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High Efficiency Antimicrobial Thiazolium and Triazolium Side-Chain Polymethacrylates Obtained by Controlled Alkylation of the Corresponding Azole Derivatives Rubén Tejero,† Daniel López,*,† Fátima López-Fabal,‡ José L. Gómez-Garcés,‡ and Marta Fernández-García*,† †

Instituto de Ciencia y Tecnología de Polímeros (ICTP-CSIC), Juan de la Cierva 3, 28006 Madrid, Spain Hospital Universitario de Móstoles, Río Júcar, s/n, 28935 Móstoles, Madrid, Spain

‡

S Supporting Information *

ABSTRACT: Two series of antimicrobial polymethacrylates (PMTAs) bearing mono and bis-cationic quaternary ammonium cations (QUATs) were prepared by controlled N-alkylation of 1,3-thiazole and 1,2,3triazole pendant groups with butyl iodide (PMTAs-BuI). The degree of quaternization (DQ) of the azole heterocycles was monitored by 1H NMR spectroscopy over a wide range of reaction times. Spectra analysis of the 1H NMR aromatic region allowed to characterize and quantify the different species involved and, therefore, to control the chemical composition distribution of the amphiphilic polycations. The polymer charge density and the hydrodynamic sizes were measured by zeta potential and dynamic light scattering (DLS), respectively. Consequently, the relationship between structure and antibacterial properties and toxicity was studied. Interestingly, these polyelectrolytes present excellent selective toxicity against bacteria being nonhemolytic even at low values of DQ. Furthermore, they were also evaluated for their microbial time-killing efficiency, presenting a 3 log-reduction in only 15 min. Additionally, the bacteria cell morphology treated with PMTAs-BuI was analyzed.

1. INTRODUCTION Microbial infections are still a public health problem worldwide,1 and a growing number of microorganisms are becoming multidrug resistant due to several factors such as the overuse of antibiotics. 2 Natural cationic amphiphilic polypeptides 3 (AMPs) as well as synthetic mimic antimicrobial polypeptides (SAMPs),4−8 especially quaternized polymeric ones (QUATs), present some advantages9 toward common antibiotics such as effective broad-spectrum activity10−13 and less toxicity. Additionally, in contrast to conventional antibiotics, these systems appear to possess different mechanisms of action,14,15 often bactericidal and, in general, by cell membrane disruption.16,17 Moreover, they often require a long exposure/contact time in order to achieve 100% bacterial death. The study and development of new kill-efficient systems are utterly required. There are numerous reported examples about the importance of several structural parameters that affect directly the antimicrobial and toxicological properties of polyelectrolites, such as the cationic charge distribution,18−21 the nature and length of the side-chain or the alkylating agent,22,23 the hydrophobicity,24 the amphiphilic balance,25,26 and the molecular weight27 or the nature of the counterions.28−31 Hence, the control on the chemical structure and the design of novel and more efficient nontoxic antimicrobial agents are currently needed.31−33 In a recent work,22 we designed six series of polymethacrylates bearing pendant 1,3-thiazole and 1,2,3-triazole hetero© XXXX American Chemical Society

cycles and spacer groups of different chemical nature and length (PMTAs) inspired by the well-known biological activities of these type of heterocyclic compounds.34−39 The N-alkylation reaction of the thiazole and triazole groups with a variety of iodoalkanes of different lengths (from methyl to hexadecyl) allowed to prepare an extensive number of amphiphilic polymers containing quaternary ammonium groups (QUATs) and controlled hydrophilic−hydrophobic balance. Overall, all these polyelectrolytes were obtained at 100% of quaternization and many of them showed promising broad-spectrum antimicrobial activity against microorganisms being, in addition, nonhemolytic against human red blood cells (RBCs). Among the strategies mentioned above that let to explore and modulate the antimicrobial and hemolytic activities of polycations and specially in QUATs,40 we decided to study the effect of the degree of quaternization (DQ) on the amphiphilic balance and hence, on the biological properties of the systems. For this purpose, two polymers of these series presenting the best global antimicrobial and nonhemolytic activities, and consequently, the best selective toxicity: N-butyl alkylated 1,3-thiazolium polymers and N-butyl alkylated 1,3-thiazolium 1,2,3-triazolium ones (PMTA1-BuI and PMTA4-BuI, respectively) were selected (see Scheme 1). Special attention has been carried Received: April 1, 2015 Revised: May 4, 2015

