**4.2 Microscopic uptake studies**

The results obtained with uptake studies were confirmed using spectral-bio-imaging. Cells were first pre-incubated with the inhibitors chlormpromazine or filipin. Therefore, the internalization routes were examined via co-localization studies with markers for the clathrin- or caveolae-dependent pathways. The DNA of the lipoplex was labeled with Rhodamine (Rh). Transferrin alexa-fluor 488 was used as a clathrin marker, Choleratoxinsubunit B-Alexa Fluor 488 was a marker for internalization via caveolae-dependent endocytosis and FITC-dextran a marker for macropinocytosis. During preparation for the microscopic experiments, cells were additionally incubated with DAPI (nucleus staining) as a control. Co-localization of the lipoplex and pathway markers was determined by biospectral-imaging.

In HAEC after one h incubation time, co-localization of lipoplexes with tf-488 (Fig. 3A) as well as with chltx-B-488 (Fig. 3B) was detected which indicates uptake via clathrin- and

Investigation of Transfection Barriers Involved in

clathrin-dependent endocytosis internalization via clathrin coated pits

golgi apparatus.

early endosome pH 5,5-6,0

> MVB pH 5,0

late endosome pH 4,5-5,0

show activity.

Non-Viral Nanoparticulate Gene Delivery in Different Cell Lines 87

Fig. 5. Cartoon of the main cellular internalization routes of lipoplexes. On the left hand the

golgi

caveosome

cytosol

endoplasmatic reticulum

transcytosis

caveolae-dependent endocytosis internalization via lipid rafts

It showed, that poor transfection efficiency of primary human aorta and smooth muscle cells (HAEC and HASMC) is not due to insufficient delivery into the cell. Uptake of lipoplex reached 20 % in HAEC and 80 % in HASMC whereas transfection was less than 3 % in both cases. Furthermore, it was shown, that plasmid DNA, transferred directly into the cytosol of the cell by electroporation effectively transfected 40 to 50% of the cells. These data suggest, that DNA, once being present in the cytosol, is able to cross the nucleolae membrane and to

In the present study it was shown, that lipoplexes are partly internalized in HAEC via caveolae mediated endocytosis. The caveolae route is known to enable transport of nanoparticulate drug delivery systems into and/or through the cell without involvement of lysosomal compartments. In case of drugs being sensitive to acidic hydrolysis or enzymatic degradation, e.g., biomacromolecules such as polypetides or nucleic acids, it is important to avoid cellular routes that transport material to lysosomes. On the other side, uptake via

clathrin-dependent pathway and on the right hand the caveolae-mediated uptake mechanisms are shown. Whereas the first pathway has been well investigated, caveolinmediated internalization has not been examined in detail to date. The first step in the clathrin-dependent pathway is the budding of clathrin-coated vesicles resulting in early or sorting endosomes. Recycling back to the membrane happens in recycling endosomes. Early endosomes transform in MVB and subsequently late endosomes are formed which end up as lysosomes. Active exchange between the golgi apparatus and late endosomes is known. Particles, internalized via caveolae from so-called caveosomes offering various processing possibilities such as transcytosis, cytosolic release, delivery to the ER or transport to the

recycling compartment

pH 6,8

lysosome

caveolae-dependent pathways. No co-localization with the macropinocytosis marker FITCdextran 70,000 could be shown.

In HASMC, Rh-labeled lipoplexes only showed co-localization with tf-488 (Fig. 4), the marker for clathrin-dependent uptake.

In both cases, spectral bio-imaging studies confirmed the results achieved by flow cytometry.

Fig. 4. HASMC were incubated for one hour with clathrin marker tf alexa-fluor 488 and Rhlipoplexes. After subsequent DAPI staining of the nucleus pictures were taken by spectral bio-imaging. I: localization of Rh-lipoplexes; II: localization of tf alexa-fluor 488; III: overlay of single colour images.

#### **4.3 Effects on transfection efficiency**

As cellular internalization of lipoplexes was successfully achieved in both HAEC and HASMC by clathrin-dependent endocytosis, which results in the transport of lipoplexes via endosomes (Fig. 5), further investigations examined possible barriers after uptake.

For these experiments DNA was introduced into the cell by electroporation. With this method, endosomal release and disintegration of the DNA from the lipoplex are no longer involved. Basically, it can be determined that the introduced DNA is not degraded in the cytosol and is able to cross the nuclear membrane to enter the nucleus. Indeed, electroporation resulted in transfection efficiencies of 43 % in HAEC and 51 % in HASMC (Table 2, Fig. 6), indicating that transfection is restricted either by insufficient endosomal release or insufficient release of DNA from the lipoplex.

To investigate insufficient endosomal release, transfection experiments with DC-30® lipoplexes were accomplished with HAEC and HASMC as described before [5]. Additionally, prior to lipoplex incubation the cells were incubated with different concentrations of chloroquine, which represses lysosomal degradation by increasing the pH [21] to facilitate endosomal escape and therefore improve transfection efficiency. Addition of chloroquine did not lead to an increase in transfection efficiency (Table 2). This shows no insufficient endosomal escape of lipoplex.

