Lysine functionalized nanodiamonds as gene carriers - Investigation of internalization pathways and intracellular trafficking

摘要

Two major determinants for effective gene delivery are the capability of the carrier to protect genetic material during extra and intracellular trafficking, and the ability to release it at the site of action in an intact form. The cellular uptake pathway is a controlling parameter regarding the fate of nanoparticle-genetic material complexes. This study elucidates the pathways involved in the cellular uptake of pristine nanodiamonds (pND), functionalized nanodiamonds (lys-NDs) and diamoplexes (ND/genetic material complexes, namely lysND-siRNA). Changes in membrane morphologies corresponding to a specific route of internalization were studied using transmission electron microscopy. Variations in cellular association of diamoplexes after general and targeted inhibition of endocytic pathways were quantified using flow cytometry. Electron micrographs reveal formation of unilateral membrane protrusions suggestive of macropinocytosis as one of the major uptake process for lys-NDs and lysND-siRNA diamoplexes. Formation of pits was also largely detected for lys-NDs but scarcely present for their diamoplexes, attributed to either clathrin-mediated or caveoli-mediated endocytosis. However, inhibition of caveoli-mediated endocytosis did not prevent internalization of the diamoplexes, suggesting the absence of this pathway. Due to the larger aggregate size, pNDs were mainly internalized through macropinocytosis, as indicated by unilateral pseudopods on their micrographs. The cells also showed bilateral pseudopods during internalization suggesting phagocytic uptake, possibly due to random functionalities on the surface of pNDs. Clathrin or caveoli mediated endocytosis was absent. Soft X-ray spectromicroscopy reveals strong sp3-carbon signals confirming that the internalized entities identified by electron microscopy are indeed NDs and their diamoplexes. These findings are the basis of further optimization of amino acid functionalized NDs towards increase of gene delivery efficacy through targeted internalization pathways and overcoming intracellular challenges.