Real-Time Nanoparticle–Cell Interactions in Physiological Media by Atomic Force Microscopy

Particle–cell interactions in physiological media are important in determining the fate and transport of nanoparticles and biological responses to them. In this work, these interactions are assessed in real time using a novel atomic force microscopy (AFM) based platform. Industry-relevant CeO2 and Fe2O3 engineered nanoparticles (ENPs) of two primary particle sizes were synthesized by the flame spray pyrolysis (FSP) based Harvard Versatile Engineering Nanomaterials Generation System (Harvard VENGES) and used in this study. The ENPs were attached on AFM tips, and the atomic force between the tip and lung epithelia cells (A549), adhered on a substrate, was measured in biological media, with and without the presence of serum proteins. Two metrics were used to assess the nanoparticle cell: the detachment force required to separate the ENP from the cell and the number of bonds formed between the cell and the ENPs. The results indicate that these atomic level ENP–cell interaction forces strongly depend on the physiological media. The presence of serum proteins reduced both the detachment force and the number of bonds by approximately 50% indicating the important role of the protein corona on the particle cell interactions. Additionally, it was shown that particle to cell interactions were size and material dependent

Heat Shock Protein Beta-1 Modifies Anterior to Posterior Purkinje Cell Vulnerability in a Mouse Model of Niemann-Pick Type C Disease

<div><p>Selective neuronal vulnerability is characteristic of most degenerative disorders of the CNS, yet mechanisms underlying this phenomenon remain poorly characterized. Many forms of cerebellar degeneration exhibit an anterior-to-posterior gradient of Purkinje cell loss including Niemann-Pick type C1 (NPC) disease, a lysosomal storage disorder characterized by progressive neurological deficits that often begin in childhood. Here, we sought to identify candidate genes underlying vulnerability of Purkinje cells in anterior cerebellar lobules using data freely available in the Allen Brain Atlas. This approach led to the identification of 16 candidate neuroprotective or susceptibility genes. We demonstrate that one candidate gene, heat shock protein beta-1 (<i>HSPB1</i>), promoted neuronal survival in cellular models of NPC disease through a mechanism that involved inhibition of apoptosis. Additionally, we show that over-expression of wild type <i>HSPB1</i> or a phosphomimetic mutant in NPC mice slowed the progression of motor impairment and diminished cerebellar Purkinje cell loss. We confirmed the modulatory effect of Hspb1 on Purkinje cell degeneration <i>in vivo</i>, as knockdown by <i>Hspb1</i> shRNA significantly enhanced neuron loss. These results suggest that strategies to promote HSPB1 activity may slow the rate of cerebellar degeneration in NPC disease and highlight the use of bioinformatics tools to uncover pathways leading to neuronal protection in neurodegenerative disorders.</p></div

PKCδ and phosphorylated HSPB1 are co-expressed in Purkinje cells in posterior lobules.

<p><b>(A)</b> Transgenic HSPB1 (HA) in the cerebellar midline of 7-week-old <i>Npc1 flox/–</i>, <i>Pcp2-Cre</i>, <i>HSPB1</i> mice. Scale bar = 200 μm. <b>(B, C)</b> Expression of phospho-HSPB1 (serine 15, <i>in green</i>, <i>panel B</i>) and PKCδ (<i>in green</i>, <i>panel C</i>) were examined in Purkinje cells (calbindin, <i>in red</i>) in the cerebellar midline of <i>Npc1 flox/–</i>, <i>Pcp2-Cre</i>, <i>HSPB1</i> mice at 7 weeks of age. Nuclei were stained by DAPI. <i>Top row</i>, lobule II; <i>bottom row</i>, lobule IX. Scale bar = 20 μm.</p

HSPB1 promotes survival in cellular models of NPC1 disease.

