Cell-penetrating compounds preferentially bind glycosaminoglycans over plasma membrane lipids in a charge density- and stereochemistry-dependent manner

- GAG charge density affects CPC binding strength.
- GAG carbohydrate composition also plays a role in CPC recognition, likely through hydrogen bonding.
- The molecular weight of CPCs affects their ability to cluster GAGs.
- CPCs bind anionic lipids but not typical plasma membrane compositions.

Cell-penetrating compounds (CPCs) are often conjugated to drugs and genes to facilitate cellular uptake. We hypothesize that the electrostatic interaction between the positively charged amines of the cell-penetrating compounds and the negatively charged glycosaminoglycans (GAGs) extending from cell surfaces is the initiating step in the internalization process. The interactions of generation 5 PAMAM dendrimer, Tat peptide and 25 kDa linear PEI with four different GAGs have been studied using isothermal titration calorimetry to elucidate structure-function relationships that could lead to improved drug and gene delivery methods to a wide variety of cell types. Detailed thermodynamic analysis has determined that CPC-GAG binding constants range from 8.7 × 103 to 2.4 × 106 M− 1 and that affinity is dependent upon GAG charge density and stereochemistry and CPC molecular weight. The effect of GAG composition on affinity is likely due to hydrogen bonding between CPC amines and amides and GAG hydroxyl and amine groups. These results were compared to the association of CPCs with lipid vesicles of varying composition as model plasma membranes to finally clarify the relative importance of each cell surface component in initial cell recognition. CPC-lipid affinity increases with anionic lipid content, but GAG affinity is higher for all cell-penetrating compounds, confirming the role these heterogeneous polysaccharides play in cellular association and clustering.