Cationic amphiphilic non-hemolytic polyacrylates with superior antibacterial activity

Article abstract of DOI:10.1039/c4cc01583e

Acrylic copolymers with appropriate compositions of counits having cationic charge with 2-carbon and 6-carbon spacer arms can show superior antibacterial activities with concomitant very low hemolytic effect. These amphiphilic copolymers represent one of the most promising synthetic polymer antibacterial systems reported. the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc01583e

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Cationic amphiphilic non-hemolytic polyacrylates
with superior antibacterial activity†
Cite this: Chem. Commun., 2014,
0, 7071
5
Ashish Punia, Edward He, Kevin Lee, Probal Banerjee and Nan-Loh Yang*
Received 3rd March 2014,
Accepted 18th May 2014
DOI: 10.1039/c4cc01583e
www.rsc.org/chemcomm
Acrylic copolymers with appropriate compositions of counits Compared with random copolymers, block copolymer architec-
4b
having cationic charge with 2-carbon and 6-carbon spacer arms ture can show lower toxicity toward RBCs. ‘‘Same centered’’
can show superior antibacterial activities with concomitant very polymers, having alkyl tail attached to their cationic center,
low hemolytic effect. These amphiphilic copolymers represent one demonstrated lower hemolytic activity in comparison with
4c
of the most promising synthetic polymer antibacterial systems ‘‘separate center’’ copolymers. Incorporation of hydrophilic
reported. poly(ethylene glycol) side groups reduced hemolytic activity in
3d
N-hexylated poly(vinyl pyridine)s. Hemolytic activity of syn-
There is a pressing need to develop new antibacterial agents to thetic amphiphilic polymers primarily arises from their hydro-
3a,b
combat the serious public health threat caused by the emer- phobic interactions with the lipid bilayer of RBCs.
Mole ratios
gence of antibiotic resistant bacteria (superbugs). The develop- and size of hydrophobic groups were varied to optimize the
3b,5a
ment of conventional antibiotics involves huge costs, and antibacterial and hemolytic activity of polymers.
rapid increase in bacterial resistance renders them ineffective In this communication, we report the synthesis of highly
1
a
within a few years. Compared to traditional antibiotics, antibacterial but selective (bacteria over RBCs) polyacrylates by
natural host defense antimicrobial peptides (AMPs) are known copolymerization of a monomer (M2) having 2-carbon spacer
to involve much lower levels of bacterial resistance develop- arm (distance from polymer backbone to cationic center) with a
1
b,c
ment.
Challenging synthesis and proteolytic degradation of 6-carbon spacer arm monomer (M6), in varying mole ratios
2a,b
AMPs have hindered their therapeutic applications.
Synthetic (Scheme 1). All copolymers in the range of 10 to 90 mole% of
amphiphilic polymers based on the design principles of AMPs M6 displayed highly selective activity toward bacteria over
have generated enormous research interest in the last decade due RBCs. A copolymer having 90 mole% of M6 demonstrated
2c
to their cost effective synthesis and structural versatility.
A
208 times more selectivity toward Escherichia coli (E. coli) over
variety of synthetic amphiphilic polymers including derivatives RBCs (Table 1 and Fig. 1).
3
a
3b
3c
based on polymethacrylates, polynorbornenes, nylon, and
We have previously found that amphiphilic polyacrylates
3d
poly(vinyl pyridine)s have displayed similar level of anti- with 2-carbon spacer arms and various lengths of alkyl tail
bacterial activities as AMPs. The inability of bacteria to gain attached to cationic center have low hemolytic activity and high
resistance toward synthetic amphiphilic polymers as opposed
4
a
to conventional antibiotics has been demonstrated.
One of the major challenges toward therapeutic applications
of synthetic amphiphilic polymers is their toxicity to mammalian
3a
cells. The biocompatibility of synthetic polymers has been
widely assessed in terms of hemolytic activity toward red blood
cells (RBCs). Some of the factors affecting the hemolytic activity
of synthetic amphiphilic polymers have been recently explored.
Center for Engineered Polymeric Materials, Department of Chemistry,
College of Staten Island, City University of New York, 2800 Victory Blvd,
Staten Island, New York 10314, USA. E-mail: NanLoh.Yang-cepm@csi.cuny.edu;
Fax: +1 718 982 4240; Tel: +1 718 982 3926
†
Electronic supplementary information (ESI) available: Experimental procedures,
Scheme 1 Synthesis of polymers from comonomers with 2- and 6-carbon
spectra, FESEM images, and biological assay protocols. See DOI: 10.1039/c4cc01583e
spacer arm lengths for charge center.
