Dendrimers-drug conjugates for tailored intracellular drug release based on glutathione levels

摘要

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-NH2 and an anionic G3.5-COOH PAMAM dendrimers with NAC payloads of 16 and 18 per dendrimer, respectively, as confirmed by ¹H-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-NH2 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.