Cell-penetrating compounds (CPCs), such as polyamidoamine (PAMAM) dendrimer, linear poly(ethyleneimine) (PEI) and trans-activating transcription factor (TAT) peptide, have been studied for use as effective drug and gene delivery agents. Successful delivery entails cargo attachment, cellular internalization and intracellular trafficking. Focusing on the internalization step, we hypothesize that electrostatic interaction occurs between the positively charged CPCs and negatively charged glycosaminoglycans (GAGs) found on all cell surfaces to different extents. The interaction of generation 5 (G5) PAMAM dendrimer, TAT peptide and linear PEI with four GAGs: heparin, heparan sulfate, dermatan sulfate and chondroitin sulfate A, has been studied using isothermal titration calorimetry (ITC), dynamic light scattering (DLS), and gel electrophoresis. DLS was used to confirm that complex aggregation occurs when charge neutrality is reached and therefore needs to be considered in the binding model. The concentration of sulfates per repeat unit of each GAG has been characterized using a colorimetric assay and reported thermodynamic parameters have been normalized per GAG sulfate group. Despite this normalization, it has been quantitatively determined that CPC-GAG affinity is dependent upon GAG charge density and stereochemistry. For example, highly sulfated heparin demonstrated stronger binding with all CPCs, 1-2 orders of magnitude greater, than that of the least sulfated heparan sulfate. All interactions are entropy-driven, however, charge density and architecture of the CPC affects both binding strength and stoichiometry. GAGs were also shown to disrupt CPC-DNA complexes, which has implications for cargo delivery at the cell surface.

Charge density and stereochemistry affect the interaction of cell-penetrating compounds with glycosaminoglycans

247th National Meeting (2014)

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