Implantable and bioresorbable chemical sensors for in-vivo monitoring of clinical-diagnostic markers

Implantable and bioresorbable chemical sensors for in-vivo monitoring of clinical-diagnostic markers Giuseppe Barillaro Information Engineering Dpt University of Pisa [email protected] Photo credit: Beckman Institute, University of Illinois and Tufts University Credits to: Prof. J. Rogers’s Group J.-K. Chang, et al. , Proc. Natl. Acad. Sci. USA 2017, 114, E5522. Bioresorbable sensing systems Zhang et al, Chem. Rev. 2023, 123, 11722−11773 Physical sensors Chemical sensors Encapsulation Dielectric Conductor Semiconductor Substrate PLGA PVA Silicon Silicon ZnO Germanium Magnesium Zinc Molibdenum Cellulose SiO2 Collagen PLGA SiO2 Current picture and challenges S. W. Hwang, et al., Nano Lett. 2015, 15, 2801. C. M. Boutry, et al. Nat. Biomed. Eng. 2019, 3, 47. G. A. Salvatore, et al., Adv. Funct. Mater. 27, (2017). H. S. Kim et al.. Adv. Healthc. Mater. 2018, 7. D1.2 Project Public Launch WP1 Project Management, Dissemination, Exploitation, Communication This project is funded by the European Union Horizon Europe programme under grant agreement No 101046946 This project has received funding from the European Union’s Horizon 2020 research and innovation programm e under grant agreem ent No 952071 D1.2 - Project Quality Handbook WP1 - Project coordination and technical management Partner: PNO Authors: Carolina Salas, Antonio M . Ortiz, Jeanett Bolther Version: v. final Date: 31.08.2020 www.resorb-project.eu chemical sensors implant resorb battery and LEDs Nanoporous silica membranes after oxidation on PLGA carrier Bioresorbability in physiological conditions M. Corsi, A. Paghi, et al., Advanced Science (2022) 14 220 h (Figure 3g-i). The PL intensity decrease can be ascribed to the degradation, chemical and/or 1 mechanical, of the fluorescent polymer multilayer, given that the silica scaffold dissolved in about 2 50 hours. The polymer brush architecture of the fluorescent multilayer after dissolution of the silica 3 scaffold might impact significantly on its degradation (Figure S14). Remarkably, full sensor 4 degradation occurred on the same timescale as the sensor operation, that is in about 100 hours. By 5 best fitting the PL reduction trend with a linear model, degradation rates of about -0.9 and -0.8 % h6 1 were consistently achieved for pH sensors on native silicon in a flow cell and on PLGA foil under 7 artificial skin, respectively. The polymer multilayer degradation was further confirmed by both 8 optical/fluorescence microscopy. No macroscopic polymer residues or residual photoluminescence 9 were found in the solution collected at the output of the flow cell or in the skin around the sensor 10 location at the end of the experiment, indicating a full degradation of the polymer and, in turn, of 11 the sensor occurred, apart from the PLGA foil that is known to degrade at a slower rate2. 12 13 14 220 h (Figure 3g-i). The PL intensity decrease can be ascribed to the degradation, chemical and/or 1 mechanical, of the fluorescent polymer multilayer, given that the silica scaffold dissolved in about 2 50 hours. The polymer brush architecture of the fluorescent multilayer after dissolution of the silica 3 scaffold might impact significantly on its degradation (Figure S14). Remarkably, full sensor 4 degradation occurred on the same timescale as the sensor operation, that is in about 100 hours. By 5 best fitting the PL reduction trend with a linear model, degradation rates of about -0.9 and -0.8 % h6 1 were consistently achieved for pH sensors on native silicon in a flow cell and on PLGA foil under 7 artificial skin, respectively. The polymer multilayer degradation was further confirmed by both 8 optical/fluorescence microscopy. No macroscopic polymer residues or residual photoluminescence 9 were found in the solution collected at the output of the flow cell or in the skin around the sensor 10 location at the end of the experiment, indicating a full degradation of the polymer and, in turn, of 11 the sensor occurred, apart from the PLGA foil that is known to degrade at a slower rate2. 12 13 Porous silica and silicon are fully degradable in physiological conditions Tunable pore size to filter out unwanted guests in vivo 10 nm 1 oxidized PSi Biotin 2 PAH coating 3 PMAA-biotin S. Mariani et al., Nat. Commun. 9(1), 5256 (2018) 15 µm 2 4 Strept detection silicon PSi air 0.01 0.1 1 10 100 1000 10000 0.01 0.1 1 IAW-IAW 0 (a.u.) Concentration (nM) 0 2 4 6 8 10 0.00 0.05 0.10 LbL functionalization Covalent functionalization IAW-IAW 0 (a.u.) Concentration (nM) noise floor LoD∼100 fM streptavidin Biofunctionalization with labelled polyelectrolytes Polyelectrolytes (PEs) are macromolecular compounds that possess minimum of 10–15% functional groups that are dissociated or dissociate in solution. 