Increasing the Antimicrobial Activity of Amphiphilic Cationic

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Increasing the antimicrobial activity of amphiphilic cationic copolymers by the facile synthesis of high molecular weight stars by SARA ATRP Madson R.E. Santos, Patrícia V. Mendonça, Mariana C. Almeida, Rita Branco, Armenio C. Serra, Paula V. Morais, and Jorge F J Coelho Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00685 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

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Biomacromolecules

35x14mm (300 x 300 DPI)

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Increasing the antimicrobial activity of

2

amphiphilic cationic copolymers by the facile

3

synthesis of high molecular weight stars by

4

SARA ATRP

5

Madson R. E. Santos,a Patrícia V. Mendonça,a Mariana C. Almeida,b Rita Branco,b

6

Arménio C. Serra,a Paula V. Moraisb and Jorge F. J. Coelhoa*

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.a CEMMPRE, Centre for Mechanical Engineering, Materials and Processes, Department

8

of Chemical Engineering, University of Coimbra, Coimbra 3030-790, Portugal

9

b

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CEMMPRE, Centre for Mechanical Engineering, Materials and Processes, Department of

Life Sciences, University of Coimbra, Coimbra 3001-401, Portugal

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12

Abstract

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Infections caused by bacteria represent great motif of concern in health area. Therefore,

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there is a huge demand for more efficient antimicrobial agents. Antimicrobial polymers

15

have attracted special attention as promising materials to prevent infectious diseases. In this

16

study, a new polymeric system exhibiting antimicrobial activity against a range of Gram1 ACS Paragon Plus Environment

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Biomacromolecules

1

positive and Gram-negative bacterial strains at micromolar concentrations (e.g., 0.8 µM)

2

was developed. Controlled linear and star-shaped copolymers, comprising hydrophobic

3

poly(butyl acrylate) (PBA) and cationic poly(3-acrylamidopropyl)trimethylammonium

4

chloride) (PAMPTMA) segments, were obtained by SARA ATRP at 30 °C. The

5

antibacterial activity of the polymers was studied by varying systematically the molecular

6

weight, hydrophilic/hydrophobic balance and architecture. The molecular weight was found

7

to exert the greatest influence on the antimicrobial activity of the polymers, with minimum

8

inhibitory concentration values decreasing with increasing molecular weight. Live/dead

9

membrane integrity assays and scanning electron microscopy analysis confirmed the

10

bactericidal character of the synthesized PAMPTMA-(b)co-PBA polymers.

11 12

Introduction

13

Infections and diseases caused by multi-resistant microbes have become a concern in

14

diverse human health-associated areas, often requiring extensive periods of treatment, high

15

medical costs or even culminating in the death of many patients.1 This context has

16

prompted researchers to seek new materials capable of effectively killing pathogens, as an

17

alternative to the common use of antibiotics, which are associated to the development of

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antimicrobial resistance. Antimicrobial polymers have been pointed out as a highly

19

promising approach to combat drug-resistant microorganisms. Polymers present several

20

advantages over the low molecular weight biocides. For example, the intensive use of

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conventional antibiotics in the last decades has dramatically increased the frequency of

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resistance among human pathogenic microorganisms.2 On the other hand, antimicrobial

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polymers can disturb the whole bacterial environment by multiple interactions, such as 2 ACS Paragon Plus Environment


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hydrophilic-hydrophobic interactions, disruption of the integrity of the lipid barrier or

2

disturbance of the transport of compounds across the membrane leading to bacteria death.3,

3

4

4

properties can be easily tuned, which allows the optimization of the antimicrobial

5

performance of the polymeric material.

Moreover, polymers could present reduced cytotoxicity, chemical stability and their

6

Since most bacteria present negatively charged cell walls, cationic polymers are usually

7

the most promising candidates as antimicrobial agents, as they can interact with bacteria

8

through electrostatic forces.5 In particular, synthetic polymers containing quaternary

9

ammonium (QAS) groups or quaternary phosphonium (QPS) groups have been widely

10

studied and applied as antimicrobial agents in diverse areas (e.g., biomedical field).6-8

11

Beside the positive charges, the antimicrobial polymers should have a proper

12

hydrophilic/hydrophobic balance, which is essential to allow the permeation through the

13

lipid bilayers of the bacteria cell wall. In addition, it is expected that the molecular weight

14

and the structure/architecture of the polymers are also important parameters that influence

15

the polymer´s antimicrobial efficiency.3 Several studies reported in the literature are

16

dedicated to the investigation of the role of each polymer feature on their antimicrobial

17

activity.9-11 However, it is difficult to establish “universal” correlations, since the

18

antimicrobial activity depends both on the polymer nature/structure and the bacterial strain

19

tested. Thus, judicious design and synthesis of well-defined macromolecules is necessary to

20

provide a clear understanding of the polymer structure/performance relationship. To this

21

end, advanced polymerization techniques namely, reversible deactivation radical

22

polymerization (RDRP) methods could be very useful tools to prepare tailor-made

23

antimicrobial polymers with stringent control over properties, such as molecular weight 3 ACS Paragon Plus Environment

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Biomacromolecules

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(MW), composition, structure and architecture, which may have great influence on the

2

biological activity of the polymers.12, 13

3

A number of recent studies have pointed out the importance of the polymer’s architecture

4

on their antimicrobial efficacy, with special relevance given to the nanostructured polymers

5

as star-shaped,

6

enhanced and indiscriminate activity towards bacterial cell.18,

7

outstanding antimicrobial activity, the strategies reported for the preparation of those

8

structures sometimes require laborious steps concerning the synthesis of the monomers,

9

reaction conditions and polymer purification.

14, 15

rod-like16 and polymer-immobilized nanoparticles17 that have shown 19

However, despite their

10

Here, a one-step strategy to obtain antimicrobial linear and star-shaped amphiphilic

11

copolymers, via supplemental activator and reducing agent atom transfer radical

12

polymerization (SARA ATRP) at near room temperature (30 °C), as a new class of

13

antimicrobial agents is reported. These polymers were designed to have (3-

14

acrylamidopropyl)trimethylammonium chloride (AMPTMA)

15

hydrophilic segment and n-butyl acrylate (n-BA) as the hydrophobic domain (Figure 1).

