Bacterial killing and the dimensions of bacterial death.

Bacteria can be dead, alive, or exhibit slowed or suspended life forms, making bacterial death difficult to establish. Here, agar-plating, microscopic-counting, SYTO9/propidium-iodide staining, MTT-conversion, and bioluminescence-imaging were used to determine bacterial death upon exposure to different conditions. Rank correlations between pairs of assay outcomes were low, indicating different assays measure different aspects of bacterial death. Principal-component analysis yielded two principal components, named "reproductive-ability" (PC1) and "metabolic-activity" (PC2). Plotting of these principal components in two-dimensional space revealed a dead region, with borders defined by the PC1 and PC2 values. Sensu stricto implies an unpractical reality that all assays determining PC1 and PC2 must be carried out in order to establish bacterial death. Considering this unpracticality, it is suggested that at least one assay determining reproductive activity (PC1) and one assay determining metabolic activity (PC2) should be used to establish bacterial death. Minimally, researchers should specifically describe which dimension of bacterial death is assessed, when addressing bacterial death.

Ellagic acid-modified gold nanoparticles to combat multi-drug resistant bacterial infections in vitro and in vivo.

The overuse of antibiotics has led to the rapid development of multi-drug resistant bacteria, making antibiotics increasingly ineffective against bacterial infections. Consequently, there is an urgent need to develop alternative strategies to combat multi-drug-resistant bacterial infections. In this study, gold nanoparticles modified with ellagic acid (EA-AuNPs) were prepared using a simple and mild one-pot hydrothermal process. EA-AuNPs demonstrated high bactericidal efficacy and broad-spectrum antimicrobial activities against clinical isolates of the antibiotic-resistant ESKAPE pathogens. Furthermore, EA-AuNPs effectively disperse biofilms of multi-drug-resistant bacteria. Additionally, EA-AuNPs mitigated inflammatory responses at the bacterial infection sites. The combined bactericidal and anti-inflammatory treatment with EA-AuNPs resulted in faster curing of peritonitis caused by Staphylococcus aureus in mice compared to treatment with free EA or gentamicin. Moreover, transcriptome analysis revealed that EA-AuNPs exhibited a multi-targeting mechanism, making resistance development in pathogens more challenging than traditional antibiotics that recognize specific cellular targets. Overall, EA-AuNPs emerged as a promising antimicrobial agent against multi-drug-resistant bacterial infections.

Nano-architectonics of Pt single-atoms and differently-sized nanoparticles supported by manganese-oxide nanosheets and impact on catalytic and anti-biofilm activities.

Hybrid-nanozymes are promising in various applications, but comprehensive comparison of hybrid-nanozymes composed of single-atoms or nanoparticles on the same support has never been made. Here, manganese-oxide nanosheets were loaded with Pt-single-atoms or differently-sized nanoparticles and their oxidase- and-peroxidase activities compared. High-resolution Transmission-Electron-Microscopy and corresponding Fast Fourier Transform imaging showed that Pt-nanoparticles (1.5 nm diameter) had no clear (111) crystal-planes, while larger nanoparticles had clear (111) crystal-planes. X-ray Photo-electron Spectroscopy demonstrated that unloaded nanosheets were composed of MnO2 with a high number of oxygen vacancies (Vo/Mn 0.4). Loading with 7.0 nm Pt-nanoparticles induced a change to Mn2O3, while loading with 1.5 nm nanoparticles increased the number of vacancies (Vo/Mn 1.2). Nanosheets loaded with 3.0 nm Pt-nanoparticles possessed similarly high catalytic activities as Pt-single-atoms. However, loading with 1.5 nm or 7.0 nm Pt-nanoparticles yielded lower catalytic activities. A model is proposed explaining the low catalytic activity of under- and over-sized Pt-nanoparticles as compared with intermediately-sized (3.0 nm) Pt-nanoparticles and single-atoms. Herewith, catalytic activities of hybrid-nanozymes composed of single-atoms and intermediately-sized nanoparticles are put a par, as confirmed here with respect to bacterial biofilm eradication. This conclusion facilitates a balanced choice between using Pt-single-atoms or nanoparticles in further development and application of hybrid-nanozymes.

Cetyltrimethylammonium-chloride assisted in situ metabolic incorporation of nano-sized ROS-generating cascade-reaction containers in Gram-positive and Gram-negative peptidoglycan layers for the control of bacterially-induced sepsis.

