Synthesis, Characterization, and Pickering Emulsifier Performance of Anisotropic Cross-Linked Block Copolymer Worms: Effect of Aspect Ratio on Emulsion Stability in the Presence of Surfactant.

Reversible addition-fragmentation chain transfer (RAFT) aqueous dispersion polymerization is used to prepare epoxy-functional PGMA-P(HPMA- stat-GlyMA) diblock copolymer worms, where GMA, HPMA, and GlyMA denote glycerol monomethacrylate, 2-hydroxypropyl methacrylate, and glycidyl methacrylate, respectively. The epoxy groups on the GlyMA residues were ring-opened using 3-aminopropyltriethoxysilane (APTES) in order to cross-link the worm cores via a series of hydrolysis-condensation reactions. Importantly, the worm aspect ratio can be adjusted depending on the precise conditions selected for covalent stabilization. Relatively long cross-linked worms are obtained by reaction with APTES at 20 °C, whereas much shorter worms with essentially the same copolymer composition are formed by cooling the linear worms from 20 to 4 °C prior to APTES addition. Small-angle X-ray scattering (SAXS) studies confirmed that the mean aspect ratio for the long worms is approximately eight times greater than that for the short worms. Aqueous electrophoresis studies indicated that both types of cross-linked worms acquired weak cationic surface charge at low pH as a result of protonation of APTES-derived secondary amine groups within the nanoparticle cores. These cross-linked worms were evaluated as emulsifiers for the stabilization of n-dodecane-in-water emulsions via high-shear homogenization at 20 °C and pH 8. Increasing the copolymer concentration led to a reduction in mean droplet diameter, indicating that APTES cross-linking was sufficient to allow the nanoparticles to adsorb intact at the oil/water interface and hence produce genuine Pickering emulsions, rather than undergo in situ dissociation to form surface-active diblock copolymer chains. In surfactant challenge studies, the relatively long worms required a thirty-fold higher concentration of a nonionic surfactant (Tween 80) to be displaced from the n-dodecane-water interface compared to the short worms. This suggests that the former nanoparticles are much more strongly adsorbed than the latter, indicating that significantly greater Pickering emulsion stability can be achieved by using highly anisotropic worms. In contrast, colloidosomes prepared by reacting the hydroxyl-functional adsorbed worms with an oil-soluble polymeric diisocyanate remained intact when exposed to high concentrations of Tween 80.

Long-Term Stability of n-Alkane-in-Water Pickering Nanoemulsions: Effect of Aqueous Solubility of Droplet Phase on Ostwald Ripening.

High-pressure microfluidization is used to prepare a series of oil-in-water Pickering nanoemulsions using sterically-stabilized diblock copolymer nanoparticles as the Pickering emulsifier. The droplet phase comprised either n-octane, n-decane, n-dodecane, or n-tetradecane. This series of oils enabled the effect of aqueous solubility on Ostwald ripening to be studied, which is the primary instability mechanism for such nanoemulsions. Analytical centrifugation (LUMiSizer instrument) was used to evaluate the long-term stability of these Pickering nanoemulsions over time scales of weeks/months. This technique enables convenient quantification of the fraction of growing oil droplets and confirmed that using n-octane (aqueous solubility = 0.66 mg dm-3 at 20 °C) leads to instability even over relatively short time periods. However, using n-tetradecane (aqueous solubility = 0.386 µg dm-3 at 20 °C) leads to significantly improved long-term stability with respect to Ostwald ripening, with all droplets remaining below 1 µm diameter after 6 weeks storage at 20 °C. In the case of n-dodecane, the long-term stability of these new copolymer-stabilized Pickering nanoemulsions is significantly better than the silica-stabilized Pickering nanoemulsions reported in the literature by Persson et al. ( Colloids Surf., A, 2014, 459, 48-57). This is attributed to a much greater interfacial yield stress for the former system, as recently described in the literature (see P. J. Betramo et al. Proc. Natl. Acad. Sci. U.S.A., 2017, 114, 10373-10378).

