Fabrication of viable cyborg cells with cyclodextrin functionality

We describe two alternative methods for surface functionalisation of Saccharomyces cerevisiae cells with cyclodextrin molecules without affecting the cell viability. The first strategy involved using epichlorohydrin as a cross-linking agent which binds covalently the cyclodextrin to the glycoproteins on the cell wall. The second strategy of interfacing of the cells with CD involved polyelectrolyte mediated deposition of cyclodextrin sulphate on the cell surface. We used the formation of host-guest inclusion complexes of a dye with the grafted cyclodextrins to estimate the average number of CD molecules grafted per cell which can reach up to hundreds of millions of CD molecules. This indicates more than one monolayer of CDs on the cell surface within the surface layer surrounding the yeast cell membrane. Fluorescein diacetate was used to check the viability of the cells after functionalisation. Living cells functionalised with CDs may find many potential applications as they can be loaded with drugs, immunosuppressants and other molecules forming inclusion complexes with their cyclodextrin interface. Therefore, we foresee such cells being used as novel selective biosorbents in polluted waters, whole cell biosensors, drug delivery, cell therapy and cell implant applications

Cyclodextrin stabilised emulsions and cyclodextrinosomes

We report the preparation of o/w emulsions stabilised by microcrystals of cyclodextrin-oil inclusion complexes. The inclusion complexes are formed by threading cyclodextrins from the aqueous phase on n-tetradecane or silicone oil molecules from the emulsion drop surface which grow further into microrods and microplatelets depending on the type of cyclodextrin (CD) used. These microcrystals remain attached on the surface of the emulsion drops and form densely packed layers which resemble Pickering emulsions. The novelty of this emulsion stabilisation mechanism is that molecularly dissolved cyclodextrin from the continuous aqueous phase is assembled into colloid particles directly onto the emulsion drop surface, i.e. molecular adsorption leads to effective Pickering stabilisation. The β-CD stabilised tetradecane-in-water emulsions were so stable that we used this system as a template for preparation of cyclodextrinosomes. These structures were produced solely through formation of cyclodextrin-oil inclusion complexes and their assembly into a crystalline phase on the drop surface retained its stability after the removal of the core oil. The structures of CD-stabilised tetradecane-in-water emulsions were characterised using optical microscopy, fluorescence microscopy, cross-polarised light microscopy and WETSEM while the cyclodextrinosomes were characterised by SEM. We also report the preparation of CD-stabilised emulsions with a range of other oils, including tricaprylin, silicone oil, isopropyl myristate and sunflower oil. We studied the effect of the salt concentration in the aqueous phase, the type of CD and the oil volume fraction on the type of emulsion formed. The CD-stabilised emulsions can be applied in a range of surfactant-free formulations with possible applications in cosmetics, home and personal care. Cyclodextrinosomes could find applications in pharmaceutical formulations as microencapsulation and drug delivery vehicles. © 2013 the Owner Societies

Self-assembly of cyclodextrins at the oil-water interface: surfactant-free emulsions and cyclodextrinosomes

We explored the self-assembly of cyclodextrins (CDs) at the oil-water interface through the formation of inclusion complexes (ICs) with the oil and further assemble into microcrystals at the oil-water interface[1-4]. We demonstrate the spontaneous formation of a dense layer of adsorbed CD-tetradecane IC microcrystals at the tetradecane-water interface whose morphology and size are dependent on the type of CD and oil used. At large oil volume fractions, this phenomenon led to the formation of a Pickering type of oil-in-water emulsion stabilised by adsorbed CD-oil microcrystals while at low oil volume fractions it completely solubilises the oil in the form of IC microcrystals. We also report the preparation of o/w emulsions stabilised by microcrystals of cyclodextrin-oil inclusion complexes. The inclusion complexes are formed by threading cyclodextrins from the aqueous phase on n-tetradecane or silicone oil molecules from the emulsion drop surface which grow further into microrods and micro-platelets depending on the type of cyclodextrin. These microcrystals remain attached at the surface of the emulsion drops and form densely packed layers. The novelty in this emulsion stabilisation mechanism is that molecularly dissolved cyclodextrin from the continuous aqueous phase is assembled into colloid particles directly onto the emulsion drop surface, i.e. molecular adsorption leads to effective Pickering stabilisation. The β-CD stabilised tetradecane-in-water emulsions were so stable that we used them as templates for preparation of cyclodextrinosomes after the removal of the core oil. We also report the preparation of CD-stabilized emulsions with a range of other oils and studied the effect of the salt concentration in the aqueous phase, the type of CD and the oil volume fraction on the type of emulsion formed. The CD-stabilized emulsions and cyclodextrinosomes can find applications in a range of surfactant-free formulations in cosmetics, home and personal care, and in pharmaceutical formulations as drug delivery vehicles. Please click Additional Files below to see the full abstract

