Mechanisms of nanotoxicity - biomolecule coronas protect pathological fungi against nanoparticle-based eradication.

Whereas nanotoxicity is intensely studied in mammalian systems, our knowledge of desired or unwanted nano-based effects for microbes is still limited. Fungal infections are global socio-economic health and agricultural problems, and current chemical antifungals may induce adverse side-effects in humans and ecosystems. Thus, nanoparticles are discussed as potential novel and sustainable antifungals via the desired nanotoxicity but often fail in practical applications. In our study, we found that nanoparticles' toxicity strongly depends on their binding to fungal spores, including the clinically relevant pathogen Aspergillus fumigatus as well as common plant pests, such as Botrytis cinerea or Penicillum expansum. Employing a selection of the model and antimicrobial nanoparticles, we found that nanoparticle-spore complex formation is influenced by the NM's physicochemical properties, such as size, identified as a key determinant for our silica model particles. Biomolecule coronas acquired in pathophysiologically and ecologically relevant environments, protected fungi against nanoparticle-induced toxicity as shown by employing antimicrobial ZnO, Ag, or CuO nanoparticles as well as dissolution-resistant quantum dots. Mechanistically, dose-dependent corona-mediated resistance was conferred via reducing the physical adsorption of nanoparticles to fungi. The inhibitory effect of biomolecules on nano-based toxicity of Ag NPs was further verified in vivo, using the invertebrate Galleria mellonella as an alternative non-mammalian infection model. We provide the first evidence that biomolecule coronas are not only relevant in mammalian systems but also for nanomaterial designs as future antifungals for human health, biotechnology, and agriculture.

Correction: Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori.

Correction for 'Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori' by Dana Westmeier et al., Nanoscale, 2018, 10, 1453-1463.

Boosting nanotoxicity to combat multidrug-resistant bacteria in pathophysiological environments.

Nanomaterials are promising novel antibiotics, but often ineffective. We found that nanomaterial-bacteria complex formation occurred with various nanomaterials. The bactericidal activity of NMs strongly depends on their physical binding to (multidrug-resistant) bacteria. Nanomaterials' binding and antibiotic effect was reduced by various pathophysiological biomolecule coronas strongly inhibiting their antibiotic effects. We show from analytical to in vitro to in vivo that nanomaterial-based killing could be restored by acidic pH treatments. Here, complex formation of negatively-charged, plasma corona-covered, nanomaterials with bacteria was electrostatically enhanced by reducing bacteria's negative surface charge. Employing in vivo skin infection models, acidic pH-induced complex formation was critical to counteract Staphylococcus aureus infections by silver nanomaterials. We explain why nano-antibiotics show reduced activity and provide a clinically practical solution.

Biomolecule-corona formation confers resistance of bacteria to nanoparticle-induced killing: Implications for the design of improved nanoantibiotics.

Multidrug-resistant bacterial infections are a global health threat. Nanoparticles are thus investigated as novel antibacterial agents for clinical practice, including wound dressings and implants. We report that nanoparticles' bactericidal activity strongly depends on their physical binding to pathogens, including multidrug-resistant primary clinical isolates, such as Staphylococcus aureus, Klebsiella pneumoniae or Enterococcus faecalis. Using controllable nanoparticle models, we found that nanoparticle-pathogen complex formation was enhanced by small nanoparticle size rather than material or charge, and was prevented by 'stealth' modifications. Nanoparticles seem to preferentially bind to Gram-positive pathogens, such as Listeria monocytogenes, S. aureus or Streptococcus pyrogenes, correlating with enhanced antibacterial activity. Bacterial resistance to metal-based nanoparticles was mediated by biomolecule coronas acquired in pathophysiological environments, such as wounds, the lung, or the blood system. Biomolecule corona formation reduced nanoparticles' binding to pathogens, but did not impact nanoparticle dissolution. Our results provide a mechanistic explanation why nano-sized antibiotics may show reduced activity in clinically relevant environments, and may inspire future nanoantibiotic designs with improved and potentially pathogen-specific activity.

Resistance to Nano-Based Antifungals Is Mediated by Biomolecule Coronas.

Fungal infections are a growing global health and agricultural threat, and current chemical antifungals may induce various side-effects. Thus, nanoparticles are investigated as potential novel antifungals. We report that nanoparticles' antifungal activity strongly depends on their binding to fungal spores, focusing on the clinically important fungal pathogen Aspergillus fumigatus as well as common plant pathogens, such as Botrytis cinerea. We show that nanoparticle-spore complex formation was enhanced by the small nanoparticle size rather than the material, shape or charge, and could not be prevented by steric surface modifications. Fungal resistance to metal-based nanoparticles, such as ZnO-, Ag-, or CuO-nanoparticles as well as dissolution-resistant quantum dots, was mediated by biomolecule coronas acquired in pathophysiological and ecological environments, including the lung surfactant, plasma or complex organic matters. Mechanistically, dose-dependent corona-mediated resistance occurred via reducing physical adsorption of nanoparticles to fungal spores. The inhibitory effect of biomolecules on the antifungal activity of Ag-nanoparticles was further verified in vivo, using the invertebrate Galleria mellonella as an A. fumigatus infection model. Our results explain why current nanoantifungals often show low activity in realistic application environments, and will guide nanomaterial designs that maximize functionality and safe translatability as potent antifungals for human health, biotechnology, and agriculture.

