Recyclable antibacterial magnetic nanoparticles grafted with quaternized poly(2-(dimethylamino)ethyl methacrylate) brushes

Highly efficient recyclable antibacterial magnetite nanoparticles consisting of a magnetic Fe(3)O(4) core with an antibacterial poly(quaternary ammonium) (PQA) coating were prepared in an efficient four-step process. The synthetic pathway included: (1) preparation of Fe(3)O(4) nanoparticles via coprecipitation of Fe(2+)/Fe(3+) in the presence of an alkaline solution; (2) attachment of an ATRP initiating functionality to the surface of the nanoparticles; (3) surface-initiated atom transfer radical polymerization (ATRP) of 2-(dimethylamino)ethyl methacrylate (DMAEMA); and (4) transformation of PDMAEMA brushes to PQA via quaternization with ethyl bromide. The success of the surface functionalization was confirmed by FT-IR, thermal gravimetric analysis (TGA), elemental analysis, and transmission electron microscopy (TEM). The PQA-modified magnetite nanoparticles were dispersed in water and exhibited a response to an external magnetic field, making the nanoparticles easy to remove from water after antibacterial tests. The PQA-modified magnetite nanoparticles retained 100% biocidal efficiency against E. coli (10(5) to 10(6)E. coli/mg nanoparticles) during eight exposure/collect/recycle procedures without washing with any solvents or water.     

Antibacterial cellulose fiber via RAFT surface graft polymerization

2-(dimethylamino)ethyl methacrylate (DMAEMA) was polymerized from cellulosic filter paper via reversible addition-fragmentation chain transfer (RAFT) polymerization. The tertiary amino groups of the grafted PDMAEMA chains were subsequently quaternized with alkyl bromides of different chain lengths (C8-C16) to provide a large concentration of quaternary ammonium groups on the cellulose surface. The antibacterial activity of the quaternized and nonquaternized PDMAEMA-grafted cellulosic fibers was tested against Escherichia coli. The antibacterial activity was found to depend on the alkyl chain length and on the degree of quaternization, i.e., the amount of quaternary amino groups present in the cellulose graft copolymers. The PDMAEMA-grafted cellulose fiber with the highest degree of quaternization and quaternized with the shortest alkyl chains was found to exhibit particularly high activity against E. coli.     

Grafting of poly[2-(tert-butylamino)ethyl methacrylate] onto polypropylene by reactive blending and antibacterial activity of the copolymer

To combine low cost, good mechanical properties, and antibacterial activity in one material, a nonquaternized polymeric biocide, i.e., poly[2-(tert-butylamino)ethyl methacrylate] (PTBAEMA), was dispersed within a commodity plastic, i.e., polypropylene (PP). The high immiscibility of the two polymers was tackled by reactive compatibilization and thus by reaction of commercially available maleic anhydride grafted polypropylene with primary amine-end-capped PTBAEMA. This reactive polymethacrylate was synthesized by atom-transfer radical polymerization with an azide-containing initiator. The azide end group was converted into a primary amine by the Huisgen [3 + 2] cycloaddition of propargylamine. The accordingly formed PP-g-PTBAEMA copolymer was melt dispersed within neat PP and processed as fibers, whose antimicrobial properties were assessed by the viable cell counting method against Escherichia coli. The antibacterial activity was long-lasting as a result of the anchoring of the PTBAEMA chains onto PP, which prevented them from being released from the surface of the fibers.     

Highly effective, water-soluble, hemocompatible 1,3-propylene oxide-based antimicrobials: poly[(3,3-quaternary/PEG)-copolyoxetanes

