Optimization of vector system 4.3.2 Composition of transfection comple

4.3.2.1. Optimal concentration of plasmid DNA

The effect of changing the concentration of plasmid DNA in the complex over a 3- fold range was investigated (see Figure 4.5).

4 0 0 0 0 0 n c ^ 3 0 0 0 0 0 - o L_ Q. 1*2 0 0 0 0 0^ _i a: 1000004

Control 1ug 2ug 3ug

Amount of plasmid DNA per well

Figure 4.5 Transfection of normal fibroblasts with transfection complexes containing different amounts of plasmid (pg).

Vector: Lipofectin- peptide 1- luciferase plasmid (weight ratio 0.75:4:1-3) Transfection time: 6h Expression time: 48h

Luciferase measurements were performed in triplicate and expressed as RLU / mg of protein. The line represents the mean of three observations. The control is untransfected cells.

Optimal transfection was observed with 1pg plasmid DNA. Increasing the amount of DNA decreased the luciferase activity and also increased the

amount of cell death as observed by examination with a light microscope. This would suggest that the cells are sensitive to the uptake or presence in the medium of larger amounts of LID complex.

4.S.2.2. Different integrin-targeting peptides

Details of the structures of the different peptides used in this study are given in Table 2.7. The original protocol had been optimised using ECV304 cells and peptide 6 in the complex. Peptide 6, [KieJGACRRETAWACG, is a specific aspi integrin ligand (Koivunen et al., 1995). Since different cells express different classes of cell surface integrins, a number of hybrid

transporter peptides with different sequences surrounding the arginine-glycine- 138

Chapter 4. Optimization of vector system

aspartic acid (RGD) or integrin-targeting domain were tried. Peptide 1 ([Ki6]GACRGDMFGCA) (Hart et al., 1994, 1995) and peptide 5

([[KieJGACDCRGDCFCA) (Kouvinen et al., 1995; Hart et al., 1997) contain RGD sequences which bind to as and av integrins. The integrin-binding domain of peptide 5 has a particularly high affinity for avps and avPs integrins (Kouvinen et al., 1995). Peptide 6 is specific for asPi integrins (Kouvinen et al., 1995). Peptide 7 is specific for integrins. Fibroblasts have been reported to express avPs(Memmo and McKeown- Longo, 1998) and asPi integrins (Dalton et al.,

1995).

Transfection complexes containing the integrin-targeting peptides 1,5,6 or 7 were prepared with pAB11 plasmid DNA and Lipofectin with a constant charge ratio. The charge ratios of LID complexes may affect their transfection efficiency and were deduced from the molecular weight and charge parameters in Table 2.8. Liposome- DNA complexes for control lipofection (i.e. without peptide) consisted of Lipofectin and DNA in a transfection- optimised weight ratio of 5:1. The complexes were used to transfect normal human fibroblasts and the level of expression of p- galactosidase activity was determined histochemically (Table 4.1).

Peptide Cells expressing p- galactosidase None 0% 1 10% 5 8% 6 5% 7 1% Lipofection 1%

Table 4.1 Transfection of normal fibroblasts with transfection complexes containing peptide 1,5, 6 or 7 or by lipofection.

Vector: Lipofectin- different peptides- luciferase plasmid (weight ratio 0.75: 4: 1).

Chapter 4.__________________________________________________Optimization of vector system

The results demonstrated that the Lipofection was ineffective with only 1% of cells expressing (3-galactosidase. Peptide 1 was found to be the most efficient in delivery of lac Z reporter gene to normal fibroblasts with a

transfection efficiency of about 10% of the cells. Other peptides showed a lower transfection efficiency in the following order; 5> 6> 7.

To check that the integrin binding ligand was mediating transfection an experiment comparing peptides 1,11 and K16 (Figure 4.6) was carried out. Kie contains 16 lysines but lacks the integrin- binding domain of the other peptides and it has no known affinity for integrins. Peptide 11 is identical to peptide 1 except it contains an RGB sequence, rather than RGD, and does not bind integrins. Complexes were made with weight ratios which produced complexes of the same charge ratio.

400000-1 c B 300000- o w. Q. 2 200000n 3 _J a: 1 0 0 0 0 0-

Control Peptide 1 K16 Peptide 11

Peptide in complex

Figure 4.6 Transfection of normal fibroblasts with transfection complexes containing peptide 1,11 or Kie.

Vector; Lipofectin- different peptides- luciferase plasmid (weight ratio 0.75: 4: 1). Incubation time: 1h Transfection time: 6h Expression time: 68h Luciferase measurements were performed in triplicate and expressed as RLU / mg of protein. The line represents the mean of three observations. The control is untransfected cells.

Transfection complexes containing the peptides lacking the integrin binding domain, peptide 11 and Kie, did not give luciferase expression. This is strong evidence that transfection with this system is mediated by ligand

interaction with integrin.

Chapter 4.__________________________________________________Optimization of vector system

4.3.2.S. Peptide: DNA ratio

The effect of changing the ratio of peptide: DNA was studied (Figure 4.7). 7 5 0 0 0 0 - c 0 2 5 0 0 0 0 0 - Q. 3 2 5 0 0 0 0 - a: Control 4:1 5:1 6:1 PeptideiDNA ratio

Figure 4.7 Transfection of normal fibroblasts with transfection complexes containing peptide: DNA plasmid ratios of 4:1, 5:1 or 6:1.

Vector: Lipofectin- peptide 1- luciferase plasmid (weight ratio 0.75: 4-6: 1). Transfection time: 6h Expression time: 4 2 h

Luciferase measurements were performed in triplicate and expressed as RLU / mg of protein. The line represents the mean of three observations. The control is untransfected cells.

The highest level of gene expression was achieved with a ratio of 4pg peptide to Ipg plasmid DNA. Increasing the ratio to 5:1 or 6:1 reduced transfection efficiency. This is probably due to an excess amount of free peptide which would compete with the transfection complex to bind to the available cell surface integrins. Reducing the ratio below 4:1 could be beneficial.

4.3.2.4. Different liposomes and liposome concentrations

Receptor- mediated gene transfer involves the internalisation of complexes into endosomes. The complex must then leave the endosome in order to deliver DNA to the nucleus. A number of agents have been reported to aid the escape of complexes from endosomes. It has been reported that the incorporation of the cationic liposome 3p[N-(N’,N’-dimethylaminoethane) carbamoyijcholesterol (DC-chol)/ dioleoyl phosphatidylethanolamine (DOPE) into antibody-poly- L- lysine conjugates increases transfection efficiency (Trubetskoy et al., 1992). This is presumed to be due to lipid-mediated

Chapter 4. Optimization of vector system

destabilisation of the endosomai membrane. The cationic liposome, Lipofectin, enhances transferrin- mediated gene delivery (Cheng, 1996) and also integrin- mediated gene delivery (Hart et al., 1998). In this study various liposome formulations were incorporated into the transfection complex to determine which is the most successful in enhancing gene expression. The structures of the liposomes used are given in Table 2.8.