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Table 1 Characterization and biological activities of polymers
MIC
mg mL
E. coli
MIC
(mg mL
S. aureus
HC50
(mg mL
RBCs
Selectivity (HC50/MIC)
À1
À1
À1
(
)
)
)
a
M6
a
M2
b
Polymer
f
f
M
w
(kDa)
M
n
(kDa)
PDI
DP
E. coli
S. aureus
PM6-0%
0
100
89
79
70
60
48
39
29
19
9
6.2
6.6
6.3
6.6
6.8
6.9
6.7
7.1
7.8
7.2
7.5
4.3
4.5
4.3
4.5
4.6
4.6
4.6
4.8
5.4
5.1
5.8
1.45
1.47
1.46
1.46
1.46
1.49
1.48
1.47
1.46
1.42
1.28
30
39
33
33
30
25
30
25
31
29
36
1428
809
250
125
62
52
41
26
16
7.8
5.8
104
62
62
52
52
52
31
31
26
16
16
42000
42000
42000
1667
41.4
42.5
48
13
26
438
449
480
4125
208
419
432
432
32
PM6-10%
PM6-20%
PM6-30%
PM6-40%
PM6-50%
PM6-60%
PM6-70%
PM6-80%
PM6-90%
PM6-100%
11
21
30
40
52
61
71
81
91
1619
31
42000
42000
42000
42000
1619
438
464
464
480
101
100
0
o1.9
o0.33
o0.12
a
1
b
1
Actual mole% of monomer in polymers, as calculated by H NMR. As calculated from H NMR (ESI).
giving polymers with compositions that closely match the
feed mole% (Table 1). The molecular weights of precursor
copolymers, before deprotection with TFA, were estimated
using gel permeation chromatography. Average degree of poly-
merization (DP) of polymers was estimated to be close to 30 for
1
all preparations based on H NMR, using end group analysis
and assuming chain transfer dominating kinetics (ESI,† p. 5).
PM6-x%, denotes the cationic amphiphilic copolymer having
x mole% (in feed) of M6 monomer.
The antibacterial activities of polymers, in terms of Minimum
Inhibitory Concentration (MIC), were determined against gram
negative E. coli (TOP 10, ampicillin resistant) and Gram positive
S. aureus (ATCC 25923), following established literature proce-
5c
dure. MIC is expressed as the minimum polymer concentration
that resulted in 100% inhibition of bacterial growth after an
incubation period of 18 hours. Hemolytic activities of polymers
Fig. 1 MIC values of polymers against (a) E. coli, and (b) S. aureus. were determined in terms of hemolytic concentration-50%
(
c) Hemolytic activity of polymers against mouse RBCs. (d) Biological activity
(
HC50), the minimum polymer concentration resulting in 50%
of polymers (MIC and HC50).
5c
lyses of mouse RBCs within an incubation period of 1 hour.
Each experiment was done in triplicate, and the values reported
5b
activity against Staphylococcus aureus (S. aureus). Kuroda and here are the averages of three sets of independent experiments
coworkers recently reported an amine functionalized poly- performed on different days.
methacrylate homopolymer with 6-carbon linear spacer arm
to be highly active against both E. coli and S. aureus. However, tially increased as the mole% of counit M6 was increased in the
Antibacterial activity of polymers toward E. coli was substan-
5
a
the hemolytic activity of this polymer was also extremely high, copolymer (Fig. 1a and Table 1). Whereas, PM6-0% is inactive
5
a
and it did not display selectivity toward bacteria over RBCs.
against E. coli; adding just 20% of M6 monomer (PM6-20%)
Thus, by copolymerizing a longer spacer arm monomer with resulted in significant reduction in MIC value. PM6-90% and
a short spacer arm monomer, copolymers with high anti- PM6-100% displayed highest antibacterial activity toward E. coli
bacterial activity and lower hemolytic activity could be in this series of polymers. The effect of increasing monomer
expected. The ‘‘snorkel effect’’ from longer spacer arms in M6 mole% on the antibacterial activity of polymers toward
which the cationic charge attach to bacterial cell membrane S. aureus was less pronounced as compared with E. coli (Fig. 1b
and long alkyl spacer arm can extend through the hydrophobic and Table 1). Compared with the MIC values of PM6-0%, PM6-
5
a
core of lipid bilayer may lead to high antibacterial activity,
90% showed 6 times lower MIC value against S. aureus, and
whereas the high cationic charge density and lower hydro- 183 times lower MIC value against E. coli. Increase in the
phobicity of short spacer arms may lower the hemolytic ability separation of side chain attach points along the homopolymers
of polymers.
backbone has been shown to result in high antibacterial
5d
We synthesized a series of polyacrylate random copolymers activity. In the present study, copolymerization of a 6-carbon
via free radical copolymerization of a monomer having spacer arm monomer (M6) with a 2-carbon spacer arm monomer
2-carbon spacer arm (M2), with a 6-carbon spacer arm monomer (M2) changes the spatial distribution of cationic charges, even
(M6). The mole% (in feed) of M6 was increased in steps of 10%, though the attach point separation distance remained the same.
