Synthetic Mimic of Antimicrobial Peptide with Nonmembrane-Disrupting Antibacterial Properties

Article (PDF Available)inBiomacromolecules 9(11):2980-3 · November 2008with28 Reads
DOI: 10.1021/bm800855t · Source: PubMed
Abstract
Polyguanidinium oxanorbornene ( PGON) was synthesized from norbornene monomers via ring-opening metathesis polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly encourage the examination of other novel guanidino containing macromolecules as powerful and selective antimicrobial agents.
Communications
Synthetic Mimic of Antimicrobial Peptide with
Nonmembrane-Disrupting Antibacterial Properties
Gregory J. Gabriel,
Ahmad E. Madkour,
Jeffrey M. Dabkowski,
Christopher F. Nelson,
Klaus Nu¨sslein,
and Gregory N. Tew*
,†
Department of Polymer Science and Engineering and Department of Microbiology, University of
Massachusetts-Amherst, Amherst, Massachusetts 01003
Received July 31, 2008; Revised Manuscript Received August 22, 2008
Polyguanidinium oxanorbornene (PGON) was synthesized from norbornene monomers via ring-opening metathesis
polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive
bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is
lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of
antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy
experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly
encourage the examination of other novel guanidino containing macromolecules as powerful and selective
antimicrobial agents.
Introduction
We report that poly guanidinium oxanorbornene (PGON)
possesses a remarkable combination of antimicrobial activity
against both Gram-negative and Gram-positive bacteria as well
as low hemolytic activity against human red blood cells (RBCs).
Standard antibacterial, bacteria cell-viability, and hemolysis
assays all give data that is encouraging for the development of
powerful, yet nontoxic, antibacterial materials.
1
Additionally,
the properties of PGON surpass that of other cationic polymers,
in particular Poly-1 and Poly-3 from our previous study.
2
Vesicle investigations show that PGON is not membrane-
disruptive, supporting a previous study indicating it also has
properties similar to poly arginine and other cell-penetrating
peptides (CPPs) like HIV-TAT.
3
As a result, PGON represents
an exciting and fundamentally novel entry in the expanding field
of synthetic mimics of antimicrobial peptides (SMAMPs).
1,2,4,5
While several antimicrobial peptides (AMPs) contain the
amino acid arginine along with other basic residues, SMAMPs
have rarely used the guanidine functionality. Instead, the focus
has been almost exclusively on amines.
2,6
In this work, a new
Boc-protected guanidinium oxanorbornene monomer was po-
lymerized via ring-opening metathesis polymerization (ROMP)
using the third generation Grubbs’ catalyst to afford synthetic
poly arginine, PGON (PDI ) 1.07, MW of the deprotected
polymer-TFA salt ) 2.5 kDa,). This strategy is in contrast to a
postpolymerization functionalization technique recently pub-
lished that afforded polynorbornenes that efficiently promote
cellular internalization.
7
Lastly, polyamine norbornenes, Poly-1
and Poly-3, with similar MWs were prepared for comparison.
Experimental Section
Materials. Solvents and reagents for the monomer synthesis were
purchased from Aldrich and used without further purification. For the
polymer synthesis the second generation Grubbs’ catalyst, tricyclo-
hexylphosphine, (1,3-dimesitylimidazolidine-2-ylidine)benzylideneru-
thenium dichloride was purchased from Aldrich. This compound was
converted to a Grubbs’ third generation catalyst by reaction with
3-bromopyridine from Aldrich. Polymerizations were performed in dry
CH
2
Cl
2
from Acros (sealed under nitrogen with molecular sieves).
Chloroform solutions of lipids were obtained from Avanti Polar Lipids.
Calcein dye was purchased from Aldrich. The BacLight Staining Kit
L-7012 was obtained from Invitrogen Corp.
Synthesis of PGON. The synthesis of PGON starts with oxanor-
bornene 1,
1
and to this salt (1.4 g, 4.34 mmol) N,N-di-Boc-1H-pyrazole-
1-carboxamidine (4.0 g, 13.02 mmol) and triethylamine (4.1 g, 40
mmol) were added with 60 mL of solvent (90% CH
3
CN, 10% H
2
O).
