Volker Mailänder

Interaction of Nanoparticles with Cells

Nanoparticles and their interaction with human cells have been a focus of many groups during the past decade. We discuss and review here the progress in the field of understanding and harnessing the interactions of polymeric nanoparticles synthesized by the miniemulsion process with different cell types. Nanotechnology and the hereby produced nanomaterials have promised to make use of specific properties of supramolecular assemblies and nanomaterials so that hitherto inaccessible effects can be exploited for new applications. Examples are superparamagnetism or the high surface area helpful for catalysis and adsorption. In biology and medicine, superparamagnetic iron oxide nanoparticles have been used for cell selection and as magnetic resonance imaging (MRI) contrast agents. Furthermore, uptake of nanoparticles into a wide variety of cells is an effect that seems to be specific for materials in the range of 50-200 nm. Surface modifications (positively or negatively charged side groups of the polymers, amino acids, or peptides/proteins) enhance this uptake. Knowledge about factors influencing cellular uptake, like size, surface properties, cell type, and endocytotic pathways, enables optimization of labeling and selection of cells and nanoparticles for applications in vitro and in vivo. For in vivo applications, we will focus on how nanoparticles can cross the blood-brain barrier.

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.

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.

Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components

Background aims The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). Methods Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. Results PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-β1 (TGF-β1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-β1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-β1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. Conclusions PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

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.

Results of Intracoronary Stem Cell Therapy After Acute Myocardial Infarction

To assess the effect of autologous bone-marrow cell (BMC) therapy in patients with acute myocardial infarction in a rigorous double-blind, randomized, placebo-controlled trial. Patients with reperfusion >6 hours after symptom onset were randomly assigned in a 2:1 ratio to receive intracoronary BMC or placebo therapy 5 to 7 days after symptom onset. The patients were stratified according to age, acute myocardial infarction localization, and left ventricular (LV) function. Rigorous double-blinding was ensured using autologous erythrocytes for the placebo preparation that was visually indistinguishable from the active treatment. Serial cardiac magnetic resonance imaging studies were performed before study therapy and after 1, 3, and 6 months. The primary end point was the difference in the LV ejection fraction from baseline to 6 months. The secondary end points included changes in the LV end-diastolic and end-systolic volume indexes and infarct size. A total of 42 patients were enrolled (29 in the BMC group and 13 in the placebo group) in the integrated pilot phase. A mean of 381 x 10(6) mononuclear BMCs were administered. The baseline clinical and cardiac magnetic resonance imaging parameters did not differ. Compared to baseline, the difference in LV ejection fraction for the placebo group versus BMC group was 1.7 +/- 6.4% versus -0.9 +/- 5.5% at 1 month, 3.1 +/- 6.0% versus 1.9 +/- 4.3% at 3 months, and 5.7 +/- 8.4% versus 1.8 +/- 5.3% at 6 months (primary end point; not significant). No difference was found in the secondary end points between the 2 groups, including changes in infarct size or LV end-diastolic and end-systolic volume indexes. In conclusion, in this rigorous double-blind, randomized, placebo-controlled trial, we did not observe an evidence for a positive effect for intracoronary BMC versus placebo therapy with respect to LV ejection fraction, LV volume indexes, or infarct size.

Amino-functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages

Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH(2)) of ∼100 nm in diameter, but not carboxyl- or nonfunctionalized particles, trigger NLRP3 inflammasome activation and subsequent release of proinflammatory interleukin 1β (IL-1β) by human macrophages. PS-NH(2) induced time-dependent proton accumulation in lysosomes associated with lysosomal destabilization, release of cathepsin B, and damage of the mitochondrial membrane. Accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. Upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (TXNIP). Liberated TXNIP, in turn, interacted with the NLRP3 protein, resulting in a conformational change of the pyrin domain of the NLRP3 protein, as was predicted by molecular modeling. Consequently, this prompted assembly of the NLRP3 inflammasome complex with recruitment and activation of caspase-1, inducing IL-1β release by cleavage of pro-IL-1β. The central role of the NLRP3 inflammasome for cytokine production was confirmed by in vitro knockdown of NLRP3 and of the adaptor protein ASC, confirming that other inflammasomes were not activated by PS-NH(2). The PS-NH(2)-mediated proinflammatory macrophage activation could be antagonized by the radical scavenger N-acetyl-L-cysteine, which prevented mitochondrial damage, caspase-1 activation, and the subsequent release of IL-1β. Our study reveals the molecular mechanism of NLRP3 inflammasome activation by amino-functionalized nanoparticles and suggests a strategy as to how such adverse effects could be antagonized.

Lysosomal degradation of the carboxydextran shell of coated superparamagnetic iron oxide nanoparticles and the fate of professional phagocytes

Contrast agents based on dextran-coated superparamagnetic iron oxide nanoparticles (SPIO) are internalized by professional phagocytes such as hepatic Kupffer cells, yet their role in phagocyte biology remains largely unknown. Here we investigated the effects of the SPIO ferucarbotran on murine Kupffer cells and human macrophages. Intravenous injection of ferucarbotran into mice led to rapid accumulation of the particles in phagocytes and to long-lasting increased iron deposition in liver and kidneys. Macrophages incorporate ferucarbotran in lysosomal vesicles containing α-glucosidase, which is capable of degrading the carboxydextran shell of the ferucarbotran particles. Intravenous injection of ferucarbotran into mice followed by incorporation of the nanoparticles into Kupffer cells triggered apoptosis and the subsequent depletion of Kupffer cells. In macrophages, the proinflammatory cytokine TNF-α increased the apoptosis rate, the reactive oxygen species production and the activation of c-Jun N-terminal kinase elicited by ferucarbotran, which might be mediated by the induction of cytoplasmic phospholipase A2 by TNF-α. Notably, the nanoparticle-induced apoptosis of murine Kupffer cells could be prevented by treatment of the mice with the radical scavenger edaravone. Thus, nanosized carboxydextran-coated SPIO-based contrast agents are retained for extended time periods by liver macrophages, where they elicit delayed cell death, which can be antagonized by a therapeutic radical scavenger.