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Longer cationic spacer arms can interact with anionic cell
The membrane rupturing mechanism of antibacterial action
membrane of bacteria via ‘‘snorkel effect’’ resulting in high of our polymers was confirmed by field-emission scanning
5a
antibacterial activity.
electron microscope analysis of bacteria cells (ESI,† Fig. S4).
Significantly, all polymers, except PM6-100%, displayed very The membranes of control bacteria cells without polymer
low hemolytic activity against RBCs (Fig. 1c and Table 1). In treatment were intact, whereas the PM6-90% treated bacteria
comparison with extremely hemolytic PM6-100%, polymer cells were found to be severely damaged.
PM6-90% was found to be 4850 times less hemolytic toward
In conclusion, we have synthesized cationic amphiphilic
RBCs, even though its antibacterial activities are similar to acrylic random copolymers having 2- and 6-carbon spacer
PM6-100%. We found that majority of our polymers have HC50 arm counits as antibacterial agents. These amphiphilic
À1
value in excess of 2000 mg mL . RBC cell membrane has copolymers (DP B 30) displayed superior antibacterial activ-
zwitterionic phospholipid head groups and thus lacks net ities with concomitant low hemolytic effect. The homopolymer
negative charge on its outer surface. Amphiphilic polymers with 6-carbon spacer arm (PM6-100%) has high cell membrane
can penetrate the RBCs’ cytoplasmic membrane through hydro- disruption ability and is non-selective. Incorporation of
3
a
phobic interactions. The observation that all of our polymers 2-carbon cationic spacer arm counits in the copolymers
in the range of PM6-0% to PM6-90% are non-hemolytic but resulted in highly selective antibacterial activity. We found that
PM6-100% is highly hemolytic, indicates that even a small with just 10 mole% of M2 counits, PM6-90% polymer displayed
mole% of the shorter spacer arm counits can prevent the drastically reduced hemolytic activity, by a factor of 850, with-
insertion of these polymer into lipid bilayer of RBCs. Monomer out serious deterioration of antibacterial activity, in compari-
M6 has a long hydrophobic spacer arm, and increasing the son with highly hemolytic PM6-100% homopolymer. Unlike the
mole% of M6 should have inevitably led to rapid increase in case of strong electrostatic interactions between negatively
hemolytic activity. Incorporation of 20–30 mole% of hydro- charged cell surface of bacteria and cationic polymers, the
phobic monomer in polymers was reported to drastically increase selectivity of cationic amphiphilic polymers toward bacteria
3
a,5c
the hemolytic activity of polymers.
In our polymers, the over RBCs may arise from the weaker electrostatic interactions
presence of positive charge on each counit and a combination between cationic polymers and zwitterionic lipid head groups
of longer and shorter cationic spacer arms have led to high of RBC membrane. Incorporation of only three M2 counits into
antibacterial but low hemolytic activity. Recently, Hedrick et al. a chain with DP of 30 can significantly impact the conformation
reported that the copolymerization of smaller spacer arm of the polymer during the process of membrane rupture. M2
monomers with longer spacer arm monomers had resulted in with four carbon shorter hydrophobic spacer arm than M6, can
reduction of hemolytic activity without significant effect on lead to a level of polymer conformation with less degree of
6
a
antibacterial activity of amphiphilic polycarbonates. Tew et al. freedom, thus substantially reducing the ‘‘snorkel effect’’ of
also reported earlier the highly selective antibacterial activity in longer spacer arm of the copolymer. This copolymer system
amphiphilic polynorbornenes obtained through the copolymeri- represents one of the most promising systems in synthetic
zation of highly antibacterial and hemolytic monomers with polymeric antibacterial agents. The control of spacer arm
6b,c
non-hemolytic monomers.
lengths and copolymer composition can serve as one of the
The selectivity (HC /MIC) of our polymers toward bacteria effective structural parameters in the synthesis of polymers
5
0
over RBCs is apparent from Fig. 1d and Table 1. PM6-100% was with highly selective (bacteria over mammalian cells) anti-
highly antibacterial as well as hemolytic, whereas PM6-90% was bacterial activity.
