Polycarbonates with Potent and Selective Antimicrobial Activity toward Gram-Positive Bacteria

Article abstract of DOI:10.1021/acs.biomac.6b01385

The resistance developed by life-threatening bacteria toward conventional antibiotics has become a major concern in public health. To combat antibiotic resistance, there has been a significant interest in the development of antimicrobial cationic polymers

Full text of DOI:10.1021/acs.biomac.6b01385

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Article
Polycarbonates with Potent and Selective
Antimicrobial Activity toward Gram-Positive Bacteria
Alekhya Nimmagadda, Xuan Liu, Peng Teng, Ma Su, Yaqiong Li, Qiao Qiao,
Nawal K Khadka, Xiaoting Sun, Jianjun Pan, Hai Xu, Qi Li, and Jianfeng Cai
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01385 • Publication Date (Web): 22 Nov 2016
Downloaded from http://pubs.acs.org on November 29, 2016
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Polycarbonates with Potent and Selective Antimicrobial Activity toward Gram-Positive
Bacteria
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Alekhya Nimmagadda,1, a Xuan Liu,2, a Peng Teng, Ma Su, Yaqiong Li, Qiao Qiao, Nawal K.
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Khadka, Xiaoting Sun, Jianjun Pan, Hai Xu, * Qi Li, * and Jianfeng Cai *
1Department of Chemistry, University of South Florida, 4202 E. Fowler Ave, Tampa, FL 33620, U.S.
2Department of Medical Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine,
Shanghai 201203, P. R. China.
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3Department of Physics, University of South Florida, 4202 E. Fowler Ave, Tampa, FL 33620, U.S.
4College of Chemistry and Chemical Engineering, Central South University, Changsha, HN 410083, P. R. China.
jianfengcai@usf.edu, Lzwf@hotmail.com, and xhisaac@csu.edu.cn
aThese authors contributed to the work equally.
ABSTRACT
The resistance developed by life-threatening bacteria toward conventional antibiotics has become
a major concern in public health. To combat antibiotic resistance, there has been a significant
interest in the development of antimicrobial cationic polymers due to the ease of synthesis and
low manufacturing cost compared to host-defense peptides (HDPs). Herein, we report the design
and synthesis of amphiphilic polycarbonates containing primary amino groups. These polymers
exhibit potent antimicrobial activity and excellent selectivity to Gram-positive bacteria,
including multi-drug resistant pathogens. Fluorescence and TEM studies suggest that these
polymers are likely to kill bacteria by disrupting bacterial membranes. These polymers also show
low tendency to elicit resistance in bacteria. Their further development may lead to new
antimicrobial agents combating drug-resistance.
INTRODUCTION
1
Bacterial infections pose a great threat to the public health. The World Health Organization
(WHO) recently acknowledged that certain bacterial pathogens have acquired significant
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resistance to most of the commercially available antibiotics. In hospitals about 50-60% of the
infections are caused by the lack of the sterility of medical devices used during surgeries,
especially due to the contamination of notorious methicillin-resistant staphylococcus aureus
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million people develop antibiotic resistance after antibiotic treatment, and 23,000 people die due
to the lack of proper treatment annually. Therefore, there is an escalating demand for the
development of new antibacterial agents to combat the emerging resistance.3
One promising approach to circumvent bacterial resistance is to develop derivatives of host-
defense peptides (HDPs). Although antimicrobial mechanisms of HDPs are still in debate, it is
widely accepted that these cationic peptides fold into discrete secondary structures such as α-
helices or β-sheets upon binding to bacterial membranes, on which distinct hydrophobic and
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cationic patches form on the peptides. The cationic groups of HDPs bind to the negatively
charged bacterial cell surface by electrostatic attraction, while the hydrophobic patch interacts
with hydrophobic lipid bilayer of the bacteria, leading to penetration of the peptides. It is known
that eukaryotic cells mainly contain zwitterionic phospholipids such as phosphatidylcholine,
phosphatidylethanolamine and sphingomyelin on their outer leaflet of membranes, whereas the
