Composited silk fibroins ensured adhesion stability and magnetic controllability of Fe3O4-nanoparticle coating on implant for biofilm treatment.

Magnetic propulsion of nano-/micro-robots is an effective way to treat implant-associated infections by physically destroying biofilm structures to enhance antibiotic killing. However, it is hard to precisely control the propulsion in vivo. Magnetic-nanoparticle coating that can be magnetically pulled off does not need precise control, but the requirement of adhesion stability on an implant surface restricts its magnetic responsiveness. Moreover, whether the coating has been fully pulled-off or not is hard to ensure in real-time in vivo. Herein, composited silk fibroins (SFMA) are optimized to stabilize Fe3O4 nanoparticles on a titanium surface in a dry environment; while in an aqueous environment, the binding force of SFMA on titanium is significantly reduced due to hydrophilic interaction, making the coating magnetically controllable by an externally-used magnet but still stable in the absence of a magnet. The maximum working distance of the magnet can be calculated using magnetomechanical simulation in which the yielding magnetic traction force is strong enough to pull Fe3O4 nanoparticles off the surface. The pulling-off removes the biofilms that formed on the coating and enhances antibiotic killing both in vitro and in a rat sub-cutaneous implant model by up to 100 fold. This work contributes to the practical knowledge of magnetic propulsion for biofilm treatment.

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.

A self-cleaning surface based on UV-activatable, AgCl micropumps for bacterial killing and removal.

We report a self-cleaning, bacterial killing surface by immobilization of AgCl microparticles on a surface, acting as chemical micropumps. The surface shows a high bacterial killing efficacy of attached bacteria and exhibits sustainable removal of bacteria as a result of UV-activatable micropumping originating from the photocatalytic reaction of AgCl microparticles. Our work provides an advance in the sustainable use of bacterial contact-killing surfaces stricto sensu through removal of dead bacteria and debris that may shield contact-killing sites.

Water in bacterial biofilms: pores and channels, storage and transport functions.

Bacterial biofilms occur in many natural and industrial environments. Besides bacteria, biofilms comprise over 70 wt% water. Water in biofilms occurs as bound- or free-water. Bound-water is adsorbed to bacterial surfaces or biofilm (matrix) structures and possesses different Infra-red and Nuclear-Magnetic-Resonance signatures than free-water. Bound-water is different from intra-cellularly confined-water or water confined within biofilm structures and bacteria are actively involved in building water-filled structures by bacterial swimmers, dispersion or lytic self-sacrifice. Water-filled structures can be transient due to blocking, resulting from bacterial growth, compression or additional matrix formation and are generally referred to as "channels and pores." Channels and pores can be distinguished based on mechanism of formation, function and dimension. Channels allow transport of nutrients, waste-products, signalling molecules and antibiotics through a biofilm provided the cargo does not adsorb to channel walls and channels have a large length/width ratio. Pores serve a storage function for nutrients and dilute waste-products or antimicrobials and thus should have a length/width ratio close to unity. The understanding provided here on the role of water in biofilms, can be employed to artificially engineer by-pass channels or additional pores in industrial and environmental biofilms to increase production yields or enhance antimicrobial penetration in infectious biofilms.

On-demand pulling-off of magnetic nanoparticles from biomaterial surfaces through implant-associated infectious biofilms for enhanced antibiotic efficacy.

Biomaterial-associated infections can occur any time after surgical implantation of biomaterial implants and limit their success rates. On-demand, antimicrobial release coatings have been designed, but in vivo release triggers uniquely relating with infection do not exist, while inadvertent leakage of antimicrobials can cause exhaustion of a coating prior to need. Here, we attach magnetic-nanoparticles to a biomaterial surface, that can be pulled-off in a magnetic field through an adhering, infectious biofilm. Magnetic-nanoparticles remained stably attached to a surface upon exposure to PBS for at least 50 days, did not promote bacterial adhesion or negatively affect interaction with adhering tissue cells. Nanoparticles could be magnetically pulled-off from a surface through an adhering biofilm, creating artificial water channels in the biofilm. At a magnetic-nanoparticle coating concentration of 0.64 mg cm-2, these by-pass channels increased the penetrability of Staphylococcus aureus and Pseudomonas aeruginosa biofilms towards different antibiotics, yielding 10-fold more antibiotic killing of biofilm inhabitants than in absence of artificial channels. This innovative use of magnetic-nanoparticles for the eradication of biomaterial-associated infections requires no precise targeting of magnetic-nanoparticles and allows more effective use of existing antibiotics by breaking the penetration barrier of an infectious biofilm adhering to a biomaterial implant surface on-demand.

Influence of interaction between surface-modified magnetic nanoparticles with infectious biofilm components in artificial channel digging and biofilm eradication by antibiotics in vitro and in vivo.