This solution was stirred at room temperature for 12 h. The solution
was then diluted with ethyl acetate and washed twice each with H
2
O
and brine. The organic layer was evaporated and purified by silica
column chromatography using a MeOH in CH
2
Cl
2
gradient to afford
the Boc-protected guanidinium oxanorbornene monomer, 2.
This monomer was polymerized by ring-opening metathesis polym-
erization (ROMP) using a derivative of the second generation Grubbs’
catalyst, [(H
2
Imes)(3-Br-py)
2
-(Cl)
2
RudCHPh].
2
The polymerization for
* To whom correspondence should be addressed. E-mail: tew@
mail.pse.umass.edu.
Department of Polymer Science and Engineering.
Department of Microbiology.
Biomacromolecules 2008, 9, 2980–29832980
10.1021/bm800855t CCC: $40.75 2008 American Chemical Society
Published on Web 10/14/2008
PGON entailed adding to a test tube monomer (100 mg) plus catalyst
(27.4 mg). The test tube was capped with a septum and purged with
N
2
for 5 min, then 1 mL dry CH
2
Cl
2
was injected. The N
2
line was
removed and the clear, brown solution was stirred at 28 °C for 30 min
after which 0.4 mL of ethyl vinyl ether was injected to terminate the
polymer. After stirring for 15 min, the solution was added dropwise to
300 mL of stirring pentane to precipitate the polymer. The pentane
solution was stirred an additional 30 min and left standing undisturbed
for an hour. The precipitate was then collected by a fine sinter funnel.
The polymer was then redissolved in 1 mL of CH
2
Cl
2
, reprecipitated,
collected, and then dried by vacuum for 8 h.
The Boc-protected polymers were deprotected by stirring 100 mg
in 8 mL of 1:1 TFA/CH
2
Cl
2
for 2 h. The solution was concentrated to
an oil by rotary evaporator set at 40 °C and residual TFA was removed
by sonicating the oil in more CH
2
Cl
2
and evaporating the solvent again
by rotary evaporator. The resulting solid was placed under vacuum for
2 h. Finally, the solid was fully dissolved in 4 mL of H
2
O and filtered
through a polyethersulphone (PES) syringe filter (Whatman, 25 mm
diameter, 0.45 µm pore) and freeze-dried for 48 h to give an eggshell
colored soft solid. Final deprotected polymers were stored at -20 °C.
Boc-Protected Guanidinium Oxanorbornene Monomer, 2.
1
H
NMR (300 MHz, DMSO-d
6
): δ 11.44 (1H, s), 8.35 (1H, t, J ) 6.0
Hz), 6.56 (2H, s), 5.10 (2H, s), 3.52 (2H, m), 3.43 (2H, m), 2.85 (2H,
s), 1.47 (9H, s), 1.36 (9H, s).
13
C NMR (75 MHz, DMSO-d
6
): δ 176.3,
156.6, 153.0, 136.5, 83.3, 80.9, 47.6, 39.0, 38.4, 28.3, 28.1. HR-MS
(FAB
+
) calcd, 451.49; found, 451.22.
Boc-Protected PGON.
1
H NMR (300 MHz, DMSO-d
6
): δ 11.51
(1H, br), 8.47 (1H, br), 5.90 (trans) and 5.70 (cis; 2H total, br), 4.89
(cis) and 4.39 (trans; 2H total, br), 1.45 (9H, s), 1.38 (9H, br), cis/
trans ratio ) 46:54.
GPC of Boc-protected PGON: M
n
) 3.2 kDa, PDI ) 1.07 (After
deprotection the TFA-salt PGON has an approximate weight of 2.5
kDa).
PGON.
1
H NMR (300 MHz, DMSO-d
6
): δ 7.88 (1H, br), 7.36 (4H,
br), 5.96 (1H, br), 5.74 (1H, br), 4.92 (1H, br), 4.43 (1H, br) 3.47 (4H,
br).
