Spontaneous organization of supracolloids into three-dimensional structured materials.

Periodic nano- or microscale structures are used to control light, energy and mass transportation. Colloidal organization is the most versatile method used to control nano- and microscale order, and employs either the enthalpy-driven self-assembly of particles at a low concentration or the entropy-driven packing of particles at a high concentration. Nonetheless, it cannot yet provide the spontaneous three-dimensional organization of multicomponent particles at a high concentration. Here we combined these two concepts into a single strategy to achieve hierarchical multicomponent materials. We tuned the electrostatic attraction between polymer and silica nanoparticles to create dynamic supracolloids whose components, on drying, reorganize by entropy into three-dimensional structured materials. Cryogenic electron tomography reveals the kinetic pathways, whereas Monte Carlo simulations combined with a kinetic model provide design rules to form the supracolloids and control the kinetic pathways. This approach may be useful to fabricate hierarchical hybrid materials for distinct technological applications.

Building Reversible Nanoraspberries.

The adsorption mechanism of small positively charged silica nanoparticles (SiO2 NPs) onto larger polystyrene latex nanoparticles (PSL NPs) forming hybrid particles was studied. CryoTEM showed the morphology of these supraparticles to be raspberry-like. After surface modification of the SiO2 NPs, the optimum pH regime to initiate the formation of nanoraspberries was determined. Thereafter, their size evolution was evaluated by dynamic light scattering for different surface charge densities. Reversibility of nanoraspberry formation was shown by cycling the pH of the mixture to make interparticle forces either attractive or repulsive, while their stability was confirmed experimentally. The number of SiO2 NPs on the PSL NPs as determined with cryoTEM matched the theoretically expected maximum number. Understanding and controlling the relevant parameters, such as size and charge of the individual particles and the Debye length, will pave the way to better control of the formation of nanoraspberries and higher-order assemblies thereof.

Flower-Like Colloidal Particles through Precipitation Polymerization of Redox-Responsive Liquid Crystals.

We report on the synthesis of monodisperse, flower-like, liquid crystalline (LC) polymer particles by precipitation polymerization of a LC mixture consisting of benzoic acid-functionalized acrylates and disulfide-functionalized diacrylates. Introduction of a minor amount of redox-responsive disulfide-functionalized diacrylates (≤10 wt %) induced the formation of flower-like shapes. The shape of the particles can be tuned from flower- to disk-like to spherical by elevating the polymerization temperature. The solvent environment also has a pronounced effect on the particle size. Time-resolved TEM reveals that the final particle morphology was formed in the early stages of the polymerization and that subsequent polymerization resulted in continued particle growth without affecting the morphology. Finally, the degradation of the particles under reducing conditions was much faster for flower-like particles than for spherical particles, likely a result of their higher surface-to-volume ratio.

Polypeptide Nanoparticles Obtained from Emulsion Polymerization of Amino Acid N-Carboxyanhydrides.

Polypeptide nanoparticles were obtained by the miniemulsion polymerization of S-(o-nitrobenzyl)-l-cysteine (NBC) N-carboxyanhydride (NCA). Through process optimization, reaction conditions were identified that allowed the polymerization of the water sensitive NCA to yield nanoparticles of about 220 nm size. Subsequent UV-irradiation of the nanoparticle emulsions caused the in situ removal of the nitrobenzyl group and particle cross-linking through disulfide bond formation accompanied by the shrinkage of the particles.

CryoTEM as an Advanced Analytical Tool for Materials Chemists.

Morphology plays an essential role in chemistry through the segregation of atoms and/or molecules into different phases, delineated by interfaces. This is a general process in materials synthesis and exploited in many fields including colloid chemistry, heterogeneous catalysis, and functional molecular systems. To rationally design complex materials, we must understand and control morphology evolution. Toward this goal, we utilize cryogenic transmission electron microscopy (cryoTEM), which can track the structural evolution of materials in solution with nanometer spatial resolution and a temporal resolution of <1 s. In this Account, we review examples of our own research where direct observations by cryoTEM have been essential to understanding morphology evolution in macromolecular self-assembly, inorganic nucleation and growth, and the cooperative evolution of hybrid materials. These three different research areas are at the heart of our approach to materials chemistry where we take inspiration from the myriad examples of complex materials in Nature. Biological materials are formed using a limited number of chemical components and under ambient conditions, and their formation pathways were refined during biological evolution by enormous trial and error approaches to self-organization and biomineralization. By combining the information on what is possible in nature and by focusing on a limited number of chemical components, we aim to provide an essential insight into the role of structure evolution in materials synthesis. Bone, for example, is a hierarchical and hybrid material which is lightweight, yet strong and hard. It is formed by the hierarchical self-assembly of collagen into a macromolecular template with nano- and microscale structure. This template then directs the nucleation and growth of oriented, nanoscale calcium phosphate crystals to form the composite material. Fundamental insight into controlling these structuring processes will eventually allow us to design such complex materials with predetermined and potentially unique properties.

