Purpose. Knowledge about the uptake mechanism and subsequent intracellular routing of non-viral gene delivery systems is important for the development of more efficient carriers. In this study we compared two established cationic polymers pDMAEMA and PEI with regard to their transfection efficiency and mechanism of cellular uptake. Materials and Methods. The effects of several inhibitors of particular cellular uptake routes on the uptake of polyplexes and subsequent gene expression in COS-7 cells were investigated using FACS and transfection. Moreover, cellular localization of fluorescently labeled polyplexes was assessed by spectral fluorescence microscopy.Results. Both pDMAEMA-and PEI-complexed DNA showed colocalization with fluorescently-labeled transferrin and cholera toxin after internalization by COS-7 cells, which indicates uptake via the clathrinand caveolae-dependent pathways. Blocking either routes of uptake with specific inhibitors only resulted in a marginal decrease in polyplex uptake, which may suggest that uptake routes of polyplexes are interchangeable. Despite the marginal effect of inhibitors on polyplex internalization, blocking the caveolae-mediated uptake route resulted in an almost complete loss of polyplex-mediated gene expression, whereas gene expression was not negatively affected by blocking the clathrin-dependent route of uptake. Conclusions. These results show the importance of caveolae-mediated uptake for successful gene expression and have implications for the rational design of non-viral gene delivery systems.

A set of polymer carriers for DNA delivery was synthesized by combining monodisperse, sequence-defined poly(amidoamine) (PAA) segments with poly(ethylene oxide) (PEO) blocks. The precise definition of the PAA segments provides the possibility of correlating the chemical structure (monomer sequence) with the resulting biological properties. Three different PAA-PEO conjugates were synthesized by solid-phase supported synthesis, and the cationic nature of the PAA segments was systematically varied. This allows for the tailoring of interactions with double-stranded plasmid DNA (dsDNA). The potential of the PAA-PEO conjugates as non-viral vectors for gene delivery is demonstrated by investigating the dsDNA complexation and condensation properties. Depending on the applied carrier, a transition in polyplex (polymer-DNA ion complex) structures is observed. This reaches from extended ring-like structures to highly compact toroidal structures, where supercoiling of the DNA is induced. An aggregation model is proposed that is based on structural investigations of the polyplexes with atomic force microscopy (AFM) and dynamic light scattering (DLS). While the cationic PAA segment mediates primarily the contact of the carrier to the dsDNA, the PEO block stabilizes the polyplex and generates a "stealth" aggregate, as was suggested by Zeta potentials that were close to zero. The controlled aggregation leads to stable, single-plasmid complexes, and stabilizes the DNA structure itself. This is shown by ethidium bromide intercalation assays and DNase digestion assays. The presented PAA-PEO systems allow for the formation of well-defined single-plasmid polyplexes, preventing hard DNA compression and strongly polydisperse polyplexes. Moreover carrier polymers and the resulting polyplexes exhibit no cytotoxicity, as was shown by viability tests; this makes the carriers potentially suitable for in vivo delivery applications.

A polymer drug-carrier was synthesized by combining a monodisperse, sequence-defined poly-(amidoamine) segment with a poly(ethylene oxide)-block (PAA-block-PEO). Between both polymer blocks a single disulfide moiety is incorporated that allows for the realization of a programmed disassembly of the carrier polymer in a reductive environment, like for instance present in certain intracellular compartments. The sequence selective positioning is realized using cystamine as a new building block for the automated synthesis of monodisperse PAAs. The controlled disassembly of the polymeric carrier was used to establish a two-phase release process, e.g., highly relevant for an effective release after successful drug delivery into a cell. The applicability of this carrier was demonstrated by analyzing the complexation behavior of the system with plasmid DNA, before and after reductive degradation of the block copolymer. The presented observations describe a transition in polyplex (polymer-DNA complex) properties from PEO-stabilized ion complexes with soft charge compensation to compact structures with more effective charge neutralization after cleavage of the PEO-block.

2008www.chemeurj.org C E U J E D 1 4 ( 7 ) 1 9 8 9 -2 2 9 2 ( 2 0 0 8 ) · I S S N 0 9 4 7 -6 5 3 9 · V o l . 1 4 · N o . 7 · 2 0 0 8 ConceptSuperelectrophiles: Charge-Charge Repulsive Effects

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