16

PAMPTMA was chosen as the cationic segment, due to the presence of a pendant

17

quaternary ammonium group, which provides a permanent cationic character to the

18

polymer regardless the pH of the medium. Quaternary ammonium salts are known to be

19

cationic membrane-active compounds possessing antibacterial activity due to electrostatic

20

interactions and consequent microbe membrane disruption.20 In addition, n-BA was chosen

21

as the hydrophobic monomer, since it could improve the mobility of the antimicrobial

22

polymer through the lipid barrier of the bacteria cell wall.21 An extensive library of

23

PAMPTMA-based polymeric structures, including linear, 4-arm stars and 6-arm stars,

as the cationic and

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consisting in homopolymers, block copolymers and random copolymers, with different

2

targeted compositions, were synthesized and evaluated against representative Gram-

3

negative and Gram positive bacterial strains (Staphylococcus aureus, Bacillus cereus,

4

Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa).

5 6

Experimental Section

7

Materials

8

Ethyl α-bromoisobutyrate (EBiB) (Sigma-Aldrich, 98%), copper wire (Sigma-Aldrich),

9

copper (II) bromide (CuBr2) (Aldrich, 99%), deuterium oxide (D2O) (Sigma-Aldrich,

10

99.9% atom D), deuterated (D6) ethanol (d6-EtOD) (Euriso-top, 99+%), N,N-

11

dimethylformamide (DMF) (Sigma-Aldrich, 99,8+%), tetrahydrofuran (THF) (HPLC

12

grade) were used as received. Ethanol (JMS, 96%) was passed though rotatory evaporator

13

before use.

14

(3-Acrylamidopropyl)trimethylammonium) chloride (AMPTMA) (TCI chemicals,

15

solution 75% in H2O) was washed with acetone in order to remove the water from the

16

solution. The pure monomer was used in the polymerizations.

17 18 19 20 21

n-Butyl acrylate (n-BA) (Sigma-Aldrich, 99+%) was passed through sand/alumina column before use in order to remove the radical inhibitor. Purified water (Milli-Q®, Millipore, resistivity >18 MΩ.cm) was obtained by reverse osmosis. Tris(2-(dimethylamino)ethyl)amine

(Me6TREN)22,

pentaerythritol

tetrakis(2-

22

bromoisobutyrate) (4f-BiB)23 and dipentaerythritol hexakis(2-bromoisobutyrate) (6f-BiB)23

23

were synthesized according the procedure described in the literature. 5 ACS Paragon Plus Environment

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Biomacromolecules

1 2

Procedures

3

Typical homopolymerization of AMPTMA by SARA ATRP

4

AMPTMA (2.5 mL, 4.8 mmol) and a solution of EBiB (9.6 mg, 48.3 µmol), CuBr2 (5.4

5

mg, 24.2 µmol) and Me6TREN (13.0 µL, 48.3 µmol) in ethanol (2.5 mL) were added to a

6

10 mL Schlenk flask equipped with a stirrer bar. Then, Cu(0) wire (l = 5cm; d = 1mm) was

7

added to the reactor, which was sealed with a glass stopper, frozen with liquid nitrogen,

8

deoxygenated with three-freeze-vacuum-thaw cycles and purged with nitrogen. The

9

reaction was allowed to proceed with stirring (360 rpm) at 30 ºC during 24 h. The monomer

10

conversion was determined by 1H NMR in D2O and the molecular weight parameters were

11

determined by SEC analysis. The polymer was dialyzed against water (cut-off 3500) and

12

the pure polymer was recovered after freeze drying.

13 14

Typical homopolymerization of n-BA by SARA ATRP

15

n-BA (2.5 mL, 17.4 mmol) and a solution of EBiB (36.9 mg, 174.0 µmol), CuBr2 (19.4

16

mg, 86.7 µmol) and Me6TREN (46.4 µL, 174.0 µmol) in ethanol (2.5 mL) were added to a

17

10 mL Schlenk flask equipped with a stirrer bar. Then, Cu(0) wire (l = 5cm; d = 1mm) was

18

added to the reactor, which was sealed with a glass stopper, frozen with liquid nitrogen,

19

deoxygenated with three-freeze-thaw cycles and purged with nitrogen. The reaction was

20

allowed to proceed with stir (360 rpm) at 30 ºC during 24 h. The monomer conversion was

21

determined by 1H NMR in CDCl3 and the molecular weight parameters were determined by

22

SEC analysis. The polymer was dissolved in THF and passed through a sand/alumina



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1

column in order to remove the catalyst. The pure polymer was obtained by precipitation in

2

cold methanol.

3 4

Typical “one-pot” block copolymerization of AMPTMA and n-BA

5

AMPTMA (3.0 mL, 14.1 mmol) and a solution of CuBr2 (15mg, 70.7µmol) and

6

Me6TREN (37.8 µL, 142.0 µmol) in ethanol (3.0 mL) were added to a 25mL Schlenk flask

7

equipped with a magnetic stirrer bar. Next, Cu(0) wire (l = 5cm; d = 1mm) was added to

8

the reactor, which was sealed with a glass stopper, frozen with liquid nitrogen,

9

deoxygenated with three freeze-vacuum-thaw cycles and purged with nitrogen. The

10

reaction was allowed to proceed with stirring (360 rpm) at 30 °C. When the monomer

11

reached more than 70 %, a degassed solution of n-BA (2.0 mL, 14.1 mmol) in ethanol (8.15

12

mL) was added to the Schlenk flask under nitrogen. The reaction was allowed to procced

13

until the maximum conversion of n-BA was achieved. The AMPTMA-b-PBA block

14

copolymer was purified through dialysis (cut-off 3500) against water, followed by ethanol

15

and then water and recovered after freeze drying. The conversion of both AMPTMA and n-

16

BA and the theoretical molecular weight of the amphiphilic block copolymer were

17

determined by 1H NMR spectroscopy.