Cascade-reaction containers generating reactive oxygen species (ROS) as an alternative for antibiotic-based strategies for bacterial infection control, require endogenous oxygen-sources and ROS-generation close to or preferably inside target bacteria. Here, this is achieved by cetyltrimethylammonium-chloride (CTAC) assisted in situ metabolic labeling and incorporation of mesoporous SiO2-nanoparticles, dual-loaded with glucose-oxidase and Fe3O4-nanoparticles as cascade-reaction containers, inside bacterial cell walls. First, azide-functionalized d-alanine (D-Ala-N3) was inserted in cell wall peptidoglycan layers of growing Gram-positive pathogens. In Gram-negatives, this could only be achieved after outer lipid-membrane permeabilization, using a low concentration of CTAC. Low concentrations of CTAC had no adverse effect on in vitro blood clotting or hemolysis nor on the health of mice when blood-injected. Next, dibenzocyclooctyne-polyethylene-glycol modified, SiO2-nanoparticles were in situ click-reacted with d-Ala-N3 in bacterial cell wall peptidoglycan layers. Herewith, a two-step cascade-reaction is facilitated inside bacteria, in which glucose-oxidase generates H2O2 at endogenously-available glucose concentrations, while subsequently Fe3O4-nanoparticles catalyze generation of •OH from the H2O2 generated. Generation of •OH inside bacterial cell walls by dual-loaded mesoporous SiO2-nanoparticles yielded more effective in vitro killing of both planktonic Gram-positive and Gram-negative bacteria suspended in 10 % plasma than SiO2-nanoparticles solely loaded with glucose-oxidase. Gram-positive or Gram-negative bacterially induced sepsis in mice could be effectively treated by in situ pre-treatment with tail-vein injected CTAC and d-Ala-N3, followed by injection of dual-loaded cascade-reaction containers without using antibiotics. This makes in situ metabolic incorporation of cascade-reaction containers as described attractive for further investigation with respect to the control of other types of infections comprising planktonic bacteria. STATEMENT OF SIGNIFICANCE: In situ metabolic-incorporation of cascade-reaction-containers loaded with glucose-oxidase and Fe3O4 nanoparticles into bacterial cell-wall peptidoglycan is described, yielding ROS-generation from endogenous glucose, non-antibiotically killing bacteria before ROS inactivates. Hitherto, only Gram-positives could be metabolically-labeled, because Gram-negatives possess two lipid-membranes. The outer membrane impedes direct access to the peptidoglycan. This problem was solved by outer-membrane permeabilization using a quaternary-ammonium compound. Several studies on metabolic-labeling perform crucial labeling steps during bacterial-culturing that in real-life should be part of a treatment. In situ metabolic-incorporation as described, can be applied in well-plates during in vitro experiments or in the body as during in vivo animal experiments. Surprisingly, metabolic-incorporation proceeded unhampered in blood and a murine, bacterially-induced sepsis could be well treated.

Beyond surface modification strategies to control infections associated with implanted biomaterials and devices - Addressing the opportunities offered by nanotechnology.

Biomaterial-associated infection (BAI) is considered a unique infection due to the presence of a biomaterial yielding frustrated immune-cells, ineffective in clearing local micro-organisms. The involvement of surface-adherent/surface-adapted micro-organisms in BAI, logically points to biomaterial surface-modifications for BAI-control. Biomaterial surface-modification is most suitable for prevention before adhering bacteria have grown into a mature biofilm, while BAI-treatment is virtually impossible through surface-modification. Hundreds of different surface-modifications have been proposed for BAI-control but few have passed clinical trials due to the statistical near-impossibility of benefit-demonstration. Yet, no biomaterial surface-modification forwarded, is clinically embraced. Collectively, this leads us to conclude that surface-modification is a dead-end road. Accepting that BAI is, like most human infections, due to surface-adherent biofilms (though not always to a foreign material), and regarding BAI as a common infection, opens a more-generally-applicable and therewith easier-to-validate road. Pre-clinical models have shown that stimuli-responsive nano-antimicrobials and antibiotic-loaded nanocarriers exhibit prolonged blood-circulation times and can respond to a biofilm's micro-environment to penetrate and accumulate within biofilms, prompt ROS-generation and synergistic killing with antibiotics of antibiotic-resistant pathogens without inducing further antimicrobial-resistance. Moreover, they can boost frustrated immune-cells around a biomaterial reducing the importance of this unique BAI-feature. Time to start exploring the nano-road for BAI-control.

