Carla Sfara

Programmable 3D silk bone marrow niche for platelet generation ex vivo and modeling of megakaryopoiesis pathologies

We present a programmable bioengineered 3-dimensional silk-based bone marrow niche tissue system that successfully mimics the physiology of human bone marrow environment allowing us to manufacture functional human platelets ex vivo. Using stem/progenitor cells, megakaryocyte function and platelet generation were recorded in response to variations in extracellular matrix components, surface topography, stiffness, coculture with endothelial cells, and shear forces. Millions of human platelets were produced and showed to be functional based on multiple activation tests. Using adult hematopoietic progenitor cells our system demonstrated the ability to reproduce key steps of thrombopoiesis, including alterations observed in diseased states. A critical feature of the system is the use of natural silk protein biomaterial allowing us to leverage its biocompatibility, nonthrombogenic features, programmable mechanical properties, and surface binding of cytokines, extracellular matrix components, and endothelial-derived proteins. This in turn offers new opportunities for the study of blood component production ex vivo and provides a superior tissue system for the study of pathologic mechanisms of human platelet production.

Human erythrocytes as nanoparticle carriers for magnetic particle imaging

The potential of red blood cells (RBCs) loaded with iron oxide nanoparticles as a tracer material for magnetic particle imaging (MPI) has been investigated. MPI is an emerging, quantitative medical imaging modality which holds promise in terms of sensitivity in combination with spatial and temporal resolution. Steady-state and dynamic magnetization measurements, supported by semi-empirical modeling, were employed to analyze the MPI signal generation using RBCs as novel biomimetic constructs. Since the superparamagnetic iron oxide (SPIO) bulk material that is used in this study contains nanoparticles with different sizes, it is suggested that during the RBC loading procedure, a preferential entrapment of nanoparticles with hydrodynamic diameter ≤60 nm occurs by size-selection through the erythrocyte membrane pores. This affects the MPI signal of an erythrocyte-based tracer, compared to bulk. The reduced signal is counterbalanced by a higher in vivo stability of the SPIO-loaded RBCs constructs for MPI applications.

Encapsulation of superparamagnetic nanoparticles into red blood cells as new carriers of MRI contrast agents.

AIMS The half-life of superparamagnetic iron oxide nanoparticles in the bloodstream is very short since they are rapidly taken up by the reticuloendothelial system. In this article, we report the encapsulation of different magnetic nanoparticles into human erythrocytes to increase their blood circulation time. MATERIALS & METHODS Newly synthesized and commercially available nanoparticles were evaluated for the encapsulation into red blood cells through the transient opening of membrane pores by controlled hypotonic dialysis and successive isotonic resealing and reannealing of cells. RESULTS Commercial superparamagnetic iron oxide nanoparticles (SHU 555A, AMI 227 and PMP-50) dextran or carboxydextran coated can be successfully loaded into red blood cells; similarly, some of the new nanomaterials, such as Np-1 nanoparticles dispersed in the Disperbyk®-190 agent, can be efficiently encapsulated into red blood cells. CONCLUSION A careful consideration of magnetic nanoparticles parameters, such as size, synthesis protocols, coating and/or dispersant agents, is required in order to obtain efficient loading through the cell membrane pores.

New Strategies to Prolong the In Vivo Life Span of Iron-Based Contrast Agents for MRI