18 19

Typical synthesis of random PAMPTMA-co-PBA copolymer

20

Several PAMPTMA-based random copolymers were synthesized by SARA ATRP, using

21

the following procedure: AMPTMA (2.0 mL, 16.7 mmol), n-BA (1.5 mL, 10.4 mmol) and

22

a solution of CuBr2 (23.5 mg, 0.10 mmol) and Me6TREN (55.0 µL, 0.21 mmol) in ethanol

23

(2.0 mL) were added to a Schlenk flask equipped with a magnetic stirrer bar. Next, Cu(0) 7 ACS Paragon Plus Environment

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Biomacromolecules

1

wire (l = 5cm; d = 1mm) was added to the Schlenk flask, which was sealed with a glass

2

stopper, frozen with liquid nitrogen deoxygenated with three freeze-vacuum-thaw cycles

3

and purged with nitrogen. The reaction was allowed to proceed with stirring (360 rpm) at

4

30°C during 15 h. The conversion of both AMPTMA and n-BA and the theoretical

5

molecular weight of the random copolymer were determined by 1H NMR spectroscopy.

6

The resulting mixture was dialyzed (cut-off 3500) against distilled water, followed by

7

ethanol and then water and the purified copolymers were obtained after freeze-drying.

8

The star-shaped PAMPTMA-co-PBA copolymers were synthesized using the procedure

9

described above but using either 4-EBiB or 6-EBiB as initiators for the preparation of 4-

10

arm or 6-arm stars, respectively.

11

The theoretical number average molecular weight of the copolymers was determined

12

following the equation: Mnth = conv.AMPMTA*MWAMPTMA*DPAMPTMA + conv.n-BA*MWn-

13

BA*DPn-BA

+ MWEBiB

14 15

Techniques

16

Polymers characterization

17

The 1H nuclear magnetic resonance (NMR) spectroscopy spectra were recorded using a

18

Bruker Avance III 400 MHz spectrometer, with a 5 mm TIX triple resonance detection

19

probe, in D2O, d6-EtOD or CDCl3 with TMS as internal standard. Conversion of the

20

monomers was determined by integration of monomer and polymers peaks using

21

MestRenova software version 6.0.2-5475.

22

PAMPTMA homopolymers were analyzed by a size exclusion chromatography (SEC)

23

system with an online degasser, a refractive index (RI) detector and a set of columns: 8 ACS Paragon Plus Environment


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Shodex OHpak SB-G guard column, OHpak SB-804HQ and OHpak SB-802.5HQ columns.

2

The polymers were eluted at a flow rate of 0.5 mL min-1 with 0.1 M Na2SO4 (aq)/1 wt%

3

acetic acid/0.02% NaN3 at 40 ºC. Before the injection (50 µL), the samples were filtered

4

through a nylon membrane with 0.20 µm pore. The system was calibrated with five narrow

5

poly(ethylene glycol) standards and the molecular weights (Mn SEC) and Đ (Mw/Mn) were

6

determined by conventional calibration using the Clarity software version 2.8.2.648.

7

PBA

homopolymers

were

analyzed

by

a

high-performance

size

exclusion

8

chromatography HPSEC; Viscotek (Viscotek TDAmax) with a differential viscometer

9

(DV), right-angle laser-light scattering (RALLS, Viscotek), low-angle laser-light scattering

10

(LALLS, Viscotek) and RI detectors. The column set consisted of a PL 10 mm guard

11

column (50 x 7.5 mm2) followed by one Viscotek T200 column (6 µm), one Viscotek

12

T3000 column (6 µm) and one Viscotek LT4000L column (7 µm). HPLC dual piston pump

13

was set with a flow rate of 1 mL min-1. The eluent (THF) was previously filtered through a

14

0.2 µm filter. The system was also equipped with an on-line degasser. The analyses were

15

done at 30 ºC using an Elder CH-150 heater. Before the injection (100 µL), the samples

16

were filtered through a PTFE membrane with 0.2 µm pore. The system was calibrated with

17

narrow polystyrene standards. The dn/dc used for PBA was 0.064.24 Molecular weight

18

(MnSEC) and Đ (Mw/Mn) of the synthesized polymers were determined by multidetectors

19

calibration using the OmniSEC software version: 4.6.1.354.

20

Dynamic light scattering (DLS) measurements were performed on Malvern Instrument

21

Zetasizer Nano-ZS (Malvern Instrument Ltd., UK). The measurement was made at 25oC, λ

22

= 632.8nm, using backward scattering angle of 173o. All samples were dissolved in LB

23

bacterial growth medium at their MIC value concentration. 9 ACS Paragon Plus Environment

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Biomacromolecules

1 2

Strains and Growth conditions

3

Bacterial indicator strains (Escherichia coli ATCC25922, Staphylococcus aureus

4

ATCC25923, Bacillus subtilis ATCC23857, Bacillus cereus 9843 and Pseudomonas

5

aeruginosa HST244P) were grown at 37 °C. Minimum inhibitory concentration (MIC)

6

determination was performed by agar diffusion test according to the Clinical & Laboratory

7

Standards Institute (CLSI)25 in Muller Hinton (MH) solid medium or by microdilution

8

procedure in microplates in Luria–Bertani (LB) medium.26

9 10

MIC determination

11

All polymer stock solutions (100 or 500 µM) were prepared in ultrapure sterile water.

12

The MIC of the polymers was evaluated using the standard broth microdilution method. In

13

this case, two-fold dilutions of the polymers were prepared in LB growth medium using 96-

14

well microliter plate. Each well containing 150 µl of polymer solution was inoculated with

15

50 µl of bacterial suspensions prepared in LB medium (106 CFU mL-1) and further

16

incubated at 37 °C for 24 h. Controls without polymer were performed for each bacterial

17

strain. Each assay was repeated in triplicate to ensure reproducibility of the experiments.

18

The MIC was the lowest concentration of polymer that completely inhibited bacterial

19

growth. MIC values in weight-based concentration are provided in supporting information

20

(Table ST1).

21 22

Scanning electron microscopy (SEM)



Page 12 of 38

1

An E. coli bacterial suspension of approximately 108 cells mL-1 in saline solution was

2

incubated in the presence or absence of polymers for 5 h at 37 °C. Co-incubation of E. coli

3

and polymers were tested at the MIC for each polymer. Cells were harvest by

4

centrifugation and washed in phosphate buffered saline (PBS). Washed cells were adhered

5

to steel slides and fixed twice with 2.5% glutaraldehyde for 10 min at room temperature.

