**3.5 In vitro transfection assay**

Cells were seeded in a 24-well cluster dish at a density of 104 cells per well 24 h prior to the experiments and cultivated in the appropriate growth medium with serum. After 24 h in culture the cells were washed with 1 ml PBS and then 400 µL growth medium containing serum was added to the cells. 200 µL of freshly prepared lipoplexes were added to the cells. After incubating for 5 h at 37 °C (5 % (v/v) CO2) the supernatants were removed and 1 ml of the appropriate growth medium was added to each well. Thereafter, the cells were cultured further for a total of 48 h at 37 °C, 5 % (v/v) CO2. In the control experiments cells were incubated with 200 µL culture medium or 200 µL TM and treated the same way as the

Investigation of Transfection Barriers Involved in

**3.8 Spectral bio-imaging** 

photobleaching effects.

software (Applied Spectral Imaging).

**4.1 Uptake experiments with inhibitors** 

after one hour of incubation with lipoplexes at 37 °C.

**4. Results** 

only 2 %.

published previously [5].

Non-Viral Nanoparticulate Gene Delivery in Different Cell Lines 81

Cells were seeded onto 12-mm coverslips in 24-well plates 2 days prior to the experiment. Lipoplexes were prepared using DC-30® and Rh-labeled plasmid. Cells were then washed twice with PBS w/o calcium and magnesium and then incubated with 400 µL medium containing the lipoplexes. Cells were incubated simultaneously with the markers for the different pathways for 30 min to 2 h (tf alexa fluor 488 (10 µg/mL), chltx-B alexa fluor 488 (2 µg/mL) and FITC dextran 70 000, (1 µg/mL)). The amount of DNA was the same as that used in the uptake experiments. Prior to microscopic examination, the cells were fixed with 4 % paraformaldehyde (500 µL, 10 min, RT) and the coverslips were mounted on glass slides with 3 µL MobiGlow (MoBiTec, Goettingen, Germany), an antifading substance to reduce

Spectral bio-imaging was performed as described previously by Huth et al. [8] with a SpectraCube SD-200H system (Applied Spectral Imaging, Migdal HaEmek, Israel). An inverted fluorescence microscope (Axiovert S 100, Zeiss, Jena Germany) equipped with a high-pressure mercury lamp (HBO 100) for excitation and a triple bandpass filter set was used. All images were taken using a 100x/1.3 oil-immersion objective lens (Plan Neofluar, Zeiss, Jena, Germany). In the spectral range from 400 to 700 nm the objective lens shows only minimal fluctuations of transmission (85-90 %). The optical head attached to the microscope is composed of a Sagnac common-path interferometer and imaging optics including a cooled CCD camera (Hamamatsu C4880-85, Japan). Microscopic images were obtained with spectral bio-imaging 2.5 software (Applied Spectral Imaging). The acquisition time of a desired image varied from 30 to 90 s depending on the brightness of the fluorescence and the image size. Cells were first incubated with only one dye to get singlecolored images. For further analysis images were then transferred to the SpectraView 1.6

For uptake experiments studied by means of flow cytometry, DNA was labeled with Cy-5. Cell experiments were performed at both 37 °C and 4 °C in order to distinguish cellular uptake from adsorption of lipoplexes to the outside of the cell membrane or their fusion with the cell membrane. It has been described that no energy dependent internalization process can take place at 4 °C [18]. As the size of the DC-30® lipoplexes was determined to be in the range of 300 and 500 nm [5], we assumed internalization to be an active process. The detection of positive fluorescent signal at 4 °C therefore refers to fusion or adsorption of lipoplex with the cell membrane. Fig. 1 represents the amount of fluorescent positive cells

The corresponding transfection efficiency is listed in Table 2 and shows that despite the fact that uptake of lipoplexes was successful in 24 % of HAEC, less than 1 % of HAEC were transfected. In HASMC, uptake reached as high as 80 %, whereas transfection was

Only 1 % of fluorescent cells were detected after incubation at 4 °C and therefore adsorption to the outside of the cell membrane or fusion with the cell membrane is negligible and the fluorescent positive cells as seen in Fig. 1 can be considered as resulting from cellular uptake (internalization) of the lipoplexes. Analysis of cytotoxicity confirmed results which were

lipoplex samples. Analysis was carried out by means of flow cytometry (see uptake and FACS analysis).