Bespoke Diblock Copolymer Nanoparticles Enable the Production of Relatively Stable Oil-in-Water Pickering Nanoemulsions.

Sterically stabilized diblock copolymer nanoparticles with an intensity-average diameter of 25 nm are prepared in the form of a concentrated aqueous dispersion using polymerization-induced self-assembly (PISA). The addition of n-dodecane followed by high-shear homogenization produces n-dodecane-in-water Pickering macroemulsions of 22-46 µm diameter. If the nanoparticles are present in sufficient excess, then subsequent processing using a high-pressure microfluidizer leads to the formation of Pickering nanoemulsions with a mean oil droplet diameter below 200 nm. The size of these Pickering nanoemulsions can be tuned by systematically varying the nanoparticle concentration, applied pressure, number of passes, and oil volume fraction. High-internal-phase emulsions can also be achieved by increasing the n-dodecane volume fraction up to 0.80. TEM studies of (dried) n-dodecane droplets confirm the presence of intact nanoparticles and suggest a relatively high surface coverage, which is consistent with model packing calculations based on radius ratios. Such Pickering nanoemulsions proved to be surprisingly stable with respect to Ostwald ripening, with no significant change in the mean DLS droplet diameter after storage for approximately 4 months at 20 °C.

Non-aqueous Isorefractive Pickering Emulsions.

Non-aqueous Pickering emulsions of 16-240 µm diameter have been prepared using diblock copolymer worms with ethylene glycol as the droplet phase and an n-alkane as the continuous phase. Initial studies using n-dodecane resulted in stable emulsions that were significantly less turbid than conventional water-in-oil emulsions. This is attributed to the rather similar refractive indices of the latter two phases. By utilizing n-tetradecane as an alternative oil that almost precisely matches the refractive index of ethylene glycol, almost isorefractive ethylene glycol-in-n-tetradecane Pickering emulsions can be prepared. The droplet diameter and transparency of such emulsions can be systematically varied by adjusting the worm copolymer concentration.

Preparation of Pickering double emulsions using block copolymer worms.

The rational formulation of Pickering double emulsions is described using a judicious combination of hydrophilic and hydrophobic block copolymer worms as highly anisotropic emulsifiers. More specifically, RAFT dispersion polymerization was utilized to prepare poly(lauryl methacrylate)-poly(benzyl methacrylate) worms at 20% w/w solids in n-dodecane and poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate)-poly(benzyl methacrylate) worms at 13% w/w solids in water by polymerization-induced self-assembly (PISA). Water-in-oil-in-water (w/o/w) double emulsions can be readily prepared with mean droplet diameters ranging from 30 to 80 µm using a two-stage approach. First, a w/o precursor emulsion comprising 25 µm aqueous droplets is prepared using the hydrophobic worms, followed by encapsulation within oil droplets stabilized by the hydrophilic worms. The double emulsion droplet diameter and number of encapsulated water droplets can be readily varied by adjusting the stirring rate employed during the second stage. For each stage, the droplet volume fraction is relatively high at 0.50. The double emulsion nature of the final formulation was confirmed by optical and fluorescence microscopy studies. Such double emulsions are highly stable to coalescence, with little or no change in droplet diameter being detected over storage at 20 °C for 10 weeks as judged by laser diffraction. Preliminary experiments indicate that the complementary o/w/o emulsions can also be prepared using the same pair of worms by changing the order of homogenization, although somewhat lower droplet volume fractions were required in this case. Finally, we demonstrate that triple and even quadruple emulsions can be formulated using these new highly anisotropic Pickering emulsifiers.

Facile phenylboronate modification of silica by a silaneboronate.

