Biozide Polymere

Tuning the Backbone of Biocidal Telechelic Poly(2-oxazoline)s
Evolution of Telechelic Antimicrobial Polymers Christian Krumm, Montasser Hijazi,

Bacterial infections are a seemingly resolved but newly emerging threat to humankind even in industrial countries (e.g. MRSA or EHEC). The reason for the massive occurrence of diseases caused by multi drug resistant bacterial pathogens is the abusive use of antibiotics and biocides. Their dilution and permanent presence in the environment results in a selection pressure on the microorganisms leading to resistant bacterial strains. We established a biocidal polymer as alternative to common biocides that can be deactivated after its application using the satellite group effect, and will thus not cause further resistant bacterial strains in the environment. Now we raised this functional concept to the next level with respect to activity, selectivity and switchability.

The satellite group effect (SG) of telechelic antimicrobial polymers has been used to create bioswitchable biocides with controlled activity based on poly(2-methyloxazoline) and the amphiphilic biocidal dodecyltrimethylammonium group (DDA). A series of such homo- and copolymers with varying monomer mixtures of 2-methyl-2-oxazoline (MOx) and 2-ethyl-2-oxazoline (EtOx), in statistical or block-wise sequence, were prepared introducing the switchable ester SG by the initiator (OP) to obtain higher activity (Figure 1).

Fig 1 Krumm Backbone

Figure 1: Schematical overview over the synthetic strategy for the preparation of the different copolymers.

The influence of the polymeric structure on the antimicrobial activity against, S. aureus and E. coli, (MIC = minimal inhibitory concentration of the polymer at which 99 % of the respective bacterial cells are inhibited in growth), their antimicrobial switching potential, and their hemocompatibility HC50 (concentration at which 50 % of red blood cells are lysed) was investigated.

Fig 2 Krumm Backbone

Figure 2: Minimal inhibitory concentration (MIC) of the prepared statisticalcopolymers based on 2-methyl-2- and 2-ethyl-2-oxazoline against the Gram-positive bacterium S. aureus (S.a.).

The polymers with the highest activity against S. aureus are PEtOx homopolymers and statistical copolymers
with 64-89 mol% of EtOx. They exhibit MIC values of 1.0-1.6 μg/mL (Figure 2). Compared to previously
reported antimicrobial telechelic poly(2-oxazolines) (40 μg/mL) this is an order of magnitude lower and
among the most active antimicrobial polymers known to literature. Additionally, the selectivity ratios (HC50/MIC)
of these polymers are tremendously improved up to 1500. Further, the SG effect makes them highly selective
for the Gram-positive strain S. aureus compared to the Gram-negative E. coli (MICE.c./MICS.a up to 750).
The switching potential of these polymers allows them to lose antimicrobial activity against S.aureus, upon
hydrolyzing the SG end group, by a factor of up to 782. This shows the excellent potential of these polymers,
which means that when the single SG is exclusively and completely hydrolyzed in the environment the whole
polymers lose their biocidal activity. The study showed that, the antimicrobial activity of telechelic polymers with a satellite group effect is greatly
influenced by the nature of the polymer backbone. Another difference to previous publications is the great selectivity
towards Gram-positive bacteria, which is smaller for polymers without SG effect. Also the HC50/MIC ratio is
much higher than that of poly(2-methyloxazoline) with a DDA and a functional SG. All these data suggest that the
SG effect is more complex than previously thought and the potential of these telechelic polymers as antibiotic
alternative is very promising. C. Krumm, H. Montasser, S. Trump, S. Saal, L. Richter, G. G. F. K. Noschmann, T.-D. Nguyen, K. Preslikoska, T. Moll, J. C. Tiller

Highly active and selective telechelic antimicrobial poly(2-oxazoline) copolymers
Polymer 118, 107-115 (2017)  Aus Scientific Highlights 2016    

Ciprofloxacin with a Tail
Polymer antibiotic conjugates with high activity

Infections caused by multi-resistant bacteria are the reason why the demand for new antibiotics is bigger than ever. Nevertheless, only one new antibiotic per year reaches the market because of exploding costs for drug development. A promising alternative to the development of new antibiotics is the formulation and derivatization of existing antibiotics. Especially, the combination of antibiotics and macromolecules moved in the focus of research because macromolecules as carriers for therapeutics afford lower toxicity, increase solubility, and prolonged activity. Moreover, few polymers possess stealth properties, which can mask the antibiotic. These properties can impede the forming of resistant germs. The here developed polymer antibiotic conjugate (PAC) are promising candidates for a new, less resistant-building antibiotics.

