Interactions of siRNA loaded dextran nanogel with blood cells

Adsorption and internalization of siRNA loaded dextran nanogels by blood cells were determined using flow cytometry. Positively and negatively charged nanogels with various PEGylation degrees were compared in order to find a formulation showing minimal interactions with blood

MMP-3 deficiency alleviates endotoxin-induced acute inflammation in the posterior eye segment

Matrix metalloproteinase-3 (MMP-3) is known to mediate neuroinflammatory processes by activating microglia, disrupting blood-central nervous system barriers and supporting neutrophil influx into the brain. In addition, the posterior part of the eye, more specifically the retina, the retinal pigment epithelium (RPE) and the blood-retinal barrier, is affected upon neuroinflammation, but a role for MMP-3 during ocular inflammation remains elusive. We investigated whether MMP-3 contributes to acute inflammation in the eye using the endotoxin-induced uveitis (EIU) model. Systemic administration of lipopolysaccharide induced an increase in MMP-3 mRNA and protein expression level in the posterior part of the eye. MMP-3 deficiency or knockdown suppressed retinal leukocyte adhesion and leukocyte infiltration into the vitreous cavity in mice subjected to EIU. Moreover, retinal and RPE mRNA levels of intercellular adhesion molecule 1 (Icam1), interleukin 6 (Il6), cytokine-inducible nitrogen oxide synthase (Nos2) and tumor necrosis factor alpha (Tnf alpha), which are key molecules involved in EIU, were clearly reduced in MMP-3 deficient mice. In addition, loss of MMP-3 repressed the upregulation of the chemokines monocyte chemoattractant protein (MCP)-1 and (C-X-C motif) ligand 1 (CXCL1). These findings suggest a contribution of MMP-3 during EIU, and its potential use as a therapeutic drug target in reducing ocular inflammation

Microfluidic interactions between red blood cells and drug carriers by image analysis techniques

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.Blood is a complex biological fluid composed of deformable cells and platelets suspended in plasma, a protein-rich liquid. The peculiar nature of blood needs to be considered when designing a drug delivery strategy based on systemically administered carriers. Here, we report on an in vitro fluid dynamic investigation of the influence of the microcapillary flow of red blood cells (RBCs) on micron sized carriers by high speed imaging methods. The experiments were carried out in a 50μm diameter glass capillary that mimicked the hydrodynamic conditions of human microcirculation. Spherical μ particles (μ-Ps), with sizes ranging between 0.5 and 3μm, were tested. Images of the flowing RBCs and μ-Ps were acquired by a highspeed/ high-magnification microscopy. The transport and distribution of rigid particles in a suspension of RBCs under shear flow were followed for: i) the migration of RBCs towards the vessel centerline due to their deformability; ii) the cross-flow migration of μ-Ps towards the vessel wall due to their hydrodynamic interactions with RBCs; iii) the radial distribution of μ-Ps in the presence of RBCs. This study suggests that the therapeutic efficacy of μ-Ps could be ultimately affected by their interactions with the flowing RBCs in the vasculature

Using neural networks for high-speed blood cell classification in a holographic-microscopy flow-cytometry system

High-throughput cell sorting with flow cytometers is an important tool in modern clinical cell studies. Most cytometers use biomarkers that selectively bind to the cell, but induce significant changes in morphology and inner cell processes leading sometimes to its death. This makes label-based cell sorting schemes unsuitable for further investigation. We propose a label-free technique that uses a digital inline holographic microscopy for cell imaging and an integrated, optical neural network for high-speed classification. The perspective of dense integration makes it attractive to ultrafast, large-scale cell sorting. Network simulations for a ternary classification task (monocytes/granulocytes/lymphocytes) resulted in 89% accuracy

High fat diet causes depletion of intestinal eosinophils associated with intestinal permeability.

The development of intestinal permeability and the penetration of microbial products are key factors associated with the onset of metabolic disease. However, the mechanisms underlying this remain unclear. Here we show that, unlike liver or adipose tissue, high fat diet (HFD)/obesity in mice does not cause monocyte/macrophage infiltration into the intestine or pro-inflammatory changes in gene expression. Rather HFD causes depletion of intestinal eosinophils associated with the onset of intestinal permeability. Intestinal eosinophil numbers were restored by returning HFD fed mice to normal chow and were unchanged in leptin-deficient (Ob/Ob) mice, indicating that eosinophil depletion is caused specifically by a high fat diet and not obesity per se. Analysis of different aspects of intestinal permeability in HFD fed and Ob/Ob mice shows an association between eosinophil depletion and ileal paracelullar permeability, as well as leakage of albumin into the feces, but not overall permeability to FITC dextran. These findings provide the first evidence that a high fat diet causes intestinal eosinophil depletion, rather than inflammation, which may contribute to defective barrier integrity and the onset of metabolic disease

Endothelial C-type natriuretic peptide maintains vascular homeostasis

PMCID: PMC4151218Wellcome Trust (084449/Z/07/Z and 078496/Z/05/Z

Intracellular delivery of nanomaterials for sub-cellular imaging and tracking of biomolecules.

