Ferucarbotran-loaded red blood cells as long circulating MRI contrast agents: first in vivo results in mice.

AIM: The encapsulation of superparamagnetic iron oxide contrast agents in red blood cells (RBCs) could overcome their rapid removal by reticulo-endothelial system improving their stability in blood circulation. MATERIALS & METHODS: Murine ferucarbotran-loaded RBCs were tested in vivo as new contrasting agents in MRI application. RESULTS: A superior visualization of organs and cerebral vessels was evidenced in ferucarbotran-loaded RBCs-treated mice compared with the controls. The signal enhancement lasted for days, while the contrast from bulk ferucarbotran disappeared after few minutes. CONCLUSION: Ferucarbotran-loaded RBCs showed to improve diagnostic imaging and their use may extend the time frame for MRI and magnetic resonance angiography since to date the time frame for data acquisition has been limited to the first pass.

Interactions of Nitroxide-Conjugated and Non-Conjugated Glycodendrimers with Normal and Cancer Cells and Biocompatibility Studies.

Poly(propyleneimine) glycodendrimers fully modified with maltose units were administered to different cancer cell lines and their effect on cell viability was evaluated by using MTS assay and flow cytometry. The mechanism of dendrimer-cell interactions was investigated by the electron paramagnetic resonance (EPR) technique by using a new nitroxide-conjugated glycodendrimer. The nitroxide groups did not modify both the biological properties (cell viability and apoptosis degree) of the dendrimers in the presence of the cells and the dendrimer-cell interactions. Since this class of dendrimers is already known to be biocompatible for human healthy cells, noncancer cells such as human peripheral blood mononuclear cells (PBMCs) and macrophages were also treated with the glycodendrimer, and EPR spectra of the nitroxide-conjugated glycodendrimer were compared for cancer and noncancer cells. It was found that this dendrimer selectively affects the cell viability of tumor cells, while, surprisingly, PBMC proliferation is induced. Moreover, H-bond-active glycodendrimer-cell interactions were different for the different cancer cell lines and noncancer cells. The nitroxide-conjugated glycodendrimer was able to interact with the cell membrane and eventually cross it, getting in contact with cytosol antioxidants. This study helps to clarify the potential anticancer effect of this class of dendrimers opening to future applications of these macromolecules as new antitumor agents.

Characterization of ferucarbotran-loaded RBCs as long circulating magnetic contrast agents.

AIM: The biomedical application of contrast agents based on superparamagnetic iron oxide nanoparticles is still limited because of their short intravascular half-life. The potential of red blood cells (RBCs) loaded with new ferucarbotran nanoparticles as magnetic contrast agents with longer blood retention time has been investigated. MATERIALS & METHODS: Ferucarbotran was loaded into RBCs by a procedure of hypotonic dialysis and isotonic resealing. Ferucarbotran amounts encapsulated in RBCs were determined by NMR. The survival of ferucarbotran-loaded RBCs and bulk ferucarbotran was evaluated in the mouse bloodstream. RESULTS: Blood retention time of these RBC constructs is longer (∼14 days) than the bulk ferucarbotran (∼1 h) with a slower Fe clearance from liver and spleen. CONCLUSION: Ferucarbotran-loaded RBCs could be used as potential contrasting agents for diagnostic applications in MRI/magnetic particle imaging.

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.

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.

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 ~ 30 nm 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 (5.3-16.7 mM Fe) higher than murine SPIO-loaded RBCs (1.4-3.55 mM 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.

Red blood cells as carriers in magnetic particle imaging.

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.

Dexamethasone restrains ongoing expression of interleukin-23p19 in peripheral blood-derived human macrophages.

BACKGROUND: Since its recent discovery, interleukin-23 has been shown to be involved in the pathogenesis of autoimmune diseases favoring the development of a T cell subset referred to as T helper 17. Glucocorticoids are widely employed in inflammatory and autoimmune diseases as they inhibit pro-inflammatory signaling and prevent production of inflammation mediators. Very limited information is available about the efficacy of synthetic glucocorticoids in containing the expression of interleukin-23 under cell activation. RESULTS: We demonstrate here that the glucocorticoid analogue dexamethasone administered to human monocyte-derived macrophages is indeed able to restrain the expression of interleukin-23 once it has been triggered by a pro-inflammatory stimulus. This effect of dexamethasone is here demonstrated being secondary to suppression of p38 MAPK activity, and involving a protein phosphatase--likely MAPK phosphatase-1 (MKP-1). CONCLUSIONS: Results reported in this paper show that a 10 nanomolar dose of dexamethasone not only prevents inflammatory activation but is also efficacious in confining active inflammation. This effect is here demonstrated not to occur through "canonical" inhibition of the NF-κB transcription factor but through a distinct cascade of down-modulation, that underlines the importance of the transactivating activity of glucocorticoid receptor in the context of its anti-inflammatory action.

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.

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.

New biomimetic constructs for improved in vivo circulation of superparamagnetic nanoparticles.

Superparamagnetic iron oxide nanoparticles (SPIOs) have been produced and used as a potent and versatile contrast media for magnetic resonance imaging (MRI). Despite a number of efforts to improve their surface chemistry and biocompatibility, the SPIOs half life in blood circulation is very short and they are rapidly taken up by the reticuloendothelial system (RES). In this paper we describe a new method that permits to avoid the rapid clearance of SPIOs. Nanoparticles are made biocompatible by encapsulation into autologous red blood cells. These biomimetic constructs preserve the main properties of the cells that escape RES clearance as well as the properties of the nanoparticles that perform even better than in blood suspension with reduced T2*. These SPIO-loaded RBCs are promising intravascular imaging contrast agents and could also be addressed to selected body compartments by an external magnetic field.