Download PAMAM Dendrimer Conjugates for Intracellular Delivery of N

PAMAM Dendrimer Conjugates for Intracellular Delivery of N-Acetyl-Cysteine
Yunus E. Kurtoglu1,2, Raghavendra S. Navath1, 2, Bing Wang2,3, Sujatha Kannan2,3, Robert Romero2,
and Rangaramanujam M. Kannan1, 2
1
Department of Chemical Engineering and Material Science, and Biomedical Engineering, Wayne
State University, Detroit, Michigan 48202, 2Perinatology Research Branch, Eunice Kennedy Shriver
National Institute of Child Health and Human Development, National Institutes of Health, DHHS,
3
Department of Pediatrics (Critical Care Medicine), Children’s Hospital of Michigan, Wayne State
University, Detroit, MI 48202.
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.
Efficacies 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. Our preliminary in vivo studies suggest that the efficacy of NAC can
be increased 10 fold by using the described PAMAM dendrimer delivery nanodevice.