A

DOI: 10.1021/acs.biomac.5b00427 Biomacromolecules XXXX, XXX, XXX−XXX

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with the aid of a Finnpipette F2 (Thermo Scientific) as a multichannel pipet and appropriately sized pipet tips previously sterilized in a Matachana SC500 Autoclave. Absorbance measurements were performed with a Triturus (Grifols) microplate reader. Sheep blood (5%) Columbia Agar plates were purchased from bioMérieux and BBL Mueller Hinton broth was purchased from Becton, Dickinson and Company and was used as a microbial growth media. American Type Culture Collection (ATCC): Gram-negative Pseudomonas aeruginosa (ATCC 27853) and Gram-positive Staphylococcus aureus (ATCC 29213) were used as bacterial strains and were purchased from Oxoid. Microorganisms were incubated at 37 °C in a Jouan IQ050 incubator. The optical density of the microorganism suspensions was measured in McFarland units proportional to microorganism concentration by a DensiCHEK Plus (VITEK, bioMérieux). Human red blood cells (RBC) from healthy donors (Hospital Universitario de Móstoles, Madrid), were centrifuged in a Kubota KS-8000 centrifuge. 2.2. Synthesis of Polymers. Briefly, MTA1 and MTA4 monomers were synthesized as previously reported.22 Then, the radical polymerization of these monomers at total conversion was carried out in dry DMSO solution (1 M) at 60 °C using AIBN as initiator (5 × 10−2 M). Polymers were purified by precipitation in distilled water and dried under vacuum. 2.3. Modification of Polymers. N-butyl quaternized PMTAs were obtained using sealed tubes containing magnetic stirring bars. The tubes were charged with the relevant polymer PMTA (1.0 equiv) and 1-iodobutane (3.5 equiv for PMTA1 and 5.0 equiv for PMTA4) in anhydrous DMF (0.1 mmol/mL). Mixtures were purged with argon for 15 min and heated at 70 °C for 140 h. Samples were withdrawn at various time intervals and purified by dialysis against distilled water and freeze-dried. The kinetics of reaction and the DQ were monitored by Nuclear Magnetic Resonance Spectroscopy (1H NMR). Spectra were recorded on a Bruker (500 MHz) spectrometer. Measurements were carried out at ambient temperature (ca. 22 °C). Chemical shifts (δ) are reported in ppm with the residual solvent signal as internal standard (dimethyl sulfoxide at 2.50 ppm). Additional calculations related to the determination of the DQ are detailed in the Supporting Information (SI). 2.3. MIC and Hemolysis Measurements. The antimicrobial activity of polymers with various DQ was tested against ATCC bacteria strains according to the Clinical Laboratory Standards Institute (CLSI) microbroth dilution reference methods.41,42 Then, the minimum inhibitory concentration (MIC) of each sample, defined as the lowest concentration of polymer required for the inhibition of the visible growth of a microorganism after a 24h incubation period at 37 °C, The hemolytic activities of the PMTA1-BuI and PMTA4-BuI polymers with different DQ against human Red Blood Cells (RBC) from healthy donors were determined by the method of Helmerhorst et al.43 and in accordance to procedures recently reported in literature (hemolytic curves are in the SI).18,23 2.4. Time-Kill Efficiency Test. Gram-negative P. aeruginosa and Gram-positive S. aureus bacteria strains were cultured according to the same procedure9,10,12 in the MIC assays mentioned above. Bacterial solutions were treated with the polymers at MIC and 2 × MIC concentrations in a sterile 96-well microplate. Null MIC was used as the positive control and samples were incubated at 37 °C under constant shaking of 100 rpm. Thus, 4 series of 10-fold dilutions were performed from bacteria solutions of each well at regular time intervals (0, 5 min, 15 min, 30 min, 60 min, 120 min, 240 min and 24 h). Twenty microliters of each diluted bacterial solution was streaked onto 5% sheep blood Columbia agar plates, and then the plates were incubated for 24 h at 37 °C. After that period, the plates were counted for visible colony-forming units (CFU).44 2.5. Morphology of Microorganisms. Bacteria strains were cultured in a similar protocol as antimicrobial measurements9,10 and treated with PMTAs-BuI at 2 × MIC and incubated for 15 min. After incubation, the microbes were fixed with formalin solution (10% neutral buffer) for 90 min, followed by washing twice with deionized water. Then, dehydration of the samples in a graded ethanol series (30−100%) was conducted. The dehydrated samples were dried at room temperature, placed onto carbon tape and imaged with field

Scheme 1. N-Alkylation of PMTAs: Synthesis of Amphiphilic Polymethacrylates with Various DQ: (A) (PMTA1)x-co(PMTA1-BuI)y and (B) (PMTA4)x-co-(PMTA4-BuI)y.

out on the control of the degree of quaternization in terms of each heterocycle of the polymer (thiazole and/or triazole) and the effect on their biological properties, that is, on the antimicrobial and hemolytic activities, the killing-efficiency, and the mechanism of action.