Macroscopic and colloidal silica surfaces were readily modified with alkoxysilaneboronate, IV, yielding silica surfaces with covalently bonded phenylboronic acid groups. XPS and neutron activation confirmed the presence of boron. The ability of these surfaces to specifically interact with polyols was demonstrated with polyol-coated latex and ARS, a dye that specifically couples to boronic acid groups immobilized on colloidal or macroscopic silica. This is a new, direct approach for introduction of phenylboronic acid groups onto silica surfaces.

Visible label-free detection of bacterial DNA using flocculation of sterically stabilised cationic latexes.

The current gold standard diagnostic for bacterial infections is the use of culture, which can be time consuming and can take up to five days for results to be reported. There is therefore an unmet clinical need for a rapid and label free alternative. This paper demonstrates a method of detecting the presence of amplified DNA from bacterial samples using a sterically-stabilised, cationic polymer latex and widely available equipment, providing an accessible alternative DNA detection technique. If DNA is present in a sample, successful amplification by polymerase chain-reaction (PCR) results in the amplified DNA inducing flocculation of the polymer latex followed by rapid sedimentation. This results in a visible and obvious change from a milky-white dispersion to a precipitated latex with a colourless and transparent supernatant, thus giving a clear visual indication of the presence or absence of amplified DNA. Specifically, the response of four polymer latexes with different morphologies to the addition of amplified bacterial DNA was investigated. Cationic latexes flocculated rapidly whereas non-ionic and anionic latexes did not, as judged by eye, disc centrifuge photosedimentometry (DCP), and UV-visible spectrophotometry. The stability of several cationic latexes with different morphologies in typical PCR reagents was investigated. It was found that unwanted flocculation occurred for a latex with a non-ionic core and a cationic corona (poly[2-vinyl pyridine-b-benzyl methacrylate], prepared by polymerisation-induced self-assembly) whereas a ∼700 nm PEGMA-stabilised P2VP latex (non-ionic stabiliser, cationic core), prepared by emulsion polymerisation remained stable. The sensitivity and rate of sedimentation of the PEGMA-stabilised P2VP latex was demonstrated by varying the sequence length and concentration of amplified DNA from Pseudomonas aeruginosa using universal bacterial primers. DNA concentrations as low as 0.78 ng µl-1 could readily be detected within 30 minutes from the addition of amplified DNA to the latex. Furthermore, the specificity of this method was demonstrated by showing a negative result occurs (no flocculation of the latex) when PCR product from a fungal (Candida albicans) sample using bacterial primers was added to the latex.

Can polymersomes form colloidosomes?

Hydroxy-functionalized polymersomes (or block copolymer vesicles) were prepared via a facile one-pot RAFT aqueous dispersion polymerization protocol and evaluated as Pickering emulsifiers for the stabilization of emulsions of n-dodecane emulsion droplets in water. Linear polymersomes produced polydisperse oil droplets with diameters of ~50 µm regardless of the polymersome concentration in the aqueous phase. Introducing an oil-soluble polymeric diisocyanate cross-linker into the oil phase prior to homogenization led to block copolymer microcapsules, as expected. However, TEM inspection of these microcapsules after an alcohol challenge revealed no evidence for polymersomes, suggesting these delicate nanostructures do not survive the high-shear emulsification process. Thus the emulsion droplets are stabilized by individual diblock copolymer chains, rather than polymersomes. Cross-linked polymersomes (prepared by the addition of ethylene glycol dimethacrylate as a third comonomer) also formed stable n-dodecane-in-water Pickering emulsions, as judged by optical and fluorescence microscopy. However, in this case the droplet diameter varied from 50 to 250 µm depending on the aqueous polymersome concentration. Moreover, diisocyanate cross-linking at the oil/water interface led to the formation of well-defined colloidosomes, as judged by TEM studies. Thus polymersomes can indeed stabilize colloidosomes, provided that they are sufficiently cross-linked to survive emulsification.

Direct observation of giant Pickering emulsion and colloidosome droplet interaction and stability.

