Understanding nanoparticle-protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticle's protein coronas, distinct proteins thereof could control nanoparticle uptake into specific cell types. Here we differentially analyzed the protein corona composition on four polymeric differently functionalized nanoparticles by label-free quantitative mass spectrometry. Next, we correlated the relative abundance of identified proteins in the corona with enhanced or decreased cellular uptake of nanoparticles into human cancer and bone marrow stem cells to identify key candidates. Finally, we verified these candidate proteins by artificially decorating nanoparticles with individual proteins showing that nanoparticles precoated with the apolipoproteins ApoA4 or ApoC3 significantly decreased the cellular uptake, whereas precoating with ApoH increased the cellular uptake.

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.

“Trees in miniemulsion” – the biopolymer lignin was used to generate biodegradable nanocontainers via an interfacial polyaddition reaction.

BSA adsorption onto negatively and positively charged polystyrene nanoparticles was investigated. The nanoparticles were characterized in terms of particle size, zeta potential, surface group density, and morphology. The adsorption behavior of BSA on the particle surface, as a function of pH and overall charge of the particle, was studied using ITC. Different thermodynamic data such as enthalpy changes upon binding and stoichiometry of the systems were determined and discussed. The degree of surface coverage with BSA was calculated using the thermodynamic data. The cellular uptake of particles before and after BSA adsorption was studied using HeLa cells in the presence and absence of supplemented FCS in the cell culture medium.

Antibacterial nanodevices could bring coatings of plastic materials and wound dressings a big step forward if the release of the antibacterial agents could be triggered by the presence of the bacteria themselves. Here, we show that novel hyaluronic acid (HA)-based nanocapsules containing the antimicrobial agent polyhexanide are specifically cleaved in the presence of hyaluronidase, a factor of pathogenicity and invasion for bacteria like Staphylococcus aureus and Escherichia coli. This resulted in an efficient killing of the pathogenic bacteria by the antimicrobial agent. The formation of different polymeric nanocapsules was achieved through a polyaddition reaction in inverse miniemulsion. After the synthesis, the nanocapsules were transferred to an aqueous medium and investigated in terms of size, size distribution, functionality, and morphology using dynamic light scattering, zeta potential measurements and scanning electron microscopy. The enzyme triggered release of a model dye and the antimicrobial polyhexanide was monitored using fluorescence and UV spectroscopy. The stability of the nanocapsules in several biological media was tested and the interaction of nanocapsules with human serum protein was studied using isothermal titration calorimetry. The antibacterial effectiveness is demonstrated by determination of the antibacterial activity and determination of the minimal bactericidal concentration (MBC).

Synthesizing nanocarriers with stealth properties and delivering a "payload" to the particular organ remains a big challenge but is the prime prerequisite for any in vivo application. As a nontoxic alternative to the modification by poly(ethylene glycol) PEG, we describe the synthesis of cross-linked hydroxyethyl starch (HES, M(w) 200,000 g/mol) nanocapsules with a size range of 170-300 nm, which do not show nonspecific uptake into cells. The specific uptake was shown by coupling a folic acid conjugate as a model targeting agent onto the surface of the nanocapsules, because folic acid has a high affinity to a variety of human carcinoma cell lines which overexpress the folate receptor on the cell surface. The covalent binding of the folic acid conjugate onto HES capsules was confirmed by FTIR and NMR spectroscopy. The coupling efficiency was determined using fluorescence spectroscopy. The specific cellular uptake of the HES nanocapsules after folic acid coupling into the folate-receptor presenting cells was studied by confocal laser scanning microscopy (CLSM) and flow cytometry.

Cross-linked potato starch nanocapsules with encapsulated dsDNA (with a defined number of base pairs, i.e., 286, 476, and 790 bp) were synthesized using the miniemulsion technique. The inverse (water-in-oil) miniemulsion system was applied to create stable aqueous nanodroplets of dissolved starch in cyclohexane as a continuous phase. The amphiphilic block copolymer poly[(ethylene-co-butylene)-b-(ethylene oxide)] was used as a surfactant to stabilize the droplets. After addition of the cross-linker, 2,4-toluene diisocyanate (TDI), the polyaddition reaction took place at the droplet's interface, resulting in the formation of a polymeric cross-linked shell. The influence of starch, surfactant, and the amount of cross-linker on the average size, size distribution, and morphology of the capsules was studied by dynamic light scattering and electron microscopy. FTIR spectroscopy was used to identify the chemical composition of the capsule shell. The permeability of the shell was studied on the fluorescent dye (i.e., sulforhodamine 101) containing capsules using fluorescence spectroscopy. High thermal stability of the cross-linked capsules allows one to perform the polymerase chain reaction inside the core. The encapsulation of dsDNA and the efficiency of the PCR were confirmed by fluorescence spectroscopy after staining with the DNA-selective dye (SYBRGreen).

The interactions between nanoparticles (NPs) and proteins in complex biological application media such as blood serum are capable of inducing aggregate formation which can lead to subsequent changes in biological activity. Here, we correlate surface charge, aggregation-tendency, and surface serum protein adsorption with cellular uptake and biodistribution in mice.Polystyrene-based NPs (80 -170 nm) with different surface functionalizations were synthesized and incubated with human serum. Interaction of NPs with serum proteins and aggregate formation were analyzed by mass spectrometryanalysis and dynamic light-scattering. Influence of surface functionalization on specific cellular uptake and organdistribution was characterized.Localization and organ targeting of intravenously applied NPs preferentially depended on their aggregationbehavior in the presence of serum. Whereas strongly aggregating particles mainly located to liver, non-aggregating particles distributed to all organs. Determination of aggregate formation of NPs in the presence of serum and further analysis of the protein corona allows for pre-selection of NPs for in vivo application. studies were done by dynamic light scattering applying a previously developed pre-in vivo screening method [10]. This enabled us to analyze the effect of surface functionalization on aggregation and protein binding in the presence of human serum under physiologically relevant conditions. To allow analyzing the cellular uptake of NPs in cells of the peripheral blood as well as in professional antigen-presenting cells, the NPs were fluorescently labeled with boron-dipyrromethene (BODIPY) for flow cytometry and immune fluorescence. In addition, the particles were loaded with a near-infrared dye (IR-Dye 780) for in vivo imaging. Using quantitative high performance liquid chromatography coupled with mass spectrometry, we determined the composition of the protein corona. Using these particles, we were able to demonstrate that the pattern of protein binding in the presence of serum correlates with the tendency to form aggregates in vitro and in vivo. In addition, the charge of the particles' surface influenced their uptake in phagocytes and endocytosis as well as their biodistribution after intravenous application in mice.