2. EXPERIMENTAL SECTION 2.1. Materials and Instrumentation. 1-Iodobutane (99%, Aldrich), anhydrous N,N-dimethylformamide (DMF, 99.8%, AlfaAesar), n-hexane (96%, Scharlau Chemie) were used as received. 2,2′Azobis(isobutyronitrile) (AIBN, 98%) was purchased from Acros and was recrystallized twice from methanol (MeOH) prior to use. Polyelectrolytes purification was performed by dialysis with a cellulose membrane with a molecular weight cut off of 12 kDa that was obtained from Spectrum. NaCl solution (0.9%, BioXtra, suitable for cell culture), dimethyl sulfoxide (DMSO; BioReagent, for molecular biology, suitable for plant cell culture, ≥ 99.9%), phosphate buffered saline powder (PBS, pH 7.4), and Triton X-100 solution (BioUltra, for molecular biology, ∼10% in H2O) were purchased from Aldrich and were used as received. The number-average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersity index (PDI) of polymers were measured by Size Exclusion Chromatography (SEC) with a chromatographic system (Waters Division Millipore) equipped with a Waters model 410 refractive-index detector. N,N-dimethylformamide (Aldrich, 99.9%) containing 0.1% of LiBr (Aldrich, 99.99%), was used as the eluent at a flow rate of 1 mL/min at 50 °C. Styragel packed columns (HR2, HR3, and HR4, Waters Division Millipore) were used. Poly(methyl methacrylate) standards (Polymer Laboratories, Laboratories, Ltd.) ranging from 2.4 × 106 to 9.7 × 102 g/mol were used to calibrate the columns. The particle size and zeta potential by dynamic light scattering (DLS) measurements were conducted using the Zetasizer Nano series ZS (Malvern Instrument Ltd., U.K.), which is equipped with a He−Ne laser beam at 658 nm. The particle size and zeta potential of polymers in deionized (DI) water was averaged over at least 5 runs. In vitro antimicrobial and cytotoxic experiments were performed in 96-well microplates (BD Biosciences) B


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Biomacromolecules Table 1. Physico-chemical Characteristics of Selected PMTAs-BuI with Different DQs PMTA1-BuI with various DQth (%) MIC (μg/mL), SId (HC50/MIC) DQth (%)

Mna (kDa)

PDIa

0 18 49 69 100

53 -

2.3 -

Dhb (nm) 270 ± 170 ± 160 ± 133 ±

PDIc (nm)

zeta potential (mV)

19 0.45 ± 0.04 12 0.41 ± 0.02 16 0.49 ± 0.03 11 0.33 ± 0.04 PMTA4-BuI with various DQth

24.2 ± 3.4 38.4 ± 4.5 45.4 ± 5.3 56.5 ± 5.3 (%) and DQtr (%)

P. aeruginosa

S. aureus

HC50 (μg/mL)

128 (12) 8 (431) 8 (>625) 8 (>625)

32 (49) 4 (862) 8 (>625) 8 (>625)

1588 ± 183 3446 ± 203 >5000 >5000

MIC (μg/mL), SIc (HC50/ MIC) DQth (%)

DQtr (%)

Mna (kDa)

PDIa

Dh (nm)

PDIb (nm)

0 21 48 71 100

0 29 62 82 100

119 -

2.5 -

185 ± 13 129 ± 19 122 ± 9 105 ± 12

0.42 0.37 0.38 0.46

± ± ± ±

0.04 0.05 0.04 0.02

zeta potential (mV)

P. aeruginosa

S. aureus

HC50 (μg/mL)

± ± ± ±

64 (43) 8 (515) 8 (>625) 4 (>1250)

32 (86) 2 (2058) 4 (>1250) 4 (>1250)

2764 ± 266 4116 ± 302 >5000 >5000

36.4 42.7 54.9 68.4

6.2 7.1 6.4 5.4

a Number-average molecular weight (Mn) and polidispersity index (PDI) of nonquaternized homopolymers determined by SEC. bHydrodynamic diameter determined by dynamic light scattering. cPolidispersity index (PDI) obtained from dynamic light scattering. dSelectivity index, SI, (HC50/ MIC) is given in parentheses.

emission scanning electron microscopy (FE-SEM) using a HITACHI SU8000 microscope.