Nanomaterials have many intriguing applications in biology and medicine. Unique properties such as enhanced electrical properties, increased chemical reactivity and resistance to degradation, novel optical properties and comparable size to that of biological systems have led to their use in various biomedical applications. The most important applications of nanomaterials for medicine are in drug delivery and imaging. This research focuses on utilizing the biocompatibility of single walled Carbon nanotubes (SWCNTs) and optical properties colloidal quantum dots (QDs) for cellular drug delivery and imaging of biomolecules. The first part of this research deals with single walled carbon nanotubes which are excellent candidates for targeted drug delivery applications due their unique structural and functional properties. However, prior to their use in therapeutics, their biocompatibility needs to be thoroughly investigated. The objectives of this research were to establish the biocompatibility of SWCNTs and demonstrate their use as drug delivery carriers into cells. Blood, a living tissue, is chosen as the biological system as it contains various cells which can potentially interact with SWCNTs during the delivery mechanism. The interactions of these cells in the blood (specifically white blood cells or leukocytes) with the SWCNTs provide vital information regarding the immune response of the host to the nanotubes. This research investigates the immune response of white blood cells due to SWCNTs via (a) direct interaction - presence of nanotubes in the blood and, (b) indirect interaction - presentation of nanotubes by antigen-presenting-cells to white blood cells. These two interactions recreate the innate and adaptive immune responses occurring in the body to any foreign substance. SWCNTs are functionalized with single stranded DNA (ss-DNA), which serves as a dispersant of nanotubes as well as a backbone for further attachment of other biomolecules of interest. Confocal microscopy and flow cytometric studies are performed to characterize the interactions. Results from this acute immune response study demonstrate the biocompatibility of SWCNTs in whole blood and also confirm the cellular delivery of single stranded DNA. The second part of the research is on colloidal quantum dots (QDs): nanometer sized semiconductor crystals typically between 1 run to 20 nm in diameter. In addition to being size comparable with many biological systems, and having large surface area for multiple biomolecules attachment, they possess high resistance to chemical and photo degradation, tunable emission based on size and composition which makes them excellent candidates for cellular delivery and imaging. The main objectives of this research was to demonstrate the use of QDs for cellular imaging as well as targeted biomolecule delivery by conjugating the QDs with an antibody to a functional protein and delivery into live cells. Conventional techniques deliver QDs as aggregates, however, a major challenge in the use of QDs for cellular imaging and biomolecule delivery is achieving freely dispersed QDs inside the cells. In this research, a new technique to deliver monodispersed QDs inside live cells was developed. The approach combines osmosis driven fluid transport into cells achieved by creating hypotonic environment and reversible permeabilization using low concentrations of cell permeabilization agents like Saponin. The results confirm that highly efficient endocytosis-free intracellular delivery of QDs can be accomplished using this method. Confocal microscopy is used to image the QDs inside the cells and flow cytometry is used for quantifying the fluorescence. To demonstrate targeted delivery, QDs are conjugated to the antibody of a protein: the nuclear transcriptional factor, NFkB (Nuclear Factor kappa-light chain-enhancer of activated B cells) using EDC/sulfo NHS chemistry methods. NFkB is a family of proteins with 5 different subunits and is involved in a variety of biological processes such as immune and inflammatory responses and cellular developmental processes. In unstimulated cells, NFkB is inactive in cytoplasm and translocates to the nucleus upon stimulation using bacterial products, viruses, radiation, and the like. QDs fluorescence could be used to monitor NFKB activity over extended periods of time in live cells

Validation of a Confocal Light Sheet Microscope using Push Broom Translation for Biomedical Applications

There exists a need for research of optical methods capable of image cytometry suitable for point-of-care technology. To propose am optical approach with no moving parts for simplification of mechanical components for the further development of the technology to the poin-of-care, a linear sensor with push broom translation method. Push broom translation is a method of moving objects by the sensor for an extended field of view. A polydimethylsiloxane (PDMS) microfluidic chamber with a syringe pump was used to deliver objects by the sensor. The volumetric rate of the pump was correlated to the integration time of the sensor to ensure images were realistically being formed, termed aspect ratio. An electro-chemical microfluidic system was then also investigated, redox-magnetohydrodynamics (R-MHD), to eliminate the mechanical syringe pump which showed deviations in linear speeds at the specimen plane. To image with adequate signal to background ratio within the deep chamber of the R-MHD device, an epitaxial light sheet confocal microscope (e-LSCM) was used to improve axial resolution. The linear sensor, having small pixels, blocked out-of-plane light while eliminating the need for a mechanical aperture which is used for traditional point-scanning confocal microscopy. The particular linear sensor used has binning modes that were used to vary the axial resolution by increasing the sensor aperture. This approach was validated by using a mirror translated in the axial direction and measuring remitted light intensity. The resulting curve estimated the real axial resolution of the microscope, which compared favorably to theoretical values. The R-MHD and the e-LSCM were then synchronized to perform continuous imaging of fluorescent microspheres and cells in suspension. This study combines epitaxial light sheet confocal microscopy and electro-chemical microfluidics as a robust approach which could be used in future point-of-care image cytometry applications