Nanoparticle decoration impacts airborne fungal pathobiology.

Airborne fungal pathogens, predominantly Aspergillus fumigatus, can cause severe respiratory tract diseases. Here we show that in environments, fungal spores can already be decorated with nanoparticles. Using representative controlled nanoparticle models, we demonstrate that various nanoparticles, but not microparticles, rapidly and stably associate with spores, without specific functionalization. Nanoparticle-spore complex formation was enhanced by small nanoparticle size rather than by material, charge, or "stealth" modifications and was concentration-dependently reduced by the formation of environmental or physiological biomolecule coronas. Assembly of nanoparticle-spore surface hybrid structures affected their pathobiology, including reduced sensitivity against defensins, uptake into phagocytes, lung cell toxicity, and TLR/cytokine-mediated inflammatory responses. Following infection of mice, nanoparticle-spore complexes were detectable in the lung and less efficiently eliminated by the pulmonary immune defense, thereby enhancing A. fumigatus infections in immunocompromised animals. Collectively, self-assembly of nanoparticle-fungal complexes affects their (patho)biological identity, which may impact human health and ecology.

Nanoparticle binding attenuates the pathobiology of gastric cancer-associated Helicobacter pylori.

Enteric bacteria may cause severe diseases, including gastric cancer-associated Helicobacter pylori. Their infection paths overlap with the oro-gastrointestinal uptake route for nanoparticles, increasingly occurring during environmental or consumer/medical exposure. By comprehensive independent analytical methods, such as live cell fluorescence, electron as well as atomic force microscopy and elemental analysis, we show that a wide array of nanoparticles (NPs) but not microparticles form complexes with H. pylori and enteric pathogens without the need for specific functionalization. The NP-assembly that occurred rapidly was not influenced by variations in physiological temperature, though affected by the NPs' physico-chemical characteristics. Improved binding was observed for small NPs with a negative surface charge, whereas binding could be reduced by surface 'stealth' modifications. Employing human gastric epithelial cells and 3D-organoid models of the stomach, we show that NP-coating did not inhibit H. pylori's cellular attachment. However, even the assembly of non-bactericidal silica NPs attenuated H. pylori infection by reducing CagA phosphorylation, cytoskeletal rearrangement, and IL-8 secretion. Here we demonstrate that NP binding to enteric bacteria may impact their pathobiology which could be further exploited to rationally modulate the (patho)biology of microbes by nanomaterials.

Nanosized food additives impact beneficial and pathogenic bacteria in the human gut: a simulated gastrointestinal study.

Nanotechnology provides the food industry with new ways to modulate various aspects of food. Hence, engineered nanoparticles (NPs) are increasingly added to food and beverage products as functional ingredients. However, the impact of engineered as well as naturally occurring NPs on both commensal and pathogenic microorganisms within the gastrointestinal tract (GI) is not fully understood. Here, well-defined synthetic NPs and bacterial models were used to probe nanoparticle-bacteria interactions, from analytical to in situ to in vitro. NP-bacteria complexation occurred most efficiently for small NPs, independent of their core material or surface charge, but could be reduced by NPs' steric surface modifications. Adsorption to bacteria could also be demonstrated for naturally occurring carbon NPs isolated from beer. Complex formation affected the (patho)biological behavior of both the NPs and bacteria, including their cellular uptake into epithelial cells and phagocytes, pathogenic signaling pathways, and NP-induced cell toxicity. NP-bacteria complex formation was concentration-dependently reduced when the NPs became coated with biomolecule coronas with sequential simulation of first oral uptake and then the GI. However, efficient NP adsorption was restored when the pH was sufficiently low, such as in simulating the conditions of the stomach. Collectively, NP binding to enteric bacteria may impact their (patho)biology, particularly in the stomach. Nanosized-food additives as well as naturally occurring NPs may be exploited to (rationally) shape the microbiome. The information contained in this article should facilitate a "safe by design" strategy for the development and application of engineered NPs as functional foods ingredients.

Synthesis and Characterization of Stimuli-Responsive Star-Like Polypept(o)ides: Introducing Biodegradable PeptoStars.

Star-like polymers are one of the smallest systems in the class of core crosslinked polymeric nanoparticles. This article reports on a versatile, straightforward synthesis of three-arm star-like polypept(o)ide (polysarcosine-block-polylysine) polymers, which are designed to be either stable or degradable at elevated levels of glutathione. Polypept(o)ides are a recently introduced class of polymers combining the stealth-like properties of the polypeptoid polysarcosine with the functionality of polypeptides, thus enabling the synthesis of materials completely based on endogenous amino acids. The star-like homo and block copolymers are synthesized by living nucleophilic ring opening polymerization of the corresponding N-carboxyanhydrides (NCAs) yielding polymeric stars with precise control over the degree of polymerization (Xn = 25, 50, 100), Poisson-like molecular weight distributions, and low dispersities (D = 1.06-1.15). Star-like polypept(o)ides display a hydrodynamic radius of 5 nm (µ2 < 0.05) as determined by dynamic light scattering (DLS). While star-like polysarcosines and polypept(o)ides based on disulfide containing initiators are stable in solution, degradation occurs at 100 × 10-3 m glutathione concentration. The disulfide cleavage yields the respective polymeric arms, which possess Poisson-like molecular weight distributions and low dispersities (D = 1.05-1.12). Initial cellular uptake and toxicity studies reveal that PeptoStars are well tolerated by HeLa, HEK 293, and DC 2.4 cells.

