Article pubs.acs.org/Biomac
Impact of Functional Satellite Groups on the Antimicrobial Activity and Hemocompatibility of Telechelic Poly(2-methyloxazoline)s Christoph P. Fik,† Christian Krumm,† Christina Muennig,† Theresa I. Baur,† Ulrich Salz,‡ Thorsten Bock,‡ and Joerg C. Tiller*,† †
Chair of Biomaterials and Polymer Science, Department of Biochemical and Chemical Engineering, TU Dortmund, Emil-Figge-Str. 66, 44227 Dortmund, Germany ‡ Ivoclar Vivadent AG, Bendererstr. 2, 9494 Schaan, Liechtenstein S Supporting Information *
ABSTRACT: Polyoxazolines with a biocidal quarternary ammonium endgroup are potent biocides. Interestingly, the antimicrobial activity of the whole macromolecule is controlled by the nature of the group at the distal end. These nonreactive groups are usually introduced via the initiator. Here we present a study with a series of polymethyloxazolines with varying satellite groups introduced upon termination of the polymerization reaction. This allowed us to introduce a series of functional satellites, including hydroxy, primary amino, and double-bond-containing groups. The resulting telechelic polyoxazolines were explored regarding their antimicrobial activity and toxicity. It was found that the functional satellite groups greatly controlled the minimal inhibitory concentrations against the bacteria Staphylococcus aureus and Escherichia coli in a range of 10 to 2500 ppm. Surprisingly, the satellite groups also controlled the hemotoxicity but in a different way than the antimicrobial efficiency.
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INTRODUCTION The increasing world population and the high traveling activity is a great challenge for the prevention of the spread of new and dangerous infectious diseases, for example, the most recent EHEC (enterohemorrhagic Escherichia coli) epidemic in Europe.1,2 Although, the number of powerful biocides, as disinfectants or antibiotics, increases,3,4 their use is more and more restricted because of safety, environmental reasons (e.g., water contamination),5 and most importantly the increasing development of stubborn biofilms6 and multidrug resistant pathogen strains,7−9 which are to become one of the greatest problems in modern medicine. Antimicrobial polymers are considered to be an alternative to environmentally problematic biocides, disinfectants, and even to antibiotics. Several recent reviews have discussed the great variety of such macromolecules.10−13 In addition to the oxidative14 or biocide releasing polymers, the majority of antimicrobial polymers are amphiphilic polycations, containing quarternary ammonium, phosphonium, or tertiary sulfonium groups and more recently primary and tertiary amino groups. As clearly pointed out by Tew, DeGrado, Lienkamp, and Barron et al.,15−17 such polymers seem to mimic the working principle of membrane active antimicrobial peptides (AMPs)18 such as magainin and other host defense peptides.19 In this regard, the hydrophobicity of the polymer plays a decisive role in its antimicrobial impact and hemolytic activity.20 Besides these polycations, it was found more recently, that polyoxazolines with an antimicrobial DDA (N,N-Dimethyldodecylammonium bromide) group are also antimicrobially active.21 © 2011 American Chemical Society
Most remarkably, their antimicrobial activity is controlled by the group distal to the biocidal function, which is introduced by using different initiators.22 This so-called satellite group affords variation of the antimicrobial activity of the whole molecule over three orders of magnitude, showing in the best case higher activity than the antimicrobial group itself.23 In recent research, the great influence of partial structural elements, end groups, and their correct spatial arrangement on the activity and mechanism of action of antimicrobial cationic peptides has been discussed,24−27 indicating the concept of the satellite group to be wider applicable. By using the initiator, mostly nonreactive neutral groups, such as alkyl chains, could be introduced in the polyoxazoline chain.23 The development of the antimicrobial initiator DDA-X28 now allows us to introduce a wider range of satellite groups via the termination, which opens the possibility to explore terminal functional groups as satellites. In this study, we present the synthesis of polyoxazolines with an antimicrobial start group and varying functional satellite groups on the polymer chain end. The molecules are explored regarding their antimicrobial activity and hemocompatibility.29−31 Received: October 10, 2011 Revised: December 7, 2011 Published: December 7, 2011 165
dx.doi.org/10.1021/bm201403e | Biomacromolecules 2012, 13, 165−172
Biomacromolecules
Article
Table 1. Analytical Data of the Telechelic Antimicrobial Poly(methyloxazoline)s (PMOx) polymer
DPset [g·mol−1]a
