Periodic Reporting for period 2 - LOGIC LAB (Molecular logic lab-on-a-vesicle for intracellular diagnostics)
Periodo di rendicontazione: 2020-11-01 al 2023-10-31
The need for analytical tools, which can assess function by sensing and quantifying intracellular chemical or physical species, has led to the development of a variety of luminescent molecular probes. However, due to the complexity of natural systems, cell signalling and function do not occur as isolated processes. Ideally, to gain truly meaningful insights into intracellular processes and perturbation to such processes induced by diseases, therapy or toxicity, multiple analytes need to be monitored simultaneously in a complex biological environment. Molecular logic sensors offer an elegant way to approach this issue by using parallel and logically connected identification of two or more analytes. A molecular logic gate processes these analytical inputs via a logic operation, producing an optical output signal. The output signal provides distinct information whether none, either of the analytes, or both of them, are present at a certain threshold concentration. Since the introduction of molecular logic gates by the group of de Silva 20 years ago, research approaches have focused on the realization of sensors and advanced drug delivery systems that exploit molecular logic gates. And, whereas the examples show the potential of molecular logic sensing for medical diagnostics, the major obstacle, which is the transfer of this sensing concept from solution to a complex biological environment such as the living cell, has not been tackled to date, but holds promise for future diagnostics.
Such tailor-made diagnostic tools are urgently needed both in fundamental research and clinical routine. As a proof-of-concept, LogicLab will develop a molecular logic sensor platform to diagnose endothelial dysfunction (ED). A dysfunction of the the cells lining our blood vessels is the primary cause of many lifestyle related diseases such as atherosclerosis, which account according to the WHO for 60% of all deaths worldwide in 2005. In atherosclerosis – a chronic medical condition that can remain undetected for decades - so called plaques deposit at the walls of blood vessels leading to stenosis and eventually to serious clinical events such as heart attack or stroke. An early-stage diagnosis of atherosclerosis would allow for intervention long before a high and thus dangerous degree of stenosis is reached. By detection of key biomarkers for ED, such as low wall shear stress and low nitric oxide (NO) concentration, LogicLab aims to develop an analytical platform to diagnose ED and the diseases it possibly provokes at a primary level. The objective of LogicLab is to explore a new concept of molecular logic sensing that can be applied in a biological environment. Not till then, molecular logic sensors will find wide-spread and important application such as in intracellular sensing, critical care diagnostics and high-throughput screening. The approach of LogicLab is a stepwise transfer of the concept from solution to membrane models to real biological environments such as cells and tissue.
Such tailor-made diagnostic tools are urgently needed both in fundamental research and clinical routine. As a proof-of-concept, LogicLab will develop a molecular logic sensor platform to diagnose endothelial dysfunction (ED). A dysfunction of the the cells lining our blood vessels is the primary cause of many lifestyle related diseases such as atherosclerosis, which account according to the WHO for 60% of all deaths worldwide in 2005. In atherosclerosis – a chronic medical condition that can remain undetected for decades - so called plaques deposit at the walls of blood vessels leading to stenosis and eventually to serious clinical events such as heart attack or stroke. An early-stage diagnosis of atherosclerosis would allow for intervention long before a high and thus dangerous degree of stenosis is reached. By detection of key biomarkers for ED, such as low wall shear stress and low nitric oxide (NO) concentration, LogicLab aims to develop an analytical platform to diagnose ED and the diseases it possibly provokes at a primary level. The objective of LogicLab is to explore a new concept of molecular logic sensing that can be applied in a biological environment. Not till then, molecular logic sensors will find wide-spread and important application such as in intracellular sensing, critical care diagnostics and high-throughput screening. The approach of LogicLab is a stepwise transfer of the concept from solution to membrane models to real biological environments such as cells and tissue.
The work performed includes the synthesis of novel fluorescent dyes for sensing of nitric oxide and calcium ions. These dyes have been used together with green and red light absorbing photosensitizers to establish a novel sensing scheme based on triplet-triplet annihilation upconversion (TTA-UC). TTA-UC enables to use red light, which penetrate cells and biological tissue more easily than blue or green light, for sensing of analytes such as biomarkers of endothelial dysfunction.