A normalized parameter for comparison of biofilm dispersants in vitro.

Dispersal of infectious biofilms increases bacterial concentrations in blood. To prevent sepsis, the strength of a dispersant should be limited to allow the immune system to remove dispersed bacteria from blood, preferably without antibiotic administration. Biofilm bacteria are held together by extracellular polymeric substances that can be degraded by dispersants. Currently, comparison of the strength of dispersants is not possible by lack of a suitable comparison parameter. Here, a biofilm dispersal parameter is proposed that accounts for differences in initial biofilm properties, dispersant concentration and exposure time by using PBS as a control and normalizing outcomes with respect to concentration and time. The parameter yielded near-identical values based on dispersant-induced reductions in biomass or biofilm colony-forming-units and appeared strain-dependent across pathogens. The parameter as proposed is largely independent of experimental methods and conditions and suitable for comparing different dispersants with respect to different causative strains in particular types of infection.

Lethal puncturing of planktonic Gram-positive and Gram-negative bacteria by magnetically-rotated silica hexapods.

Planktonic bacterial presence in many industrial and environmental applications and personal health-care products is generally countered using antimicrobials. However, antimicrobial chemicals present an environmental threat, while emerging resistance reduces their efficacy. Suspended bacteria have no defense against mechanical attack. Therefore, we synthesized silica hexapods on an α-Fe2O3 core that can be magnetically-rotated to inflict lethal cell-wall-damage to planktonic Gram-negative and Gram-positive bacteria. Hexapods possessed 600 nm long nano-spikes, composed of SiO2, as shown by FTIR and XPS. Fluorescence staining revealed cell wall damage caused by rotating hexapods. This damage was accompanied by DNA/protein release and bacterial death that increased with increasing rotational frequency up to 500 rpm. Lethal puncturing was more extensive on Gram-negative bacteria than on Gram-positive bacteria, which have a thicker peptidoglycan layer with a higher Young's modulus. Simulations confirmed that cell-wall-puncturing occurs at lower nano-spike penetration levels in the cell walls of Gram-negative bacteria. This approach offers a new way to kill bacteria in suspension, not based on antimicrobial chemicals.

Ciprofloxacin-Loaded, pH-Responsive PAMAM-Megamers Functionalized with S-Nitrosylated Hyaluronic Acid Support Infected Wound Healing in Mice without Inducing Antibiotic Resistance.

Antimicrobial-resistant bacterial infections threaten to become the number one cause of death by the year 2050. Since the speed at which antimicrobial-resistance develops is exceeding the pace at which new antimicrobials come to the market, this threat cannot be countered by making more, new and stronger antimicrobials. Promising new antimicrobials should not only kill antimicrobial-resistant bacteria, but also prevent development of new bacterial resistance mechanisms in strains still susceptible. Here, PAMAM-dendrimers are clustered using glutaraldehyde to form megamers that are core-loaded with ciprofloxacin and functionalized with HA-SNO. Megamers are enzymatically disintegrated in an acidic pH, as in infectious biofilms, yielding release of ciprofloxacin and NO-generation by HA-SNO. NO-generation does not contribute to the killing of planktonic Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa, but in a biofilm-mode of growth short-lived NO-assisted killing of both ciprofloxacin-susceptible and ciprofloxacin-resistant bacterial strains by the ciprofloxacin released. Repeated sub-culturing of ciprofloxacin-susceptible bacteria in presence of ciprofloxacin-loaded and HA-SNO functionalized PAMAM-megamers does not result in ciprofloxacin-resistant variants as does repeated culturing in presence of ciprofloxacin. Healing of wounds infected by a ciprofloxacin-resistant S. aureus variant treated with ciprofloxacin-loaded, HA-SNO functionalized megamers proceed faster through NO-assisted ciprofloxacin killing of infecting bacteria and stimulation of angiogenesis.

Magnetically-targetable outer-membrane vesicles for sonodynamic eradication of antibiotic-tolerant bacteria in bacterial meningitis.

