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] Courtesy of: Medtronics, BostonScientific, MEDEL Traditional implantable devices 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. Example of a bioresorbable electronic circuit In-vivo Si-based bioresorbable electronics[28] Si foundrycompatible bioresorbable electronics[66] In-vitro fullybioresorbable organic transistor[79] In-vivo photovoltaic cells[113] In-vivo physical sensors[93] In-vitro chemical sensor[97] In-vivo implanted neural electrodes[100] In-vitro piezoelectric harvester[72] In-vitro bioresorbable pressure sensor[88] In-vitro electrochemical battery[102] In-vivo triboelectric harvester[110] In-vivo optical fibers for optogenetics[119] In-vitro fullybioresorbable LED[36] Optoelectronic system for in-vivo spectroscopy[131] In-vivo RF antenna[28] Electronic component Power/light sourceSensor In-vivo glucose biosensors[89] In-vivo Si transistor on bioresorbable substrate[64] Optical component Bioresorbable system In-vitro polymeric waveguides[118] 2009 2012 2013 2014 2015 2016 2017 2018 2019 2022 A. A. La Mattina, S. Mariani, G. Barillaro,Advanced Science, 1902872 (2020). Where we are Open challenges A. A. La Mattina, S. Mariani, G. Barillaro, Advanced Science, 1902872 (2020). Physical sensors Chemical sensors Encapsulation Dielectric Conductor Semiconductor Substrate PLGA Silicon Silicon ZnO Magnesium Zinc Cellulose SiO2 Collagen PLGA SiO2 Materials Examples of bioresorbable sensors 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 Tuning the pore size to filter out unwanted guests 10 nm Nanostructured porous silica (nPSiO2) 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 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 Ex-vivo pH sensing and sensor degradation M. Corsi, A. Paghi, et al., Advanced Science (2022) In-vivo pH sensing and dissolution 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 M. Corsi, A. Paghi, et al., Advanced Science (2022) 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 data of 3 mice CTR= 6 mice, implant=6 mice In-vivo biocompatibility tests at 2 months from implant M. Corsi, A. Paghi, et al., Advanced Science (2022) 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 skin liver 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 harvested organs 18 1 Figure 4. Assessment of pH sensing in-vivo, bioresorbability and biocompatibility. a) Sketch of 2 sensor implant in the animal model. b) In-vivo fluorescent images acquired through skin on one of 3 the animals implanted with the pH sensor on their back, (excitation 520-560 nm, collection 620 nm) 4 in 1) physiological conditions and 2) after local injection of a solution at pH 4. c) In-vivo real-time 5 response of the pH sensor measured through skin on mice implanted with the sensor in 6 physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant location (n=3 7 mice). 1) d) In-vivo fluorescence intensity values measured through skin on mice implanted with 8 the sensor in physiological conditions, and after a pH 4 and 7.5 PBS injection around the implant 9 location (n=3 mice). * p < 0.01 two-tailed Student’s t-test. e) In-vivo fluorescence intensity 10 measured through skin on mice implanted with the sensor (n=3 mice) and control mice (n=5 mice) 11 at different time points, before sacrifice. * p < 0.01 two-tailed Student’s t-test . f) In-vivo 12 fluorescence images acquired through skin right after implant and after two months from implant. 13 g) In-vivo b/w and fluorescent images of explanted organs (brain, heart, liver, spleen, lung, and 14 kidney) of control mice (n=5 mice) and mice implanted with the sensor (n=6 mice), acquired after 2 15 months from implant. h) Mean fluorescence emission intensity for each organ for the two groups of 16 mice (control and implanted with pH sensor). i) Optical microscope images of skin and liver 17 cryosections labeled with eosin and hematoxylin staining of a control mouse and a mouse implanted 18 with the sensor after sacrifice at 2 months from implant. Panels 1-2 and 3-4 show, respectively, skin 19 and liver from control and mice with implanted sensor. Data are presented as mean (± s.d). 20 21 Conclusions 22 CTR= 6 mice, implant=6 mice 015 30 45 60 75 7 6 5 4 3 Measured pH Time (h) Real pH 3 4 5 6 7 8 2 3 4 5 6 7 8 Measured pH Real pH Slope: 1.01 A. Paghi, M. Corsi, et al., Advanced Materials Technologies (2023) Wireless measurement/trasmission of pH in real-time •In-vivo pharmaco-kinetic study of doxorubicin with the biosensors is on-going •Bioresorbable batteries & LEDs have been fabricated and tested by the RESORB partner’s •Assembling of all the components into an impantable biodegradable system is on-going 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 Collaborators: -M. Corsi, A. Paghi, S. Mariani, A. La Mattina, S. Surdo, @Barillaro’s grou -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 position 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