Magnetic targeting of antimicrobial-loaded magnetic nanoparticles to micrometer-sized infectious biofilms is challenging. Bacterial biofilms possess water channels that facilitate transport of nutrient and metabolic waste products, but are insufficient to allow deep penetration of antimicrobials and bacterial killing. Artificial channel digging in infectious biofilms involves magnetically propelling nanoparticles through a biofilm to dig additional channels to enhance antimicrobial penetration. This does not require precise targeting. However, it is not known whether interaction of magnetic nanoparticles with biofilm components impacts the efficacy of antibiotics after artificial channel digging. Here, we functionalized magnetic-iron-oxide-nanoparticles (MIONPs) with polydopamine (PDA) to modify their interaction with staphylococcal pathogens and extracellular-polymeric-substances (EPS) and relate the interaction with in vitro biofilm eradication by gentamicin after magnetic channel digging. PDA-modified MIONPs had less negative zeta potentials than unmodified MIONPs due to the presence of amino groups and accordingly more interaction with negatively charged staphylococcal cell surfaces than unmodified MIONPs. Neither unmodified nor PDA-modified MIONPs interacted with EPS. Concurrently, use of non-interacting unmodified MIONPs for artificial channel digging in in vitro grown staphylococcal biofilms enhanced the efficacy of gentamicin more than the use of interacting, PDA-modified MIONPs. In vivo experiments in mice using a sub-cutaneous infection model confirmed that non-interacting, unmodified MIONPs enhanced eradication by gentamicin of Staphylococcus aureus Xen36 biofilms about 10 fold. Combined with the high biocompatibility of magnetic nanoparticles, these results form an important step in understanding the mechanism of artificial channel digging in infectious biofilms for enhancing antibiotic efficacy in hard-to-treat infectious biofilms in patients.

Homogeneous Distribution of Magnetic, Antimicrobial-Carrying Nanoparticles through an Infectious Biofilm Enhances Biofilm-Killing Efficacy.

Magnetic, antimicrobial-carrying nanoparticles provide a promising, new and direly needed antimicrobial strategy against infectious bacterial biofilms. Penetration and accumulation of antimicrobials over the thickness of a biofilm is a conditio sine qua non for effective killing of biofilm inhabitants. Simplified schematics on magnetic-targeting always picture homogeneous distribution of magnetic, antimicrobial-carrying nanoparticles over the thickness of biofilms, but this is not easy to achieve. Here, gentamicin-carrying magnetic nanoparticles (MNPs-G) were synthesized through gentamicin conjugation with iron-oxide nanoparticles and used to demonstrate the importance of their homogeneous distribution over the thickness of a biofilm. Diameters of MNPs-G were around 60 nm, well below the limit for reticuloendothelial rejection. MNPs-G killed most ESKAPE-panel pathogens, including Escherichia coli, equally as well as gentamicin in solution. MNPs-G distribution in a Staphylococcus aureus biofilm was dependent on magnetic-field exposure time and most homogeneous after 5 min magnetic-field exposure. Exposure of biofilms to MNPs-G with 5 min magnetic-field exposure yielded not only homogeneous distribution of MNPs-G, but concurrently better staphylococcal killing at all depths than that of MNPs, gentamicin in solution, and MNPs-G, or after other magnet-field exposure times. In summary, homogeneous distribution of gentamicin-carrying magnetic nanoparticles over the thickness of a staphylococcal biofilm was essential for killing biofilm inhabitants and required optimizing of the magnetic-field exposure time. This conclusion is important for further successful development of magnetic, antimicrobial-carrying nanoparticles toward clinical application.

Artificial Channels in an Infectious Biofilm Created by Magnetic Nanoparticles Enhanced Bacterial Killing by Antibiotics.

The poor penetrability of many biofilms contributes to the recalcitrance of infectious biofilms to antimicrobial treatment. Here, a new application for the use of magnetic nanoparticles in nanomedicine to create artificial channels in infectious biofilms to enhance antimicrobial penetration and bacterial killing is proposed. Staphylococcus aureus biofilms are exposed to magnetic-iron-oxide nanoparticles (MIONPs), while magnetically forcing MIONP movement through the biofilm. Confocal laser scanning microscopy demonstrates artificial channel digging perpendicular to the substratum surface. Artificial channel digging significantly (4-6-fold) enhances biofilm penetration and bacterial killing efficacy by gentamicin in two S. aureus strains with and without the ability to produce extracellular polymeric substances. Herewith, this work provides a simple, new, and easy way to enhance the eradication of infectious biofilms using MIONPs combined with clinically applied antibiotic therapies.

Synergy in thrombin-graphene sponge for improved hemostatic efficacy and facile utilization.