Microbial and Hemolysis Assays. Bacteria suspensions of Ec, Sm,
Sa, and Bs,(Escherichia coli D31, Serratia marcescens ATCC 43861,
Staphylococcus aureus ATCC 25923, and Bacillus subtilis ATCC 8037)
were grown in Mueller-Hinton Broth (MHB) overnight at 37 °C,
diluted with fresh MHB to an optical density of 0.001 at 600 nm
(OD
600
). This OD
600
gave an initial bacterial concentration of 10
5
cells/mL. This suspension was mixed with different concentrations of
freshly prepared polymer solutions dissolved in DMSO (40 mg/mL
stock solution) by serial dilutions in a 96-well plate, and incubated for
6hat37°C. The OD
600
was measured for bacteria suspensions that
were incubated in the presence of polymer solution or only MHB.
Antibacterial activity was expressed as minimal inhibitory concentration
(MIC), the concentration at which more than 90% inhibition of growth
was observed after 6 h. All experiments were run in quadruplicate.
Hemolytic activity measurements were performed on all polymers.
Freshly drawn human red blood cells (RBC, 30 µL), were suspended
in 10 mL of Tris saline (10 mM Tris, 150 mM NaCl, pH 7.2, filtered
through PES membrane with 0.22 µm pore size) and rinsed three times
by centrifugation (5 min at 1500 rpm) and resuspended in Tris saline.
Polymer solutions were prepared in DMSO at 40 mg/mL and further
diluted as necessary. Freshly prepared polymer solutions with different
concentrations were added to 100 µL of the above-prepared RBC
suspension to reach a final volume of 200 µL on a 96-well plate. The
resulting mixture was kept at 37 °C for 30 min on a stirring plate.
Then the plate was centrifuged (IEC Centra-4B, 10 min at 1500 rpm),
and the supernatant in each well was transferred to a new plate.
Hemolysis was monitored by measuring the absorbance of the released
hemoglobin at 414 nm. Hemolysis (100%) was obtained by adding 10
µL of Triton-X (polyoxyethylene isooctylphenyl ether from Sigma-
Aldrich) solution (20 vol % in DMSO), a strong surfactant, to the above-
prepared HRBC suspension. The upper limit of polymer concentration
that was required to cause 50% hemolysis is reported as HC
50
, and the
absorbance from Tris saline containing no polymer was used as 0%
hemolysis. All experiments were run in quadruplicate.
Bactericidal Kinetics Studies. Bactericidal kinetics studies (also
known as time-kill assays) were performed on Sa to differentiate growth
inhibition from cell death. The protocol was adapted from literature.
3
Cells were grown in MHB to midexponential phase with an OD
600
0.3, and then adjusted to an OD
600
of 0.001. This OD
600
gave an
initial bacterial concentration of 10
5
cells/mL, the same as in the MIC
studies. Cells were either untreated or treated to PGON at 1× MIC or
four times the MIC
90
concentration and then placed under rotary
agitation at 37 °C. Aliquots were removed at different time points (0,
15, 30, 60, 120, and 240 min) and placed back at 37 °C with agitation.
Cells were diluted immediately to remove any effect of PGON. Serial
10-fold dilutions were spread on MHB agar plates and incubated at 37
°C for 18 h to determine viable colony counts. Viable cell counts were
plotted on a log-scale graph (CFU/mL vs time) to determine log-
reduction of bacterial growth compared to the untreated control.
Dye-Leakage from Large Unilamellar Vesicles. The following is
a general procedure to make large unilamellar vesicles (LUV) having
a hydrodynamic radius, R
h
120 nm (by dynamic light scattering).
Chloroform solutions of POPE (3.23 mg, 0.0045 mmol) and POPG
(1.15 mg, 0.0015 mmol; Avanti Polar Lipids, Inc.) were mixed in a 10
mL round-bottom flask, and the chloroform was removed at room
temperature by rotary evaporator to form a uniform film. The flask
was placed under vacuum for an additional 6 h. The dried film was
hydrated with 1 mL of a 40 mM aqueous calcein (Sigma-Aldrich)
solution in Tris buffer without added NaCl. The calcein dye solution
was adjusted to pH ) 7 prior to adding it to the film. The solution was
subjected through five freeze/thaw cycles using liquid nitrogen and
warm water. The entire volume of 1 mL of solution was subjected to
extrusion (a total of 15 passes) through two stacked 400 nm pore PC
membranes (Avanti Polar Lipids, Inc.) at room temperature. Finally,
the solution was pressurized through a small column packed with
Sephadex-25 (Sigma-Aldrich), eluting with Tris-saline buffer to remove
nontrapped calcein dye. Fractions (5 drops each) were collected. The
vesicle solution can be stored in a vial at 4 °C and diluted as needed
for up to 4 days.