affording 100% of quaternization at ca. 98 h. In addition, the reaction fits a first-order kinetic reaction as detailed in the SI. On the other hand, looking deeper into the expanded view of the aromatic region of the spectra (Figure 1B), it is observed that the peaks linearly shift downfield with the DQth (Figure 2B). This circumstance is possibly due to the increment of positively charged heterocycles as the quaternization reaction proceeds. Accordingly, a significant deshielding effect is caused by the positive global charge density of the polycations.48 It is worth mentioning that no splitting of the peaks throughout the reaction is observed in the 1H NMR spectra, although it can be noticed a broadening of the peaks and a suppression of symmetry at high values of DQth. These facts can be due to configurational or compositional effects as well as to the increment of viscosity49,50 due to interactions arising from quaternized polyelectrolytes. Having in mind that PMTAs were obtained by conventional radical polymerization, thus yielding atactic polymers,51 along with the fact that quaternization was carried out at high temperature with excess of the alkylating agent, randomly modified polymers could possibly be formed.52,53 In the case of the modification reaction of the bisheterocyclic PMTA4, it is known that triazoles groups were regioselectively alkylated at the more electron rich N3 position (when 1,2,3triazoles are alkylated by soft alkylating reagents, only 1,2,3triazolium salt products are obtained),54−56 and four species of repeating units during the N-alkylation reaction are expected as it is depicted in Scheme 2. These species include nonquaternized heterocycles (Species 1), monocatonic triazolium and thiazole (Species 2), triazole and monocationic thiazolium (Species 3), and both quaternized (bis-cationic) heterocycles (Species 4). Therefore, peaks corresponding to aromatic protons of both quaternized and nonquaternized heterocycles (A protons for thiazole and B protons for triazole) in each of the four species (a total of eight different peaks, see Scheme 2 for assignments), should be expected. Looking at the 1H NMR spectra of the N-alkylation reaction (Figure 3A), especially at the aromatic region (Figure 3B), the appearance of an

3. RESULTS AND DISCUSSION 3.1. Polymer Modification. The N-butyl alkylation of PMTA1 and PMTA4 polymers was carried out as described in the Experimental Section. Since the degree of polymerization does not change with the course of the reaction, molecular weights were only determined for PMTA1 and PMTA4 polymers by SEC chromatography (see Table 1). In order to obtain polyelectrolytes of different and controlled chemical compositions, samples were withdrawn at various time intervals, purified by dialysis against distilled water and analyzed by 1H NMR. The general synthetic procedure and NMR analysis are gathered in the SI. All the spectra were referenced to the solvent peak (DMSO-d6, 2.50 ppm) and normalized to the intensity of the OCH2 signal of methacrylate group around 4.00 ppm, the intensity of which does not vary with the course of the reaction. The area of interest for the analysis was the aromatic region (above 7 ppm). Protons on the nonquaternized heterocycles (1,3-thiazole and 1,2,3-triazole) as well as the quaternized ones (1,3-thiazolium and 1,2,3-triazolium) appear at that region and show different chemical shifts (δ), which make them distinguishable and quantifiable. In addition, protons on the positively charged heterocycles are shifted downfield in relation to the nonquaternized ones.45,46 In the case of the modification of PMTA1 (see Figure 1A), the peaks corresponding to the aromatic protons (A1 for the thiazole and A2 for the thiazolium) do not overlap with each other. Thus, a comprehensive relationship between the areas of the peaks can be applied to calculate DQ, which in this case corresponds to the thiazole modification, DQth (eq 1). DQth (%) =

Integral (Thiazolium proton A 2) Integral (Thiazolium proton A 2 + Thiazole proton A1) × 100

(1)

The representation of the calculated DQth of PMTA1 versus reaction time displays an exponential behavior47 (Figure 2A) in which the DQth significantly increases at low reaction times C


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Figure 2. (A) Degree of quaternization, DQth, of PMTA1 versus reaction time. (B) Chemical shift of the A1 protons (thiazole) and A2 (thiazolium) of the aromatic rings versus DQth.