6

The slides were dehydrated using a series of graded ethanol solutions (70, 80, 90, 95 and

7

100%) for 15 min at room temperature before drying. The dehydrated samples were gold-

8

coated for 20 sec and imaged under high vacuum using a Zeiss Merlin FEG-SEM

9

microscope.

10 11

Live/dead membrane integrity assay

12

Similarly, to the SEM sample preparation, an E. coli bacterial suspension of 108 cells mL-

13

1

14

incubation of E. coli and polymers was tested at the MIC for each polymer. Cells were

15

harvest by centrifugation and washed in 1x PBS. Washed cells were stained with 3 µL of

16

SYTO 9 and propidium iodine (PI) mixed solution (L7007 LIVE/DEAD BacLight

17

Bacterial Viability Kit, Invitrogen) and incubated at room temperature for 15 min in dark

18

conditions. 5 µL of dyed bacterial suspension was placed on a glass slide and covered with

19

glass cover slip. Cells were visualized under a fluorescence Axioskop 2 Plus microscope

20

using FITC and Rhodamine filters. SYTO 9 (green dye) stains both live and dead cells,

21

while PI (red dye) stains only dead cells. At least 100 cells in triplicates were counted in

22

each experiment.

in PBS was incubated in the presence or absence of polymer for 5 h at 37 °C. Co-

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Biomacromolecules

1

Statistical Analysis

2

Statistical analysis was performed using the software Prism 6. Two-way analysis of

3

variance (ANOVA) was performed for live/dead assays. At least 100 cells in triplicate for n

4

= 2 independent experiments were quantified. A p value of 0.05 or less was considered

5

significant.

6 7

Results and discussion

8

Polymer synthesis and characterization

9

It is known from the literature3 that a proper balance of hydrophilic cationic segments

10

and hydrophobic regions is a key factor for a polymer to achieve its maximum

11

antimicrobial activity. Therefore, the control over the polymer composition is essential for

12

the preparation of antimicrobial polymers with enhanced performance, which can be

13

achieved by using RDRP techniques. In this work, SARA ATRP was explored for the

14

preparation of cationic amphiphilic antimicrobial copolymers, based on PAMPTMA as the

15

cationic segment and PBA as the hydrophobic domain. Prior work reported by our research

16

group showed that star-shaped PMA-b-PAMPTMA block copolymers could be

17

successfully prepared using an eco-friendly SARA ATRP system.27 Here, the same

18

catalytic system was used for the polymerization of both AMPMTA and n-BA. Due to the

19

very different nature of the monomers (AMPTMA is a highly hydrophilic monomer

20

whereas n-BA is very hydrophobic), the first step was to select an appropriate solvent for

21

the copolymerization. In this case, ethanol enabled homogeneous polymerization,

22

dissolving all monomers and polymers.



Page 14 of 38

1

The successful polymerization of both AMPTMA and n-BA by ATRP techniques has

2

already been reported.28, 29 However, to best of our knowledge, this is the first time that

3

methods have been developed for the controlled synthesis of PAMPTMA-b-PBA block

4

copolymers by “one-pot” SARA ATRP (Figure 1).

5 6

Figure 1. General structures of the amphiphilic linear and star-shaped (PAMPTMA-co-

7

PBA or PAMPTMA-b-PBA) copolymers prepared by SARA ATRP.

8

First experiments were dedicated to the homopolymerization of either AMPTMA (linear

9

and star-shaped) or n-BA (linear) by SARA ATRP in ethanol in order to define the reaction

10

conditions for a controlled and compatible synthesis of both polymers (Figures SF1 to

11

SF3). Then, the reaction conditions were optimized for the preparation of the PMAPTMA-

12

co-PBA (block)copolymers. Figure 2 shows a typical 1H NMR spectrum of a PMAPTMA-

13

co-PBA copolymer, successfully prepared by SARA ATRP in ethanol at 30°C. The

14

recorded spectrum shows the characteristic signals of the cationic segment appearing at 13 ACS Paragon Plus Environment

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Biomacromolecules

1

3.38 ppm, corresponding to the quaternary ammonium group, and an isolated signal of the

2

PBA segment at 4.55 ppm, corresponding to the -OCH2- protons of the ester group.30, 31

3 4

Figure 2. 400 MHz 1H NMR spectrum in d6-EtOD, of purified PAMPTMA50-co-PBA15

5

copolymer (Mnth = 12.4 x 103) obtained by SARA ATRP.

6

Using the developed SARA ATRP method, a library of different amphiphilic

7

(co)polymers was prepared by targeting several parameters: MW, mole fraction of the

8

hydrophobic segment (PBA) and architecture of the polymers (Table 1 and Table ST2).

9

PAMPMTA and PBA homopolymers were also synthesized and used as control samples. It

10

is worth mentioning that the systematic study of the antimicrobial efficiency requires the

11

preparation of polymers with diverse characteristics (e.g., composition, architecture, etc.),

12

which can only be achieved by means of advanced polymerization techniques, such as the

13

SARA ATRP, ARGET, photo-ATRP, eATRP and sono-ATRP.32-37 The samples were

14

labeled according to the architecture and composition of the (co)polymers to facilitate the

15

interpretation of the results: L – linear random (co)polymer; Lb – linear block copolymer; 14 ACS Paragon Plus Environment


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1

4S – 4-arm star; 6S – 6-arm star; A – PAMPMTA segment followed by the correspondent

2

DP value; B – PBA segment followed by the correspondent DP value. As an example, the

3

sample LA27B33 (Table 1, entry 12) is a linear random copolymer with 27 units of

4

PAMPMTA and 33 units of PBA.

5 6

Table 1. Characteristics of the antimicrobial polymers prepared by SARA ATRP.

