Cationic Polyamidoamine Dendrimers As Modulators Of Egfr Signaling In Vitro And In Vivo

Cationic polyamidoamine (PAMAM) dendrimers are branch-like spherical polymers being investigated for a variety of applications in nanomedicine including nucleic acid drug delivery. Emerging evidence suggests they exhibit intrinsic biological and toxicological effects but little is known of their interactions with signal transduction pathways. We previously showed that the activated (fragmented) generation (G) 6 PAMAM dendrimer, Superfect (SF), stimulated epidermal growth factor receptor (EGFR) tyrosine kinase signaling - an important signaling cascade that regulates cell growth, survival and apoptosis-in cultured human embryonic kidney (HEK 293) cells. Here, we firstly studied the in vitro effects of Polyfect (PF), a non-activated (intact) G6 PAMAM dendrimer, on EGFR tyrosine kinase signaling via extracellular-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) in cultured HEK 293 cells and then compared the in vivo effects of a single administration (10mg/kg i.p) of PF or SF on EGFR signaling in the kidneys of normal and diabetic male Wistar rats. Polyfect exhibited a dose- and time-dependent inhibition of EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK-293 cells similar to AG1478, a selective EGFR inhibitor. Administration of dendrimers to non-diabetic or diabetic animals for 24h showed that PF inhibited whereas SF stimulated EGFR phosphorylation in the kidneys of both sets of animals. PF-mediated inhibition of EGFR phosphorylation as well as SF or PF-mediated apoptosis in HEK 293 cells could be significantly reversed by co-treatment with antioxidants such as tempol implying that both these effects involved an oxidative stress-dependent mechanism. These results show for the first time that SF and PF PAMAM dendrimers can differentially modulate the important EGFR signal transduction pathway in vivo and may represent a novel class of EGFR modulators. These findings could have important clinical implications for the use of PAMAM dendrimers in nanomedicine

Comparison of the <i>in vivo</i> effects of Superfect (SF) and Polyfect (PF) PAMAM dendrimers on EGFR phosphorylation and histopathology in the non-diabetic rat kidney.

<p>A single dose of SF or PF (10mg/kg) or AG1478 (1mg/kg) was administered by i.p. injection 24 h prior to sacrifice and subsequent analysis. Panel a) shows representative Western blots and panels b-d) shows the densitometric histograms showing the relative intensity levels of phosphorylated (p-) EGFR bands relative to the actin loading control or to total EGFR protein in the non-diabetic rat kidney. Error bars represent mean ± SEM. N = 4. * indicates statistically significant from untreated controls. Panel e) shows representative photomicrographs of the morphology of the kidneys taken from non-diabetic control (C) animals and those treated with PF or SF or AG1478 as indicated. The kidneys were fixed in formalin and mid-transverse sections taken following paraffin embedding and hematoxylin-eosin staining.</p

The effect of AG1478 or PF and SF PAMAM dendrimers on rat body weight and blood glucose following acute 24 hr <i>in vivo</i> administration in non-diabetic controls (C) and diabetic animals (D).

<p>Asterix (*) indicates significantly different from controls (p<0.05).</p><p>(Means ± SEM; N = 4).</p

Superfect (SF) and Polyfect (PF) PAMAM dendrimer-mediated apoptosis is inhibited by the antioxidant, Tempol, in HEK293 cells.

<p>The % of cells undergoing apoptosis is shown as a) histograms and b) representative flow cytometry data plots for cells treated with PF alone, SF alone or together with Tempol (+ TEMP) and AG1478 alone. Error bars represent mean ± SEM. N = 5. * indicates statistically significant from control cells # indicates statistically significant from the respective dendrimer alone treatment.</p

Effects of Polyfect (PF) PAMAM dendrimer dose on phosphorylation of ERK1/2 and p38 MAPK in HEK293 cells.

<p>Cells were treated with the stated doses of PF for 4h and analyzed after 24h or with AG1478 for 24h as per the Methods. Panel a) shows representative Western blots and panels b) shows the densitometric histograms showing the relative intensity levels of phosphorylated (p-) ERK1/2 and phosphorylated p38 MAPK (labelled as p-P-38) bands relative to the actin loading control. Error bars represent mean ± SEM. N = 5. * indicates statistically significant from untreated controls.</p

Dose-dependent effects of Polyfect (PF) PAMAM dendrimer on phosphorylated (p-) and total (t-) EGFR levels in HEK293 cells.

<p>Cells were treated with the stated doses of PF for 4h and analyzed after 24h or with AG1478 (10 μM) for 24h as per the Methods. Panel a) shows representative Western blots and panels b-d show densitometric histograms showing the relative intensity levels of pEGFR (b), total EGFR (c) bands relative to the actin loading control and the ratio of the two (panel d). Error bars represent mean ± SEM. N = 5. (Asterix) * indicates statistically significant from untreated controls.</p

The effect of acute <i>in vivo</i> administration PF and SF PAMAM dendrimers on morphological changes in non-diabetic and diabetic rat kidneys.

<p><b>Key:</b> Morphology was graded as either (0) none, (+) mild, (++) moderate or (++++) severe.</p><p>^<b>$</b>GS: diffuse glomerular sclerosis; EX: extracelluar matrix deposition (or interstitial fibrosis); FTI: fibrosis in tubulointerstitium; HPAS: hyperplastic arteriolosclerosis; IIVCIH: intensity and incidence of vacuolations, cellular infiltration and hypertrophy.</p><p>The effect of acute <i>in vivo</i> administration PF and SF PAMAM dendrimers on morphological changes in non-diabetic and diabetic rat kidneys.</p

Dose-dependent effects of Polyfect (PF) PAMAM dendrimer on HEK 293 cell viability.

<p>Error bars represent mean ± SEM. * indicates statistically significant from untreated controls. N = 5.</p

The effect of anti-oxidants apocynin (APO), Tempol (TEMP) and catalase (CAT) on PF PAMAM dendrimer-induced EGFR, ERK1/2 and p38 MAPK phosphorylation in HEK 293 cells.

<p>Cells were pretreated for 30 min with the stated antioxidant and then co-treated with PF (40 μg/ml) for 4h in serum-free media. Panel a) shows a representative Western blot and panel b-d) shows the densitometric histograms showing the relative intensity levels of phosphorylated (p-) EGFR, ERK1/2 and p38 MAPK (labelled as p-P38) bands relative to the actin loading control. Error bars represent mean ± SEM. N = 5. * indicates statistically significant from untreated controls # indicates statistically significant from PF alone treatment.</p

The <i>in vivo</i> effects of Superfect (SF) and Polyfect (PF) PAMAM dendrimers on EGFR phosphorylation and histopathology in the kidney of diabetic rats.

<p>After 4 weeks of diabetes, a single dose of SF or PF (10mg/kg) or AG1478 (1mg/kg) was administered by i.p. injection 24 h prior to sacrifice and subsequent analysis. Panel a) shows representative Western blots and panels b-d) shows the densitometric histograms showing the relative intensity levels of phosphorylated (p-) EGFR bands relative to the actin loading control or to total EGFR protein in the non-diabetic rat kidney. Error bars represent mean ± SEM. N = 4. * indicates statistically significant from untreated controls and # indicates statistically significant from diabetes. Panel e) shows representative photomicrographs of the morphology of the kidneys taken from diabetic control (D) animals and those treated with PF or SF or AG1478 as indicated. The kidneys were fixed in formalin and mid-transverse sections taken following paraffin embedding and hematoxylin-eosin staining.</p