Control of nanoparticle penetration into biofilms through surface design.
ABSTRACT: Quantum dots were used as fluorescent probes to investigate nanoparticle penetration into biofilms. The particle penetration behavior was found to be controlled by surface chemical properties.
Project description:Orally administrated probiotic bacteria can aid antibiotic treatment of intestinal infections, but their arrival at their intestinal target site is hampered by killing in the gastrointestinal tract and by antibiotics solely intended for pathogen killing. Carbon-quantum-dots are extremely small nanoparticles and can be derived from different sources, including bacteria. Here, we hypothesize that carbon-quantum-dots inherit antibacterial activity from probiotic source bacteria to fulfill a similar role as live probiotics in intestinal infection therapy. Physico-chemical analyses indicated that carbon-quantum-dots, hydrothermally derived from <i>Bifidobacterium breve</i> (B-C-dots), inherited proteins and polysaccharides from their source-bacteria. B-C-dots disrupted biofilm matrices of <i>Escherichia coli</i> and <i>Salmonella typhimurium</i> biofilms through extensive reactive-oxygen-species (ROS)-generation, causing a decrease in volumetric bacterial-density in biofilms. Decreased bacterial densities leave more open space in biofilms and have enhanced ciprofloxacin penetration and killing potential in an <i>E. coli</i> biofilm pre-exposed to probiotic B-C-dots. Pathogenic carbon-quantum-dots hydrothermally derived from <i>E. coli</i> (E-C-dots) did not disrupt pathogenic biofilms nor enhance <i>E. coli</i> killing potential by ciprofloxacin. B-C-dots were biosafe in mice upon daily administration, while E-C-dots demonstrated a decrease in white blood cell and platelet counts and an increase in C-reactive protein levels. Therefore, the way is paved for employing probiotic carbon-quantum-dots instead of viable, probiotic bacteria for synergistic use with existing antibiotics in treating intestinal infections.
Project description:Biofilm-related infections can develop everywhere in the human body and are rarely cleared by the host immune system. Moreover, biofilms are often tolerant to antimicrobials, due to a combination of inherent properties of bacteria in their adhering, biofilm mode of growth and poor physical penetration of antimicrobials through biofilms. Current understanding of biofilm recalcitrance toward antimicrobial penetration is based on qualitative descriptions of biofilms. Here we hypothesize that stress relaxation of biofilms will relate with antimicrobial penetration. Stress relaxation analysis of single-species oral biofilms grown in vitro identified a fast, intermediate and slow response to an induced deformation, corresponding with outflow of water and extracellular polymeric substances, and bacterial re-arrangement, respectively. Penetration of chlorhexidine into these biofilms increased with increasing relative importance of the slow and decreasing importance of the fast relaxation element. Involvement of slow relaxation elements suggests that biofilm structures allowing extensive bacterial re-arrangement after deformation are more open, allowing better antimicrobial penetration. Involvement of fast relaxation elements suggests that water dilutes the antimicrobial upon penetration to an ineffective concentration in deeper layers of the biofilm. Next, we collected biofilms formed in intra-oral collection devices bonded to the buccal surfaces of the maxillary first molars of human volunteers. Ex situ chlorhexidine penetration into two weeks old in vivo formed biofilms followed a similar dependence on the importance of the fast and slow relaxation elements as observed for in vitro formed biofilms. This study demonstrates that biofilm properties can be derived that quantitatively explain antimicrobial penetration into a biofilm.
Project description:Inefficient delivery of macromolecules and nanoparticles to intracellular targets is a major bottleneck in drug delivery, genetic engineering, and molecular imaging. Here we apply live-cell single-quantum-dot imaging and tracking to analyze and classify nanoparticle states after intracellular delivery. By merging trajectory diffusion parameters with brightness measurements, multidimensional analysis reveals distinct and heterogeneous populations that are indistinguishable using single parameters alone. We derive new quantitative metrics of particle loading, cluster distribution, and vesicular release in single cells, and evaluate intracellular nanoparticles with diverse surfaces following osmotic delivery. Surface properties have a major impact on cell uptake, but little impact on the absolute cytoplasmic numbers. A key outcome is that stable zwitterionic surfaces yield uniform cytosolic behavior, ideal for imaging agents. We anticipate that this combination of quantum dots and single-particle tracking can be widely applied to design and optimize next-generation imaging probes, nanoparticle therapeutics, and biologics.
Project description:Current Food and Drug Administration-approved cancer nanotherapeutics, which passively accumulate around leaky regions of the tumor vasculature because of an enhanced permeation and retention (EPR) effect, have provided only modest survival benefits. This suboptimal outcome is likely due to physiological barriers that hinder delivery of the nanotherapeutics throughout the tumor. Many of these nanotherapeutics are ? 100 nm in diameter and exhibit enhanced accumulation around the leaky regions of the tumor vasculature, but their large size hinders penetration into the dense collagen matrix. Therefore, we propose a multistage system in which 100-nm nanoparticles "shrink" to 10-nm nanoparticles after they extravasate from leaky regions of the tumor vasculature and are exposed to the tumor microenvironment. The shrunken nanoparticles can more readily diffuse throughout the tumor's interstitial space. This size change is triggered by proteases that are highly expressed in the tumor microenvironment such as MMP-2, which degrade the cores of 100-nm gelatin nanoparticles, releasing smaller 10-nm nanoparticles from their surface. We used quantum dots (QD) as a model system for the 10-nm particles because their fluorescence can be used to demonstrate the validity of our approach. In vitro MMP-2 activation of the multistage nanoparticles revealed that the size change was efficient and effective in the enhancement of diffusive transport. In vivo circulation half-life and intratumoral diffusion measurements indicate that our multistage nanoparticles exhibited both the long circulation half-life necessary for the EPR effect and the deep tumor penetration required for delivery into the tumor's dense collagen matrix.
