Oriented immobilization of Pep19-2.5 on antifouling brushes suppresses the development of Staphylococcus aureus biofilms

Bacterial colonization of indwelling medical devices poses a danger to the patient and is a tremendous economic burden that costs billions of dollars to the healthcare system. Thus, it is essential to develop an effective mechanism that prevents the attachment of bacteria to the surface in combination with bactericidal strategies to kill them in direct contact. In this work, we combine the repellent/antifouling properties of polymer brushes with the antimicrobial activity of the synthetic peptide Pep19-2.5 and test its efficacy to inhibit Staphylococcus aureus biofilm formation. To tackle this, we utilized hierarchical polymer brushes, where the bottom block provides an effective barrier against adhesion while the top block provides functional groups for the immobilization of active molecules. Further, these polymer brushes were decorated with dibenzocyclooctine (DBCO)-functionalized Pep19-2.5 using strain-promoted alkyne-azide cycloaddition (SPAAC). This click chemistry proceeds very fast and does not require any catalyst, which is crucial for biomedical applications. The obtained coating was not only able to decrease the number of freely planktonic bacteria in the surrounding media (by 52.5%) but also inhibit the development of S. aureus biofilm by reducing the number of total, viable, and viable but non-culturable (VBNC) cells (up to 58%, 66%, and 70% respectively) and reduce the biovolume and thickness. Conversely, this coating does not exert any cytotoxic effect on Normal Human Dermal Fibroblasts (NHDF) cells. Thus, the combination of hierarchical polymer brushes with Pep19-2.5 is a promising approach to fight medical biofilms without affecting surrounding tissues

Controlled Surface Adhesion of Macrophages via Patterned Antifouling Polymer Brushes

Macrophages will play an important role in future diagnostics and immunotherapies of cancer. However, this demands to selectively capture and sort different subpopulations, which remains a challenge due to their innate ability to bind to a wide range of interfaces indiscriminately. The main obstacle here is the lack of interfaces combining sufficient antifouling properties with the display of specific binding sites allowing sorting and quantification. Herein, as a proof of principle means, it is introduced to pattern interfaces to locally and selective capture macrophages. The repellent coating is based on antifouling polymer brushes, which can be functionalized. Arrays of binding sites are constructed by microchannel cantilever spotting. Those structures are tested for the isolation of different macrophage subtypes, especially polarized anti‐inflammatory macrophages of the M2 type which can be found associated to tumors (“tumor associated macrophages”; TAMs). Using macrophages as a model system, it is demonstrated that the newly developed surfaces and patterns are efficient for specifically trapping targeted cells and can be useful for further development of therapeutic or diagnostic purposes in the future

Dendrimersome Synthetic Cells Harbor Cell Division Machinery of Bacteria

The integration of active cell machinery with synthetic building blocks is the bridge toward developing synthetic cells with biological functions and beyond. Self-replication is one of the most important tasks of living systems, and various complex machineries exist to execute it. In Escherichia coli, a contractile division ring is positioned to mid-cell by concentration oscillations of self-organizing proteins (MinCDE), where it severs membrane and cell wall. So far, the reconstitution of any cell division machinery has exclusively been tied to liposomes. Here, the reconstitution of a rudimentary bacterial divisome in fully synthetic bicomponent dendrimersomes is shown. By tuning the membrane composition, the interaction of biological machinery with synthetic membranes can be tailored to reproduce its dynamic behavior. This constitutes an important breakthrough in the assembly of synthetic cells with biological elements, as tuning of membrane-divisome interactions is the key to engineering emergent biological behavior from the bottom-up

Sensitive and rapid detection of aflatoxin M1 in milk utilizing enhanced SPR and p(HEMA) brushes

The rapid and sensitive detection of aflatoxin M-1 (AFM(1)) in milk by using surface plasmon resonance (SPR) biosensor is reported. This low molecular weight mycotoxin is analyzed using an indirect competitive immunoassay that is amplified by secondary antibodies conjugated with Au nanoparticles. In order to prevent fouling on the sensor surface by the constituents present in analyzed milk samples, an interface with poly(2-hydroxyethyl methacrylate) p(HEMA) brush was employed. The study presents a comparison of performance characteristics of p(HEMA)-based sensor with a regularly used polyethylene glycol -based architecture relying on mixed thiol self-assembled monolayer. Both sensors are characterized in terms of surface mass density of immobilized AFM(1) conjugate as well as affinity bound primary and secondary antibodies. The efficiency of the amplification strategy based on Au nanoparticle is discussed. The biosensor allowed for highly sensitive detection of AFM(1) in milk with a limit of detection (LOD) as low as 18 pg mL(-1) with the analysis time of 55 min. (C) 2016 Elsevier B.V. All rights reserved

Surface plasmon resonance-based aptasensor for direct monitoring of thrombin in a minimally processed human blood

Optical affinity biosensors are pursued for timely monitoring of thrombin in human blood, which is of urgent need in tailored anticoagulation therapies. However, the unspecific deposition of molecules, cells, and aggregates from the blood at their surface (also termed fouling) severely hinders their development and impedes the deploying of this technology to everyday clinical practice. We addressed this challenge by designing surface plasmon resonance (SPR) sensor chip with an antifouling polymer brush architecture and incorporated thrombin aptamer bioreceptors. Poly[(N-(2-hydroxypropyl)-methacrylamide)-co-(carboxybetaine methacrylamide)] brushes were synthesized on gold sensor chip surface via photoinduced single-electron transfer living radical polymerization and postmodified with three thrombin aptamers (HD1 short, HD1 and HD22). The affinity interaction of the aptamer bioreceptors with thrombin (as well as with other molecules present in the blood) was investigated and changes in their performance when incorporated into the polymer brushes were characterized. The combination of brushes and aptamer bioreceptors allowed for the analysis of medically relevant concentrations of thrombin in the 10% blood by direct SPR detection format. This is the first time that the optical affinity biosensor is demonstrated for label-free analysis of biomarkers in a minimally processed human blood without a need for pre-separation steps. We believe that this system constitutes a basis for the future affinity biosensor applications that are suitable for the clinical settings and can be readily adapted to detect a range of important biological markers

Grafting of functional methacrylate polymer brushes by photoinduced SET-LRP

Photoinduced surface-initiated single electron transfer living radical polymerization (SET-LRP) is a versatile technique for the preparation of polymer brushes. The vast diversity of compatible functional groups, together with a high end-group fidelity that enables precise control of the architecture, makes this approach an effective tool for tuning the properties of surfaces. We report the application of photoinduced SET-LRP for the surface-initiated grafting of polymer brushes from a wide range of methacrylate monomers for the first time. The living character of the process was demonstrated by the linear evolution of the polymer brush thickness in time, the ability to reinitiate the polymerization for the preparation of well-defined block copolymers, and also by X-ray photoelectron spectroscopy depth profiling. The surface patterning with these brushes could be achieved simply by restricting the irradiated area. The ability of poly(methacrylate) brushes prepared in this way to prevent non-specific protein adsorption is also demonstrated, indicating the suitability of this procedure for advanced applications