Phage Display Panning on Silica: Optimization of Elution Conditions for Selection of Strong Binders.

Phage display panning is a powerful tool to select strong peptide binders to a given target, and when applied to inorganic materials (e.g., silica) as a target, it provides information on binding events and molecular recognition at the peptide-mineral interface. The panning process has limitations with the phage chemical elution being affected by bias toward positively charged binders, resulting in the potential loss of information on binder diversity; the presence of fast growing phages with an intrinsic growth advantage; and the presence of false positives from target unrelated peptides. To overcome some of these limitations, we developed a panning approach based on the sequential use of different eluents (Gly-HCl, pH-2.2; MgCl2, pH-6.1; and TEA, pH-11.0), or pH conditions (Gly-HCl 2.2 < pH < 11.0) that allows the identification of a diverse and comprehensive pool of strong binders. We have assessed and tested the authenticity of the identified silica binders via a complementary experimental (in vivo phage recovery rates and TEM imaging) and bioinformatics approach. We provide experimental evidence of the nonspecificity of the Gly-HCl eluent as typically used. Using a fluorimetric assay, we investigate in vitro binding of two peptides that differ by pI-S4 (HYIDFRW, pI 7.80) and S5 (YSLKQYQ, pI 9.44)─modified at the C terminal with an amide group to simulate net charges in the phage display system, confirming the vital role of electrostatic interactions as driving binding forces in the phage panning process. The presented optimized phage panning approach provides an opportunity to match known surface interactions at play with suitable elution conditions; to select only sequences relevant to a particular interfacial system. The approach has the potential to open up avenues to design interfacial systems to advance our understanding of peptide-assisted mineral growth, among other possibilities.

Nanoarchitectonics of Bactericidal Coatings Based on CaCO3-Nanosilver Hybrids.

Antimicrobial coatings provide protection against microbes colonization on surfaces. This can prevent the stabilization and proliferation of microorganisms. The ever-increasing levels of microbial resistance to antimicrobials are urging the development of alternative types of compounds that are potent across broad spectra of microorganisms and target different pathways. This will help to slow down the development of resistance and ideally halt it. The development of composite antimicrobial coatings (CACs) that can host and protect various antimicrobial agents and release them on demand is an approach to address this urgent need. In this work, new CACs based on microsized hybrids of calcium carbonate (CaCO3) and silver nanoparticles (AgNPs) were designed using a drop-casting technique. Polyvinylpyrrolidone and mucin were used as additives. The CaCO3/AgNPs hybrids contributed to endowing colloidal stability to the AgNPs and controlling their release, thereby ensuring the antibacterial activity of the coatings. Moreover, the additives PVP and mucin served as a matrix to (i) control the distribution of the hybrids, (ii) ensure mechanical integrity, and (iii) prevent the undesired release of AgNPs. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FTIR) techniques were used to characterize the 15 µm thick CAC. The antibacterial activity was determined against Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and Pseudomonas aeruginosa, three bacteria responsible for many healthcare infections. Antibacterial performance of the hybrids was demonstrated at concentrations between 15 and 30 µg/cm2. Unloaded CaCO3 also presented bactericidal properties against MRSA. In vitro cytotoxicity tests demonstrated that the hybrids at bactericidal concentrations did not affect human dermal fibroblasts and human mesenchymal stem cell viability. In conclusion, this work presents a simple approach for the design and testing of advanced multicomponent and functional antimicrobial coatings that can protect active agents and release them on demand.

Vaterite vectors for the protection, storage and release of silver nanoparticles.

Silver nanoparticles (AgNPs) have found widespread commercial applications due to their unique physical and chemical properties. However, their relatively poor stability remains a main problem. An ideal way to improve the stability of AgNPs is not only to endow colloidal stability to individual nanoparticles but also to protect them from environmental factors that induce their agglomeration, like variation of ionic strength and pH, presence of macromolecules, etc. Mesoporous calcium carbonate vaterite crystals (CaCO3 vaterite) have recently attracted significant attention as inexpensive and biocompatible carriers for the encapsulation and controlled release of both drugs and nanoparticles. This work aimed to develop an approach to load AgNPs into CaCO3 vaterite without affecting their properties. We focused on improving the colloidal stability of AgNPs by using different capping agents, and understanding the mechanism behind AgNPs loading and release from CaCO3 crystals. Various methods were applied to study the AgNPs and CaCO3 crystals loaded with AgNPs (CaCO3/AgNPs hybrids), such as scanning and transmission electron microscopy, X-ray diffraction, infrared and mass spectrometry. The results demonstrated that polyvinylpyrrolidone and positively charged diethylaminoethyl-dextran can effectively keep the colloidal stability of AgNPs during co-precipitation with CaCO3 crystals. CaCO3/AgNPs hybrids composed of up to 4 % weight content of nanoparticles were produced, with the loading mechanism being well-described by the Langmuir adsorption model. In vitro release studies demonstrated a burst release of stable AgNPs at pH 5.0 and a sustained release at pH 7.5 and 9.0. The antibacterial studies showed that these hybrids are effective against Escherichia coli, methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa, three important bacteria responsible for nosocomial infections. The developed approach opens a new way to stabilise, protect, store and release AgNPs in a controlled manner for their use as antimicrobial agents.

