Dynamic Manipulation of Cell Membrane Curvature by Light-Driven Reshaping of Azopolymer.

Local curvatures on the cell membrane serve as signaling hubs that promote curvature-dependent protein interactions and modulate a variety of cellular processes including endocytosis, exocytosis, and the actin cytoskeleton. However, precisely controlling the location and the degree of membrane curvature in live cells has not been possible until recently, where studies show that nanofabricated vertical structures on a substrate can imprint their shapes on the cell membrane to induce well-defined curvatures in adherent cells. Nevertheless, the intrinsic static nature of these engineered nanostructures prevents dynamic modulation of membrane curvatures. In this work, we engineer light-responsive polymer structures whose shape can be dynamically modulated by light and thus change the induced-membrane curvatures on-demand. Specifically, we fabricate three-dimensional azobenzene-based polymer structures that change from a vertical pillar to an elongated vertical bar shape upon green light illumination. We observe that U2OS cells cultured on azopolymer nanostructures rapidly respond to the topographical change of the substrate underneath. The dynamically induced high membrane curvatures at bar ends promote local accumulation of actin fibers and actin nucleator Arp2/3 complex. The ability to dynamically manipulate the membrane curvature and analyze protein response in real-time provides a new way to study curvature-dependent processes in live cells.

Effect of microneedles shape on skin penetration and transdermal drug administration.

Microneedle (MN) patches are highly efficient and versatile tools for transdermal drug administration, in particular for pain-free, self-medication and rapid local applications. Diffraction ultraviolet (UV) light lithography offers an advanced method in fabricating poly(ethylene glycol)-based MNs with different shapes, by changing both the UV-light exposure time and photomask design. The exposure time interval is limited at obtaining conical structures with aspect ratio < 1:3, otherwise MNs exhibit reduced fracture load and poor indentation ability, not suitable for practical application. Therefore, this work is focused on a systematic analysis of the MN's base shapes effects on the structural characteristics, skin penetration and drug delivery. Analyzing four different base shapes (circle, triangle, square and star), it has been found that the number of vertices in the polygon base heavily affects these properties. The star-like MNs reveal the most efficient skin penetration ability (equal to 40 % of -their length), due to the edges action on the skin during the perforation. Furthermore, the quantification of the drug delivered by the MNs through ex-vivo porcine skin shows that the amounts of small molecules released over 24 h by star-like MNs coated by local anesthetic (Lidocaine) and an anti-inflammatory (Diclofenac epolamine) drugs are 1.5× and 2× higher than the circular-MNs, respectively.

One-Shot Fabrication of Polymeric Hollow Microneedles by Standard Photolithography.

Microneedles (MNs) are an emerging technology in pharmaceutics and biomedicine, and are ready to be commercialized in the world market. However, solid microneedles only allow small doses and time-limited administration rates. Moreover, some well-known and already approved drugs need to be re-formulated when supplied by MNs. Instead, hollow microneedles (HMNs) allow for rapid, painless self-administrable microinjection of drugs in their standard formulation. Furthermore, body fluids can be easily extracted for analysis by a reverse use of HMNs, thus making them perfect for sensing issues and theranostics applications. The fabrication of HMNs usually requires several many-step processes, increasing the costs and consequently decreasing the commercial interest. Photolithography is a well-known fabrication technique in microelectronics and microfluidics that fabricates MNs. In this paper, authors show a proof of concept of a patented, easy and one-shot fabrication of two kinds of HMNs: (1) Symmetric HMNs with a "volcano" shape, made by using a photolithographic mask with an array of transparent symmetric rings; and (2) asymmetric HMNs with an oblique aperture, like standard hypodermic steel needles, made by using an array of transparent asymmetric rings, defined by two circles, which centers are slightly mismatched. Simulation of light propagation, fabrication process, and preliminary results on ink microinjection are presented.

Photoactive Interfaces for Spatio-Temporal Guidance of Mesenchymal Stem Cell Fate.

Patterned surfaces have proved effective in guiding stem cells commitment to a specific lineage by presenting highly ordered biophysical/biochemical cues at the cellmaterial interface. Their potency in controlling cell fate can be significantly empowered by encoding logic of space and time control of signal presentation. Here, azopolymeric photoactive interfaces are proposed to present/withdraw morphophysical signals to living cells using a green light trigger in a non-invasive spatio-temporal controlled way. To assess the potency of these dynamic platforms in controlling cell decision and fate, topography changes are actuated by light at specific times to reverse the fate of otherwise committed human mesenchymal stem cells (hMSC) toward osteoblastic lineage. It is first proved by dynamic change from ordered parallel patterning to flat or grid surfaces, that it is possible to induce cyclic cellular and nuclear stretches. Furthermore, by culturing hMSCs on a specific pattern known to prime them toward osteoblast lineage, the possibility to reroute or reverse stem cell fate decision by dynamic modulation of morphophysical signal is proved. To conclude, dynamic topographies can control the spatial conformation of hMSCs, modulate lineage reversal even after several weeks of culture and redirect lineage specification in response to light-induced changes in the microenvironment.

Dynamic azopolymeric interfaces for photoactive cell instruction.

The ability to affect a wide range of biophysical properties through the use of light has led to the development of dynamic cell instructive materials. Using photoresponsive materials such as azopolymers, smart systems that use external, minimally damaging, light irradiation can be used to trigger specific surface morpho-physical properties in the presence of living cells. The interaction of light with an azopolymer film induces a mass migration phenomenon, allowing a variety of topographic patterns to be embossed on the polymeric film. Photoisomerization induces conformational changes at the molecular and macroscopic scale, resulting in light-induced variations of substrate morphological, physical, and mechanical properties. In this review, we discuss the photoactuation of azopolymeric interfaces to provide guidelines for the engineering and design of azopolymer films. Laser micropatterning for the modulation of azopolymer surfaces is examined as a way to diversify the capabilities of these polymers in cellular systems. Mass migration effects induced by azopolymer switching provides a foundation for performing a broad range of cellular manipulation techniques. Applications of azopolymers are explored in the context of dynamic culture systems, gaining insight into the complex processes involved in dynamic cell-material interactions. The review highlights azopolymers as a candidate for various applications in cellular control, including cell alignment, migration, gene expression, and others. Recent advances have underlined the importance of these systems in applications regarding three-dimensional cell culture and stem cell morphology. Azopolymers can be used not only to manipulate cells but also to probe for mechanistic studies of cellular crosstalk in response to chemical and mechanical stimuli.

Three-Dimensional Microstructured Azobenzene-Containing Gelatin as a Photoactuable Cell Confining System.

In materials science, there is a considerable interest in the fabrication of highly engineered biomaterials that can interact with cells and control their shape. In particular, from the literature, the role played by physical cell confinement in cellular structural organization and thus in the regulation of its functions has been well-established. In this context, the addition of a dynamic feature to physically confining platforms aiming at reproducing in vitro the well-known dynamic interaction between the cells and their microenvironment would be highly desirable. To this aim, we have developed an advanced gelatin-based hydrogel that can be finely micropatterned by two-photon polymerization and stimulated in a controlled way by light irradiation thanks to the presence of an azobenzene cross-linker. Light-triggered expansion of gelatin microstructures induced an in-plane nuclear deformation of physically confined NIH-3T3 cells. The microfabricated photoactuable gelatin shown in this work paves the way to new "dynamic" caging culture systems that can find applications, for example, as "engineered stem cell niches".