General method for the quantification of drug loading and release kinetics of nanocarriers

Macromolecular nanostructures that are used as drug carriers are characterized by their loading and release kinetics. Release studies commonly employ the dialysis method, in which a cellulose membrane separates the solution of released drug from the nanocarrier solution. We demonstrate that it is necessary to take the effect of the dialysis membrane on the release kinetics into account. Using a two-step approach, consisting of a calibration experiment of drug diffusion through the dialysis membrane in the absence of nanocarriers, and an experiment in the presence of nanocarriers, we are able to determine all kinetic rates and in particular to disentangle kinetic dialysis membrane properties from kinetic nanocarrier properties. We apply our general approach to experimental dexamethasone release data from core-multishell nanocarriers and demonstrate that our method yields a consistent description of the nanocarrier release kinetics

Hydration effects turn a highly stretched polymer from an entropic into an energetic spring

Polyethylene glycol (PEG) is a structurally simple and nontoxic water-soluble polymer that is widely used in medical and pharmaceutical applications as molecular linker and spacer. In such applications, PEG’s elastic response against conformational deformations is key to its function. According to text-book knowledge, a polymer reacts to the stretching of its end-to-end separation by a decrease in entropy that is due to the reduction of available conformations, which is why polymers are commonly called entropic springs. By a combination of single-molecule force spectroscopy experiments with molecular dynamics simulations in explicit water, we show that entropic hydration effects almost exactly compensate the chain conformational entropy loss at high stretching. Our simulations reveal that this entropic compensation is due to the stretching-induced release of water molecules that in the relaxed state form double hydrogen bonds with PEG. As a consequence, the stretching response of PEG is predominantly of energetic, not of entropic, origin at high forces and caused by hydration effects, while PEG backbone deformations only play a minor role. These findings demonstrate the importance of hydration for the mechanics of macromolecules and constitute a case example that sheds light on the antagonistic interplay of conformational and hydration degrees of freedom

Force Response of Polypeptide Chains from Water-Explicit MD Simulations

Using molecular dynamics simulations in explicit water, the force–extension relations for the five homopeptides polyglycine, polyalanine, polyasparagine, poly(glutamic acid), and polylysine are investigated. From simulations in the low-force regime the Kuhn length is determined, from simulations in the high-force regime the equilibrium contour length and the linear and nonlinear stretching moduli, which agree well with quantum-chemical density-functional theory calculations, are determined. All these parameters vary considerably between the different polypeptides. The augmented inhomogeneous partially freely rotating chain (iPFRC) model, which accounts for side-chain interactions and restricted dihedral rotation, is demonstrated to describe the simulated force–extension relations very well. We present a quantitative comparison between published experimental single-molecule force–extension curves for different polypeptides with simulation and model predictions. The thermodynamic stretching properties of polypeptides are investigated by decomposition of the stretching free energy into energetic and entropic contributions

Opposing temperature dependence of the stretching response of single PEG and PNiPAM polymers

The response of switchable polymer blends and coatings to temperature variation is important for the development of high-performance materials. Although this has been well studied for bulk materials, a proper understanding at the molecular level, in particular for high stretching forces, is still lacking. Here we investigate the molecular details of the temperature-dependent elastic response of two widely used water-soluble polymers, namely, polyethylene glycol (PEG) and poly(N-isopropylacrylamide) (PNiPAM) with a combined approach using atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) experiments and molecular dynamics (MD) simulations. SMFS became possible by the covalent attachment of long and defined single polymers featuring a functional end group. Most interestingly, varying the temperature produces contrasting effects for PEG and PNiPAM. Surprising as these results might occur at first sight, they can be understood with the help of MD simulations in explicit water. We find that hydration is widely underestimated for the mechanics of macromolecules and that a polymer chain has competing energetic and entropic elastic components. We propose to use the temperature dependence to quantify the energetic behavior for high stretching forces. This fundamental understanding of temperature-dependent single polymer stretching response might lead to innovations like fast switchable polymer blends and coatings with polymer chains that act antagonistically

