Yung Pin Chen

Antimicrobial metallopolymers and their bioconjugates with conventional antibiotics against multidrug-resistant bacteria.

Bacteria are now becoming more resistant to most conventional antibiotics. Methicillin-resistant Staphylococcus aureus (MRSA), a complex of multidrug-resistant Gram-positive bacterial strains, has proven especially problematic in both hospital and community settings by deactivating conventional β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems, through various mechanisms, resulting in increased mortality rates and hospitalization costs. Here we introduce a class of charged metallopolymers that exhibit synergistic effects against MRSA by efficiently inhibiting activity of β-lactamase and effectively lysing bacterial cells. Various conventional β-lactam antibiotics, including penicillin-G, amoxicillin, ampicillin, and cefazolin, are protected from β-lactamase hydrolysis via the formation of unique ion-pairs between their carboxylate anions and cationic cobaltocenium moieties. These discoveries could provide a new pathway for designing macromolecular scaffolds to regenerate vitality of conventional antibiotics to kill multidrug-resistant bacteria and superbugs.

Amphipathic antibacterial agents using cationic methacrylic polymers with natural rosin as pendant group

We prepared a class of novel cationic polymers as antimicrobial agents: quaternary ammonium-containing poly(N,N-dimethylaminoethyl methacrylate) with natural rosin as the pendant group (PDMAEMA-g-rosin). Different from most other amphipathic antimicrobial polymeric systems reported in the literature, our approach sandwiched the hydrophilic cationic group between the polymer backbone and bulky hydrophobic hydrophenanthrene side groups. A simple quaternization reaction was used to link the rosin ester chloride and PDMAEMA homopolymers. Both the Gram-positive bacterium Staphylococcus aureus (S. aureus) and Gram-negative bacterium Escherichia coli (E. coli) were tested against the PDMAEMA-g-rosin copolymers. PDMAEMA-g-rosin copolymers with the amphipathic structure exhibited effective antimicrobial activity against both E. coli and S. aureus. Both the degree of quaternization of rosin group and the molecular weight of PDMAEMA played roles in antimicrobial activities. Our results also indicated that conformation of hydrophobic group (particularly steric hindrance) played a role in dictating antibacterial efficacy. Scanning electron microscopy and confocal laser scanning microscopy were used to characterize morphological changes of bacteria after exposure with PDMAEMA-g-rosin copolymers. Possible mechanisms on a combination of ionic and hydrophobic interactions between bacterial cells and polymers are discussed.

Rapid Screening of Quorum-Sensing Signal N-Acyl Homoserine Lactones by an In Vitro Cell-Free Assay

ABSTRACT A simple, sensitive, and rapid cell-free assay system was developed for detection of N-acyl homoserine lactone (AHL) autoinducers involved in bacterial quorum sensing (QS). The present approach improves upon previous whole-cell biosensor-based approaches in its utilization of a cell-free assay approach to conduct bioassays. The cell-free assay was derived from the AHL biosensor bacterium Agrobacterium tumefaciens NTL4(pCF218)(pCF372), allowing the expression of β-galactosidase upon addition of exogenous AHLs. We have shown that β-galactosidase expression is possible in cell-free solution [lysate from Agrobacterium tumefaciens NTL4(pCF218)(pCF372) culture]. Assay detection limits with the use of chromogenic substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) ranged from approximately 100 nM to 300 nM depending on the specific AHL. Replacement (of X-Gal) with the luminescent substrate Beta-Glo increased sensitivity to AHLs by 10-fold. A major advantage of the cell-free assay system is elimination of time-consuming steps for biosensor cell culture conditioning, which are required prior to whole-cell bioassays. This significantly reduced assay times from greater than 24 h to less than 3 h, while maintaining high sensitivity. Assay lysate may be prepared in bulk and stored (−80°C) over 6 months for future use. Finally, the present protocol may be adapted for use with other biosensor strains and be used in high-throughput AHL screening of bacteria or metagenomic libraries.