Scheme 2. Side-Chain Species during Modification of PMTA4: (1) Nonquaternized Heterocycles, (2) Monocatonic Triazolium and Thiazole, (3) Triazole and Monocationic Thiazolium, and (4) Biscatonic Quaternized Heterocycles

Figure 1. (A) N-alkylation reaction of PMTA1 monitored by 1H NMR spectroscopy in DMSO-d6 at 500 MHz and 25 °C and at various selected time intervals (from bottom to top). (B) Expanded view of the aromatic region.

important number of different peaks as a consequence of the quaternization reaction can be observed. These peaks ought to be assigned to the aromatic protons involved in the four different species, that is, A1, A2, A3, A4 for the thiazole heterocycles and B1, B2, B3, B4 for the triazole ones. Particularly, the assignment of the peaks corresponding to the monocationic Species 2 and 3 is made based on their neighboring chemical environment: protons of nonquaternized heterocycles should be shifted downfield because they are situated next to another positive charged heterocycle. Similarly, those quaternized heterocycles located near to nonquaternized ones are less downfield than in the case of Species 4. In fact, the biscationic Species 4 having both heterocycles quaternized present the most deshielded protons. Therefore, there is a significant “neighboring” shielding or deshielding effect. In a similar way as PMTA1 modification, most of the peaks are also gradually shifted downfield during the course of the reaction for PMTA4 (Figure 4B) may be due to the positive global charge density deshielding effect of the quaternized macromolecules. Some peaks are slightly overlapped; consequently, the deconvolution of the spectra (see SI) was carried out.

The assignment of the bands corresponding to the different species allows not only the determination of the global degree of quaternization of the polyelectrolyte, but also the composition, that is, the mole percentage of the different species in the modified polymer as the quaternization reaction proceeds. DQth (%) =

Integral (Thiazolium A4 + A3) × 100 Integral (Thiazolium A4 + A3 + Thiazole A 2 + A1)

(2) D


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Figure 3. (A) N-alkylation reaction of PMTA4 monitored by 1H NMR spectroscopy in DMSO-d6 at 500 MHz at 25 °C and at various selected time intervals (from bottom to top). (B) Expanded view of the aromatic region showing the splitting protons of thiazolium (A4, A3), triazolium (B4, B2), thiazole (A2, A1) and triazole (B3, B1) rings and the deshielding effect on their chemical shift. DQtr (%) =

Figure 4. (A) Degrees of quaternization (DQth and DQtr) of PMTA4 versus reaction time. (B) Chemical shift of the aromatic protons of each species versus DQth.

Integral (Triazolium B4 + B2) × 100 Integral (Triazolium B4 + B2 + Triazole B3 + B1)

triazole peaks present lower or worst mobility than thiazole ones, as can be seen in nonquaternized PMTA4 and completely quaternized PMTA4-BuI-100. In fact, triazolium peaks (B2 and B4) are fully overlapped throughout the reaction. Nevertheless, it is possible to know the proportion of the corresponding units thanks to the contribution of the other heterocycle involved in each species.

(3)

The global DQ of each heterocycle separately (DQth and DQtr, for thiazole and triazole groups) were easily determined applying eqs 2 and 3, considering the relationship between the global integrals of quaternized (A4 + A3 and B4 + B2) and nonquaternized heterocycles (A1 + A2 and B1 + B3), respectively. The evolution of the DQs with reaction time describes well-defined exponential curves for both triazole and thiazole units, as shown in Figure 4A, and, therefore, first-order kinetic reactions are found (see SI). Following the course of the reaction, it is easy to notice that triazole rings are quaternized faster than thiazole groups due to the more nucleophilic character of these heterocycles. According to the peak assignments achieved, eqs 4−7 are applied to determine the composition of each involved species in the quaternization reaction. It is worth pointing out that there are two series of possible equations that can be used for the determination of the chemical composition of the polyelectrolyte since both of the heterocycles are linked in every repeating unit, which allows checking the correct integration of the peaks. That means that the series of peaks A1 to A4 or B1 to B4 can be used to determine the chemical composition with the same experimental result. In particular,

Species 1 (%) =

Species 1 (%) =

Integral (Triazole B1) Integral (Triazolium B2 + B4 + Triazole B3 + B1) × 100 or Integral (Thiazole A1) Integral (Thiazolium A4 + A3 + Thiazole A 2 + A1) × 100

(4) Species 2 (%) =

Species 2 (%) =

Integral (Triazolium B2) Integral (Triazolium B2 + B4 + Triazole B3 + B1) × 100 or Integral (Thiazole A 2) Integral (Thiazolium A4 + A3 + Thiazole A 2 + A1) × 100

(5) E


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Biomacromolecules Species 3 (%) =

Species 3 (%) =

the biscatonic triazolium−thiazolium Species 4, its content increases linearly with the reaction time being the main component at DQs higher than 60%. The quantity of monocationic Species 2 and 3 increases at low reaction times (low DQths) and becomes the main component when the nonquaternized Species 1 is almost consumed (content around 50−60%). At that point, the contribution of both Species (2 and 3) decreases linearly until the end of reaction, globally describing an asymmetric parabolic curve. Accordingly, the quaternization takes place preferentially in Species 1 than in repeating units that have already been quaternized (Species 2 and 3) may be due to the repulsion of the positive environment and the more steric hindrance. 3.2. Biological Assays. 3.2.1. Antimicrobial and Hemolytic Activities. Two series of copolymers from both families, PMTA1 and PMTA4, at different degrees of quaternization from 10 to 100% were selected, and the influence of DQth on the antimicrobial and hemolytic activities of the polyelectrolytes was analyzed. The results are summarized in Table 1 and Figure 6.