Entry

Polymer code

Topology

Composition

DP PAMPTMA*

DP PBA*

Mnthx10-3

% PBA (molar)

1

6SA114B0

6-arm star

homopolymer

114

0

142.0

0

2

6SA69B41

6-arm star

random copolymer

69

41

117.4

37

3

6SA79B24

6-arm star

random copolymer

79

24

117.3

23

4

6SA95B0

6-arm star

homopolymer

95

0

117.0

0

5

6SA22B0

6-arm star

homopolymer

22

0

28.1

0

6

6SA9B0

6-arm star

homopolymer

9

0

12.7

0

7

4SA93B0

4-arm star

homopolymer

93

0

77.6

0

8

4SA84B20

4-arm star

random copolymer

84

20

80.5

20

9

LA38B2

linear

random copolymer

38

2

8.3

5

10

LA34B9

linear

random copolymer

34

9

8.4

20

11

LA23B23

linear

random copolymer

23

23

8.0

50

12

LA27B33

linear

random copolymer

27

33

10.0

56

13

LA50B4

linear

random copolymer

50

4

11.0

7

14

LA45B15

linear

random copolymer

45

15

11.5

24

15

LA39B47

linear

random copolymer

39

47

14.2

55

16

LA115B0

linear

homopolymer

115

0

24.0

0

17

LA98B29

linear

random copolymer

98

29

24.2

23

18

LA59B44

linear

random copolymer

59

44

18.2

42

19

LbA79B47

linear

block copolymer

79

47

22.5

37


Page 17 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Biomacromolecules

20

LbA78B63

linear

block copolymer

78

63

24.4

44

21

LbA68B40

linear

block copolymer

68

40

19.4

37

22

LbA264B33

linear

block copolymer

264

33

59.0

10

23

LA335B0

linear

homopolymer

335

0

69.4

0

24

LA0B49

linear

homopolymer

0

49

6.4

100

*

Degree of polymerization (DP) value per star arm

2 3

For the determination of the MW parameters of the copolymers, SEC analysis using a

4

refractive index detector and ethanol as the eluent were carried out. However,

5

unfortunately, it was found that the cationic segment (PAMPTMA) interacted with the

6

column (Shodex Asahipak® 5 µm GF-510 HQ, LC Column 300 x 7.5 mm) either in the

7

form of homopolymer or as a segment of the copolymers, which avoided the acquisition of

8

chromatograms. Even with the addition of stabilizers (e.g., amines, LiBr, etc.) to the eluent,

9

there was no separation of the polymers. Only the PBA homopolymer eluted properly in the

10

conditions described. Therefore, for this study, the molecular weight used for the

11

characterization of the polymers was the Mnth (Table 1), taking into account that the

12

polymerization of both monomers is well-controlled and gives polymers with MnSEC close

13

to the theoretical ones.

14 15

3.2. Influence of MW and hydrophobicity on polymers’ antimicrobial activity

16

The MW of an antimicrobial polymer has been previously found to play an important

17

role in bacterial death induction.38 Recent studies have demonstrated bacterial inactivation

18

and/or death by low MW polymers,9, 39, 40 based on the fact that such structures can easily

19

diffuse through the lipid membrane. However, since the antibacterial activity largely 16 ACS Paragon Plus Environment


Page 18 of 38

1

depends on polycationic charges, high-MW can provide a higher number of cationic units

2

thus, improving the electrostatic forces and simultaneously, the bactericidal effects.38, 41 On

3

the other hand, solubility issues, aggregation and interaction with the cell wall barrier could

4

become crucial with MW increase. Nevertheless, these factors are dependent on both the

5

polymeric system employed and the type of bacteria tested. Here, three series of

6

PAMPTMA-co-PBA linear random copolymers with targeted MW values around 8000,

7

12000 and 24000 were synthesized (Table 1, entries 9-18, except entries 15 and 16). In

8

each series, the content of PBA was varied from 0% to 50% in order to evaluate the

9

influence of the hydrophobicity of the polymers on their antimicrobial activity. The

10

efficiencies were investigated by determining the MIC of the polymers (Table 2) against a

11

range of Gram-positive (S. aureus, B. subtilis and B. cereus) and Gram-negative (E. coli

12

and P. aeruginosa) bacteria in liquid medium.

13 14

Table 2. Characteristics of linear PAMPTMA-co-PBA copolymers with different MW

15

values and hydrophobic characters and respective MIC values obtained in liquid medium. MIC (µM) DPAMPT MA / DPnBA

% PBA (molar)

LA38B2

38 / 2

5

LA34B9

34 / 9

LA23B23

Mnth 10-3

x

S. aureus

B. subtilis

B. cereus

E. coli

P. aeruginosa

8.3

>200

3.1

200

>200

>200

20

8.4

>200

0.8

200

200

>200

23 / 23

50

8.0

>200

28.1

112.5

56.2

>200

LA50B4

50/4

7

11.0

25

0.8

50

200

200

LA45B15

46/ 15

24

11.5

>200

1.5

>200

200

>200

LA27B33

27 / 33

56

10.0

>200

200

200

100

200

Polymer code


Page 19 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

LA39B47

39 / 47

55

14.2

200

6.2

25

200

200

LA98B29

100 / 28

21

24.4

200

0.8

200

200

3.1

LA59B44

59 / 44

42

18.2

>200

0.8

>200

100

100

LA115B0*

115 / 0

0

24.0

25

6.2

12.5

200

200

LA335B0*

328/0

0

68.0

2.5

1.2

10

> 40

40

1

*

2

For the copolymers with a low percentage of PBA (LA38B2 vs. LA50B4in Table 2), the

3

increase of the MW led to an enhancement of the antimicrobial activity for all Gram-

4

positive and Gram-negative bacteria tested. The same behavior was observed for the

5

PAMPMTA homopolymers used as control samples (LA115B0 and LA335B0 in Table 2),

6

as well as for the copolymers with 20% PBA in a less pronounced variation of the MIC

7

values (LA34B9 vs. LA45B15 vs. LA98B29 in Table 2). However, for the highest contents

8

of PBA studied (up to 50 %), the antimicrobial activity of the copolymers was dependent

9

on the bacterial species tested (LA23B23vs. LA27B33 vs. LA39B47 in Table 2). In general

10

terms, the amphiphilic copolymers investigated were more active towards B. subtilis

11

bacteria, as jugged by the lower MIC values.