Interactions between Metal Oxides and Biomolecules: from Fundamental Understanding to Applications.

Metallo-oxide (MO)-based bioinorganic nanocomposites promise unique structures, physicochemical properties, and novel biochemical functionalities, and within the past decade, investment in research on materials such as ZnO, TiO2, SiO2, and GeO2 has significantly increased. Besides traditional approaches, the synthesis, shaping, structural patterning, and postprocessing chemical functionalization of the materials surface is inspired by strategies which mimic processes in nature. Would such materials deliver new technologies? Answering this question requires the merging of historical knowledge and current research from different fields of science. Practically, we need an effective defragmentation of the research area. From our perspective, the superficial accounting of material properties, chemistry of the surfaces, and the behavior of biomolecules next to such surfaces is a problem. This is particularly of concern when we wish to bridge between technologies in vitro and biotechnologies in vivo. Further, besides the potential practical technological efficiency and advantages such materials might exhibit, we have to consider the wider long-term implications of material stability and toxicity. In this contribution, we present a critical review of recent advances in the chemistry and engineering of MO-based biocomposites, highlighting the role of interactions at the interface and the techniques by which these can be studied. At the end of the article, we outline the challenges which hamper progress in research and extrapolate to developing and promising directions including additive manufacturing and synthetic biology that could benefit from molecular level understanding of interactions occurring between inanimate (abiotic) and living (biotic) materials.

Aqueous Peptide-TiO2 Interfaces: Isoenergetic Binding via Either Entropically or Enthalpically Driven Mechanisms.

A major barrier to the systematic improvement of biomimetic peptide-mediated strategies for the controlled growth of inorganic nanomaterials in environmentally benign conditions lies in the lack of clear conceptual connections between the sequence of the peptide and its surface binding affinity, with binding being facilitated by noncovalent interactions. Peptide conformation, both in the adsorbed and in the nonadsorbed state, is the key relationship that connects peptide-materials binding with peptide sequence. Here, we combine experimental peptide-titania binding characterization with state-of-the-art conformational sampling via molecular simulations to elucidate these structure/binding relationships for two very different titania-binding peptide sequences. The two sequences (Ti-1, QPYLFATDSLIK; Ti-2, GHTHYHAVRTQT) differ in their overall hydropathy, yet via quartz-crystal microbalance measurements and predictions from molecular simulations, we show these sequences both support very similar, strong titania-binding affinities. Our molecular simulations reveal that the two sequences exhibit profoundly different modes of surface binding, with Ti-1 acting as an entropically driven binder while Ti-2 behaves as an enthalpically driven binder. The integrated approach presented here provides a rational basis for peptide sequence engineering to achieve the in situ growth and organization of titania nanostructures in aqueous media and for the design of sequences suitable for a range of technological applications that involve the interface between titania and biomolecules.

Correction: Preparation of hexagonal GeO2 particles with particle size and crystallinity controlled by peptides, silk and silk-peptide chimeras.

Correction for 'Preparation of hexagonal GeO2 particles with particle size and crystallinity controlled by peptides, silk and silk-peptide chimeras' by Estefania Boix et al., Dalton Trans., 2014, 43, 16902-16910.

Preparation of hexagonal GeO2 particles with particle size and crystallinity controlled by peptides, silk and silk-peptide chimeras.

We demonstrate the use of silk based proteins to control the particle/crystallite size during GeO2 formation, using a bio-mimetic approach at circumneutral pH and ambient temperature. Multicrystalline GeO2 was prepared from germanium tetraethoxide (TEOG) in the presence of different silk-based proteins: Bombyx mori silk (native silk) and two chimeric proteins prepared by linking a germania binding peptide (Ge28: HATGTHGLSLSH) with Bombyx mori silk via chemical coupling at different peptide loadings (silk-Ge28 10% and silk-Ge28 50%). The mineralisation activity of the silk-based proteins was compared with that of peptide Ge28 as a control system. GeO2 mineralisation was investigated in water and in citric acid/bis-tris propane buffer at pH 6. Morphology, particle size, crystallinity, water and organic content of the materials obtained were analysed to study the effect of added biomolecules and mineralisation environment on material properties. In the presence of silk additives well-defined cube-shape hybrid materials composed of hexagonal germania and up to ca. 5 wt% organic content were obtained. The cubic particles ranged from 0.4 to 1.4 µm in size and were composed of crystalline domains in the range 35-106 nm depending on the additive used and synthesis conditions. The organic material incorporated in the mineral did not appear to affect the unit cell dimensions. The silk and chimeric proteins in water promote material formation and crystal growth, possibly via an effective ion-channelling mechanism, however further studies are needed to assert to what extent the presence of the silk impacts on nucleation and growth stages. The germania binding peptide alone did not have any significant effect on reaction rate, yield or the material's properties compared to the blank. Interestingly, the peptide content in the silk chimeras tested did not affect mineralisation. The presence of buffer inhibited mineral condensation rate and yield. The use of silk-based biomolecules allows control of crystallite/particle size of hybrid materials opening up opportunities for bio-inspired approaches to be applied for the synthesis of functional germania based devices and materials.