The antibiotic ciprofloxacin (CIP) was covalently attached to poly(2-methyloxazoline) (PMOx), poly(2-ethyloxazoline) (PEtOx) and poly(ethylene glycol) (PEG) via end group modification. A polymer analog implementation was developed, in order to introduce a spacer between polymer and CIP (Figure 1). The structures of the different PACs were proven by 1H NMR and electron spray ionization mass spectrometry.
The antimicrobial activity of these conjugates was tested against different bacteria, three Gram negative strains (Escherichia coli, Pseudomonas aeruginosa and Kleisella pneumoniae) and two Gram positive strains
(Staphylococcus aureus and Streptococcus mutans).

2015Schmidt Fig1

Figure 1: Chemical structure of polymer antibiotic conjugates.

The Me-PMOx30-EDA-CIP conjugate shows the same activity against Gram positive strains as CIP. In case of the three Gram negative strains, the conjugate exhibits a drastacally lower activity than CIP (Figure 2). The MIC values of the poly(2-ethyloxazoline) and poly(ethylene glycol conjugates are generally lower than those of the respective Me-PMOx30-EDA-CIP, but show a similar trend for the different bacteria.

2015Schmidt Fig 2

Figure 2: Molar MIC values of the different PACs against S. aureus (S.a.), S. mutans (S.m.), E. coli (E.c.), P. aeruginosa (P.a.) and K. pneumoniae (K.p.) in comparison to the molar MIC value of CIP against each strain (white columns).

The conjugates demonstrate excellent activity against E. coli and the two Gram positive strains. The data show further that the nature on the polymer backbone has a strong influence on the antimicrobial activity. The activity of the conjugates increases in the order of PMOx<PEtOx<PEG. The greatest effect was found for K. pneumoniae. Future work will explore the properties of the conjugates with respect to biodegradability, blood plasma halve life, and building up of bacterial resistances. M. Schmidt, S. Harmuth, E. R. Barth, E. Wurm, R. Fobbe, A. Sickmann, C. Krumm, J.C. Tiller, Bioconjugate Chem. 26, 1950-1962 (2015) Aus Scientific Highlights 2015      

Non-cytotoxic antimicrobial polymers: Hydrophilic Polyionenes
A new class of antimicrobial polymers
Antimicrobial polymers are an alternative to low molecular weight biocides and potentially even to antibiotics. According to the literature antimicrobial polymers are supposed to be amphiphilic cations, which makes them inevitably toxic to mammalian cells, because they act at the cell membrane. Here, we could show that hydrophilic polycations are not only very quick killing biocides but also show no toxicity towards blood cells, which suggests that they act differently compared to amphiphilic polycations.

