Periodic Reporting for period 1 - Multi-IF (Multicolor Fluorescent Ultrashort Carbon Nanotubes for Multiplexed immunofluorescence in Cancer Diagnostics)
Berichtszeitraum: 2021-11-01 bis 2023-10-31
Heterogeneity is a common indication of cancer. In order to fight cancer pathology, a successful and more effective diagnosis is the essential criteria to adopt optimum treatment and subsequent clinical management. Histopathology, based upon immunohistochemistry (IHC), is the well-established and classical tool of choice for diagnosis of many cancers. However, this technique needs the usage of a lot of tissue biopsy and commonly used labels organic fluorophores suffer from severe photobleaching and preventing simultaneous multiplexed IHC/immunofluorescence due to spectral overlaps. “Multi-IF” proposed to investigate an innovative approach combining nanoscience, click chemistry, surface (bio-)functionalization and fluorescence microscopy for the advancement of cancer histopathology with the aim to divulge the expression level of proteins of interest. Our overall objective was the proof-of-concept that single walled carbon nanotubes (SWCNTs) can be used as novel fluorescent immuno-probes and thus open new strategies for multiplexed immunofluorescence. The use of SWCNTs finds its roots in coordinator’s expertise on optical microscopy due to their strong photoluminescence (PL) in short wave infrared (SWIR) window with high brightness and photostability. This project involved in the preparation and characterization of novel immuno-labels using ultrashort carbon nanotubes (usCNTs), the development of a microscope dedicated to SWIR single nano-labels imaging and the proof-of-concept of this on liver tissue for cancer diagnostic.
The project started with the setting up of a strategy for the preparation of usCNTs in high yield and their full characterizations. More precisely, SWCNTs were first covalently functionalized by sp3 defects (p-nitro aryl groups) using a diazonium chemistry in oleum, followed by chemical oxidation at the defect sites by hydrogen peroxide to produce ultrashort ones keeping its bright fluorescent properties. The spectroscopy characterizations (e.g. PL) have confirmed the presence of sp3 defects and the structural analysis (Atomic Force Microscopy) demonstrate that they are really shorter in length (average length of ~ 40 nm).
This multidisciplinary project also involved the design and development of an optical microscopy setup to allow for the excitation and visualization of different laser excitation wavelengths on the same imaging apparatus for multiplexed imaging. With this dedicated home-built fluorescence microscope, operating in the SWIR window, we have successfully identified and quantified the photophysical properties (photostability, brightness etc.) of usCNTs down to the single nanotube level leading to confirm that these sp3 functionalized usCNTs are bright enough to be tested on biological environment (cells or thick tissue). For that, we introduced surface bio-functionalization to couple them with an antibody (IgG) to be used as diagnostic biomarkers in cancer. We tested them on 2D cells before going into the more complex environment of liver tissue. The PL microscopic images of A431 cells (which express abnormally high levels of the Epidermal growth factor receptor/EGFr), immune-stained by this immuno-labels, clearly show marking around the membrane regions which confirms that EGFr at the membrane are immune-stained. These results were an important first step to go further into the liver cancer tissue. In parallel, we tested the accessibility of functionalized usCNTs in biological tissues. We could detect the movements of CNTs in the intricate maze of the interstitial space in brain tissue via real-time imaging technique.
The results of the project were presented in two major international conferences as well as in several scientific journals. More precisely, this work has been disseminated at the most important conferences in the field of nanoscience and low-dimensional materials (ChemOnTube and NT’23). In addition, two publications have already been published in top-class scientific journals, and at least two more are on preparation for imminent submission.
This multidisciplinary project also involved the design and development of an optical microscopy setup to allow for the excitation and visualization of different laser excitation wavelengths on the same imaging apparatus for multiplexed imaging. With this dedicated home-built fluorescence microscope, operating in the SWIR window, we have successfully identified and quantified the photophysical properties (photostability, brightness etc.) of usCNTs down to the single nanotube level leading to confirm that these sp3 functionalized usCNTs are bright enough to be tested on biological environment (cells or thick tissue). For that, we introduced surface bio-functionalization to couple them with an antibody (IgG) to be used as diagnostic biomarkers in cancer. We tested them on 2D cells before going into the more complex environment of liver tissue. The PL microscopic images of A431 cells (which express abnormally high levels of the Epidermal growth factor receptor/EGFr), immune-stained by this immuno-labels, clearly show marking around the membrane regions which confirms that EGFr at the membrane are immune-stained. These results were an important first step to go further into the liver cancer tissue. In parallel, we tested the accessibility of functionalized usCNTs in biological tissues. We could detect the movements of CNTs in the intricate maze of the interstitial space in brain tissue via real-time imaging technique.
The results of the project were presented in two major international conferences as well as in several scientific journals. More precisely, this work has been disseminated at the most important conferences in the field of nanoscience and low-dimensional materials (ChemOnTube and NT’23). In addition, two publications have already been published in top-class scientific journals, and at least two more are on preparation for imminent submission.
The results and their interpretation in this interdisciplinary project have taken the research carried out beyond the state-of-the-art in various scientific fields. SWIR microscopy allowed to detect functionalized usCNTs at single nanotubes level. Functionalized usCNTs thus have been used to track some specific regions of the deep biological tissues at millisecond timescale where the long functionalized CNTs cannot access to that region, opening up the novel possibility of targeting immuno-targets in thick tissue biopsy and detecting the immuno-labels immersed in these tissue due to their strong PL in the SWIR domain.
The implications of this novel work in understanding the accessibility of tissue environment by the usCNTs is significant. Changing the length of SWCNTs in addition to the sp3 defects opens the route for the investigation on physiological and pathological questions such as the diagnosis of cancer by fully using the near-infrared transparency window of biological tissue (e.g. biopsies). We can also envisage other applications such as the study of brain tissue structures in the context of neurodegenerative diseases like Alzheimer, Parkinson and Huntington diseases.
The implications of this novel work in understanding the accessibility of tissue environment by the usCNTs is significant. Changing the length of SWCNTs in addition to the sp3 defects opens the route for the investigation on physiological and pathological questions such as the diagnosis of cancer by fully using the near-infrared transparency window of biological tissue (e.g. biopsies). We can also envisage other applications such as the study of brain tissue structures in the context of neurodegenerative diseases like Alzheimer, Parkinson and Huntington diseases.