Preparation and attachment of liquid-infused porous supra-particles to liquid interfaces

© 2016 The Royal Society of Chemistry. We prepared model porous composite supra-particles and investigated the effect of the initial infused fluid phase on their attachment at the liquid-fluid interface. We used a simple method for fabrication of millimetre-sized spherical porous supra-particles from much smaller monodisperse latex microparticles as building blocks by evaporation of a polystyrene sulphate latex suspension on a hot super-hydrophobic surface. We annealed the dried supra-particles at the polymer's glass transition temperature to fuse partially their latex particle building blocks. Spherical porous supra-particles were produced above 40 wt% initial concentration of the latex particles in the suspension, which had a rough surface, with a porous and amorphous structure. We controlled the supra-particle size by varying the initial volume of the latex suspension drop, the latex particle concentration and the drop evaporation temperature. This preparation technique allowed limited control over the porosity of the supra-particles by varying the initial concentration of the latex particle suspension, the rate of evaporation and the annealing temperature. We characterised the surface morphology and the inner structure of supra-particles by SEM imaging. We report for the first time results of an MRI study of supra-particles attached to an air-water or an oil-water interface, which indicated that only the surface layer of the building block particles attaches to the liquid interface while the pore fluid was not displaced by the outer fluid. We observed that supra-particles infused with water had different wettability and attachment positions at the oil-water interface compared with the same particles infused with oil. Similarly, the infusion of the porous supra-particles with water led to a different attachment at the air-water interface compared to the attachment of the same supra-particle when dry. The fundamental importance of this result is that the porous particles (or colloid particle agglomerates) may give an oil-in-water or water-in-oil Pickering emulsion depending on whether they are initially impregnated with oil or water. The results of this study are relevant for particle-stabilised emulsions and foams in a range of pharmaceutical, food and cosmetic formulations as well as ore flotation

Attachment of composite porous supra-particles to air–water and oil–water interfaces: theory and experiment

We developed and tested a theoretical model for the attachment of fluid-infused porous supra-particles to a fluid–liquid interface. We considered the wetting behaviour of agglomerated clusters of particles, typical of powdered materials dispersed in a liquid, as well as of the adsorption of liquid-infused colloidosomes at the liquid–fluid interface. The free energy of attachment of a composite spherical porous supra-particle made from much smaller aggregated spherical particles to the oil–water interface was calculated. Two cases were considered: (i) a water-filled porous supra-particle adsorbed at the oil–water interface from the water phase, and, (ii) an oil-filled porous supra-particle adsorbed at the oil–water interface from the oil-phase. We derived equations relating the three-phase contact angle of the smaller “building block” particles and the contact angle of the liquid-infused porous supra-particles. The theory predicts that the porous supra-particle contact angle attached at the liquid interface strongly depends on the type of fluid infused in the particle pores and the fluid phase from which it approaches the liquid interface. We tested the theory by using millimetre-sized porous supra-particles fabricated by evaporation of droplets of polystyrene latex suspension on a pre-heated super-hydrophobic surface, followed by thermal annealing at the glass transition temperature. Such porous particles were initially infused with water or oil and approached to the oil–water interface from the infusing phase. The experiment showed that when attaching at the hexadecane–water interface, the porous supra-particles behaved as hydrophilic when they were pre-filled with water and hydrophobic when they were pre-filled with hexadecane. The results agree with the theoretically predicted contact angles for the porous composite supra-particles based on the values of the contact angles of their building block latex particles measured with the Gel Trapping Technique. The experimental data for the attachment of porous supra particles to the air–water interface from both air and water also agree with the theoretical model. This study gives important insights about how porous particles and particle aggregates attach to the oil–water interface in Pickering emulsions and the air–water surface in particle-stabilised aqueous foams relevant in ore flotation and a range of cosmetic, pharmaceutical, food, home and personal care formulations