Mutual A domain interactions in the force sensing protein von Willebrand factor

The von Willebrand factor (VWF) is a glycoprotein in the blood that plays a central role in hemostasis. Among other functions, VWF is responsible for platelet adhesion at sites of injury via its A1 domain. Its adjacent VWF domain A2 exposes a cleavage site under shear to degrade long VWF fibers in order to prevent thrombosis. Recently, it has been shown that VWF A1/A2 interactions inhibit the binding of platelets to VWF domain A1 in a force-dependent manner prior to A2 cleavage. However, whether and how this interaction also takes place in longer VWF fragments as well as the strength of this interaction in the light of typical elongation forces imposed by the shear flow of blood remained elusive. Here, we addressed these questions by using single molecule force spectroscopy (SMFS), Brownian dynamics (BD), and molecular dynamics (MD) simulations. Our SMFS measurements demonstrate that the A2 domain has the ability to bind not only to single A1 domains but also to VWF A1A2 fragments. SMFS experiments of a mutant [A2] domain, containing a disulfide bond which stabilizes the domain against unfolding, enhanced A1 binding. This observation suggests that the mutant adopts a more stable conformation for binding to A1. We found intermolecular A1/A2 interactions to be preferred over intramolecular A1/A2 interactions. Our data are also consistent with the existence of two cooperatively acting binding sites for A2 in the A1 domain. Our SMFS measurements revealed a slip-bond behavior for the A1/A2 interaction and their lifetimes were estimated for forces acting on VWF multimers at physiological shear rates using BD simulations. Complementary fitting of AFM rupture forces in the MD simulation range adequately reproduced the force response of the A1/A2 complex spanning a wide range of loading rates. In conclusion, we here characterized the auto-inhibitory mechanism of the intramolecular A1/A2 bond as a shear dependent safeguard of VWF, which prevents the interaction of VWF with platelets

Elasticity of Proteins and Polymers from Molecular Dynamics Simulations

Understanding the mechanical response of polymers to external force gives crucial insights into physiological processes. In this thesis we present the force extension relation of homo- and polypeptides as well as two synthetic polymer examples. Our findings rely on a combination of molecular or Brownian dynamics simulations, analytical modeling, and comparison to experimental results. First, we present the force−extension relations for the five homopeptides from molecular dynamics simulations in explicit water. The Kuhn length, equilibrium contour length and linear and nonlinear stretching moduli are deduced. An augmented freely rotating chain model, which accounts for side-chain interactions and restricted dihedral rotation, is shown to describe the simulated force−extension relations very well. We present a comparison between published experimental single-molecule force−extension curves for different polypeptides with simulation and model predictions. The simulations allow for the disentanglement of energetic and entropic contributions to the stretching energy of the polypeptides. Secondly, molecular dynamics simulations of a coiled coil linker present in photoreceptor histidine kinases are evaluated in terms of three different mechanical modes which are candidates for signal transmission. The levels of the output signals of shift, splay, and twist on one end of the coiled coil linker are quantified as a function over a wide range of frequencies for the driving force input on the other end of the coiled coil linker by investigation of response functions. Thirdly, the opposite temperature dependence of polyethylene glycol and poly(N-isopropylacrylamide) is investigated from a basis of single molecule force spectroscopy and molecular dynamics simulations in explicit water. Energetic and entropic contributions are deduced from simulations and compared for PEG and PNiPAM. Hydration effects are shown to explain the different temperature dependent responses. Finally, the response of the glycoprotein von Willebrand factor to linear shear flow is examined by a coarse-grained model in Brownian dynamics simulations including long range hydrodynamic interactions. Tensile forces and the shear-rate-dependent globular-coil transition are investigated. The scaling of the critical shear rate for the globular-coil transition with the monomer number is inverse for the grafted and non-grafted scenarios. This implicates that for the grafted scenario, larger chains have a decreased critical shear rate, while for the non-grafted scenario higher shear rates are needed to unfold larger chains

Hydrodynamic Shear Effects on Grafted and Non-Grafted Collapsed Polymers

We study collapsed homo-polymeric molecules under linear shear flow conditions using hydrodynamic Brownian dynamics simulations. Tensile force profiles and the shear-rate-dependent globular-coil transition for grafted and non-grafted chains are investigated to shine light on the different unfolding mechanisms. The scaling of the critical shear rate, at which the globular-coil transition takes place, with the monomer number is inverse for the grafted and non-grafted scenarios. This implicates that for the grafted scenario, larger chains have a decreased critical shear rate, while for the non-grafted scenario higher shear rates are needed in order to unfold larger chains. Protrusions govern the unfolding transition of non-grafted polymers, while for grafted polymers, the maximal tension appears at the grafted end