Homopolymer for control

12

While the cationic segments of the polymers provide electrostatic interaction with the

13

negatively-charged bacterial cell wall, the presence of hydrophobic domains within the

14

copolymer structure is also important, since it can allow for a better permeation of the

15

polymer through the bacteria lipid membrane.42 Several studies have shown that the

16

antimicrobial activity of amphiphilic structures is higher than that of hydrophilic (cationic)

17

analogues18,

18

structures.9, 14, 44 However, in this work the variation of the percentage of the hydrophobic

19

PBA segment in the copolymers did not influence their antimicrobial activity. Only a small

43

and it increases with the hydrophobicity character of the amphiphilic



Page 20 of 38

1

improvement was observed for the samples with MW around 8000 (LA38B2 vs. LA34B9

2

vs. LA23B23 in Table 2), for B. cereus and E. coli, suggesting that only low MW polymers

3

that could permeate the bacteria lipid membrane can benefit from having hydrophobic

4

domains. Possibly, the majority of the polymers investigated in this work have high MW (>

5

10000), preventing them from penetrating into the bacterial membrane. In fact, the

6

PAMPTMA homopolymers (LA115B0 and LA335B0 in Table 2) used as control samples

7

(no hydrophobic domains) showed the broadest spectrum of antimicrobial activity, along

8

with some of the lowest MIC values, compared to the amphiphilic PAMPTMA-co-PBA

9

copolymers. Besides the high MW of the samples, it was hypothesized that the behavior

10

observed in this work might be attributed to the fact that the increase of the hydrophobicity

11

could lead to different self-assembled polymeric structures in aqueous media.43 To

12

investigate this, DLS analyses of three different representatives amphiphilic copolymers

13

(highest content of PBA (LbA68B40 and LA39B47); low content of PBA (LbA264B33)),

14

at their respective MIC values in the growth medium used for the antimicrobial activity

15

assays, were performed. The results (Figure SF4) showed size distributions in the range of

16

1 nm up to 10 nm, which suggest that these polymers do not form aggregates but exist as

17

single chains (unimers) at their MIC. Therefore, in this case the effect of polymer

18

aggregation on the antimicrobial activity can be ruled out. The PBA homopolymer

19

(LA0B49 in Table 1) did not show any antimicrobial activity against the bacteria strains

20

investigated. This result highlights the need for a cationic segment in the structure of a

21

polymer in order to exhibit good antimicrobial activity.


Page 21 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

In summary, this set of results indicates that the antimicrobial activity of the studied

2

copolymers does not show dependence with hydrophobicity, but it is positively influenced

3

by the increase of the MW of the polymers.

4 5

Influence of architecture on polymers’ antimicrobial activity

6

The SARA ATRP method is a very useful tool to prepare polymers with distinct

7

architectures, which can result in materials with different antimicrobial performances. Star-

8

shaped polymers have been pointed out as promising antimicrobial agents, with a

9

considerable higher activity than their linear analogues.18 Here, new PAMPMTA

10

homopolymer stars and PAMPTMA-co-PBA star copolymers were tailored to have

11

different number of arms, MW values and hydrophobic contents to investigate the influence

12

of the architecture on their antimicrobial performance (Table 1, entries 1-8). The MIC

13

values of the polymers were obtained in liquid medium and the results are shown in Table

14

3.

15

Table 3. Characteristic of linear and star-shaped PAMPTMA-co-PBA copolymers and MIC

16

values obtained in liquid medium. MIC (µM)

Polymer code

DPAMPTMA / DPn-BA*

% PBA (molar)

Mnth x 10-3

S. aureus

B. subtilis

B. cereus

E. coli

P. aeruginosa

6SA114B0

114 / 0

0

142.0

2.5

1.2

10

40

10

6SA22B0

22 / 0

0

28.1

40

5

10

> 40

> 40

6SA9B0

9/0

0

12.7

> 40

40

> 40

> 40

> 40

6SA79B24

79 /24

23

117.3

20

1.2

> 40

> 40

10

6-arms star



Page 22 of 38

69 / 41

37

117.4

20

0.6

> 40

40

10

4SA93B0

93 / 0

0

77.6

2.5

1.2

10

40

20

4SA84B20

84 / 20

20

80.5

20

1.2

> 40

40

40

LA115B0

115/0

0

24.0

25

6.2

12.5

200

200

LA335B0

328/0

0

68.0

2.5

1.2

10

> 40

40

LA98B29

98/29

23

24.2

200

0.8

200

200

3.1

6SA69B41 4-arms star

Linear

1

*

DP value per star arm

2 3

In general, the MIC values either increased or remained constant with PBA content

4

increase, as previously observed for the linear copolymers. Compared to the star-shaped

5

cationic PAMPTMA homopolymers, the amphiphilic star-shaped copolymer analogues

6

were much less active against bacteria. This effect was highly visible for S. aureus, with

7

MIC values at least 8-fold higher than those of the homopolymers (Table 3, 6SA114B0 vs.

8

6SA69B41 and 6SA79B24; 4SA93B0 vs. 4SA84B20).

9

When compared to its star copolymer analogues (4SA84B20 and 6SA79B24 in Table 3),

10

the linear copolymer (LA98B29 in Table 3) presented higher MIC values, except for

11

P. aeruginosa. This decrease in antimicrobial efficacy might be associated with the initial

12

adsorption/binding interaction between polymer and bacteria, which is stronger for star-

13

shaped compared to linear polymers.19 Moreover, it is expected that star-shaped polymers

14

present high charge density which provides increased interactions between these polymeric

15

structures and the anionic compounds of the bacterial cell wall.45 This behavior has been

16

previously observed by other authors for peptide stars18 or nanoparticles formed by self21 ACS Paragon Plus Environment

Page 23 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Biomacromolecules

1

assembly of linear peptides.46 However, for this conclusion it is worth mentioning that the

2

star-shaped polymers have higher molecular weight (MWstar ≈ MWlinear x number of arms)

3

than their linear analogues, since the linear polymers studied are equivalent in MW to one

4

arm of the star. This rationalization has been also used by other authors and could be

5

argued due the importance of the amounts of positive charges in polymer activity.18 The

6

increase of antimicrobial activity from linear to star, in these cases, could be then associated

7

with the increase of the MW of the polymers, as previously observed.

