Our team works on developing ultrathin optoelectronic devices for biomedical implants that can stimulate biophysical processes [1-9]. All these devices rely on near infrared irradiation in the tissue transparency window to actuate nanoscale organic semiconductor components. Our motivation is to provide a minimalistic wireless implant which can perform the duty of standard implantable electrodes, but without invasive wiring [3]. The devices we fabricate are not only wireless, but also 100-1000 times thinner than most existing technologies. Making implants have as small as possible mechanical footprint improves the efficacy of bioelectronic medical treatments by minimizing the risk for inflammation and making surgical implantation less invasive.

While organic semiconductors provide a promising platform for cellular photostimulation, understanding of the mechanisms occurring at a semiconductor/electrolyte interface is a complex problem. Organic semiconductor thin films can afford charging of electrolytic double layers or faradaic reactions. The magnitude of these two effects will depend on the thermodynamics of the materials used in the devices, in particular the nature of the cathodic and anodic components of the device, as well as the capacitance. Through judicious selection of materials one can obtain high photovoltages which can either drive efficient charging of double layer capacitors or faradaic reactions. The former is used to generate displacement currents which can capacitively couple with the cell membrane potential of nearby cells – this can be used to stimulate action potentials. Our experiment and model converge to create a detailed picture of how such devices, known as organic electrolytic photocapacitors, work to affect the gating of ion channels. This device can mimic biphasic current-pulse neurostimulation and thus transduces an optical signal into directly-evoked action potentials in neurons. On the other hand, the other block of our research efforts is directed at devices which, when stimulated with light, perform faradaic chemistry. We focus on the delivery of controlled amounts of reactive oxygen species (mostly peroxide). We aim to study the effects of photoelectrochemically-generated peroxides on physiological processes, with the hope of developing novel therapeutic approaches to neurodegenerative diseases.

Use of light for selective and spatio-temporally resolved control of cell functions (photoceutics) is emerging as a valuable alternative to standard electrical and chemical methods. Here, we propose the use of organic semiconductors as efficient and biocompatible optical transducers, and we focus in particular on breakthrough applications in the field of regenerative medicine.

Devices able to selectively and precisely modulate the fate of living cells, from adhesion to proliferation, from differentiation up to specific function, upon visible light will be presented. Examples of practical applications, recently reported by our group, include optical modulation of the activity of both excitable and non-excitable cells, control of essential cellular switches like transient receptor potential channels and other cationic channels, as well as effective modulation of intracellular calcium signalling for precise control of cell metabolic processes.

We will describe fabrication and optimization of micro- and nano-structured polymeric interfaces, in the form of beads and 3D scaffolds, with different cell models.

As representative examples, we report on (i) a novel strategy to gain optical control of Endothelial Progenitor Cell (EPC) fate and to optically induce angiogenesis in vitro; (ii) optical modulation of mesenchymal stem cells and human-induced pluripotent stem cells physiological pathways; (iii) effects of light-sensitive 3D scaffolds on neuronal differentiation and astrocyte cell models.

The above mentioned study-cases demonstrate groundbreaking applications of organic semiconductors for effective modulation of the cell fate driven by light, with promising perspectives in cell-based therapies. Future opportunities for further implementation of the optoceutics technique and its applications in regenerative medicine will be evaluated in the conclusions.

Bioelectronic medicine, which targets the regeneration of cells and biological matter by delivering local electrical cues, has shown unprecedented potential for developing new therapies. Bioelectronic light actuators transduce light signals into electrical cues, which can then affect the function of living systems. This approach is used to control different types of tissue in a wireless fashion, both in vitro and in vivo. In general, light actuation strategies include the direct excitation of tissue with artificial light sources, the genetic modification of cells to promote photosensitivity, and the use of light sensitive materials and/or devices to mediate photon/cell interaction. These concepts are opening exciting future possibilities in biomedical science, spanning artificial vision to wireless stimulation of the nervous system, as well as tissue regeneration via phototherapy.

