Temperature, concentration, and surface modification influence the cellular uptake and the protein corona of polystyrene nanoparticles.

The composition of the protein corona varies depending on several parameters and influences the cellular fate of the nanocarriers. Here, we investigated the influence of three key parameters (surface charge, temperature, and plasma concentration) on the formation and composition of the protein corona of polystyrene nanoparticles and ultimately on the cellular uptake of pre-coated nanoparticles. At a fixed temperature and concentration, the surface charge, and surfactant influence its composition. We observed that the composition of the corona formed at low temperatures (4°C) is different from that formed at physiological temperatures (37°C). At low plasma concentrations (up to 25%), the corona consists of more diverse proteins than at higher concentrations. Finally, we concluded that regardless of the nanoparticle formulation, the degree of uptake by model cancer and endothelial cells of the nanoparticles decreased when pre-coated at increasing temperature or plasma concentration. STATEMENT OF SIGNIFICANCE: Drug delivery through nanocarriers is an increasingly important concept in research and medicine. One problem in the application of nanocarriers in medicine is the protein corona that forms around the nanocarriers when they get in contact with protein-containing fluids. So far, several factors have been identified that influence the composition of the protein corona and thus the biological identity of the particles. However, lacking comparability remains between the studies because different concentrations or temperatures of the protein solutions are used. In this study we demonstrate how the incubation temperature or the concentration of plasma influences the protein corona and thus the cellular uptake of polystyrene nanoparticles.

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. STATEMENT OF SIGNIFICANCE: Currently, 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.

Stimuli-responsive protection of optically excited triplet ensembles against deactivation by molecular oxygen.

Herein we demonstrate temperature-dependent sacrificial singlet oxygen scavenging properties of N-butyl-2-pyridone, ensuring efficient stimuli-responsive protection of densely populated excited triplet state ensembles against deactivation by molecular oxygen. As an acting external stimulus the temperature was chosen: it will be shown that at low temperature the concentration of singlet oxygen will be substantially lowered; in contrast, at elevated temperatures singlet oxygen will not be captured, and thus the optically excited densely populated triplet ensembles will be effectively depopulated. The singlet oxygen scavenging ability of N-butyl-2-pyridone demonstrates long-term protection of a triplet-triplet annihilation upconversion process against photooxidation.

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.

Coating nanoparticles with tunable surfactants facilitates control over the protein corona.

Nanoparticles with long blood circulation time are a prerequisite for targeted drug delivery. To make the nanoparticles invisible for phagocytizing cells, functional moieties on the particle surface are believed to be necessary to attract specific so-called 'stealth' proteins forming a protein 'corona'. Currently, covalent attachment of those moieties represents the only way to achieve that attraction. However, that approach requires a high synthetic effort and is difficult to control. Therefore, we present the coating of model nanoparticles with biodegradable polymeric surfactants as an alternative method. The thermodynamic parameters of the coating process can be tuned by adjusting the surfactants' block lengths and hydrophilicity. Consequently, the unspecific protein adsorption and aggregation tendency of the particles can be controlled, and stealth proteins inhibiting cell uptake are enriched on their surface. This non-covalent approach could be applied to any particle type and thus facilitates tuning the protein corona and its biological impact.

Protein source and choice of anticoagulant decisively affect nanoparticle protein corona and cellular uptake.

Protein adsorption on nanoparticles has been a focus of the field of nanocarrier research in the past few years and more and more papers are dealing with increasingly detailed lists of proteins adsorbed to a plethora of nanocarriers. While there is an urgent need to understand the influence of this protein corona on nanocarriers' interactions with cells the strong impact of the protein source on corona formation and the consequence for interaction with different cell types are factors that are regularly neglected, but should be taken into account for a meaningful analysis. In this study, the importance of the choice of protein source used for in vitro protein corona analysis is concisely investigated. Major and decisive differences in cellular uptake of a polystyrene nanoparticle incubated in fetal bovine serum, human serum, human citrate and heparin plasma are reported. Furthermore, the protein compositions are determined for coronas formed in the respective incubation media. A strong influence of heparin, which is used as an anticoagulant for plasma generation, on cell interaction is demonstrated. While heparin enhances the uptake into macrophages, it prevents internalization into HeLa cells. Taken together we can give the recommendation that human plasma anticoagulated with citrate seems to give the most relevant results for in vitro studies of nanoparticle uptake.