Polymer grafted recyclable magnetic nanoparticles

This work reports on a new combination of recyclable magnetic nanoparticles, polymers and antibiotics that show increased effectiveness in combating bacterial infections. The direct-coprecipitation of iron salts strategy was used to generate superparamagnetic nanoparticles with a saturation magnetization of 59.5 emu g−1. A silica coating was applied and used to stabilize the magnetic nanoparticles and create a convenient platform for further functionalization. A variety of PMAA brushes with different lengths and densities were prepared on the magnetic nanoparticles with an average diameter size as small as 10 nm via surface-initiated reversible addition fragmentation chain transfer (RAFT) polymerization of methacrylic acid. The polymer grafted magnetic nanoparticles were removed from water solutions after antimicrobial testing using a magnet, thereby avoiding nano-based pollution of the environment. The bioactivity of an antibiotic (penicillin-G) over bacteria (Staphylococcus aureus and Escherichia coli) was significantly enhanced when physically bound to the PMAA grafted magnetic nanoparticles. The inhibition activity of the penicillin-nanoparticle complex was retained using recycled magnetic nanoparticles that had been reloaded with penicillin-G.

Surface-functionalization effects on uptake of fluorescent polystyrene nanoparticles by model biofilms.

A study was conducted to investigate the role of nanoparticle (NP) surface functionalization/charge on their uptake by biofilms. Biofilms, bacterial colonies attached to surfaces via extracellular polymers, are effective at removing suspended nanomaterials from the aqueous phase. However, the mechanisms regulating particle uptake are unknown. Here, it was shown that the mechanism was strongly dependent on the nanoparticle surface ionization, and not the core composition of the NP. Uptake experiments were conducted using laboratory-cultured biofilms. The biofilms were incubated in the presence of fluorescent polystyrene NPs with either negatively-charged surfaces (i.e. functionalized with sulfated (SO4−-NP) or carboxylated (COO−-NP) groups) or positively-charged surfaces (functionalized with primary amines, Amine-P). Particles with negatively-charged sulfated surfaces associated most strongly to biofilms across all experimental conditions. Associations of positively-charged amine particles with biofilms were greatest at high ionic conditions resembling those of seawater, but were sensitive to changes in ionic strength. Sorption of COO−-NPs was lowest, relative to other particle types, and was not sensitive to ionic strength. The results of this study support an emerging precedent that biofilms may be an effective player in the binding and sequestration of nanoparticles in aqueous systems.

Bio-inspired resin acid-derived materials as anti-bacterial resistance agents with unexpected activities

Methicillin-resistant Staphylococcus aureus (MRSA), a complex of multidrug resistant Gram-positive bacterial strains, has proven especially problematic in both hospital and community-settings, resulting in increased mortality rates and hospitalization costs. Emergence of resistance even to vancomycin, the standard reference for MRSA treatment, builds up pressure for the search of novel alternatives. We report potent natural resin acid-based cationic antimicrobial compounds and polymers that exhibit surprising antimicrobial activity against a range of MRSA strains, yet are largely non-toxic against mammalian cells. Molecular dynamics simulations and dye-leakage assays with anionic phospholipid membrane mimics of bacteria demonstrate a membrane-lysing effect induced by unique fused ring structures of resin acids that may constitute the principal mechanism of action for selective lysis of bacterial cells over mammalian cells. Our antimicrobial materials are derived from an unlikely yet abundant natural source, and offer a novel alternative to currently-used approaches.

Charged Metallopolymer-Grafted Silica Nanoparticles for Antimicrobial Applications

Inappropriate and frequent use of antibiotics has led to the development of antibiotic-resistant bacteria, which cause infectious diseases that are difficult to treat. With the rising threat of antibiotic resistance, the need to develop effective new antimicrobial agents is prominent. We report antimicrobial metallopolymer nanoparticles, which were prepared by surface-initiated reversible addition-fragmentation chain transfer polymerization of a cobaltocenium-containing methacrylate monomer from silica nanoparticles. These particles are capable of forming a complex with β-lactam antibiotics, such as penicillin, rejuvenating the bactericidal activity of the antibiotic. Disk diffusion assays showed significantly increased antibacterial activities against both Gram-positive and Gram-negative bacteria. The improved efficiencies were attributed to the inhibition of hydrolysis of the β-lactam antibiotics and enhancement of local antibiotics concentration on a nanoparticle surface. In addition, hemolysis evaluations demonstrated minimal toxicity to red blood cells.