Composites are attractive for its potential synergistic effects that can result in high-performance, but the synergy depends on subtle design. In this study, a hemostatic composite, a thrombin/cross-linked graphene sponge (TCGS), was developed through a facile gradient composite strategy. The porous structure of the CGS assures that the thrombin is stably embedded in the TCGS, avoiding a burst release but maintaining its bioactivity. In the synergy between proper thrombin stimulation and the fast absorption of the sponge, TCGS exhibits outstanding hemostatic performance, ultrafast bleeding cessation, within 100s, which is superior to both CGS and equal amounts of native thrombin. Lower or excessive thrombin dosages prolong the bleeding time. The study revealed that the balance between plasma absorption and thrombin stimulation at the interface is critical for improving hemostatic efficacy. TCGS is also highlighted for its biosafety and stability, even after 6 months of storage in environment. This potentially ultra-long shelf life is conducive to its practical applications. Therefore, TCGS not only provides a new strategy for developing a hemostatic composite but also provides a new method and understanding for the design of hemostatic materials.

Antifungal graphene oxide-borneol composite.

Although antibacterial activities of graphene oxide (GO) and its derivatives have been investigated comprehensively, their antifungal properties are still less reported. Yet, fungal contamination seriously threatens the public health. Herein, we present a design of graphene oxide-borneol (GOB) composite, and report its great antifungal effect. This GOB composite is prepared by esterification of borneol with thiomalic-acid-modified GO sheets, where the linker molecule is used to increase surface carboxyl groups. As a result, the antifungal activity displays a dramatically conversion from no activity of GO and its derivatives to distinct antifungal adhesion and growth inhibition of the GOB. Under microscopy, few spores can be found on the GOB surface, while large numbers of sporangia and spores adhere and grow on the control groups. It is also worth noting that on the GOB sample the fallen spore does not germinate even after 5days, demonstrating a long-term antifungal effect of the GOB composite. Further studies confirm that carbon stereochemistry rather than wettability plays a crucial role on the antifungal adhesion properties. This study not only highlights a promising GOB composite as a candidate of graphene-based antifungal agent, but also provides us with in-depth understanding of the interactions between fungi and graphene-based materials.

Graphene-Montmorillonite Composite Sponge for Safe and Effective Hemostasis.

Montmorillonite (MMT) is considered to be the most effective hemostat among natural phyllosilicates. However, there is a barrier against using MMT for the commercial hemostatics because the invaded MMT powders might cause thrombosis in vessel. Until now, it is still a challenge to manage the release of MMT and eliminate its side effect. Herein, we present a graphene-MMT composite sponge (GMCS), synthesized under a hydrothermal reaction, fixing MMT powders into the cross-linked graphene sheets. We demonstrate that only a few embedded MMT can evoke remarkable platelet stimulation at the sponge interface, while maintaining fast plasma absorbency of the innate sponge. In the synergy of the above hemostatic mechanisms, the GMCS can rapidly stop bleeding in approximately 85 s in rabbit artery injury test. More importantly, computed tomography angiography certifies that the GMCS does not cause thrombus or blood clot in vessels. Cytotoxicity assay further highlights its biocompatibility. In-depth analysis proposes that two-dimensional graphene overmatches one-dimensional linear polymers in the composite construction, and dimension transformation of blood distribution plays a crucial role for reinforcing the hemostatic performance. This GMCS hemostat not only opens a new perspective for graphene composite, but also makes a new chance of using clays for trauma therapy.

Diaminopropionic Acid Reinforced Graphene Sponge and Its Use for Hemostasis.

2,3-Diaminopropionic acid (DapA), a medicinal amino acid, is used for the first time to prepare a DapA cross-linked graphene sponge (DCGS) for hemostasis treatment. In a comparison with the reported ethanediamine (EDA) cross-linked graphene sponge (CGS), this carboxyl-functionalized DCGS can not only quickly absorb plasma, but also stimulate erythrocytes and platelets to change their normal form and structure at the interface, which largely affects a cell's metabolism and biofunction, thus further promoting blood coagulation. Whole blood clotting and rat-tail amputation tests indicated that on the basis of the additional interfacial stimulation, the hemostatic efficiency of the DCGS has been significantly improved in comparison with that of the CGS control (P < 0.05). In-depth insight revealed that the increased oxidation degree and the negative charge density play the crucial rule in the enhanced hemostatic performance. The chiral effect contributes mainly to the selective adhesion of erythrocytes and platelets rather than practical hemostasis. Nevertheless, this presentation demonstrated that, on the premise of keeping the fast absorbability, this is an effective method to improve the hemostatic efficiency by enhancing the cell/graphene interface interaction.

Black hemostatic sponge based on facile prepared cross-linked graphene.

In this study, we demonstrate for the first time the remarkable hemostatic performance of a cross-linked graphene sponge (CGS) as a superb hemostat. The CGS can absorb plasma immediately (<40 ms) to form a blood cell layer and promotes subsequent clotting. The interaction between the interface of the CGS and blood cells reveals that the fast blood coagulation is primarily attributed to the enrichment of hemocytes and platelets on the wound surface. An in vitro dynamic whole-blood clotting test further highlights the effectiveness of the CGS. Considering the facile preparation, low cost, nontoxicity, and long shelf life of the portable black sponge, the CGS has great potential for trauma treatment.