Redefining the functions of nanocapsule materials.

Nanocapsules are key components in new technologies related to biomedicine and materials science. However, their long-term fate after use is still largely ignored. We discuss here a sustainable approach where the products of degradation of the nanoparticles play a significant role in their application because they are also functional molecules. The polymer shell of the nanocapsules is chemically engineered so that the degradation products formed upon chemical damage are useful after their normal use.

Complementary analysis of the hard and soft protein corona: sample preparation critically effects corona composition.

Here we demonstrate how a complementary analysis of nanocapsule-protein interactions with and without application media allows gaining insights into the so called hard and soft protein corona. We have investigated how both human plasma and individual proteins (human serum albumin (HSA), apolipoprotein A-I (ApoA-I)) adsorb and interact with hydroxyethyl starch (HES) nanocapsules possessing different functionalities. To analyse the hard protein corona we used sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and a protein quantitation assay. No significant differences were observed with regards to the hard protein corona. For analysis of the soft protein corona we characterized the nanocapsule-protein interaction with isothermal titration calorimetry (ITC) and dynamic light scattering (DLS). DLS and ITC measurements revealed that a high amount of plasma proteins were adsorbed onto the capsules' surface. Although HSA was not detected in the hard protein corona, ITC measurements indicated the adsorption of an HSA amount similar to plasma with a low binding affinity and reaction heat. In contrast, only small amounts of ApoA-I protein adsorb to the capsules with high binding affinities. Through a comparison of these methods we have identified ApoA-I to be a component of the hard protein corona and HSA as a component of the soft corona. We demonstrate a pronounced difference in the protein corona observed depending on the type of characterization technique applied. As the biological identity of a particle is given by the protein corona it is crucial to use complementary characterization techniques to analyse different aspects of the protein corona.

Direct visualization of the interfacial position of colloidal particles and their assemblies.

A method for direct visualization of the position of nanoscale colloidal particles at air-water interfaces is presented. After assembling hard (polystyrene, poly(methyl methacrylate), silica) or soft core-shell gold-hydrogel composite (Au@PNiPAAm) colloids at the air-water interface, butylcyanoacrylate is introduced to the interface via the gas phase. Upon contact with water, an anionic polymerization reaction of the monomer is initiated and a film of poly(butylcyanoacrylate) (PBCA) is generated, entrapping the colloids at their equilibrium position at the interface. We apply this method to investigate the formation of complex, binary assembly structures directly at the interface, to visualize soft, nanoscale hydrogel colloids in the swollen state, and to visualize and quantify the equilibrium position of individual micro- and nanoscale colloids at the air-water interface depending of the amount of charge present on the particle surface. We find that the degree of deprotonation of the carboxyl group shifts the air-water contact angle, which is further confirmed by colloidal probe atomic force microscopy. Remarkably, the contact angles determined for individual colloidal particles feature a significant distribution that greatly exceeds errors attributable to the size distribution of the colloids. This finding underlines the importance of accessing soft matter on an individual particle level.

Iron-loaded PLLA nanoparticles as highly efficient intracellular markers for visualization of mesenchymal stromal cells by MRI.

