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Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) are among the most employed (co)polymers for the preparation of drug nanocarriers for the treatment of cancer and infectious diseases. Before considering any clinical use, it is necessary to understand the interactions between polymeric nanoparticles (NPs) and their physiological environment, especially immune cells. Here, we propose a simple, yet precise method to assess NPs internalization kinetics in macrophages, based on the direct analysis of the cell culture media after different incubation times. The proof of concept is given here by using fluorescent PLGA NPs. Nanoparticle tracking analysis (NTA) was a method of choice, enabling detecting each individual NP and analyzing its trajectory while in Brownian motion. As compared to dynamic light scattering (DLS), NTA enabled a more precise determination of NP size distribution. The uptake process was rapid: in one hour, around a third of the NPs were internalized. In addition, the internalized NPs were visualized by confocal microscopy. The fluorescent cellular stacks were analyzed using a freely available macro for ImageJ software, Particle_In_Cell-3D. The internalized objects were localized and counted. This methodology could serve for further studies while analyzing the effects of NPs size, shape and surface properties on their interaction with various cell lines.

The ABCA3 lipid transporter is located in the limiting membrane of lamellar bodies (LBs) in type-II-pneumocytes. Mutations within the ABCA3 gene may functionally impair the transporter, causing lung diseases in newborns, children and adults. Assays to quantify volume and lipid filling of the LBs on the level of the vesicular structures and thereby assess the function of ABCA3 are still lacking. In the present study human influenza haemagglutinin- (HA-) tagged wild type and mutant ABCA3 proteins were stably expressed in lung A549 cells. Fluorescently-labelled TopFluor phosphatidylcholine (TopF-PC) incorporated in surfactant-like liposomes was delivered to the cells and visualized by confocal microscopy. Subsequently, a comprehensive image analysis method was applied to quantify volume and fluorescence intensity of TopF-PC in ABCA3-HA-positive vesicles. TopF-PC accumulated within the vesicles in a time and concentration-dependent manner, whereas the volume remained unchanged, suggesting active transport into preformed ABCA3 containing vesicles. Furthermore, this finding was supported by a decrease of the fluorescence intensity within the vesicles when either the ATPase of the transporter was inhibited by vanadate, or when a disease-causing mutation (K1388N) close to the ABCA3-nucleotide binding domain 2 was introduced. Conversely, a mutation (E292V) located in the first cytoplasmic loop of ABCA3 did not significantly affect lipid transport, but rather resulted in smaller vesicles. In addition to these findings, the assay used in this work for analysing the PC-lipid transport into ABCA3 positive vesicles will be useful to screen for compounds susceptible to restore function in mutated ABCA3 protein.

Mesoporous silica nanoparticles (MSNs) attract increasing interest in the field of gene and drug delivery due to their versatile features as a multifunctional drug delivery platform. Here, we describe poly(amidoamine) (PAMAM) dendron-functionalized MSNs that fulfill key prerequisites for a controllable intracellular drug release. In addition to high loading capacity, they offer 1) low cytotoxicity, showing no impact on the metabolism of endothelial cells, 2) specific cancer cell targeting due to receptor-mediated cell uptake, 3) a redox-driven cleavage of disulfide bridges allowing for stimuli-responsive cargo release, and most importantly, 4) a specific internal trigger based on the high buffering capacity of PAMAM dendrons to provide endosomal escape.

Effective and controlled drug delivery systems with on-demand release and targeting abilities have received enormous attention for biomedical applications. Here, we describe a novel enzyme-based cap system for mesoporous silica nanoparticles (MSNs) that is directly combined with a targeting ligand via bio-orthogonal click chemistry. The capping system is based on the pH-responsive binding of an aryl-sulfonamide-functionalized MSN and the enzyme carbonic anhydrase (CA). An unnatural amino acid (UAA) containing a norbornene moiety was genetically incorporated into CA. This UAA allowed for the site-specific bio-orthogonal attachment of even very sensitive targeting ligands such as folic acid and anandamide. This leads to specific receptor-mediated cell and stem cell uptake. We demonstrate the successful delivery and release of the chemotherapeutic agent Actinomycin D to KB cells. This novel nanocarrier concept provides a promising platform for the development of precisely controllable and highly modular theranostic systems., 40 pages, Article including Supporting Information

Static conditions represent an important shortcoming of many in vitro experiments on the cellular uptake of nanoparticles. Here, we present a versatile microfluidic device based on acoustic streaming induced by surface acoustic waves (SAWs). The device offers a convenient method for introducing fluid motion in standard cell culture chambers and for mimicking capillary blood flow. We show that shear rates over the whole physiological range in sample volumes as small as 200 μL can be achieved. A precise characterization method for the induced flow profile is presented and the influence of flow on the uptake of Pt-decorated CeO2 particles by endothelial cells (HMEC-1) is demonstrated. Under physiological flow conditions the particle uptake rates for this system are significantly lower than at low shear conditions. This underlines the vital importance of the fluidic environment for cellular uptake mechanisms.