Diluted calcein-loaded vesicle fractions that afforded fluorescence
intensities <80 au without surfactant and >800 au after addition of
surfactant, were used (Ex. ) 490 nm used and Em. ) 515 nm
monitored). A total of 50 µL of 0.2% Triton-X was used as the strong
surfactant, which causes complete vesicle disruption and leakage.
Typically, chosen vesicle fractions were diluted 6-fold and 25 µLof
this stock solution is added to 2 mL of Tris-saline buffer. A fluorescence
intensity of <80 au was verified to be stable over a minute before 25
µL of a polymer stock solution was added (2.5 µg/mL final concentra-
tion in cuvette), agitated briefly with the pipettor tip, then monitored
at 515 nm. After 5 min, Triton-X was added and the corresponding
fluorescence was taken as 100% leakage.
Fluorescence Microscopy of Stained Bacteria Cells. An Olympus
BX51 Reflected Fluorescence Microscope (Optical Analysis Corp.
Nashua, NH) with a 100 W Mercury Lamp (Chin Technical Corp.)
was used for fluorescence studies. A BacLight Kit L-7012 (Invitrogen
Corp.) was used as the fluorescence dye in a mixture of 1:1 propidium
iodide:SYTO9 to examine Sa in the presence of PGON. It is important
to mention that an initial bacterial concentration of 10
8
cells/mL was
used for microscopy studies (3 orders of magnitude higher than the
MIC and bactericidal kinetics studies) for ease of visualization. The
dye mixture was incubated with the bacteria at room temperature for
15 min before adding polymer solution (75 µg/mL final concentration).
Solution of cells, dye, and polymer were allowed to sit for 30 min
before 50 µL was placed on a slide, fitted with a coverslip, and
visualized. Bacteria were viewed under a green filter (excitation/
emission, 420-480 nm/520-800 nm) or a red filter (480-550 nm/
590-800 nm). To confirm that these experiments agreed with the time-
kill experiments discussed above and to confirm that Poly-1 is not lethal
Communications Biomacromolecules, Vol. 9, No. 11, 2008 2981
or static and that Poly-3 is lethal, cell counts were taken directly from
the microscopy experiments. They confirmed that Poly-1 is not active
with no reduction in cell count compared to the control (at 30 min and
two hours), while Poly-3 and PGON showed a reduction of 8 logs in
less than two hours. At the 30 min time point, Poly-3 and PGON
showed log reductions of 8 and 4, respectively.
Results and Discussion
Results from microbiological assays
8
showed that PGON had
an MIC of 6 µg/mL against E. coli (see Table 1 for abbrevia-
tions). 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. PGON’s 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 (HC
50
) 1500
µg/mL). As a result of PGON’s high HC
50
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 HC
50
/MIC ratio.
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 1).
Starting with 10
5
cells/mL, aliquots were removed periodically
and viable cells counted via plating techniques. The results
clearly showed PGON’s ability to kill bacteria (bactericidal
activity) not just inhibit growth (bacteriostatic activity).
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 and Poly-3 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
Poly-1 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 unfortu-
nately 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, Poly-3, 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) phospho-
lipids, 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 2). 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.
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 3). 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.
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 3A). In contrast, Poly-3 led to gross
aggregation and clear membrane damage inferred from the
dramatic increase in red fluorescence intensity (Figure 3D),
while under the green filter the cells now appeared more yellow
compared to the Poly-1 or PGON trials (compare Figure 3C to
3A and 3E). Cell viability assays confirmed Poly-3 is lethal
under these conditions.