Treatment of acute bacterial meningitis is difficult due to the impermeability of the blood-brain barrier, greatly limiting the antibiotic concentrations that can be achieved in the brain. Escherichia coli grown in presence of iron-oxide magnetic nanoparticles secrete large amounts of magnetic outer-membrane vesicles (OMVs) in order to remove excess Fe from their cytoplasm. OMVs are fully biomimetic nanocarriers, but can be inflammatory. Here, non-inflammatory magnetic OMVs were prepared from an E. coli strain in which the synthesis of inflammatory lipid A acyltransferase was inhibited using CRISPR/Cas9 mediated gene knockout. OMVs were loaded with ceftriaxone (CRO) and meso-tetra-(4-carboxyphenyl)porphine (TCPP) and magnetically driven across the blood-brain barrier for sonodynamic treatment of bacterial meningitis. ROS-generation upon ultrasound application of CRO- and TCPP-loaded OMVs yielded similar ROS-generation as by TCPP in solution. In vitro, ROS-generation by CRO- and TCPP-loaded OMVs upon ultrasound application operated synergistically with CRO to kill a hard-to-kill, CRO-tolerant E. coli strain. In a mouse model of CRO-tolerant E. coli meningitis, CRO- and TCPP-loaded OMVs improved survival rates and clinical behavioral scores of infected mice after magnetic targeting and ultrasound application. Recurrence did not occur for at least two weeks after arresting treatment.

Rapid Bacterial Detection and Gram-Identification Using Bacterially Activated, Macrophage-Membrane-Coated Nanowired-Si Surfaces in a Microfluidic Device.

Bacterially induced sepsis requires rapid bacterial detection and identification. Hours count for critically ill septic patients, while current culture-based detection requires at least 10 h up to several days. Here, we apply a microfluidic device equipped with a bacterially activated, macrophage-membrane-coating on nanowired-Si adsorbent surfaces for rapid, bacterial detection and Gram-identification in bacterially contaminated blood. Perfusion of suspensions of Gram-negative or Gram-positive bacteria through a microfluidic device equipped with membrane-coated adsorbent surfaces detected low (<10 CFU/mL) bacterial levels. Subsequent, in situ fluorescence-staining yielded Gram-identification for guiding antibiotic selection. In mixed Escherichia coli and Staphylococcus aureus suspensions, Gram-negative and Gram-positive bacteria were detected in the same ratios as those fixed in suspension. Results were validated with a 100% correct score by blinded evaluation (two observers) of 15 human blood samples, spiked with widely different bacterial strains or combinations of strains, demonstrating the potential of the platform for rapid (1.5 h in total) diagnosis of bacterial sepsis.

Zwitterionic poly(sulfobetaine methacrylate)-based hydrogel coating for drinking water distribution systems to inhibit adhesion of waterborne bacteria.

Presence of biofilms in drinking water distribution systems (DWDS) can be a nuisance, leading to several operational and maintenance issues (i.e., increased secondary disinfectants demand, pipe damage or increased flow resistance), and so far, no single control practice was found to be sufficiently effective. Here, we propose poly (sulfobetaine methacrylate) (P(SBMA))-based hydrogel coating application as a biofilm control strategy in DWDS. The P(SBMA) coating was synthetized through photoinitiated free radical polymerization on polydimethylsiloxane with different combinations of SBMA as a monomer, and N, N'-methylenebis (acrylamide) (BIS) as a cross-linker. The most stable coating in terms of its mechanical properties was obtained using 20% SBMA with a 20:1 SBMA:BIS ratio. The coating was characterized using Scanning Electron Microscopy, Energy Dispersive X-Ray Spectroscopy, and water contact angle measurements. The anti-adhesive performance of the coating was evaluated in a parallel-plate flow chamber system against adhesion of four bacterial strains representing genera commonly identified in DWDS biofilm communities, Sphingomonas and Pseudomonas. The selected strains exhibited varying adhesion behaviors in terms of attachment density and bacteria distribution on the surface. Despite these differences, after 4 h, presence of the P(SBMA)-based hydrogel coating significantly reduced the number of adhering bacteria by 97%, 94%, 98% and 99%, for Sphingomonas Sph5, Sphingomonas Sph10, Pseudomonas extremorientalis and Pseudomonas aeruginosa, respectively, compared to non-coated surfaces. These findings motivate further research into a potential application of a hydrogel anti-adhesive coating as a localized biofilm control strategy in DWDS, especially on materials known to promote excessive biofilm growth.