This study focuses on the solution antimicrobial effectiveness of a novel class of copolyoxetanes with quaternary ammonium and PEG-like side chains. A precursor P[(BBOx-m)(ME2Ox)] copolyoxetane was prepared by cationic ring-opening copolymerization of 3-((4-bromobutoxy)methyl)-3-methyloxetane (BBOx) and 3-((2-(2-methoxyethoxy)ethoxy)methyl)-3-methyloxetane (ME2Ox) to give random copolymers with 14-100 (m) mol % BBOx. Reaction of P[(BBOx-m)(ME2Ox)] with dodecyl dimethylamine gave the corresponding quaternary P[(C12-m)(ME2Ox)] polycation salts, designated C12-m, as viscous liquids in 100% yield. BBOx/ME2Ox and C12/ME2Ox ratios were obtained by (1)H NMR spectroscopy. C12-m molecular weights (M(n), 3.5-21.9 kDa) were obtained from (1)H NMR end group analysis. DSC studies up to 150 °C showed only thermal transitions between -69 and -34 °C assigned to T(g) values. Antibacterial activity for the C12-m copolyoxetanes was tested by determining minimum inhibitory concentrations (MICs) against Gram(+) Staphylococcus aureus and Gram(-) Escherichia coli and Pseudomonas aeruginosa . MIC decreased with increasing C12 mol percent, reaching a minimum in the range C12-43 to C12-60. Overall, the antimicrobial with consistently low MICs for the three tested pathogenic bacteria was C12-43: (bacteria, MIC, μg/mL) E. coli (6), S. aureus (5), and P. aeruginosa (33). For C12-43, minimum biocidal concentration (MBC) to reach 99.99% kill in 24 h required 1.5× MIC for S. aureus and 2× MIC for E. coli and P. aeruginosa . At 5× MIC against a challenge of 10(8) cfu/mL, C12-43 kills ≥99% S. aureus , E. coli , and P. aeruginosa within 1 h. C12-m copolyoxetane cytotoxicity toward human red blood cells was low, indicating good prospects for biocompatibility. The tunability of C12-m copolyoxetane compositions, effective antimicrobial behavior against Gram(+) and Gram(-) bacteria, and promising biocompatibility offer opportunities for further modification and potential applications as therapeutic agents.     

Immobilization of Escherichia coli Cells Containing Aspartase Activity with Polyurethane and Its Application for l-Aspartic Acid Production

Whole cells of Escherichia coli containing aspartase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol). The immobilized cell preparation was used to convert ammonium fumarate to l-aspartic acid. Properties of the immobilized E. coli cells containing aspartase were investigated with a batch reactor. A 1.67-fold increase in the l-aspartic acid production rate was observed at 37 degrees C as compared to 25 degrees C operating temperature. The pH optimum was broad, ranging from 8.5 to 9.2. Increasing the concentration of ammonium fumarate to 1.5 M from 1.0 M negatively affected the reaction rate. l-Aspartic acid was produced at an average rate of 2.18 x 10 mol/min per g (wet weight) of immobilized E. coli cells with a 37 degrees C substrate solution consisting of 1.0 M ammonium fumarate with 1 mM Mg (pH 9.0).     

Polyelectrolyte complexes between (cross-linked) N-carboxyethylchitosan and (quaternized) poly[2-(dimethylamino)ethyl methacrylate]: preparation, characterization, and antibacterial properties

Novel polyelectrolyte complexes (PECs) between N-carboxyethylchitosan (CECh) and well-defined (quaternized) poly[2-(dimethylamino)ethyl methacrylate] (PDMAEMA) have been obtained. The modification of chitosan into CECh allows the preparation of PECs in a pH range in which chitosan cannot form complexes. The CECh/PDMAEMA complex is formed in a narrow pH range around 7. The quaternization of the tertiary amino groups of PDMAEMA enables complex formation with CECh both in neutral and in alkaline medium. Cross-linked CECh is also capable of forming complexes with (quaternized) PDMAEMA. The antibacterial activity of (cross-linked) CECh, (quaternized) PDMAEMA, and their complexes against Escherichia coli has been evaluated. In contrast to (quaternized) PDMAEMA, (cross-linked) CECh exhibits no antibacterial activity. The complex formation between cross-linked CECh and (quaternized) PDMAEMA results in a loss of the inherent antibacterial activity of the latter in neutral medium. In acidic medium, the complexes exhibit strong antibacterial activity due to complex disintegration and release of (quaternized) PDMAEMA.     

Deposition gene transfection using bioconjugates of DNA and thermoresponsive cationic homopolymer

A poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics was applied to a vector for use in deposition transfection. PDMAEMA with a molecular weight of 2.5 × 10(5) g mol(-1) was synthesized by photoinduced radical polymerization. Polyplexes approximately 750 nm in size were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA. The polyplexes had a lower critical solution temperature (LCST) of approximately 30 °C. In addition, they exhibited excellent adsorption and durability on a polystyrene surface, as confirmed by a surface chemical compositional analysis. When HeLa cells and primary cells were cultured on a substrate coated with the polyplexes, high transgene expression and cell viability of more than 90% were obtained at low charge ratios (PDMAEMA/plasmid DNA ratio) ranging from 2 to 8. In addition, transgene expression was sustained for over 2 weeks post-transfection whereas decreased expression was observed 5 days post-transfection when the conventional solution-mediated transfection method was used. Thus, high and sustained transgene expression as well as high cell viability can be realized by using small amounts of PDMAEMA as a deposition transfection material.     