8
Interestingly, PGON, which is signifi
-
cantly bactericidal, as demonstrated by cell viability assays, did
not aggregate bacteria nor allow propidium iodide to enter the
Table 1. Biological Activities
MIC
a
(µg/mL)
polymer (kDa) Ec Sm Sa Bs HC
50
Sel.
b
Poly-1 (3.3) 400
d
400 400 200
d
2150 5
Poly-3 (2.9) 25
d
25 25 12
d
1 0.04
PGON (2.5) 6 50 12 12 1500 250
AMP (2.5)
c
12 >256 12 2 120 10
a
MIC ) minimum inhibitory concentration, HC
50
) hemolytic concen
-
tration, Ec ) Escherichia coli, Sm ) Serratia marcescens, Sa )
Staphylococcus aureus, and Bs ) Bacillus subtilis.
b
Selectivity for Ec over
RBCs shown (Sel. ) HC
50
/MIC).
c
Previously reported MIC values
measured for Ec,
4d
Sm,
9
Sa,
4d
and Bs
5e
used derivatives of antimicrobial
peptide Magainin.
d
Previously reported values.
2
Figure 1. 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.
Figure 2. Vesicle dye leakage after polymer addition.
2982 Biomacromolecules, Vol. 9, No. 11, 2008 Communications
cells. These results agreed completely with the dye leakage
experiments in which Poly-1 and PGON 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 was inside.
8
Accordingly, these results suggest that PGON’s 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,d,12,13
Recently, we demon
-
strated the ability of these PGON’s, 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.
Conclusions
There is no doubt that PGON is potently antimicrobial
without gross membrane disruption. This leads to a lack of RBC
lysis and thus improved selectivity compared to AMPs and
previous primary amine containing SMAMPs. The recent
demonstration that PGON-like polymers are effective trans-
duction domains implies intercellular targets are responsible for
the antimicrobial properties and lack of mammalian toxicity.
7,14
Although no data is yet available on these targets, it is clear
that these PGONs represent an interesting and potentially
extremely important new class of molecules. At the same time,
highly selective antimicrobial polymers, such as PGON, can
be extremely valuable in materials designed to prevent Biofilm
formation. Targets of opportunity include cardiovascular, or-
thopedic, and other implants that account for 1 million implant-
associated infections and 3 billion U.S. dollars of healthcare
costs annually.
1d
Acknowledgment. The National Institutes of Health and the
Office of Naval Research are acknowledged for their generous
support.
Supporting Information Available. Synthetic schemes,
instrumentation, and dye-labeled polymer synthesis. This mate-
rial is available free of charge via the Internet at http://
pubs.acs.org.
References and Notes
(1) (a) Madkour, A. E.; Tew, G. N. Polym. Int. 2008, 57, 6–10. (b) Gabriel,
G. J.; Som, A.; Madkour, A. E.; Eren, T.; Tew, G. N. Mater. Sci.
Eng., R 2007, 57, 28–64. (c) Klibanov, A. M. J. Mater. Chem. 2007,
17, 2479–2482. (d) Hetrick, E. M.; Schoenfisch, M. H. Chem. Soc.
ReV. 2006, 35, 780–789.
(2) Ilker, M. F.; Nu¨sslein, K.; Tew, G. N.; Coughlin, E. B. J. Am. Chem.
Soc. 2004, 126, 15870–15875.
(3) (a) Miyatake, T.; Nishihara, M.; Matile, S. J. Am. Chem. Soc. 2006,
128, 12420–12421. (b) Henriques, S. T.; Melo, M. N.; Castanho,
M. A. R. B. Biochem. J. 2006, 399, 1–7. (c) Rothbard, J. B.; Kreider,
E.; VanDeusen, C. L.; Wright, L.; Wylie, B. L.; Wender, P. A. J. Med.
Chem. 2002, 45, 3612–3618. (d) Futaki, S.; Suzuki, T.; Ohashi, W.;
Yagami, T.; Tanaka, S.; Ueda, K.; Sugiura, Y. J. Biol. Chem. 2001,
276, 5836–5840. (e) Mitchell, D. J.; Kim, D. T.; Steinman, L.;
Fathman, C. G.; Rothbard, J. B. J. Pept. Res. 2000, 56, 318–325.
