Cytochemical methods for tracking nanoparticles and monitoring drug delivery inside the cell

Chitosan nanoparticles (ChiNPs) have been attracting increasing interest for their potential in biomedical applications as drug delivery systems since they are able to protect the encapsulated drugs and/or improve their efficacy by making them able to cross biological barriers and reach their intracellular targets. Detecting the intracellular location of ChiNPs and monitoring the release of the loaded molecules is crucial for designing drug delivery strategies. By using fluorescently- labelled ChiNPs and diaminobenzidine photoconversion to correlate fluorescence and transmission electron microscopy we precisely described the intracellular fate of ChiNPs in neuronal cells in vitro. We demonstrated that ChiNPs enter neuronal cells by endocytosis; in the cytoplasm they occur both inside membrane-bounded vesicles and free in the cytosol, and accumulate around the cell nucleus. The efficacy of ChiNPs in delivering D-Ala2-D-Leu5-enkephalin (DADLE) to the same neuronal cell line was then tested. DADLE is a hypometabolising synthetic opiod potentially useful for biomedical applications, but its short half-life makes a systemic administration inefficient. We demonstrate by immunoelectron microscopy that ChiNPs are effective opioid delivery carriers to neuronal cells, protecting incorporated molecules from enzymatic degradation and prolonging their intracellular effects.