Monitoring of the fate of cells after injection appears paramount for the further development of cell therapies. In this context magnetic resonance imaging (MRI) is increasing in relevance owing to its unique tissue visualization properties. For assessment of cell trafficking and homing, the cells have to be labeled to become MR visible. The rather low sensitivity of MRI demands dedicated intracellular markers with high payloads of MR contrast agents for ensuring sensitive detection of local cell aggregations. In the presented work the application of custom-designed nanometer-sized iron oxide loaded poly-(l-lactide) (iPLLA) nanoparticles was investigated. The particles were synthesized by the mini-emulsion process and evaluated for labeling of mesenchymal stromal cells (MSCs). The efficient cellular uptake and long intracellular retention times of the particles as well as their nontoxicity are demonstrated. The average cellular iron content was 55 pg iron per cell. Further incorporation of, for example, fluorescent dye enables the generation of multireporter particles, providing the great potential for multimodal imaging. The efficiency of these nanoparticles as MRI contrast agent was evaluated in vitro using relaxation rate mapping, yielding relaxivities r2 = 273.3, r2 (*) = 545.1 mm(-1) s(-1) at 3 T and r2 = 415.7, r2 (*) = 872.3 mm(-1) s(-1) at 11.7 T. The high r2 (*) relaxivity of the iPLLA nanoparticles enabled visualization of a single labeled cell in vitro at 50-µm spatial resolution. In vivo evaluation in a rat injury model revealed the potential of the iPLLA particles to efficiently label MSCs for MRI monitoring of ~20 000-40 000 injected cells at 11.7 T. In conclusion the presented work demonstrates the applicability of iPLLA particles as efficient intracellular marker for MSC labeling for monitoring the fate of the cells by MRI.

Switching light with light--advanced functional colloidal monolayers.

Colloidal monolayers comprising of highly ordered two dimensional crystals are of high interest to generate surface patterns for a variety of different applications. Mostly, unfunctionalized polymer or silica colloids are assembled into monolayers. However, the incorporation of functional molecules into such colloids offers a convenient possibility of implementing additional properties to the two-dimensional crystal. Here, we present the formation of novel functional colloidal monolayers with photoswitchable fluorescence. The miniemulsion polymerization technique was used to incorporate an appropriate dye system of a perylene-based fluorophore and a bis-arylethene as a photochrome in polymeric colloids in defined ratios. Upon irradiation with UV or visible light the photochrome reversibly isomerizes from the ring-closed form, which is able to absorb light of the emission wavelength of the fluorescent dye and the ring-open form, which is not. The fluorescence emission of the dye can thus be reversibly switched on and off with light even when embedded in colloids. The colloids were self-assembled at the air-water interface to produce hexagonally ordered functional monolayers and more complex binary crystals. We investigate in detail the influence of the polymeric matrix on the switching properties of the fluorophore/photochrome system and find that the rate constants for the photoswitching, which all lie in the same range, are less influenced by the polymeric environment than expected. We demonstrate the reversible switching of the fluorescence emission in self-assembled colloidal monolayers. The arrangement of broadly distributed functional colloids into ordered monolayers with high addressability was obtained by the formation of binary colloidal monolayers.

High surface area crystalline titanium dioxide: potential and limits in electrochemical energy storage and catalysis.

Titanium dioxide is one of the most intensely studied oxides due to its interesting electrochemical and photocatalytic properties and it is widely applied, for example in photocatalysis, electrochemical energy storage, in white pigments, as support in catalysis, etc. Common synthesis methods of titanium dioxide typically require a high temperature step to crystallize the amorphous material into one of the polymorphs of titania, e.g. anatase, brookite and rutile, thus resulting in larger particles and mostly non-porous materials. Only recently, low temperature solution-based protocols gave access to crystalline titania with higher degree of control over the formed polymorph and its intra- or interparticle porosity. The present work critically reviews the formation of crystalline nanoscale titania particles via solution-based approaches without thermal treatment, with special focus on the resulting polymorphs, crystal morphology, surface area, and particle dimensions. Special emphasis is given to sol-gel processes via glycolated precursor molecules as well as the miniemulsion technique. The functional properties of these materials and the differences to chemically identical, non-porous materials are illustrated using heterogeneous catalysis and electrochemical energy storage (battery materials) as example.

Criteria impacting the cellular uptake of nanoparticles: a study emphasizing polymer type and surfactant effects.

A detailed understanding of the particle-cell interaction is essential and of immense interest in order to create a "specific carrier" for each particular application. In this paper, the effect of the surfactant type (non-ionic vs ionic) and polymer nature on the cellular uptake of fluorescent polystyrene and poly(L-lactide) nanoparticles was studied on HeLa cells. Nanoparticles in a size range from 100 to 160 nm were synthesized by the miniemulsion process. The particles were detected in cells by confocal laser scanning fluorescence microscopy and flow cytometry. It was found that the influence of the surface charge is greater than that of the polymer type itself. In fact, particles stabilized with cationic surfactant were incorporated in a large number irrespective of polymer type. Cellular pathways at ultrastructural level were studied by transmission electron microscopy in more detail to shed light on the particle-cell interaction based on the material properties. The criteria governing the cellular uptake of nanoparticles based on the polymer and surfactant types are finally established.

