Anthony Dellinger

Application of fullerenes in nanomedicine: an update

Fullerenes are carbon spheres presently being pursued globally for a wide range of applications in nanomedicine. These molecules have unique electronic properties that make them attractive candidates for diagnostic, therapeutic and theranostic applications. Herein, the latest research is discussed on developing fullerene-based therapeutics as antioxidants for inflammatory diseases, their potential as antiviral/bacterial agents, utility as a drug delivery device and the promise of endohedral fullerenes as new MRI contrast agents. The recent discovery that certain fullerene derivatives can stabilize immune effector cells to prevent or inhibit the release of proinflammatory mediators makes them potential candidates for several diseases such as asthma, arthritis and multiple sclerosis. Gadolinium-containing endohedral fullerenes are being pursued as diagnostic MRI contrast agents for several diseases. Finally, a new class of fullerene-based theranostics has been developed, which combine therapeutic and diagnostic capabilities to specifically detect and kill cancer cells.

A potential new target for asthma therapy: a disintegrin and metalloprotease 10 (ADAM10) involvement in murine experimental asthma.

To cite this article: Mathews JA, Ford J, Norton S, Kang D, Dellinger A, Gibb DR, Ford AQ, Massay H, Kepley CL, Scherle P, Keegan AD, Conrad DH. A potential new target for asthma therapy: A Disintegrin and Metalloprotease 10 (ADAM10) involvement in murine experimental asthma. Allergy 2011; 66: 1193–1200.

Fullerene nanomaterials inhibit phorbol myristate acetate-induced inflammation

Abstract:  Inflammation is a natural biological response that occurs when vascular tissues are subjected to harmful stimuli. This process may be beneficial to the host during wound healing and infections but can be detrimental if left unchecked. Oxidative stress, the generation of reactive oxygen species, is thought to be one component of this response. Fullerenes can counteract reactive oxygen species due to their potent antioxidant capabilities. Thus, we hypothesized that these molecules may inhibit inflammation. To test this hypothesis we used an in vivo model of phorbol 12‐myristate 13‐acetate (PMA)‐induced inflammation and examined the effects fullerenes have on mitigating this response. We show that PMA‐induced inflammation and oedema is dramatically inhibited when fullerenes are given prior to challenge. Thus, fullerene derivatives may be a novel way to blunt certain inflammatory conditions and facilitate faster recovery of damaged tissue.

Uptake and distribution of fullerenes in human mast cells

Fullerenes are carbon cages of variable size that can be derivatized with various side chain moieties resulting in compounds that are being developed into nanomedicines. Although fullerene use in several preclinical in vitro and in vivo models of disease has demonstrated their potential as diagnostic and therapeutic agents, little is known about how they enter cells, what organelles they target, and the time course for their cellular deposition. Fullerenes (C(70)) that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and the duration they can be detected in situ. We show that C(70)-TR are nonspecifically endocytosed into MCs, where they are shuttled throughout the cytoplasm, lysosomes, mitochondria, and into endoplasmic reticulum at different times. No nuclear or secretory granule localization was observed. The C(70)-TR remained detectable within cells at 1 week. These studies show that MCs endocytose fullerenes, where they are shuttled to organelles involved with calcium and reactive oxygen species production, which may explain their efficacy as cellular inhibitors. From the clinical editor: Fullerenes are carbon cages of variable size that have already been shown to be potent inhibitors of mast cell (MC)-mediated allergic inflammation. These were conjugated with Texas red (TR) and used in conjunction with confocal microscopy to determine mechanisms of uptake, the organelle localization, and duration, demonstrating that MCs endocytose fullerenes, which are shuttled to organelles involved with calcium and reactive oxygen species production. This intracellular trafficking may explain the efficacy of fullerenes as cellular inhibitors.

IL-33/ST2 axis promotes mast cell survival via BCLXL

Significance Mast cells (MC) are long-lived cells that accumulate in inflamed tissues. IL-33 has been characterized extensively as a MC activator, but a contribution of IL-33 and its receptor IL-1 receptor-related protein ST2 to MC survival remains unappreciated. Here, we show that IL-33 attenuates apoptosis of human and murine MC, principally via the antiapoptotic molecule B-cell lymphoma-X large (BCLXL). In vivo, IL-33 and ST2 can confer a cell-intrinsic survival advantage to murine MC, particularly in the context of inflammation. These results identify the IL-33/ST2 axis as an important pathway supporting MC persistence in tissues, raising the possibility that therapeutic targeting of IL-33 could limit the contribution of MC to chronic inflammatory diseases. Mast cells (MC) are potent innate immune cells that accumulate in chronically inflamed tissues. MC express the IL-33 receptor IL-1 receptor-related protein ST2 at high level, and this IL-1 family cytokine both activates MC directly and primes them to respond to other proinflammatory signals. Whether IL-33 and ST2 play a role in MC survival remains to be defined. In skin-derived human MC, we found that IL-33 attenuated MC apoptosis without altering proliferation, an effect mediated principally through the antiapoptotic molecule B-cell lymphoma-X large (BCLXL). Murine MC demonstrated a similar mechanism, dependent entirely on ST2. In line with these observations, St2−/− mice exhibited reduced numbers of tissue MC in inflamed arthritic joints, in helminth-infected intestine, and in normal peritoneum. To confirm an MC-intrinsic role for ST2 in vivo, we performed peritoneal transfer of WT and St2−/− MC. In St2−/− hosts treated with IL-33 and in WT hosts subjected to thioglycollate peritonitis, WT MC displayed a clear survival advantage over coengrafted St2−/− MC. IL-33 blockade specifically attenuated this survival advantage, confirming IL-33 as the relevant ST2 ligand mediating MC survival in vivo. Together, these data reveal a cell-intrinsic role for the IL-33/ST2 axis in the regulation of apoptosis in MC, identifying thereby a previously unappreciated pathway supporting expansion of the MC population with inflammation.