Superparamagnetic iron oxide (SPIO) and ultra small superparamagnetic iron oxide (USPIO) nanoparticles have been developed as magnetic resonance imaging (MRI) contrast agents. Iron oxide nanoparticles, that become superparamagnetic if the core particle diameter is ~ 30nm or less, present R1 and R2 relaxivities which are much higher than those of conventional paramagnetic gadolinium chelates. Generally, these magnetic particles are coated with biocompatible polymers that prevent the agglomeration of the colloidal suspension and improve their blood distribution profile. In spite of their potential as MRI blood contrast agents, the biomedical application of iron oxide nanoparticles is still limited because of their intravascular half-life of only few hours; such nanoparticles are rapidly cleared from the bloodstream by macrophages of the reticulo-endothelial system (RES). To increase the life span of these MRI contrast agents in the bloodstream we proposed the encapsulation of SPIO nanoparticles in red blood cells (RBCs) through the transient opening of cell membrane pores. We have recently reported results obtained by applying our loading procedure to several SPIO nanoparticles with different chemical physical characteristics such as size and coating agent. In the current investigation we showed that the life span of iron-based contrast agents in the mice bloodstream was prolonged to 12 days after the intravenous injection of murine SPIO-loaded RBCs. Furthermore, we developed an animal model that implicates the pretreatment of animals with clodronate to induce a transient suppression of tissue macrophages, followed by the injection of human SPIO-loaded RBCs which make it possible to encapsulate nanoparticle concentrations (5.3-16.7mM Fe) higher than murine SPIO-loaded RBCs (1.4-3.55mM Fe). The data showed that, when human RBCs are used as more capable SPIO nanoparticle containers combined with a depletion of tissue macrophages, Fe concentration in animal blood is 2-3 times higher than iron concentration obtained by the use of murine SPIO-loaded RBCs.

USPIO-loaded red blood cells as a biomimetic MR contrast agent: a relaxometric study.

Red blood cells (RBCs) loaded with iron oxide nanoparticles have been proposed as biomimetic constructs with long half-life (ca. 20 days) in the blood compartment and potentially interesting properties (such as relaxivity) as intravascular contrast agents for magnetic resonance imaging. However, the encapsulation of nanoparticles into RBCs might affect their magnetic properties and relaxivity, which may be significantly different from the native suspension. Here, we present a relaxometric study of P904, a novel ultra small iron oxide nanoparticle developed by Guerbet, enclosed in human RBCs. We measured longitudinal (r1 ) and transverse (r2 ) relaxivity over a wide range of Larmor frequencies (0.01-300 MHz) in samples of P904-loaded RBCs, and in control samples with P904 nanoparticles dispersed in blood. Internalization of P904 into RBCs resulted in smaller r1 , and in a very high r2 /r1 ratio (232) at the highest field. Moreover, a shift of the Curie peak to high fields was observed in P904-loaded RBCs, possibly the result of nanoparticle size selection caused by the internalization process. High r2 relaxivity together with a high r2 /r1 ratio and a very long blood half-life make P904-loaded RBCs a promising blood-pool negative contrast agent for MR diagnostic applications.

Red blood cells as carriers in magnetic particle imaging.

Abstract Red blood cells (RBCs) represent intravascular carriers for drugs, biologics, and other therapeutic agents, characterized by their unique longevity in the bloodstream, availability, considerable surface and volume, high biocompatibility, and natural mechanisms for safe elimination. Recently, the potential of RBCs loaded with superparamagnetic iron oxide (SPIO) nanoparticles as a tracer material for magnetic particle imaging (MPI) to realize a blood-pool tracer agent with longer blood retention time for imaging of the circulatory system, has been investigated. MPI is a new tomographic imaging approach that can quantitatively map magnetic nanoparticle distributions in vivo. However, SPIO contrast agents, such as Resovist, have a short blood half-life due to rapid uptake by the reticuloendothelial system, which limits the applicability of such compounds for certain applications such as long-term monitoring. Here, we report the in vitro magnetic characterization study of human SPIO-loaded RBCs and the first MPI results obtained after intravenous injection of murine SPIO-loaded RBCs in an in vivo MPI experiment.

Effect of the redox state on HIV-1 tat protein multimerization and cell internalization and trafficking

The redox state of the cysteine-rich region of the HIV Tat protein is known to play a crucial role in Tat biological activity. In this article, we show that Tat displays two alternative functional states depending on the presence of either one or three reduced sulphydryl groups in the cysteine-rich region, respectively. Using different approaches, a disulfide pattern has been defined for the Tat protein and a specific DTT-dependent breaking order of disulfide bonds highlighted. The Tat redox state deeply influences macrophage protein uptake. Immunoistochemistry analysis shows that the oxidized protein does not enter cells, whereas partially reduced protein reaches the cytosol and, to a limited extent, the nucleus. Finally electrophoretic analysis shows Tat high-molecular weight multi-aggregation, resulting in the loss of biological activity. This is due to strong electrostatic and metal-binding interactions, whereas Tat dimerization involves metal-binding interactions as well as disulfide bond formation.