Project description:Multidrug resistant bacterial infections threaten to become the number one cause of death by the year 2050. Development of antimicrobial dendritic polymers is considered promising as an alternative infection control strategy. For antimicrobial dendritic polymers to effectively kill bacteria residing in infectious biofilms, they have to penetrate and accumulate deep into biofilms. Biofilms are often recalcitrant to antimicrobial penetration and accumulation. Therefore, this work aims to determine the role of compact dendrons with different peripheral composition in their penetration into <i>Pseudomonas aeruginosa</i> biofilms. Red fluorescently labeled dendrons with pH-responsive NH<sub>3</sub><sup>+</sup> peripheral groups initially penetrated faster from a buffer suspension at pH 7.0 into the acidic environment of <i>P. aeruginosa</i> biofilms than dendrons with OH or COO<sup>-</sup> groups at their periphery. In addition, dendrons with NH<sub>3</sub><sup>+</sup> peripheral groups accumulated near the top of the biofilm due to electrostatic double-layer attraction with negatively charged biofilm components. However, accumulation of dendrons with OH and COO<sup>-</sup> peripheral groups was more evenly distributed across the depth of the biofilms than NH<sub>3</sub><sup>+</sup> composed dendrons and exceeded accumulation of NH<sub>3</sub><sup>+</sup> composed dendrons after 10 min of exposure. Unlike dendrons with NH<sub>3</sub><sup>+</sup> groups at their periphery, dendrons with OH or COO<sup>-</sup> peripheral groups, lacking strong electrostatic double-layer attraction with biofilm components, were largely washed-out during exposure to PBS without dendrons. Thus, penetration and accumulation of dendrons into biofilms is controlled by their peripheral composition through electrostatic double-layer interactions, which is an important finding for the further development of new antimicrobial or antimicrobial-carrying dendritic polymers.
Project description:Biofilm cells are less susceptible to antimicrobials than their planktonic counterparts. While this phenomenon is multifactorial, the ability of the matrix to reduce antibiotic penetration into the biofilm is thought to be of limited importance studies suggest that antibiotics move fairly rapidly through biofilms. In this study, we monitored the transport of two clinically relevant antibiotics, tobramycin and ciprofloxacin, into non-mucoid Pseudomonas aeruginosa biofilms. To our surprise, we found that the positively charged antibiotic tobramycin is sequestered to the biofilm periphery, while the neutral antibiotic ciprofloxacin readily penetrated. We provide evidence that tobramycin in the biofilm periphery both stimulated a localized stress response and killed bacteria in these regions but not in the underlying biofilm. Although it is unclear which matrix component binds tobramycin, its penetration was increased by the addition of cations in a dose-dependent manner, which led to increased biofilm death. These data suggest that ionic interactions of tobramycin with the biofilm matrix limit its penetration. We propose that tobramycin sequestration at the biofilm periphery is an important mechanism in protecting metabolically active cells that lie just below the zone of sequestration.
Project description:<h4>Background</h4>Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.<h4>Methodology/principal findings</h4>The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50-200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.<h4>Conclusions/significance</h4>This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.
Project description:Mature biofilms are highly resistant to antimicrobial agents due to the presence of extracellular polymeric substances (EPS), which inhibit the penetration of external molecules. In this study, we developed a coordination compound consisting of zinc chloride and erythritol that exhibits penetrating and bactericidal activity against Streptococcus mutans biofilms. An in vitro biofilm model was established in microplates, and bactericidal activity against biofilms was evaluated using an Alamar blue assay. The cause of the antimicrobial activity of the zinc-erythritol mixture on mature biofilms was demonstrated using fast atom bombardment-mass spectrometry, confocal laser scanning microscopy and atomic force microscopy. We demonstrated that zinc chloride spontaneously formed cationic complexes with erythritol in water. The zinc-erythritol complexes reduced intra- and inter-molecular interactions between bacterial exopolysaccharides, a major component of EPS. This activity was confirmed by measuring the attenuation of the hardness of dried polysaccharides isolated from S. mutans biofilms. The reduction in the interactions between polysaccharides allowed the complexes to penetrate into biofilms and kill the embedded bacteria. While approximately 13% of biofilm-associated microbes were killed by a 10?min treatment with 6.6?mM zinc chloride, 45% were killed when a solution containing 19.8?mM erythritol and 6.6?mM zinc chloride was used. This strategy of leveraging the coordination properties of metal ions with sugar alcohols provides a simple way to effectively remove mature biofilms using only conventional substances without the need for intricate chemical synthesis processes.
Project description:Bacterial biofilms are widely associated with persistent infections. High resistance to conventional antibiotics and prevalent virulence makes eliminating these bacterial communities challenging therapeutic targets. We describe here the fabrication of a nanoparticle-stabilized capsule with a multicomponent core for the treatment of biofilms. The peppermint oil and cinnamaldehyde combination that comprises the core of the capsules act as potent antimicrobial agents. An in situ reaction at the oil/water interface between the nanoparticles and cinnamaldehyde structurally augments the capsules to efficiently deliver the essential oil payloads, effectively eradicating biofilms of clinically isolated pathogenic bacteria strains. In contrast to their antimicrobial action, the capsules selectively promoted fibroblast proliferation in a mixed bacteria/mammalian cell system making them promising for wound healing applications.
Project description:Water-dispersible amphiphilic surface-engineered quantum dots (QDs) were found to be strongly accumulated within discrete zones of the exopolymer network of Shewanella oneidensis MR-1 biofilms, but not on the cell surfaces. These microdomains showed a patterned distribution in the exopolymer matrix, which led to a restricted diffusion of the amphiphilic nanoparticles.