Periodic Reporting for period 1 - LIGHTEST (LIght-responsive GrapHene-based inTerfaces for Electrical STimulation)
Reporting period: 2020-05-01 to 2022-04-30
Neurological disorders are one of the world’s leading medical and societal challenges, affecting approximately 2.6 billion people worldwide. In Europe, the economic burden of neurodegenerative diseases is rapidly growing, with estimated costs to exceed €350 billion/year by 2050. The European Commission has strongly invested in novel strategies to prevent and treat neurological disorders, which constitutes a major challenge towards the United Nations’ Sustainable Development Goal for 2030 on Good Health and Well Being. Because of the lack of efficiency of pharmacological options, alternatives such as electrical stimulation using implanted electrodes are becoming increasingly popular. However, this strategy lacks spatiotemporal control over the stimulation of specific neural circuits. Although light has been employed in the modulation of the electrical activity of single cells with unprecedented resolution, this strategy required genetic modification to express membrane proteins sensitive to visible light. Another caveat of clinically approved devices consists of their adverse effects, which are commonly associated with the rigidity and redox reactions associated with Faradaic charge conduction in metal electrodes.
Non-genetic, light-mediated stimulation could offer a promising alternative to transform the treatment of neurological disorders and promote brain tissue regeneration. In order to realise that, biointerfaces need to be designed with flexible materials with low electrochemical impedance, in order to minimise electrode degradation and tissue damage. Graphene is an ideal candidate for this application because not only fits these requirements but also electrically responds to light. However, in order to develop minimally invasive devices, the use of tissue-penetrating near-infrared (NIR) radiation may be limited by graphene’s capability of converting it to heat.
This project aims to incorporate lanthanide-doped upconversion nanoparticles (UCNPs) capable of absorbing NIR radiation and emitting visible light. Graphene was therefore conjugated with UCNPs in order to generate electrodes capable of enhancing electrical conductivity upon NIR activation.
Non-genetic, light-mediated stimulation could offer a promising alternative to transform the treatment of neurological disorders and promote brain tissue regeneration. In order to realise that, biointerfaces need to be designed with flexible materials with low electrochemical impedance, in order to minimise electrode degradation and tissue damage. Graphene is an ideal candidate for this application because not only fits these requirements but also electrically responds to light. However, in order to develop minimally invasive devices, the use of tissue-penetrating near-infrared (NIR) radiation may be limited by graphene’s capability of converting it to heat.
This project aims to incorporate lanthanide-doped upconversion nanoparticles (UCNPs) capable of absorbing NIR radiation and emitting visible light. Graphene was therefore conjugated with UCNPs in order to generate electrodes capable of enhancing electrical conductivity upon NIR activation.
This project aimed to developed a graphene-based biointerface for electrical stimulation using NIR radiation. First, the electrochemical response of graphene printed electrodes to light at different wavelengths was investigated using cyclic voltammetry and electrochemical resonance spectroscopy (Figure 1). Graphene enhanced its capacitive charge conduction in the presence of light, irrespective of the used wavelength. This effect was instead dictated by the laser power, reaching an optimal response around 100 mW/cm2 using NIR radiation at 780 nm. Using these settings, a UCNP library synthesised using a Design of Experiments approach was screened on graphene electrodes in the presence of NIR radiation (780 nm or 980 nm). Electrochemical characterisation revealed that the intrinsic capacitive charge conduction of graphene could be improved by 3-fold depending on the chemical composition of the anchored UCNPs. Two different UCNP formulations responding to either of the tested NIR wavelengths were then covalently attached to graphene (Figure 2). The generated nanocomposites were characterised with respect to their size and chemical composition. The chemical reaction did not significantly affect the morphology of graphene, resulting in stable complexes with average size between 1-2 μm and minor agglomeration despite the reduced oxygen content. UCNPs were attached to graphene with an estimated yield of 18.5%±0.7.
Finally, the biological impact of these nanocomposites was investigated in the presence of NIR radiation (Figure 3). Conjugation of UCNPs to graphene (GU) resulted in a biocompatible material with minimal cytotoxicity and oxidative stress, irrespective of their activation with NIR radiation (980 nm, 100 mW/cm2). SH-5YSY human neuroblastoma cells were used as a model to interface with graphene-based substrates prepared by spray coating onto PET films. Conjugation of graphene with UCNPs significantly increased the number of mitotic cells, evidencing increased cell proliferation (Figure 3). Interestingly, this effect was further enhanced by NIR radiation at 980 nm but not at 780 nm. Immunocytochemical analysis showed that glial and neuronal markers (e.g. GFAP, DCX) decreased with exposure to NIR radiation. Future work will investigate what (de)differentiation mechanisms might be activated, in order to better understand the mechanisms behind neurostimulation.
Finally, the biological impact of these nanocomposites was investigated in the presence of NIR radiation (Figure 3). Conjugation of UCNPs to graphene (GU) resulted in a biocompatible material with minimal cytotoxicity and oxidative stress, irrespective of their activation with NIR radiation (980 nm, 100 mW/cm2). SH-5YSY human neuroblastoma cells were used as a model to interface with graphene-based substrates prepared by spray coating onto PET films. Conjugation of graphene with UCNPs significantly increased the number of mitotic cells, evidencing increased cell proliferation (Figure 3). Interestingly, this effect was further enhanced by NIR radiation at 980 nm but not at 780 nm. Immunocytochemical analysis showed that glial and neuronal markers (e.g. GFAP, DCX) decreased with exposure to NIR radiation. Future work will investigate what (de)differentiation mechanisms might be activated, in order to better understand the mechanisms behind neurostimulation.
Overall, this project will generate a platform to modulate cell activity using electrical stimuli controlled by light, which may be applied in minimally invasive strategies for regenerative medicine. This project demonstrated for the first time that NIR radiation could be used to elicit generation of electric current from graphene under acceptable conditions for application in the brain. This could ultimately improve the quality of life of patients suffering from neurodegenerative diseases and traumatic injuries including stroke, in order to promote the recovery of damaged tissues.