Tuning the Surface of Nanoparticles: Impact of Poly(2-ethyl-2-oxazoline) on Protein Adsorption in Serum and Cellular Uptake.

Due to the adsorption of biomolecules, the control of the biodistribution of nanoparticles is still one of the major challenges of nanomedicine. Poly(2-ethyl-2-oxazoline) (PEtOx) for surface modification of nanoparticles is applied and both protein adsorption and cellular uptake of PEtOxylated nanoparticles versus nanoparticles coated with poly(ethylene glycol) (PEG) and non-coated positively and negatively charged nanoparticles are compared. Therefore, fluorescent poly(organosiloxane) nanoparticles of 15 nm radius are synthesized, which are used as a scaffold for surface modification in a grafting onto approach. With multi-angle dynamic light scattering, asymmetrical flow field-flow fractionation, gel electrophoresis, and liquid chromatography-mass spectrometry, it is demonstrated that protein adsorption on PEtOxylated nanoparticles is extremely low, similar as on PEGylated nanoparticles. Moreover, quantitative microscopy reveals that PEtOxylation significantly reduces the non-specific cellular uptake, particularly by macrophage-like cells. Collectively, studies demonstrate that PEtOx is a very effective alternative to PEG for stealth modification of the surface of nanoparticles.

The concept of bio-corona in modulating the toxicity of engineered nanomaterials (ENM).

Besides the wide use of engineered nanomaterials (ENM) in technical products, their application spectrum in biotechnology and biomedicine is steadily increasing. In complex physiological environments the physico-chemical properties and the behavior of nanoparticles (NPs) are challenging to characterize. Biomolecules rapidly adsorb to the nanomaterial, leading to the formation of the protein/biomolecule corona, which critically affects the nanomaterials' (patho)biological and technical identities. This formation can trigger an immune response and affect nanoparticles' toxicity and targeting capabilities. In this review, we provide a survey of recent findings on the (protein)corona-nanoparticle interaction and discuss how the corona modulates both cytotoxicity and the immune response as well as to improve the efficacy of targeted delivery of nanocarriers.

No king without a crown--impact of the nanomaterial-protein corona on nanobiomedicine.

Besides the wide use of nanomaterials in technical products, their application spectrum in biotechnology and biomedicine is steadily increasing. Whereas the physico-chemical properties and behavior of nanomaterials can be engineered and characterized accurately under idealized conditions, this is no longer the case in complex physiological environments. In biological fluids, proteins rapidly bind to nanomaterials forming the protein corona, critically affecting the nanomaterials' biological identity. As the corona impacts in vitro and/or in vivo nanomaterial applications, we here review the concept of the protein corona and its analytical dissection. We comment on how corona signatures may be linked to effects at the nano-bio interface and conclude how such knowledge is offering novel opportunities for improved nanomedicine.

The protein corona protects against size- and dose-dependent toxicity of amorphous silica nanoparticles.

Besides the lung and skin, the gastrointestinal (GI) tract is one of the main targets for accidental exposure or biomedical applications of nanoparticles (NP). Biological responses to NP, including nanotoxicology, are caused by the interaction of the NP with cellular membranes and/or cellular entry. Here, the physico-chemical characteristics of NP are widely discussed as critical determinants, albeit the exact mechanisms remain to be resolved. Moreover, proteins associate with NP in physiological fluids, forming the protein corona potentially transforming the biological identity of the particle and thus, adding an additional level of complexity for the bio-nano responses. Here, we employed amorphous silica nanoparticles (ASP) and epithelial GI tract Caco-2 cells as a model to study the biological impact of particle size as well as of the protein corona. Caco-2 or mucus-producing HT-29 cells were exposed to thoroughly characterized, negatively charged ASP of different size in the absence or presence of proteins. Comprehensive experimental approaches, such as quantifying cellular metabolic activity, microscopic observation of cell morphology, and high-throughput cell analysis revealed a dose- and time-dependent toxicity primarily upon exposure with ASP30 (Ø = 30 nm). Albeit smaller (ASP20, Ø = 20 nm) or larger particles (ASP100; Ø = 100 nm) showed a similar zeta potential, they both displayed only low toxicity. Importantly, the adverse effects triggered by ASP30/ASP30L were significantly ameliorated upon formation of the protein corona, which we found was efficiently established on all ASP studied. As a potential explanation, corona formation reduced ASP30 cellular uptake, which was however not significantly affected by ASP surface charge in our model. Collectively, our study uncovers an impact of ASP size as well as of the protein corona on cellular toxicity, which might be relevant for processes at the nano-bio interface in general.