(4) For polymeric examples, see: (a) Sambhy, V.; Peterson, B. R.; Sen,
A. Angew. Chem., Int. Ed. 2008, 47, 1250–1254. (b) Mowery, B. P.;
Lee, S. E.; Kissounko, D. A.; Epand, R. F.; Epand, R. M.; Weisblum,
B.; Stahl, S. S.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 15474–
15476. (c) Kuroda, K.; DeGrado, W. F. J. Am. Chem. Soc. 2005, 127,
4128–4129. (d) Arnt, L.; Nu¨sslein, K.; Tew, G. N. J. Polym. Sci.,
Part A: Polym. Chem. 2004, 42, 3860–3864. (e) Tew, G. N.; Liu, D.;
Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll, P. J.; Klein, M. L.;
DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 5110–5114.
(5) For peptidomimetic examples, see (a) Gabriel, G. J.; Tew, G. N. Org.
Biomol. Chem. 2008, 6, 417–423. (b) Schmitt, M. A.; Weisblum, B.;
Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 417–428. (c) Tew, G. N.;
Clements, D.; Tang, H.; Arnt, L.; Scott, R. W. Biochim. Biophys. Acta
2006, 1758, 1387–1392. (d) Patch, J. A.; Barron, A. E. J. Am. Chem.
Soc. 2003, 125, 12092–12093. (e) Porter, E. A.; Wang, X.; Lee, H.-
S.; Weisblum, B.; Gellman, S. H. Nature 2000, 404, 565–565.
(6) Radzishevsky, I. S.; Rotem, S.; Bourdetsky, D.; Navon-Venezia, S.;
Carmeli, Y.; Mor, A. Nat. Biotechnol. 2007, 25, 657–659.
(7) Kolonko, E. M.; Kiessling, L. L. J. Am. Chem. Soc. 2008, 130, 5626–
5627.
(8) See Supporting Information for details.
(9) Ge, Y.; MacDonald, D. L.; Holroyd, K. J.; Thornsberry, C.; Wexler,
H.; Zasloff, M. Antimicrob. Agents Chemother. 1999, 43, 782–788.
(10) Brogden, K. A. Nat. ReV. Microbiol. 2005, 3, 238–250.
(11) Som, A.; Tew, G. N. J. Phys. Chem. B 2008, 112, 3495–3502.
(12) Sakai, N.; Futaki, S.; Matile, S. Soft Matter 2006, 2, 636–641.
(13) (a) Pantos, A.; Tsogas, I.; Paleos, C. M. Biochim. Biophys. Acta 2008,
1778, 811–823. (b) Schroeder, T.; Niemeier, N.; Afonin, S.; Ulrich,
A. S.; Krug, H. F.; Braese, S. J. Med. Chem. 2008, 51, 376–379. (c)
Deglane, G.; Abes, S.; Michel, T.; Prevot, P.; Vives, E.; Debart, F.;
Barvik, I.; Lebleu, B.; Vasseur, J.-J. ChemBioChem 2006, 7, 684–
692. (d) Fillon, Y. A.; erson, J. P.; Chmielewski, J. J. Am. Chem.
Soc. 2005, 127, 11798–11803. (e) Funhoff, A. M.; Van Nostrum, C. F.;
Lok, M. C.; Fretz, M. M.; Crommelin, D. J. A.; Hennink, W. E.
Bioconjugate Chem. 2004, 15, 1212–1220.
(14) Hennig, A.; Gabriel, G. J.; Tew, G. N.; Matile, S. J. Am. Chem. Soc.
2008, 130, 10338-10344.
BM800855T
Figure 3. Fluorescence microscopy of S. aureus (cells 1 µmin
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 the identical
field using a red filter.