Integral (Triazole B3) Integral (Triazolium B2 + B4 + Triazole B3 + B1) × 100 or Integral (Thiazolium A3) Integral (Thiazolium A4 + A3 + Thiazole A 2 + A1) × 100

(6) Species 4 (%) =

Species 4 (%) =

Integral (Triazolium B4 ) Integral (Triazolium B2 + B4 + Triazole B3 + B1) × 100 or Integral (Thiazolium A4) Integral (Thiazolium A4 + A3 + Thiazole A 2 + A1) × 100

(7)

Very useful information can be obtained about the chemical composition of the polyelectrolytes throughout the whole reaction by representing the mole percentage of the different repeating units as a function of time (Figure 5A) or as a function of the DQth (Figure 5B).

Figure 6. Effect of the DQth on the antimicrobial and hemolytic activities of PMTAs-BuI toward microorganisms and human RBCs: (A) PMTA1-BuI and (B) PMTA4-BuI.

The microbroth dilution reference method was applied,41,42 obtaining the MIC values. Two opportunistic57,58 bacterial strains were chosen: Gram-positive S. aureus and the highly resistant Gram-negative P. aeruginosa.59 At the same time, the hemotoxicity of these systems toward RBC was studied by their capability to produce 50% of hemolysis at a given concentration (HC50) as well as the selectivity index (SI = HC50/MIC) against microorganisms versus RBCs. The results of these studies are shown in Figure 6. Interestingly, both series of mono and bis-cationic side-chain copolymers exhibited the pronounced antimicrobial activity detected for polymers at 100% of quaternization22 (MIC values of 8 μg/mL for PMTA1-BuI and 4 μg/mL for PMTA4-BuI) as shown in Figure 6. This activity along with the low hemotoxicity (the data are later described) is unexpected because mainly high activity is directly related to high toxicity.18 Additionally, these polymers present high molecular weights (Mn of 50−120 kDa), and it is reported that the cytotoxicity increases with Mn.60,61 Some works have suggested the positive influence25,62 of increasing molecular weights on antimicrobial

Figure 5. (A) Percentage of species during the N-alkylation reaction of PMTA4 versus reaction time and (B) versus DQth.

In Figure 5A, the evolution of nonquaternized heterocycles (Species 1) describes an exponential decay during reaction time as is expected, since both aromatic rings are being constantly quaternized. It can also be observed that the monocationic triazolium−thiazole, Species 2, is predominant over triazole-thiazolium Species 3, which agrees well with the more nucleophilic character of the triazole group. In the case of F


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Biomacromolecules properties, while others claim the opposite behavior.4,12,63,64 For example, the antimicrobial activity of polycarbonates against the Gram-positive S. aureus increases as the molecular weight decreases from 23 800 g/mol to 8170 g/mol.12 On the contrary, the activity of polymethacrylate derivatives increases with molecular weight from 10100 g/mol to 1300 g/mol.62 On the other hand, two facts can be distinguished in the evolution of the MIC values with DQ. At DQth values from 0 to 50−60% moderate antibacterial activities are observed. In this range, minimum inhibitory concentration (MIC) values continuously decrease from ca. 128 μg/mL to around 10 μg/ mL as the DQ increases for both series of copolymers. At DQth values higher than 60% the antimicrobial activity reaches a constant MIC value at 8 μg/mL. This behavior suggests that cationic charge density plays a key role on the antimicrobial activity of the polycations, probably due to the interactions between them and the negatively charged bacterial cell walls.65 Similar effects have been observed in several reported systems bearing ammonium groups including dendrimers,28 copolyoxetanes66 or SAMPs.29,67 Yet, not only does charge density influence antimicrobial activity, but also the distribution and the position of cationic charges.18,29,68 As it was stated before, low MIC values are obtained for both families of polymers at DQth values of about 50−60% and higher. However, the density of charges is approximately twice in the case of PMTA4 than in the case of PMTA1 series. The variation of zeta potential as a function of QDth and MIC values is represented in Figure 7 for

