Svenja Morsbach

The Transferability from Animal Models to Humans: Challenges Regarding Aggregation and Protein Corona Formation of Nanoparticles

Nanomaterials are interesting candidates for applications in medicine as drug delivery or diagnostic agents. For safe application, they have to be evaluated in in vitro and in vivo models to finally be translated to human clinical trials. However, often those transfer processes fail, and it is not completely understood whether in vitro models leading to these animal models can reliably be compared to the situation in humans. In particular, the interaction of nanomaterials with components from different blood plasma sources is difficult to compare, and the outcomes of those interactions with respect to body distribution and cell uptake are unclear. Therefore, we investigated the interactions of differently functionalized polymeric and inorganic nanoparticles with human, mouse, rabbit, and sheep plasma. The focus was put on the determination of aggregation events of the nanoparticles occurring in concentrated plasma and the correlation with the respectively formed protein coronas. Both the stability in plasma as well as the types of adsorbed proteins were found to strongly depend on the plasma source. Thus, we suggest evaluating the potential use of nanocarriers always in the plasma source of the chosen animal model for in vitro studies as well as in human plasma to pin down differences and eventually enable transfer into clinical trials in humans.

Beyond the protein corona – lipids matter for biological response of nanocarriers

The interaction of nanocarriers with blood plasma components influences the biological response, and therefore, it needs to be controlled. Whereas protein adsorption to nanocarriers has been investigated to a large extent, the role of lipid interaction for drug delivery and its biological effect is not yet clear. However, lipids represent an important constituent of blood plasma and are usually bound in the form of lipoproteins. Because already for many nanocarrier systems an enrichment of apolipoproteins in their protein corona was reported, we examine the interaction of lipoproteins with nanocarriers. If interaction occurs in terms of lipoprotein adsorption, two scenarios are possible: adsorption of intact lipoprotein complexes or disintegration of the complexes with adsorption of the single components. To investigate the interaction and clarify which scenario occurs, polymeric model nanoparticles and different lipoprotein types have been studied by isothermal titration calorimetry, transmission electron microscopy, and other methods. Our data indicate that upon contact with polymeric nanoparticles, disintegration of lipoproteins and adsorption of lipids occurs. Further, the effect of lipoprotein adsorption on cell uptake has been examined, and a major effect of the lipoproteins has been found.nnnSTATEMENT OF SIGNIFICANCEnIt is now well accepted that nanomaterials developed as diagnostic or therapeutic carrier systems need to be well characterized in terms of biological responses inside an organism. Many studies have already shown that proteins adsorb to the surface of a nanomaterial and create a new interface that define the identity of the material. However, the presence of other surface-active components of the blood plasma and how they interact with nanomaterials has been much less investigated. Thus, this study aims at providing a significant contribution to understanding the interaction mechanism between lipoproteins and nanomaterials. Since lipoproteins transport a high amount of lipids, which are surface-active molecules, the demonstrated interactions can go as far as complete lipoprotein disintegration.

Exploiting the biomolecular corona: pre-coating of nanoparticles enables controlled cellular interactions

Formation of the biomolecular corona ultimately determines the successful application of nanoparticles in vivo. Adsorption of biomolecules such as proteins is an inevitable process that takes place instantaneously upon contact with physiological fluid (e.g. blood). Therefore, strategies are needed to control this process in order to improve the properties of the nanoparticles and to allow targeted drug delivery. Here, we show that the design of the protein corona by a pre-formed protein corona with tailored properties enables targeted cellular interactions. Nanoparticles were pre-coated with immunoglobulin depleted plasma to create and design a protein corona that reduces cellular uptake by immune cells. It was proven that a pre-formed protein corona remains stable even after nanoparticles were re-introduced to plasma. This opens up the great potential to exploit protein corona formation, which will significantly influence the development of novel nanomaterials.

Understanding the elusive protein corona of thermoresponsive nanogels

AIMnWe analyzed the protein corona of thermoresponsive, poly(N-isopropylacrylamide)- or poly(N-isopropylmethacrylamide)-based nanogels.nnnMATERIALS & METHODSnTraces of protein corona detected after incubation in human serum were characterized by proteomics and dynamic light scattering in undiluted serum.nnnRESULTSnApolipoprotein B-100 and albumin were the main components of the protein coronae. For dendritic polyglycerol-poly(N-isopropylacrylamide) nanogels at 37°C, an increase in adsorbed immunoglobulin light chains was detected, followed by partially reversible nanogel aggregation. All nanogels in their hydrophilic state are colloidally stable in serum and bear a dysopsonin-rich protein corona.nnnCONCLUSIONnWe observed strong changes in NG stability upon slight alterations in the composition of the protein coronae according to nanogel solvation state. Nanogels in their hydrophilic state possess safe protein coronae.

