**3. Methods**

78 Non-Viral Gene Therapy

nucleases and finally low intra-nuclear DNA delivery have to be overcome. A promising strategy for increasing the efficiency of non-viral vectors is to target certain uptake pathways that improve the efficient delivery of particles. Such a strategy requires thorough investigation of the different internalization pathways and the subsequent intracellular

In this work we concentrate on the first and second major barrier to improved transfection efficiency in human primary cells. Two different cell types were chosen because of their relevance in cardiovascular diseases: human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC). Both cells are involved in the unwanted re-narrowing (restenosis) of cardiac vessels after angioplasty and therefore are the primary targets in a

The distinct uptake mechanism of cationic lipid/DNA aggregates had to be clarified in order to gain knowledge about further intracellular processing and other barriers which still had to be overcome. To distinguish between the different endocytic pathways involved in lipoplex internalization of the Rhodamine-labeled DC-30 lipoplexes (Rh-DC-30), both general and specific inhibitors of endocytic routes were monitored in the presence of lipoplexes and analyzed by means of flow cytometry. Table 1 represents an overview of the inhibitors used. To gain more insight into the intracellular fate of lipoplexes, investigation of their co-localization with a variety of molecules was carried out using spectral bio-imaging. Transferrin alexa fluor 488 (tf) was used as a marker for clathrin-mediated uptake [9-16], cholera toxin B alexa fluor 488 (chltx-B) was used as marker for internalization via caveolae and related membrane structures [8, 17], and FITC-dextran was used as a marker for

It was subsequently investigated whether the failure of the endosomal release of the lipoplex or the desaggregation of the complex was responsible for the low transfection efficiency. Therefore, plasmid DNA was additionally introduced into the cytosol by

Chlorpromazine (chlp) Clathrin [11]

DC-30 was purchased from Avanti Polar Lipids (Birmingham, AL, USA). The Cy-5- and Rh-DNA Labeling Kits, Transferrin Alexa Fluor conjugate 488, Cholera Toxin Subunit B Alexa Fluor conjugate 488 and DAPI were purchased from Molecular Probes (Leiden, The Netherlands). Cells were purchased from ATCC (American Type Culture Collection),

Inhibitor Uptake route Reference

(mbCD) Clathrin and caveolae [22-24] Filipin (fil) Caveolae [25-27] Genistein (gen) Caveolae [28] LY 29004 (Ly) Macropinocytosis [29, 30] Wortmannin (wm) Macropinocytosis [29, 30] Nocodazole (noco) Microtubule depolymerization [31] Cytochalasin-D (cch-D) Actin skeleton disruptor [8]

events involved in each case.

macropinocytosis.

electroporation.

**2. Materials** 

Methyl-ß-cyclodextrin

Table 1. Inhibitors for the specific endocytic routes

strategy for cardiovascular gene-therapy.
