ENDONANO aims at developing new concepts and methods for the unbiased and quantitative evaluation of bacterial endotoxin in complex matrices, and at laying the groundwork for developing new commercially exploitable assays thereof.
Safety regulations for drugs and medical devices encompass proving the absence of contamination by the toxic bacterial molecule endotoxin (lipopolysaccharide, LPS). Endotoxin is a large molecule present on the outer membrane of Gram-negative bacteria. Activation of innate immune cells such as monocytes and macrophages by LPS is a major step of the defensive inflammatory response against bacteria, but excessive activation leads to a burst of inflammation and to cell death.
Endotoxin is highly toxic to humans and responsible of life-threatening conditions.Therefore, intravenous (IV) drugs, surgical instruments, wound dressing materials must be proven not only sterile but also endotoxin-free, to avoid toxicity due to contaminating LPS.
The maximum permissible endotoxin levels in IV drugs have been declared and regulated by the United States Food and Drug Administration (FDA) as early as in 1985 and updated thereafter. This regulation obliges pharmaceutical companies to control their IV drugs for bacterial endotoxin contamination, and to place on the market only endotoxin-free products. Procedures for “depyrogenation”, include methods that are not easily applicable to all materials without changing or destroying their properties. For this reason, particularly in the case of medical products, new biomaterials and nanotechnological products, endotoxin-free synthesis and preparation procedures are the choice method for obtaining endotoxin-free products that fully maintain the pristine characteristics. In order to develop protocols for producing endotoxin-free biomaterials and nanomaterials, robust and reliable assessment of endotoxin levels in such materials should be available.
Several methods have been used in the last decades for measuring endotoxin in drugs for human use: the rabbit pyrogen test (RPT), the Limulus Amoebocyte Lysate (LAL) assay, the recombinant Horseshoe Crab Factor C assay and the monocyte activation-type pyrogen test (MAT).
All these kits present disadvantages for detecting endotoxin or pyrogens, whose the main are the use of animals in RPT and LAL, and the non-specificity for endotoxin in RPT and MAT. Moreover, all these methods present disadvantages when formulations of inorganic nanoparticles (NP) have to be tested for endotoxin contamination due to the intrinsic chemical-physical properties of NPs themselves that can interfere with the assay readouts.
It is in this context that nanotechnology emerges also as a “disruptive technology” with a great potential to contribute to improve a generation of new diagnostic and therapeutic products. NP behave as “artificial atoms” since their high density of electronic states -which controls many physical properties- can be extensively and easily tuned by adjusting composition, size, shape and surface state, and used in biological environments. Organic functionalisation allows mimicking biomolecular structures. Therefore, nanotechnology’s ability to shape matter at the scale of molecules is opening the door to a new generation of diagnostics, imaging agents and drugs.
Another powerful set of tools is being used to explore the molecular bases of biological functions, based on the interactions of proteins with ligands such as ions, other macromolecules, and metabolites. Since proteins mediate with high specificity almost all the interactions and reactions in cells, they are well suited to act as the most important components of biosensors, provided their interactions with ligands can be monitored. Since it is now possible, by protein engineering, to modify the properties of proteins and produce them in good quantity, the use of proteins and enzymes as specific sensors for biochemical analysis is becoming an important tool for modern biotechnology.
In this context, 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