Conjugation of gold nanoparticles with multidentate surfactants for enhanced stability and biological properties.

This work originated from the need to functionalize surfactant-coated inorganic nanoparticles for biomedical applications, a process that is limited by excess unbound surfactant. These limitations are connected to the bioconjugation of targeting molecules that are often in equilibrium between the free aliquot in solution and that which binds the surface of the nanoparticles. The excess in solution can play a role in the biocompatability in vitro and in vivo of the final nanoparticles stock. For this purpose, we tested the ability of common surfactants - monothiolated polyethylene glycol and amphiphilic polymers - to colloidally stabilize nanoparticles as excess surfactant is removed and compared them to newly appearing multidentate surfactants endowed with high avidity for inorganic nanoparticles. Our results showed that monothiolated polyethylene glycol or amphiphilic polymers have an insufficient affinity to the nanoparticles and as the excess surfactant is removed the colloidal stability is lost, while multidentate high-avidity surfactants excel in the same regard, possibly allowing improvement in an array of nanoparticle applications, especially in those stated.

Specific immunosuppressive role of nanodrugs targeting calcineurin in innate myeloid cells.

Calcineurin (CN) inhibitors currently used to avoid transplant rejection block the activation of adaptive immune responses but also prevent the development of tolerance toward the graft, by directly inhibiting T cells. CN, through the transcription factors of the NFAT family, plays an important role also in the differentiation dendritic cells (DCs), the main cells responsible for the activation of T lymphocytes. Therefore, we hypothesized that the inhibition of CN only in DCs and not in T cells could be sufficient to prevent T cell responses, while allowing for the development of tolerance. Here, we show that inhibition of CN/NFAT pathway in innate myeloid cells, using a new nanoconjugate capable of selectively targeting phagocytes in vivo, protects against graft rejection and induces a longer graft acceptance compared to common CN inhibitors. We propose a new generation of nanoparticles-based selective immune suppressive agents for a better control of transplant acceptance.

Development of an Effective Tumor-Targeted Contrast Agent for Magnetic Resonance Imaging Based on Mn/H-Ferritin Nanocomplexes.

Magnetic resonance imaging (MRI) is one of the most sophisticated diagnostic tools that is routinely used in clinical practice. Contrast agents (CAs) are commonly exploited to afford much clearer images of detectable organs and to reduce the risk of misdiagnosis caused by limited MRI sensitivity. Currently, only a few gadolinium-based CAs are approved for clinical use. Concerns about their toxicity remain, and their administration is approved only under strict controls. Here, we report the synthesis and validation of a manganese-based CA, namely, Mn@HFn-RT. Manganese is an endogenous paramagnetic metal able to produce a positive contrast like gadolinium, but it is thought to result in less toxicity for the human body. Mn ions were efficiently loaded inside the shell of a recombinant H-ferritin (HFn), which is selectively recognized by the majority of human cancer cells through their transferrin receptor 1. Mn@HFn-RT was characterized, showing excellent colloidal stability, superior relaxivity, and a good safety profile. In vitro experiments confirmed the ability of Mn@HFn-RT to efficiently and selectively target breast cancer cells. In vivo, Mn@HFn-RT allowed the direct detection of tumors by positive contrast enhancement in a breast cancer murine model, using very low metal dosages and exhibiting rapid clearance after diagnosis. Hence, Mn@HFn-RT is proposed as a promising CA candidate to be developed for MRI.

Inositol 1,4,5-trisphosphate 3-kinase B promotes Ca2+ mobilization and the inflammatory activity of dendritic cells.

Innate immune responses to Gram-negative bacteria depend on the recognition of lipopolysaccharide (LPS) by a receptor complex that includes CD14 and TLR4. In dendritic cells (DCs), CD14 enhances the activation not only of TLR4 but also that of the NFAT family of transcription factors, which suppresses cell survival and promotes the production of inflammatory mediators. NFAT activation requires Ca2+ mobilization. In DCs, Ca2+ mobilization in response to LPS depends on phospholipase C γ2 (PLCγ2), which produces inositol 1,4,5-trisphosphate (IP3). Here, we showed that the IP3 receptor 3 (IP3R3) and ITPKB, a kinase that converts IP3 to inositol 1,3,4,5-tetrakisphosphate (IP4), were both necessary for Ca2+ mobilization and NFAT activation in mouse and human DCs. A pool of IP3R3 was located on the plasma membrane of DCs, where it colocalized with CD14 and ITPKB. Upon LPS binding to CD14, ITPKB was required for Ca2+ mobilization through plasma membrane-localized IP3R3 and for NFAT nuclear translocation. Pharmacological inhibition of ITPKB in mice reduced both LPS-induced tissue swelling and the severity of inflammatory arthritis to a similar extent as that induced by the inhibition of NFAT using nanoparticles that delivered an NFAT-inhibiting peptide specifically to phagocytic cells. Our results suggest that ITPKB may represent a promising target for anti-inflammatory therapies that aim to inhibit specific DC functions.

Modeling the interaction of amphiphilic polymer nanoparticles with biomembranes to Guide rational design of drug delivery systems.

Nanoparticle assisted drug delivery to the cytoplasm is limited by sequestration of nanoparticles in endosomes. Endosomal escape through polymer-induced membrane destabilization is one of a few well characterized mechanisms to overcome it. Aiming to utilize this method in vivo, it is necessary to understand how modulating the structural and chemical features of the polymer and the presence of proteins in biological fluids can affect this activity. Here, as a model for the endosomal membrane, we use the membrane of red blood cells to evaluate the membrane destabilization ability of a model amphiphilic polymer in the presence of blood plasma using a hemolysis assay. This allows determination of red blood cells membrane permeabilization through the quantification of hemoglobin leakage. Our results showed a strong inhibitory effect of plasma, and that hemolytic activity can be improved by chemical modification of the polymeric micelle, reducing its interaction with plasma proteins. Finally, a second mechanism of pH-induced direct diffusion is proposed and tested using an oil/water partitioning model. These results offer a body of knowledge to improve delivery of drugs across biological membranes using carefully designed polymeric nanocarriers.

Are nanotechnological approaches the future of treating inflammatory diseases?

The current treatments for chronic inflammatory diseases cause severe side effects due to nonspecific drug accumulation. Nanotechnology opens the way to new therapeutic strategies that exploit the ability of immune cells, and especially of phagocytes, to internalize nanoparticles. The cellular uptake of nanoparticles requires specific interactions and is affected by the chemical and physical properties of the carriers. Therefore, optimizing these properties is crucial for designing nanodrugs for immunotherapy. In perspective, we discuss the nanoparticle-based approaches that have been proposed to induce tolerance in autoimmune disorders and lessen the symptoms of inflammatory diseases.

Tumour homing and therapeutic effect of colloidal nanoparticles depend on the number of attached antibodies.

Active targeting of nanoparticles to tumours can be achieved by conjugation with specific antibodies. Specific active targeting of the HER2 receptor is demonstrated in vitro and in vivo with a subcutaneous MCF-7 breast cancer mouse model with trastuzumab-functionalized gold nanoparticles. The number of attached antibodies per nanoparticle was precisely controlled in a way that each nanoparticle was conjugated with either exactly one or exactly two antibodies. As expected, in vitro we found a moderate increase in targeting efficiency of nanoparticles with two instead of just one antibody attached per nanoparticle. However, the in vivo data demonstrate that best effect is obtained for nanoparticles with only exactly one antibody. There is indication that this is based on a size-related effect. These results highlight the importance of precisely controlling the ligand density on the nanoparticle surface for optimizing active targeting, and that less antibodies can exhibit more effect.