Cell uptake mechanisms of glycosylated cationic pDNA–cyclodextrin nanoparticles

Abstract

The incorporation of carbohydrate functional elements in the architecture of polycationic amphiphilic cyclodextrins (paCDs) provides glycosylated paCDs (pGaCDs) that form transfectious nanocomplexes (glycoCDplexes) with pDNA. In this study, we aimed at elucidating the internalization mechanisms at play and their incidence in transfection efficiency for glycoCDplexes formulated with 6-amino-6-deoxy-b-Dglucopyranosyl-appended pGaCDs in comparison with mannosylated and non-glycosylated congeners.Preliminary data showed a relatively high uptake of the 6-aminoglucosylated nanocomplexes by BNL-CL2 hepatocytes that correlated with a strong affinity towards the galactose-specific peanut agglutinin (PNA) lectin, suggesting that the galactose-binding asialoglycoprotein receptor at the surface of hepatocytes might be involved in glycoCDplex internalization. Transfection kinetics, internalization rates and protein expression data in BNL-CL2 ASGPR-expressing cells and COS-7 ASGPR-devoid epithelial cells in the absence and presence of different inhibitors of clathrin-dependent (chlorpromazine), caveolae-dependent (genistein) and macropinocytosis (amiloride) endocytic routes evidenced significant differences in cell uptake pathways and fate of glycoCDplexes as compared with CDplexes. Most importantly, such differences were dependent on the cell type and on the carbohydrate coating moiety.Clathrin-mediated uptake in BNLCL-2 cells is particularly favored for the 6-amino-6-deoxyglucose CDplexes, supporting the interplay of specific recognition phenomena. Competitive uptake and transfection experiments conducted in the presence of asialofetuin or of a polyclonal ASGPR-antibody, as well as siRNA-mediated ASGPR-specific gene knockdown, supported the involvement of ASGPR, firmly demonstrating the dual role of the 6-amino-6-deoxyglucose motif as DNA and lectin receptor ligand. The results reinforce the use of carbohydrates in glycoCDplexes to modulate cellular uptake and transfection capabilities in a cell-dependent manner.

Plasmid‐Templated Control of DNA–Cyclodextrin Nanoparticle Morphology through Molecular Vector Design for Effective Gene Delivery

“…Importantly, no toxicity was observed for any of the CDplex formulation assayed in this work (see the Supporting Information), compared with 60–70 % cell viability for bPEI polyplexes. The results evidenced that the relative transfection efficiencies are cell dependent, which probably arises from dissimilarities in cell‐uptake routes as previously observed for CDplexes generated from monomeric paCDs . Nevertheless, some common features emerged.…”

“…The results evidencedt hat the relative transfection efficiencies are cell dependent, which probablya rises from dissimilarities in cell-uptake routes as previously observed for CDplexes generated fromm onomeric paCDs. [81,82] Nevertheless, some common features emerged. Dimeric vectors generally performed better than the monomeric controli nb oth cell lines.…”

Cyclodextrin-Based Functional Glyconanomaterials

“…A homologous heptaconjugate exposing 2,6-diamino-2,6-dideoxyglucoside motifs formed a solid precipitate in the presence of pDNA and was not further pursued. Most interestingly, the 6-amino-6-deoxyglucoside pGaCD was found to selectively bind the galactose-specific lectin peanut agglutinine (PNA) and preferentially internalize BNL-CL2 hepatocytes by ASGPR-mediated endocytosis [171], highlighting this iminosugar as a dual nucleic acid/lectin receptor binder when presented in multivalent form (Figure 17). Jiménez Blanco, Di Giorgio and coworkers proposed an alternative pGaCD vector design that displayed aminoglucosyl units attached at all primary positions of the per(O-2,O-3)-hexanoylated βCD scaffold, instead of bearing amine groups and sugar ligands at separate branches [170].…”

Recent Advances in the Chemistry of Glycoconjugate Amphiphiles

“…A β-cyclodextrin scaffold has been used to synthesize a multivalent polycationic glyco-amphiphile cyclodextrin (pGaCD) as a gene delivery system [ 51 , 52 ]. The upper rim of the CD is functionalized with a glyco-cationic moiety while the lower rim exhibits lipophilic tails ( Scheme 3 ).…”