Effect of Targeted Poly IC on 10-d-Old Intracranial Tumor Models

For this assay, 10,000 U87MGwtEGFR cells per animal were stereotactically implanted into the brains of 35 nude mice as described [8]. Five animals were sacrificed 10 d later to evaluate the sizes of the tumors [8]. Then, 200 ll of Alzet (Alzet, Cupertino, California, United States) osmotic micropumps with intra-tumoral catheters were installed in 20 mice. Ten mice received (poly IC)PEI-PEG-EGFþPEI-Mel complex dissolved in HBG buffer [9] at 0.1 lg poly IC/ll buffer (the dose of poly IC was 0.8 lg/h or 19.2 lg/d), and ten others received equivalent doses of PEI-PEG-EGFþPEI-Mel complexes only (no poly IC). Ten remaining control animals did not receive any treatment. The pumps were replaced twice every 24 h. At day 20 after cell implantation (10 d after treatment initiation), five animals from each group were sacrificed to evaluate tumor size as described [8]. Five other animals in each group were retained for survival analysis [8]. All animal experiments were conducted in accordance with the Hebrew University guidelines for the care of laboratory animals.

In Vivo Bystander Effect

For this assay, 5,000 U87MGwtEGFR cells were mixed with 5,000 U87MGDEGFR cells in 5 ll of PBS per animal. The cells were stereotactically implanted into the brains of 14 nude mice as described [8]. 10 d later, 200-ll Alzet osmotic micropumps with intra-tumoral catheters were installed in 12 mice. Six mice received (poly IC)PEI-PEG-EGFþPEI-Mel (poly IC/conjugate) complexes dissolved in HBG buffer at the indicated dose (two animals for each dose), and six others received an equivalent dose of PEI-PEG-EGFþPEI-Mel complexes without poly IC (Conjugate only). The pumps were replaced twice every 24 h. Survival of the animals was analyzed as above.

In Vivo Apoptosis Detection

For this assay, 10,000 U87MGwtEGFR cells per animal were stereotactically implanted into the brains of 12 mice as above. 14 d later, 200-ll Alzet osmotic micropumps with intratumoral catheters were installed in six U87MGwtEGFRbearing mice. Three animals received formulated poly IC (19.2 lg total per animal for 24 h), and three mice received complexes without poly IC. Six remaining animals received no treatment. Immediately after termination of infusion (15 d after cell implantation), animals were sacrificed, the brains were fixed with 4% formalin, embedded in paraffin, and ultra-thin slices were prepared. Slices were then de-paraffinized and analyzed by fluorescent immunohistochemistry to determine apoptosis with the Cell Death Detection kit-TMR Red, Roche (red fluorescence) and EGFR expression with FITC conjugated EGFR (internal domain) antibody (green fluorescence), Biosource (Camarillo, California, United States).

Synthesis of Mel-PEI25-PEG-mEGF

The synthesis of mEGF-PEG-PEI25 is described elsewhere [6,7]. mEGF-PEG-PEI25 (83 nmol PEI) was mixed with SPDP (664 nmol in 100% ethanol) under argon. After 3 h at room temperature the mixture of about 2 ml was loaded on a gel filtration column (Sephadex G25 superfine; HR10/30; 20mM HEPES [pH 7.1], 0.5M NaCl; Amersham Biosciences). The purified PDP-functionalized conjugate (5 ml) containing 309 nmol of PDP was concentrated to 1.5 ml by speed vac. For the reaction with Melittin 464 nmol of Mel was weighed out and dissolved in 0.5 ml of 0.5 M NaCl, 100mM HEPES [pH 7.4] degassed with argon. Both components were mixed under argon. After 20 h at room temperature mEGF-PEG-PEI25-Mel was purified by gel filtration. To gel filtrate the conjugate, a Superdex 75 prep grade column 10/30, conditioned with PEI25br (10 mg PEI25/60 ml gel material) was used. After dialysis overnight (MWCO 14000; Visking type 27/32; Roth, Karlsruhe, Germany) against HBS 6 ml of mEGF-PEG-PEI25- Mel conjugate were obtained; these contained 66 nmol PEI (1.64 mg), 350 nmol Mel, and 70 nmol EGF.

A431 Xenograft Treatment

Two million A431 cells were implanted subcutaneously into the left flanks of 15 nude CD-1 mice. When palpable tumors (average size 10.1 mm3) had developed, animals were divided into three groups of five animals. The complexes were delivered by intra-tumoral injection twice per day for 6 d. Five mice received poly IC-Melittin-PEI25-PEG-EGF (poly ICMPPE) complex dissolved in HBG buffer at 0.1 lg poly IC/ll buffer (the dose of poly IC was 15 lg/injection or 30 lg/d), and five others received equivalent doses of MPPE complexes only (no poly IC). Remaining animals did not receive any treatment. Length (a) and width (b) of the tumors were measured daily with caliper and the tumor size was calculated as ab2/2. Control animals were sacrificed at day 33 after treatment initiation. Poly IC treated animals were left for follow up study to detect recurrence of the tumors.

MDA-MB-468 Xenograft Treatment

Two million MDA-MB-468 cells were implanted over the mammary fat pad of 15 SCID/non-obese diabetic (NOD) mice. When palpable tumors (average size 9.8 mm3) had developed, animals were divided into three groups of five animals each. The complexes were delivered by intra-tumoral injection once per day for 6 d. Five mice received (poly IC)MPPE complex dissolved in HBG buffer at 0.1 lg poly IC/ll buffer (the dose of poly IC was 20 lg/d), and five others received an equivalent doses of MPPE complexes only (no poly IC). Tumor size was calculated as above. Control animals were sacrificed at day 33 after treatment initiation. Poly IC-treated animals were left for follow-up study to detect recurrence of the tumors.

Results

Fast and Selective Killing of EGFR

Over-Expressing GBM Cells In Vitro The PEI25-PEG-EGF complexes efficiently delivered poly IC, killing up to 85% of U87MGwtEGFR cells, which overexpress EGFR (;1 3 106 receptors [10]), within 1 h of transfection (Figure 1A). At poly IC concentrations of up to 10 lg/ml, no significant effect was observed on the parental U87MG cells, which express 100,000 of EGFR per cell [10], on cells that over-express the mutated D(2–7)EGFR (U87MGDEGFR), or on glioma cells lacking the EGFR (U138MG [11]) (Figure 1A). At the high concentration of 20 lg/ml poly IC, the survival of U87MG and U87MGDEGFR cells, was inhibited by 20%. U138MG cells, which completely lack EGFR, were not inhibited at all. The killing effect on U87MGwtEGFR cells was enhanced 8- to 10-fold when PEI25- PEG-EGF was partially replaced with the polyethylenimine (2 kDa)-Melittin conjugate PEI2-Melittin (Figure 1A), and more than 95% of U87MGwtEGFR died within an hour of transfection, again, with no effect on the other glioma cell lines (Figure 1A). Melittin is a bee venom peptide that facilitates the release of nucleic acids from the endosome into the cytoplasm [9,12], thus enhancing the release of poly IC from the endocytosed vesicle. No significant toxic effect of the complexes without poly IC on any of the cell lines was detected (Figure S1). Annexin V and TUNEL staining showed that the majority (70%–90%) of the U87MGwtEGFR cells died by apoptosis within 1 h of transfection (Figure 1B and 1C). Although fast apoptotic death is not common, it has occasionally been detected in other cell lines where, like here, a number of pro-apoptotic pathways are activated simultaneously [13,14].