Nanogel-based coating as an alternative strategy for biofilm control in drinking water distribution systems.

Biofilm formation and detachment in drinking water distribution systems (DWDS) can lead to several operational issues. Here, an alternative biofilm control strategy of limiting bacterial adhesion by application of a poly(N-isopropylmethacrylamide)-based nanogel coating on DWDS pipe walls was investigated. The nanogel coatings were successfully deposited on surfaces of four polymeric pipe materials commonly applied in DWDS construction. Nanogel-coated and non-coated pipe materials were characterized in terms of their surface hydrophilicity and roughness. Four DWDS relevant bacterial strains, representing Sphingomonas and Pseudomonas, were used to evaluate the anti-adhesive performance of the coating in 4 h adhesion and 24 h biofilm assays. The presence of the nanogel coating resulted in adhesion reduction up to 97%, and biofilm reduction up to 98%, compared to non-coated surfaces. These promising results motivate further investigation of nanogel coatings as a strategy for biofilm prevention in DWDS.

Identification of Potential Antimicrobial Targets of Pseudomonas aeruginosa Biofilms through a Novel Screening Approach.

Pseudomonas aeruginosa is an opportunistic pathogen of considerable medical importance, owing to its pronounced antibiotic tolerance and association with cystic fibrosis and other life-threatening diseases. The aim of this study was to highlight the genes responsible for P. aeruginosa biofilm tolerance to antibiotics and thereby identify potential new targets for the development of drugs against biofilm-related infections. By developing a novel screening approach and utilizing a public P. aeruginosa transposon insertion library, several biofilm-relevant genes were identified. The Pf phage gene (PA0720) and flagellin gene (fliC) conferred biofilm-specific tolerance to gentamicin. Compared with the reference biofilms, the biofilms formed by PA0720 and fliC mutants were completely eliminated with a 4-fold-lower gentamicin concentration. Furthermore, the mreC, pprB, coxC, and PA3785 genes were demonstrated to play major roles in enhancing biofilm tolerance to gentamicin. The analysis of biofilm-relevant genes performed in this study provides important novel insights into the understanding of P. aeruginosa antibiotic tolerance, which will facilitate the detection of antibiotic resistance and the development of antibiofilm strategies against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen of high medical importance and is one of the main pathogens responsible for the mortality of patients with cystic fibrosis. In addition to inherited antibiotic resistance, P. aeruginosa can form biofilms, defined as communities of microorganisms embedded in a self-produced matrix of extracellular polymeric substances adhering to each other and/or to a surface. Biofilms protect bacteria from antibiotic treatments and represent a major reason for antibiotic failure in the treatment of chronic infections caused by cystic fibrosis. Therefore, it is crucial to develop new therapeutic strategies aimed at specifically eradicating biofilms. The aim of this study was to generalize a novel screening method for biofilm research and to identify the possible genes involved in P. aeruginosa biofilm tolerance to antibiotics, both of which could improve the understanding of biofilm-related infections and allow for the identification of relevant therapeutic targets for drug development.

A Heterocatalytic Metal-Organic Framework to Stimulate Dispersal and Macrophage Combat with Infectious Biofilms.

Eradication of infectious biofilms is becoming increasingly difficult due to the growing number of antibiotic-resistant strains. This necessitates development of nonantibiotic-based, antimicrobial approaches. To this end, we designed a heterocatalytic metal-organic framework composed of zirconium 1,4-dicarboxybenzene (UiO-66) with immobilized Pt nanoparticles (Pt-NP/UiO-66). Pt-NP/UiO-66 enhanced singlet-oxygen generation compared with Pt nanoparticles or UiO-66, particularly in an acidic environment. Singlet-oxygen generation degraded phosphodiester bonds present in eDNA gluing biofilms together and therewith dispersed biofilms. Remaining biofilms possessed a more open structure. Concurrently, Pt-NP/UiO-66 stimulated macrophages to adapt a more M1-like, "fighting" phenotype, moving faster toward their target bacteria and showing increased bacterial killing. As a combined effect of biofilm dispersal and macrophage polarization, a subcutaneous Staphylococcus aureus biofilm in mice was more readily eradicated by Pt-NP/UiO-66 than by Pt nanoparticles or UiO-66. Therewith, heterocatalytic Pt-NP/UiO-66 metal-organic frameworks constitute a nonantibiotic-based strategy to weaken protective matrices and disperse infectious biofilms, while strengthening macrophages in bacterial killing.