Within this multi-disciplinary symposium, we aim to bridge the gap between biology, engineering, and materials science to promote a holistic overview on light actuating concepts and biomedical platforms. We aim to bring together researchers with diverse expertise across various fields, and from around the world, to share their knowledge on light sensitive interfaces that are used to stimulate living systems. We hope that by opening the communication between established light actuating concepts, with less mature but promising approaches, will in turn pave the way for major advancements of light actuating systems for therapeutic and diagnostics.

Nongenetic optical control of neurons is a powerful technique to study and manipulate the function of the nervous system. Herein we have benchmarked the performance of organic electrolytic photocapacitors (OEPCs) at the level of single mammalian cells. These optoelectronic devices use nontoxic organic pigments that form a planar semiconductor on top of ITO and act as an extracellular stimulation electrode driven by deep red light.

Light stimulation and signal propagation require close contacts between cell membranes and pigments. We could biochemically prove cell viability and show with SEM imaging that cell culture cell lines adhere to the surface and neuronal networks establish and exhibit neurite outgrowth.

Our electrophysiological recordings show that millisecond light-stimulation of OEPCs shifted heterologous expressed voltage-gated K⁺ channel activation by ~ 30 mV. We further demonstrate a time-dependent increase in voltage-gated channel conductivity in response to OEPC stimulation and compared our experimental findings with a mathematical model of this bioelectronic-cell system.

In a further step we cultured primary hippocampal neurons on OEPCs and found that millisecond optical stimuli trigger repetitive action potentials in these neurons. Our findings demonstrate that OEPC devices enable the manipulation of neuronal signaling activities with high precision. OEPCs can therefore be integrated into novel in vitro electrophysiology protocols, and the findings can inspire new in vivo applications for the regeneration of axonal sprouting in damaged neuronal tissues.

Implantable neural prostheses are devices exploited to recover impaired or lost functions, such as vision. In this talk I will present our group effort to develop novel visual prostheses. I will cover aspects spanning from materials, to manufacturing methods and preclinical validation. In particular, I will focus on wireless solutions for stimulation.

A common design constraint in neural implants is the presence of cables connecting the electrode-tissue interface to implantable electronic units. The presence of wires and connectors is a significant disadvantage for neural prostheses. They are weak points often leading to failure, they exert mechanical forces and tractions on the implant and the tissue, and they might lead to post-surgical complications, such as infection. Also, the use of implantable electronic units is another disadvantage due to constraints in power consumption, heat generation, and high risk of failure in a wet environment due to leakage. In neurotechnology, truly wireless electrodes are highly desirable.

POLYRETINA is a wireless retinal prosthesis allowing wide-field and high-resolution stimulation of the retina. First, I will describe our recent results related to POLYRETINA testing. Then, I will discuss how materials and solutions adopted for POLYRETINA are now applied to new devices for artificial vision and other applications.

Tissue regeneration is one of the most fascinating biological capabilities of multicellular organisms. During animal evolution this capability has been progressively lost while primitive organisms such as cnidarian are able to regenerate amputated body parts starting from tiny piece of tissues. After injury, various intracellular pathways and intercellular communication must be activated to establish a new tissue integrity and homeostasis. Alongside biochemical and genetic networks, in the last decade bioelectrical signaling has gained an important role as a biophysical master regulator, controlling cell behaviours and driving proliferation, differentiation, migration processes. Here we show the possibility to use of organic semiconducting nanoparticles to modulate the regeneration process of the invertebrate polyps Hydra. We compare the diverse effects of two different polymer photovoltaic materials (P3HT and PCPDTBT). By integrating animal, cellular, molecular and biochemical approaches we suggest a mechanism underlying the cell responses to the nanoparticle photostimulation which could be exploited to augment the tissue regenerative capacity or to inhibit the proliferation potential, opening the path to novel approaches for the optical modulation of various biologic functions.

Electrophysiological investigations reveal a great deal about the organization and function of the retina. In particular, investigations of explanted retinas with multi electrode arrays are widely used for basic and applied research purposes, offering highresolution and detailed information about connectivity and structure. Low-resolution, non-invasive approaches are also widely used. Owing to its delicate nature, highresolution electrophysiological investigations of the intact retina until now are sparse. In this presentation I will discuss progress, challenges and opportunities for electrode arrays suitable for high-resolution, multisite electrophysiological interfacing with the intact retina. In particular, our recent progress towards bi-directional electrophysiological investigation of the intact retina will be discussed.