Communications Biomacromolecules, Vol. 9, No. 11, 2008 2983
    • "However, too much PEG induced a drastic loss of antibacterial activity due to the shielding effect of the PEG preventing the hexamethyleneamine units from interacting with the bacterial cell surface. A number of studies have also investigated the incorporation of functional groups that mimic specific amino acids such as arginine (Budhathoki-Uprety et al. 2012; Locock et al. 2013; Gabriel et al. 2008) and tryptophan (Trp) (Locock et al. 2014) instead of the traditional mimics of lysine (amine-based cationic groups). Trp residue has been identified at high concentrations in various AMPs including indolicidin, tritrpticin, and lactoferrampin and has the unique ability to insert into membranes and associate with the positively charged choline headgroups of the lipid bilayer (Chan et al. 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: Antimicrobial peptides (AMPs), or more generally host defense peptides, have broad-spectrum antimicrobial activity and use nonspecific interactions to target generic features common to the membranes of many pathogens. As a result, development of resistance to such natural defenses is inhibited compared to conventional antibiotics. The disadvantage of AMPs, however, is that they are often not very potent. In contrast, traditional antibiotics typically have strong potency, but due to a broad range of bacterial defense mechanisms, there are many examples of resistance. Here, we explore the possibility of combining these two classes of molecules. In the first half of this chapter, we review the fundamentals of membrane curvature generation and the various strategies recently used to mimic this membrane activity of AMPs using different classes of synthetic molecules. In the second half, we show that it is possible to impart membrane activity to molecules with no previous membrane activity, and summarize some of our recent works which aim to combine advantages of traditional antibiotics and AMPs into a single molecule with multiple mechanisms of killing as well as multiple mechanisms of specificity.
    Chapter · Jan 2016 · Colloids and surfaces B: Biointerfaces
    • "Compared to low-molecular-weight QAS/QPS, polymeric QAS/QPS have higher positive charge density which promotes initial adsorption onto the negatively charged bacterial surfaces and disruption of cellular membranes, resulting in significantly enhanced antibacterial activity [43,44]. Benefiting from the rapid development of characterization technology, various advanced technologies, including AFM [45,46], fluorescence correlation spectroscopy [47,48], and/or tracking the leakage of cellular constituents [49], have been applied to investigate the action mode of antimicrobial materials. These studies provide intuitive and persuasive evidence for supporting the hypothesis about the antimicrobial mechanism of cationic biocides. "
    [Show abstract] [Hide abstract] ABSTRACT: Polymeric materials containing quaternary ammonium and/or phosphonium salts have been extensively studied and applied to a variety of antimicrobial-relevant areas. With various architectures, polymeric quaternary ammonium/phosphonium salts were prepared using different approaches, exhibiting different antimicrobial activities and potential applications. This review focuses on the state of the art of antimicrobial polymers with quaternary ammonium/phosphonium salts. In particular, it discusses the structure and synthesis method, mechanisms of antimicrobial action, and the comparison of antimicrobial performance between these two kinds of polymers.
    Full-text · Article · Feb 2015
    • "ROMP is a well-known synthetic method that has been commonly practiced to produce well-defined polymers with controlled molecular weights and low polydispersities (PDI). In particular , Tew et al. have produced different assets of ROMP for the preparation of several highly potent antibacterial polymers [25,33343536. These polynorbonene derivatives contained primary amine-trifluoroacetic acid salts and various alkyl moieties as pendant groups. "
    [Show abstract] [Hide abstract] ABSTRACT: The purpose of this study is to understand the antibacterial properties of cationic polymers on solid surfaces by investigating the structure-activity relationships. The polymer synthesis was carried via ring opening metathesis polymerization (ROMP) of oxanorbornene derivatives. Modulation of molecular weights and alkyl chain lengths of the polymers were studied to investigate the antibacterial properties on the glass surface. Fluorescein (Na salt) staining contact angle measurements were used to characterize the positive charge density and hydrophobicity on the polymer coated surfaces. Positive charge density for the surface coated polymers with molecular weights of 3,000 and 10,000 g mol−1 are observed to be in the range of 2.3–28.5 nmol cm−2. The ROMP based cationic pyridinium polymer with hexyl unit exhibited the highest bactericidal efficiency against E. coli on solid surface killing 99% of the bacteria in 5 minutes. However, phenyl and octyl functionalized quaternary pyridinium groups exhibited lower biocidal properties on the solid surfaces compared to their solution phase biocidal properties. Studying the effect of threshold polymer concentrations on the antibacterial properties indicated that changing the concentrations of polymer coatings on the solid surface dramatically influences antibacterial efficiency.
    Full-text · Article · Jan 2015
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