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.

Bimodal Latex Effect on Spin-Coated Thin Conductive Polymer-Single-Walled Carbon Nanotube Layers.

We synthesize two differently sized poly(methyl methacrylate-co-tert-butyl acrylate) latexes by emulsion polymerization and mix these with a sonicated single-walled carbon nanotube (SWCNT) dispersion, in order to prepare 3% SWCNT composite mixtures. We spin-coat these mixtures at various spin-speed rates and spin times over a glass substrate, producing a thin, transparent, solid, conductive layer. Keeping the amount of SWCNTs constant, we vary the weight fraction of our smaller 30-nm latex particles relative to the larger 70-nm-sized ones. We find a maximum in the electrical conductivity up to 370 S/m as a function of the weight fraction of smaller particles, depending on the overall solid content, the spin speed, and the spin time. This maximum occurs at 3-5% of the smaller latex particles. We also find a more than 2-fold increase in conductivity parallel to the radius of spin-coating than perpendicular to it. Atomic force microscopy points at the existence of lanes of latex particles in the spin-coated thin layer, while large-area transmission electron microscopy demonstrates that the SWCNTs are aligned over a grid fixed on the glass substrate during the spin-coating process. We extract the conductivity distribution on the surface of the thin film and translate this into the direction of the SWCNTs in it.

Phase-Separated Lipid-Based Nanoparticles: Selective Behavior at the Nano-Bio Interface.

The membrane-protein interface on lipid-based nanoparticles influences their in vivo behavior. Better understanding may evolve current drug delivery methods toward effective targeted nanomedicine. Previously, the cell-selective accumulation of a liposome formulation in vivo is demonstrated, through the recognition of lipid phase-separation by triglyceride lipases. This exemplified how liposome morphology and composition can determine nanoparticle-protein interactions. Here, the lipase-induced compositional and morphological changes of phase-separated liposomes-which bear a lipid droplet in their bilayer- are investigated, and the mechanism upon which lipases recognize and bind to the particles is unravelled. The selective lipolytic degradation of the phase-separated lipid droplet is observed, while nanoparticle integrity remains intact. Next, the Tryptophan-rich loop of the lipase is identified as the region with which the enzymes bind to the particles. This preferential binding is due to lipid packing defects induced on the liposome surface by phase separation. In parallel, the existing knowledge that phase separation leads to in vivo selectivity, is utilized to generate phase-separated mRNA-LNPs that target cell-subsets in zebrafish embryos, with subsequent mRNA delivery and protein expression. Together, these findings can expand the current knowledge on selective nanoparticle-protein communications and in vivo behavior, aspects that will assist to gain control of lipid-based nanoparticles.

Phase-Separated Liposomes Hijack Endogenous Lipoprotein Transport and Metabolism Pathways to Target Subsets of Endothelial Cells In Vivo.

Plasma lipid transport and metabolism are essential to ensure correct cellular function throughout the body. Dynamically regulated in time and space, the well-characterized mechanisms underpinning plasma lipid transport and metabolism offers an enticing, but as yet underexplored, rationale to design synthetic lipid nanoparticles with inherent cell/tissue selectivity. Herein, a systemically administered liposome formulation, composed of just two lipids, that is capable of hijacking a triglyceride lipase-mediated lipid transport pathway resulting in liposome recognition and uptake within specific endothelial cell subsets is described. In the absence of targeting ligands, liposome-lipase interactions are mediated by a unique, phase-separated ("parachute") liposome morphology. Within the embryonic zebrafish, selective liposome accumulation is observed at the developing blood-brain barrier. In mice, extensive liposome accumulation within the liver and spleen - which is reduced, but not eliminated, following small molecule lipase inhibition - supports a role for endothelial lipase but highlights these liposomes are also subject to significant "off-target" by reticuloendothelial system organs. Overall, these compositionally simplistic liposomes offer new insights into the discovery and design of lipid-based nanoparticles that can exploit endogenous lipid transport and metabolism pathways to achieve cell selective targeting in vivo.

Strategy for optimizing experimental settings for studying low atomic number colloidal assemblies using liquid phase scanning transmission electron microscopy.

Observing processes of nanoscale materials of low atomic number is possible using liquid phase electron microscopy (LP-EM). However, the achievable spatial resolution (d) is limited by radiation damage. Here, we examine a strategy for optimizing LP-EM experiments based on an analytical model and experimental measurements, and develop a method for quantifying image quality at ultra low electron dose De using scanning transmission electron microscopy (STEM). As experimental test case we study the formation of a colloidal binary system containing 30 nm diameter SiO2 nanoparticles (SiONPs), and 100 nm diameter polystyrene microspheres (PMs). We show that annular dark field (DF) STEM is preferred over bright field (BF) STEM for practical reasons. Precise knowledge of the material's density is crucial for the calculations in order to match experimental data. To calculate the detectability of nano-objects in an image, the Rose criterion for single pixels is expanded to a model of the signal to noise ratio obtained for multiple pixels spanning the image of an object. Using optimized settings, it is possible to visualize the radiation-sensitive, hierarchical low-Z binary structures, and identify both components.

