Periodic Reporting for period 5 - NanoPokers (Deciphering cell heterogeneity in tumors using arrays of nanowires to controllably poke single cells in longitudinal studies)
Periodo di rendicontazione: 2022-09-01 al 2023-08-31
Cancer is a systemic disease, involving many cells that interact and evolve over time in a complex manner, which current technologies cannot grasp. It is therefore crucial to investigate cancer at the single cell level and over time. To address this issue, the Nanopokers project aims at achieving global monitoring of individual cell responses to stimuli/perturbation in real-time. This is being pursued by combining super resolution live-cell imaging and the development of sensors, as well as injection devices based on vertical nanostructure arrays. The newly developed technology will bring new knowledge on cancer, such as the origin of cancer relapse and how individual tumor cells respond to treatment.
We have developed methods involving 1 D nanostructures to achieve longitudinal single cell interrogation and manipulation. We have worked with phase holographic microscopy to monitor proliferation and motility of immortalized cancer cells cultured on nanowire arrays at the single cell level. We have shown that subpopulations were drowned in average population data and that single cell analysis revealed these populations. This was published in J. Mater. Chem. B, 2018, 6, 7042-7049.
We have shown that the photovoltaic properties of pn nanowire junctions could be used to send cancer cells in dormancy, which can be used to study cell dormancy in cancer research. The results were published in Nanoscale, 2020,12, 14237-14244.
We have, in collaboration with the group of Stefan Hell, MPI Göttingen, developed a method to image nanowires using super resolution microscopy. With this method, we achieved a 5 fold higher resolution compared to confocal microscopy. The results have been published in Nano Letters (Oracz et al. Nano Lett, 2017, 17, 4, 2652-2659)
We have also worked in a collaboration where we demonstrated that gallium phosphide nanowires could be used as lightguiding nanostructures, guiding the light emitted by fluorophores on the nanowire surface along the nanowire axis. The light is emitted at the nanowire tip in a directional way leading to signal quality enhancement. We have characterized the light guiding effect as a function of nanowire diameter and fluorophore wavelength, shedding light on how to best optimize nanowires for biosensing. The results were published in Nano Lett. 2018, 18, 8, 4796–4802. We also wrote an invited review on the subject: Nanotechnology 30 (2019) 214003.
In parallel, we also investigated the safety of nanowires in cells and tissues and a big part of the project dealt with interfacing cells and tissue with nanowires. We have shown that nanowires are internalized via phagocytosis and micropinocytosis and that this has no effect on cell viability, proliferation and motility (PLoS ONE 14(6): e0218122, 2019). In contrast, we have shown that nanowire inhalation elicits an inflammatory and allergic reaction (J Nanobiotechnol 21, 322, 2023).
In order to manipulate a great number of individual cells simultaneously, we have developed a nanostraw platform, consisting of nanotubes protruding from a substrate with fluidic connection to both sides of the substrate. Cells are cultured on top of the nanostraws and mild electrical pulses are applied across the substrate, which leads to a local opening of the cell membrane on top of the nanostraws and the transport of charged cargos inside the cells using electrophoresis.
We were able to show, using STED video rate imaging, that the cell membrane opens on top of the nanostraws upon applications of the electrical pulses and that the membrane seals again 30-60 min after switching off the electrical field. We have used nanostraws to demonstrate transfection of cd34+ cells with GFP mRNA, while demonstrating the non-invasiveness of the method, leaving cells fully functional after nanostraw transfection. The results were published in PNAS, 117 (35) 21267-21273, 2020.
The nanostraws were used to inject fluorescent nanodiamonds inside cells, which are promising nanoparticles for the long-time monitoring of living cells. Until our contribution, fluorescent nanodiamonds were not easily transported to the cytosol and mostly ended up in lysosomes. Using nanostraws, compared to standard delivery methods, we achieved transport of 5 times more nanodiamonds inside cells, most of them in the cytosol. The results were published in Small 2021, 17, 2006421.
We have also demonstrated that immortalized T cells could be guided to microwells with nanostraws at the bottom using electrophoresis, thereby limiting the number of free nanostraws, which is an important parameter when interfacing cells using nanowires. The results were published in RSC Adv., 2022, 12, 30295-30303.
We have shown that the photovoltaic properties of pn nanowire junctions could be used to send cancer cells in dormancy, which can be used to study cell dormancy in cancer research. The results were published in Nanoscale, 2020,12, 14237-14244.
We have, in collaboration with the group of Stefan Hell, MPI Göttingen, developed a method to image nanowires using super resolution microscopy. With this method, we achieved a 5 fold higher resolution compared to confocal microscopy. The results have been published in Nano Letters (Oracz et al. Nano Lett, 2017, 17, 4, 2652-2659)
We have also worked in a collaboration where we demonstrated that gallium phosphide nanowires could be used as lightguiding nanostructures, guiding the light emitted by fluorophores on the nanowire surface along the nanowire axis. The light is emitted at the nanowire tip in a directional way leading to signal quality enhancement. We have characterized the light guiding effect as a function of nanowire diameter and fluorophore wavelength, shedding light on how to best optimize nanowires for biosensing. The results were published in Nano Lett. 2018, 18, 8, 4796–4802. We also wrote an invited review on the subject: Nanotechnology 30 (2019) 214003.
In parallel, we also investigated the safety of nanowires in cells and tissues and a big part of the project dealt with interfacing cells and tissue with nanowires. We have shown that nanowires are internalized via phagocytosis and micropinocytosis and that this has no effect on cell viability, proliferation and motility (PLoS ONE 14(6): e0218122, 2019). In contrast, we have shown that nanowire inhalation elicits an inflammatory and allergic reaction (J Nanobiotechnol 21, 322, 2023).
In order to manipulate a great number of individual cells simultaneously, we have developed a nanostraw platform, consisting of nanotubes protruding from a substrate with fluidic connection to both sides of the substrate. Cells are cultured on top of the nanostraws and mild electrical pulses are applied across the substrate, which leads to a local opening of the cell membrane on top of the nanostraws and the transport of charged cargos inside the cells using electrophoresis.
We were able to show, using STED video rate imaging, that the cell membrane opens on top of the nanostraws upon applications of the electrical pulses and that the membrane seals again 30-60 min after switching off the electrical field. We have used nanostraws to demonstrate transfection of cd34+ cells with GFP mRNA, while demonstrating the non-invasiveness of the method, leaving cells fully functional after nanostraw transfection. The results were published in PNAS, 117 (35) 21267-21273, 2020.
The nanostraws were used to inject fluorescent nanodiamonds inside cells, which are promising nanoparticles for the long-time monitoring of living cells. Until our contribution, fluorescent nanodiamonds were not easily transported to the cytosol and mostly ended up in lysosomes. Using nanostraws, compared to standard delivery methods, we achieved transport of 5 times more nanodiamonds inside cells, most of them in the cytosol. The results were published in Small 2021, 17, 2006421.
We have also demonstrated that immortalized T cells could be guided to microwells with nanostraws at the bottom using electrophoresis, thereby limiting the number of free nanostraws, which is an important parameter when interfacing cells using nanowires. The results were published in RSC Adv., 2022, 12, 30295-30303.
The project demonstrates the development of a platform for cell manipulation, which is totally non-invasive and preserves the cell functions. The project also shows that single cell analysis is bringing new knowledge on cancer cell subpopulations which are not visible in ensemble average.