PEGylated gambogenic acid nanostructured lipid carriers enhance intracellular delivery in A549 cells via clathrin-, caveolae-mediated endocytosis and macropinocytosis.
TL;DR
P-GNA-NLC enters cells via the Cav-ME, macropinocytosis, and CDE pathways, which may account for the stronger antitumor effect of P-GNA-NLC than GNA.
Abstract
Objective
To investigate the transport mechanism of gambogenic acid (GNA) and PEGylated gambogenic acid nanostructured lipid carriers (P-GNA-NLC) in A549 cells and to quantify their intracellular contents.
Methods
The GNA content in A549 cells was determined by high-performance liquid chromatography (HPLC). The mechanisms of GNA and P-GNA-NLC entry were elucidated using various experimental conditions, including different temperatures, concentrations, times, and endocytosis inhibitors. Initially, A549 cells were obtained through cell culture, and HPLC was employed to establish the quantitative analysis of GNA inside the cells. Subsequent experimentation involved the determination of cellular uptake profiles, the execution of carrier saturation experiments, investigations into the effects of varying temperatures and concentrations on uptake efficiency, and the implementation of endocytosis inhibitor treatment experiments. In the latter, the inhibitor's potential for inducing cellular toxicity was determined by means of an MTT assay, and the inhibitor concentration that exhibited minimal effect on cellular activity was selected for subsequent experimentation.
Results
Both GNA and P-GNA-NLC entered A549 cells via active transport. GNA entered mainly through caveolae-mediated endocytosis (Cav-ME) and macropinocytosis, whereas P-GNA-NLC could enter the cells through clathrin-dependent endocytosis (CDE) pathway in addition to Cav-ME and macropinocytosis. This may account for the stronger antitumor effect of P-GNA-NLC than GNA. Furthermore, the entry of P-GNA-NLC into cells in the form of nanolipid carriers may have improved the intracellular pharmacokinetics of GNA, thus enhancing the efficacy of GNA.
Conclusion
P-GNA-NLC enters cells via the Cav-ME, macropinocytosis, and CDE pathways. These findings provide a preliminary understanding of the intracellular transport mechanisms of GNA and P-GNA-NLC and provide an experimental basis for the clinical application of GNA antitumor drugs.