Organic electrochemical transistors (OECTs) based on poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) are widely used in biosensors with high sensitivity. As a three-terminal device, they can be efficiently gated by non-polarizable electrodes such as Ag/AgCl. When appropriate gate or channel functionalization strategies are employed, they can be used for bio-sensing and bioelectronic readout applications. When used as implantable sensors, these devices would ideally operate fully wirelessly, being wirelessly powered, gated and having the ability for a wireless readout. Additionally, they should be fabricated on flexible and stretchable substrates and be able to conform well to differently shaped tissues. Organic photovoltaic (OPV) powered OECTs fabricated on flexible and stretchable substrates were previously reported [1]. Wireless readout strategies based on conventional technologies were also reported [2], however more reliable and practical readout technologies remain a worthy goal for the near future.
We will demonstrate our approach to wirelessly gating OECTs by gate electrode modified with an organic capacitively coupled photovoltaic-like stack based on a bilayer of metal-free phthalocyanine (H₂PC) and N,N′-dimethyl perylenetetracarboxylic diimide (PTCDI), modified with PEDOT:PSS for increased electrode capacitance. We will show how said devices could be fabricated on 3D structured flexible and stretchable substrates and wirelessly powered by OPVs. In addition, we will propose wireless readout strategies which don't rely on complicated implanted electronic components.

Current implant technology exploits electrical signaling at the electrode-neural interface.  This approach has fundamental problems which limit both the performance and safety of the implants, bearing high invasiveness. Inducing light sensitivity in living organisms is an alternative approach that provides ground breaking opportunities in neuroscience. Optogenetics is a spectacular demonstration of this, yet limited by the viral transfection of exogenous genetic material. In this talk I will describe alternative approaches aimed at NON-genetically inducing light sensitivity in cells or organism by using light-responsive nanostructures (0.1-1 um) or molecular actuators that trigger signaling cascades.  The photophysics of the actuators is fully characterized, both in vitro as well in vivo, and their effect on prokaryiotic and eukaryiotic cells investigated.

Photosynthetic microorganisms produce a wide variety of functional micro/nano structures optimized in billions of years of evolution for interaction with sun light. Combining such specialized structures with tailored molecules paves new ways towards design of sustainable materials for optoelectronic and photonic devices [1].

The following examples will be discussed in the lecture.

i) Photoconverters with photosynthetic bacterial enzymes. Chemical modifications are introduced to boost the performance of hybrid constructs vis-à-vis the native proteins [2] and biocompatible interfaces enable to assemble photoenzymes onto electrodes resulting in active materials for optoelectronics [3].

ii) Nanostructures obtained by in vitro and/or in vivo functionalization of ornate biosilica shells of diatoms unicellular algae  with functional organic molecules with intriguing photonic properties [4].

iii) Intact photosynthetic bacteria cells used as living materials in photoelectrochemical cells for solar energy conversion [5].

The lecture will discuss the logic behind designing and synthesizing the biohybrid micro/nano assemblies, highlighting the challenges raised by the controlled functionalization and integration in devices. New concepts for photoresponsive materials and devices can be envisaged by combining the biotechnological production and modification of photosynthetic microorganisms with photonic and optoelectronic engineering.

The distinctive properties of single-walled carbon nanotubes (SWCNTs) have inspired the development of many novel applications in the field of cell nanobiotechnology. However, studies thus far have not explored the effect of SWCNT functionalization on transport across the cell walls of prokaryotes. We explore the uptake of SWCNTs in Gram-negative cyanobacteria and demonstrate a passive length-dependent and selective internalization of SWCNTs decorated with positively charged biomolecules. We show that lysozyme-coated SWCNTs spontaneously penetrate the cell walls of a unicellular strain and a multicellular strain. A custom-built spinning-disc confocal microscope was used to image the distinct near-infrared SWCNT fluorescence within the autofluorescent cells, revealing a highly inhomogeneous distribution of SWCNTs. Real-time near-infrared monitoring of cell growth and division reveal that the SWCNTs are inherited by daughter cells. Moreover, these nanobionic living cells retained photosynthetic activity and showed an improved photo-exoelectrogenicity when incorporated into bioelectrochemical devices.