All newly synthesized compounds have been spectroscopically and theoretically characterized for their optical and electronic properties in solution in order to tune them for application in a biological environment. In parallel, stable vesicles and liposomes have been prepared and evaluated regarding their stability in buffer. These vesicles have been functionalized with photosensitzers and annihilator molecules and TTA-UC within the lipid membrane of the vesicle has been demonstrated. This serves as a proof of concept on the way to supramolecular logic sensing. Moreover, the functionalized vesicles have been demonstrated to fuse with two-dimensional (2D) models of the cell membrane. Cell cultures and co-cultures of different endothelial cell lines have been cultivated both in classical petri dish cell cultures and in microfluidic cell cultures.These 3D cell cultures cultivated inside a microfluidic channel represent a model of an artery and will be used to evaluate the supramolecular logic gates and molecules which release specific doses of nitric oxide upon light irradiation. To implement a therapeutic function into the vesicles, a light-activatable compound (photoNORM) for release of therapeutic amounts of nitric oxide has been synthesized, spectroscopically characterized and evaluated towards light-triggered NO release.
All newly synthesized compounds have been spectroscopically and theoretically characterized for their optical and electronic properties in solution in order to tune them for application in a biological environment. In parallel, stable vesicles and liposomes have been prepared and evaluated regarding their stability in buffer. These vesicles have been functionalized with photosensitzers and annihilator molecules and TTA-UC within the lipid membrane of the vesicle has been demonstrated. This serves as a proof of concept on the way to supramolecular logic sensing. Moreover, the functionalized vesicles have been demonstrated to fuse with two-dimensional (2D) models of the cell membrane. Cell cultures and co-cultures of different endothelial cell lines have been cultivated both in classical petri dish cell cultures and in microfluidic cell cultures.These 3D cell cultures cultivated inside a microfluidic channel represent a model of an artery and will be used to evaluate the supramolecular logic gates and molecules which release specific doses of nitric oxide upon light irradiation. To implement a therapeutic function into the vesicles, a light-activatable compound (photoNORM) for release of therapeutic amounts of nitric oxide has been synthesized, spectroscopically characterized and evaluated towards light-triggered NO release.
Novel sensor fluorophores have been established and studied for their sensing properties in solution as well as in lipid vesicles. We expect more sensor molecules e.g. to probe reactive oxygen species and viscosity to be synthesized and applied in TTA-UC sensing. Moreover, the project has created so far unknown TTA-UC systems with exceptionally high efficiency, which can be used for biomedical application. Different new spectroscopic techniques and theoretical models have been developed that allow for detailed studies of the systems in solution and in lipid membranes. In the next year, we expect to implement various TTA-UC sensing systems in vesicles and hence build the supramolecular logic gates. A novel microfluidic model of a blood vessel (artery-on-a-chip) consisting of different cell types has been developed and evaluated. A metabolomic assay has been developed that indicates the markers of ED and can be used to monitor effects of novel treatments. In the next year, the development and characterization of of a diseased/inflamed artery-on-a-chip is planned. Comparing healthy and diseased models will help to look deeper into disease progression of ED and to evaluate the supramolecular logic gates and light-activated NO releasing molecules. A new label-free method to image disease progressing has been established, using astaxanthin as a Raman reporter for lipids in endothelial cells. For the second project period, we combined this method with other spectroscopic techniques in order to obtain detailed insights into the behavior of the new probes inside a biological environment. The results achieved so far and expected results impact the development of a new supramolecular logic sensing concept that might be used for biomedical diagnostic application in future (e.g. an early-stage detection of ED). The research and training programme has a strong impact on the career perspectives and employability of the ESRs. The individual potential and career perspectives of each ESR are promoted by the acquired scientific methods, the strong multidisciplinarity of the project with active participation of private companies and excellent complementary training. LogicLab provided the ESRs with a broad and well-founded scientific background, though leaving room for own creativity and specialization. The individual potential and career perspectives of each ESR are promoted by the acquired scientific methods, the strong multidisciplinarity of the project with active participation of private companies, excellent complementary training and a regularly reviewed career development
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