Supplementary information
Supplementary Figure 1The in vitro transfection activities of MEND in other cell lines
The in vitro transfection activity was also evaluated with three additional types of cells. The expression level of MMP-2 in the supernatant of the three cell lines was evaluated by an ELISA. MMP-2 was expressed highly in (a) MG-63 (68.7 ± 2.85 ng/ml) and (b) HOS (28.7 ± 4.52 ng/ml) and poorly in (c) PC-3 (7.09 ± 3.06 ng/ml). In the MMP-rich cell lines (MG-63 and HOS), the transfection activity of PPD-MEND was more than 10 times higher than that of PEG-MEND, whereas the enhancement was less prominent in the MMP-poor cells (PC-3). These data also support the conclusion that PPD-MEND was activated in response to the MMP expression. Luciferase activities of MEND, PEG-MEND and PPD-MEND are expressed as RLU per mg of protein. A value of 106 RLU represents the luciferase activity of 5 ng luciferase protein.
Figure 2 Effect of co-incubation of MMP-2 and conditioned medium from HT1080 on the gene expression of PPD-MEND in HEK293
The transfection activity of PPD-MEND to MMP-negative HEK293 cells was increased by a co-incubation with conditioned medium from MMP-positive HT1080 cells or with recombinant MMP-2. Luciferase activities of MENDs in HEK293 without supplement (open bars), with conditioned medium from HT1080 cells (gray bars) and with recombinant MMP-2 at 14 nM (closed bars). To obtain the conditioned medium, 4 x104 HT1080 cells were seeded in a 24-well plate, as described in the Materials and methods. After 24 h, the supernatant of the HT1080 cells was collected and centrifuged to remove the cell debris. The MEND was transfected with 0.25 ml of conditioned medium. Alternatively, recombinant MMP-2 was added to 0.25 ml DMEM in the supernatant of HEK293 cells, and then the MEND was applied to the supernatant. The luciferase reporter assay was performed as described in the Materials and methods. As a result, the transfection activity of PPD-MEND was promoted by these supplements more than 14-fold, indicating that PPD-MEND was activated in response to the soluble MMP secreted from HT1080 cells.Luciferase activities of MEND, PEG-MEND and PPD-MEND are expressed as RLU per mg of protein. A value of 106 RLU represents the luciferase activity of 5 ng luciferase protein. Each bar represents the mean ± SD, n = 3.
Table 1 AUC1→24 of MENDs
AUC1→24 was calculated as described previously35. Briefly, the blood concentrations at the indicated times (C(t))are expressed as %ID per ml of blood radioactivity. Pharmacokinetic parameters were determined by fitting the C(t) to a mono-exponential equation, usinga non-linear regression analysis by the MULTI program as follows:
The AUC1→24values were calculated based on the following equation:
Table 2 Fractions of PEG-MEND and PPD-MEND free in plasma
At 24 h after the i.v. injection of [3H]MEND, blood was collected and centrifuged at 12000 rpm for 10 sec, to separate the plasma and the blood cells. The radioactivity in the plasma and the total blood was measured, and the fraction free in the plasma was calculated, based on the assumption that the hematocrit value was 0.55. As a result, the fraction of 5% PEG-MEND free in the plasma was 4.2 times higher than that of 5% PPD-MEND. Similarly, the fraction of 15% PEG-MEND free in the plasma was 5.0 times higher than that of 15% PPD-MEND. Therefore, PPD-MEND is susceptible to binding the blood cells, and this may contribute to the poor tumor accumulation of PPD-MEND (Figure 6b).