Recent advances in synthesis, characterization of hydroxyapatite/polyurethane composites and study of their biocompatible properties.

The development of engineered biomaterials that mimic bone tissues is a promising research area that benefits from a growing interest. Polymers and polymer-ceramic composites are the principle materials investigated for the development of synthetic bone scaffolds thanks to their proven biocompatibility and biostability. Several polymers have been combined with calcium phosphates (mainly hydroxyapatite) to prepare nanocomposites with improved biocompatible and mechanical properties. Here, we report the hydrothermal synthesis in high pressure conditions of nanostructured composites based on hydroxyapatite and polyurethane functionalized with carboxyl and thiol groups. Cell-material interactions were investigated for potential applications of these new types of composites as coating for orthopedic implants. Physical-chemical and morphological characteristics of hydroxyapatite/polyurethane composites were evaluated for different compositions, showing their dependence on synthesis parameters (pressure, temperature). In vitro experiments, performed to verify if these composites are biocompatible cell culture substrates, showed that they are not toxic and do not affect cell viability.

Nanoparticle encapsulation in red blood cells enables blood-pool magnetic particle imaging hours after injection.

Magnetic particle imaging (MPI) is a new medical imaging approach that is based on the nonlinear magnetization response of super-paramagnetic iron oxide nanoparticles (SPIOs) injected into the blood stream. To date, real-time MPI of the bolus passage of an approved MRI SPIO contrast agent injected into the tail vein of living mice has been demonstrated. However, nanoparticles are rapidly removed from the blood stream by the mononuclear phagocyte system. Therefore, imaging applications for long-term monitoring require the repeated administration of bolus injections, which complicates quantitative comparisons due to the temporal variations in concentration. Encapsulation of SPIOs into red blood cells (RBCs) has been suggested to increase the blood circulation time of nanoparticles. This work presents first evidence that SPIO-loaded RBCs can be imaged in the blood pool of mice several hours after injection using MPI. This finding is supported by magnetic particle spectroscopy performed to quantify the iron concentration in blood samples extracted from the mice 3 and 24 h after injection of SPIO-loaded RBCs. Based on these results, new MPI applications can be envisioned, such as permanent 3D real-time visualization of the vessel tree during interventional procedures, bleeding monitoring after stroke, or long-term monitoring and treatment control of cardiovascular diseases.

Glucocorticoid resistance in Crohn's disease and ulcerative colitis: an association study investigating GR and FKBP5 gene polymorphisms.

The aim of this study is to investigate the role of single-nucleotide polymorphisms (SNPs) of the glucocorticoid receptor (GR) and of the related co-chaperone FKBP5 genes in the development of glucocorticoid (GC) resistance in Crohn's disease (CD) and ulcerative colitis (UC) patients. We have developed a high-resolution DNA melting method that allows simultaneous identification of GR (BclI, N363S and ER22/23EK) and FKBP5 (rs3800373, rs1360780 and rs4713916) polymorphisms. Genotype frequencies were determined in 100 consecutive CD and 100 UC patients under GCs therapy (50 responders and 50 resisters). The variation of FKBP5 polymorphism rs4713916 (G/A), in the putative promoter region of FKBP5, is significantly associated with resistance to GC treatment in CD (responder=17% versus resister=35%; P=0.0043). No significant differences were found in UC patients. If these preliminary findings will be confirmed, the combination of GR and FKBP5 mutational analyses could help to identify subgroups of CD patients with higher chances to benefit from GC treatment.

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.

Improved cellular uptake of functionalized single-walled carbon nanotubes.

Single-walled carbon nanotubes (SWNTs) due to their unique structural and physicochemical properties, have been proposed as delivery systems for a variety of diagnostic and therapeutic agents. However, SWNTs have proven difficult to solubilize in aqueous solution, limiting their use in biological applications. In an attempt to improve SWNTs' solubility, biocompatibility, and to increase cell penetration we have thoroughly investigated the construction of carbon scaffolds coated with aliphatic carbon chains and phospholipids to obtain micelle-like structures. At first, oxidized SWNTs (2370 ± 30 nmol mg(-1) of SWNTs) were covalently coupled with an alcoholic chain (stearyl alcohol, C(18)H(37)OH; 816 nmol mg(-1) of SWNTs). Subsequently, SWNTs-COOC(18)H(37) derivatives were coated with phosphatidylethanolamine (PE) or -serine (PS) phospholipids obtaining micelle-like structures. We found that cellular uptake of these constructs by phagocytic cells occurs via an endocytotic mechanism for constructs larger than 400 nm while occurs via diffusion through the cell membrane for constructs up to 400 nm. The material that enters the cell by phagocytosis is actively internalized by macrophages and localizes inside endocytotic vesicles. In contrast the material that enters the cells by diffusion is found in the cell cytosol. In conclusion, we have realized new biomimetic constructs based on alkylated SWNTs coated with phospholipids that are efficiently internalized by different cell types only if their size is lower than 400 nm. These constructs are not toxic to the cells and could now be explored as delivery systems for non-permeant cargoes.

Effect of macrophage depletion on viral DNA rebound following antiretroviral therapy in a murine model of AIDS (MAIDS).

In the attempt to eradicate HIV-1 infection, a strategy to eliminate macrophages, one of the most important cellular reservoirs in sustaining virus replication during HAART, could be of great benefit in the suppression of viral rebound. Aware of the ability of clodronate to cause macrophage depletion, the effect of the administration of clodronate encapsulated in erythrocytes on disease progression and on viral rebound was evaluated in a murine model of AIDS (MAIDS). One group of LP-BM5 retroviral complex-infected C57BL/6 mice received oral administrations of azidothymidine and dideoxyinosine daily for 12 weeks; two other groups received in addition, either clodronate-loaded erythrocytes or free clodronate at 7-10 day intervals. At the end of the treatment, the three groups maintained parameters characterizing disease progression similar to those of uninfected mice and showed a significantly lower level of BM5d DNA than infected mice in all organs and cells tested. To assess the viral rebound, some animals were left for an additional 4 month period without any treatment. After this time, the BM5d DNA content in blood leukocytes increased in all groups, but the group having received clodronate-loaded erythrocytes, in addition to transcriptase inhibitors, showed a significant delay in viral rebound.