Results from microbiological assays(8
) showed that PGON
had an MIC of 6 μg/mL against E. coli
(see Table for abbreviations). Activity against an additional Gram-negative bacteria, S. marcescens
, which is not typically susceptible to AMPs, and two Gram-positive bacteria, S. aureus
and B. subtilis
, were also examined attesting to the broad activity of PGON
. For comparison, the Magainin derivative, MSI-78, had an MIC of 12 μg/mL against E. coli
ʼs close structural analogue, Poly-1
, having a primary amino group instead of a guanidino group, had MICs of 200−400 μg/mL and was thus considered to be relatively inactive. In contrast, the MICs of the more hydrophobic, isobutylidene-derivatized Poly-3
were found to be between 12−25 μg/mL for all bacteria, representing good activity. However, and in sharp contrast to Poly-3
, broadly active PGON
possessed a lack of toxicity toward RBCs (HC50
= 1500 μg/mL). As a result of PGON
ʼs high HC50
value, its selectivity of 250 is significantly better than other polymeric SMAMPs.1,2,4
Again, as a comparison, MSI-78 had a selectivity of 10 for E. coli
over RBCs using the HC50
Bactericidal kinetics studies (also known as time-kill assays) were performed on S. aureus to differentiate growth inhibition from cell death and showed that PGON was lethal at 4× the MIC with a 5-log reduction in less than 60 min (Figure ). Starting with ~105 cells/mL, aliquots were removed periodically and viable cells counted via plating techniques. The results clearly showed PGONʼs ability to kill bacteria (bactericidal activity) not just inhibit growth (bacteriostatic activity).
Time kill plots for S. aureus. CFU = colony forming unit; squares = untreated cells; white circles = treated with PGON at 1× MIC; and black circles = at treated with PGON 4× MIC.
Many AMPs and their structural mimics have been shown to be membrane-active with the balance of their hydrophobic/hydrophilic properties critical to their mode of action.(10
) Similarly, the biological properties of Poly-1
were previously explained based on their amphiphilicity, in agreement with the concept that the proper balance of hydrophobic and hydrophilic groups is often essential for the activity of AMPs.(2
was the most hydrophilic and consequently not membrane-active, presumably because it can not sufficiently disrupt the lipid bilayer. In contrast, Poly-3
, being significantly more hydrophobic, exhibited good antibacterial activity but unfortunately showed high hemolysis. With its outstanding biological properties, it appeared that PGON
had attained a favorable amphiphilicity.
High performance liquid chromatography (HPLC) retention times are routinely exploited in the evaluation of a peptide’s, or peptidomimetic’s relative amphiphilicity.(5b
) Retention times for Poly-1
, and PGON
, were 19.9, 25.5, and 22.4 min, respectively, indicating that the guanidinium groups of PGON
resulted in a polymer with intermediate hydrophobic character. Therefore, from the microbiology and HPLC data, it was expected that PGON
would exhibit strong membrane-lysis in dye-leakage assays using vesicles composed of phosphatidyl ethanolamine (PE) and phosphatidyl glycerol (PG) phospholipids, which are typical of Gram-negative bacteria.(11
However, these assays showed that membrane-disruption induced by Poly-1 and Poly-3 did reflect their MICs, while PGON was completely inactive (Figure ). In other words, Poly-1, which displayed negligible antibacterial activity, caused little dye leakage (16%), while Poly-3, with good antibacterial activity, had significant dye release (87%). On the other hand, vesicles exposed to PGON were seemingly undisturbed (4% leakage). This lack of membrane disruption activity for PGON was unexpected given its high antibacterial efficiency and the fact that its hydrophobicity fell in between the two amine containing polymers.
Vesicle dye leakage after polymer addition.
While dye leakage assays are simple models and quite useful in many studies,1b,11
caution should be taken when correlating these results to biological activity. Thus, the effects of these polymers on S. aureus
were directly observed using a two-component fluorescence stain (Figure ). This stain uses a green-emitting SYTO9 dye that aids visualization of all cells and red-emitting propidium iodide, which only enters cells with compromised membranes.
Figure 3 Fluorescence microscopy of S. aureus (cells ~ 1 μm in diameter) incubated with a stain to visualize membrane-disruption and polymer for 30 min. Top row shows emission from S. aureus using a green filter setting, while the bottom row shows (more ...)
From these microscopy studies, Poly-1
appeared to be inactive (also confirmed by cell viability assays under the same microscopy conditions),(8
) and showed green intact individual S. aureus
cells (Figure A). In contrast, Poly-3
led to gross aggregation and clear membrane damage inferred from the dramatic increase in red fluorescence intensity (Figure D), while under the green filter the cells now appeared more yellow compared to the Poly-1
trials (compare Figure C to A and E). Cell viability assays confirmed Poly-3
is lethal under these conditions.(8
) Interestingly, PGON
, which is significantly bactericidal, as demonstrated by cell viability assays, did not aggregate bacteria nor allow propidium iodide to enter the cells. These results agreed completely with the dye leakage experiments in which Poly-1
caused little to no membrane damage relative to Poly-3
. This apparent ability to transverse the membrane without disruption is also consistent with CPP-like activity.(14
) At the same time, studies with a dye-labeled version of PGON
did support interactions between PGON
and bacterial cells under the same conditions but due to the small size of bacteria (nominally 1 μm), it was not possible to see if PGON
Accordingly, these results suggest that PGON
ʼs ability to effectively kill bacteria likely occurs via a different mechanism than gross membrane trauma. Likely targets would then be anionic macromolecules such as essential membrane proteins or RNA/DNA. Although no data is yet available on these other targets, CPPs and polyarginine, are well-known to efficiently cross membranes and bind DNA.3a,3d,12,13
Recently, we demonstrated the ability of these PGON
ʼs, with various chain lengths, to traverse membranes as well as other properties reminiscent of CPPs.(14
) Therefore, the proposal of intercellular targets is within reason.