Porous Semiconducting Polymer Nanoparticles as Intracellular Biophotonic Mediators to Modulate the Reactive Oxygen Species Balance.

The integration of nanotechnology with photoredox medicine has led to the emergence of biocompatible semiconducting polymer nanoparticles (SPNs) for the optical modulation of intracellular reactive oxygen species (ROS). However, the need for efficient photoactive materials capable of finely controlling the intracellular redox status with high spatial resolution at a nontoxic light density is still largely unmet. Herein, highly photoelectrochemically efficient photoactive polymer beads are developed. The photoactive material/electrolyte interfacial area is maximized by designing porous semiconducting polymer nanoparticles (PSPNs). PSPNs are synthesized by selective hydrolysis of the polyester segments of nanoparticles made of poly(3-hexylthiophene)-graft-poly(lactic acid) (P3HT-g-PLA). The photocurrent of PSPNs is 4.5-fold higher than that of nonporous P3HT-g-PLA-SPNs, and PSPNs efficiently reduce oxygen in an aqueous environment. PSPNs are internalized within endothelial cells and optically trigger ROS generation with a >1.3-fold concentration increase with regard to nonporous P3HT-SPNs, at a light density as low as a few milliwatts per square centimeter, fully compatible with in vivo, chronic applications.

Nongenetic Optical Modulation of Pluripotent Stem Cells Derived Cardiomyocytes Function in the Red Spectral Range.

Optical stimulation in the red/near infrared range recently gained increasing interest, as a not-invasive tool to control cardiac cell activity and repair in disease conditions. Translation of this approach to therapy is hampered by scarce efficacy and selectivity. The use of smart biocompatible materials, capable to act as local, NIR-sensitive interfaces with cardiac cells, may represent a valuable solution, capable to overcome these limitations. In this work, a far red-responsive conjugated polymer, namely poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl]] (PCPDTBT) is proposed for the realization of photoactive interfaces with cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Optical excitation of the polymer turns into effective ionic and electrical modulation of hPSC-CMs, in particular by fastening Ca2+ dynamics, inducing action potential shortening, accelerating the spontaneous beating frequency. The involvement in the phototransduction pathway of Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) and Na+ /Ca2+ exchanger (NCX) is proven by pharmacological assays and is correlated with physical/chemical processes occurring at the polymer surface upon photoexcitation. Very interestingly, an antiarrhythmogenic effect, unequivocally triggered by polymer photoexcitation, is also observed. Overall, red-light excitation of conjugated polymers may represent an unprecedented opportunity for fine control of hPSC-CMs functionality and can be considered as a perspective, noninvasive approach to treat arrhythmias.

Bimodal modulation of in vitro angiogenesis with photoactive polymer nanoparticles.

Angiogenesis is a fundamental process in biology, given the pivotal role played by blood vessels in providing oxygen and nutrients to tissues, thus ensuring cell survival. Moreover, it is critical in many life-threatening pathologies, like cancer and cardiovascular diseases. In this context, conventional treatments of pathological angiogenesis suffer from several limitations, including low bioavailability, limited spatial and temporal resolution, lack of specificity and possible side effects. Recently, innovative strategies have been explored to overcome these drawbacks based on the use of exogenous nano-sized materials and the treatment of the endothelial tissue with optical or electrical stimuli. Here, conjugated polymer-based nanoparticles are proposed as exogenous photo-actuators, thus combining the advantages offered by nanotechnology with those typical of optical stimulation. Light excitation can achieve high spatial and temporal resolution, while permitting minimal invasiveness. Interestingly, the possibility to either enhance (≈+30%) or reduce (up to -65%) the angiogenic capability of model endothelial cells is demonstrated, by employing different polymer beads, depending on the material type and the presence/absence of the light stimulus. In vitro results reported here represent a valuable proof of principle of the reliability and efficacy of the proposed approach and should be considered as a promising step towards a paradigm shift in therapeutic angiogenesis.

