Understanding and improving drug release kinetics from dendrimer-drug conjugates is a key step to improving their in vivo efficacy. N-Acetylcysteine (NAC) is an anti-inflammatory agent with significant potential for clinical use in the treatment of neuroinflammation, stroke and cerebral palsy. There is a need for delivery of NAC which can enhance its efficacy, reduce dosage and prevent it from binding plasma proteins. For this purpose, a poly(amidoamine) dendrimer-NAC conjugate that contains a disulfide linkage was synthesized and evaluated for its release kinetics in the presence of glutathione (GSH), Cysteine (Cys), and bovine serum albumin (BSA) at both physiological and lysosomal pH. The results indicate that the prepared conjugate can deliver ~60% of its NAC payload within 1 hour at intracellular GSH concentrations at physiological pH, whereas the conjugate did not release any drug at plasma GSH levels. The stability of the conjugate in the presence of bovine serum albumin at plasma concentrations was also demonstrated. The efficacy of the dendrimer-NAC conjugate was measured in activated microglial cells (target cells in vivo) using the reactive oxygen species (ROS) assay. The conjugates showed an order of magnitude increase in anti-oxidant activity compared to free drug. When combined with intrinsic and ligand-based targeting with dendrimers, these types of GSH sensitive nanodevices can lead to improved drug release profiles and in vivo efficacy.

N-Acetyl-L-Cysteine (NAC) is an anti-oxidant and anti-inflammatory agent with significant potential in clinical applications including stroke and neuroinflammation. The drug shows high plasma binding upon IV administration, requiring high doses and associated side effects. Through the use of an appropriate delivery vehicle, the stability and efficacy of NAC can be significantly improved. Dendrimers are an emerging class of nanoscale drug delivery vehicles, which enable high drug payloads and intracellular delivery. Poly(amidoamine) dendrimer-NAC conjugates having cleavable disulfide linkages are designed for intracellular delivery based on glutathione levels. We have successfully synthesized two conjugates of with a cationic G4-NH 2 and an anionic G3.5-COOH PAMAM dendrimers with NAC payloads of 16 and 18 per dendrimer, respectively, as confirmed by 1 H-NMR and MALDI-TOF analysis. NAC release kinetics of the conjugates at intracellular and extracellular Glutathione (GSH) concentrations were evaluated by reverse phase HPLC (RP-HPLC) analysis, and ~70% of NAC payload was released within one hour at intracellular GSH concentrations (~10 mM), whereas negligible NAC release was observed at extracellular GSH levels (2 µM). FITC-labeled conjugates showed that they enter cells rapidly and localize in the cytoplasm of lipopolysaccharide (LPS)-activated microglial cells (the target cells in vivo). The efficacies of dendrimer-NAC conjugates in activated microglial cells was confirmed by measuring the nitrite inhibition in the cell culture medium, which is an indication of the anti-oxidative property of the drug.Both G4-NH 2 and G3.5-COOH conjugates showed more significantly better nitrite inhibition both at 24 and 72 hours compared to free NAC, by as much as a factor of 16. The results indicate that PAMAM dendrimer conjugates produce a higher local NAC concentration inside the cells, with GSH-sensitive disulfide linker enabling efficient and rapid cellular release of the drug.

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