Tiny, yet impactful: Detection and oxidative stability of very small oil droplets in surfactant-stabilized emulsions.

HYPOTHESIS: The shelf life of multiphase systems, e.g. oil-in-water (O/W) emulsions, is severely limited by physical and/or chemical instabilities, which degrade their texture, macroscopic appearance, sensory and (for edible systems) nutritional quality. One prominent chemical instability is lipid oxidation, which is notoriously complex. The complexity arises from the involvement of many physical structures present at several scales (1-10,000 nm), of which the smallest ones are often overlooked during characterization. EXPERIMENTS: We used cryogenic transmission electron microscopy (cryo-TEM) to characterize the coexisting colloidal structures at the nanoscale (10-200 nm) in rapeseed oil-based model emulsions stabilized by different concentrations of a nonionic surfactant. We assessed whether the oxidative and physical instabilities of the smallest colloidal structures in such emulsions may be different from those of larger colloidal structures. FINDINGS: By deploying cryo-TEM, we analyzed the size of very small oil droplets and of surfactant micelles, which are typically overlooked by dynamic light scattering when larger structures are concomitantly present. Their size and oil content were shown to be stable over incubation, but lipid oxidation products were overrepresented in these very small droplets. These insights highlight the importance of the fraction of "tiny droplets" for the oxidative stability of O/W emulsions.

Time-Resolved Cryo-TEM Study on the Formation of Iron Hydroxides in a Collagen Matrix.

The mineralization of collagen via synthetic procedures has been extensively investigated for hydroxyapatite as well as for silica and calcium carbonate. From a fundamental point of view, it is interesting to investigate whether collagen could serve as a generic mineralization template for other minerals, like iron oxides. Here, bio-inspired coprecipitation reaction, generally leading to the formation of magnetite, is used to mineralize collagen with iron hydroxides. Platelet-shaped green rust crystals form outside the collagen matrix, while inside the collagen, nanoparticles with a size of 2.6 nm are formed, which are hypothesized to be iron (III) hydroxide. Mineralization with nanoparticles inside the collagen solely occurs in the presence of poly(aspartic acid) (pAsp). In the absence of pAsp, magnetite particles are formed around the collagen. Time-resolved cryo-TEM shows that during the coprecipitation reaction, initially a beam-sensitive phase is formed, possibly an Fe3+-pAsp complex. This beam-sensitive phase transforms into nanoparticles. In a later stage, sheet-like crystals are also found. After 48 h of mineralization, ordering of the nanoparticles around one of the collagen sub-bands (the a-band) is observed. This is very similar to the collagen-hydroxyapatite system, indicating that mineralization with iron hydroxides inside collagen is possible and proceeds via a similar mechanism as hydroxyapatite mineralization.

Photoactivated nanomotors via aggregation induced emission for enhanced phototherapy.

Aggregation-induced emission (AIE) has, since its discovery, become a valuable tool in the field of nanoscience. AIEgenic molecules, which display highly stable fluorescence in an assembled state, have applications in various biomedical fields-including photodynamic therapy. Engineering structure-inherent, AIEgenic nanomaterials with motile properties is, however, still an unexplored frontier in the evolution of this potent technology. Here, we present phototactic/phototherapeutic nanomotors where biodegradable block copolymers decorated with AIE motifs can transduce radiant energy into motion and enhance thermophoretic motility driven by an asymmetric Au nanoshell. The hybrid nanomotors can harness two photon near-infrared radiation, triggering autonomous propulsion and simultaneous phototherapeutic generation of reactive oxygen species. The potential of these nanomotors to be applied in photodynamic therapy is demonstrated in vitro, where near-infrared light directed motion and reactive oxygen species induction synergistically enhance efficacy with a high level of spatial control.

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Hybrid Biodegradable Nanomotors through Compartmentalized Synthesis.

Designer particles that are embued with nanomachinery for autonomous motion have great potential for biomedical applications; however, their development is highly demanding with respect to biodegradability/compatibility. Previously, biodegradable propulsive machinery based on enzymes has been presented. However, enzymes are highly susceptible to proteolysis and deactivation in biological milieu. Biodegradable hybrid nanomotors powered by catalytic inorganic nanoparticles provide a proteolytically stable alternative to those based upon enzymes. Herein we describe the assembly of hybrid biodegradable nanomotors capable of transducing chemical energy into motion. Such nanomotors are constructed through a process of compartmentalized synthesis of inorganic MnO2 nanoparticles (MnPs) within the cavity of organic stomatocytes. We show that the nanomotors remain active in cellular environments and do not compromise cell viability. Effective tumor penetration of hybrid nanomotors is also demonstrated in proof-of-principle experiments. Overall, this work represents a new prospect for engineering of nanomotors that can retain their functionality within biological contexts.

Designing stable, hierarchical peptide fibers from block co-polypeptide sequences.