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 An implantable bioresorbable pH sensor 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 Layer-by-layer coating of porous silica scaffold with fluorescent polyelectrolytes, namely, polyallylaminehydrochloride (PAH:Rh) and poly-methacrylic-acid (PMAA:Rh) 9 7 PMAA:Rh layers in the stack was then varied from (PAH:Rh/PMAA:Rh)1 to 1 (PAH:Rh/PMAA:Rh)3+(PAH:Rh)1. The thickness of the polymer stack increased linearly with the 2 number of layers reaching a maximum value of about 8 nm, thus leaving the pores sufficiently open 3 to ensure effective H+ diffusion within the porous scaffold (Figure S6a). Conformal assembling of 4 the polymer stack in the porous scaffold is supported by the cumulative red-shift of the nPSiO2 5 effective optical thickness (EOT) with the number of layers (Figure S6b,c). 6 7 Figure 1. Preparation of the bioresorbable fluorescence pH sensor. a) Sketch of the pH sensor 8 architecture and operation principle. The sensor consists of a micrometer-thick nanostructured 9 porous silicon oxide (nPSiO2) membrane coated with a nanometer-thick pH-responsive stack of two 10 polymers labelled with Rhodamine (Rh) fluorophores (PAH:Rh and PMAA:Rh). b) Main 11 fabrication steps of the pH sensor on PLGA substrate: 1) preparation of a ~5-μm-thick nPSi 12 membrane via two-steps electrochemical silicon etching; 2) thermal oxidation of the nPSi 13 membrane to nPSiO2; 3) transfer-printing of nPSiO2 membrane onto a ~40-μm-thick PLGA film; 4) 14 layer-by-layer conformal coating of the nPSiO2 scaffold with a nanometer-tick multilayer stack of 15 PAH:Rh and PMAA:Rh. c) 1) Picture of the bioresorbable pH sensor highlighting the Rh-coated 16 nPSiO2 membrane (pink area) on the PLGA foil; 2) bright-field and 3) fluorescence optical 17 microscope images of the cross-section of the nPSiO2 scaffold LbL-coated with Rh-labelled 18 polyelectrolytes. Scale bar is 10µm. d) Photoluminescence intensity at 580 nm vs. number of Rh-19 labelled polyelectrolyte layers assembled in the nPSiO2 scaffold either transfer-printed on PLGA 20 foil or left on the native Si chip, as well as on flat SiO2 substrate used as control. The inset 21 In-vivo tracking of doxorubicin concentration n=9 mice n=9 mice Patent pending – Paper under review Dose: 10 mg/kg In-vivo biocompatibility at 3 months Patent pending – Paper under review Skin histology and blood analysis at 3 months Patent pending – Paper under review More to come with the use of synthetic receptors… E. Mazzotta et al., Small, 2302274 (2023) PPy-based MIP •Bioresorbable batteries & LEDs have been fabricated and tested by the RESORB partner’s •Assembling and testing of all the components into an impantable biodegradable system is ongoing D1.2 Project Public Launch WP1 Project Management, Dissemination, Exploitation, Communication This project is funded by the European Union Horizon Europe programme under grant agreement No 101046946 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreem ent No 952071 D1.2 - Project Quality Handbook WP1 - Project coordination and technical management Partner: PNO Authors: Carolina Salas, Antonio M . Ortiz, Jeanett Bolther Version: v. final Date: 31.08.2020 www.resorb-project.eu What’s next? Acknoledgments -M. Corsi, A. Paghi, S. Mariani, S. Surdo @Barillaro’s group -Dr. L. Dahne @Surflay Nanotec, Berlin -Prof. D. Giuliani and group @University of Modena e Reggio Emilia -Prof. E. Mazzotta and group @University of Salento -People @ab medica s.r.l. Open positions availlable for talented and motivated PhD students and postdoc. If interested, please send your CV and motivation letter to: [email protected] Acknoledgments D1.2 Project Public Launch WP1 Project Management, Dissemination, Exploitation, Communication This project is funded by the European Union Horizon Europe programme under grant agreement No 101046946 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreem ent No 952071 D1.2 - Project Quality Handbook WP1 - Project coordination and technical management Partner: PNO Authors: Carolina Salas, Antonio M . Ortiz, Jeanett Bolther Version: v. final Date: 31.08.2020 www.resorb-project.eu Thank You D1.2 Project Public Launch WP1 Project Management, Dissemination, Exploitation, Communication This project is funded by the European Union Horizon Europe programme under grant agreement No 101046946 This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreem ent No 952071 D1.2 - Project Quality Handbook WP1 - Project coordination and technical management Partner: PNO Authors: Carolina Salas, Antonio M . Ortiz, Jeanett Bolther Version: v. final Date: 31.08.2020 www.resorb-project.eu