Trio Act of Boronolectin with Antibiotic-Metal Complexed Macromolecules toward Broad-Spectrum Antimicrobial Efficacy

Bacterial infections, particularly by Gram-negative pathogens, have become a serious threat to global healthcare due to the diminishing effectiveness of existing antibiotics. We report a nontraditional therapy to combine three components in one macromolecular system, in which boronic acid adheres to peptidoglycan or lipopolysaccharide via boron-polyol based boronolectin chemistry, cationic metal polymer frameworks interact with negatively charged cell membranes, and β-lactam antibiotics are reinstated with enhanced vitality to attack bacteria via evading the detrimental enzyme-catalyzed hydrolysis. These macromolecular systems exhibited high efficacy in combating pathogenic bacteria, especially Gram-negative strains, due to synergistic effects of multicomponents on interactions with bacterial cells. In vitro and in vivo cytotoxicity and hemolysis evaluation indicated that these multifunctional copolymers did not induce cell death by apoptosis, as well as did not alter the phenotypes of immune cells and did not show observable toxic effect on red blood cells.

Aflatoxin-Exposure of Vibrio gazogenes as a Novel System for the Generation of Aflatoxin Synthesis Inhibitors.

Aflatoxin is a mycotoxin and a secondary metabolite, and the most potent known liver carcinogen that contaminates several important crops, and represents a significant threat to public health and the economy. Available approaches reported thus far have been insufficient to eliminate this threat, and therefore provide the rational to explore novel methods for preventing aflatoxin accumulation in the environment. Many terrestrial plants and microbes that share ecological niches and encounter the aflatoxin producers have the ability to synthesize compounds that inhibit aflatoxin synthesis. However, reports of natural aflatoxin inhibitors from marine ecosystem components that do not share ecological niches with the aflatoxin producers are rare. Here, we show that a non-pathogenic marine bacterium, Vibrio gazogenes, when exposed to low non-toxic doses of aflatoxin B1, demonstrates a shift in its metabolic output and synthesizes a metabolite fraction that inhibits aflatoxin synthesis without affecting hyphal growth in the model aflatoxin producer, Aspergillus parasiticus. The molecular mass of the predominant metabolite in this fraction was also different from the known prodigiosins, which are the known antifungal secondary metabolites synthesized by this Vibrio. Gene expression analyses using RT-PCR demonstrate that this metabolite fraction inhibits aflatoxin synthesis by down-regulating the expression of early-, middle-, and late- growth stage aflatoxin genes, the aflatoxin pathway regulator, aflR and one global regulator of secondary metabolism, laeA. Our study establishes a novel system for generation of aflatoxin synthesis inhibitors, and emphasizes the potential of the under-explored Vibrio’s silent genome for generating new modulators of fungal secondary metabolism.

Recyclable magnetic nanoparticles grafted with antimicrobial metallopolymer-antibiotic bioconjugates

Over-prescription and improper use of antibiotics has led to the emergence of bacterial resistance, posing a major threat to public health. There has been significant interest in the development of alternative therapies and agents to combat antibiotic resistance. We report the preparation of recyclable magnetic iron oxide nanoparticles grafted with charged cobaltocenium-containing metallopolymers by surface-initiated reversible addition-fragmentation chain transfer (RAFT) polymerization. β-Lactam antibiotics were then conjugated with metallopolymers to enhance their vitality against both Gram-positive and Gram-negative bacteria. The enhanced antibacterial activity was a result of synergy of antimicrobial segments that facilitate the inhibition of hydrolysis of antibiotics and local enhancement of antibiotic concentration on a nanoparticle surface. These magnetic nanoparticles can be recycled numerous times without losing the initial antimicrobial potency. Studies suggested negligible toxicity of metallopolymer-grafted nanoparticles to red blood cells and minimal tendency to induce resistance in bacteria.