Preservation of dendritic cell function upon labeling with amino functionalized polymeric nanoparticles.

Dendritic cells (DCs) are key players in eliciting immunity against antigens, therefore making them the focus of many investigations on immune responses in infections, cancer and autoimmune diseases. Nanosized materials have just recently been investigated for their use as carriers of antigens and as labeling agents for DCs. For this later use nanoparticles should be non-toxic and should most importantly not alter the physiological functions of DCs. Here we demonstrate that by the use of polymeric fluorescent nanoparticles as synthesized by the miniemulsion process immature DCs (iDCs) can be efficiently labeled intracellularly. Amino functionalized nanoparticles are more effective than carboxy functionalized ones. Even after 8 days 95% of DCs have retained nanoparticles with a fluorescence intensity of 67% compared to day 1. Nanoparticle labeling does not influence expression of cell surface molecules on mature DCs (mDCs) like HLA-DR, CD80/83/86, CCR7, CD11c nor does it influence the immunostimulatory capacity of mDCs. This procedure does also not impair the capability of DCs for uptake, processing and presentation of viral antigens as demonstrated by interferon-gamma ELISPOT on T cells stimulated with viral antigens presented by DCs. Therefore polymeric nanoparticles are a promising tool to study migration and homing of DCs in animal studies.

Disposition of charged nanoparticles after their topical application to the skin.

The objective of this preliminary investigation was to examine the disposition of charged nanoparticles on and within the skin following their topical application and to explore whether the formulations have potential utility for the local delivery of an associated 'active' substance. Three nanoparticles (approx. 100 nm in diameter) were investigated: cationic amino-functionalized polystyrene, an anionic carboxyl-functionalized polystyrene and anionic poly-(L-lactide), into each of which the fluorophore N-(2,6-diisopropylphenyl)perylene-3,4-dicarboximine (PMI) was incorporated. Formulations were applied to excised porcine skin in vitro for 6 h. After cleaning the skin surface following treatment, the skin was either examined by laser scanning confocal microscopy or subjected to repeated tape-stripping and subsequent analysis of the removed stratum corneum (SC) for the presence of PMI. The cationic nanoparticles showed clear affinity for the negatively charged skin surface (in contrast to the anionic carriers) and delivered a significantly greater amount of the model 'active' agent (PMI) into the SC.

Poly(N-isopropylacrylamide) grafted on plasma-activated poly(ethylene oxide): thermal response and interaction with proteins.

Thermoresponsive polymer layers offer the possibility of preparing smart surfaces with properties that are switchable through a phase transition, usually close to the lower critical solution temperature of the polymer. In particular, poly( N-isopropylacrylamide) (pNIPAM) has gained a great deal of attention because it has such a phase transition in a physiologically interesting temperature range. We have prepared ultrathin thermoresponsive coatings by grafting pNIPAM on a plasma-CVD-deposited, poly(ethylene oxide)-like polymer substrate that was activated in an Ar plasma discharge to initiate the grafting. The presence and integrity of pNIPAM was verified by XPS and ToF-SIMS, and a dramatic change in the wettability during the phase transition was identified by temperature-dependent contact angle measurements. The transition from the hydrated to the collapsed conformation was analyzed by temperature-dependent QCM measurements and by AFM. An unusual, reversible behavior of the viscoelastic properties was seen directly at the phase transition from the swollen to the collapsed state. The phase transition leads to a switching from protein repulsion to a state that allows the adsorption of proteins.

Single molecule chemistry with polymers and colloids: a way to handle complex reactions and physical processes?

Incorporation of polymers in compartments slightly larger than their size allows chemistry and physics on single entities, for example the formation of single-chain polymer crystals by single-molecule chemistry. Recent results and possibilities are discussed.