the way toward an in situ and real-time investigation of the material-cytoskeleton crosstalk caused by the intrinsic properties of azopolymers.

Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

Development of an Activity-Based Probe for Autotaxin

Autotaxin (ATX), or ecto‐nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted lysophospholipase D that hydrolyses lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX has been implicated in tumour progression and inflammation, and might serve as a biomarker. Here we describe the development of a fluorescent activity‐based probe that covalently binds to the active site of ATX. The probe consists of a lysophospholipid‐based backbone linked to a trapping moiety that becomes reactive after phosphate ester hydrolysis, and a Cy5 fluorescent dye to allow visualisation of active ATX. The probe reacts specifically with the three known isoforms of ATX, it competes with small‐molecule inhibitors for binding to ATX and allows ATX activity in plasma to be determined. Our activity‐based reporter will be useful for monitoring ATX activity in biological fluids and for inhibitor screening.

Light-responsive polymer brushes: active topographic cues for cell culture applications

In the call for novel stimuli-responsive biomaterials, azobenzene-containing polymer brushes entail a remarkable potential. In fact, their ability to be patterned at the micro- and nanoscale using interference lithography (IL) might be exploited for the realisation of cell-instructive materials (CIMs). In this work, Disperse Red 1 (DR1)-based photoresponsive polymer brushes were synthesised using a controlled radical polymerisation technique. A sinusoidal pattern was inscribed on the azopolymer brush samples using Lloyds IL configuration. Interestingly, we found that seeded human umbilical vein endothelial cells (HUVECs) oriented in the pattern direction. Furthermore, using a non-cytotoxic ultrasonication treatment, pattern erasure was achieved. Hence, we envisage the possibility of using these surfaces as reconfigurable cell-instructive biomaterials for tissue engineering applications.

Biological evaluation of a Porphyrin-SPION nanoconjugate as an antimicrobial magnetic photosensitizer

The present work describes the use of a magnetic porphyin (5-(4-carboxy-phenyl)-10,15,20-triphenyl-21H, 23H-porphyrin TPP) nanoconjugate (SPION-TPP) for destroying pathogenic bacteria followed by the recovery of the magnetic photosensitizer. SPION-TPP was tested for its activity against two different gram-positive bacterial strains (Staphylococcus aureus and Steptoccoccus mutans). It is observed that SPION-TPP at a very low concentration of 0.5 μM is effective in destroying gram-positive bacteria (107–108 CFU ml-1) S. aureus with several orders reduction and few orders in S. mutans. The aim of this work is to combine photoactivity against microorganisms imparted by the photosensitizer with the possibility of recovering the nanoconstruct with magnets for disposal/reuse.

Imaging and characterization of surface relief gratings on azopolymer by digital holographic microscopy

Azopolymer materials belong to family special materials, which are subject to photo-isomerization when illuminated by appropriate light wavelength. Optical characterization of azopolymer materials is interesting because they can be patterned when illuminated by coherent polarized light with potentially interesting applications in the biotechnology, photonic elements, molding templates, etch masks and micro-nanochannels. The interference lithography is an excellent tool to trigger the isomerization reaction on the material. During this work, switchable patterns were fabricated by means of a well established holographic set-up: surface relief gratings (SRGs) were realized with Lloyd’s mirror system. Moreover, optical characterization of the material was performed, starting from a commercial one and using a new way to analyse SRGs by means of Digital Holography Microscopy, to determine relevant parameters for the realization of the patterns with different shape and size. Some preliminary results of the influence of such patterns on the cell behavior were shown.