Macrophage-membrane coated nanowired surfaces for diagnosing and cleansing of infected blood

Extracorporeal devices to cleanse septic blood from infecting bacteria, PAMPs and excess cytokines are based upon the use of hemofiltration membranes or adsorbent surfaces, but the current generation of devices has variable and inconclusive therapeutic efficacy. Nanostructured surfaces are consistently called “promising” to control bacterial adhesion and therewith also classify as potential adsorbent surfaces in extracorporeal blood cleansing. Therefore, we first review the interactions between bacteria and nanostructured surfaces. An easy distinction between nanostructured surfaces can be made on basis of periodic- or random-occurrence of nanostructured features, although often nanostructured surfaces are microstructured due to merging of their nanofeatures. Characterization of nanostructured surfaces is not trivial due to the myriad of different nanoscaled morphologies. Both superhydrophobic and hydrophilic, nanostructured surfaces generally yield low bacterial adhesion as compared with smooth surfaces. This thesis is based on the hypothesis that nanostructured surfaces will also impact the properties of adsorbed cell membranes due to their small adhesion forces and may therewith yield a highly biomimetic surface. It aims to verify the hypothesis that (bacterially-)activated macrophage membranes with natural recognition abilities for bacteria, pathogen-associated molecular patterns, cytokines and endotoxins, adsorbed to a nanostructured surface can be used as adsorbent surfaces in a microfluidic device for extracorporeal cleansing of blood

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

PAMAM dendrimers with dual-conjugated vancomycin and Ag-nanoparticles do not induce bacterial resistance and kill vancomycin-resistant Staphylococci

The effective life-time of new antimicrobials until the appearance of the first resistant strains is steadily decreasing, which discourages incentives for commercialization required for clinical translation and application. Therefore, development of new antimicrobials should not only focus on better and better killing of antimicrobial-resistant strains, but as a paradigm shift on developing antimicrobials that prevent induction of resistance. Heterofunctionalized, poly-(amido-amine) (PAMAM) dendrimers with amide-conjugated vancomycin (Van) and incorporated Ag nanoparticles (AgNP) showed a 6-7 log reduction in colony-forming-units of a vancomycin-resistant Staphylococcus aureus strain in vitro, while not inducing resistance in a vancomycin-susceptible strain. Healing of a superficial wound in mice infected with the vancomycin-resistant S. aureus was significantly faster and more effective by irrigation with low-dose, dual-conjugated Van-PAMAM-AgNP dendrimer suspension than by irrigation with vancomycin in solution or a PAMAM-AgNP dendrimer suspension. Herewith, dual-conjugation of vancomycin together with AgNPs in heterofunctionalized PAMAM dendrimers fulfills the need for new, prolonged life-time antimicrobials killing resistant pathogens without inducing resistance in susceptible strains. Important for clinical translation, this better use of antibiotics can be achieved with currently approved and clinically applied antibiotics, provided suitable for amide-conjugation. Statement of significance Stringent regulations, high development costs and shorter effective life-times of new antimicrobials before the first resistant strains appear, make development of new antimicrobials commercially little attractive. Considering the steadily shortening effective life-time of novel antibiotics, development of novel antimicrobials should focus on infection-control strategies that kill infectious bacteria without inducing bacterial antimicrobial-resistance. The heterofunctionalized dual-antimicrobial dendrimers described in this study, killed vancomycin-resistant staphylococci, while not inducing resistance in a vancomycin-susceptible strain. Significantly, these heterofunctionalized dendrimers can be prepared with currently available antibiotics. Therewith they allow to make better use of existing antibiotics, provided amenable to amideconjugation and their pathway to clinical translation is short. (C) 2021 Acta Materialia Inc. Published by Elsevier Ltd

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.</p

Coating of a Novel Antimicrobial Nanoparticle with a Macrophage Membrane for the Selective Entry into Infected Macrophages and Killing of Intracellular Staphylococci

Internalization of Staphylococcus aureus by macrophages can inactivate bacterial killing mechanisms, allowing intracellular residence and dissemination of infection. Concurrently, these staphylococci can evade antibiotics that are frequently unable to pass mammalian cell membranes. A binary, amphiphilic conjugate composed of triclosan and ciprofloxacin is synthesized that self-assemble through micelle formation into antimicrobial nanoparticles (ANPs). These novel ANPs are stabilized through encapsulation in macrophage membranes, providing membrane-encapsulated, antimicrobial-conjugated NPs (Me-ANPs) with similar protein activity, Toll-like receptor expression and negative surface charge as their precursor murine macrophage/human monocyte cell lines. The combination of Toll-like receptors and negative surface charge allows uptake of Me-ANPs by infected macrophages/monocytes through positively charged, lysozyme-rich membrane scars created during staphylococcal engulfment. Me-ANPs are not engulfed by more negatively charged sterile cells possessing less lysozyme at their surface. The Me-ANPs kill staphylococci internalized in macrophages in vitro. Me-ANPs likewise kill staphylococci more effectively than ANPs without membrane-encapsulation or clinically used ciprofloxacin in a mouse peritoneal infection model. Similarly, organ infections in mice created by dissemination of infected macrophages through circulation in the blood are better eradicated by Me-ANPs than by ciprofloxacin. These unique antimicrobial properties of macrophage-monocyte Me-ANPs provide a promising direction for human clinical application to combat persistent infections

