Clara De Palma

HER2 Expression in Breast Cancer Cells Is Downregulated Upon Active Targeting by Antibody-Engineered Multifunctional Nanoparticles in Mice

Subcellular destiny of targeted nanoparticles in cancer cells within living organisms is still an open matter of debate. By in vivo and ex vivo experiments on tumor-bearing mice treated with antibody-engineered magnetofluorescent nanocrystals, in which we combined fluorescence imaging, magnetic relaxation, and trasmission electron microscopy approaches, we provide evidence that nanoparticles are effectively delivered to the tumor by active targeting. These nanocrystals were demonstrated to enable contrast enhancement of the tumor in magnetic resonance imaging. In addition, we were able to discriminate between the fate of the organic corona and the metallic core upon cell internalization. Accurate immunohistochemical analysis confirmed that hybrid nanoparticle endocytosis is mediated by the complex formation with HER2 receptor, leading to a substantial downregulation of HER2 protein expression on the cell surface. These results provide a direct insight into the pathway of internalization and degradation of targeted hybrid nanoparticles in cancer cells in vivo and suggest a potential application of this immunotheranostic nanoagent in neoadjuvant therapy of cancer.

Assessing the in vivo targeting efficiency of multifunctional nanoconstructs bearing antibody-derived ligands

A great challenge in nanodiagnostics is the identification of new strategies aimed to optimize the detection of primary breast cancer and metastases by the employment of target-specific nanodevices. At present, controversial proof has been provided on the actual importance of surface functionalization of nanoparticles to improve their in vivo localization at the tumor. In the present paper, we have designed and developed a set of multifunctional nanoprobes, modified with three different variants of a model antibody, that is, the humanized monocolonal antibody trastuzumab (TZ), able to selectively target the HER2 receptor in breast cancer cells. Assuming that nanoparticle accumulation in target cells is strictly related to their physicochemical properties, we performed a comparative study of internalization, trafficking, and metabolism in MCF7 cells of multifunctional nanoparticles (MNP) functionalized with TZ or with alternative lower molecular weight variants of the monoclonal antibody, such as the half-chain (HC) and scFv fragments (scFv). Hence, to estimate to what extent the structure of the surface bioligand affects the targeting efficiency of the nanoconjugate, three cognate nanoconstructs were designed, in which only the antibody form was differentiated while the nanoparticle core was maintained unvaried, consisting of an iron oxide spherical nanocrystal coated with an amphiphilic polymer shell. In vitro, in vivo, and ex vivo analyses of the targeting efficiency and of the intracellular fate of MNP-TZ, MNP-HC, and MNP-scFv suggested that the highly stable MNP-HC is the best candidate for application in breast cancer detection. Our results provided evidence that, in this case, active targeting plays an important role in determining the biological activity of the nanoconstruct.

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.

Endothelial Nitric Oxide Synthase Activation by Tumor Necrosis Factor α Through Neutral Sphingomyelinase 2, Sphingosine Kinase 1, and Sphingosine 1 Phosphate Receptors: A Novel Pathway Relevant to the Pathophysiology of Endothelium

Objective—Tumor necrosis factor α (TNF-α), a key proinflammatory cytokine acting on the endothelium, activates endothelial nitric oxide synthase (eNOS). We have examined the signaling pathway leading to this activation and its biological role in endothelium, which are still unknown. Methods and Results—In human endothelial cells, we found that eNOS activation by TNF-α is time dependent and requires activation of Akt, a known eNOS activator. eNOS activation was preceded by sequential activation of neutral-sphingomyelinase-2 (N-SMase2) and sphingosine-kinase-1 (SK1) and generation of sphingosine-1-phosphate (Sph1P). Inhibition of N-SMase2 inhibited Sph1P formation, whereas inhibition of SK1 did not affect N-SMase2 activation by TNF-α. Blockade of N-SMase2, SK1, or the Sph1P receptors S1P1 and S1P3, either by silencing or pharmacological inhibitors, prevented eNOS activation. Thus, eNOS is activated by TNF-α via S1P receptors, activated by Sph1P generated through N-SMase2 and SK1 activation. We found that nitric oxide generated through this pathway has a biological role, because it inhibits the expression of E-selectin and the adhesion of dendritic cells to the endothelium stimulated by TNF-α. Conclusions—This study establishes a previously undescribed link among TNF-α, Sph1P, and eNOS in a same signaling pathway of biological relevance in the process of endothelial cell activation by TNF-α.

