**1. Introduction**

76 Non-Viral Gene Therapy

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endothelial cells by lipoplexes and polyplexes in the presence of nuclear targeting NLS-PEG-acridine conjugates. *Bioconjugate Chemistry*. Vol. 20, No. 1, (January 2009), In order to improve the delivery efficiency of genetic material into cells both *in vitro* and *in vivo*, the development of effective non viral vectors for optimized gene transfer into target cells has become an important objective. Non-viral vector systems in particular, such as cationic lipids and polymers, have been widely investigated as to their suitability as a delivery system [1-4]. In cell lines, non-viral gene transfer mediated by cationic lipid/DNA aggregates has been accomplished efficiently showing no immunogenicity and low cytotoxicity [5]. Unfortunately, non-viral gene transfer into primary cells is still inefficient and results in low transgene expression *in vivo* [6]. In contrast to the transfection of most cell lines, which can be successfully performed using a variety of methods, the introduction of foreign DNA into primary cells requires careful selection of the gene transfer technique. Whereas viral strategies are involved in immunogenic risks, non-viral methods have proved to be inefficient for most primary cell types. This might be due to the fact that biological barriers have to be overcome in order to achieve successful gene delivery.

Therefore, knowledge about the uptake mechanism and the subsequent intracellular processing of non-viral gene delivery systems is important for the development of efficient gene delivery systems. Moreover, in understanding the internalization of particles into cells, distinct pathways might be targeted.

Multiple processes are thought to be involved in the cellular internalization of particles [7], whereas clathrin-dependent uptake is the one which has been investigated the most. However, other internalization pathways such as the caveolae-dependent pathway, macropinocytosis, phagocytosis and the non-clathrin-non-caveolae dependent pathway are possible ways for gene delivery and further processing in the cells as well [8].

The mode of internalization may affect the kinetics of intracellular processing as well as transfection efficiency. Depending on the uptake mechanism a variety of obstacles could be the reason for low transfection efficiency. Also depending on the mode of cellular uptake, internalization may lead to either lysosomal degradation and digestion, recycling back to the membrane, transcytotic transport across the epithelial barrier or delivery to other compartments.

Once having been released into the cytosol, additional barriers such as insufficient desaggregation of the complex, poor cytoplasmic transport, cytosolic digestion by means of

Investigation of Transfection Barriers Involved in

atmosphere with 5 % (v/v) CO2.

260/280 nm and by gel electrophoresis.

(Molecular Probes, Leiden, Netherlands).

**3.2 Plasmid preparation** 

**3.3 DNA labeling** 

**3.4 Lipoplex formation** 

**3.5 In vitro transfection assay** 

**3. Methods 3.1 Cell culture** 

Non-Viral Nanoparticulate Gene Delivery in Different Cell Lines 79

Manassas, USA and pEGFP from BD Clontech Germany, Heidelberg. Unless otherwise stated, all other chemicals were purchased from Sigma/Fluka (Deisenhofen, Germany) and were of analytical grade. Additional material is described in the appropriate method section.

Human vascular smooth muscle cells (HASMC) were cultivated in smooth muscle cell medium 2 (Promocell, Heidelberg, Germany) supplemented with 5 % (v/v) FCS and human aortic endothelial (HAEC) were maintained in endothelial cell medium MV (Promocell, Heidelberg, Germany) on 100 mm culture plates. For HAEC, tissue culture plates were first coated with sterile-filtered 2 % gelatine solution for 10 min and washed twice with PBS w/o calcium and magnesium before seeding. Cells were cultivated at 37 °C in a humidified

The plasmid pEGFP carries the green fluorescent protein coding region under the control of the cytomegalovirus immediate-early promoter region. It was isolated from Escherichia coli (Stratagene, Amsterdam, NL) with the Maxi-Prep Kit from Qiagen (Hilden, Germany). Isolated DNA was stored in TE buffer (100 mM NaCl, 10 mM Tris-HCl) at a concentration of 1 mg/mL at -20 °C after its purity was verified by determining the ratio of absorbance at

Cy-5-labeled and Rh-labeled DNA was prepared as described for the Mirus Labeling Kit

Enhanced green fluorescent plasmid pEGFP (either labeled for uptake studies or unlabeled for transfection studies) was mixed with DC-30® in a lipid/DNA ratio (w/w) of 5:1 according to the following protocol: lyophilized DC-30® was redispersed in sterile transfection medium (TM; 250 mM saCcharose, 25 mM NaCl) at a concentration of 1 mg/ml and incubated for 30 min at room temperature. Plasmid DNA and the respective amount of DC-30®-dispersion were diluted separately into equal volumes of TM in order to achieve the desired lipoplex concentration in 200 µL of lipoplex preparation. The dilutions were combined discontinuously by pipetting the plasmid into the liposome solution, gently mixing and incubating for at least

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

30 min at room temperature to allow the formation of the lipoplexes.

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 events involved in each case.

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 strategy for cardiovascular gene-therapy.

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 macropinocytosis.

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 electroporation.


Table 1. Inhibitors for the specific endocytic routes