Semiconducting Polymer Nanoporous Thin Films as a Tool to Regulate Intracellular ROS Balance in Endothelial Cells.

The design of soft and nanometer-scale photoelectrodes able to stimulate and promote the intracellular concentration of reactive oxygen species (ROS) is searched for redox medicine applications. In this work, we show semiconducting polymer porous thin films with an enhanced photoelectrochemical generation of ROS in human umbilical vein endothelial cells (HUVECs). To achieve that aim, we synthesized graft copolymers, made of poly(3-hexylthiophene) (P3HT) and degradable poly(lactic acid) (PLA) segments, P3HT-g-PLA. In a second step, the hydrolysis of sacrificial PLA leads to nanometer-scale porous P3HT thin films. The pore sizes in the nm regime (220-1200 nm) were controlled by the copolymer composition and the structural arrangement of the copolymers during the film formation, as determined by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The porous P3HT thin films showed enhanced photofaradaic behavior, generating a higher concentration of ROS in comparison to non-porous P3HT films, as determined by scanning electrochemical microscopy (SECM) measurements. The exogenous ROS production was able to modulate the intracellular ROS concentration in HUVECs at non-toxic levels, thus affecting the physiological functions of cells. Results presented in this work provide an important step forward in the development of new tools for precise, on-demand, and non-invasive modulation of intracellular ROS species and may be potentially extended to many other physiological or pathological cell models.

Conjugated polymers mediate intracellular Ca2+ signals in circulating endothelial colony forming cells through the reactive oxygen species-dependent activation of Transient Receptor Potential Vanilloid 1 (TRPV1).

Endothelial colony forming cells (ECFCs) represent the most suitable cellular substrate to induce revascularization of ischemic tissues. Recently, optical excitation of the light-sensitive conjugated polymer, regioregular Poly (3-hexyl-thiophene), rr-P3HT, was found to stimulate ECFC proliferation and tube formation by activating the non-selective cation channel, Transient Receptor Potential Vanilloid 1 (TRPV1). Herein, we adopted a multidisciplinary approach, ranging from intracellular Ca2+ imaging to pharmacological manipulation and genetic suppression of TRPV1 expression, to investigate the effects of photoexcitation on intracellular Ca2+ concentration ([Ca2+]i) in circulating ECFCs plated on rr-P3HT thin films. Polymer-mediated optical excitation induced a long-lasting increase in [Ca2+]i that could display an oscillatory pattern at shorter light stimuli. Pharmacological and genetic manipulation revealed that the Ca2+ response to light was triggered by extracellular Ca2+ entry through TRPV1, whose activation required the production of reactive oxygen species at the interface between rr-P3HT and the cell membrane. Light-induced TRPV1-mediated Ca2+ entry was able to evoke intracellular Ca2+ release from the endoplasmic reticulum through inositol-1,4,5-trisphosphate receptors, followed by store-operated Ca2+ entry on the plasma membrane. These data show that TRPV1 may serve as a decoder at the interface between rr-P3HT thin films and ECFCs to translate optical excitation in pro-angiogenic Ca2+ signals.

High Aspect Ratio and Light-Sensitive Micropillars Based on a Semiconducting Polymer Optically Regulate Neuronal Growth.

Many nano- and microstructured devices capable of promoting neuronal growth and network formation have been previously investigated. In certain cases, topographical cues have been successfully complemented with external bias, by employing electrically conducting scaffolds. However, the use of optical stimulation with topographical cues was rarely addressed in this context, and the development of light-addressable platforms for modulating and guiding cellular growth and proliferation remains almost completely unexplored. Here, we develop high aspect ratio micropillars based on a prototype semiconducting polymer, regioregular poly(3-hexylthiophene-2,5-diyl) (P3HT), as an optically active, three-dimensional platform for embryonic cortical neurons. P3HT micropillars provide a mechanically compliant environment and allow a close contact with neuronal cells. The combined action of nano/microtopography and visible light excitation leads to effective optical modulation of neuronal growth and orientation. Embryonic neurons cultured on polymer pillars show a clear polarization effect and, upon exposure to optical excitation, a significant increase in both neurite and axon length. The biocompatible, microstructured, and light-sensitive platform developed here opens up the opportunity to optically regulate neuronal growth in a wireless, repeatable, and spatio-temporally controlled manner without genetic modification. This approach may be extended to other cell models, thus uncovering interesting applications of photonic devices in regenerative medicine.

