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.

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.

Low frequency electric current noise in glioma cell populations.

Measuring the electrical activity of large and defined populations of cells is currently a major technical challenge to electrophysiology, especially in the picoampere-range. For this purpose, we developed and applied a bidirectional transducer based on a chip with interdigitated gold electrodes to record the electrical response of cultured glioma cells. Recent research determined that also non-neural brain glia cells are electrically active and excitable. Their transformed counterparts, e.g. glioma cells, were suggested to partially retain these electric features. Such electrophysiological studies however are usually performed on individual cells and are limited in their predictive power for the overall electrical activity of the multicellular tumour bulk. Our extremely low-noise measuring system allowed us to detect not only prominent electrical bursts of neuronal cells but also minute, yet constantly occurring and functional, membrane capacitive current oscillations across large populations of C6 glioma cells, which we termed electric current noise. At the same time, tumour cells of non-brain origin (HeLa) proved to be electrically quiescent in comparison. Finally, we determined that the glioma cell activity is primarily caused by the opening of voltage-gated Na+ and K+ ion channels and can be efficiently abolished using specific pharmacological inhibitors. Thus, we offer here a unique approach for studying electrophysiological properties of large cancer cell populations as an in vitro reference for tumour bulks in vivo.

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.

Resistance of ex vivo expanded CD3+CD56+ T cells to Fas-mediated apoptosis.

A variety of malignancies express Fas ligand (FasL), which can induce apoptosis in effector lymphocytes and may limit the success of cellular immunotherapy. Our laboratory has been investigating a population of ex vivo activated T cells, termed cytokine-induced killer (CIK) cells. These cells share functional and phenotypic properties with natural killer cells and a subset of cytolytic cells have the phenotype CD3+CD56+. CIK cells expand in culture, have significant antitumor activity and are presently being tested in phase I/II clinical trials. In this study, we investigated the sensitivity of CIK cells to Fas-mediated apoptosis. Fas engagement leads to apoptosis in small numbers of CIK cells and does not significantly influence antitumor cytotoxicity. CIK cells will undergo apoptosis following Fas engagement when protein synthesis is inhibited, suggesting the expression of antiapoptotic genes. Evaluation of antiapoptotic gene transcripts shows an upregulation in the expression of cFLIP, Bcl-2, Bcl-xL, DAD1 and survivin. Resistance to Fas-mediated apoptosis may come about through an in vitro selection for Fas resistance, since CIK cells synthesize FasL and supernatant from CIK cultures contains biologically active soluble FasL, which can be inhibited with Fas:Fc. These results indicate that CIK cells are a suitable form of immunotherapy against FasL-positive tumors.

Visualizing the kinetics of tumor-cell clearance in living animals.

Evaluation of potential antineoplastic therapies would be enhanced by noninvasive detection of tumor cells in living animals. Because light is transmitted through mammalian tissues, it was possible to use bioluminescence to monitor (both externally and quantitatively) growth and regression of labeled human cervical carcinoma (HeLa) cells engrafted into immunodeficient mice. The efficacy of both chemotherapy and immunotherapeutic treatment with ex vivo expanded human T cell-derived effector cells was evaluated. In the absence of therapy, animals showed progressive increases in signal intensity over time. Animals treated with cisplatin had marked reductions in tumor signal; 5'-fluorouracil was less effective, and cyclophosphamide was ineffective. Immunotherapy dramatically reduced signals at high effector-to-target cell ratios, and significant decreases were observed with lower ratios. This model system allowed sensitive, quantitative, real-time spatiotemporal analyses of the dynamics of neoplastic cell growth and facilitated rapid optimization of effective treatment regimens.

Novel muscle chloride channel mutations and their effects on heterozygous carriers.

Mutations within CLCN1, the gene encoding the major skeletal muscle chloride channel, cause either dominant Thomsen disease or recessive Becker-type myotonia, which are sometimes difficult to discriminate, because of reduced penetrance or lower clinical expressivity in females. We screened DNA of six unrelated Becker patients and found four novel CLCN1 mutations (Gln-74-Stop, Tyr-150-Cys, Tyr-261-Cys, and Ala-415-Val) and a previously reported 14-bp deletion. Five patients were homozygous for the changes (Gln-74-Stop, Ala-415-Val, and 14-bp deletion), four of them due to parental consanguinity. The sixth patient revealed compound heterozygosity for Tyr-150-Cys and Tyr-261-Cys. Heterozygous carriers of the Becker mutations did not display any clinical symptoms of myotonia. However, all heterozygous males, but none of the heterozygous females, exhibited myotonic discharges in the electromyogram suggesting (i) a gene dosage effect of the mutations on the chloride conductance and (ii) male predominance of subclinical myotonia. Furthermore, we report a novel Gly-200-Arg mutation resulting in a dominant phenotype in a male and a partially dominant phenotype in his mother. We discuss potential causes of the gender preference and the molecular mechanisms that may determine the mode of inheritance.

Myotonia levior is a chloride channel disorder.

The group of dominant non-dystrophic myotonias, comprising disorders characterized by clinically similar forms of myogenic muscle stiffness, is genetically inhomogeneous. Dominant myotonia congenita (Thomsen's disease) is linked to CLCN1, the gene encoding the major muscle chloride channel, localized on chromosome 7q35. In contrast, dominant myotonias sensitive to potassium are caused by point mutations in SCN4A on chromosome 17q, the gene for the alpha subunit of the adult skeletal muscle sodium channel. No linkage or molecular genetic data are as yet available on 'myotonia levior' characterized by milder symptoms and later onset of myotonia than in Thomsen's disease, and absence of muscle hypertrophy. We report a CLCN1 Gln-552-Arg substitution for a family with dominant inheritance previously diagnosed to have myotonia levior. Thus, this disorder appears as a variant of Thomsen's disease due to mutations leading to low clinical expressivity. In addition, we report a novel Ile-290-Met CLCN1 mutation for a typical Thomsen pedigree. In another family previously diagnosed as having Thomsen's disease, we unexpectedly found a CLCN1 14 bp deletion known to cause recessive myotonia, and a rare Trp-118-Gly polymorphism.

Effect of heavy metals on cellular growth, metabolism and integrity of cultured Chinese hamster kidney cells.

The effects of CdCl2, Na2CrO4, NaAsO2 and NiSO4 on cultured Chinese hamster kidney cells were studied over a concentration range for 48 h. Release of lactate dehydrogenase, a parameter of cell viability, was not closely related to cell proliferation except for Na2CrO4. A better correlation was obtained between glucose consumption and lactate production, and cellular growth. When studying toxic effects of metals in cell-culture systems, metabolic parameters should be determined in addition to cell viability and cellular growth. The results indicate that Chinese hamster kidney cells in culture might be useful to study mechanisms of metal-induced toxicity.