Adsorption of carboxylic modified latex particles at liquid interfaces studied by the gel trapping technique

We have studied how carboxylic modified latex (CML) microparticles adsorb at liquid surfaces and the preferred type of emulsion they can stabilise depending on the particle size and the surface density of carboxylic groups. We measured the particle contact angle by using the gel trapping technique (GTT) for CML particles adsorbed at air–water and oil–water interfaces. Using this method we obtained scanning electron microscopy (SEM) micrographs of polydimethylsiloxane (PDMS) replicas of the liquid interface with the particles, where the PDMS replicates the non-polar phase and measured the particle contact angle. We discovered that the particle wettability correlates well with the surface density of the carboxylic groups but is not very sensitive to the presence of electrolyte in the aqueous phase and the value of the particle zeta potential. We demonstrated that CML microparticles with a high surface density of COOH groups stabilise oil-in-water (O/W) emulsions while those with the lowest coverage of COOH groups favour the formation of water-in-oil (W/O) emulsions. We found that this corresponds to a change of the CML particle contact angle from lower than 90° to higher than 90° upon decrease of the surface density of COOH groups. The findings confirm that the surface density of polar groups has a much bigger effect on the particle wettability and the preferred emulsion than the particle surface charge and zeta potential. Our results on the type of stabilised Pickering emulsion agree with other experimental studies with different particle materials. We propose an alternative explanation for the link between the particle contact angle and the type of stabilised Pickering emulsion

Nanotoxicity of polyelectrolyte-functionalized titania nanoparticles towards microalgae and yeast: Role of the particle concentration, size and surface charge

We studied the nanotoxicity of titania nanoparticles (TiO₂NPs) of various hydrodynamic diameters and crystallite sizes towards C. reinhardtii microalgae and S. cerevisiae (yeast) upon illumination with UV and visible light. The cell viability was assessed for a range of nanoparticle concentrations and incubation times. We found that bare TiO₂NPs affect the C. reinhardtii cell viability at much lower particle concentrations than for yeast. We observed an increase of the TiO₂NPs toxicity upon illumination with UV light compared with that in dark conditions due to the oxidative stress of the produced reactive oxygen species. We also found an increased TiO₂NPs nanotoxicity upon illumination with visible light which indicates that they may also interfere with the microalgae's photosynthetic system leading to decreased chlorophyll content upon exposure to TiO₂NPs. The results indicate that the larger the hydrodynamic diameter of the TiO₂NPs the lower is their nanotoxicity, with anatase TiO₂NPs generally being more toxic than rutile TiO₂NPs. We also prepared a range of polyelectrolyte-coated TiO₂NPs using a layer by-layer method and studied their nanotoxicity towards yeast and microalgae. We found that the toxicity of the coated TiO₂NPs changes with their surface charge. TiO₂NPs coated with cationic polyelectrolyte as an outer layer exhibit much higher nanotoxicity than the ones with an outer layer of anionic polyelectrolyte. TEM images of sectioned microalgae and yeast cells exposed to different polyelectrolyte-coated TiO₂NPs confirmed the formation of a significant build-up of nanoparticles on the cell surface for bare and cationic polyelectrolyte-coated TiO₂NPs. The effect comes from the increased adhesion of cationic nanoparticles to the cell walls. Significantly, coating the TiO₂NPs with anionic polyelectrolyte as an outer layer led to a reduced adhesion and much lower nanotoxicity due to electrostatic repulsion with the cell walls. This suggest a new way of making cationic TiO₂NPs safer for use in different formulations by pre-coating them with anionic polyelectrolytes. The results of this study give important insights into the various factors controlling the nanotoxicity of TiO₂NPs

Cell shape recognition by colloidal cell imprints: Energy of the cell-imprint interaction