Antimicrobial surfaces of viologen-quaternized poly((2-dimethyl amino)ethyl methacrylate)-Si(100) hybrids from surface-initiated atom transfer radical polymerization

To improve the antimicrobial ability of silicon-based bioelectronics and to tailor the silicon surfaces for inhibiting biofilm formation, well-defined functional polymer-Si(100) hybrids, consisting of nearly monodispersed poly((2-dimethylamino)ethyl methacrylate) (P(DMAEMA)) covalently tethered on the silicon surface and functionalized by viologen moieties, were prepared. P(DMAEMA)-Si hybrids were prepared via surface-initiated atom transfer radical polymerization (ATRP) of (2-dimethylamino)ethyl methacrylate (DMAEMA) on the hydrogen-terminated Si(100) surfaces (Si−H surfaces). The tertiary amino groups of the covalently immobilized (Si−C bonded) P(DMAEMA) brushes on the silicon substrates were quaternized by an alkyl halide to produce a high concentration of quaternary ammonium groups with biocidal functionality. Alternatively, covalent coupling of viologen moieties to the tertiary amino groups of P(DMAEMA) brushes produced the quaternized P(DMAEMA)-Si(100) hybrids with substantially enhanced antimicrobial capability, as well as capability to effectively inhibit biofilm formation. Thus, the viologen-quaternized P(DMAEMA)-Si(100) hybrids possess good antibacterial surface properties and are potentially useful to the silicon-based bioelectronics to ensure their efficiency, durability and reliability.     

From multifunctionalized poly(ethylene imine)s toward antimicrobial coatings

Primary amine groups of branched poly(ethylene imine) (PEI) were functionalized with quaternary ammonium groups, alkyl chains of different length, allylic and benzylic groups in a one-step reaction, using a carbonate coupler. The structure of the obtained amphiphilic polymers was determined by means of 1H and 13C NMR spectroscopy. Depending on their hydrophilic/hydrophobic balance, the obtained polymers can be used as water-soluble disinfectants and for antimicrobial coating materials. The bactericidal properties of some of the amphiphilic polymers against Gram-negative and Gram-positive bacteria were investigated. Minimal inhibitory concentrations (log 4 reduction of bacterial growth) against Escherichia coli and Bacillus subtilis were determined in the range of 0.3-0.4 mg/mL and 0.03-0.04 mg/mL for water-soluble polymers. Glass slides coated with functionalized PEIs showed a reduction of colony forming units of at least 95%, at best 99.9%, against E. coli and B. subtilis.     

Antialgal activity of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes against the marine alga Ulva

Marine biofouling has detrimental effects on the environment and economy, and current antifouling coatings research is aimed at environmentally benign, non-toxic materials. The possibility of using contact-active coatings is explored, by considering the antialgal activity of cationic poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) brushes. The antialgal activity was investigated via zoospore settlement and sporeling growth assays of the marine algae Ulva linza and U. lactuca. The assay results for PDMAEMA brushes were compared to those for anionic and neutral surfaces. It was found that only PDMAEMA could disrupt zoospores that come into contact with it, and that it also inhibits the subsequent growth of normally settled spores. Based on the spore membrane properties, and characterization of the PDMAEMA brushes over a wide pH range, it is hypothesized that the algicidal mechanisms are similar to the bactericidal mechanisms of cationic polymers, and that further development could lead to successful contact-active antialgal coatings.     

Grafting of antibacterial polymers on stainless steel via surface‐initiated atom transfer radical polymerization for inhibiting biocorrosion by Desulfovibrio desulfuricans

To enhance the biocorrosion resistance of stainless steel (SS) and to impart its surface with bactericidal function for inhibiting bacterial adhesion and biofilm formation, well‐defined functional polymer brushes were grafted via surface‐initiated atom transfer radical polymerization (ATRP) from SS substrates. The trichlorosilane coupling agent, containing the alkyl halide ATRP initiator, was first immobilized on the hydroxylated SS (SS‐OH) substrates for surface‐initiated ATRP of (2‐dimethylamino)ethyl methacrylate (DMAEMA). The tertiary amino groups of covalently immobilized DMAEMA polymer or P(DMAEMA), brushes on the SS substrates were quaternized with benzyl halide to produce the biocidal functionality. Alternatively, covalent coupling of viologen moieties to the tertiary amino groups of P(DMAEMA) brushes on the SS surface resulted in an increase in surface concentration of quaternary ammonium groups, accompanied by substantially enhanced antibacterial and anticorrosion capabilities against Desulfovibrio desulfuricans in anaerobic seawater, as revealed by antibacterial assay and electrochemical studies. With the inherent advantages of high corrosion resistance of SS, and the good antibacterial and anticorrosion capabilities of the viologen‐quaternized P(DMAEMA) brushes, the functionalized SS is potentially useful in harsh seawater environments and for desalination plants. Biotechnol. Bioeng. 2009;103: 268–281. © 2009 Wiley Periodicals, Inc.     

