Interactions between extracellular vesicles, nanoparticles and cells in the presence of a protein corona
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Abstract
Controlling the in vivo targeting of synthetic nanocarriers is one of the major obstacles impairing their clinical translation. This is mainly caused by the high level of complexity involved in the targeting, the dynamic change of the in vivo environment, and a lack of knowledge concerning the molecular determinants guiding this process. After injection of nanocarriers into the bloodstream a layer of biomolecules, mostly proteins, rapidly adsorb to the surface of the nanocarrier. This layer, called protein corona, adds another level of complexity as it is determined by the physico-chemical properties of the carrier and in turn influences its behavior in the in vivo environment. Thus, when designing a nanocarrier, the influence on the protein corona composition needs to be considered as well. Here, PEGylation, which increases the hydrophilicity of the carrier surface, is often used to prolong the blood circulation time. This is accompanied by the reduction of unspecific uptake into immune cells, which is in favor of targeted delivery. As a next step in the targeting process, the nanocarriers need to accumulate at the target site, for example in the tumor tissue. To achieve this a plethora of targeting ligands for many different target sites have been developed. Finally, the nanocarrier must overcome several biological barriers such as tissue barriers, the cell membrane, or intracellular barriers, which led to the development of functional moieties that facilitate barrier crossing. One approach to designing a multi-functional nanocarrier is to use a modular building principle and attach several different functional moieties to the nanocarrier surface.
An alternative that does not require a rational design of the carrier surface is using biologically derived membranes as nanocarrier surface coating. Such membranes can be derived from cells but recently small vesicles, termed extracellular vesicles, became of interest as drug delivery vehicles. EVs serve as a transport system for biological cargo between distant cells. Therefore, it is hypothesized that they are naturally equipped with a variety of functionalities needed for navigating the in vivo environment. In this work, we aimed to explore different aspects of using EV mem-branes as a multi-functional surface coating for drug delivery nanocarriers.
Sections 3.1 and 3.2 focus on developing a strategy to package synthetic nanocarriers into EVs without impairing their membrane integrity as it is crucial to their functionality. Here, we developed a strategy to utilize the cellular EV biogenesis pathway to directly package the NPs during the biogenesis of EVs. We hypothesize that this prevents damage to the EV membrane or the embedded proteins. While we used polystyrene NPs as model NPs for developing a general packaging protocol in Section 3.1, we used this protocol as a blueprint for packaging therapeutically relevant silica nanocapsules in Section 3.2. The purification protocol developed here is based on size exclusion chromatography, which is a commonly used gentle extra-cellular vesicle purification method. The success of our packaging strategy was confirmed by fluorescence cross-correlation spectroscopy, which identified a packaging rate of 3-7%.
Section 3.3 aimed to understand the influence of the protein corona on the exocytosis of silica NPs as exocytosis is a pre-requisite for harvesting NPs packaged in extracellular vesicles. Therefore, the presence or absence of a protein corona could be one parameter for optimization of the packaging protocol. Here, we found that the presence of a plasma protein corona enhances exocytosis in an NP diameter-dependent manner. The exocytosis of larger silica NPs (100 nm) was enhanced by the presence of a corona, whereas the exocytosis of smaller silica NPs (10 nm) was not affected. Aside from gaining insight into parameters relevant to the optimization of packaging, this section also contributes important insights into the relation between protein corona formation and NP exocytosis. This aspect has not yet been addressed by NP protein corona research.
In the last section, we aimed to investigate the protein corona composition of the extracellular vesicles used for packaging the NPs in sections 3.1 and 3.2. Here, we compared the protein corona of extracellular vesicles to liposomes as this is the synthetic nanocarrier type most similar to extracellular vesicles. We found that the protein corona of extracellular vesicles increased the uptake in immune cells similar to the effect observed for liposomes. In vivo, this is associated with rapid blood clearance and impairs efficient drug delivery to target cells. These findings have implications for using extracellular vesicles as surface coating and suggest that further modifications of the EV membrane might be needed. Beyond the usage of extracellular vesicles for drug delivery, these findings are valuable to understanding the basic biology of extracellular vesicles.