The results presented in this study are aimed at the theoretical estimate of the interactions between a spherical microbial cell and the colloidal cell imprints in terms of the Derjaguin, Landau, Vervey, and Overbeek (DLVO) surface forces. We adapted the Derjaguin approximation to take into account the geometry factor in the colloidal interaction between a spherical target particle and a hemispherical shell at two different orientations with respect to each other. We took into account only classical DLVO surface forces, i.e., the van der Waals and the electric double layer forces, in the interaction of a spherical target cell and a hemispherical shell as a function of their size ratio, mutual orientation, distance between their surfaces, their respective surface potentials, and the ionic strength of the aqueous solution. We found that the calculated interaction energies are several orders higher when match and recognition between the target cell and the target cell imprint is achieved. Our analysis revealed that the recognition effect of the hemispherical shell towards the target microsphere comes from the greatly increased surface contact area when a full match of their size and shape is produced. When the interaction between the surfaces of the hemishell and the target cell is attractive, the recognition greatly amplifies the attraction and this increases the likelihood of them to bind strongly. However, if the surface interaction between the cell and the imprint is repulsive, the shape and size match makes this interaction even more repulsive and thus decreases the likelihood of binding. These results show that the surface chemistry of the target cells and their colloidal imprints is very important in controlling the outcome of the interaction, while the shape recognition only amplifies the interaction. In the case of nonmonotonous surface-to-surface interaction we discovered some interesting interplay between the effects of shape match and surface chemistry which is discussed in the paper. The results from this study establish the theoretical basis of cell shape recognition by colloidal cell imprints which, combined with cell killing strategies, could lead to an alternative class of cell shape selective antimicrobials, antiviral, and potentially anticancer therapies

An ultra melt-resistant hydrogel from food grade carbohydrates

© 2017 The Royal Society of Chemistry. We report a binary hydrogel system made from two food grade biopolymers, agar and methylcellulose (agar-MC), which does not require addition of salt for gelation to occur and has very unusual rheological and thermal properties. It is found that the storage modulus of the agar-MC hydrogel far exceeds those of hydrogels from the individual components. In addition, the agar-MC hydrogel has enhanced mechanical properties over the temperature range 25-85 °C and a maximum storage modulus at 55 °C when the concentration of methylcellulose was 0.75% w/v or higher. This is explained by a sol-gel phase transition of the methylcellulose upon heating as supported by differential scanning calorimetry (DSC) measurements. Above the melting point of agar, the storage modulus of agar-MC hydrogel decreases but is still an elastic hydrogel with mechanical properties dominated by the MC gelation. By varying the mixing ratio of the two polymers, agar and MC, it was possible to engineer a food grade hydrogel of controlled mechanical properties and thermal response. SEM imaging of flash-frozen and freeze-dried samples revealed that the agar-MC hydrogel contains two different types of heterogeneous regions of distinct microstructures. The latter was also tested for its stability towards heat treatment which showed that upon heating to temperatures above 120 °C its structure was retained without melting. The produced highly thermally stable hydrogel shows melt resistance which may find application in high temperature food processing and materials templating

High-throughput fabrication of hepatic cell clusteroids with enhanced growth and functionality for tissue engineering applications

Culturing of cells as three-dimensional (3D) clusters can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome and expensive than two-dimensional (2D) cultures. Current methods for the preparation of tissue spheroids require complex materials, involve tedious facilities and are generally not scalable. We report a novel inexpensive and up-scalable method for the preparation of large quantities of viable cell clusters (clusteroids) of hepatocytes (Hep-G2). The method has a high throughput potential and is based on an aqueous two-phase system (ATPS) of stable water-in-water (w/w) Pickering emulsions, formed of dextran (DEX) drops and poly ethylene oxide (PEO) continuous phase stabilized with whey protein particles. This system enabled the rapid fabrication of cell clusteroids from Hep-G2 cells. Here, the interfacial tension of the aqueous phase in the emulsion droplets, where the cells partition preferentially, is used to wrap the cells in separate compartments, and then the droplets are shrank by changing the balance of ATPS, thus rapidly driving the cells from larger and loosely packed DEX drops to mostly spherical clusters. Cell-Cell adhesion was strongly promoted within the w/w Pickering emulsion droplets which helped the formation of the 3D clusteroids. These were collected after subsequent dilution of the emulsion with culture media. The collected hepatic clusteroids were incorporated into an alginate hydrogel in media to study their proliferation and the cell function compared with individual cells under the same conditions. Our results confirmed that urea and albumin production, which are both linked to hepatocyte cell function, was strongly reinforced in the clusteroid based tissues compared to the one formulated with individual cells. This methodology could potentially extend the w/w emulsion cell clustering platform in tissue generation and preparation of large quantities of organoids for drug tests and replacement of animal models