The interactions of two 2-mm pendant oil droplets grown in the presence of an aqueous solution of poly(glycerol monomethacrylate)-stabilized polystyrene latex particles was observed using a high-speed video camera. The coalescence behavior was monitored as a function of oil type (n-dodecane versus sunflower oil) and particle size (135 versus 902 nm), as well as in the presence and absence of an oil-soluble cross-linker [tolylene 2,4-diisocyanate-terminated poly(propylene glycol)]. The damping coefficient of the coalescing n-dodecane droplets was found to increase in the presence of the latex, demonstrating particle adsorption. Coalescence times increased when the oil phase was changed from n-dodecane to sunflower oil, because of the much higher viscosity of the latter oil. In addition, increasing the adsorbed particle size from 135 to 902 nm led to longer coalescence times because of the greater distance separating the oil droplets. Coalescence times observed in the presence of the larger 902-nm particles indicated that two different modes of contact can occur prior to a coalescence event (bilayer or bridging monolayer of particles in the film). Addition of an oil-soluble surface-active cross-linker to the sunflower oil phase to react with the hydroxy groups of the particle stabilizer reduced the interfacial elasticity and ultimately prevented coalescence after cross-linking for 20 min at 25 °C. Such giant colloidosomes can remain in contact for several hours without undergoing coalescence, which demonstrates their high stability. Furthermore, coalescence is prevented even if the cross-linker is present in only one of the pendant droplets. Finally, evidence for cross-linker diffusion from one pendant droplet to another was indicated by a visible filament connecting the two droplets upon retraction.

Control of Staphylococcal-mediated endogenous protease activity alters wound closure time in a complex wound model.

BACKGROUND: Elevated protease activity is a characteristic feature of chronic wounds, where the inflammatory phase of wound healing is prolonged. The choice of dressings in treatment of chronic wounds can change the nature of the wound base and have a significant impact on healing. OBJECTIVE: To evaluate the impact of oxidised regenerated cellulose/collagen dressings on Staphylococcal-mediated protease activity in an inflamed wound model. METHODS: We developed an in vitro 3D inflamed wound model, and simulated inflammation by exposing the models to Staphylococcal spent culture supernatant. Protease activity and wound healing were assessed in the presence/absence of the dressings. RESULTS: Histological analysis of the wound model revealed two distinct layers, an epidermal and dermal layer, similar to the organisation of human skin. Inflammation with Staphylococcal spent culture supernatant elevated protease levels by 1.7x and consequently prevented the wound from progressing to the proliferative phase of healing, without having a negative effect on cell viability. Adding a collagen dressing, known to have non-specific protease modulating properties, reduced Staphylococcal-mediated protease activity back to baseline, with a concomitant reduction in wound closure time. Inflamed wounds thus resembled unwounded skin after 10 days of treatment with the dressings. CONCLUSION: Our findings support the further evaluation and use of oxidised regenerated cellulose/collagen dressings for inflamed, non-healing wounds in the clinical setting. The model used in this study has the potential to be applied in preclinical research; to test wound dressing performance, such as healing and cell viability, and to also assess key markers of inflammation.

Borate binding to polyol-stabilized latex.

Borate or 4-carboxyphenylboronate anions condense with diol units on poly(glycerol monomethacrylate) stabilizer chains at the surface of polystyrene latex, increasing the latex charge density. Combining Leibler's equilibrium binding model with Ohshima's hydrogel electrophoresis model simulated the primary experimental observation: the electrophoretic mobility of this latex becomes much more negative above pH 9.5 because of borate anion binding. There is an unusual inverse relation between the electrophoretic mobility and the density of borate anions bound to the latex particles. Very high solution concentrations of borate ions and hence high ionic strengths are required to give high densities of bound borate ions. Thus, mobilities decrease in spite of increasing charge density with borate addition because of increased screening at high ionic strength.

Controlling deposition and release of polyol-stabilized latex on boronic acid-derivatized cellulose.