#### **5. Discussion**

This study demonstrates, that the development of efficient non-viral gene delivery systems requires better understanding of the mechanism of the delivery and of the intracellular processing. Endocytosis is the major pathway of entry, but the involvement of specific endocytic pathways is still poorly defined. This study contributes to further clarify uptake mechanisms and barriers in gene delivery in primary cells.

caveolae-dependent pathways. No co-localization with the macropinocytosis marker FITC-

In HASMC, Rh-labeled lipoplexes only showed co-localization with tf-488 (Fig. 4), the

In both cases, spectral bio-imaging studies confirmed the results achieved by flow

Fig. 4. HASMC were incubated for one hour with clathrin marker tf alexa-fluor 488 and Rhlipoplexes. After subsequent DAPI staining of the nucleus pictures were taken by spectral bio-imaging. I: localization of Rh-lipoplexes; II: localization of tf alexa-fluor 488; III: overlay

As cellular internalization of lipoplexes was successfully achieved in both HAEC and HASMC by clathrin-dependent endocytosis, which results in the transport of lipoplexes via

For these experiments DNA was introduced into the cell by electroporation. With this method, endosomal release and disintegration of the DNA from the lipoplex are no longer involved. Basically, it can be determined that the introduced DNA is not degraded in the cytosol and is able to cross the nuclear membrane to enter the nucleus. Indeed, electroporation resulted in transfection efficiencies of 43 % in HAEC and 51 % in HASMC (Table 2, Fig. 6), indicating that transfection is restricted either by insufficient endosomal

To investigate insufficient endosomal release, transfection experiments with DC-30® lipoplexes were accomplished with HAEC and HASMC as described before [5]. Additionally, prior to lipoplex incubation the cells were incubated with different concentrations of chloroquine, which represses lysosomal degradation by increasing the pH [21] to facilitate endosomal escape and therefore improve transfection efficiency. Addition of chloroquine did not lead to an increase in transfection efficiency (Table 2). This shows no

This study demonstrates, that the development of efficient non-viral gene delivery systems requires better understanding of the mechanism of the delivery and of the intracellular processing. Endocytosis is the major pathway of entry, but the involvement of specific endocytic pathways is still poorly defined. This study contributes to further clarify uptake

endosomes (Fig. 5), further investigations examined possible barriers after uptake.

10µm

SUN-picture

dextran 70,000 could be shown.

cytometry.

marker for clathrin-dependent uptake.

cholera toxin-B alexa-fluor 488 Rh-DC-30 lipoplexe

**4.3 Effects on transfection efficiency** 

insufficient endosomal escape of lipoplex.

**5. Discussion** 

release or insufficient release of DNA from the lipoplex.

mechanisms and barriers in gene delivery in primary cells.

of single colour images.

Fig. 5. Cartoon of the main cellular internalization routes of lipoplexes. On the left hand the clathrin-dependent pathway and on the right hand the caveolae-mediated uptake mechanisms are shown. Whereas the first pathway has been well investigated, caveolinmediated internalization has not been examined in detail to date. The first step in the clathrin-dependent pathway is the budding of clathrin-coated vesicles resulting in early or sorting endosomes. Recycling back to the membrane happens in recycling endosomes. Early endosomes transform in MVB and subsequently late endosomes are formed which end up as lysosomes. Active exchange between the golgi apparatus and late endosomes is known. Particles, internalized via caveolae from so-called caveosomes offering various processing possibilities such as transcytosis, cytosolic release, delivery to the ER or transport to the golgi apparatus.

It showed, that poor transfection efficiency of primary human aorta and smooth muscle cells (HAEC and HASMC) is not due to insufficient delivery into the cell. Uptake of lipoplex reached 20 % in HAEC and 80 % in HASMC whereas transfection was less than 3 % in both cases. Furthermore, it was shown, that plasmid DNA, transferred directly into the cytosol of the cell by electroporation effectively transfected 40 to 50% of the cells. These data suggest, that DNA, once being present in the cytosol, is able to cross the nucleolae membrane and to show activity.

In the present study it was shown, that lipoplexes are partly internalized in HAEC via caveolae mediated endocytosis. The caveolae route is known to enable transport of nanoparticulate drug delivery systems into and/or through the cell without involvement of lysosomal compartments. In case of drugs being sensitive to acidic hydrolysis or enzymatic degradation, e.g., biomacromolecules such as polypetides or nucleic acids, it is important to avoid cellular routes that transport material to lysosomes. On the other side, uptake via

Investigation of Transfection Barriers Involved in

Pharm Res 21 (2004) 1009-1017.

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Non-Viral Nanoparticulate Gene Delivery in Different Cell Lines 89

beta-cyclodextrine; chlp, chlorpromazine; chtx-B, cholera toxin subunit B; tf, human transferrin; wm, wortmannin; gen, genistein; fil, filipin; noco, nocodazole; Ly, Ly294002 ; Rh,

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Fig. 6. HAEC (A) and HASMC (B) after electroporation of pEGFP and 48 h incubation. Lefthanded: brightfield; Right handed: fluorescent picture (GFP), middle position: overlay of pictures.

caveolae does not automatically result in effective transfection as shown in this study. Although uptake in HAEC was partly achieved by the caveolae route, no transfection occurred. This is important to consider, because obviously it is not enough to focus the research on modifying particulate drug carriers, e.g., by attaching specific ligands, introducing surface charge or pH-sensitivity, or changing the particle size or elasticity in order to trigger the caveolae mediated endocytosis.

The use of different methods (flow cytometry, spectral bio-imaging, electroporation) and materials (markers and inhibitors of endocytosis pathways) contributed to the evidence that transfection of primary human aorta endothelium and smooth muscle cells was not effective due to insufficient dissociation of plasmid DNA from the lipid/DNA delivery system. This finding is of great importance for future drug deliver development in the field of gene therapy.