In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ∼50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ∼150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine.

The uptake of nanoparticles into cells often involves their engulfment by the plasma membrane and a fission of the latter. Understanding the physical mechanisms underlying these uptake processes may be achieved by the investigation of simple model systems that can be compared to theoretical models. Here, we present experiments on a massive uptake of silica nanoparticles by giant unilamellar lipid vesicles (GUVs). We find that this uptake process depends on the size of the particles as well as on the thermodynamic state of the lipid membrane. Our findings are discussed in the light of several theoretical models and indicate that these models have to be extended in order to capture the interaction between nanomaterials and biological membranes correctly.

Aim: To assess the role of the endothelial glycocalyx (eGCX) for the uptake of nanoparticles by endothelial cells. Methods: The expression of the eGCX on cultured human umbilical vein endothelial cells was determined by immunostaining of heparan sulfate. Enzymatic degradation of the eGCX was achieved by incubating the cells with eGCX-shedding enzymes. The uptake of 50-nm polystyrene nanospheres was quantified by confocal microscopy. Results: Human umbilical vein endothelial cells expressed a robust eGCX when cultured for 10 days. The uptake of both carboxylated and aminated polystyrene nanospheres was significantly increased in cells in which the glycocalyx was enzymatically degraded, while it remained at a low level in cells with an intact glycocalyx. Conclusion: The eGCX constitutes a barrier against the internalization of blood-borne nanoparticles by endothelial cells.

The growing use of nanoparticles in a variety of applications calls for detailed studies of their toxicology, which in turn require understanding the interactions between nanoparticles and living cells. Since simulating the interaction with real cell membranes is rather complex, Langmuir monolayers (LMs) have been used to mimic the first barrier encountered by a nanoparticle as it approaches a biological membrane to assess molecular-level interactions. In this study, we show how oppositely charged gold nanoparticles (Au-NPs) interact with monolayers of the zwitterionic dipalmitoylphosphatidyl choline (DPPC) and negatively charged dipalmitoylphosphatidyl glycerol (DPPG). The monolayers were spread on subphases containing two concentrations of either negatively charged Au-NPs coated with citrate anions or positively charged Au-NPs functionalized with the cationic polyelectrolyte poly(allylamine hydrochloride) (PAH). For DPPG, electrostatic effects dominated which depended strongly on the NPs capping agent, being obviously larger for the positive nanoparticles. The in-plane elasticity for DPPG monolayers within the surface pressure range corresponding to real cell membranes increased with adsorption of positively charged NPs, but decreased with the negative ones. For the zwitterionic DPPC, on the other hand, significant effects only occurred for negatively charged NPs, including a decrease in elasticity. Therefore, it is concluded that the nature, namely the charge of the capping agents, is crucial for the interaction of charged NPs with the cell membrane. (c) 2013 Elsevier B.V. All rights reserved.

In this study, it is shown that the cytotoxic response of cells as well as the uptake kinetics of nanoparticles (NPs) is cell type dependent. We use silica NPs with a diameter of 310 nm labeled with perylene dye and 304 nm unlabeled particles to evaluate cell type-dependent uptake and cytotoxicity on human vascular endothelial cells (HUVEC) and cancer cells derived from the cervix carcinoma (HeLa). Besides their size, the particles are characterized concerning homogeneity of the labeling and their zeta potential. The cellular uptake of the labeled NPs is quantified by imaging the cells via confocal microscopy in a time-dependent manner, with subsequent image analysis via a custom-made and freely available digital method, Particle_in_Cell-3D. We find that within the first 4 h of interaction, the uptake of silica NPs into the cytoplasm is up to 10 times more efficient in HUVEC than in HeLa cells. Interestingly, after 10 or 24 h of interaction, the number of intracellular particles for HeLa cells by far surpasses the one for HUVEC. Inhibitor studies show that these endothelial cells internalize 310 nm SiO₂ NPs via the clathrin-dependent pathway. Remarkably, the differences in the amount of taken up NPs are not directly reflected by the metabolic activity and membrane integrity of the individual cell types. Interaction with NPs leads to a concentration-dependent decrease in mitochondrial activity and an increase in membrane leakage for HUVEC, whereas HeLa cells show only a reduced mitochondrial activity and no membrane leakage. In addition, silica NPs lead to HUVEC cell death while HeLa cells survive. These findings indicate that HUVEC are more sensitive than HeLa cells upon silica NP exposure.

