Inhibition of bacterial growth and intramniotic infection in a guinea pig model of chorioamnionitis using PAMAM dendrimers

Abstract

Dendrimers have emerged as topical microbicides to treat vaginal infections. This study explores the in vitro, in vivo antimicrobial activity of PAMAM dendrimers, and the associated mechanism. Interestingly, topical cervical application of 500 microg of generation-4 neutral dendrimer (G(4)-PAMAM-OH) showed potential to treat the Escherichia coli induced ascending uterine infection in guinea pig model of chorioamnionitis. Amniotic fluid collected from different gestational sacs of infected guinea pigs posttreatment showed absence of E. coli growth in the cultures plated with it. The cytokine level [tumor necrosis factor (TNFalpha) and interleukin (IL-6 and IL-1beta)] in placenta of the G(4)-PAMAM-OH treated animals were comparable to those in healthy animals while these were notably high in infected animals. Since, antibacterial activity of amine-terminated PAMAM dendrimers is known, the activity of hydroxyl and carboxylic acid terminated PAMAM dendrimers was compared with it. Though the G(4)-PAMAM-NH(2) shows superior antibacterial activity, it was found to be cytotoxic to human cervical epithelial cell line above 10 microg/mL, while the G(4)-PAMAM-OH was non-cytotoxic up to 1mg/mL concentration. Cell integrity, outer (OM) and inner (IM) membrane permeabilization assays showed that G(4)-PAMAM-OH dendrimer efficiently changed the OM permeability, while G(4)-PAMAM-NH(2) and G(3.5)-PAMAM-COOH damaged both OM and IM causing the bacterial lysis. The possible antibacterial mechanism are G(4)-PAMAM-NH(2) acts as polycation binding to the polyanionic lipopolysaccharide in E. coli, the G(4)-PAMAM-OH forms hydrogen bonds with the hydrophilic O-antigens in E. coli membrane and the G(3.5)-PAMAM-COOH acts as a polyanion, chelating the divalent ions in outer cell membrane of E. coli. This is the first study which shows that G(4)-PAMAM-OH dendrimer acts as an antibacterial agent.

Fig. 1

Bacterial growth inhibition assays. E.coli was treated with the indicated concentration of G₄-PAMAM-NH₂ (A) and (B), G₄-PAMAM-OH (C) and (D), G_3.5-PAMAM-COOH (E) and (F) dendrimers for 18 h. The initial concentration used for bacterial seeding was 5×10⁵ CFU/mL. Three samples were in each group. Bacterial growth was measured by turbidity as the optical density at 650 nm using a microplate reader. * P<0.05, ** P<0.01, *** P<0.001 VS Positive control.

Fig. 2

SEM images of E.coli. (A) untreated E.coli (B) 8h treatment of G_3.5-PAMAM-COOH (C) 8h treatment of G₄-PAMAM-OH (D) 8h treatment of G₄-PAMAM-NH₂. Magnification 20000 ×. Scale bars indicate 5 µm. The treatment with dendrimers shows the damage to the bacterial cell wall.

Fig. 3

Release of intracellular components of E. coli suspensions treated with (A) G_3.5-PAMAM-COOH, (B) G₄-PAMAM-OH and (C) G₄-PAMAM-NH₂. Four samples were evaluated in each group. The increase in the absorbance is an indicator of the compromised cell integrity resulting in leaching of the nuclear components which are absorbed at 260nm.

Fig. 4

Uptake of NPN by E.coli suspensions treated with (A) G_3.5-PAMAM-COOH, (B) G₄-PAMAM-OH and (C) G₄ -PAMAM-NH₂. Four samples were in each group.

Fig. 5

Release of cytoplasmic β-galactosidase of E.coli treated with (A) G_3.5-PAMAM-COOH, (B) G₄-PAMAM-OH and (C) G₄-PAMAM-NH₂. Four samples were in each group.

Fig. 6

Cytotoxicity assay. (A) Human cervical epithelial End1/E6E7 cells and (B) mouse microglial cells were treated with the G₄-PAMAM-OH, G3.5-PAMAM-COOH and G₄-PAMAM-NH₂ dendrimers at concentrations indicated for MIC values. Three samples were in each group. Cell viability was assessed by MTT method. The proportion of viable cells in the treated group was compared to that of negative control.

Fig. 7

Flow cytometry of the cell entry dynamics of (A) G₄-PAMAM-OH-FITC in E. coli and (B) BV-2 microglial cell line. The log of FITC absorption intensity (FL1-H on x-axis) is plotted against the number of cells (counts on y-axis). The maximum uptake of G₄-PAMAM-OH-FITC in E. coli occurs at 3h. The rapid cellular uptake of G4-PAMAM-OH-FITC within 15 min in microglial cells is evident. The transport of conjugate into microglial cell increased with increasing time. Confocal microscopy images (400×) showed that G₄-PAMAM-OH-FITC appeared to be mainly localized in the cytoplasm of BV-2 cells while the nucleus appeared to be relatively free of the presence of any fluorescence at this time point (C).

Fig. 8

The placental tissue (0.3 g) was homogenized in 1 ml RIPA lysis buffer. The homogenate was kept on ice for 30 min and the protein concentration of supernatant was determined. Cytokines concentrations were measured in the total protein fraction using ELISA. (A) TNFα measurements in normal, E.coli infected and G4-PAMAM-OH treated guinea pigs (B) IL-6 measurements in normal, E.coli infected and G4-PAMAM-OH treated guinea pigs (C) IL-1β measurements in normal, E.coli infected and G4-PAMAM-OH treated guinea pigs * P<0.05, *** P<0.001 Vs. Normal control. ▲▲ P<0.01, ▲▲▲ P<0.001 Vs. E.coli group.