Nonleaching antimicrobial films prepared from surface-modified microfibrillated cellulose

We have prepared potentially permanent antimicrobial films based on surface-modified microfibrillated cellulose (MFC). MFC, obtained by disintegration of bleached softwood sulfite pulp in a homogenizer, was grafted with the quaternary ammonium compound octadecyldimethyl(3-trimethoxysilylpropyl)ammonium chloride (ODDMAC) by a simple adsorption-curing process. Films prepared from the ODDMAC-modified MFC were characterized by Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) and tested for antibacterial activity against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa. The films showed substantial antibacterial capacity even at very low concentrations of antimicrobial agent immobilized on the surface. A zone of inhibition test demonstrated that no ODDMAC diffused into the surroundings, verifying that the films were indeed of the nonleaching type.     

The activity and purification of membrane-associated thioredoxin reductase from human metastatic melanotic melanoma

Electron spin resonance spectroscopy has been used to measure the reduction of nitroxide radicals on a spin-labeled quaternary ammonium substrate by plasma membrane-associated thioredoxin reductase (EC 1.6.4.5) at the surface of cutaneous and subcutaneous melanoma metastases from one patient (B.M.). Enzyme activity in these metastases was shown to be hyperactive compared to normal skin and was subject to inhibition by calcium. From the remainder of the tissue (50.6 g), plasma membrane-associated thioredoxin reductase has been isolated and its molecular properties were compared with the same enzyme purified from the cytosol of rat liver and Escherichia coli. The enzyme from melanoma possessed an identical molecular weight to that from rat liver as determined by SDS-polyacrylamide gel electrophoresis (Mr 58,000). Upon fluorescence spectroscopic examination, the enzyme from melanoma was shown to contain flavin adenine dinucleotide as previously shown in the enzymes from E. coli and rat liver. The increased activities in plasma membrane-associated thioredoxin reductase in metastases of malignant melanotic melanoma are discussed in terms of the cellular functions of this important enzyme.     

Direct capture of plasmid DNA from non-clarified bacterial lysate using polycation-grafted monoliths

Monolith columns from macroporous polyacrylamide gel were grafted with polycations, poly(N,N-dimethylaminoethyl methacrylate) (polyDMAEMA), (2-(methacryloyloxy)ethyl)-trimethyl ammonium chloride (polyMETA) and partially quaternized polyDMAEMA prepared via treating polyDMAEMA-grafted columns with propylbromide. The polymer grafting degrees varied between 34 and 110%. The polycation-grafted monolithic columns are able to capture plasmid DNA directly from alkaline lysate of Escherichia coli cells. Due to the large pore size in macroporous monoliths the particulate material present in non-clarified feeds did not block the columns. The captured plasmid DNA was eluted with 1M NaCl as particulate-free preparation with significantly reduced content of protein and RNA as compared to the applied lysate.     

Synthesis of well-defined graft copolymers by combination of enzymatic polycondensation and "click" chemistry

Aliphatic polyesters having pendant azide groups were prepared by enzymatic polycondensation in the presence of lipase from Candida antarctica type B (CAL-B). The grafting reaction to the N(3)-functional polyester was carried out quantitatively at room temperature using copper-catalyzed azide-alkyne cycloaddition (CuAAC, "click" reaction) with monoalkyne-functional poly(ethylene oxide) (alkyne-PEO, M(n) = 750 g/mol). Furthermore, both enzymatic polycondensation and "click" reaction were carried out successfully in sequential one-pot reaction. The graft copolymer was surface-active and self-assembled in water. The graft copolymer had a critical aggregation concentration (cac) of 3 × 10(-2) μM in water determined by surface tension measurements. Above cac, the graft copolymer formed single chains and aggregates having a hydrodynamic radius of ～75 nm. Furthermore, the surface activity of the polymers at the air-water interface was studied by Langmuir trough measurements. The Langmuir isotherm of the graft polymer showed a pseudoplateau resulting from desorption of PEO chains into the subphase upon compression.     

新型脱氢松香基季铵盐类抗菌剂的合成、结构表征及抗菌活性研究

以天然松香酸衍生物——脱氢松香胺为原料,合成了6种脱氢松香基季铵盐。对合成化合物结构进行了元素分析、紫外、红外光谱及核磁共振谱鉴定。药理实验结果表明,合成产品对金黄色葡萄球菌及大肠杆菌具有一定抗菌活性,其最低抑菌浓度分别为7. 18～31. 25μ/ml和250～500μg/ml。同时对产品结构对抗菌活性影响也进行了比较研究。     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Influence of polymer architecture and molecular weight of poly(2-(dimethylamino)ethyl methacrylate) polycations on transfection efficiency and cell viability in gene delivery

Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.