Activation of Acid Sphingomyelinase and Its Inhibition by the Nitric Oxide/Cyclic Guanosine 3′,5′-Monophosphate Pathway: Key Events in Escherichia coli-Elicited Apoptosis of Dendritic Cells

Depletion of dendritic cells (DCs) via apoptosis contributes to sepsis-induced immune suppression. The mechanisms leading to DC apoptosis during sepsis are not known. In this study we report that immature DCs undergo apoptosis when treated with high numbers of Escherichia coli. This effect was mimicked by high concentrations of LPS. Apoptosis was accompanied by generation of ceramide through activation of acid sphingomyelinase (A-SMase), was prevented by inhibitors of this enzyme, and was restored by exogenous ceramide. Compared with immature DCs, mature DCs expressed significantly reduced levels of A-SMase, did not generate ceramide in response to E. coli or LPS, and were insensitive to E. coli- and LPS-triggered apoptosis. However, sensitivity to apoptosis was restored by addition of exogenous A-SMase or ceramide. Furthermore, inhibition of A-SMase activation and ceramide generation was found to be the mechanism through which the immune-modulating messenger NO protects immature DCs from the apoptogenic effects of E. coli and LPS. NO acted through formation of cGMP and stimulation of the cGMP-dependent protein kinase. The relevance of A-SMase and its inhibition by NO/cGMP were confirmed in a mouse model of LPS-induced sepsis. DC apoptosis was significantly higher in inducible NO synthase-deficient mice than in wild-type animals and was significantly reduced by treatment ex vivo with NO, cGMP, or the A-SMase inhibitor imipramine. Thus, A-SMase plays a central role in E. coli/LPS-induced DC apoptosis and its inhibition by NO, and it might be a target of new therapeutic approaches to sepsis.

Single-domain protein A-engineered magnetic nanoparticles: toward a universal strategy to site-specific labeling of antibodies for targeted detection of tumor cells

Highly monodisperse magnetite nanocrystals (MNC) were synthesized in organic media and transferred to the water phase by ultrasound-assisted ligand exchange with an iminodiacetic phosphonate. The resulting biocompatible magnetic nanoparticles were characterized by transmission electron microscopy, dynamic light scattering, and magnetorelaxometry, indicating that this method allowed us to obtain stable particle dispersions with narrow size distribution and unusually high magnetic resonance T(2) contrast power. These nanoparticles were conjugated to a newly designed recombinant monodomain protein A variant, which exhibited a convincingly strong affinity for human and rabbit IgG molecules. Owing to the nature of antibody-protein A binding, tight antibody immobilization occurred through the Fc fragment thus taking full advantage of the targeting potential of bound IgGs. If necessary, monoclonal antibodies could be removed under controlled conditions regenerating the original IgG-conjugatable MNC. As a proof of concept of the utility of our paramagnetic labeling system of human IgGs for biomedical applications, anti-HER-2 monoclonal antibody trastuzumab was immobilized on hybrid MNC (TMNC). TMNC were assessed by immunoprecipitation assay and confocal microscopy effected on HER-2-overexpressing MCF-7 breast cancer cells, demonstrating excellent recognition capability and selectivity for the target membrane receptor.

Nitric oxide in myogenesis and therapeutic muscle repair.