negative charged lipids are largely sequestered in the inner leaflet of the membranes. As a result,
these cells generally carry a net neutral charge on their surface. In contrast, both Gram-positive
and Gram-negative bacteria bear a negative charge on their membrane surface due to the
presence of phospholipids including phosphatidylglycine, phosphatidylserine and cardiolipin,
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making them more selective to cationic HDPs over mammalian cells due to charge attraction. In
addition, Gram-positive bacteria have a thick peptidoglycan layer embedded with techoic and
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lipotechoic acids, whereas lipopolysaccharides (LPS) are an important component on the outer
membranes of Gram-negative bacteria. These molecules all further contribute to the overall
negative charge on the bacterial membranes. Another factor aiding to the selectivity of HDPs
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towards bacterial cells is that bacteria lack cholesterol in their membranes, whereas mammalian
cells have 25% cholesterol or more in their membranes, which stabilizes their membrane
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integrity. As such, disruption of bacterial membranes is relatively less challenging. The
disruption of the bacterial cell membranes causes the leakage of the cytoplasmic contents and
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ultimately cell death. As this biophysical interaction lacks defined membrane proteins and other
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targets, development of resistance is believed to be more challenging. Therefore, HDPs and
their derivatives have been extensively explored for antimicrobial development.10,10b However,
HDPs have intrinsic drawbacks such as tedious and costly synthesis and purification. The
pharmacokinetic properties and chemical instability of the peptides are other obstacles that
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hamper therapeutic applications of HDPs. Moreover, HDPs exhibit moderate selectivity and are
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reported to be toxic toward mammalian cells. One of the successful examples of HDPs is
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magainin II, which exhibits broad-spectrum but weak antimicrobial activity against bacteria.
The synthetic analog of magainin, Pexiganan (also known as MSI-78) has shown much improved
activity and entered Phase III clinical trials for the treatment of diabetic foot ulcers. However, it
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failed eventually due to its moderate in vivo efficacy and high cost. Due to drawbacks of
HDPs, considerable effort has been extended to develop cationic antimicrobial polymers that
mimic the function of HDPs, in the hope to supplement the potential application of HDPs.
Compared to HDPs, polymers have some apparent advantages. The procedure for the preparation
of polymers is generally very straightforward. Most of the polymer synthesis involves one pot
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polymerization reaction, which makes it easy to scale up to obtain products in large quantity. In
addition, polymers are cost-effective. Examples of antimicrobial polymers include poly (α-amino
acid)s,13 metallopolymers,14 nylon-3 polymers,15
polyacrylates,16 polyvinyl pyridines,17
polystyrenes,18 polycarbonates,19 etc. Similar to HDPs, these polymers generally exert their
activity by acting on bacterial membranes.
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Yang and Hedrick et al recently reported cationic antimicrobial polycarbonates containing
quaternary ammonium salts.20,21 These quaternary ammonium moieties were introduced through
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post-modification. Our previously findings suggest that antimicrobial agents having primary
amino groups could have potent antimicrobial activity.10,10a It is thus intriguing to study
antibacterial activity of polycarbonates bearing same groups. Herein, we report the design and
investigation of antimicrobial polycarbonates containing primary amino groups. These
polycarbonates eliminate the post-modification step in their preparation. Surprisingly, they
display potent and selective antimicrobial activity against clinically relevant Gram-positive
bacteria, and show virtually no toxicity to blood cells under the tested condition.
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MATERIALS AND METHODS
Synthesis of the monomer 1
The 5-methyl-2-oxo-1,3-dioxane-5-carbonyl chloride (MDC) was prepared according to the
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previous reported work by Yang and Hedrick. To synthesize the monomer 1, MDC (6.8 g, 38
mmol) was dissolved in 30 mL DCM in a 100 mL round bottom flask, to which a solution of
Boc-protected ethanolamine (6.15 g, 38 mmol) and TEA (7.85 mL, 57 mmol) in 10 mL DCM
was added drop wise. The reaction was allowed to continue for 4 h in an ice bath. The solution
was washed with 1 N HCl (100 mL×3), water (75 mL×3), brine (50 mL×1), and then dried over
sodium sulfate. The solvent was removed in vacuo to give a yellow colored oil, which was
further purified by flash chromatography (ethyl acetate/hexane 2:1) to give the final product
monomer 1 (6.9 g, 22 mol, 60 %) as a white solid.
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O
O
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NHBoc
HO
TEA, DCM
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NHBoc
Cl
Monomer 1
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Scheme 1. Synthesis of the monomer 1.
Synthesis of the monomer 2
The monomer 2 was prepared according to the previous reported protocol by Yang and
Hedrick.22
O
O
OH OH
OH OH
O
O
O
O
DMF, KOH
00C, 80%
Cl
O
Br
7
THF, 85%
TEA
O
OH
O
O
Monomer 2
Scheme 2. Synthesis of the monomer 2.
Synthesis of polycarbonate polymers
All the polymers were prepared in a similar fashion using ring opening polymerization21
(Scheme 3 and Table 1). In order to synthesize Boc-protected random copolymer polycarbonate
P6’ which was formed by the mixed monomers of 1 and 2, the initiator benzyl alcohol (0.05 g,
0
.46 mmol) was dissolved in 10 mL of DCM in a N purged round bottom flask. Hydrophobic
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monomer 2 (2.31 g, 9.0 mmol, 20 eq) and hydrophilic monomer 1 (2.8 g, 9.0 mmol, 20 eq) were
added to the flask together (Scheme 3A), followed by addition of 1-(3,5-bis(trifluoromethyl)-
phenyl)-3-cyclohexyl-2-thiourea catalyst (TU) (0.34 g, 0.9 mmol, 2 eq) and (1,8-diazabicyclo
[
5.4.0] undec-7- ene (DBU) (0.14 g, 0.9 mmol, 2 eq). The reaction was allowed to stir for 4 h
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under nitrogen, and then quenched benzoic acid (1.1 eq, 0.06 g, 0.5 mmol). The other Boc-
protected random copolymers were prepared in the similar way.
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For the synthesis of Boc-protected di-block polymers P4’, the two monomers 1 and 2 were
added in two batches separately in order to form two segments in the polymers (Scheme 3B). In
brief, the Boc protected monomer 1 was first added to the reaction vessel in the presence of TU
and DBU in 20 mL DCM and the reaction was allowed to run for 4 h under nitrogen.
Subsequently, the hydrophobic monomer 2 was added and the reaction was allowed to continue
for another 4 h. The reaction was quenched by benzoic acid at the end.
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Table 1. Structures of the synthesized polymers. based on NMR integration of boc-protected
polymers P1’-P4’.
Hydro-
phobic
units
Hydro-
philic
units
Molecular weight
Type of co-
polymer
Compound
Theoretical Observeda
4.1 × 102
P1
P2
P3
P4
P5
P6
P7
P8
Single
0
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4168
5653
4638
4638
5888
9168
6903
7138
Di-block
Random
Di-block
Random
Random
Random
Random
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4.6 × 102
4.6 × 102
5.9 × 102
9.0 × 102
6.9 × 102
7.1 × 102
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O
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H
m
+
n
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H
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O m+n
TFA hydrolysis
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O
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HO
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O
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A
B
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eq
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Boc
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DCM
O
P6'
P6
Boc HN
S
O
O
O
O
H N
NH2
2
H
n
H
m eq
Monomer 1 Monomer 2
n eq
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Om
O
O
O
O
Om
O
O
n
TFA
hydrolysis
N
O
O
O
O
O
O
O
O
N
HN
NH2
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Boc
P4
P4'
Scheme 3. Synthesis of the amphiphilic polycarbonates. A, synthesis of random polymer P6’ and
P6, in which the monomer 1 and 2 were added in one batch; B, synthesis of di-block polymer P4’
and P4, in which the monomer 1 and 2 were added in two batches.
Dialysis of polymers P1’-P8’
The polymers were dissolved in 2 mL methanol and dialyzed against methanol (dialysis
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tubing MWCO=3000) for three days, with methanol being replaced twice a day. After dialysis,
the solvent was evaporated to yield the products as colorless sticky oils, which were
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characterized by H NMR (supporting information).
Preparation of polymers P1-P8
Polymers P1’-P8’ were treated with 50 % TFA in 10 mL DCM for 2 h (Scheme 3). The
solvent was removed in vacuo and the residue was dissolved in DMSO and dialyzed against
methanol for three days. After methanol was removed, the samples were dissolved in 5 mL of
water and lyophilized to give the final polymers as colorless sticky oils.
MIC (Minimum Inhibitory Concentration)/Antimicrobial Activity
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Minimum inhibitory concentration (MIC) was used to measure the efficacy of the
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synthesized polycarbonates against bacteria. MIC is defined as the lowest concentration of the
compound by which it completely inhibits the growth of the bacteria for a period of 20 h. Three
clinically relevant Gram-positive strains, Methicillin-resistant S. epidermidis (MRSE, RP62A),
Vancomycin-resistant E. faecalis (VREF, ATCC 700802), and Methicillin-resistant S. aureus
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(
MRSA, ATCC 33591) were used in the assay. Briefly, a single colony was isolated from the
agar plate and allowed to grow in 4 mL TSB solution overnight in a shaking incubator at 37 °C.
The culture was diluted by 100-fold and the diluted culture was shaken for 6 h in order for the
bacteria to grow to the mid-logarithm phase. In a 96-well plate 50 µL of the polymer solution in
2-fold serial dilutions (50 µg/mL to 0.8 µg/mL) were added to the wells. Next, aliquots of the
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bacterial solution (50 µL, 1 × 10 CFU/mL) were added to those polymer solutions, respectively.
The plate was incubated at 37 °C for 20 h, and the absorbance was read at 600 nm wavelength on
a Biotek Synergy HT microtiter plate reader. Results were repeated at least three times in
duplicates each time.24
Hemolytic Activity Study
To determine the selectivity of the polymers, the compounds were incubated with the human
red blood cells and the HC50 for the compounds was calculated. HC50 is defined as the
concentration that causes 50% hemolysis of the human red blood cells (hRBCs). 25,26,27 In this
assay, freshly drawn blood was centrifuged,, and erythrocytes were separated and washed a
couple of times with PBS buffer, and the supernatant was removed. The polymer samples (50
µL) of various concentrations were placed in a 96 well plate using 2-fold serial dilution
technique. The erythrocytes were diluted to a final concentration of 5% (v/v) in PBS buffer. 50
µL of the abovementioned diluted erythrocyte solution were added to the serial-diluted polymer
solutions and incubated for 1 h at 37 °C. The 96 well plate was centrifuged at 3500 rpm for 10
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min, and 30 µL supernatant liquid was transferred to a new 96 well plate containing 100 µL of
PBS in each vial. The absorbance of these solutions was taken at a wavelength of 540 nm. For
this assay PBS was used as negative control and 0.1% Triton x-100 was used as positive control.
Hemolysis is calculated using the formula below:
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%
hemolysis = [(Abs of Sample-Abs of PBS negative control)/ (Abs of positive Control-Abs of
PBS negative control)] ×100
Drug Resistance Assay
MICs of the samples were obtained by the method described above. After determination of
the MIC, the bacterial solution from the well that contained the polymer at one-half
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concentration of the MIC value was used to dilute to 1 × 10 CFU/mL. Next, 50 µL of this
bacterial solution was added to 50 µL of 2-fold serial-diluted polymer samples, and the new MIC
was measured. This assay was repeated for 14 passages. If the polymer had virtually the same
MIC after every passage it indicated that the polymer did not developed resistance in the
bacteria.28
Fluorescence Microscopy:
DAPI (4’, 6-Diamidino-2-phenylindole dihydrochloride) and PI (Propidium iodide) were
used in the assay.29,29b DAPI is the dye that stains all dead and living bacteria, whereas the PI dye
only stains the dead bacteria with damaged membranes as itself is not cell permeable and it has
to interact with the nucleic acids of the bacteria and fluoresce in bright red color. Briefly, the
bacteria were allowed to grow to the mid logarithmic phase and then incubated with the polymer
P6 (10 µg/mL) at 37 °C for 3 h. The solution was centrifuged at 10,000 g for 10 min in an
Eppendorf tube. The supernatant was removed and the bacterial pellets were washed with PBS
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three to four times. PI (5 µg/mL) was added and incubated for 15 min in dark at 0 °C. The excess
of the dye was removed by PBS washes (×3). Next, the cells were incubated with DAPI (10
µg/mL in water) for 15 min in dark at 0 °C and excess of the dye was removed, followed by PBS
washes (×3). The bacteria were then examined under oil-immersion objective (100×) by using
the Zeiss Axio Imager Z1optical microscope.30
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Time Kill Assay:
This assay determines bacterial killing kinetics for the polymer. Different concentrations of
the polymer were incubated with bacterial suspension, at time intervals of 0 min, 10 min, 30 min,
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h, and 2 h. At these time points, 100 µL of the solution were taken and diluted 10 to 10 times
and then spread on respective agar plates for incubation at 37 °C. After 20 h, the bacterial
colonies were counted. The assay was repeated at least three times.31
Dead Bacteria TEM:
The control and the polymer treated bacterial samples were made in the similar way as in
fluorescence microscopy assay. The samples were spread on 200-mesh copper grids and were
left for 1 h for adsorption onto the grid, and stained by 1% uranyl acetate for 30 sec. The grids
were analyzed at 60 kV with FEI Morgagni 268D TEM instrument.29b
RESULTS AND DISCUSSION
Synthesis of cationic polycarbonate polymers containing primary amino groups
Yan and Hedrick et al developed antimicrobial polycarbonates containing quaternary
20
ammonium salts that show good activity towards a range of Gram-positive bacteria. However,
the formation of quaternary ammonium salts involves post-modification. Based on our previous
findings, we envisioned that polycarbonates with primary amino groups should also be active
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and maybe even more selective toward bacteria. As such, we designed and synthesized a series
of such type of polymers (P1-P8) and investigated their antibacterial activity. The synthesis is
straightforward as described earlier. The polymers were characterized by NMRs of the polymers
P1’-P8’ which contain boc groups. (Table 1, Figure 1, and supporting information).
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Figure 1. HNMR (400 MHz, CDCl
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) of P6’.
Antimicrobial activity of the polymers:
In order to understand the effect of sequence composition on the antimicrobial activity of
the polymers, we synthesized both random (in which hydrophilic and hydrophobic monomers
were randomly arranged) and diblock (in which there are defined hydrophobic and hydrophilic
segments) polycarbonate copolymers. These amphiphilic polycarbonate polymers (P1 - P8) were
tested for their antimicrobial activity against three different Gram-positive bacterial strains,
Methicillin-resistant S. epidermidis (MRSE, RP62A), Vancomycin-resistant E. faecalis (VREF,
ATCC 700802), and Methicillin-resistant S. aureus (MRSA, ATCC 33591), all of which are
clinically relevant threatening strains (Table 2). The polymer P1, containing only hydrophilic
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amino groups but no hydrophobic groups, failed to show any activity against the bacterial strains
under tested concentrations. In contrast, the other polymers containing both hydrophilic and
hydrophobic groups all show good antibacterial activity. This data clearly demonstrates that
although positive charges are necessary for initial recognition and selective association with
bacteria, cationic groups alone are not sufficient for bacterial killing. Hydrophobic groups are
critical for membrane interaction and disruption, and ultimate death of the bacteria.32 The
diblock copolymers P2 and P4, differing in the number of hydrophobic groups, show similar
activity against three different Gram-positive bacterial strains. We hypothesized that diblock
copolymers may form stable nanomicelle structures in solution, in which their hydrophobic
groups are sequestered, leading to comparable antimicrobial activity. To test our hypothesis, we
synthesized the random polymer P3 that contains same number of hydrophobic and hydrophilic
groups as P4. Consistent to our hypothesis, the antibacterial activity of P3 was better than P4.
Although the activity against VREF remained the same for both P3 and P4, P3 exhibited good
activity of 2.2 µM and 2.6 µM against MRSA and MRSE, which is 2-fold more potent than P4
toward these two strains. We thus focused on the study of activity of random polymers, and
further investigated how the ratio of hydrophobic and hydrophilic groups affect the activity of
this type of polymers. With the same number of hydrophilic groups, increasing the number of
hydrophobic groups enhanced antimicrobial activity, as seen for polymers P3, P5 and P8.
Containing 20 hydrophobic groups, P8 exhibited an excellent activity of 0.7 µM, 1.4 µM and 2.8
µM against MRSA, MRSE and VREF respectively. Similar trend was also revealed with the
change of positively charged groups, which led to the discovery of the most potent polymer P6.
Containing 20 hydrophilic and 20 hydrophobic groups, the random copolymer P6 showed potent
activity against all three bacterial strains, with the activity of 0.17 µM, 0.55 µM and 0.55 µM
against MRSA, MRSE and VREF respectively. It is reasonable because the sequences containing
more hydrophobic and hydrophilic groups have stronger interaction with bacterial membranes.
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The findings again suggested that both cationic and hydrophobic groups are essential for the
development of antimicrobial polymers, and their arrangement in the sequences plays a very
important role. It should be noted that P6 is much more active compared with magainin II (Table
1
2
3
4
5
6
7
8
9
2
), a well-known HDP, augmenting the promise of this class of polycarbonates for antimicrobial
applications.
Hemolysis is the lysis of red blood cells, and it is the mostly identified side effect of HDPs
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and polymers. Thus, hemolytic assay is the common method to measure the toxicity of HDPs
and polymers including polycarbonates.20,21 One can evaluate the potential application of
cationic polymers by comparing their antimicrobial activity and hemolytic activity.
We next conducted the hemolytic assay to evaluate the selectivity of the polymers (Table
2
). Surprisingly, none of the polymers was toxic at the tested condition even up to 1 mg/mL. The
most potent polymer P6 displayed a selectivity of at least 600 fold for MRSA. The results
indicate that these polymers are highly selective toward bacteria and thus hold great promise for
the development of potent antibacterial agents.
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Table 2. Antibacterial activity of polycarbonates. Magainin II, a host-defense peptide, was
included as a positive control.
Type of Hydro Hydro
Selectivity
µg/mL (µM)
Hemolysis
Compound
co-
polymer
phobic philic
units units
Index
(HC50/MIC
of MRSA)
(HC50
)
(µg/mL)
MRSA MRSE
>50 >50
VREF
>50
P1
P2
P3
P4
P5
Single
0
20
>1000 >20
Di-block 10
Random 10
Di-block 10
Random 15
15
10
10
10
25 (4.47) 25 (4.47) 25 (4.47) >1000
10 (2.2) 12 (2.6) 25 (5.45) >1000
25 (5.44) 20 (4.35) 25 (5.44) >1000
10 (1.7) 20 (3.4) 20 (3.4) >1000
>60
>100
>40
>100
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P6
P7
P8
Random 20
Random 15
Random 20
20
15
10
1.6 (0.17) 5.0 (0.55) 5.0 (0.55) >1000
10 (1.45) 20 (2.89) 20 (2.89) >1000
>625
>100
>200
>62.5
5 (0.7)
16 (40)
10 (1.4)
>50
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>50
>1000
>1000
Magainin II HDP
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Morphology of polymers
As we predicted that the antimicrobial activity of the polymers is related to their
nanomorphology, we next conducted Transmission Electron Microscopy (TEM) experiment to
test our hypothesis. As expected, most of the polymers formed micelles in water with the size
ranging from 80 to 200 nm (Figure 2). We particularly compared the morphology of P3, P4 and
P6. These micelles show hydrophobic cores and hydrophilic coronas. The size of the particle
increases as the hydrophobic and hydrophilic entities increase, as observed for P3 and P6 (Figure
2
A and 2B). The diblock copolymer P4 (Figure 2C) was found to be larger than the random
copolymer P3 (Figure 2A) which comprised of same number of hydrophobic and hydrophilic
entities. This may be because P4 forms more stable core-shell micelle structure due to its diblock
structure, and therefore more sequences can self-assemble together to form large-sized micelles.
In the contrast, although P3 also contains both hydrophobic and hydrophilic groups, their
random arrangement renders less ability of the sequences to assembly into defined nanostructure.
This is also demonstrated by their zeta potentials (Table 3). P4 has a zeta potential of 58.98 mv,
which is much larger than P3 and P6. This is consistent to our hypothesis, that random polymers
are more active to bacteria than diblock polymers as they can quickly dissociate into smaller
aggregates or single molecules, and as a result, they effectively interact and disrupt bacterial
membranes.
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A: P3
B: P6
C: P4
Figure 2. TEM micrographs showing the morphology of the polycarbonate nanomicelles.
Type of
co-
polymer
Z-Average (d.nm)
Zeta Potential
(mV)
Hydro
phobic units
Hydro
philic units
Compound
P1
P2
P3
P4
P5
P6
P7
P8
Single
0
20
255.7
44.2
Di-block 10
Random 10
Di-block 10
Random 15
Random 20
Random 15
Random 20
15
10
10
10
20
15
10
154.0
81.20
212.0
209.0
288.4
328.7
435.1
54.7
26.1
58.9
31.7
16.1
9.70
7.73
Table 3. Size and Zeta potential of polycarbonates.
Antimicrobial mechanism
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The mode of action of the polymers against bacteria was initially evaluated by fluorescence
microscopy using double staining method (Figure 3).33 The most potent sequence P6 was
selected for the study. The control, which was just MRSA bacteria themselves, show blue
fluorescence under DAPI channel (a1). They were not observed under PI channel (a2) because
their membranes were intact, and therefore they could not be stained by DAPI. In contrast, after
MRSA were treated with P6, they were stained by both PI and DAPI (b1, b2), and observable
under both channels, indicating MRSA membranes were disrupted.
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Figure 3. Fluorescence micrographs of MRSA treated with 10 µg/mL of P6.
The mechanism of action was further supported by TEM (Figure 4), under which the
morphology of untreated MRSA and the polymer P6 treated MRSA was revealed. The control
(bacteria without treatment) show spherical shape with intact cell membranes (Figure 4A), a
typical morphology of MRSA. However, after the treatment of bacteria with P6, most bacteria
lost their spherical morphology. Instead, the leakage of the contents from the bacterial cells was
observed (Figure 4B). These results suggest that bacterial membranes were disrupted by the
polymer P6.
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Figure 4. TEM micrographs of Control MRSA (A) and MRSA treated with 10 µg/mL of P6 (B).
Based on antimicrobial activity and nanomorphology of the polymers, we propose their
mechanism of action (Figure 5). The polymers are initially believed to exist in micelle
conformation in solution. As polymers approach the bacterial surface, due to change in the
electrostatic interactions, the polymers lose their stability and start to dissociate into small
entities or free polymer chains, which could bind to the bacterial membranes more effectively.
The free polymer chain penetrates the surface of the bacteria due to its amphipathic nature and
ultimately disrupts the bacterial membrane, leading to bacterial cell death. It is anticipated that
the mechanism of action might be more complex as many other proteins, carbohydrates and
lipids are present. It is plausible that the presence of other biological molecules may further
destabilize the micelle structures of polymers, leading to enhanced activity in bacterial
eradication.
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Figure 5. Proposed mechanism of action of the polymers on Gram-positive bacteria.
Time-kill assay
To determine the time of action and efficacy of the polymers, the most potent polymer P6
was chosen for time kill study (Figure 6). Cell viability was determined by the colony county
method on agar plate at regular intervals of 30 min, 1 h, 2 h, 3 h, 6 h, 9 h, 12 h and 24 h. At the
concentration of 50 µg/mL and 25 µg/mL, the bacteria were completely eradiated after 6 h,
suggesting that the polymer has the bactericidal mechanism and the action is rapid.
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Figure 6. Time kill study of P6 for MRSA.
Drug resistance study
The TEM and florescence microscopy suggest the polymers act by membrane disruption
mechanism on the bacteria. Under this mechanism, it is accepted that the bacteria are less prone
to develop resistance. To evaluate the probability of the polymer to induce resistance in bacteria,
drug resistance study was conducted for the most potent compound P6 against MRSA. As shown
in the Figure 7, the MIC values were virtually constant even after 14 passages, indicating that
the bacteria do not develop resistance readily towards the polymer.
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Figure 7. Drug Resistance Study of P6 for MRSA.
CONCLUSION:
We reported the development of potent and highly selective antimicrobial polycarbonate
polymers containing primary amino groups. Although they do not show strong activity toward
Gram-negative bacteria, they display remarkable antimicrobial activity toward multi-drug
resistant Gram-positive bacteria. Our results suggest that amphiphilic nature is necessary for
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bacterial killing, and random block polymers are more potent than diblock polymers, possibly
due to stable nanostructures of diblock polymers which prevent them from interacting with
bacterial membranes more effectively. The fluorescence microscopy and the TEM data suggest
that these polymers have the mechanism of action of bacterial membrane disruption.
Remarkably, these polymers are highly selective towards bacterial cells and show no discernable
hemolytic activity. In vivo study of these polymers on the mouse model to evaluate their efficacy
is currently underway.
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ASSOCIATED CONTENT
Supporting Information
NMR spectra of polymers. The Supporting Information is available free of charge on the ACS
Publications website.
AUTHOR INFORMATION
Corresponding Authors
* Department of Chemistry, University of South Florida, 4202 E. Fowler Ave, Tampa, FL
3
3620, USA. E-mail: jianfengcai@usf.edu ; Tel: 1-813-974-9506. Department of Medical
Oncology, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai
201203, P. R. China. E-mail: Lzwf@hotmail.com ; College of Chemistry and Chemical
Engineering, Central South University, Changsha, HN 410083, P. R. China. E-mail:
xhisaac@csu.edu.cn
ACKNOWLEDGEMENTS
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This work was partially supported by NSF CAREER 1351265 (JC) and NIH 1R01GM112652-
01A1 (JC), NIH R15GM117531 (JP), National Natural Science Foundation of China
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(
81520108031), the Science Foundation for Shanghai Committee of Science Project
14430722900), the Program of Shanghai Academic Research Leader (16XD1403600).
(
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NOTES
The authors declare no competing financial interest.
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Table of Contents
Polycarbonates with Potent and Selective Antimicrobial Activity toward Gram-Positive Bacteria
Alekhya Nimmagadda,1, a Xuan Liu, Peng Teng, Ma Su, Yaqiong Li, Qiao Qiao, Nawal K.
2,a
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Khadka, Xiaoting Sun, Jianjun Pan, Hai Xu, * Qi Li, * and Jianfeng Cai *
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