In the last 2–3 decades, gene therapy represented a promising option for hepatocellular carcinoma (HCC) treatment. However, the design of safe and efficient gene delivery systems is still one of the major challenges that require solutions. In this study, we demonstrate a versatile method for covalent conjugation of glycyrrhizin acid (GL) or glycyrrhetinic acid (GA) to increase the transfection efficiency of Polyethyleneimine (PEI, Mw 1.8K) and improve their targeting abilities of hepatoma carcinoma cells. GA and GL targeting ligands were grafted to PEI via N-acylation, and we systematically investigated their biophysical properties, cytotoxicity, liver targeting and transfection efficiency, and endocytosis pathway trafficking. PEI-GA0.75, PEI-GL10.62 and PEI-GL20.65 conjugates caused significant increases in gene transfection efficiency and superior selectivity for HepG2 cells, with all three conjugates showing specific recognition of HepG2 cells by the free GA competition assay. The endocytosis inhibition and intracellular trafficking results indicated that PEI-GA0.75 and GL10.62 conjugates behaved similarly to SV40 virus, by proceeding via the caveolae- and clathrin-independent mediated endocytosis pathway and bypassing entry into lysosomes, with an energy independent manner, achieving their high transfection efficiencies. In the HepG2 intraperitoneal tumor model, PEI-GA0.75 and PEI-GL10.62 carrying the luciferase reporter gene gained high gene expression, suggesting potential use for in vivo application.

Amides with b-carboxylic acid groups are stable and negatively charged at physiological pH, but hydrolyze back to the corresponding amines and anhydrides once in an acidic environment. We thereby developed charge-reversal nanocarriers for drug delivery. In these systems, the anhydrides served only as groups to amidize the amines in cationic polymers and had no therapeutic use after being cleaved from the carrier in the cells. Herein, we utilized the characteristic anhydride structure of norcantharidin (NCTD) and the pH-dependent hydrolysis of its b-carboxylic amides and developed novel acid-labile conjugates for the targeted delivery of NCTD. In this study, NCTD was used as both an anticancer drug and an acid-labile anhydride to react with cationic polymers including PEI and PLL to form bcarboxylic amides. The obtained conjugates (PEI-NCTD and PLL-NCTD) had not only significantly improved NCTD solubility and high drug loading contents (as high as 72.3% and 56.8%, respectively, for PEI-NCTD and PLL-NCTD), but also favorable acid-labile capabilities. These conjugates were stable and negatively charged at neutral pH, but once in acidic environments (e.g. endo/lysosomes), they hydrolyzed and regenerated not only NCTD as an antitumor drug, but also the PEI or PLL, which could assist the endo/lysosomal escape and release of the drugs. Further, functionalizing the acid-labile conjugates with targeting moiety folic acid (FA) increased the cellular uptake of the conjugates into folate receptor-overexpressing tumor cells and thereby the in vitro cytotoxicity. These targeted acidlabile conjugates may help to reduce the side effects of NCTD and improve its clinical applications.