Nitric oxide is a short-lived intracellular and intercellular messenger. The first realisation that nitric oxide is important in physiology occurred in 1987 when its identity with the endothelium-derived relaxing factor was discovered. Subsequent studies have shown that nitric oxide possesses a number of physiological functions that are essential not only to vascular homeostasis but also to neurotransmission, such as in the processes of learning and memory and endocrine gland regulation, as well as inflammation and immune responses. The discovery in 1995 that a splice variant of the neuronal nitric oxide synthase is localised at the sarcolemma via the dystrophin–glycoprotein complex and of its displacement in Duchenne muscular dystrophy has stimulated a host of studies exploring the role of nitric oxide in skeletal muscle physiology. Recently, nitric oxide has emerged as a relevant messenger also of myogenesis that it regulates at several key steps, especially when the process is stimulated for muscle repair following acute and chronic muscle injuries. Here, we will review briefly the mechanisms and functions of nitric oxide in skeletal muscle and discuss its role in myogenesis, with specific attention to the promising nitric oxide-based approaches now being explored at the pre-clinical and clinical level for the therapy of muscular dystrophy.

Nitric Oxide Confers Therapeutic Activity to Dendritic Cells in a Mouse Model of Melanoma

Susceptibility of dendritic cells (DCs) to tumor-induced apoptosis reduces their efficacy in cancer therapy. Here we show that delivery within exponentially growing B16 melanomas of DCs treated ex vivo with nitric oxide (NO), released by the NO donor (z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO), significantly reduced tumor growth, with cure of 37% of animals. DETA-NO-treated DCs became resistant to tumor-induced apoptosis because DETA-NO prevented tumor-induced changes in the expression of Bcl-2, Bax, and Bcl-xL; activation of caspase-9; and a reduction in the mitochondrial membrane potential. DETA-NO also increased DC cytotoxic activity against tumor cells and DC ability to trigger T-lymphocyte proliferation. All of the effects of DETA-NO were mediated through cGMP generation. NO and NO-generating drugs may therefore be used to increase the anticancer efficacy of DCs.

The low-affinity receptor for neurotrophins p75NTR plays a key role for satellite cell function in muscle repair acting via RhoA.

Regeneration of muscle fibers, lost during pathological muscle degeneration or after injuries, is mediated by the production of new myofibres. This process, sustained by the resident stem cells of the muscle, the satellite cells, is finely regulated by local cues, in particular by cytokines and growth factors. Evidence in the literature suggests that nerve growth factor (NGF) is involved in muscle fiber regeneration; however, its role and mechanism of action were unclear. We have investigated this issue in in vivo mouse models of muscle regeneration and in primary myogenic cells. Our results demonstrate that NGF acts through its low-affinity receptor p75(NTR) in a developmentally regulated signaling pathway necessary to myogenic differentiation and muscle repair in vivo. We also demonstrate that this action of NGF is mediated by the down-regulation of RhoA-GTP signaling in myogenic cells.

Synergism of nitric oxide and maturation signals on human dendritic cells occurs through a cyclic GMP-dependent pathway

Nitric oxide (NO), generated by phagocytes at inflammation sites, contributes to regulate immune responses through autocrine and paracrine actions on bystander cells. Among the latter are dendritic cells (DCs). Little is known about regulation of DC function by NO, especially in the human system. We exposed human monocyte‐derived DCs to the NO donor (z)‐1‐[2‐(2‐aminoethyl)‐N‐(2‐ammonioethyl)amino] diazen‐1‐ium‐1,2 diolate (DETA‐NO) during their maturation process induced by treatment with tumor necrosis factor α or lipopolysaccharide or by CD40 activation. We report here that after exposure to DETA‐NO, DCs exhibit a significantly increased ability to activate T lymphocytes stimulated by mycobacterial antigens, Staphylococcus aureus Cowen strain B, allo‐antigens, or cross‐linking of the CD3–T cell receptor complex. This effect persists after removal of DETA‐NO, depends on the generation of cyclic guanosine 5′‐monophosphate, and is a result of enhanced release by DCs of soluble factors, in particular interleukin (IL)‐12. This modulation of DC function is a result of a synergism between NO and the various maturation stimuli, as neither enhanced T cell activation nor IL‐12 release was observed after DC exposure to DETA‐NO only. These results provide the first evidence that NO acts as a cosignaling molecule regulating human DC response to maturation stimuli.