How Low Can You Go? Low Densities of Poly(ethylene glycol) Surfactants Attract Stealth Proteins

It is now well-established that the surface chemistry and stealth surface functionalities such as poly(ethylene glycol) (PEG) chains of nanocarriers play an important role to decrease unspecific protein adsorption of opsonizing proteins, to increase the enrichment of specific stealth proteins, and to prolong the circulation times of the nanocarriers. At the same time, PEG chains are used to provide colloidal stability for the nanoparticles. However, it is not clear how the chain length and density influence the unspecific and specific protein adsorption keeping at the same time the stability of the nanoparticles in a biological environment. Therefore, this study aims at characterizing the protein adsorption patterns depending on PEG chain length and density to define limits for the amount of PEG needed for a stealth effect by selective protein adsorption as well as colloidal stability during cell experiments. PEG chains are introduced using the PEGylated Lutensol AT surfactants, which allow easy modification of the nanoparticle surface. These findings indicate that a specific enrichment of stealth proteins already occurs at low PEG concentrations; for the decrease of unspecific protein adsorption and finally the colloidal stability a full surface coverage is advised.

Quantification of fluorescent dyes in organ tissue samples via HPLC analysis

The determination of regional blood flow via the accumulation of fluorescent microspheres is a concept regularly used in medical research. Typically, the microbeads get extracted from the tissue of interest and are then quantified by measuring the absorption or fluorescence of the incorporated dyes without further separation from the medium. However, in that case the absorption spectra of different dyes can overlap when used simultaneously, leading to an overestimation of the concentration. Additionally, background absorption from the medium can be problematic. Therefore, a high performance liquid chromatography method for the simultaneous detection of four dyes (orange, crimson, yellow-green and red) incorporated in different microbeads in samples from biological media such as organ tissue (brain, heart and kidneys) was developed. Since for biological samples often a large sample size is required for sufficient statistics, the method was optimized to yield very short run times. With this method it was possible to detect very low concentrations of only one microsphere per gram of organ tissue. By applying this sensitive quantification technique, it was demonstrated that the application of microbeads for perfusion measurements might not be reliable due to different organ distributions in each animal.

Polymer tube nanoreactors via DNA-origami templated synthesis

We describe the stepwise synthesis of precise polymeric objects programmed by a 3D DNA tube transformed from a common 2D DNA tile as a precise biotemplate for atom transfer radical polymerization.

Denaturation via Surfactants Changes Composition of Protein Corona

The use of nanocarriers as drug delivery vehicles brings them into contact with blood plasma proteins. Polymeric nanocarriers require some sort of surfactant to ensure colloidal stability. Formation of the protein corona is therefore determined not only by the intrinsic properties of the nanocarrier itself but also by the accompanying surfactant. Although it is well-known that surfactants have an impact on protein structure, only few studies were conducted on the specific effect of surfactants on the composition of protein corona of nanocarriers. Therefore, we analyzed the composition of the protein corona on stealth nanoparticles with additional surfactant (cetyltrimethylammonium chloride, CTMA-Cl) after plasma incubation. Additional CTMA-Cl led to an enrichment of apolipoprotein-A1 and vitronectin in the corona, while less clusterin could be found. Further, the structural stability of apolipoprotein-A1 and clusterin was monitored for a wide range of CTMA-Cl concentrations. Clusterin turned out to be more sensitive to CTMA-Cl, with denaturation occurring at lower concentrations.

Engineering Proteins at Interfaces: From Complementary Characterization to Material Surfaces with Designed Functions

Abstract Once materials come into contact with a biological fluid containing proteins, proteins are generally—whether desired or not—attracted by the materials surface and adsorb onto it. The aim of this Review is to give an overview of the most commonly used characterization methods employed to gain a better understanding of the adsorption processes on either planar or curved surfaces. We continue to illustrate the benefit of combining different methods to different surface geometries of the material. The thus obtained insight ideally paves the way for engineering functional materials that interact with proteins in a predetermined manner.

Preservation of the soft protein corona in distinct flow allows identification of weakly bound proteins

Nanocarriers that are used for targeted drug delivery come in contact with biological liquids and subsequently proteins will adsorb to the nanocarriers surface to form the so called protein corona. The protein corona defines the biological identity and determines the biological response towards the nanocarriers in the body. To make nanomedicine safe and reliable it is required to get a better insight into this protein corona and, therefore, the adsorbed proteins have to be characterized. Currently, centrifugation is the common method to isolate the protein corona for further investigations. However, with this method it is only possible to investigate the strongly bound proteins, also referred to as hard protein corona. Therefore, we want to introduce a new separation technique to separate nanoparticles including the soft protein corona containing also loosely bound proteins for further characterization. The used separation technique is the asymmetric flow field-flow fractionation (AF4). We were able to separate the nanoparticles with proteins forming the soft protein corona and were able to show that in our system only the hard protein corona directly influenced the cell uptake behavior.nnnSTATEMENT OF SIGNIFICANCEnCurrently, there is an ongoing debate whether only strongly bound proteins (hard corona) or also loosely bound proteins (soft corona) contribute to the biological identity of nanocarriers, because up to now isolation of the soft corona was not possible. Here, asymmetric flow field-flow fractionation was used to isolate nanoparticles with a preserved soft corona from the biological medium. This enabled the characterization of the soft corona composition and to evaluate its influence on cellular uptake. For our system we found that only the strongly bound proteins (hard corona) determined cell internalization. This method can now be used to evaluate the impact of the soft corona further and to characterize nanomaterials that cannot be separated from blood plasma by other means.