Transendothelial migration is a crucial step during metastasis. Before circulating tumor cells enter the endothelium, they face the glycocalyx. While invasive migration of cancer cells is well studied, few investigations exist regarding their interaction with the glycocalyx. Here, the interaction of three breast cell lines with an endothelial glycocalyx is studied. Benign MCF-10A, noninvasive malign MCF-7, and invasive MDA-MB-231 cells penetrate the glycocalyx, just adhere to it or approach without even attaching to it. Remarkable fluctuations in these interaction modes are detected by time-resolved interaction profiles. Adhesion, migration, and invasion characteristics as well as combinations of interaction modes, cell shapes, and cell extensions are studied. The motility and penetration depth into the glycocalyx are analyzed. The invasive cells are the most flexible, penetrating the glycocalyx mostly with a round shape and feet-like membrane extensions. Noninvasive cancer cells penetrate the glycocalyx the deepest over time and benign cells integrate more likely into the endothelial cell layer underneath the glycocalyx.

Background: ABCA3 is an ATP-dependent lipid transporter involved in surfactant production and is located in the limiting membrane of lamellar bodies (LB) in type II pneumocytes. Mutations within the ABCA3-gene cause respiratory distress syndrome in newborns and chronic interstitial lung diseases in children and adults. In order to develop correctors that can rescue mutant ABCA3, a reliable method is needed to measure ABCA3 transport activity. Methods: Liposomes of surfactant-like composition were prepared and fluorescent Top-Fluor-Phosphatidylcholine (TopF-PC) was incorporated. A549 cells stably expressing ABCA3-HA were incubated with these liposomes. ATP-dependence of transport was evaluated using an ATPase-inhibitor. Cells were imaged using confocal laser scanning microscopy and fluorescence intensity was analysed using the Fiji-Plugin Particle_in_cell-3D , which has been modified for the analysis of vesicular transport. Results: We show that TopF-PC fluorescence can be used to estimate the amount of lipids present in single LBs. Moreover, we found differences in the TopF-PC fluorescence intensity between wild type-cells and cells expressing mutations, which are supposed to be functional ones. We were able to demonstrate an increase of fluorescence in the LBs over different time slots from 0 to 72 hours. In concordance with active transport of TopF-PC by ABCA3, we found a decrease in ABCA3-function after treatment with the ATPase-inhibitor. Summary: This method presents an important step towards a functional assay to analyse transport of lipids by ABCA3 and its impairment by mutations. The assay will enable us to identify correctors, which can improve lipid transport function of mutated ABCA3.

Until now, the potential effects of titanium dioxide (TiO2) nanoparticles on endothelial cells are not well understood, despite their already wide usage. Therefore, the present work characterizes six TiO2 nanoparticle samples in the size range of 19 × 17 to 87 × 13 nm, which are commonly present in sun protection agents with respect to their physicochemical properties (size, shape, ζ-potential, agglomeration, sedimentation, surface coating, and surface area), their interactions with serum proteins and biological impact on human microvascular endothelial cells (relative cellular dehydrogenase activity, adenosine triphosphate content, and monocyte chemoattractant protein-1 release). We observed no association of nanoparticle morphology with the agglomeration and sedimentation behavior and no variations of the ζ-potential (−14 to −19 mV) in dependence on the surface coating. In general, the impact on endothelial cells was low and only detectable at concentrations of 100 μg/ml. Particles containing a rutile core and having rod-like shape had a stronger effect on cell metabolism than those with anatase core and elliptical shape (relative cellular dehydrogenase activity after 72 h: 60 vs. 90 %). Besides the morphology, the nanoparticle shell constitution was found to influence the metabolic activity of the cells. Upon cellular uptake, the nanoparticles were localized perinuclearly. Considering that in the in vivo situation endothelial cells would come in contact with considerably lower nanoparticle amounts than the lowest-observable adverse effects level (100 μg/ml), TiO2 nanoparticles can be considered as rather harmless to humans under the investigated conditions. Electronic supplementary material The online version of this article (doi:10.1007/s11051-013-2130-3) contains supplementary material, which is available to authorized users.