Liposomal formulation of amphiphilic fullerene antioxidants.

Novel amphiphilic fullerene[70] derivatives that are rationally designed to intercalate in lipid bilayers are reported, as well as its vesicular formulation with surprisingly high loading capacity up to 65% by weight. The amphiphilic C(70) bisadduct forms uniform and dimensionally stable liposomes with auxiliary natural phospholipids as demonstrated by buoyant density test, particle size distribution, and (31)P NMR. The antioxidant property of fullerenes is retained in the bipolarly functionalized C(70) derivative, amphiphilic liposomal malonylfullerene[70] (ALM), as well as in its liposomal formulations, as shown by both electron paramagnetic resonance (EPR) studies and in vitro reactive oxygen species (ROS) inhibition experiments. The liposomally formulated ALM efficiently quenched hydroxyl radicals and superoxide radicals. In addition, the fullerene liposome inhibited radical-induced lipid peroxidation and maintained the integrity of the lipid bilayer structure. This new class of liposomally formulated, amphipathic fullerene compounds represents a novel drug delivery system for fullerenes and provides a promising pathway to treat oxidative stress-related diseases.

Functionalization of gadolinium metallofullerenes for detecting atherosclerotic plaque lesions by cardiovascular magnetic resonance

BackgroundThe hallmark of atherosclerosis is the accumulation of plaque in vessel walls. This process is initiated when monocytic cells differentiate into macrophage foam cells under conditions with high levels of atherogenic lipoproteins. Vulnerable plaque can dislodge, enter the blood stream, and result in acute myocardial infarction and stroke. Imaging techniques such as cardiovascular magnetic resonance (CMR) provides one strategy to identify patients with plaque accumulation.MethodsWe synthesized an atherosclerotic-targeting contrast agent (ATCA) in which gadolinium (Gd)-containing endohedrals were functionalized and formulated into liposomes with CD36 ligands intercalated into the lipid bilayer. In vitro assays were used to assess the specificity of the ATCA for foam cells. The ability of ATCA to detect atherosclerotic plaque lesions in vivo was assessed using CMR.ResultsThe ATCA was able to detect scavenger receptor (CD36)-expressing foam cells in vitro and were specifically internalized via the CD36 receptor as determined by focused ion beam/scanning electron microscopy (FIB-SEM) and Western blotting analysis of CD36 receptor-specific signaling pathways. The ATCA exhibited time-dependent accumulation in atherosclerotic plaque lesions of ApoE −/− mice as determined using CMR. No ATCA accumulation was observed in vessels of wild type (C57/b6) controls. Non-targeted control compounds, without the plaque-targeting moieties, were not taken up by foam cells in vitro and did not bind plaque in vivo. Importantly, the ATCA injection was well tolerated, did not demonstrate toxicity in vitro or in vivo, and no accumulation was observed in the major organs.ConclusionsThe ATCA is specifically internalized by CD36 receptors on atherosclerotic plaque providing enhanced visualization of lesions under physiological conditions. These ATCA may provide new tools for physicians to non-invasively detect atherosclerotic disease.

Inhibition of Inflammatory Arthritis Using Fullerene Nanomaterials

Inflammatory arthritis (e.g. rheumatoid arthritis; RA) is a complex disease driven by the interplay of multiple cellular lineages. Fullerene derivatives have previously been shown to have anti-inflammatory capabilities mediated, in part, by their ability to prevent inflammatory mediator release by mast cells (MC). Recognizing that MC can serve as a cellular link between autoantibodies, soluble mediators, and other effector populations in inflammatory arthritis, it was hypothesized that fullerene derivatives might be used to target this inflammatory disease. A panel of fullerene derivatives was tested for their ability to affect the function of human skin-derived MC as well as other lineages implicated in arthritis, synovial fibroblasts and osteoclasts. It is shown that certain fullerene derivatives blocked FcγR- and TNF-α-induced mediator release from MC; TNF-α-induced mediator release from RA synovial fibroblasts; and maturation of human osteoclasts. MC inhibition by fullerene derivatives was mediated through the reduction of mitochondrial membrane potential and FcγR-mediated increases in cellular reactive oxygen species and NF-κB activation. Based on these in vitro data, two fullerene derivatives (ALM and TGA) were selected for in vivo studies using K/BxN serum transfer arthritis in C57BL/6 mice and collagen-induced arthritis (CIA) in DBA/1 mice. Dye-conjugated fullerenes confirmed localization to affected joints in arthritic animals but not in healthy controls. In the K/BxN moldel, fullerenes attenuated arthritis, an effect accompanied by reduced histologic inflammation, cartilage/bone erosion, and serum levels of TNF-α. Fullerenes remained capable of attenuating K/BxN arthritis in mast cell-deficient mice Cre-Master mice, suggesting that lineages beyond the MC represent relevant targets in this system. These studies suggest that fullerene derivatives may hold promise both as an assessment tool and as anti-inflammatory therapy of arthritis.