Poly(glycerol monomethacrylate)-stabilized polystyrene (PGMA-PS) latex particles undergo specific, pH-dependent adsorption onto regenerated cellulose film bearing surface phenylboronic acid groups (cellulose-PBA). Deposition occurs at pH 10 and is driven by the boronate ester formation with the polyol latex surface coating. In contrast, no deposition occurs at pH 4, and previously deposited particles can be readily desorbed at this lower pH. In control experiments, conventional anionic sulfate-stabilized polystyrene latex did not deposit onto the hydrophilic cellulose surface. The distribution of boronate groups in the cellulose was determined by exposure to Alizarin Red S dye, which forms a fluorescent complex with phenylboronic acid; confocal microscopy was used to determine a surface density of 3 nm(2) per boronic acid group on the cellulose surface. Although the boronic acid binding constant with PGMA is relatively low (5.4 L/mol), the cooperative interactions between multiple PBA surface sites and the many PGMA chains per latex particle are sufficient to induce specific latex adsorption, providing a convenient new tool for controlling nanoparticle deposition on surfaces.

Preparation of biocompatible sterically stabilized latexes using well-defined poly(2-(methacryloyloxy)ethyl phosphorylcholine) macromonomers.

A range of well-defined methacrylic macromonomers based on the biomimetic monomer 2-(methacryloyloxy)ethyl phosphorylcholine (MPC) were synthesized by atom-transfer radical polymerization (ATRP) in alcoholic media using 2-(dimethylamino)ethyl-2-bromoisobutyrylamide. This tertiary amine-functionalized initiator was used to produce homopolymer precursors of various chain lengths via ATRP. These polymerizations were relatively well controlled (M(w)/M(n) < or = 1.30), provided that the target degree of polymerization (DP) did not exceed 30. For higher target DPs, polymerization was only poorly controlled and characterized by broad molecular weight distributions (M(w)/M(n) = 1.50-2.31). The tertiary amine end-group of each nearly monodisperse homopolymer precursor was then quaternized using 4-vinylbenzyl chloride (4-VBC) to afford the corresponding styrene-functionalized macromonomers. PMPC(30) macromonomer proved to be an effective reactive steric stabilizer for the formation of polystyrene latexes when employed at 10 w/w % on the basis of the styrene monomer. Nearly monodisperse submicrometer-sized and micrometer-sized latexes were prepared by aqueous emulsion and alcoholic dispersion polymerization, respectively, as judged by scanning electron microscopy and dynamic light scattering studies. In contrast, attempted alcoholic dispersion polymerization conducted either in the presence of the PMPC(30) homopolymer precursor or in the absence of any macromonomer always resulted in macroscopic precipitation. Such control experiments confirmed the importance of the terminal styrene groups on the macromonomer chains for successful latex formation. FTIR spectroscopy indicated the presence of the PMPC(30) macromonomer within the polystyrene latex, and XPS studies indicated that these stabilizer chains are located at (or very near) the latex surface, as expected. Using PMPC(20) and PMPC(10) macromonomers for the alcoholic dispersion polymerization of styrene led to latexes with substantially broader size distributions compared to those produced using the PMPC(30) macromonomer under the same conditions. Finally, these new sterically stabilized latexes exhibit excellent freeze-thaw stability and salt tolerance.

Time-Resolved SAXS Studies of the Kinetics of Thermally Triggered Release of Encapsulated Silica Nanoparticles from Block Copolymer Vesicles.