polymeric families. The best values of MIC are therefore obtained for polymers with a 40−60% of quaternization of the thiazole groups irrespectively of the modification degree of the triazole ones. This is consistent with the results of DLS measurements (see Table 1) showing that above 50% of DQth all polymers present similar hydrodynamic sizes (from 100 to 170 nm) and positive Zeta potential values (38−68 mV), directly related with the antimicrobial activity, indicating that polycations form dispersions with moderate to good stability. Slightly higher MIC values are found for PMTA4-BuI (Figure 6B) copolymers at different DQth against the Gramnegative bacteria P. aeruginosa than for the Gram-positive S. aureus, which is probably due to the more-complex P. aeruginosa cell membrane with an extra outer membrane,58 although this effect is not observed in PMTA1-BuI series (Figure 6A). In addition, MIC values toward Gram-positive S. aureus of the bis-cationic PMTA4-BuI series (4 μg/mL) compared to PMTA1-BuI (8 μg/mL) with DQth above 30% may indicate that the charge density in this case could play a slightly improvement of the antibacterial activity. Another intriguing result is the low MIC values observed, and therefore the good antibacterial properties, at very low degrees of quaternization, especially in the case of PMTA4 series. For example, PMTA4-BuI at 30% DQth presents a MIC of 16 μg/mL, which is rather high in relation to other QUATs with similar charge density.69−71 This result suggests a synergic effect due to some sort of cooperation between the unquaternized heterocycles that are highly polar72−74 and the quaternized ones. The reason for these cooperative effect may be due to a compensation of the amphiphilic balance, as some polar heterocycles may be counterweighing the absence of net charges and hydrophobic butyl groups.75 As a matter of fact, there exist natural76 thiopeptides macrolides with thiazole moieties in their backbone with known antimicrobial activities, even antimalaria.77 The mode of antibacterial action of these thiazolyl macrolides is generally within the cell wall via the inhibition of ribosomal protein synthesis and depends on the macrocycle size. Nevertheless, those macrolides with the largest number of thiazole units (>35 rings) possess an unknown mechanism.78 Furthermore, some of them also exhibit antifungal activity by binding to chitin of the cell walls of fungi.79 Additionally, polyelectrolytes were found to be nonhemolytic irrespective of the DQth, presenting HC50 values close to 1000 μg/mL at low DQth and above 5000 μg/mL at higher DQth, thus maintaining excellent selectivity index defined as HC50/ MIC. In fact, some of them present selectivity values ranging from 600 to 2000. PMTA4-BuI at DQth above 50% showed slightly less hemolytic character compared to PMTA1-BuI series. Also, it is manifest that when the DQth increases (i.e., charge density and hydrophilic character) both series of polycations are slightly less hemolytic in agreement with some examples found in the literature that point to hemolytic activity increases with hydrophobicity.16,24,67,80,81 The low hemotoxicity shown by this QUATS at low DQth, regardless of their low cationic charge densites (i.e., hydrophilicity), can also be explained due to the presence of polar and less lipophilic unquaternized thiazole and triazole groups. 3.2.2. Bactericidal Kinetics and Mechanism. The antimicrobial efficiency of these polycations against Gram-positive S. aureus (Figure 8A,B) and Gram-negative P. aeruginosa (Figures 8C,D) was evaluated by time-kill experiments

Figure 7. Representation of relationships between modification degree (QDth), MIC, and zeta potential values.

both series. It can be clearly observed that PMTA4 series have higher outer positive charge than PMTA1 when similar DQth are compared. Besides, it is also clear that MIC values decrease as zeta potential data increase, that is, with the positive charge. The maximum antimicrobial activity is obtained for PMTA1 at around 50% DQth, whose density of charges corresponds to 23% DQth for the PMTA4 series (see Figure 5B), which at this modification degree has only a moderate antimicrobial activity. At a DQth value of 23% for PMTA4 the distribution of species is approximately 50% nonquaternized units, 27% monocationic triazolium units, 14% monocationic thiazolium, and 9% biscationic triazolium thiazolium units. Therefore, it seems as if the antimicrobial activity is determined not by the absolute charge density but for the type of charged unit (triazolium or thiazolium) and its distribution in the polymer chain. These results point out to a greater influence of thiazolium units than triazoliun ones in the antimicrobial properties of these G


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Figure 8. Microbial time-killing efficiency of the systems with various DQth at MIC and 2 × MIC against S. aureus (A and B) and P. aeruginosa (C and D).

