(203a) Bone Target N Acetylcysteine Loaded in PLGA-ALE Nanoparticle to Osteoporosis Treatment. an in Vitro Test

N acetylcysteine (NAC) is a derivative of cysteine which is a precursor of well known antioxidant glutathione. This kind of components have shown the potential to attenuate osteoclasts activity in-vitro experiments. Osteoporosis is an illness characterized by an imbalance between osteoclast and osteoblast activity, so a component like NAC could be used as a therapeutic agent for its treatment. However, studies on the osteoporosis treatment with this agent have not yet shown beneficial effects. Despite there is no a conclusive explanation for this lack of activity, a possible cause is the low bioavailability of NAC (6% oral), preventing the required NAC doses to be delivered in the bone tissue to get a therapeutic effect.

In order to increase the bioavailability of NAC, and to selectively target the bone tissue, conjugated alendronate PLGA nanoparticles (PLGA-ALE) were used as carrier for NAC. This carrier should not react or destabilize the active compound, particularly as NAC denature by internally disulfide bond formation. Because of that, it is necessary to verify if the active compound retain its biological action after the loading process.

To confirm how suitable carrier are the PLGA-ALE nanoparticles to maintain NAC stability, this work developed an in vitro test to evaluate the NAC activity on osteoclast-cells like, previously generated by polyethylene glycol (PEG) induced macrophage fusion.

Synthesis of PLGA-ALE was done through carbodiimide chemistry. Nanoparticles were produced by the nanoprecipitation method and the in vitro test was done by fusion of U937 cells in the presence of PEG, following a fusion hybridoma protocol. Fused Cells were seeded on sterile bone slices for 4 to 6 hours, to attach the cells to the support. After that, a fetal bovine serum and free NAC supplemented culture medium was added and loaded within nanoparticles. Finally cells were incubated during 4 days, and subjected to reactive oxygen species (ROS), bone resorption pits and immunohistochemistry analysis. As osteoclastic activity surrogates, presence of different proteins were evaluated: matrix metalloproteinase (MMP), integrin aVb3 receptor (IB3), subunit of vacuolar ATPase (ATPase), DC-STAMP, Carbonic Anhydrase II (CAII), Cathepsin K, ADAM-12 and Calcitonin Receptor.

According with results both, free or polymer-loaded NAC, caused a decrease expression of the different osteoclast proteins (free NAC 74% and loaded NAC 71%). This experiments indicated that the polymer helps maintaining the stability of the active molecule up to 4 days. Taking into account that previous experiments demonstrated a high affinity of the polymer nanoparticles for the bone tissue, it is expected that a loaded NAC osteoporosis treatment to be more active than a free NAC one. Still in-vivo tests are necessary to confirm our findings.

As a conclusion, NAC loaded in PLGA-ALE nanoparticles retain the antioxidants properties of free NAC. The carrier did not have cytotoxic properties because cells grew in the present of it, and the entrapment process did not affect NAC stability.