A novel gadolinium-based trimetasphere metallofullerene for application as a magnetic resonance imaging contrast agent.

ObjectiveMacromolecular contrast agents for magnetic resonance imaging (MRI) are useful blood-pool agents because of their long systemic half-life and have found applications in monitoring tumor vasculature and angiogenesis. Macromolecular contrast agents have been able to overcome some of the disadvantages of the conventional small-molecule contrast agent Magnevist (gadolinium-diethylenetriaminepentaacetic acid), such as rapid extravasation and quick renal clearance, which limits the viable MRI time. There is an urgent need for new MRI contrast agents that increase the sensitivity of detection with a higher relaxivity, longer blood half-life, and reduced toxicity from free Gd3+ ions. Here, we report on the characterization of a novel water-soluble, derivatized, gadolinium-enclosed metallofullerene nanoparticle (Hydrochalarone-1) in development as an MRI contrast agent. Materials and MethodsThe physicochemical properties of Hydrochalarone-1 were characterized by dynamic light scattering (hydrodynamic diameter), atomic force microscopy (particle height), &zgr; potential analysis (surface charge), and inductively coupled plasma-mass spectrometry (gadolinium concentration). The blood compatibility of Hydrochalarone-1 was also assessed in vitro through analysis of hemolysis, platelet aggregation, and complement activation of human blood. In vitro relaxivities, in vivo pharmacokinetics, and a pilot in vivo acute toxicity study were also performed. ResultsAn extensive in vitro and in vivo characterization of Hydrochalarone-1 is described here. The hydrodynamic size of Hydrochalarone-1 was 5 to 7 nm depending on the dispersing media, and it was negatively charged at physiological pH. Hydrochalarone-1 showed compatibility with blood cells in vitro, and no significant hemolysis, platelet aggregation, or complement activation was observed in vitro. In addition, Hydrochalarone-1 had significantly higher r1 and r2 in vitro relaxivities in human plasma in comparison with Magnevist and was not toxic at the doses administered in an in vivo pilot acute-dose toxicity study in mice.In vivo MRI pharmacokinetic analysis after a single intravenous injection of Hydrochalarone-1 (0.2 mmol Gd/kg) showed that the volume of distribution at steady state was approximately 100 mL/kg, suggesting prolonged systemic circulation. Hydrochalarone-1 also had a long blood half-life (88 minutes) and increased relaxivity, suggesting application as a promising blood-pool MRI contrast agent. ConclusionsThe evidence suggests that Hydrochalarone-1, with its long systemic half-life, may have significant utility as a blood-pool MRI contrast agent.

Effects of Novel Nanomaterials on Allergic Mediator Release from Human Mast Cells and Basophils through Non-Ige Mediated Pathways

Mast cells (MC) and peripheral blood basophils (PBB) are well known for their role in the allergic response mediated through high affinity IgE receptors (FceRI). However, these cells can also be stimulated by other non-allergic secretagogues to release their inflammatory mediators. Certain fullerene derivatives (FD) have already been shown to stabilize FceRI-mediated MC/PBB responses, but it is not know if they also stabilize these cells through non-IgEmediated mechanisms. A panel of FD was synthesized and tested for their ability to inhibit non-FceRI mediated release from human MC and PBB. It was found that specifically engineered FD could significantly inhibit calcium ionophore, compound 48/80, somatostatin, and poly L-lysine induced MC degranulation and cytokine production, as well as blunt degranulation and cytokine production from N-formyl-methionine-leucine-phenylalanine (fMLP), poly L-lysine, and calcium ionophore stimulated PBB. The mechanism of inhibition was due in part to the prevention of secretagogueinduced increases in cellular reactive oxygen species (ROS) and calcium levels as well as the reduced activation of the MAPK signaling intermediates ERK1/ERK2 and LAT. Additionally, preincubation of MC with FD blunted the prostaglandin D2 (PGD2) production upon exposure to inflammatory stimuli. In both cell types, the extent of inhibition of mediator release in response to each secretagogue was dependent on the moieties/side chains attached to the carbon cage. These results further extend the utility of fullerene nanomaterials to control mediator release through non-IgE mediated pathways in MC/PBB.