performed by colony counting method.82 Copolymers of both families PMTA1 and PMTA4 at 100% and 50% of quaternization (DQth) were selected. Bacteria solutions were treated at two inhibitory concentrations (Null MIC as control, MIC and 2 × MIC) and at various time intervals for each family. Excitingly, PMTAs presented very high killing-efficiency (>3log reduction) tested against both microorganisms at very low exposure times, achieving 100% killing in less than 1 h for both PMTA series and, in the case of the PMTA4 series, in less than 15 min at 2 × MIC. The fact that a high level of bacterial death is achieved at only 5 min of exposure, together with PMTA4 polymers being substantially more efficient than the PMTA1 series, indicate that biscatonic side chain groups play an important role not only in delivering better selectivities, as stated before, but also in producing a superior effectiveness. Additional, PMTA4-BuI with 50% of DQth reduces the 100% of either bacteria (Grampositive or Gram-negative) at only 5 min, as can be seen in Figure 8B and 8D. This result makes this polymer can be considered as a potent antiseptic with bactericidal activity. To the best of our knowledge, there are only a few examples that have been reported about polycations presenting fastkilling antimicrobial activities in solution.11,12 For example, biodegradable polycarbonates at 2 × MIC concentration are able to eliminate S. aureus in 2.5 h, which gives an idea of PMTA1-BuI and PMTA4-BuI series strengthens. These results suggest that the polyelectrolytes presented here are not only bacteriostatic, inhibiting the growing of microorganisms at a given concentration, but also strong bactericidal systems.

To support the mechanism of bactericidal action, the cell morphology of S. aureus and P. aeruginosa was examined by FESEM before and after the treatment with PMTAs-BuI at 2 × MIC for 15 min (see Figure 9). After that period of time, some important changes on their cell surface are found for the treated samples compared to the smooth cell surfaces of the intact controls. In the case of Gram-positive S. aureus bacteria, the surface seems to be wrinkled and with some blebs after treatment with PMTAs-BuI as can be observed in Figure 9A. Regarding P. aeruginosa bacteria (Figure 9B), they show an intensified aggregation between cells like membrane-fusion events occurred after treatment with polycations (Figure 9, b2 (ii)). Besides, the surface is severely damaged, presenting a distorted and collapsed cell surface (Figure 9, b2 (iii)). These results suggest a plausible bacterial membrane disruption mechanism of action induced by polycationic PMTAs.9,10,12,83

4. CONCLUSIONS Two series of amphiphilic polycations has been prepared through the controlled quaternization reaction of thiazole and triazole side-chain polymethacrylates. The kinetics of the modification reaction has been followed by 1H NMR spectroscopy allowing for the study of the different species involved during the quaternization processes. Since this methodology is based on the aromatic region of the 1H NMR spectra, it can be extended to any alkylating agent with any of the polymer series developed in our previous work.22 The controlled N-alkylation of QUATS allows tuning the charge density, and the charge density distribution in the quaternized polymers, and therefore their amphiphilic properH


Article

Biomacromolecules Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been supported by MINECO (MAT201017016, MAT2011-24797 and MAT2013-47902). R.T. acknowledges CSIC for his JAE-Pre grant.

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Figure 9. FE-SEM images of (A) Gram-positive S. aureus (a1 and expanded view a2) and (B) Gram-negative P. aeruginosa (b1 and expanded view b2) before (i) and after 15 min treatment with PMTA1-BuI (ii) and PMTA4-BuI (iii) at 2 × MIC.

ties. This has permitted us to develop a straightforward method to study the relationship structure/antimicrobial properties of these systems. Our results show that high values of degree of quaternization are not required for these series of polymers to exhibit strong and very fast bactericidal behavior at low exposure times being, in addition, nonhemolytic. Particularly, PMTA4-BuI-50 at 2 × MIC, can produce 100% of bacterial death at only 5 min even at a quaternization degree of 50%. In summary, these new types of thiazole and bisheterocyclic thiazole-triazole derivatives represent a novel class of highly effective antimicrobial polymers and may serve as antibacterial agents in different biomedical devices or processes such as wound healing. Further studies to improve the performance of these biomaterials changing the chemical nature of the macromolecular main chain by copolymerization with either hydrophobic and hydrophilic monomers as well as further assays in order to understand the mode of action of these QUATs will be developed.

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ASSOCIATED CONTENT

* Supporting Information S

Deconvolution of 1H NMR spectra of the PMTA4-BuI polyelectrolytes and biological assays procedures. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.5b00427.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (D.L.). *E-mail: [email protected] (M.F.G.). I


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