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

A model study for tethering of (bio)active molecules to biomaterial surfaces through arginine

A new approach for tethering of bioactive molecules via arginine is proposed and validated on collagen 2D matrices. The method involves the introduction of a methyl ketone on arginine side-chains, followed by reaction with model alkoxyamino derivatives

Carbohydrate-functionalized collagen matrices: design and characterization of a novel neoglycosylated biomaterial

Collagen matrices have been neoglycosylated with lactose by reductive amination at lysine side chains. AFM analysis highlights that the chemical does not affect molecular assembly into fibrils. Moreover, ELLA biochemical assays show that the glycan moiety is efficiently exposed on the matrix surface for receptor recognition

Optimization of a detergent-based protocol for membrane proteins purification from mammalian cells

Membrane proteins constitute around 20–30 % of the proteins encoded by mammalian genes, are involved in many cell functions, and represent the majority of drug targets. However, the isolation of membrane proteins is challenging because of their partial hydrophobicity, requiring detergents to extract them from cell membranes and stabilize them in solution. Many commercial kits use this principle, but they are expensive, and their chemical composition is not known. In this work, we propose a fast, detergent-based protocol for the purification of membrane proteins from murine and human cells. This protocol is based on three steps: cell washing to remove cell culture medium proteins, cells permeabilization using digitonin to remove the intracellular components, and cell membranes disruption using Triton X-100 to solubilize membrane proteins and keep them in solution. We measured the total protein yield using our protocol with two different detergent concentrations and compared it to a commercial kit. We further assessed membrane protein enrichment by comparing markers for specific cellular components using SDS-PAGE/western blot and identifying specific proteins by qualitative mass spectrometry. Our protocol led to a final protein yield analogous to the commercial kit and similar membrane protein purity, while resulting significantly cheaper compared to the commercial kit. Furthermore, this process can be applied to a different number and types of cells, resulting scalable, versatile, and robust. The possibility to perform downstream mass spectrometry analysis is of particular importance since it enables the use of “omics” techniques for protein discovery and characterization. Our approach could be used as a starting point for the isolation of membrane proteins for pharmacological and biochemical studies, or for the discovery of new druggable or prognostic markers

Effects of the protein corona on liposome–liposome and liposome–cell interactions

Claudia Corbo,1,2 Roberto Molinaro,1 Francesca Taraballi,1 Naama E Toledano Furman,1 Michael B Sherman,3 Alessandro Parodi,1 Francesco Salvatore,2,4 Ennio Tasciotti1,5 1Center for Biomimetic Medicine, Houston Methodist Research Institute, Houston, TX, USA; 2CEINGE-Biotecnologie Avanzate s.c.a r.l., Naples, Italy; 3Department of Biochemistry and Molecular Biology, Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX, USA; 4Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Naples, Italy; 5Department of Orthopedics, Houston Methodist Hospital, Houston, TX, USA Abstract: A thorough understanding of interactions occurring at the interface between nanocarriers and biological systems is crucial to predict and interpret their biodistribution, targeting, and efficacy, and thus design more effective drug delivery systems. Upon intravenous injection, nanoparticles are coated by a protein corona (PC). This confers a new biological identity on the particles that largely determines their biological fate. Liposomes have great pharmaceutical versatility, so, as proof of concept, their PC has recently been implicated in the mechanism and efficiency of their internalization into the cell. In an attempt to better understand the interactions between nanocarriers and biological systems, we analyzed the plasma proteins adsorbed on the surface of multicomponent liposomes. Specifically, we analyzed the physical properties and ultrastructure of liposome/PC complexes and the aggregation process that occurs when liposomes are dispersed in plasma. The results of combined confocal microscopy and flow cytometry experiments demonstrated that the PC favors liposome internalization by both macrophages and tumor cells. This work provides insights into the effects of the PC on liposomes’ physical properties and, consequently, liposome–liposome and liposome–cell interactions. Keywords: liposomes, protein corona, macrophages, cancer cell

Mesoporous silica nanoparticles trigger mitophagy in endothelial cells and perturb neuronal network activity in a size- and time-dependent manner

Antonina Orlando,1 Emanuela Cazzaniga,1 Maria Tringali,2 Francesca Gullo,3 Andrea Becchetti,3 Stefania Minniti,1 Francesca Taraballi,4,5 Ennio Tasciotti,4,5 Francesca Re1 1Nanomedicine Center, School of Medicine and Surgery, University of Milano-Bicocca, Monza, 2Department of Environmental Sciences, 3Department of Biotechnologies and Biosciences, University of Milano-Bicocca, Milan, Italy; 4Center for Biomimetic Medicine, Houston Methodist Research Institute (HMRI), 5Department of Orthopedics and Sports Medicine, Houston Methodist Hospital, Houston, TX, USA Purpose: Mesoporous silica nanoparticles (MSNPs) are excellent candidates for biomedical applications and drug delivery to different human body areas, the brain included. Although toxicity at cellular level has been investigated, we are still far from using MSNPs in the clinic, because the mechanisms involved in the cellular responses activated by MSNPs have not yet been elucidated.Materials and methods: This study used an in vitro multiparametric approach to clarify relationships among size, dose, and time of exposure of MSNPs (0.05–1 mg/mL dose range), and cellular responses by analyzing the morphology, viability, and functionality of human vascular endothelial cells and neurons.Results: The results showed that 24 hours of exposure of endothelial cells to 250 nm MSNPs exerted higher toxicity in terms of mitochondrial activity and membrane integrity than 30 nm MSN at the same dose. This was due to induced cell autophagy (in particular mitophagy), probably consequent to MSNP cellular uptake (>20%). Interestingly, after 24 hours of treatment with 30 nm MSNPs, very low MSNP uptake (<1%) and an increase in nitric oxide production (30%, P<0.01) were measured. This suggests that MSNPs were able to affect endothelial functionality from outside the cells. These differences could be attributed to the different protein-corona composition of the MSNPs used, as suggested by sodium dodecyl sulfate polyacrylamide-gel electrophoresis analysis of the plasma proteins covering the MSNP surface. Moreover, doses of MSNPs up to 0.25 mg/mL perturbed network activity by increasing excitability, as detected by multielectrode-array technology, without affecting neuronal cell viability.Conclusion: These results suggest that MSNPs may be low-risk if prepared with a diameter <30 nm and if they reach human tissues at doses <0.25 mg/mL. These important advances could help the rational design of NPs intended for biomedical uses, demonstrating that careful toxicity evaluation is necessary before using MSNPs in patients. Keywords: mesoporous silica nanoparticles, nanotoxicity, endothelial cells, neurons, brain&nbsp

Smart biomaterials: The contribution of glycoscience

Examples of material functionalisation ("biodecoration") with signalling and relevant glycidic scaffolds will be outlined. Recent research concerning the development of smart biomaterials for Tissue Engineering (TE) applications will be considered. © The Royal Society of Chemistry 2012