Anna Musyanovych

Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology

In biological fluids, proteins bind to the surface of nanoparticles to form a coating known as the protein corona, which can critically affect the interaction of the nanoparticles with living systems. As physiological systems are highly dynamic, it is important to obtain a time-resolved knowledge of protein-corona formation, development and biological relevancy. Here we show that label-free snapshot proteomics can be used to obtain quantitative time-resolved profiles of human plasma coronas formed on silica and polystyrene nanoparticles of various size and surface functionalization. Complex time- and nanoparticle-specific coronas, which comprise almost 300 different proteins, were found to form rapidly (<0.5 minutes) and, over time, to change significantly in terms of the amount of bound protein, but not in composition. Rapid corona formation is found to affect haemolysis, thrombocyte activation, nanoparticle uptake and endothelial cell death at an early exposure time.

Protein Corona of Nanoparticles: Distinct Proteins Regulate the Cellular Uptake

Understanding nanoparticle-protein interactions is a crucial issue in the development of targeted nanomaterial delivery. Besides unraveling the composition of the nanoparticles 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.

Differential Uptake of Functionalized Polystyrene Nanoparticles by Human Macrophages and a Monocytic Cell Line

Tumor cell lines are often used as models for the study of nanoparticle-cell interactions. Here we demonstrate that carboxy (PS-COOH) and amino functionalized (PS-NH2) polystyrene nanoparticles of ∼100 nm in diameter are internalized by human macrophages, by undifferentiated and by PMA-differentiated monocytic THP-1 cells via diverse mechanisms. The uptake mechanisms also differed for all cell types and particles when analyzed either in buffer or in medium containing human serum. Macrophages internalized ∼4 times more PS-COOH than THP-1 cells, when analyzed in serum-containing medium. By contrast, in either medium, THP-1 cells internalized PS-NH2 more rapidly than macrophages. Using pharmacological and antisense in vitro knockdown approaches, we showed that, in the presence of serum, the specific interaction between the CD64 receptor and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization in THP-1 cells occurred via dynamin II-dependent endocytosis. PMA-differentiated THP-1 cells differed in their uptake mechanism from macrophages and undifferentiated THP-1 cells by internalizing the particles via macropinocytosis. In line with our in vitro data, more intravenously applied PS-COOH particles accumulated in the liver, where macrophages of the reticuloendothelial system reside. By contrast, PS-NH2 particles were preferentially targeted to tumor xenografts grown on the chorioallantoic membrane of fertilized chicken eggs. Our data show that the amount of internalized nanoparticles, the uptake kinetics, and its mechanism may differ considerably between primary cells and a related tumor cell line, whether differentiated or not, and that particle uptake by these cells is critically dependent on particle opsonization by serum proteins.

Uptake Mechanism of Oppositely Charged Fluorescent Nanoparticles in HeLa Cells

The endocytotic mechanisms involved in the uptake of charged polystyrene nanoparticles into HeLa cells were investigated. Uptake experiments were done in the presence or absence of drugs known to inhibit various factors in endocytosis. Independent of the particle charge, endocytosis is highly dependent on dynamin, F-actin, and tyrosine-specific protein kinases, which suggests a dynamin-dependent and lipid raft-dependent mechanism. However, cholesterol depletion did not hinder particle uptake. Regarding positively charged particles, macropinocytosis, the microtubule network, and cyclooxygenases are also involved. The clathrin-dependent pathway plays a minor role.

Carboxylated superparamagnetic iron oxide particles label cells intracellularly without transfection agents

Cell labeling by superparamagnetic iron oxide particles (SPIO) has emerged as a potentially powerful tool to monitor trafficking of transplanted cells by magnetic resonance tomography, e.g., in studies for tissue repair. However, intracellular labeling is mostly achieved by transfection agents not approved for clinical use. In this work, the feasibility and efficiency of labeling human mesenchymal stem cells (MSC) and HeLa cells with two commercially available SPIOs (Resovist® and Feridex®) without transfection agents was evaluated. In both cell types, Resovist® without a transfection agent was more efficiently taken up than Feridex®. Increasing the concentration of Resovist® can yield similar amounts of iron in cells as SPIOs with transfection agents. This offers the opportunity to omit transfection agents from the labeling protocol when Resovist® is used. Intracellular localization of the contrast agents is found by light microscopy and confirmed by electron microscopy. Coagulation of the SPIO nanoparticles, which is problematic for the quantification of the intracellular iron content, was observed and analyzed with a fluorescent activated cell sorter. As Resovist® consists of a carboxydextran shell in contrast to Feridex® which is composed of a dextran shell, we synthesized fluorescent polymeric nanoparticles as model systems with different amounts of carboxyl groups on the surface by the miniemulsion process. A steady increase in uptake of nanoparticles was detected with a higher density of carboxyl groups showing the relevance of charged groups as in the case of Resovist®. Aggregation of these polymeric nanoparticles was not found.