The cellular endosomal sorting complex required for transport (ESCRT) machinery is involved in membrane budding processes, such as multivesicular biogenesis and cytokinesis. In HIV-infected cells, HIV-1 hijacks the ESCRT machinery to drive HIV release. Early in the HIV-1 assembly process, the ESCRT-I protein Tsg101 and the ESCRT-related protein ALIX are recruited to the assembly site. Further downstream, components such as the ESCRT-III proteins CHMP4 and CHMP2 form transient membrane associated lattices, which are involved in virus-host membrane fission. Although various geometries of ESCRT-III assemblies could be observed, the actual membrane constriction and fission mechanism is not fully understood. Fission might be driven from inside the HIV-1 budding neck by narrowing the membranes from the outside by larger lattices surrounding the neck, or from within the bud. Here, we use super-resolution fluorescence microscopy to elucidate the size and structure of the ESCRT components Tsg101, ALIX, CHMP4B and CHMP2A during HIV-1 budding below the diffraction limit. To avoid the deleterious effects of using fusion proteins attached to ESCRT components, we performed measurements on the endogenous protein or, in the case of CHMP4B, constructs modified with the small HA tag. Due to the transient nature of the ESCRT interactions, the fraction of HIV-1 assembly sites with colocalizing ESCRT complexes was low (1.5%-3.4%). All colocalizing ESCRT clusters exhibited closed, circular structures with an average size (full-width at half-maximum) between 45 and 60 nm or a diameter (determined using a Ripley’s L-function analysis) of roughly 60 to 100 nm. The size distributions for colocalizing clusters were narrower than for non-colocalizing clusters, and significantly smaller than the HIV-1 bud. Hence, our results support a membrane scission process driven by ESCRT protein assemblies inside a confined structure, such as the bud neck, rather than by large lattices around the neck or in the bud lumen. In the case of ALIX, a cloud of individual molecules surrounding the central clusters was often observed, which we attribute to ALIX molecules incorporated into the nascent HIV-1 Gag shell. Experiments performed using YFP-tagged Tsg101 led to an over 10-fold increase in ESCRT structures colocalizing with HIV-1 budding sites indicating an influence of the fusion protein tag on the function of the ESCRT protein., Author Summary Viruses hijack the cellular machinery to complete their life cycle. In the case of HIV-1, the endosomal sorting complex required for transport (ESCRT) is recruited by nascent viruses to release themselves from infected cells. Currently, there has been an intense amount of research on how the ESCRT machinery induces viral release. Using super-resolution imaging with endogenous ESCRT proteins or ESCRT proteins containing a small tag, we are able to provide insight into how ESCRT leads to budding of HIV-1. Super resolution imaging of the early ESCRT factors Tsg101 and ALIX, as well as later factors CHMP4B and CHMP2A, also showed condensed, circular structures with diameters of roughly 60 to 90 nm. The cluster sizes were significantly smaller than that of the HIV-1 bud and the distribution of cluster sizes that colocalized with nascent HIV-1 assembly sites were narrower than for non-colocalizing structures. This indicates that the point of interaction between the ESCRT machinery and the HIV-1 assembly site is in the bud neck.

The question whether nanoparticles (NPs) are toxic to humans has attracted increasing attention during the last years. As NPs have unique chemical and physical properties they are extensively used in industry and added to commercial products. One commonly used type are silica-nanoparticles (SiO2-NPs). They occur e.g. as carriers in catalysis, drug delivery systems or food additives leading to a constant exposure of humans. Therefore a strong need for more detailed and systematic investigations exists concerning NP uptake into human cells and the resulting impact on cellular viability.To gain a closer insight into NP-cell interaction and fundamental mechanisms of cytotoxicity we determined the uptake efficiency and uptake pathways of SiO2-NPs in HeLa and HUVEC cells by live-cell-imaging and correlated the results with observed cytotoxic effects. First, the SiO2-NPs were extensively characterized concerning size, shape and labeling. We then incubated SiO2-NPs with cells for varying time periods and monitored the NP uptake by highly sensitive spinning-disc-confocal-microscopy. By measuring stacks of confocal cross-sections of individual living cells and subsequent 3D reconstructions and digital image alanysis, NPs that are in contact with the cell membrane and NPs localized inside the cytoplasm can be differentiated and quantified. We furthermore investigated the NP uptake while blocking individual uptake pathways with specific inhibitors. Thereby we gained valuable information about the uptake pathways used by the NPs. We found that both HeLa and HUVEC cells internalized the SiO2-NPs via the clathrin dependant pathway. Interestingly the NP uptake by HUVEC cells is significantly more efficient compared to HeLa cells. We furthermore compared the cytotoxic response of both cell lines to the SiO2-NPs and found that this difference in uptake efficiency is reflected by the toxic response.