Tiago Santos

Effects of Transport Inhibitors on the Cellular Uptake of Carboxylated Polystyrene Nanoparticles in Different Cell Lines

Nanotechnology is expected to play a vital role in the rapidly developing field of nanomedicine, creating innovative solutions and therapies for currently untreatable diseases, and providing new tools for various biomedical applications, such as drug delivery and gene therapy. In order to optimize the efficacy of nanoparticle (NP) delivery to cells, it is necessary to understand the mechanisms by which NPs are internalized by cells, as this will likely determine their ultimate sub-cellular fate and localisation. Here we have used pharmacological inhibitors of some of the major endocytic pathways to investigate nanoparticle uptake mechanisms in a range of representative human cell lines, including HeLa (cervical cancer), A549 (lung carcinoma) and 1321N1 (brain astrocytoma). Chlorpromazine and genistein were used to inhibit clathrin and caveolin mediated endocytosis, respectively. Cytochalasin A and nocodazole were used to inhibit, respectively, the polymerisation of actin and microtubule cytoskeleton. Uptake experiments were performed systematically across the different cell lines, using carboxylated polystyrene NPs of 40 nm and 200 nm diameters, as model NPs of sizes comparable to typical endocytic cargoes. The results clearly indicated that, in all cases and cell types, NPs entered cells via active energy dependent processes. NP uptake in HeLa and 1321N1 cells was strongly affected by actin depolymerisation, while A549 cells showed a stronger inhibition of NP uptake (in comparison to the other cell types) after microtubule disruption and treatment with genistein. A strong reduction of NP uptake was observed after chlorpromazine treatment only in the case of 1321N1 cells. These outcomes suggested that the same NP might exploit different uptake mechanisms to enter different cell types.

Nanoparticle-mediated brain drug delivery: Overcoming blood–brain barrier to treat neurodegenerative diseases

The blood-brain barrier (BBB) is a vital boundary between neural tissue and circulating blood. The BBBs unique and protective features control brain homeostasis as well as ion and molecule movement. Failure in maintaining any of these components results in the breakdown of this specialized multicellular structure and consequently promotes neuroinflammation and neurodegeneration. In several high incidence pathologies such as stroke, Alzheimers (AD) and Parkinsons disease (PD) the BBB is impaired. However, even a damaged and more permeable BBB can pose serious challenges to drug delivery into the brain. The use of nanoparticle (NP) formulations able to encapsulate molecules with therapeutic value, while targeting specific transport processes in the brain vasculature, may enhance drug transport through the BBB in neurodegenerative/ischemic disorders and target relevant regions in the brain for regenerative processes. In this review, we will discuss BBB composition and characteristics and how these features are altered in pathology, namely in stroke, AD and PD. Additionally, factors influencing an efficient intravenous delivery of polymeric and inorganic NPs into the brain as well as NP-related delivery systems with the most promising functional outcomes will also be discussed.

Experimental and theoretical comparison of intracellular import of polymeric nanoparticles and small molecules: toward models of uptake kinetics

UNLABELLED Central to understanding how nanoscale objects interact with living matter is the need for reproducible and verifiable data that can be interpreted with confidence. Likely this will be the basis of durable advances in nanomedicine and nanomedical safety. To develop these fields, there is also considerable interest in advancing the first generation of theoretical models of nanoparticle (NP) uptake into cells, and NP biodistribution in general. Here we present an uptake study comparing the outcomes for free molecular dye and NPs labeled with the same dye. A simple flux-based approach is presented to model NP uptake. We find that the intracellular NP concentration grows linearly in time, and that the uptake is essentially irreversible, with the particles accumulating in lysosomes. A wide range of practical challenges, from labile dye release to NP aggregation and the need to account for cell division, are addressed to ensure that these studies yield meaningful kinetic information. FROM THE CLINICAL EDITOR The authors present an uptake study comparing the outcomes for free molecular dye and NPs labeled with the same dye. A wide range of practical challenges are addressed including labile dye release, NP aggregation and the need to account for cell division with the goal that these studies yield meaningful kinetic information.

Quantitative Assessment of the Comparative Nanoparticle-Uptake Efficiency of a Range of Cell Lines

The mechanism(s) of nanoparticle-cell interactions are still not understood. At present there is little knowledge of the relevant length- and timescales for nanoparticle intracellular entry and localization within cells, or the cell-specificity of nanoparticle uptake and localisation. Here, the effect of particle size on the in-vitro intracellular uptake of model fluorescent carboxyl-modified polystyrene nanoparticles is investigated in various cell lines. A range of micro- and nanoparticles of defined sizes (40 nm to 2 μm) are incubated with a series of cell types, including HeLa and A549 epithelial cells, 1321N1 astrocytes, HCMEC D3 endothelial cells, and murine RAW 264.7 macrophages. Techniques such as confocal microscopy and flow cytometry are used to study particle uptake and subcellular localisation, making significant efforts to ensure reproducibility in a semiquantitative approach. The results indicate that internalization of (nano)particles is highly size-dependent for all cell lines studied, and the kinetics of uptake for the same type of nanoparticle varies in the different cell types. Interestingly, even cells not specialized for phagocytosis are able to internalize the larger nanoparticles. Intracellular uptake of all sizes of particles is observed to be highest in RAW 264.7 cells (a specialized phagocytic cell line) and the lowest in the HeLa cells. These results suggest that (nano)particle uptake might not follow commonly defined size limits for uptake processes, and highlight the variability of uptake kinetics for the same material in different cell types. These conclusions have important implications for the assessment of the safety of nanomaterials and for the potential biomedical applications of nanoparticles.