A dynamic flow model mimicking duodenoscope reprocessing after bacterial contamination for translational research.

Objective: Duodenoscopy-associated infections and outbreaks are reported globally despite strict adherence to duodenoscope reprocessing protocols. Therefore, new developments in the reprocessing procedure are needed. Design: We evaluated a novel dynamic flow model for an additional cleaning step between precleaning and manual cleaning in the reprocessing procedure. Methods: A parallel plate flow chamber with a fluorinated ethylene propylene bottom plate was used to mimic the duodenoscope channels. The flow chamber was inoculated with a suspension containing Klebsiella pneumoniae to simulate bacterial contamination during a duodenoscopic procedure. After inoculation the flow chamber was flushed with a detergent mimicking precleaning. Subsequently the flow chamber was subjected to different interventions: flow with phosphate-buffered saline (PBS), flow with 2 commercial detergents, flow with sodium dodecyl sulfate with 3 different concentrations, and flow with microbubbles. Adhering bacteria were counted using phase-contrast microscopy throughout the experiment, and finally, bacterial viability was assessed. Results: During precleaning both PBS and 1% (v/v) Neodisher Mediclean Forte were able to desorb bacteria, but neither proved superior. After precleaning only sodium dodecyl sulfate could desorb bacteria. Conclusions: Flushing during precleaning is an essential step for reducing adhering luminal bacteria, and sodium dodecyl sulfate is a promising detergent for bacterial desorption from duodenoscope channels after precleaning.

Proton-mediated burst of dual-drug loaded liposomes for biofilm dispersal and bacterial killing.

Exposure of infectious biofilms to dispersants induces high bacterial concentrations in blood that may cause sepsis. Preventing sepsis requires simultaneous biofilm dispersal and bacterial killing. Here, self-targeting DCPA(2-(4-((1,5-bis(octadecenoyl)1,5-dioxopentan-2-yl)carbamoyl)pyridin-1-ium-1-yl)acetate) liposomes with complexed water were self-assembled with ciprofloxacin loaded in-membrane and PEGylated as a lipid-membrane component, together with bromelain loaded in-core. Inside biofilms, DCPA-H2O and PEGylated ciprofloxacin became protonated, disturbing the balance in the lipid-membrane to cause liposome-burst and simultaneous release of bromelain and ciprofloxacin. Simultaneous release of bromelain and ciprofloxacin enhanced bacterial killing in Staphylococcus aureus biofilms as compared with free bromelain and/or ciprofloxacin. After tail-vein injection in mice, liposomes accumulated inside intra-abdominal staphylococcal biofilms. Subsequent liposome-burst and simultaneous release of bromelain and ciprofloxacin yielded degradation of the biofilm matrix by bromelain and higher bacterial killing without inducing septic symptoms as obtained by injection of free bromelain and ciprofloxacin. This shows the advantage of simultaneous release from liposomes of bromelain and ciprofloxacin inside a biofilm.

High-Yield, Magnetic Harvesting of Extracellular Outer-Membrane Vesicles from Escherichia coli.

Extracellular outer-membrane vesicles (OMVs) are attractive for use as drug nanocarriers, because of their high biocompatibility and ability to enter cells. However, widespread use is hampered by low yields. Here, a high-yield method for magnetic harvesting of OMVs from Escherichia coli is described. To this end, E. coli are grown in the presence of magnetic iron-oxide nanoparticles (MNPs). Uptake of MNPs by E. coli is low and does not increase secretion of OMVs. Uptake of MNPs can be enhanced through PEGylation of MNPs. E. coli growth in the presence of PEGylated MNPs increases bacterial MNP-uptake and OMV-secretion, accompanied by upregulation of genes involved in OMV-secretion. OMVs containing MNPs can be magnetically harvested at 60-fold higher yields than achieved by ultracentrifugation. Functionally, magnetically-harvested OMVs and OMVs harvested by ultracentrifugation are both taken-up in similar numbers by bacteria. Uniquely, in an applied magnetic field, magnetically-harvested OMVs with MNPs accumulate over the entire depth of an infectious biofilm. OMVs harvested by ultracentrifugation without MNPs only accumulate near the biofilm surface. In conclusion, PEGylation of MNPs is essential for their uptake in E. coli and yields magnetic OMVs allowing high-yield magnetic-harvesting. Moreover, magnetic OMVs can be magnetically targeted to a cargo delivery site in the human body.