From binary AB to ternary ABC supraparticles.

Control over the assembly and morphology of nanoscale functional building blocks is of great importance to hybrid and porous nanomaterials. In this paper, by combining different types of spherical nanoparticles with different size ratios in a hierarchical assembly process which allows us to control the final structure of multi-component assemblies, we discuss self-assembly of an extensive range of supraparticles, labelled as AB particles, and an extension to novel ternary particles, labelled as ABC particles. For supraparticles, the organization of small nanoparticles is known to be inherently related to the size ratio of building blocks. Therefore, we studied the formation of supraparticles prepared by colloidal self-assembly using small silica nanoparticles (SiO2 NPs) attached on the surface of large polystyrene latex nanoparticles (PSL NPs) with a wide size ratio range for complete and partial coverage, by controlling the electrostatic interactions between the organic and inorganic nanoparticles and their concentrations. In this way hierarchically ordered, stable supraparticles, either fully covered or partially covered, were realized. The partially covered, stable AB supraparticles offer the option to create ABC supraparticles of which the fully covered shell contains two different types of nanoparticles. This has been experimentally confirmed using iron oxide (Fe3O4) nanoparticles together with silica nanoparticles as shell particles on polystyrene core particles. Cryo-electron tomography was used to visualize the AB binary and ABC ternary supraparticles and to determine the three-dimensional structural characteristics of supraparticles formed under different conditions.

Anionic Lipid Nanoparticles Preferentially Deliver mRNA to the Hepatic Reticuloendothelial System.

Lipid nanoparticles (LNPs) are the leading nonviral technologies for the delivery of exogenous RNA to target cells in vivo. As systemic delivery platforms, these technologies are exemplified by Onpattro, an approved LNP-based RNA interference therapy, administered intravenously and targeted to parenchymal liver cells. The discovery of systemically administered LNP technologies capable of preferential RNA delivery beyond hepatocytes has, however, proven more challenging. Here, preceded by comprehensive mechanistic understanding of in vivo nanoparticle biodistribution and bodily clearance, an LNP-based messenger RNA (mRNA) delivery platform is rationally designed to preferentially target the hepatic reticuloendothelial system (RES). Evaluated in embryonic zebrafish, validated in mice, and directly compared to LNP-mRNA systems based on the lipid composition of Onpattro, RES-targeted LNPs significantly enhance mRNA expression both globally within the liver and specifically within hepatic RES cell types. Hepatic RES targeting requires just a single lipid change within the formulation of Onpattro to switch LNP surface charge from neutral to anionic. This technology not only provides new opportunities to treat liver-specific and systemic diseases in which RES cell types play a key role but, more importantly, exemplifies that rational design of advanced RNA therapies must be preceded by a robust understanding of the dominant nano-biointeractions involved.

Monodisperse Liquid Crystalline Polymer Shells with Programmable Alignment and Shape Prepared by Seeded Dispersion Polymerization.

Monodisperse, micrometer-sized liquid crystalline (LC) shells are prepared by seeded dispersion polymerization. After polymerizing LC monomer mixtures in the presence of non-crosslinked polymer seeds, hollow LC polymer shells with programmable alignment and shape are prepared by removing the seeds. The LC alignment in the LC polymer shells can be easily manipulated by the polymer seeds, as a radial alignment is observed with amorphous poly(phenyl methacrylate) seeds and a bipolar alignment is observed with bipolar LC polymer seeds. After removal of the seeds, the radially aligned samples give radially aligned shells with small dimples. The resulting bipolar LC polymer shells collapse into a biconcave shape. Polarized optical microscopy and transmission electron microscopy indicate that the collapse occurs at the defect points in the shell. In the case of a lower crosslink density, LC polymer hollow shells with larger dimples are obtained, resulting in cup-shaped polymer particles. Biconcave LC polymer shells based on other LC mixtures have also been prepared, showing the versatility of the seeded dispersion polymerization method.

Morphology Control of Liposome - RAFT Oligomer Precursors to Complex Polymer Nanostructures.

Different types of butyl acrylate (BA)-co-acrylic acid (AA) oligomers were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization and mixed with extruded 200 nm dimethyldioctadecylammonium bromide vesicles. The resulting precursor structures form the basis for subsequent vesicle-templated polymerizations. Systematic variations in temperature, pH, oligomer length, and oligomer composition and their effects on precursor morphology were studied. Surprisingly, different morphologies were obtained, including capsules, protruded capsules, solid spheres, and multicompartment structures. For example, capsules and multicompartment structures were found to result from higher AA contents, and protruded capsules and solid particles resulted from lower AA contents. Subsequent chain extension of the RAFT oligomers resulted in polymer nanostructures resembling the precursor morphologies.

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.