8

The real influence of the architecture could be then evaluated by comparing linear and

9

star-shaped polymers with the similar MW values. The results presented in Table 3 show

10

that the MIC values of linear and star-shaped PAMPTMA homopolymers (6SA22B0 vs.

11

LA115B0 and 4SA93B0 vs. LA335B0) are in the same range for the bacteria investigated,

12

suggesting that in fact the architecture itself might not play a key role in the antimicrobial

13

activity. In this case, it seems that the MW of the polymers has a greater influence on their

14

performance, than the particular shape. The results obtained with star shape polymers seem

15

to be only related to the increase in the molecular weight of the polymer (considering the

16

same DP for the star arm). On this matter, the preparation of stars by SARA ATRP is still

17

an advantageous strategy since it is a straightforward route for the synthesis of very high

18

MW polymers, under controlled conditions (Ð < 1.5). The most effective polymers

19

prepared in this work (e.g., 6SA114B0 and 4SA93B0) were active for all the bacteria

20

strains investigated and presented lower MIC values, in particular for Gram positive, than

21

amphiphilic peptide nanoparticles reported in the literature,46 making them excellent

22

candidates for antimicrobial applications. Besides the good performance of the polymers



Page 24 of 38

1

presented in this work, the synthesis via SARA ATRP is much more affordable than the

2

chemistry involving the preparation of polypeptides.

3 4

Influence of the structure copolymers on the antimicrobial activity

5

As previously mentioned, several studies investigated the role of key factors that control

6

the bactericidal activity of polymers.19, 39, 47 Structural parameters such as amphiphilicity,48

7

MW, type and density of cationic charges,19 have been shown to influence the biological

8

activity of different polymers. However, there are relatively few reports investigating

9

amphiphilic block copolymers.47,

49

Here, the MIC values of amphiphilic PAMPTMA-

10

co(b)-PBA random and block copolymers with similar MW and molar ratio of n-BA was

11

evaluated in order to understand if the distribution of the hydrophobic domains within the

12

copolymers affects their antimicrobial activity.

13

The antimicrobial activity of block copolymers was higher against Gram positive bacteria

14

when compared to random copolymers with similar MW and composition (LA59B44 and

15

LbA68B40 in Table 4). Nonetheless, for E. coli, the random copolymer has a better

16

performance, showing a lower MIC value than the analogue block copolymer. These results

17

are in sharp contrast with previous data reported in the literature, showing that the

18

bactericidal activity of random and block amphiphilic poly(vinyl ether)s copolymers was

19

similar against E. coli.47 Based on these observations, it seems that the structure of the

20

copolymers could influence their antimicrobial activity depending on the bacterial strain

21

considered and the nature of the monomers. It is then important to investigate the

22

performance of antimicrobial polymers in conditions mimicking those of the targeted

23

application of the materials (e.g., target bacteria strains, in vivo or ex vivo application, etc.). 23 ACS Paragon Plus Environment

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Biomacromolecules

1

Similarly, to what was observed for the random copolymers, the decrease of the

2

hydrophobic content in the block copolymers and the increase in the MW led to an increase

3

of the antimicrobial activity (LbA264B33 sample in Table 4 exhibited the lowest MIC

4

values). Overall, block copolymers showed the broadest spectrum of activity and lower

5

MIC values.

6 7

Table 4. Characteristics of PAMPTMA-co-PBA and PAMPTMA-b-PBA copolymers and

8

MIC values obtained in liquid medium. MIC (µM) DPAMPTMA / DPn-BA*

% PBA (molar)

Mnth x 10-3

S. aureus

B. subtilis

B. cereus

E. coli

P. aeruginosa

LbA264B33

264 / 33

10

59.0

20

1.2

> 40

> 40

40

LbA68B40

68 / 40

37

19.4

225

0.8

28.1

225

112.5

LbA78B63

78 / 63

44

24.4

200

50

200

200

200

LA59B44

59 / 44

42

18.2

> 200

1.5

> 200

100

100

LA98B29

98/29

23

24.2

200

0.8

200

200

3.1

Polymer code Block copolymers

Random copolymers

9

* DP value per star arm

10 11

Preliminary studies on cell-polymer interactions

12

In order to evaluate whether the antimicrobial effect of polymers was due to loss of

13

bacterial membrane integrity, two distinct approaches to assess membrane damage were

14

used: SEM and fluorescence microscopy, as described in the experimental section. For this

15

study, representative PAMPTMA homopolymers and PAMPTMA-co-PBA copolymers of

16

each architecture (linear and star-shaped) were selected based on their antimicrobial 24 ACS Paragon Plus Environment


Page 26 of 38

1

performance. In both techniques, E. coli cells were incubated for 5 h with 4-arm star

2

(4SA84B20 and 4SA93B0, Table 1), 6-arm star (6SA114B0 and 6SA69B41, Table 1) or

3

linear (LA115B0 and LA98B29, Table 1) polymers at a concentration of MIC.

4

As shown in Figure 3, untreated E. coli cells exhibited rod-shaped morphology and

5

smooth surface. Treatment of the cells with the star-shaped polymers (4SA84B20,

6

4SA93B0, 6SA114B0 and 6SA69B41, Table 1) caused increased granules at the cell

7

surface (cell debris) and lysed cell morphologies, regardless the number of arms of the

8

stars. Treatment with linear polymers (LA115B0 and LA98B29, Table 1) also cause

9

increased cell debris granules at the cell surface, but to a less extent than the star-shaped

10

polymers. To further investigate whether the antimicrobial activity of these polymers is in

11

fact due to direct damage of the bacterial membrane, a second approach using live (Syto

12

09)/dead (propidium iodide) staining was used to assess membrane damage after polymer

13

treatment. In agreement with the SEM results, all tested polymers displayed a significant

14

increase in cell death by membrane damage compared with untreated cells (p < 0.0001)

15

(Figure 3). In particular, the 6-arm star polymers with the highest MW values (6SA114B0

16

and 6SA69B41, Table 1), exhibited the highest death by membrane damage (85.1 % and

17

90.5 %, respectively) compared with 2.5 % death in E. coli untreated cells. 4SA84B20 and

18

4SA93B0 (4-arm star polymers, Table 1) also showed increased of cell death by membrane

19

damage (72.0 % and 62.1 %, respectively). Linear polymers (LA115B0 and LA98B29,

20

Table 1) showed the lowest cell death among all polymer architecture types, but still as

21

high as 58.9 % and 52.7 % for the homopolymer and random copolymer, respectively.

22

When comparing bacterial membrane damage by different amphiphilic polymers, cell

23

damage was higher for copolymers, except in linear ones. This result suggests that 25 ACS Paragon Plus Environment

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Biomacromolecules

1

polymers with amphiphilic character and high MW increase membrane damage leading to

2

cell death.

3 4

Figure 3. SEM and fluorescence microscopy images of E. coli cells before and after

5

treatment with 6-arm, 4-arm star-shaped and linear PAMPMTA homopolymers and

6

PAMPTMA-co-PBA copolymers. Conditions: 5 h incubation at concentration of MIC.

7

Live/dead staining shows live (SYTO 09 labeled) green cells and dead (propidium iodide

8

labeled) in red. Percentage of death by membrane damage for untreated, 6SA114B0,

9

6SA69B41, 4SA84B20, 4SA93B0, LA115B0 and LA98B29-treated E. coli cells. At least

10

100 cells were counted in triplicates for at least two independent experiments. *** p =

11

0.0001; **** p < 0.0001.

12

Conclusions

13

An extensive library of effective well-defined PAMPTMA-based antimicrobial

14

(co)polymers, with different architectures, MW and compositions was prepared by a simple 26 ACS Paragon Plus Environment


Page 28 of 38

1

and scalable SARA ATRP method at near room temperature. Despite the difficulty to

2

establish general correlations between polymer features and antimicrobial activity, the

3

results showed that: (i) generally amphiphilic structures were more effective towards B.

4

subtilis bacteria; (ii) Increasing hydrophobicity (PBA segment) had no influence on the

5

antimicrobial activity; (iii) The presence of the cationic segment is mandatory for the

6

antimicrobial properties of the (co)polymers; (iv) Star-shaped polymers with the similar

7

MW as linear analogues presented similar antimicrobial activity and (v) The antimicrobial

8

activity was enhanced by the increase of the MW. The synthesis of star-shaped polymers

9

represents a convenient route to obtain high molecular weight polymers with remarkable

10

antimicrobial activity. Preliminary SEM and fluorescence microscopy studies on the

11

interaction between polymers and bacteria revealed that both cationic homopolymers and

12

amphiphilic copolymers were able to kill E. coli bacteria by membrane damage.

13 14

ASSOCIATED CONTENT

15

Supporting Information.

16

The following files are available free of charge.

17

Results of the homopolymerization of both AMPMTA and n-BA by SARA ATRP in

18

ethanol MIC values in weight-based concentration and DLS volume distributions of

19

representatives PAMPTMA-co(b)-PBA.

20 21

Corresponding Author

22

E-mail: [email protected] 27 ACS Paragon Plus Environment

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Biomacromolecules

1

Author Contributions

2

The manuscript was written through contributions of all authors. All authors have given

3

approval to the final version of the manuscript.

4 5

ACKNOWLEDGMENT

6

Patrícia V. Mendonça acknowledges her post-doctoral grant (SFRH/BPD/117589/2016)

7 8 9 10 11

supported by the Portuguese Foundation for Science and Technology (FCT). Madson R. E. Santos acknowledges CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) for his doctoral fellowship (202484/2015-7). Rita Branco acknowledges her post-doctoral grant (SFRH/BPD/110807/2015) supported by the Portuguese Foundation for Science and Technology (FCT).

12

Mariana C. Almeida acknowledges her post-doctoral grant within the Project

13

ReNATURE - Valorization of the Natural Endogenous Resources of the Centro Region

14

(Centro 2020, Centro-01-0145-FEDER-000007).

15 16 17 18

The authors thank Postnova for the valuable technical discussions about the molecular weight characterization of polymers. The authors thank Portuguese Foundation for Science and Technology (FCT) for funding the Safesurf project (PTDC/CTMPOL/ 6138/2014).

19

The 1H NMR data was collected at the UC-NMR facility which is supported in part by

20

FEDER –European Regional Development Fund through the COMPETE Programme 28 ACS Paragon Plus Environment


Page 30 of 38

1

(Operational Programme for Competitiveness) and by National Funds through FCT –

2

Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and

3

Technology) through grants REEQ/481/QUI/2006, RECI/QEQ-QFI/0168/2012, CENTRO-

4

07-CT62-FEDER-002012, and Rede Nacional de Ressonância Magnética Nuclear

5

(RNRMN).

6 7 8 9 10 11

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Ng, V. W. L.; Chan, J. M. W.; Sardon, H.; Ono, R. J.; García, J. M.; Yang, Y. Y.;

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Hedrick, J. L., Antimicrobial hydrogels: A new weapon in the arsenal against multidrug-

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resistant infections. Adv. Drug Del. Rev. 2014, 78, 46-62.

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Santos, R. M.; Fonseca, C. A.; Mendonça, V. P.; Branco, R.; Serra, C. A.; Morais,

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V. P.; Coelho, F. J., Recent Developments in Antimicrobial Polymers: A Review. Materials

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2016, 9, (7), 599.

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Parachin, N. S.; Franco, O. L., New edge of antibiotic development: antimicrobial

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Xue, Y.; Xiao, H.; Zhang, Y., Antimicrobial Polymeric Materials with Quaternary

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Biointerfaces 2014, 122, 294-300.

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Koromilas, N. D.; Lainioti, G. C.; Vasilopoulos, G.; Vantarakis, A.; Kallitsis, J. K.,

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Synthesis of antimicrobial block copolymers bearing immobilized bacteriostatic groups.

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Table of contents

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Increasing the Antimicrobial Activity of Amphiphilic Cationic

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