Silica-loaded poly(glycerol monomethacrylate)-poly(2-hydroxypropyl methacrylate) diblock copolymer vesicles are prepared in the form of concentrated aqueous dispersions via polymerization-induced self-assembly (PISA). As the concentration of silica nanoparticles present during the PISA synthesis is increased up to 35% w/w, higher degrees of encapsulation of this component within the vesicles can be achieved. After centrifugal purification to remove excess non-encapsulated silica nanoparticles, SAXS, DCP, and TGA analysis indicates encapsulation of up to hundreds of silica nanoparticles per vesicle. In the present study, the thermally triggered release of these encapsulated silica nanoparticles is examined by cooling to 0 °C for 30 min, which causes in situ vesicle dissociation. Transmission electron microscopy studies confirm the change in diblock copolymer morphology and also enable direct visualization of the released silica nanoparticles. Time-resolved small-angle X-ray scattering is used to quantify the extent of silica release over time. For an initial silica concentration of 5% w/w, cooling induces a vesicle-to-sphere transition with subsequent nanoparticle release. For higher silica concentrations (20 or 30% w/w) cooling only leads to perforation of the vesicle membranes, but silica nanoparticles are nevertheless released through the pores. For vesicles prepared in the presence of 30% w/w silica, the purified silica-loaded vesicles were cooled to 0 °C for 30 min, and SAXS patterns were collected every 15 s. A new SAXS model has been developed to determine both the mean volume fraction of encapsulated silica within the vesicles and the scattering length density. Satisfactory data fits to the experimental SAXS patterns were obtained using this model.

Bespoke contrast-matched diblock copolymer nanoparticles enable the rational design of highly transparent Pickering double emulsions.

We report the preparation of highly transparent oil-in-water Pickering emulsions using contrast-matched organic nanoparticles. This is achieved via addition of judicious amounts of either sucrose or glycerol to an aqueous dispersion of poly(glycerol monomethacrylate)56-poly(2,2,2-trifluoroethyl methacrylate)500 [PGMA-PTFEMA] diblock copolymer nanoparticles prior to high shear homogenization with an equal volume of n-dodecane. The resulting Pickering emulsions comprise polydisperse n-dodecane droplets of 20-100 µm diameter and exhibit up to 96% transmittance across the visible spectrum. In contrast, control experiments using non-contrast-matched poly(glycerol monomethacrylate)56-poly(benzyl methacrylate)300 [PGMA56-PBzMA300] diblock copolymer nanoparticles as a Pickering emulsifier only produced conventional highly turbid emulsions. Thus contrast-matching of the two immiscible phases is a necessary but not sufficient condition for the preparation of highly transparent Pickering emulsions: it is essential to use isorefractive nanoparticles in order to minimize light scattering. Furthermore, highly transparent oil-in-water-in-oil Pickering double emulsions can be obtained by homogenizing the contrast-matched oil-in-water Pickering emulsion prepared using the PGMA56-PTFEMA500 nanoparticles with a contrast-matched dispersion of hydrophobic poly(lauryl methacrylate)39-poly(2,2,2-trifluoroethyl methacrylate)800 [PLMA39-PTFEMA800] diblock copolymer nanoparticles in n-dodecane. Finally, we show that an isorefractive oil-in-water Pickering emulsion enables fluorescence spectroscopy to be used to monitor the transport of water-insoluble small molecules (pyrene and benzophenone) between n-dodecane droplets. Such transport is significantly less efficient than that observed for the equivalent isorefractive surfactant-stabilized emulsion. Conventional turbid emulsions do not enable such a comparison to be made because the intense light scattering leads to substantial spectral attenuation.

Colloidosomes: synthesis, properties and applications.

Colloidosomes represent a rapidly expanding field with various applications in microencapsulation, including the triggered release of cargoes. With self-assembled shells comprising colloidal particles, they offer significant flexibility with respect to microcapsule functionality. This review explores the various types of particles and techniques that have been employed to prepare colloidosomes. The relative advantages and disadvantages of these routes are highlighted and their potential as microcapsules for both small molecule and macromolecular actives is evaluated.

From well-defined macromonomers to sterically-stabilised latexes to covalently cross-linkable colloidosomes: exerting control over multiple length scales.

A new route to covalently cross-linkable colloidosomes has been developed via the assembly of sterically stabilised latex particles onto oil droplets. These latexes can be cross-linked from within the oil droplets via the steric stabiliser chains on the inner-particle surface using an oil-soluble polymeric diisocyanate. Such microcapsules can survive both surfactant and alcohol challenges.