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Quantitative detection of bacterial endotoxin by novel nanotechnological approaches

Periodic Reporting for period 1 - ENDONANO (Quantitative detection of bacterial endotoxin by novel nanotechnological approaches)

Reporting period: 2019-01-01 to 2020-12-31

ENDONANO addresses a key regulatory and safety issue, i.e. the unbiased quantitative detection of bacterial endotoxin in products for medical use and in drug development and toxicological studies. The currently adopted LAL assays are reliable only in limited conditions and prone to interference at several levels, and therefore cannot be applied to all products and substances. ENDONANO will exploit new concepts, based on the capacity of metal nanoparticles to adsorb endotoxin, and new detection methods, based on molecular beacons, for developing novel assays to quantitatively detect endotoxin in complex matrices and in a wide range of conditions. The scientific and technological goals of ENDONANO include: 1. Investigating the capacity of endotoxin to specifically inducing inflammatory reactions in human primary blood cells; 2. Developing new methods based on endotoxin capture by metal nanoparticles in complex matrices (biological fluids, emulsions, gels, etc.); 3. Designing and implementing signal generation and detection methods for the quantitative endotoxin measurement; 4. Planning assay prototypes to be developed and validated for commercial purposes.
The work performed in the first 24 months and the results achieved so far are detailed below.
All of the fourteen ESRs have been successfully recruited and have been enrolled in Medical Biology PhD Program of Paris-Lodron University Salzburg, one of academic partners. Due to a huge amendment that had to be implemented at the very beginning of the project and restrictions due to Covid-19 pandemic, the ESRs have started their research activities with some delays and difficulties. However, they have collected preliminary results on their individual projects. The research activities mainly addressed: 1. Interaction of endotoxin with biological entities/molecules; 2.Interaction of endotoxin with particulate matter; 3.NP functionalisation for optimal endotoxin capture/scavenging in different media; 4.Synthesis of detection probes based on fluorescence total internal reflection for optimal detection and quantitative signalling of endotoxin presence. They addressed the interaction of endotoxin from different bacteria with cells, such as human monocytic cell lines, human primary immune cells, and human embryonic kidney cells transiently transfected with the known LPS receptor components and co-transfected with NFkB luciferase to receive a luminescence signal depending on LPS induced signaling. New method based on magnetic microbeads has been set up to uncover novel LPS binding molecules from promising sources such as cell lysate. Biological molecules antibodies, proteins, aptamers and anti-microbial peptides have been identified as potential new biosensors and for NP optimization/conjugation. Conjugation conditions (molecule/nanoparticle ratio, buffer, time and temperature) have been optimised in order to establish the stability of gold nanoparticles complexed with selected antibodies and proteins. Fluorescence spectroscopy method has been chosen to study the interaction between endotoxin and selected biomolecules. Total Internal Reflection Fluorescence (TIRF) configuration was established and silver nanoparticles deposited on glass surfaces has been identified to be used in the development of LPS immunoassay.
ENDONANO aims at identifying and developing new knowledge-based tools and unbiased methods for measuring endotoxin in complex matrices. Innovation is based on the harmonised mix between fundamental research and advanced biosensing and detection nanotechnologies for proposing and implementing original solutions to the problem. The kits currently in use have been developed before the advent of nanotechnologies, so they have not been finely designed to test LPS contamination in nanoparticle formulations for medical devices or to avoid technical interferences due to the chemical-physical characteristics of nanoparticles themselves. Moreover, these kits have never been sufficiently validated and investigated to test contamination in any type of biological matrix / fluid but they are quite generic. The nature of the samples to be tested may be quite different and kits that might work for some may fail on others. Furthermore, differentially from the kits already in use, the originality of the project is to exploiting the ability of NPs to interact with proteins and use this interaction in order to develop more specific and sensitive systems/kits for endotoxin detection suitable for every kind of matrix/sample. Considering the growing application of nanoparticles for medical devices and consumer products the development of new kits tailored specifically for endotoxin detection in NP formulations is extremely needed.
ENDONANO’s originality and innovation lies in the new ideas and technical advancement that the partners will deploy towards the development of novel unbiased methods for endotoxin detection, which could be applied to reliably measuring endotoxin also in complex matrices. The technological objectives of ENDONANO are innovative inasmuch they aim at identifying, by applying modern tools of cellular and molecular biology, material science and advanced optics, new integrated ways of endotoxin sensing and quantitative evaluation. The final innovative objective is that of translating the identified features of endotoxin detection and measurement into new knowledge for future robust and valid assays for endotoxin measurement in complex matrices. The complementarity of the expertise within the ENDONANO consortium (including industries and academic partners) will create the optimal synergy for reaching the common research training goals. The plan of fellows’ secondments will be the basis for ensuring the effectiveness of networking. The eventual research training goal, i.e. the creation of a core group of technologically advanced immuno-toxicologists, goes well beyond current programmes. ENDONANO aims at merging immunology, nanotechnology and physics into a broader approach to address toxicology, safety and related regulatory issues that is expected to attain a deeper vision of the problem, and to propose valid tools and realistic solutions.