Polymeric Nanoparticles to Control the Differentiation of Neural Stem Cells in the Subventricular Zone of the Brain

Herein, we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors, decreased stemness, and increased proneurogenic gene expression. Importantly, this work reports for the first time a nanoparticle formulation able to modulate in vivo the subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases.

Controlling the Neuronal Differentiation of Stem Cells by the Intracellular Delivery of Retinoic Acid-Loaded Nanoparticles

The manipulation of endogenous stem cell populations from the subventricular zone (SVZ), a neurogenic niche, creates an opportunity to induce neurogenesis and influence brain regenerative capacities in the adult brain. Herein, we demonstrate the ability of polyelectrolyte nanoparticles to induce neurogenesis exclusively after being internalized by SVZ stem cells. The nanoparticles are not cytotoxic for concentrations equal or below 10 μg/mL. The internalization process is rapid, and nanoparticles escape endosomal fate in a few hours. Retinoic acid-loaded nanoparticles increase the number of neuronal nuclear protein (NeuN)-positive neurons and functional neurons responding to depolarization with KCl and expressing NMDA receptor subunit type 1 (NR1). These nanoparticles offer an opportunity for in vivo delivery of proneurogenic factors and neurodegenerative disease treatment.

Histamine modulates microglia function

BackgroundHistamine is commonly acknowledged as an inflammatory mediator in peripheral tissues, leaving its role in brain immune responses scarcely studied. Therefore, our aim was to uncover the cellular and molecular mechanisms elicited by this molecule and its receptors in microglia-induced inflammation by evaluating cell migration and inflammatory mediator release.MethodsFirstly, we detected the expression of all known histamine receptor subtypes (H1R, H2R, H3R and H4R), using a murine microglial cell line and primary microglia cell cultures from rat cortex, by real-time PCR analysis, immunocytochemistry and Western blotting. Then, we evaluated the role of histamine in microglial cell motility by performing scratch wound assays. Results were further confirmed using murine cortex explants. Finally, interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by ELISA measurements to determine the role of histamine on the release of these inflammatory mediators.ResultsAfter 12 h of treatment, 100 μM histamine and 10 μg/ml histamine-loaded poly (lactic-co-glycolic acid) microparticles significantly stimulated microglia motility via H4R activation. In addition, migration involves α5β1 integrins, and p38 and Akt signaling pathways. Migration of microglial cells was also enhanced in the presence of lipopolysaccharide (LPS, 100 ng/ml), used as a positive control. Importantly, histamine inhibited LPS-stimulated migration via H4R activation. Histamine or H4R agonist also inhibited LPS-induced IL-1β release in both N9 microglia cell line and hippocampal organotypic slice cultures.ConclusionsTo our knowledge, we are the first to show a dual role of histamine in the modulation of microglial inflammatory responses. Altogether, our data suggest that histamine per se triggers microglia motility, whereas histamine impedes LPS-induced microglia migration and IL-1β release. This last datum assigns a new putative anti-inflammatory role for histamine, acting via H4R to restrain exacerbated microglial responses under inflammatory challenge, which could have strong repercussions in the treatment of CNS disorders accompanied by microglia-derived inflammation.

Neuropeptide Y inhibits interleukin-1β-induced phagocytosis by microglial cells.

BackgroundNeuropeptide Y (NPY) is emerging as a modulator of communication between the brain and the immune system. However, in spite of increasing evidence that supports a role for NPY in the modulation of microglial cell responses to inflammatory conditions, there is no consistent information regarding the action of NPY on microglial phagocytic activity, a vital component of the inflammatory response in brain injury. Taking this into consideration, we sought to assess a potential new role for NPY as a modulator of phagocytosis by microglial cells.MethodsThe N9 murine microglial cell line was used to evaluate the role of NPY in phagocytosis. For that purpose, an IgG-opsonized latex bead assay was performed in the presence of lipopolysaccharide (LPS) and an interleukin-1β (IL-1β) challenge, and upon NPY treatment. A pharmacological approach using NPY receptor agonists and antagonists followed to uncover which NPY receptor was involved. Moreover, western blotting and immunocytochemical studies were performed to evaluate expression of p38 mitogen-activated protein kinase (MAPK) and heat shock protein 27 (HSP27), in an inflammatory context, upon NPY treatment.ResultsHere, we show that NPY inhibits phagocytosis of opsonized latex beads and inhibits actin cytoskeleton reorganization triggered by LPS stimulation. Co-stimulation of microglia with LPS and adenosine triphosphate also resulted in increased phagocytosis, an effect inhibited by an interleukin-1 receptor antagonist, suggesting involvement of IL-1β signaling. Furthermore, direct application of LPS or IL-1β activated downstream signaling molecules, including p38 MAPK and HSP27, and these effects were inhibited by NPY. Moreover, we also observed that the inhibitory effect of NPY on phagocytosis was mediated via Y1 receptor activation.ConclusionsAltogether, we have identified a novel role for NPY in the regulation of microglial phagocytic properties, in an inflammatory context.

Neuropeptide Y inhibits interleukin-1 beta-induced microglia motility.

J. Neurochem. (2012) 120, 93–105.

Histamine Stimulates Neurogenesis in the Rodent Subventricular Zone

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell‐based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1 genes. Biocompatible poly (lactic‐co‐glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine‐loaded microparticles facilitated neuronal differentiation of SVZ‐GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial‐derived vehicles may represent a new tool for brain repair strategies. STEM CELLS 2012; 30:773–784