MNMR [g·mol−1]b
DPNMRc
Mn,SEC [g·mol−1]
Mw,SEC [g·mol−1]
DPSEC
PDI
yield [%]d
f i/f t [%]e
DDA-X-PMOX23−OH 1 DDA-X-PMOX35−OH 2 DDA-X-PMOX48−OH 3 DDA-X-PMOX77−OH 4 DDA-X-PMOX35-EDA 5 DDA-X-PMOX48-EDA 6 DDA-X-PMOX81-EDA 7 DDA-X-PMOX77-EDA 8 DDA-X-PMOX23-DDA 9 DDA-X-PMOX31-DDA 10 DDA-X-PMOX37-DDA 11 DDA-X-PMOX53-DDA 12 DDA-X-PMOX28-AMA 13
20 30 50 70 30 50 60 80 20 30 30 50 20
2400 3400 4600 7050 3400 4600 7400 7100 2650 3350 3850 5200 3000
22 35 48 77 35 48 81 77 23 31 37 53 28
1800 3600 4600 6700 3600 4600 5800 6700 2400 3300 3300 5100 2700
2050 4200 5400 8000 4200 5400 7000 8000 2800 3900 4300 6400 3200
21 37 49 73 37 49 63 73 20 31 31 52 24
1.14 1.15 1.16 1.19 1.15 1.16 1.20 1.19 1.15 1.16 1.28 1.25 1.19
86 90 84 92 86 81 90 89 79 81 85 89 82
n. d. n. d. n. d. n. d. >94 >94 >90 >92 96 94 90 92 94
Calculated from [M0]·[DDA-X]−1 ratio with associated polymerization times between 2 (DPset = 20) and 16 h (DPset = 80). bDPNMR (n ± 1) was determined as the ratio of the characteristic initiator signal at δ 0.84 (t, 3H, −CH3) versus the average integral value of the PMOx backbone signal around δ 3.3 (b, n4H, N(CH2)2) and δ 2.0 (b, n3H, NCOCH3) cCalculated assuming the postulated telechelic polymer structure. dYield after multiple reprecipitation and dialysis against methanol, determined versus used initial weights of the biocidal initiator DDA-X and the monomer MOx. eFunctionalization ratios, calculated from amino group assay (5−8), and the initiating f i versus terminating agent f t signals in respective 1H NMR spectra. a
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The white byproduct precipitate was removed completely by sedimentation; the supernatant solution was concentrated in vacuum to give 18.80 g (79%) of the slightly yellow highly viscous (honey-like) product, which was fully characterized by 1H/13C NMR and GC-MS. 1 H NMR (CDCl3, δ): 7.63 (d, 2H, BrCH2C(CHar)2(CHar)2C), 7.38 (d, 2H, BrCH 2 C(CH ar ) 2 (CH ar ) 2 C), 5.11 (s, 2H, C(CH ar ) 2 (CHar)2CCH2N+(CH3)2), 4.42 (s, 2H, BrCH2‑C(CHar)2(CHar)2C), 3.49 (t, 2H, N + (CH 3 ) 2 CH 2 CH 2 C 9 H 18− CH 3 ), 3.23 (s, 6H, N + (CH 3 ) 2− CH 2 CH 2 C 9 H 18 CH 3 ), 1.73 (m, 2H, N + (CH 3 ) 2 CH2CH2C9H18CH3), 1.21 (m, 18H, N+(CH3)2−CH2CH2C9H18CH3), 0.82 (t, 3H, N+(CH3)2CH2−CH2C9H18CH3). 13C NMR (CDCl3, δ): 1 4 0. 2 ( B rC H 2 C a r ( C H ) 2 ( C H ) 2 C a r ) , 1 3 3 . 6 ( C a r ( C H ) 2 − (CH)2CarCH2N+(CH3)2), 129.5 (BrCH2Car(CH)2(CH)2Car), 127.4 (Car(CH)2(CH)2CarCH2−N+(CH3)2), 67.4 (CarCH2N+(CH3)2CH2), 66.5 (CarCH2N+(CH3)2CH2), 63.7 (N+(CH3)2CH2CH2−C8H16CH2CH3), 49.4 (N+(CH3)2), 32.0−26.1 (N+(CH3)2CH2CH2C8H16CH2CH3), 22.5 (N+(CH3)2CH2CH2C8H16CH2CH3), 14.9 (N+(CH3)2CH2CH2C8H16CH2CH3). GC-MS (toluene), m/z = 264.3 (9%, CCHarCarCH2N+(CH3)2C12H25+), 185.1 (97%, 81Br− CH2−Ar−CH2+), 183.0 (98%, 79Br−CH2−Ar−CH2+), 104.1 (100%, CH2−Ar−CH2.+), 77.0 (14%, Ar−H+), 58.1 (18%, CH2N+(CH3)2+), 51.0 (15%, CCHarCarCH2+). Standard Procedure of the Polymerization. The required concentration of the biocidal initiator (0e, DDA-X) was calculated with the desired degree of polymerization DPset and the startconcentration of monomer [M 0 ] according to [DDA-X] = [M0]·DPset−1. The resulting amount of DDA-X (0.21 to 0.84 g, 0.44 to 1.76 mmol) was dissolved in chloroform (12 mL) at room temperature, and the monomer methyloxazoline (MOx) was added (3.00 g, 3.00 mL, 35.25 mmol). The microwave-assisted polymerizations were carried out in CEM Discover synthesis microwaves; reaction temperature was constantly monitored with a vertically focused IR temperature sensor. The closed vessels were heated to 100 °C under magnetic stirring/without active cooling using the maximum available power. Immediately after reaching 100 °C, power was reduced to a basis level maintaining the target temperature. To avoid short-term temperature peaks, the vessels were counter-cooled by compressed air shocks within the next 2 to 3 min. The corresponding reaction times varied with DPset in the range of 2−16 h. Termination and Purification. A 20-fold molar excess (based on the initiator amount) of the respective terminating agent was added to the living polymers and heated at 42 °C for 24 h. The hydroxyterminals in the case of the PMOx (1−4) were introduced on the living polymers by contacting the reaction mixtures with a saturated, aqueous K2CO3 solution (12 h, 25 °C). In the case of N-[3-
EXPERIMENTAL PART
Materials. All reactions, purifications, and polymerizations were carried out under an argon atmosphere. Chloroform (AppliChem) was shaken with conc H2SO4, washed with water and dried by passage through a column of activated alumina resulting in residual moisture 2500 >2500 >5000 19.5 >5000
>9400 >40 000 >11 700 >29 000 40.8 >1700
>2500 >5000 >2500 >5000 39.1 >5000
>9400 >80 000 >11 700 >29 000 81.8 >1700
800 >2500 1150 1600 100 >1250
>3000 >40 000 5400 9400 209 >400
