Effect of integrin targeting and PEG shielding on polyplex micelle internalization studied by live-cell imaging.

α(v)ß(3) and α(v)ß(5) integrins are attractive target structures for cancer therapy as they are upregulated in tumor and tumor associated host cells and play a pivotal role for tumor growth and metastasis. Gene vectors such as polyplex micelles consisting of thiolated PEG-block-poly(lysine) copolymers complexed with plasmid DNA can be targeted to these specific integrins by equipment with a cyclic RGD peptide. In this study, we analyzed the effect of the RGD ligand on micelle endocytosis by comparing fluorescently labeled, targeted and untargeted micelles in live-cell imaging experiments with highly sensitive fluorescence microscopy and flow cytometry. Two micelle types with 12 kDa (PEG12) and 17 kDa (PEG17) PEG shell layers were examined to evaluate the influence of surface shielding on the internalization characteristics. Our results reveal three major effects: First, the RGD ligand accelerates the internalization of micelles into integrin expressing HeLa cells without changing the uptake pathway of the micelles. Both targeted as well as untargeted micelles are predominantly internalized via clathrin mediated endocytosis. Second, the PEG shielding of micelles has an important effect on their targeting specificity. At high PEG shielding selective endocytosis of integrin targeted micelles occurs, whereas at low PEG shielding targeted and untargeted micelles show comparable internalization. In addition, PEG17 RGD(+) micelles induce the highest reporter gene expression. Third, our data demonstrate a clear influence of the applied micelle dose on the internalization of integrin targeted micelles. We propose that PEG17 shielded micelles equipped with a cyclic RGD ligand are the favored system of choice for clinical therapy as they exhibit higher transgene expression, a higher specificity for integrin-dependent endocytosis compared to PEG12 shielded micelles, and are functional at low doses as well.

Visualizing uptake and intracellular trafficking of gene carriers by single-particle tracking.

Single-particle microscopy und live-cell single-particle tracking are powerful tools to follow the cellular internalization pathway of individual nanoparticles such as viruses and gene carriers and investigate their interaction with living cells. Those single-cell and single-particle methods can elucidate the "black box" between application of the gene carrier to the cell and the final gene expression and allow the essential bottlenecks to be identified in great detail on the cellular level. In this review we will give a short introduction into single-particle tracking microscopy and present an overview of the mechanisms of DNA delivery from attachment to the cell membrane over internalization towards nuclear entry unraveled by single-particle methods.

Dynamics of magnetic lipoplexes studied by single particle tracking in living cells.

Magnetofection, gene delivery under the influence of a magnetic field, is a technique to increase transfection efficiency by enforcing gene vector contact with a target cell. Mechanisms of magnetic lipoplex internalization and intracellular details of magnetofection are still unknown. In this study, cellular dynamics of magnetic lipoplexes were examined in real time by means of highly sensitive dual-color fluorescence microscopy. Single particle tracking of magnetic lipoplexes provided trajectories representing the movement of the lipoplexes during internalization and subsequent intracellular processes. Magnetic lipoplexes show a three-phase behavior similar to polyplexes. During phase I lipoplexes are attached to the cell surface and show slow cooperative transport behavior. Phase II takes place inside the cell and was characterized by anomalous and confined diffusion. Phase III represented active transport along microtubules inside the cell. The majority of lipoplexes were internalized via endocytosis during phase I. On later time scales the formation of a perinuclear ring was observed. Persisting colocalization of fluid phase marker and lipoplexes after 24 h indicated slow endosomal release. In short, the internalization characteristics of magnetic lipoplexes are very similar to that of polyplexes. Furthermore our results suggest that the magnetic field induces an increased concentration of magnetic complexes on the cell surface resulting in higher transfection efficiency.

Dynamics of photoinduced endosomal release of polyplexes.

Endosomal escape is a well-known bottleneck for successful delivery of macromolecular drugs and genes. Photochemical disruption of endosomal membranes is an approach to overcome this bottleneck. In this study, we used the photosensitizer disulphonated meso-tetraphenylporphine with sulfonate groups on adjacent phenyl rings (TPPS(2a)) to investigate photoinduced endosomal release in living cells with high resolution fluorescence wide-field microscopy in real time. We studied the release dynamics of 10 kDa dextran and polyplexes consisting of DNA condensed with the cationic polymers linear polyethyleneimine (LPEI), poly-(L)-lysine (PLL) or poly-(D)-lysine (PDL). By means of dual-color microscopy and the use of double-labeled polyplexes DNA and polymer were imaged simultaneously. We show that the characteristics of the cationic polymer significantly influence the release behavior of the polyplexes. The release of dextran occurred within 100 ms. For LPEI/DNA particles, LPEI quickly spread throughout the cytosol similar to dextran, whereas DNA was released slowly (within 4 s) and remained immobile thereafter. In case of PLL particles, both DNA and polymer showed quick release. PDL particles remained condensed upon photosensitizer activation. In addition, we demonstrate that TPPS(2a) has biological side effects. Besides stop of microtubule dynamics in the dark, the movement of endosomes ceased after photosensitizer activation.

Four-dimensional imaging of transvacuolar strand dynamics in tobacco BY-2 cells.

The vacuole is a characteristic organelle of plant cells and fulfills several important functions related to metabolism and growth of the cell. To shed light on the details of vacuolar structural changes in plant cells, we explored the three-dimensional organization and dynamics of living Nicotiana tabacum L. cv. Bright Yellow 2 cell vacuoles by real-time confocal time-lapse imaging. For imaging, the cells were pulse-labeled with the amphipathic styryl dye FM1-43 (N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide), which is delivered to the plant vacuole by endocytic uptake and then incubated overnight. Imaging of the membrane-labeled vacuole revealed a complex vacuole morphology underlaid by constant remodeling. The vacuole is traversed by multiple transvacuolar strands which move along each other and fuse in multiple manners. New strands were created by fission of large membrane sheets. Endocytic vesicle trafficking was followed within the dynamic transvacuolar strands. The movement occurred in a stop-and-go fashion with an average vesicle velocity of 0.46 microm/s and a peak velocity of 0.82 microm/s. Transvacuolar-strand reduction and creation is a characteristic event observed during mitosis. Here we propose a mechanistic model for the alteration of the number of transvacuolar strands, on the basis of their fusion and fission.