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Engineered Carbon Nanostructures for Preventing Alzheimer’s Disease

Alzheimer’s disease (AD) is an aging-associated devastating disease that affects over 5 million Americans and imposes an annual economic burden of $236 billion. Currently, all efforts to develop remedies to AD have failed. This unfortunate situation calls for the development of new research directions. One of the common pathological features of AD is the decline in endogenous production of hydrogen sulfide (H2S), a gaseous signaling molecule. H2S in the brain is historically believed to be solely derived via enzymatic production and deficiencies in H2S producing enzymes, such as cystathionine-γ-lyase (CGL) and cystathionine-β-synthase (CBS) are associated with neurodegenerative diseases. Exogenous administration of H2S and H2S donors have shown to attenuate Alzheimer’s and Parkinson’s disease phenotypes in rodent models. However, exogenous H2S gas and donor molecules are short-lived, highly reactive, and display acute toxicity at elevated levels, and delivery to the intended tissue may be challenging to achieve in the clinic. Thus, discovering new technologies that regulate and/or augment endogenous enzymatic H2S pose novel scientific and therapeutic avenues particularly for the treatment of neurodegenerative disorders such as Alzheimer’s disease.
 We are designing enzyme-mimicking carbon nanostructures (EMCNs) that can augment the production of H2S using biological substrates present in the body and compensate for the declining enzyme activity and prevent the progression of Alzheimer’s disease. Carbon nanostructures with sp2 carbons are composed of multiple electron-deficient double bonds, which can be systematically replaced with functional groups to create a heterogeneous surface that is fundamentally different from that originally present. Based on the sp2 site engineering concept, we selected and synthesized EMCNs capable of interacting with the sulfur-containing biomolecules, catalyzing their breakdown into H2S and other products, thus mimicking CGL and CBS enzymes.
 The ability of EMCNs to mimic enzymes was evaluated with lead acetate saturated paper-based assay. EMCNs break down cysteine to produce H2S, which reacts with lead acetate to form lead sulfide. The concentration of H2S generated is calculated by using sodium hydrosulfide as an equimolar H2S donor for calibration. The H2S generation from EMCNs can be matched with the CBS enzyme. Further, EMCNs are stable and demonstrate long-term activity. In a repeated exposure experiment, H2S production was constant over a 20 day period where the substrate was added every day without replenishing ECMNs. If EMCNs were a reactant, the H2S production should have decreased with repeated substrate addition until all the EMCN was consumed.
 The efficacy of EMCNs to produce H2S in vitro was demonstrated with murine neuronal cells. P3 multi-photon fluorescent probe was utilized to detect intracellular H2S levels. The fluorescence intensity of the neuronal cells exposed to EMCN increased in a concentration-dependent manner. Neither cells nor EMCN exhibited auto-fluorescence under multi-photon microscopy. Beta-amyloid (Aβ) plaques, a hallmark of brain pathology in Alzheimer's disease patients, is formed by aggregation of β-amyloid peptides. The role of EMCNs in the AD model was evaluated with β-amyloid toxicity assay using Aβ1-42, which is selectively toxic to neurons. Preliminary studies suggest that EMCNs protect neuronal cells against the toxicity induced by Aβ1-42 by 19%. The ability of EMCNs to produce H2S under physiological conditions thus offers potential for rescuing enzyme deficiencies in Alzheimer’s disease.