found to be 208 times more selective toward E. coli over RBCs,
and 101 times selective toward S. aureus over RBCs. PM6-80% is neered Polymeric Materials, CUNY Graduate Center, and CUNY
125 times selective toward E. coli over RBCs and 480 times RF 66617-0044. We thank Ms Sultana Begum for providing
We acknowledge financial support from Center for Engi-
4
more selective toward S. aureus over RBCs. Both polymers freshly drawn mouse RBCs.
displayed high antibacterial activity similar to PM6-100%. All
copolymers in the range of 0 to 90 mole% of M6 monomer
manifested highly selective antibacterial activity. Moreover,
polymers containing 0 to 60% of M6 monomer, displayed
selective antibacterial activity against S. aureus over E. coli.
Notes and references
1
(a) D. Jabes, Curr. Opin. Microbiol., 2011, 14, 1; (b) A. Som,
S. Vemparala, I. Ivanov and G. N. Tew, Biopolymers, 2008, 90, 83;
c) M. Zasloff, Nature, 2002, 415, 389.
(
PM6-0%, a homopolymers of M2, is inactive against E. coli, 2 (a) A. K. Marr, W. J. Gooderham and R. E. W. Hancock, Curr. Opin.
Pharmacol., 2006, 6, 468; (b) J. L. Fox, Nat. Biotechnol., 2013, 31, 379;
but it displayed high activity against S. aureus. The double
membrane structure of E. coli is more difficult to penetrate
(
c) A. C. Engler, N. Wiradharma, Z. Y. Ong, D. J. Coady, J. L. Hedrick
and Y. Yang, Nano Today, 2012, 7, 201.
than the single membrane structure of S. aureus. Also, S. aureus 3 (a) K. Kuroda and G. A. Caputo, Wiley Interdiscip. Rev.: Nanomed.
Nanobiotechnol., 2012, 5(1), 49; (b) K. Lienkemp and G. N. Tew,
Chem. – Eur. J., 2009, 15, 11784; (c) B. P. Mowery, A. H. Lindner,
has a 15–80 nm thick negatively charged murein layer
(peptidoglycans) covering the lipid bilayer, whereas E. coli has
B. Weisblum, S. S. Stahl and S. H. Gellman, J. Am. Chem. Soc., 2009,
131, 9735; (d) P. H. Sellenet, B. Allison, B. M. Applegate and J. P.
Youngblood, Biomacromolecules, 2007, 8, 19.
(a) I. Sovadinova, E. F. Palermo, M. Urban, P. Mpiga, G. A. Caputo and
K. Kuroda, Polymers, 2011, 3, 1512; (b) Y. Oda, S. Kanaoka, T. Sato,
S. Aoshima and K. Kuroda, Biomacromolecules, 2011, 12(10), 3581;
a thin 6 nm thick peptidoglycan layer, sandwiched between
the outer and inner membranes. This may result in higher
coulombic interactions between PM6-0% and S. aureus, as
4
6
d
compared with E. coli.
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(
c) V. Sambhy, B. R. Peterson and A. Sen, Angew. Chem., 2008, 6 (a) A. C. Engler, J. P. K. Tan, Z. Y. Ong, D. J. Coady, V. W. L. Ng,
1
20, 1270.
(a) E. F. Palermo, S. Vemparala and K. Kuroda, Biomacromolecules,
012, 13, 1632; (b) A. Punia, P. R. Debata and N.-L. Yang, Polym.
Y. Y. Yang and J. L. Hedrick, Biomacromolecules, 2013, 14(12), 4331;
(b) M. F. Ilker, K. N u¨ sslein, G. N. Tew and E. B. Coughlin, J. Am. Chem.
Soc., 2004, 126, 15870; (c) K. Lienkamp, A. E. Madkour, A. Musante,
C. F. Nelson, K. N u¨ sslein and G. N. Tew, J. Am. Chem. Soc., 2008,
130, 9836; (d) K. Lienkemp, K.-N. Kumar, A. Som, K. N u¨ sslein and
G. N. Tew, Chem. – Eur. J., 2009, 15, 11710.
5
2
Prepr., 2012, 53(2), 382; (c) K. Kuroda and W. F. DeGrado, J. Am. Chem.
Soc., 2005, 127, 4128; (d) A. Song, S. G. Walker, K. A. Parker and
N. S. Sampson, ACS Chem. Biol., 2011, 6(6), 590.
7074 | Chem. Commun., 2014, 50, 7071--7074
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