Reference:

Antonucci, A., Reggente, M., Roullier, C. et al. Carbon nanotube uptake in cyanobacteria for near-infrared imaging and enhanced bioelectricity generation in living photovoltaics. Nat. Nanotechnol. (2022). 

Organic bioelectronics has experienced significant growth over the past twenty years due to the increasing attention to the design and synthesis of innovative organic smart materials that allowed the development of a plethora of devices suitable for both electrical recording and stimulation[1].  Among all bioelectronics devices, organic transistors emerge as one of the most promising platforms allowing a wide range of applications from electrophysiological signal recording to chemical and biological sensing and neuromorphic devices[2]. In the last decade, efforts have been devoted to the fabrication of photoresponsive remote-controllable devices capable of modulation of the output signal, with potential applications ranging from non-volatile memory devices to logic gates and photodetectors[3]. Indeed, the integration of photoswitchable molecules into Organic Field Effect Transistors (OFETs) and Organic Thin Film transistor (OTFTs) has been already explored suggesting a new and sensitive methodology for tuning the detectable electrical signals depending on the conformational changes of the molecules [4] . The capability of a light-associated modulation of the output current has been demonstrated also for Organic electrochemical transistors (OECTs) showing the possibility to regulate the device response. However, few examples of photoresponsive OECTs are reported in literature and involve the use of metals, sophisticated nanostructures and tedious synthetic approaches while the integration of photoswitchable molecules in OECTs architecture remains unexplored. In this work, a simple and effective synthesis of a light-responsive PEDOT:PSS bearing photoswitchable azobenzenes moieties (azo-tz-PEDOT:PSS) via Cu(I)-catalysed azide-alkyne Huisgen [3+2] cycloaddition (click reaction) has been reported. The full electrochemical and morphological characterization of this innovative material confirmed both its photodynamic behaviour and the electrical modulation induced by light irradiation. Moreover, the successful integration of the azo-tz-PEDOT:PSS in a fully organic OECT as a light-responsive planar gate has been also demonstrated.

The ability to probe the activity of neuronal networks can allow deciphering the fundamental processes in the brain and, eventually, help with the diagnosis and treatment of neurological disorders. Optogenetics is currently a leader among optical stimulation methods. However, optogenetic stimulation is a complex phenomenon that inevitably affects cells due to the need for high expression levels of exogenous light-sensitive ion channels, their gating kinetics, and the activity of specific ions conducted by optogenetic actuators. Therefore, in certain cell systems (e.g., stem cell-derived neurons), it is not desirable to express exogenous proteins that might affect physiology of differentiating and maturing cells.

   We pioneered an alternative optical stimulation method that enables fast and reversible optical stimulation of genetically intact neurons by taking advantage of optoelectronic properties of graphene. Previously, graphene materials have been successfully interfaced with various cell types in cell scaffolds, where graphene merely provides the passive structural support. In contrast, in this study, due to its ability to efficiently convert light to electricity, graphene can play an active role in interactions with live cells.

Here we present a novel optoelectrical biointerface for graphene-mediated optical stimulation (GraMOS) of cells via external light-controlled electric field [1]. Our GraMOS biointerface does not interfere with either genetic make-up of cells or their structural integrity, thus providing truly non-invasive stimulation. By performing imaging and electrophysiological experiments on hiPSC-derived neurons, we demonstrated that the GraMOS biointerface exhibits the excellent biocompatibility and the ability to optically trigger action potentials in neurons. Using geometrically patterning of GraMOS biointerfaces, we are evaluating the connectivity of neuronal networks under various experimental conditions. Graphene-mediated optical stimulation is a powerful new method that can be used 1) to probe the existing neuronal activity, decipher the fundamental processes in the brain and, eventually, help with the diagnosis and treatment of neurological disorders; and 2) to elevate the neuronal activity to the new level that could enable activity-dependent neurogenesis, restore vision, and assist with deep-brain stimulation.