Antimicrobial, amphiphilic polycations follow the general motiv of the antimicrobial membrane-active peptide Magai-nin. Due to their relatively high toxicity, they are rarely FDA-approved. Interestingly, hydrophilic polycations are also known to be antimicrobially active, but do not structurally mimic Magainin. We proposed that they may act differently and are therefore not cytotoxic towards mammals. In order to investigate this poly(4en,4en-ionene), poly(6,6-ionene) and poly(3,4en-ionene) (PBIn) were synthesized by polyaddition of N,N,N´,N´-tetramethyldiamines and the respective α,ω-dibromoalkanes. All polyionenes showed similar very good antimicrobial activities against E. coli, S. aureus and S. mutans in terms of minimal inhibitory concentration (MIC), the concentration where 99% of all bacterial cells are inhibited in growth. The microbial cells were even killed by more than 99.99% after only one min contact at the respective MIC. That is very efficient and ultrafast. Additional, no lysis (less than 1 %) of porcine red blood cells (RBC) was measured even at concentrations of 40000 µg/mL. Typically, hemotoxicity is given as concentration, where 50 % of the cells are lysed (HC50). This result shows that the variation of the alkyl groups in the main chain from propyl to hexyl makes no significant difference in biological activity. Measurements of the novel polymer PBIn against various Gram-positive and Gram-negative bacteria strains revealed that it kills microbial cells with very high HC50/MIC selectivities of more than 20000 for S. epidermidis, which is the highest ever recorded value for antimicrobial polymers. To obtain insights in the structure-activity-relation of the PBIn, we prepared defined macromolecules with narrowly distributed molecular weights and controlled end groups (bromine, amine, alkyl) up to 5000 g/mol by sequential synthesis and tested their antimicrobial potential and cytotoxicity for RBCs. The investigations of the compounds showed that the antimicrobial activity increases with growing molecular weight and the nature of the end groups influences the activity particularly for smaller molecular weights (Figure 1). Further, the antimicrobial activity of the PBIn is specific for the investigated bacteria strains. The identified influences can be used to create various non-hemotoxic biocides with broad spectrum or microorganism-specific activity. Moreover, PBI can be used to detoxify antimicrobial monomers such as DTAC (dodecyltrimethylammonium chloride) without losing the antimicrobial activity. The found structure-activity-relations of PBI as representative of hydrophilic, antimicrobial polymers, is profoundly different from amphiphilic, antimicrobial polymers. Particularly, the high selectivity for mammalian cells calls the commonly proposed working mechanism of membrane disruption in question. Thus, hydrophilic polycations were identified as a unique class of antimicrobial polymers.

2015Strassburg SciHigh 1

Figure 1: Chemical structures (1a) of PBIn with specific end groups (bromine and amine) and their dependency of the antimicrobial activity (1b) against E. coli and S. aureus (below) of the sequential synthesized PBIn. Additionally, the MIC of PBIn (-.) and the maximal concentration of MIC (….., 5000 µg/mL) are shown as a dotted lines A. Strassburg, F. Kracke, J. Wenners, A. Jemeljanova, J. Kuepper, H. Petersen, J.C. Tiller, Macromolecular Bioscience 15 (12), 1710-1723 (2015). Aus Scientific Highlights 2015

Antimicrobial bioswitchable Poly(2-oxazoline)s
How to control the antimicrobial activity of polymeric biocides by altering end groups

Biocides are killing pathogen germs in medical environments and protect surfaces from fouling. Unfortunately, the massive use of biocides and antibiotics helps the built up of resistant pathogenic bacteria, which are presenting one of the greatest challenges in modern medicine. Further, they are found everywhere and cause unpredictable long-term environmental problems. Thus, a biocide which can be deactivated after its application would be a tremendous progress to reduce the spread of microbial resistances. We have designed such a modern biocide based on antimicrobial poly(2-methyloxazoline)s by using a bio-renderable end group, the so-called satellite group (SG).

The antimicrobial activity of polymers with a biocidal group at the end of biologically inert PMOx is greatly controlled by the nature of the group at the other end of the macromolecule. We proposed that this so-called satellite group effect might be used to switch the antimicrobial activity of a macromolecular chain by just changing the satellite group. Figure 1 shows the concept of this approach on the example of a PMOx with an ester SG and the biocidal group N,N-dimethyldodecylamine DDA.

Fig. 1. Schematic representation of the membrane-active biocidal PMOx controlled by the cleavable satellite group before and after hydrolysis.

A series of such polymers was synthesized introducing the ester by the initiators for the cationic ring-opening polymerization of 2-methyl-2-oxazoline and terminating the polymer with DDA. The ester SG was hydrolyzed by treatment with aqueous NaOH. NMR spectroscopy and electron spray ionization mass spectrometry revealed that the ester cleavage is quantitative and no other group of the macromolecule or its mass itself is affected by the procedure, proving selective cleavage.

Fig. 2 Minimal inhibitory concentrations of the antimicrobial telechelic PMOx before and after hydrolysis of the satellite group.