MEPE/OF45 protects cells from DNA damage induced killing via stabilizing CHK1

Matrix extracellular phosphoglycoprotein/osteoblast factor 45 (MEPE/OF45) was cloned in 2000 with functions related to bone metabolism. We identified MEPE/OF45 for the first time as a new co-factor of CHK1 in mammalian cells to protect cells from DNA damage induced killing. We demonstrate here that MEPE/OF45 directly interacts with CHK1. Knocking down MEPE/OF45 decreases CHK1 levels and sensitizes the cells to DNA damage inducers such as ionizing radiation (IR) or camptothicin (CPT)-induced killing. Over-expressing wild-type MEPE/OF45, but not the mutant MEPE/OF45 (depleted the key domain to interact with CHK1) increases CHK1 levels in the cells and increases the resistance of the cells to IR or CPT. MEPE/OF45, interacting with CHK1, increases CHK1 half-life and decreases CHK1 degradation through the ubiquitine-mediated pathway. In addition, the interaction of MEPE/OF45 with CHK1 decreases CHK1 levels in the ubiquitin E3 ligases (Cul1 and Cul4A) complex, which suggests that MEPE/OF45 competes with the ubiquitin E3 ligases binding to CHK1 and thus decreases CHK1 from ubiquitin-mediated proteolysis. These findings reveal an important role of MEPE/OF45 in protecting cells from DNA damage induced killing through stabilizing CHK1, which would provide MEPE/OF45 as a new target for sensitizing tumor cells to radiotherapy or chemotherapy

Extensive RPA2 hyperphosphorylation promotes apoptosis in response to DNA replication stress in CHK1 inhibited cells

The replication protein A (RPA)-ssDNA complex formed at arrested replication forks recruits key proteins to activate the ATR-CHK1 signalling cascade. When CHK1 is inhibited during DNA replication stress, RPA2 is extensively hyperphosphorylated. Here, we investigated the role of RPA2 hyperphosphorylation in the fate of cells when CHK1 is inhibited. We show that proteins normally involved in DNA repair (RAD51) or control of RPA phosphorylation (the PP4 protein phosphatase complex) are not recruited to the genome after treatment with CHK1 and DNA synthesis inhibitors. This is not due to RPA2 hyperphosphorylation as suppression of this response does not restore loading suggesting that recruitment requires active CHK1. To determine whether RPA2 hyperphosphorylation protects stalled forks from collapse or induction of apoptosis in CHK1 inhibited cells during replication stress, cells expressing RPA2 genes mutated at key phosphorylation sites were characterized. Mutant RPA2 rescued cells from RPA2 depletion and reduced the level of apoptosis induced by treatment with CHK1 and replication inhibitors however the incidence of double strand breaks was not affected. Our data indicate that RPA2 hyperphosphorylation promotes cell death during replication stress when CHK1 function is compromised but does not appear to be essential for replication fork integrity

Clearance of ESKAPE Pathogens from Blood Using Bacterially Activated Macrophage Membrane-Coated Silicon Nanowires

Extracorporeal devices to cleanse blood from infecting bacteria are based upon bacterial capture to surfaces, but the current generation of capture devices has variable and inconclusive therapeutic efficacy. Here, a microfluidic device equipped with a Si capture surface with a highly periodic nanowired structure is designed. Nanowired Si surfaces are coated with macrophage membranes to benefit from the natural blood compatibility and ligand-receptor binding of macrophages. When macrophages are activated by uptake of Staphylococcus aureus or Escherichia coli, zeta potentials of activated macrophage membrane coatings become less negative than those of nonactivated ones, stimulating nonspecific bacterial capture. In addition, Toll-like receptors in bacterially activated membrane coatings on nanowired surfaces that are absent in nonactivated membrane coatings contribute to specific bacterial capture. These two factors, together with the maintenance of fluidity in activated membrane coatings, cause broad spectrum, high capture efficiencies of all six ESKAPE member pathogens, considered most threatening to human health. Surfaces with such broad-spectrum capture efficiencies have not been previously described, but are clinically most relevant because blood cleansing should start as soon as possible after a septic patient becomes symptomatic, when the causative bacterial strain is still unknown