Interactions at the silica-peptide interface: the influence of particle size and surface functionality.

The variety of interactions that can occur at the silica/aqueous interface makes silica nanoparticles (SiNPs) attractive materials for technological applications. Despite their importance, interfacial interactions are not fully understood. In this contribution, we investigate the effect of (1) particle size and (2) surface functionalization on the adsorption of small biomolecular binders on SiNPs. Small silica binding peptides with different properties (charge, pI, and amino acid composition) were used as binders, while a range of fully characterized SiNPs of diameters ranging between 28 and 500 nm (pristine silica) and SiNPs of ca. 500 nm functionalized with cationic 3-aminopropyl groups and hydrophobic methyl groups was used as binding substrates. Adsorption and binding affinity were investigated by a fluorimetric assay at pH 7.4. A detailed characterization of the surface chemistry of the particles showed that the extent of surface functionalization on modified silica was well below monolayer coverage [by X-ray photoelectron spectroscopy (XPS), ca. 2 and 18 atomic % for the amino- and methyl-modified surfaces, respectively]. Although peptide binding is generally moderated by the physicochemical characteristics of the adsorbing peptide, the introduction of such a small degree of functionality onto silica particles was sufficient to produce drastic changes in adsorption at the silica/aqueous interface. In addition, an increase in peptide adsorption with an increasing particle size, independent of the nature and properties of the peptide, was observed. This particle size effect is attributed to a shift of the dominant binding mechanism toward electrostatic interactions on larger SiNPs. The data presented demonstrate that particle size and surface functionality are both parameters that can substantially influence (bio)molecule uptake via modulation or selection of specific binding modes at the silica/peptide interface. These results are applicable to the design and development of interfaces with specific adsorption/affinity response for biomedical applications, where uptake is important, such as drug delivery. Further, they provide important insights on how peptide affinity and selection during biopanning can be determined by small changes in surface chemistry of the surface of a target that can, in some instances, be associated with the presence of impurities.

Titania binding peptides as templates in the biomimetic synthesis of stable titania nanosols: insight into the role of buffers in peptide-mediated mineralization.

In this Article, we report the unusual behavior of two peptides (Ti-1 (QPYLFATDSLIK) and Ti-2 (GHTHYHAVRTQT)) with high affinity for titania that efficiently promote titania mineralization from an aqueous titanium bisammonium lactatodihydroxide (TiBALDH) solution, yielding small (ca. 4 nm) titania nanoparticles. As a result, we were able to produce for the first time using a biomimetic approach highly stable sub-10-nm titania sols. Both sequences show a high titania mineralization activity per unit peptide concentration and a capacity to control particle size and stabilize nanoparticles through specific surface interactions. We also show that phosphate ions disrupt the controlled particle formation and stabilization achieved in the presence of the two peptides. The products obtained from phosphate buffered solutions are titanium-containing materials (not pure oxide) with poor morphological control similar to those previously reported by others. Our results provide important insights into understanding the mechanism of titania mineralization in a range of different aqueous media (water, Tris, and phosphate buffer).

Peptide adsorption on silica nanoparticles: evidence of hydrophobic interactions.

Molecular recognition and interactions at the interface between biomolecules and inorganic materials determine important phenomena such as protein adsorption, cell adhesion to biomaterials, or the selective response of biosensors. Events occurring at the biomolecule-inorganic interface, despite their importance, are still poorly understood, thus limiting control of interfacial properties and response. In this contribution, using well-characterized silica nanoparticles and a series of peptides having heterogeneous physicochemical properties (S1: KLPGWSG, S2: AFILPTG, and S3: LDHSLHS) identified from biopanning against the same particles, we identify the driving forces that govern peptide-silica binding. Binding isotherms obtained by fluorimetric assay under different pH conditions allowed us to demonstrate the impact of binding environment (pH) on adsorption behavior of a given peptide-surface silica nanoparticle. Our experimental data suggest a multistep adsorption mechanism leading to the formation of multilayers on silica, in which the prevailing interactions (i.e., electrostatic or hydrophobic/hydrogen bonding) and their relative contribution to the binding event are governed by the identity of the peptide itself, the substrate's surface functionality (hydrophilic or hydrophobic), and the peptide bulk concentration and solution bulk pH. Our studies show how it is possible to modulate peptide uptake on silica, or in fact on any particle, by changing either the surface properties or, more simply, the binding environment. In addition, the data reveal an intrinsic bias toward positively charged sequences in the elution conditions used in the biopanning protocol with much information about strong binder sequence diversity being lost during panning.

Novel one step hydrothermal synthesis of TiO2/WO3 nanocomposites with enhanced photocatalytic activity.

A novel and simple one step hydrothermal process is used to prepare TiO(2)/WO(3) nanocomposites, in which WO(3) and TiO(2) are present in hexagonal and anatase crystalline forms, respectively, and have enhanced photocatalytic activity towards trichloroethylene degradation in the gas phase.