Synergy between pH- and hypoxia-responsiveness in antibiotic-loaded micelles for eradicating mature, infectious biofilms.

Antibiotic-loaded PEG/PAE-based micelles are frequently considered for eradicating infectious biofilms. At physiological pH, PEG facilitates transport through blood. Near an acidic infection-site, PAE becomes protonated causing micellar targeting to a biofilm. However, micellar penetration and accumulation is confined to the surface region of a biofilm. Especially matured biofilms also possess hypoxic regions. We here designed dual-responsive PEG/PAE-b-P(Lys-NBCF) micelles, responding to both acidity and low oxygen-saturation level in matured biofilms. Dual, pH- and hypoxia-responsive micelles targeted and accumulated evenly over the depth of 7- to 14-days old biofilms. Delineation demonstrated that pH-responsiveness was responsible for targeting of the infection-site and accumulation of micelles in the surface region of the biofilm. Hypoxia-responsiveness caused deep penetration in the biofilm. Dual, pH- and hypoxia-responsive micelles loaded with ciprofloxacin yielded more effective, synergistic eradication of 10-days old, matured Staphylococcus aureus biofilms underneath an abdominal imaging-window in living mice than achieved by ciprofloxacin in solution or single, pH- or hypoxia responsive micelles loaded with ciprofloxacin. Also, wound-healing after removal of window and its frame proceeded fastest after tail-vein injection of ciprofloxacin-loaded, dual, pH- and hypoxia-responsive micelles. Concluding, pH- and hypoxia-responsiveness are both required for eradicating mature biofilms and advancing responsive antibiotic nanocarriers to clinical application. STATEMENT OF SIGNIFICANCE: pH-responsive antibiotic nanocarriers have emerged as a possible new strategy to prevent antimicrobial-resistant bacterial infections from becoming the leading cause of death. In this paper, we show that commonly studied, pH-responsive micellar nanocarriers merely allow self-targeting to an infectious biofilm, but do not penetrate deeply into the biofilm. The dual-responsive (acidic pH- and hypoxia) antibiotic-loaded micelles designed here not only self-target to an infectious biofilm, but also penetrate deeply. The in vitro and in vivo advantages of dual-responsive nanocarriers are most obvious when studied in infectious biofilms grown for 10 viz a viz the 2 days, usually applied in the literature. Significantly, clinical treatment of bacterial infection usually starts more than 2 days after appearance of the first symptoms.

Viscoelastic properties of plasma-agarose hydrogels dictate favorable fibroblast responses for skin tissue engineering applications.

Dermal wound healing relies on the properties of the extracellular matrix (ECM). Thus, hydrogels that replicate skin ECM have reached clinical application. After a dermal injury, a transient, biodegradable fibrin clot is instrumental in wound healing. Human plasma, and its main constituent, fibrin would make a suitable biomaterial for improving wound healing and processed as hydrogels albeit with limited mechanical strength. To overcome this, plasma-agarose (PA) composite hydrogels have been developed and used to prepare diverse bioengineered tissues. To date, little is known about the influence of variable agarose concentrations on the viscoelastic properties of PA hydrogels and their correlation to cell biology. This study reports the characterization of the viscoelastic properties of different concentrations of agarose in PA hydrogels: 0 %, 0.5 %, 1 %, 1.5 %, and 2 % (w/v), and their influence on the cell number and mitochondrial activity of human dermal fibroblasts. Results show that agarose addition increased the stiffness, relaxation time constants 1 (τ1) and 2 (τ2), and fiber diameter, whereas the porosity decreased. Changes in cell metabolism occurred at the early stages of culturing and correlated to the displacement of fast (τ1) and intermediate (τ2) Maxwell elements. Fibroblasts seeded in low PA concentrations spread faster during 14 d than cells cultured in higher agarose concentrations. Collectively, these results confirm that PA viscoelasticity and hydrogel architecture strongly influenced cell behavior. Therefore, viscoelasticity is a key parameter in the design of PA-based implants.