N-Acetyl-L-Cysteine (NAC) is a drug frequently used as a mucolytic agent as well as acetaminophen overdose cases. The determination of its antioxidant, radical-scavenging properties, gave rise to investigation of its other therapeutic applications. Even though NAC proved to be a very therapeutic drug, delivery problems remain. The stability of NAC is low which increases the effective dose required. The high protein binding is another problem associated with NAC therapies since it affects activity and elimination of the drug. We proposed that the efficacy of NAC could be increased by protecting the drug from degradation, preventing protein binding and enhancing the cellular uptake, in addition to targeting it to tissue of interest in the body. For this purpose, we designed Polyamidoamine (PAMAM) dendrimer NAC conjugates as delivery devices that can address these issues. Evaluation of the nanodevices indicates that we successfully engineered Glutathione sensitive PAMAM dendrimer conjugates with high payload that release NAC specifically inside the cells and have higher in vitro efficacy compared to free NAC.

PAMAM dendrimers are highly hyper-branched synthetic polymers with well defined spherical structure and size. They have shown prospect as drug delivery vehicles with active and passive targeting capabilities, drug solubility and stability enhancement and vast opportunities for innovative designs at molecular level. In this study, we developed PAMAM dendrimer conjugates containing disulfide linkages. The choice of disulfide linkage was aimed at enhancing the intracellular availability of NAC by protecting it from degradation and protein binding and finally selectively releasing it intracellularly employing differences in Glutathione (GSH) levels. GSH is the most abundant thiol species in the cytoplasm and the major reducing agent in biochemical processes. The intracellular GSH concentration (1-10 mM) is substantially higher than extracellular levels (2uM in plasma), thus provides opportunities for intracellular cleavage of disulfide linkages.

In the present investigation, we have successfully synthesized two conjugates of NAC with G4-NH2 and G3.5-COOH PAMAM dendrimers. Additionally Fluorescein Isothiocyanate (FITC) was also conjugated for imaging and cellular uptake studies. The cell studies were carried out on Mouse microglial cells (BV-2). G4-NH2 and G3.5-COOH conjugates contained 16 and 18 NAC molecules per dendrimer respectively as determined by 1H-NMR and MALDI-TOF analysis. The drug release kinetics of the conjugates was evaluated at intracellular and extracellular GSH concentrations. The release mechanism was determined to be via disulfide exchange reactions with the thiol group of GSH. The conjugates released ~50% of their NAC payload within 1 hour at intracellular GSH concentrations whereas they were quite stable at extracellular GSH levels and did not release significant levels of NAC. The release rates indicate that the disulfide bond was easily and rapidly cleaved by GSH to a high extent within a short amount of time. These results have significant implications in achieving controlled release in dendrimer-based delivery systems as well as demonstrating that GSH can be used as a reliable in vivo releasing agent.

Cellular entry of FITC labeled G4-NH2-NAC conjugate was evaluated using flow cytometry. It was determined that most of the G4-NH2-NAC conjugate entered BV-2 cells within 15 minutes after incubation and uptake continued slowly for another 45 minutes. The cellular entry was also visualized by using Confocal Laser Scanning Microscopy. It is evident that the FITC labeled G4-NH2-NAC conjugate localized mostly in the cytoplasm and lysosomes, while the nucleus appears to be relatively free of the presence of any fluorescence two hours after incubation. The results indicate that PAMAM dendrimer conjugates are transported inside the cells efficiently by endocytosis mechanism.

Efficacy of conjugates were evaluated by measuring the Nitrite in BV-2 cell culture medium which indicates the level of anti-oxidation. Free NAC inhibited nitrite release in dose-dependent manner after 72 hours incubation, while only high concentration of free NAC (8mM) inhibited nitrite release significantly after 24 hour incubation. On the other hand both G4-NH2 and G3.5-COOH conjugates had more significant inhibition both at 24 and 72 hours compared to the same concentration of free NAC. The results indicated both dendrimer-NAC conjugates are more effective anti-oxidants compared to equivalent free NAC, especially at lower NAC doses. The results indicate that PAMAM dendrimer conjugates produce a higher local NAC concentration inside the cells by enhancing cellular uptake and blocking degradation of the drug.