The polymers were investigated with regard to their antimicrobial activity against the GRAM-positive bacterium Staphylococcus aureus. The PMOx with the octyl ester SG revealed the highest antimicrobial activity (156 µg·mL-1). After ester cleavage the antimicrobial activity of the polymers showed that the carboxylic acid SG completely diminishes the antimicrobial activity of the polymers against the microorganism (Fig 2). We prepared an OP-PMOx-DDA with a shorter chain length of 30 repeating units, and it was found that this polymer is more active against S. aureus (MIC 40 µg·mL-1) and the hydrolysis of the SG group of the macromolecule resulted in an MIC against S. aureus of 1250 µg·mL-1 indicating an activity decrease by a factor of 30.
The hydrolysis of the SG was performed additional with lipase under physiological conditions. After 3 and 7 days of contact with lipase the MIC values were found to be 156 and 312 µg·mL-1, respectively, while the polymer treated with the buffer without lipase remained fully antimicrobial active. This shows that lipase indeed deactivates the antimicrobial activity of the polymer.
Further, the octyl ester SG-PMOx-DDA polymer showed a very low toxicity for pork blood cells (HC50 ≈5000g/l), which reults in a selectivity value of HC50/MIC (S. aureus) of more than 100, This shos that teh polymer is not only enironmentally friendly but also of low toxicity to mammalians.

C. Krumm, S. Harmuth, M. Hijazi, B. Neugebauer, A.-L. Kampmann, H. Geltenpoth,  A. Sickmann and J.C. Tiller
Antimicrobial Poly(2-methyloxazoline)s with Bioswichable Activity through Satellite Group Modification
Angewandte Chemie Int. Ed. 2014, 53, 3830-3834

Aus Scientific Highlights 2013

Biocidal Polymers with Satellite Groups
Influence of functional satellite groups on antimicrobial activity and hemotoxicity

Antimicrobial polymers are considered as alternative to environmentally problematic biocides, disinfectants and even to antibiotics. It was found recently, that poly(2-oxazoline)s with an antimicrobial end group are also antimicrobially active.Most remarkably, their antimicrobial activity is controlled by the group distal to the biocidal function. Due to a new synthesis strategy, we were able to introduce functional satellite groups. These satellite groups also control the antimicrobial activity and even the hemotoxicity of the polymers.

Polyoxazolines with a biocidal quarternary ammonium end-group are potent biocides. The activity-controlling satellite groups had to be introduced via the initiator, and thus were non-functional, such as alkyl chains. The synthesis of a new antimicrobial initiator DDA-X allowed great variety of new satellites. Here, we present a study with a series of poly(2-methyloxazoline)s with varying functional satellite groups introduced upon termination of the polymerization reaction.    

Figure 1. Telechelic poly(2-methyloxazoline)s (PMOx) with biocidal initiator (DDA-X) at the start position and varying functional satellites: (OH= hydroxyl, EDA= ethane-1,2-diamine, DDA= N,N-dimethyl-dodecylammonium bromide, AMA= 3-(methacryloyl-amino)-N,N-dimethyl-propan-1-ammonium bromide) at the respective terminal position.

This allowed introducing a series of functional satellites, including hydroxy, primary amino, and double bond containing groups. The resulting telechelic poly(2-oxazoline)s were explored
regarding their antimicrobial activity and hemotoxicity. 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. 


 Figure 2. Selectivity values of the telechelic polymethyloxazolines DDA-X-PMOx-SATELLITE with varying satellite groups (=OH, EDA, DDA, AMA) in comparison to the biocidal low molecular weight initiator DDA-X. Calculated from HC50·MIC-1 ratios for each, S. aureus and E. coli.

It could be demonstrated that the introduction of one antimicrobial group (DDA-X) and one hydrophilic cationic satellite group (EDA) results in a highly active antimicrobial, less hemotoxic polymer, because both groups combine their different functions, membrane penetration and surface attraction, symbiotically in one polymer. Obviously, this kind of satellite groups might be useful for targeting multiple biological functions.
C.P. Fik, C. Krumm, C. Münnig, T.I. Baur, U. Salz, T. Bock, J.C. Tiller
Impact of Functional Satellite Groups on the Antimicrobial Activity and Hemocompatibility of Telechelic Poly(2-methyloxazoline)s
Biomacromolecules 13 (1), 165-172 (2012)

 Aus Scientific Highlights 2011