BSA Adsorption on Differently Charged Polystyrene Nanoparticles using Isothermal Titration Calorimetry and the Influence on Cellular Uptake

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.

Specific Effects of Surface Amines on Polystyrene Nanoparticles in their Interactions with Mesenchymal Stem Cells

We have investigated the uptake of cationic polystyrene nanoparticles by mesenchymal stem cells (MSCs) using confocal fluorescence microscopy and flow cytometry. Two types of nanoparticles of about 100 nm diameter with similar zeta potentials were employed in this study, plain polystyrene (PS) nanoparticles and amino-functionalized polystyrene (NPS) nanoparticles, each carrying about 6000 amino groups on the surface. To assess the relative importance of specific endocytosis mechanisms, uptake was observed in the presence of the drugs dynasore and chlorpromazine. NPS nanoparticles were rapidly internalized and accumulated to a much higher level in MSCs than PS nanoparticles, predominantly via the main clathrin-mediated pathway. PS nanoparticles were internalized mainly via clathrin-independent endocytosis. The pronounced difference in the internalization behavior of PS and NPS nanoparticles points to specific interactions of the amino groups on the nanoparticle surface with the endocytosis machinery of the cells.

Synthesis and optimization of gelatin nanoparticles using the miniemulsion process

A convenient synthetic route based on the concept of nanoreactors using the versatility of the miniemulsion technique to synthesize glutardialdehyde cross-linked gelatin nanoparticles with tailored properties is reported. It is demonstrated that, independent of the molecular weight distribution of the gelatin used, stable nanoparticles can be produced with a small amount of surfactant. The amount of gelatin and the cross-linking degree in the particle can be well controlled. Different types of gelatin have been used without purification or fractionation. The stability of the dispersion, particle size, and the efficiency of cross-linking have been studied. Such nanoparticles with varying gelatin concentration and cross-linking density have high potential to be used for drug delivery applications, as nanoenvironment or template for synthesizing inorganic materials.

Polymer micro- and nanocapsules as biological carriers with multifunctional properties.

The design and development of multifunctional polymer capsules with controlled chemical composition and physical properties has been the focus of academic and industrial research in recent years. Especially in the biomedical field, the formulation of novel polymer-based encapsulation systems for the early-stage disease diagnostic and effective delivery of bioactive agents represent one of the most rapidly advancing areas of science. The stimuli-responsive release of cargo molecules from the carrier gains remarkable attention for in vitro and in vivo delivery of contrast agents, genes, and pharmaceutics. In this Review, the current status and the challenges of different polymer-based micro- and nanocapsule formulations are considered, emphasizing on their potential biological application as carriers for specific drug targeting and controlled release upon applying of external stimulus.

Targeted lipid-coated nanoparticles: Delivery of tumor necrosis factor-functionalized particles to tumor cells

Polymeric nanoparticles displaying tumor necrosis factor on their surface (TNF nanocytes) are useful carrier systems capable of mimicking the bioactivity of membrane-bound TNF. Thus, TNF nanocytes are potent activators of TNF receptor 1 and 2 leading to a striking enhancement of apoptosis. However, in vivo applications are hampered by potential systemic toxicity. Here, using TNF nanocytes as a model system, we developed a procedure to generate targeted lipid-coated particles (TLP) in which TNF activity is shielded. The TLPs generated here are composed of an inner single-chain TNF (scTNF)-functionalized, polymeric nanoparticle core surrounded by a lipid coat endowed with polyethylene glycol (PEG) for sterical stabilization and a single-chain Fv (scFv) fragment for targeting. Using a scFv directed against the tumor stroma marker fibroblast activation protein (FAP) we show that TLP and scTNF-TLP specifically bind to FAP-expressing, but not to FAP-negative cells. Lipid coating strongly reduced nonspecific binding of particles and scTNF-mediated cytotoxicity towards FAP-negative cells. In contrast, an increased cytotoxicity of TLP was observed for FAP-positive cells. Thus, through liposome encapsulation, nanoparticles carrying bioactive molecules, which are subject to nonselective uptake and activity towards various cells and tissues, can be converted into target cell-specific composite particles exhibiting a selective activity towards antigen-positive target cells. Besides safe and targeted delivery of death ligands such as TNF, TLP should be suitable for various diagnostic and therapeutic applications, which benefit from a targeted delivery of reagents embedded into the particle core or displayed on the core particle surface.