Implantable and bioresorbable nanostructured fluorescence sensor for in-vivo pH monitoring

Implantable and Bioresorbable Nanostructured Fluorescence Sensor for In vivo pH Monitoring M. Corsi1, A. Paghi1, S. Mariani1, G. Golinelli2, A. Debrassi3, G. Egri3, G. Leo4, E. Vandini4, A. Vilella4, L. Dahne3, D. Giuliani4and G. Barillaro1 1: Department of Information Engineering, University of Pisa, Via G. Caruso 16, 56122, Pisa, Italy 2: Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Via del Pozzo 71, 41124, Modena, Italy 3: Surflay Nanotec GmbH, Max-Planck-Straße 3, 12489 Berlin, Germany 4: Department of Biomedical, metabolic and Neural Sciences, University of Modena and Reggio Emilia, via G. Campi 287, 41125, Modena Italy 31 October, 2022 Martina Corsi 1 31 October, 2022 Martina Corsi 2 Biocompatibility •Citotossicity •Tissue immune response Biodegradability •Degradation mechanism •Degradation regulation •Fluctuations of the pH level in blood induce regulatory effects at the level of the cell, organ, and organism. Furthermore, changes of the pH level in body fluids are predictive of cancer growth [1] and cardiac disease, [2] among others. •In-situ and in-vivo bioresorbable fluorescence pH sensors were fabricated with application to preliminary diagnosis of diseases and to in skin wound monitoring, that during the lengthy healing process, is invariably exposed to bacteria, which can colonize the wound bed and form biofilms respectively. •Bioresorbable sensors leveraging the use of biocompatible and biodegradable materials are fabricated to guarantee their functionality from days to week once implanted in the body, then they fully degrade in vivo with safe by-products [3, 4]. [1] S. W. Hwang et al., Adv. Mater., 2014. [2] M. T. Ghoneim et al., Chem. Rev. 2019. [3] G. D. Cha et al. Adv. Healthc. Mater., 2019. [4] A. A. La Mattina et al., Adv. Sci., 2020. 31 October, 2022 Martina Corsi 3 How is it made up? nPSi fabrication Emission peak of Rh at 580 nm Excitation peak of Rh at 520 nm Layer-by-Layer deposition Transfer printing on PLGA film 31 October, 2022 Martina Corsi 4 nPSiO2 PAH:Rh PMAA:Rh Swelling @ Low pH Shrinking @ High pH How does it work? The inner surface of the pores was coated by electrostatic layer-by-layer technique with pH-responsive polyelectrolytes engineered with Rhodamine-6G (Rh). The polymer ionization degree (and therefore cohesion of the polymer) changes with pH value, leading to shrinking/swelling of the assembly and, therefore to the fluorophore distance variation. The variation of the fluorophore distance produces a fluorophore quenching and reduces the photoluminescence intensity at basic pH. 31 October, 2022 Martina Corsi 5 Why PSi? Porous silicon was chosen for its huge specific surface (500 times hight than flat substrate) which allows to accommodate a huge quantity of Rh per unit area compared to the flat silicon and therefore the intensity of light emission for each pH value. The homogeneity of the LbL nanoassembly was evaluated by fluorescence optical microscope analysis with a 520 nm emission laser. Bright field nPSiO2 Fluorescence nPSiO2 10 m 31 October, 2022 Martina Corsi 6 pH sensors on PLGA were tested at physiological temperature (37 °C) in physiological PBS buffer and in interstitial fluid simulant at different pH, ranging from 4 to 7.5 pH Sensing in vitro 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 10k 20k 30k 40k 50k pH sensor in ISF Photoluminescence Intensity (counts) pH PBS ISF 0 2k 4k 6k 8k 10k |Sensitivity| (counts/pH) 31 October, 2022 Martina Corsi 7 050 100 150 200 250 0 20 40 60 80 100 PL/PL0 (%) Time (h) sensor operation sensor dissolution •The PL intensity in physiological conditions was constant for the first 100 hours of operation, with an average value of 96.6 ± 2.2%. Remarkably, the calibration curves of the pH sensor measured at the beginning of the experiment, as well as after 50 and 95 hours of operation in the pH range from 4 to 7.5 were well superposed. •The performance of the pH sensor was evaluated over >200 hours in PBS buffer at 37°C. After 100 hours, the PL intensity in physiological conditions steadily reduced over time due to degradation of the fluorescent polymer stack, then vanished in about 220 hours once complete degradation of the multilayer stack occurred. In vitro dissolution 31 October, 2022 Martina Corsi 8 Sensors were implanted in mice under the dermis to carry out in vivo tests at different pH values. After 30 minutes of pH variation (from physiological pH to pH 4 with intradermal injection) the signal increases of a factor ~2.8. Injection pH 4Injection pH 7.5 Physiological pH pH Sensing in vivo 31 October, 2022 Martina Corsi 9 In vivo degradation Implant 2 mo 12 Radiant efficiency x 108 (p s-1 μW) 0.8 0.6 0.4 0.2 1 1.2 Implant 1w 2mo 0 1 2 3 4 5 6 7 8 9 10 Implant CTR TRE x 108(p cm² s-1µW-1) o o* * In-vivo dissolution of implanted sensor was investigated monitoring the fluorescence intensity through skin at the implant site over a period of 2 months. The fluorescence signal decreased down to the value of tissue autofluorescence of control mice after a week, retaining the same value for two months. After two months, animals with and without sensor implanted were sacrificed to verify biocompatibility and bioresorbability of the pH sensor.