Red Blood Cells as Magnetic Carriers for MPI Applications

Magnetic Particle Imaging (MPI), a method that takes advantage of the non-linear magnetization curve of superparamagnetic iron oxide (SPIO) nanoparticles, promises to deliver high spatial and temporal resolution with a sensitivity exceeding that of magnetic resonance imaging (MRI). However, SPIO nanoparticles have a short blood retention time which limits the applicability of such compounds for MPI. We propose the use of red blood cells (RBCs) as carriers of SPIO nanoparticles to realize a blood pool tracer with longer blood retention time. Previously, we described a method of SPIO nanoparticle encapsulation into RBCs. The loading procedure consists of a hypotonic dialysis of cells in the presence of magnetic nanoparticles and successive resealing and reannealing of cells using isotonic solutions. Here, we report for the first time Magnetic Particle Spectroscopy (MPS) and MPI results obtained after intravenous administration of murine Resovist-loaded RBCs in an in vivo MPI experiment.

Intravascular contrast agents in diagnostic applications: Use of red blood cells to improve the lifespan and efficacy of blood pool contrast agents

In medicine, discrimination between pathologies and normal areas is of great importance, and in most cases, such discrimination is made possible by novel imaging technologies. Numerous modalities have been developed to visualize tissue vascularization in cardiovascular diseases or during angiogenic and vasculogenic processes. Here, we report the recent advances in vasculature imaging, providing an overview of the current non-invasive approaches in biomedical diagnostics and potential future strategies for prognostic assessment of vessel diseases, such as aneurysms and coronary artery occlusion leading to myocardial infarction. There are several contrast agents (CAs) available to improve the visibility of specific tissues at the early stage of diseases, allowing for rapid treatment. However, CAs are also hampered by numerous limitations, including rapid diffusion from blood vessels into the interstitial space, toxicity, and low sensitivity. Extravasation from blood vessels leads to a rapid loss of the image. If the contrast medium can fully be confined to the vascular space, high-resolution structural and functional vascular imaging could be obtained. Many scientists have contributed new materials and/or new carrier systems. For example, the use of red blood cells (RBCs) as CA-delivery systems appears to provide a scalable alternative to current procedures that allows adequate vascular imaging. Recognition and removal of CAs from the circulation can be prevented and/or delayed by using RBCs as biomimetic CA-carriers, and this technology should be clinically validated.

Magnetic red blood cells as new contrast agents for MRI applications

Superparamagnetic iron oxide (SPIO) nanoparticles have been produced and used successfully as potent contrast agents for Magnetic Resonance Imaging (MRI). However, a significant challenge associated with the biological application of SPIO-tracer agents is their behavior in vivo since their efficacy is often compromised due to a rapid recognition and clearance by the reticuloendothelial system (RES) which limits the applicability of such compounds in MRI. The advances in nanotechnology and molecular cell biology had lead to improve stability and biocompatibility of these nanoparticles, but despite a number of efforts, the SPIO half-life in blood circulation is very short. In this contest, the potential of red blood cells (RBCs) loaded with SPIO nanoparticles as a tracer material for MRI has been investigated in order to realize a blood pool tracer with longer blood retention time. Previously, we have proposed the encapsulation into RBCs of superparamagnetic iron oxide nanoparticles carboxydextran coated, such as Resovist contrast agent. This approach led to a nanoparticle reduction in uptake by the RES, increasing the blood circulation half-life of nanoparticles. Recently, the loading procedure was applied to a new contrast agent, the P904 ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles coated by hydrophilic derivatives of glucose, recently developed by Guerbet Laboratories. The results evidenced that this nanomaterial can be efficiently loaded into human and murine RBCs at concentrations ranging from 1.5 to 12 mM Fe. In vivo experiments performed in mice have showed an increased survival in the mouse vascular system of P904 encapsulated into RBCs respect to free P904 sample intravenously injected at the equivalent amounts.