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PHOTOCHEMICAL TREATMENT STIMULATES CATIONIC POLYMER-MEDIATED, BUT NOT CATIONIC LIPID-MEDIATED TRANSFECTION
Lina Prasmickaite, Anders Høgset, Marit Hellum, Birgit Ø. Engesæter, Vibeke M. Olsen, Carl J. Wheeler*, Kristian Berg
Department of Biophysics, Inst. for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway
*Vical Inc., 9373 Towne Centre Drive, San Diego, CA 92121, USA
One of the biggest barriers for gene transfer by vectors delivering transgene by the endocytic pathway is inefficient escape from the endocytic vesicles. To improve endosomal release we have developed a new technology, named photochemical internalisation (PCI), based on photochemical reactions initiated by photosensitizers localised in endocytic vesicles and inducing rupture of these vesicles upon light exposure. This technology constitutes an efficient light-inducible gene transfer method in vitro, which potentially can be developed into a site-specific method for gene delivery in vivo.
In the present work the effect of the photochemical treatment on transfection mediated by different transfection vectors has been studied in human melanoma THX and human colon carcinoma HCT 116 cells in vitro, employing a transgene encoding enhanced green fluorescence protein (EGFP). We have shown that the photochemical effect on transfection is dependent on the transfection agent used for delivering the gene. Thus, photochemical treatment by the photosensitizer aluminium phthalocyanine (AlPcS2a) substantially improved transfection (polyfection) mediated by cationic polymers (poly-L-lysine, polyarginine, transferrin-polylysine, polyethylenimine) that in the absence of light transfected only few percent of cells. In contrast, photochemical treatment reduced transfection (lipofection) with cationic lipids (1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Lipofectin and others). Moreover, the photochemical inhibition of lipofection was a general phenomenon for all photosensitizers irrespective of their localisation, whereas photochemical stimulation of polyfection was observed only for photosensitizers like AlPcS2a localised in the membranes of endocytic vesicles. Thus, the results have revealed important differences between polyfection and lipofection, reflecting differences in the mechanism of action for two types of transfection agents.
INTRODUCTION
Most non-viral gene transfection vectors deliver the transfecting DNA into cells through the endocytic pathway. Inefficient escape from endocytic vesicles in many cases constitutes a major barrier for delivery of a functional transgene, which is unable to reach the cytosol and further be transported to the nucleus, but rather is trapped in the vesicles and finally degraded in lysosomes.
Photochemical internalisation (PCI) is a new endosome-disruptive strategy to achieve light-inducible permeabilisation of endocytic vesicles. The technology is based on photochemical reactions initiated by photosensitizers localised in endocytic vesicles and inducing rupture of these vesicles upon light exposure (Fig 1).
TASK
To study the effect of photochemical treatment on transfection mediated by different non-viral transfection vectors.
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MATERIALS AND METHODS
Cells - human melanoma THX and human colon carcinoma HCT 116 cells.
Transfecting DNA and transfection vectors - The plasmid pEGFP-N1, carrying a gene for enhanced green fluorescence protein (EGFP) that fluoresces after synthesis and folding, has been used as a model. The plasmid (either 5mg or 1mg) was delivered into the cell as a complex with different transfection agents such as cationic polypeptides, cationic polymers and cationic lipids named in Abbreviations section.
Photosensitizers - most of the experiments were carried out using the photosensitizer AlPcS2a, which localises in the membranes of endocytic vesicles and is activated by light of 670nm.
Other photosensitizers used and their intracellular localisation are shown in the Abbreviations section. Light of appropriate wavelength for excitation of these photosensitizers was used.
Transfection efficiency was evaluated measuring EGFP expression by flow cytometry. Cells which possessed elevated green fluorescence were designated as positive for EGFP synthesis (Fig. 2, cells to the right side of the drawn line).
Photochemical transfection (scheme):
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RESULTS AND DISCUSSION
The effect of photochemical treatment on gene transfection is dependent on the transfection vector used to deliver a transgene.
Photochemical treatment by AlPcS2a and light substantially improved transfection mediated by cationic polypeptides (polyfection) such as polylysines (Fig.2, Fig. 3B, D, red bars) or PEI (green bars) that in the absence of light showed very low efficiency in both cell lines tested (Fig. 3A, C, red and green bars).
Photochemical treatment had an inhibitory or only negligible positive effect on transfection mediated by cationic lipids (lipofection) (Fig.2, Fig. 3B, D, yellow bars) or Tf-PEI (green bars) that were more efficient transfection vectors in the absence of light (Fig. 3A, C, yellow and green bars).
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AlPcS2a and TPPS2a, photosensitizers localising in the membranes of endocytic vesicles, substantially improved polyfection with pEGFP-N1/PLL upon light exposure (Fig.4A, red bars). Photosensitizers localising elsewhere had lower or no effect on polyfection, indicating that photochemical permeabilisation of vesicular membranes is the mechanism underlying PCI and photochemical transfection.
All the photosensitizers irrespective of their localisation hindered lipofection with pEGFP-N1/DOTAP (Fig.4B, red bars), indicating some general inhibitory photochemical effects on lipofection.
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CONCLUSIONS
• Photochemical transfection gives a possibility to achieve a light-inducible increase in transfection efficiency. However, the photochemical effect on transfection depends on the transfection vector used to deliver the transgene. Photochemical treatment with AlPcS2a stimulates polyfection, but not lipofection.
• The photochemical effect on polyfection depends on the properties of the photosensitizer. Amphiphilic photosensitizers localised in the membranes of endocytic vesicles show the strongest stimulating effect on transfection.
• Photochemical inhibition of lipofection is a general phenomenon for all the photosensitizers irrespective of their localisation.
Localised in the membranes of endocytic vesicles:
AlPcS2a - aluminum phthalocyanine with 2 sulfonate groups on adjacent phthalate rings;
TPPS2a - meso-tetraphenylporphine with 2 sulfonate groups on adjacent phenyl rings;
Localised in the matrix of endocytic vesicles:
TPPS4 - meso-tetraphenylporphine with 4 sulfonate groups;
TMPyP - meso-tetra(N-methyl-4-pyridyl)porphine;
Localised not in endocytic vesicles:
3THPP - tetra(3-hydroxyphenyl)porphyrin;
Ala - 5-aminolevulinic acid.
Transfection agents
Cationic polypeptides:
L-PLL - poly-L-lysine;
D-PLL - poly-D-lysine;
Tf-PLL - transferrin-polylysine;
Cationic polymers:
PEI - polyethylenimine;
Tf-PEI - transferrin-polyethylenimine;
Cationic lipids:
DOTAP - 1,2-dioleoyl-3-trimethylammonium-propane;
DMRIE - dimyristoyl Rosenthal inhibitor ether;
DOPE - dioleoylphosphatidylethanolamine;
bAE-DMRIE - ß-aminoethyl-DMRIE;
bAE-DMRIE-DOPE - ß-aminoethyl-DMRIE-DOPE.