<p><b>(A)</b> Expression of Hspb1 <i>(left panel)</i> and calbindin <i>(right panel</i>, Purkinje cells) in cerebellar midline of <i>Npc1 flox/-</i>, <i>Pcp2-Cre</i> mice 7 weeks. Scale bar = 200 μm. <b>(B)</b> (<i>Upper panel</i>) HeLa cells were transfected with non-targeted (NT, lanes 1 and 3) or <i>HSPB1</i> siRNA (lanes 2 and 4), then treated with vehicle (lanes 1–2) or 1 mg/ml U18666A (lanes 3–4) for 24 hr. HSPB1 expression was determined by western blot. GAPDH controls for loading. (<i>Lower panel</i>) Caspase-3 in HeLa cell lysates. Data are mean ± SEM. *<i>p</i><0.05. <b>(C)</b> NPC1 patient fibroblasts were transfected with non-targeted or <i>HSPB1</i> siRNA. Cells were stained with Hoechst, and the percentage of cells with condensed chromatin was scored. Data are mean ± SEM. **<i>p</i><0.01. <b>(D)</b> Primary mouse cortical neurons were transduced with wild type <i>HSPB1</i>, <i>HSPB1</i>^<i>3A</i>, <i>HSPB1</i>^<i>3E</i>, or empty vector, and then treated with 2.5 μg/ml U18666. XTT assay was performed 72 hrs post U18666A. Neuron survival is reported relative to vehicle treated cells. Data are mean ± SEM. ***<i>p</i><0.001.</p

Schematic of gene expression analysis.

<p><b>(A)</b> Calbindin staining of cerebellum from 20-week old <i>Npc1 flox/-</i>, <i>Pcp2-Cre</i> mouse, demonstrating survival pattern of Purkinje cells in midline. Scale bar = 500 μm. <b>(B)</b> Schematic of differential vulnerability of Purkinje cell subpopulations. Regions of interest (ROI) were selected to include the population that experiences the most rapid neurodegeneration (lobules II and III, ROI #1) or the population that does not degenerate (lobule X, ROI #2). <b>(C)</b> Approach to gene expression analysis. Allen Brain Atlas data was downloaded and consolidated into a single gene expression matrix. Each row represents gene expression data from a single series of <i>in situ</i> hybridization data, while each column represents a single voxel within the mouse brain. The data set was then narrowed to include only voxels lying within the defined regions of interest. To identify genes differentially expressed between regions of interest, expression data was treated analogously to replicate microarrays and subjected to standard statistical tests (<i>t</i>-test and Significance Analysis for Microarrays). The top 1000 most significant genes were accepted for manual curation to verify expression in Purkinje cells. The gene list was further narrowed to include only genes with absolute expression differences between regions of interest, and expression matching the pattern of Purkinje cell survival or death throughout the entire cerebellum.</p

Genes differentially expressed in Purkinje cells in anterior or posterior lobules.

<p>Genes differentially expressed in Purkinje cells in anterior or posterior lobules.</p

PKCδ and HSPB1 phosphorylation increase cell viability.

<p><b>(A)</b> HeLa cells were transfected with non-targeted (NT) or <i>PKCδ</i> siRNA and then treated with 3 μg/ml U18666A or vehicle for 24 hrs. XTT assay was performed to measure cell viability. Data are mean ± SD, **<i>p</i><0.01. <b>(B)</b> <i>PKCδ</i> expression was determined by quantitative real-time PCR. Data are mean ± SD, ***<i>p</i><0.001. <b>(C, D)</b> 6-week-old <i>Npc1 flox/-</i>, <i>Pcp2-Cre</i> mice were injected with AAV2 expressing HSPB1-3E or control vector and then examined at 10 weeks of age. Quantification of Purkinje cell density in lobules VI <b>(C)</b> and VII <b>(D)</b> of midline cerebellar sections. Data are mean ± SEM, <i>n</i> = 4 mice/group. *<i>p</i><0.05.</p

Optimizing the properties of the protein corona surrounding nanoparticles for tuning payload release

10.1021/nn404166qACS Nano71110066-1007