Reply to the 'Comment on "Relating side chain organization of PNIPAm with its conformation in aqueous methanol"' by N. van der Vegt and F. Rodriguez-Ropero, Soft Matter, 2017, 13, DOI: 10.1039/C6SM02139E.

In a comment van der Vegt and Rodriguez-Ropero (vdVRR) challenged our explanation of the co-non-solvency effect of PNIPAm in aqueous methanol solutions. They argue, based on a careful selection of published studies including some of their own, that direct repulsions between the different constituents are sufficient to understand this phenomenon. According to vdVRR, the emerging view of entropic collapse, put forward by Flory (1910-1985) to explain common polymers in poor solvents, would be enough to explain co-non-solvency. In this reply we attempt to bring this discussion into firmer grounds. We provide a more comprehensive view of available experimental, numerical and theoretical results and review basic concepts of physical chemistry and of statistical mechanics of polymer collapse that show how methanol mediated attractions between chain monomers are required to understand this fascinating behavior.

Small Surfactant Concentration Differences Influence Adsorption of Human Serum Albumin on Polystyrene Nanoparticles.

Surfactants, even in miniscule amounts, are often used for the synthesis and especially the stabilization of nanomaterials, which is essential for in vivo applications. In this study, we show that the interaction between nanoparticles and proteins strongly depends on the type of stabilizing surfactants and their (small) concentration changes. The reaction between human serum albumin and polystyrene nanoparticles stabilized by an ionic or nonionic surfactant-sodium dodecyl sulfate or Lutensol AT50, respectively-was monitored using isothermal titration calorimetry. It was found that the amount of surfactant molecules on the surface significantly determines the protein binding affinity and adsorption stoichiometry, which is important for all nanomaterials coming into contact with biological components such as blood plasma proteins. Thus after synthesizing nanomaterials for in vivo applications as drug delivery agents, it is crucial to perform a detailed analysis of the obtained surface chemistry that accounts for the presence of minimal amounts of stabilizing agents.

Carbohydrate-Based Nanocarriers Exhibiting Specific Cell Targeting with Minimum Influence from the Protein Corona.

Whenever nanoparticles encounter biological fluids like blood, proteins adsorb on their surface and form a so-called protein corona. Although its importance is widely accepted, information on the influence of surface functionalization of nanocarriers on the protein corona is still sparse, especially concerning how the functionalization of PEGylated nanocarriers with targeting agents will affect protein corona formation and how the protein corona may in turn influence the targeting effect. Herein, hydroxyethyl starch nanocarriers (HES-NCs) were prepared, PEGylated, and modified on the outer PEG layer with mannose to target dendritic cells (DCs). Their interaction with human plasma was then studied. Low overall protein adsorption with a distinct protein pattern and high specific affinity for DC binding were observed, thus indicating an efficient combination of "stealth" and targeting behavior.

Fluorescence labels may significantly affect the protein adsorption on hydrophilic nanomaterials.

Fluorescently labelled proteins are often used to study processes in vitro, e.g. the binding of proteins to cell surfaces or the adsorption of plasma proteins on drug nanocarriers. However, the fact that the fluorescent labelling may affect the protein properties is frequently neglected. On the example of a simple model system, we reiterate the importance of this issue by showing that even a single label may perturb interactions between hydrophilic starch-based nanocapsules and serum albumin and thus prevent binding.

Protein adsorption is required for stealth effect of poly(ethylene glycol)- and poly(phosphoester)-coated nanocarriers.

The current gold standard to reduce non-specific cellular uptake of drug delivery vehicles is by covalent attachment of poly(ethylene glycol) (PEG). It is thought that PEG can reduce protein adsorption and thereby confer a stealth effect. Here, we show that polystyrene nanocarriers that have been modified with PEG or poly(ethyl ethylene phosphate) (PEEP) and exposed to plasma proteins exhibit a low cellular uptake, whereas those not exposed to plasma proteins show high non-specific uptake. Mass spectrometric analysis revealed that exposed nanocarriers formed a protein corona that contains an abundance of clusterin proteins (also known as apolipoprotein J). When the polymer-modified nanocarriers were incubated with clusterin, non-specific cellular uptake could be reduced. Our results show that in addition to reducing protein adsorption, PEG, and now PEEPs, can affect the composition of the protein corona that forms around nanocarriers, and the presence of distinct proteins is necessary to prevent non-specific cellular uptake.