Authors:	Simon, Johanna
Title:	Proteomic characterization of the biomolecular corona and its impact on cellular uptake
Online publication date:	30-Jul-2019
Year of first publication:	2019
Language:	english
Abstract:	Once nanoparticles are exposed to a biological milieu, blood components rapidly interact with the nanoparticles´ surface, hereby giving the nanoparticle a biological fingerprint (´biomolecular corona´). This process determinately affects the pharmacological profile and therapeutic efficiency of any nanoparticle. Despite the great effort in the development of nanoparticles with a protein-repellent surface (for example surface functionalization with PEG), it is still under debate whether it is possible to completely prevent protein adsorption. Therefore, a thorough characterization of the adsorbed protein layer is needed to improve the biological properties of nanoparticles applied as drug delivery vehicles. This work aimed to shed light onto the multiple interactions occurring at the nano-bio-interface. In a correlative analysis using TEM and liquid chromatography coupled to mass spectrometry (LC-MS) it was possible to monitor the evolution of the protein corona. It was shown that the protein corona had a non-uniform structure and was not - as supposed - dense layer. For meaningful protein corona analysis, it is of great importance to perform in vitro studies under physiologically relevant conditions coming as close as possible to the in vivo situation. Here, as an example, a widely performed cell culture technique known as heat inactivation was investigated. Commonly, the protein source supplemented within the cell culture medium is heated up to 56 °C prior to use in order to avoid interference with in vitro assays. However, it was demonstrated that this procedure strongly affects the nanoparticle-protein interactions and further alters their cellular uptake behavior. Clincial trials are the final step before nanoparticles are approved for therapeutic treatment. Beforehand, mouse studies are performed in order to evaluate the nanoparticles´ behavior in vivo. Based on this, it was investigated how the interspecies protein composition (mouse vs. human) influences the interaction with nanoparticles. Here, it was found that there is a severe discrepancy in the protein corona depending on the protein source. This needs to be considered in order to transfer knowledge gained from in vivo mouse experiments to clinical studies in human. In nanomedicine the surface of the nanoparticles is commonly functionalized with hydrophilic polymers (e.g. PEG) in order to prolong the circulation of nanoparticles in blood. This is known as the stealth effect, which is an established method since the early developments of nanoparticles applied as drug delivery vehicles. However, even up to now there is a limited knowledge about the principal mechanism behind this effect. To unravel this, the influence of polymer hydrophilicity on the protein adsorption pattern was investigated. This was further correlated with the cellular interactions towards macrophages. It was shown that a defined surface hydrophilicity triggers a selective protein adsorption and that actually distinct proteins mediate the stealth effect. This knowledge now opens up the great potential to exploit protein corona formation. As an example, it was demonstrated that an engineered protein corona can prevent unspecific cellular uptake towards macrophages. The overall goal of nanomedicine is the targeted transport of nanoparticles to the body region of interest (e.g. cancer cells). Therefore, the surface of the nanoparticles needs to be functionalized with ligands to guide the nanoparticles´ way. Literature reports described that adsorbed proteins can completely cover up the targeting moiety, hereby preventing cellular recognition of the ligand on the nanoparticles´ surface and the cellular receptor. Therefore, in this work two different methods were developed, which allow an efficient surface functionalization and thereby enable targeted cell interactions even in the presence of the protein corona. It was found that nanoparticles can be functionalized with antibodies through adsorption depending on the pH of the buffer system. Antibody functionalized nanoparticles were able to reach the targeted cell even after exposure to blood plasma and were able to withstand extended periods of incubation in a complex protein surroundings. Further, an alternative strategy is highlighted, which takes advantage of surfactants for the non-covalent functionalization of nanoparticles. Surfactants are surface-active substances and rapidly adsorb onto nanoparticles. To investigate whether this method allows targeted cell interactions, different surfactants were modified with a mannose ligand. It was shown that mannose modified surfactants can be used as a universal approach for the non-covalent functionalization of a broad range of nanoparticle to achieve targeted cell interactions even after incubation with blood plasma.
DDC:	540 Chemie 540 Chemistry and allied sciences
Institution:	Johannes Gutenberg-Universität Mainz
Department:	Externe Einrichtungen
Place:	Mainz
ROR:	https://ror.org/023b0x485
DOI:	http://doi.org/10.25358/openscience-963
URN:	urn:nbn:de:hebis:77-diss-1000029855
Version:	Original work
Publication type:	Dissertation
License:	In Copyright
Information on rights of use:	https://rightsstatements.org/vocab/InC/1.0/
Extent:	304 Seiten
Appears in collections:	JGU-Publikationen