Light-Triggered Electron Transfer between a Conjugated Polymer and Cytochrome C for Optical Modulation of Redox Signaling.

Protein reduction/oxidation processes trigger and finely regulate a myriad of physiological and pathological cellular functions. Many biochemical and biophysical stimuli have been recently explored to precisely and effectively modulate intracellular redox signaling, due to the considerable therapeutic potential. Here, we propose a first step toward an approach based on visible light excitation of a thiophene-based semiconducting polymer (P3HT), demonstrating the realization of a hybrid interface with the Cytochrome c protein (CytC), in an extracellular environment. By means of scanning electrochemical microscopy and spectro-electrochemistry measurements, we demonstrate that, upon optical stimulation, a functional interaction between P3HT and CytC is established. Polymer optical excitation locally triggers photoelectrochemical reactions, leading to modulation of CytC redox activity, either through an intermediate step, involving reactive oxygen species formation, or via a direct photoreduction process. Both processes are triggered by light, thus allowing excellent spatiotemporal resolution, paving the way to precise modulation of protein redox signaling.

High-Aspect-Ratio Semiconducting Polymer Pillars for 3D Cell Cultures.

Hybrid interfaces between living cells and nano/microstructured scaffolds have huge application potential in biotechnology, spanning from regenerative medicine and stem cell therapies to localized drug delivery and from biosensing and tissue engineering to neural computing. However, 3D architectures based on semiconducting polymers, endowed with responsivity to visible light, have never been considered. Here, we apply for the first time a push-coating technique to realize high aspect ratio polymeric pillars, based on polythiophene, showing optimal biocompatibility and allowing for the realization of soft, 3D cell cultures of both primary neurons and cell line models. HEK-293 cells cultured on top of polymer pillars display a remarkable change in the cell morphology and a sizable enhancement of the membrane capacitance due to the cell membrane thinning in correspondence to the pillars' top surface, without negatively affecting cell proliferation. Electrophysiology properties and synapse number of primary neurons are also very well preserved. In perspective, high aspect ratio semiconducting polymer pillars may find interesting applications as soft, photoactive elements for cell activity sensing and modulation.

Photocatalytic Activity of Polymer Nanoparticles Modulates Intracellular Calcium Dynamics and Reactive Oxygen Species in HEK-293 Cells.

Optical modulation of living cells activity by light-absorbing exogenous materials is gaining increasing interest, due to the possibility both to achieve high spatial and temporal resolution with a minimally invasive and reversible technique and to avoid the need of viral transfection with light-sensitive proteins. In this context, conjugated polymers represent ideal candidates for photo-transduction, due to their excellent optoelectronic and biocompatibility properties. In this work, we demonstrate that organic polymer nanoparticles, based on poly(3-hexylthiophene) conjugated polymer, establish a functional interaction with an in vitro cell model (Human Embryonic Kidney cells, HEK-293). They display photocatalytic activity in aqueous environment and, once internalized within the cell cytosol, efficiently generate reactive oxygen species (ROS) upon visible light excitation, without affecting cell viability. Interestingly, light-activated ROS generation deterministically triggers modulation of intracellular calcium ion flux, successfully controlled at the single cell level. In perspective, the capability of polymer NPs to produce ROS and to modulate Ca2+ dynamics by illumination on-demand, at non-toxic levels, may open the path to the study of biological processes with a gene-less approach and unprecedented spatio-temporal resolution, as well as to the development of new biotechnology tools for cell optical modulation.