Natural materials, such as collagen, can assemble with multiple levels of organization in solution. Achieving a similar degree of control over morphology, stability and hierarchical organization with equilibrium synthetic materials remains elusive. For the assembly of peptidic materials the process is controlled by a complex interplay between hydrophobic interactions, electrostatics and secondary structure formation. Consequently, fine tuning the thermodynamics and kinetics of assembly remains extremely challenging. Here, we synthesized a set of block co polypeptides with varying hydrophobicity and ability to form secondary structure. From this set we select a sequence with balanced interactions that results in the formation of high-aspect ratio thermodynamically favored nanotubes, stable between pH 2 and 12 and up to 80 °C. This stability permits their hierarchical assembly into bundled nanotube fibers by directing the pH and inducing complementary zwitterionic charge behavior. This block co-polypeptide design strategy, using defined sequences, provides a straightforward approach to creating complex hierarchical peptide-based assemblies with tunable interactions.

Reversible Restructuring of Silver Particles during Ethylene Epoxidation.

The restructuring of a silver catalyst during ethylene epoxidation under industrially relevant conditions was investigated without and with vinyl chloride (VC) promotion. During non-VC-promoted ethylene epoxidation, the silver particles grow and voids are formed at the surface and in the bulk. Electron tomography highlighted the presence of voids below the Ag surface. A mechanism is proposed involving reconstruction of the silver lattice and defect sites induced by oxygen adsorption on the external surface and grain boundaries, which finally create pores. Promotion of the catalytic reaction by VC suppresses to a significant extent void formation. The use of VC also redisperses silver particles, initially grown during ethylene epoxidation without VC. This process is rapid as the average size decreased from 172 to 136 nm within 2 h. These insights emphasize the dynamic nature of the silver particles during the ongoing ethylene epoxidation reaction and indicate that particle size and morphology strongly depend on reaction conditions.

Native Chemical Ligation for Cross-Linking of Flower-Like Micelles.

In this study, native chemical ligation (NCL) was used as a selective cross-linking method to form core-cross-linked thermosensitive polymeric micelles for drug delivery applications. To this end, two complementary ABA triblock copolymers having polyethylene glycol (PEG) as midblock were synthesized by atom transfer radical polymerization (ATRP). The thermosensitive poly isopropylacrylamide (PNIPAM) outer blocks of the polymers were copolymerized with either N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys), P(NIPAM- co-HPMA-Cys)-PEG-P(NIPAM- co-HPMA-Cys) (PNC) or N-(2-hydroxypropyl)methacrylamide-ethylthioglycolate succinic acid (HPMA-ETSA), P(NIPAM- co-HPMA-ETSA)-PEG-P(NIPAM- co-HPMA-ETSA) (PNE). Mixing of these polymers in aqueous solution followed by heating to 50 °C resulted in the formation of thermosensitive flower-like micelles. Subsequently, native chemical ligation in the core of micelles resulted in stabilization of the micelles with a Z-average of 65 nm at body temperature. Decreasing the temperature to 10 °C only affected the size of the micelles (increased to 90 nm) but hardly affected the polydispersity index (PDI) and aggregation number ( Nagg) confirming covalent stabilization of the micelles by NCL. CryoTEM images showed micelles with an uniform spherical shape and dark patches close to the corona of micelles were observed in the tomographic view. The dark patches represent more dense areas in the micelles which coincide with the higher content of HPMA-Cys/ETSA close to the PEG chain revealed by the polymerization kinetics study. Notably, this cross-linking method provides the possibility for conjugation of functional molecules either by using the thiol moieties still present after NCL or by simply adjusting the molar ratio between the polymers (resulting in excess cysteine or thioester moieties) during micelle formation. Furthermore, in vitro cell experiments demonstrated that fluorescently labeled micelles were successfully taken up by HeLa cells while cell viability remained high even at high micelle concentrations. These results demonstrate the potential of these micelles for drug delivery applications.

Coiled coil driven membrane fusion between cyclodextrin vesicles and liposomes.

Controlled fusion events between natural membranes composed of phospholipids with synthetic unnatural membranes will yield valuable fundamental information on the mechanism of membrane fusion. Here, fusion between vastly different phospholipid liposomes and cyclodextrin amphiphile based vesicles (CDVs) controlled by a pair of coiled coil forming lipidated peptides was investigated. Fusion events were characterized using lipid and content mixing assays and the resulting hybrid assemblies were characterized with cryo-TEM imaging. The secondary/quaternary structure of the lipidated peptides at the membrane interface was studied using circular dichroism spectroscopy. This is the first example of targeted fusion between natural and non-natural bilayer membranes and the in situ formation of hybrid CDV-liposome structures is of interest as it yields fundamental information about the mechanism through which fusion proceeds.

The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors.

Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals. In the periodic 67 nm cross-striated pattern of the collagen fibril, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow. This process is believed to be directed by highly acidic non-collagenous proteins; however, the role of the collagen matrix during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation.