for experiments. For mimicking molecular effects such as molecular crowding and establishing new routines, FBS is a good alternative to human serum or plasma.
4.5 Viscosity of Mixtures of Fetal Bovine Serum and Phos-phate Buffered Saline
There is a certain trend towards applying FCS in the area of medicine, e.g. to characterize drug nanocarriers, or clinical applications such as diagnostics [22, 165, 166]. Measure-ments in plasma, serum or mixtures of biofluids and buffer are often used to perform experiments under closer to physiological conditions. In Chapter 5 of this thesis, FBS is added to precoated NPs to study the reversibility of protein binding and in Chapter 6 the influence of biofluids from different species on the release profile of thermosensitive liposomes (TSLs) is investigated. It has been stated that FCS is suitable to measure in complex fluids such as plasma or serum, but there is no systematic study that considers all the various aspects that have to be taken into account when measuring in biofluids.
Measuring in complex biofluids provides additional challenges in the evaluation of data.
Scattering and hydrodynamic effects due to crowding have to be considered [167–169].
Crowding occurs when high concentrations of macromolecules are present and that this presence can alter the properties and interactions of biomolecules in solution. Properly determined values of the viscosity in literature are rare. Often important information on the underlying experiments are missing such as the ionic strength of the dilution buffer or the temperature at which the viscosity or density of a medium was determined.
Although temperature-dependency of media and diffusion is known, it is not always considered properly in analysis. In order to ensure proper results from FCS measure-ments and analysis of experimeasure-ments in mixtures of FBS and PBS, physical properties of these solutions, such as density and viscosity, are determined in a systematic way and at experimentally relevant temperatures: room temperature 22◦C and body temperature 37◦C. GFP is used as a test molecule, since no binding to serum components was ob-served in previous experiments. Crowding leads to an increase of the dynamic viscosity and a slowing down of the diffusion. This effect has to be considered in the analysis, especially for the conversion from diffusion constantDto the hydrodynamic size using the Stokes-Einstein equation (Equation 2.35), where the correct value for the viscosity of the surrounding mediumη(T)has to be used. The density of mixtures of FBS and PBS
0 20 40 60 80 100 1.000
1.004 1.008 1.012 1.016 1.020
density[g/cm³]
percentage of FBS [%]
Figure 4.2: Density of mixtures of PBS and FBS ranging from 0 to 100% FBS at room temperature22◦C(blue squares) and physiological temperature37◦C(red circles). Data is the average of 30 measurements. Linear fits to guide the eye. Error bars represent the standard deviation.
was measured at 22 and 37◦C using a Gay-Lussac pycnometer (Figure 4.2). The density is an input parameter for viscosity measurements. The viscosity was determined with a capillary viscometer with a heating element (Anton Paar).
The dependence of the viscosity on temperature and the fraction of FBS is shown in Figure 4.3. These results are clearly contradictory to statements such as “Although the viscosity of plasma is variable and higher than that of PBS, which could alter the diffusion time, 1:1 or greater dilution is sufficient to normalize the viscosity to that of water” [165].
The suitability of estimations should be questioned carefully, and in case of doubt be rechecked, before use. Assuming a viscosity of water of η(22◦C) = 0.955 mPa s and η(37◦C) = 0.692 mPa s, a deviation of up to 13-15% is observed for 40% FBS at the corre-sponding temperature. The impact of a crowded solution on the structure parameter was evaluated in a study of crowded vesicle solutions by Engelke et. al [170]. The conclusions of this paper are transferable to measurements in biofluids. The importance of control measurements and a well-defined calibration cannot be emphasized enough for FCS measurements in complex media [170, 171]. In an appropriate calibration measurement, the distortion of the focal volume due to scattering is independent and can thus be determined separately. A correct calibration can compensate for several artifacts, making FCS measurements in complex and scattering media reliable.
4.5 Viscosity of Mixtures of Fetal Bovine Serum and Phosphate Buffered Saline 45
0 20 40 60 80 100
0.7 0.8 0.9 1.0 1.1 1.2 1.3
viscosity[mPas]
percentage of FBS [%]
Figure 4.3: Viscosity of mixtures of PBS and FBS ranging from 0 to 100% FBS at room temperature22◦C(red circles) and body temperature37◦C(blue squares). Data is the average of at least 50 measurements. Linear fits to guide the eye. Error bars represent the standard deviation.
Chapter 5
Interaction of Proteins with Solid Nanoparticles
The objective of the work described in this chapter is to gain insights into the interactions of proteins and solid NPs from a fundamental point of view. Knowledge of these processes is relevant for understanding how NPs interact with living organisms. As outlined in Chapter 4, NPs acquire a biomolecular corona when they are in contact with biological fluids. The interaction with and the uptake by cells is dominated by the corona, for instance dictating the biocompatibility and efficacy of nanotherapeutics. In order to be able to elucidate how the biomolecular corona affects these reactions, one has to first understand the corona itself, in particular its kinetics, meaning the types of biomolecules that form it, their abundance and their residence times. To this end, four representative blood plasma proteins, that are presented in Section 4.1, and several types of NPs are chosen. The used NPs are characterized in the following section.
5.1 Solid Nanoparticles
Many different types of nanoparticles are available to address the diverse and evolving needs of research. Polymer, silica and further NPs are available with different surface chemistries and in a range of sizes. A NP is defined as a particle with a spherical size between 1 and 100 nm in diameter, in a broader definition NPs can have a size of up to 10,000 nm. Nano-sized objects behave in a very unique fashion in many respects, compared to their bulk form and therefore have to be characterized separately.
5.1.1 Silica
The application of silica NPs is widely spread in all areas of industry. Silica particles are inherently hydrophilic and negatively charged. NPs made of silica are very stable com-pared to polymer microspheres. These NPs are available with various chemical surface modifications that allow to tune their physico-chemical properties. Their mesoporous structure makes them suited to load drugs into them and use them as biomedical devices [172–174]. Silica NPs are supposed to have a large specific surface area. Small-diameter spheres present more surface area per unit mass, while larger spheres present more surface area per bead.
5.1.2 Polystyrene Latex
Polystyrene NPs are as widely distributed as silica. Due to their hydrophobicity, they strongly bind hydrophobic molecules such as proteins or nucleic acids. Thus, these NP can be easily coated with specific proteins by covalent binding, reducing the capacity to bind biomolecules non-specifically. Polystyrene NPs are available with various sur-face chemistries. In particular, particles with carboxyl (PSCOOH) sursur-faces are popular.
PSCOOH particles are negatively charged, sensitive to low concentrations and multi-valent cations. These beads are made by the formation of many single chain polymers which may be likened to a ball of “wool”. Thus, the determination of the surface area is complicated and this area may be much greater that predicted. This is important for protein binding and charge calculations.
5.1.3 Further Nanoparticles
The following particles in suspension were kindly provides from the JRC Nanomaterials repository NanoMILE project.
Fe dex SPION
Superparamagnetic iron oxide nanoparticles (SPION) coated with dextran (Dextran-SPION) are widely applied for clinical magnetic resonance imaging of cancer tumors and have been utilized to detect metastases and to delineate primary tumors [175]. Moreover Dextran-SPION have been used for imaging inflammatory components of atherosclerosis [176]. These NPs are injected directly into circulation. Due to their common application
5.2 Understanding the Kinetics of Protein-Nanoparticle Corona Formation 49