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Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in condensed, phase separated droplets which exhibit a range of liquid-like properties driven by transient intermolecular interactions. Understanding the organization within these condensates is crucial for deciphering their material properties and functions. This study explores the distinct nanoscale networks and interfaces in the condensate samples using a modified cryo-electron microscopy (cryo-EM) method. The method involves initiating condensate formation on electron microscopy grids to limit droplet growth as large droplet sizes are not ideal for cryo-EM imaging. The versatility of this method is demonstrated by imaging three different classes of condensates. We further investigate the condensate structures using cryo-electron tomography which provides 3D reconstructions, uncovering porous internal structures, unique core-shell morphologies, and inhomogeneities within the nanoscale organization of protein condensates. Comparison with dry-state transmission electron microscopy emphasizes the importance of preserving the hydrated structure of condensates for accurate structural analysis. We correlate the internal structure of protein condensates with their amino acid sequences and material properties by performing viscosity measurements that support that more viscous condensates exhibit denser internal assemblies. Our findings contribute to a comprehensive understanding of nanoscale condensate structure and its material properties. Our approach here provides a versatile tool for exploring various phase-separated systems and their nanoscale structures for future studies.

Bioreducible polymeric mRNA carriers are an emerging family of vectors for gene delivery and vaccine development. A few bioreducible systems have been generated through aqueous-phase ring-opening polymerization of lipoic acid derivatives, however this methodology limits hydrophobic group incorporation and functionality into resulting polymers. Herein, a poly(active ester)disulfide polymer is synthesized that can undergo facile aminolysis with amine-containing substrates under stoichiometric control and mild reaction conditions to yield a library of multifunctional polydisulfide polymers. Functionalized polydisulfide polymer species form stable mRNA-polymer nanoparticles for intracellular delivery of mRNAs in vitro. Alkyl-functionalized polydisulfide-RNA nanoparticles demonstrate rapid cellular uptake and excellent biodegradability when delivering EGFP and OVA mRNAs to cells in vitro. This streamlined polydisulfide synthesis provides a new facile methodology for accessing multifunctional bioreducible polymers as biomaterials for RNA delivery and other applications., (© 2022 Wiley-VCH GmbH.)

Amphiphilic triblock copolymers containing Fe(III) -catecholate complexes formulated as spherical- or cylindrical-shaped micellar nanoparticles (SMN and CMN, respectively) are described as new T1-weighted agents with high relaxivity, low cytotoxicity, and long-term stability in biological fluids. Relaxivities of both SMN and CMN exceed those of established gadolinium chelates across a wide range of magnetic field strengths. Interestingly, shape-dependent behavior is observed in terms of the particles' interactions with HeLa cells, with CMN exhibiting enhanced uptake and contrast via magnetic resonance imaging (MRI) compared with SMN. These results suggest that control over soft nanoparticle shape will provide an avenue for optimization of particle-based contrast agents as biodiagnostics. The polycatechol nanoparticles are proposed as suitable for preclinical investigations into their viability as gadolinium-free, safe, and effective imaging agents for MRI contrast enhancement., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Single chain polymer nanoparticles (SCNPs) have been formed using polystyrenes decorated with pendent norbornenes and a bifunctional tetrazine crosslinker. Characterisation by size exclusion chromatography and (1)H NMR gives evidence for the formation of SCNPs by the tetrazine-norbornene reaction, whilst light scattering, neutron scattering, transmission electron microscopy and atomic force microscopy show that discrete well-defined nanoparticles are formed and their size in solution calculated.

There has been much interest in the construction of soft nanomaterials in solution due to a desire to emulate the exquisite structure and function of Nature's equivalents (e.g. enzymes, viruses, proteins and DNA). Nature's soft nanomaterials are capable of selectivity, precision and efficiency in areas such as information storage and replication, transportation and delivery, and synthesis and catalysis. To this end, the use of small molecules, amphiphiles, colloids, and polymers have been investigated for the development of advanced materials in myriad fields of biomedicine and synthetic chemistry. Two major challenges are faced in this area of research: the reproducible, scalable and precise synthesis of such constructs and the reliable, accurate and in-depth analysis of these materials. This tutorial review will focus on this second aspect and provide a guide for the characterisation and analysis of soft nanomaterials in solution using scattering and microscopic techniques.

In this paper we present in situ transmission electron microscopy of synthetic polymeric nanoparticles with emphasis on capturing motion in a solvated, aqueous state. The nanoparticles studied were obtained from the direct polymerization of a Pt(II)-containing monomer. The resulting structures provided sufficient contrast for facile imaging in situ. We contend that this technique will quickly become essential in the characterization of analogous systems, especially where dynamics are of interest in the solvated state. We describe the preparation of the synthetic micellar nanoparticles together with their characterization and motion in liquid water with comparison to conventional electron microscopy analyses.

The use of functional core-shell micelles as asymmetric catalytic nanoreactors for organic reactions in water is presented. An unprecedented increase in rate of reaction was achieved, which is proposed to be associated with the ability of the nanostructures to effectively concentrate the reagents in the catalytically active micelle core.

Unusual self-assembly behavior is observed for a range of hydrophilic homopolymers. This self-assembly behavior is contrary to the expected behavior of such hydrophilic polymers and instead mimics more commonly reported amphiphilic block copolymers. It is proposed that the unique combination of hydrophobic end groups at both the α and ω chain end accounts for this unusual self-assembly behavior. Complex internal polymer micelles are spontaneously formed when hydrophilic homopolymer polyelectrolytes and neutral polymers (with a weight fraction of the hydrophobic end groups <10 wt%) are directly dissolved in water. The homopolymers, poly[2-(diethylamino)ethyl methacrylate], poly(N-isopropylacrylamide), and poly(ethoxyethylacrylate) are synthesized by reversible addition-fragmentation chain-transfer (RAFT) polymerization using S'-1-dodecyl-(S')-(α,α'-dimethyl-α″-acetic acid) trithiocarbonate (DDMAT) and its derivatives as chain transfer agents (CTAs). A range of polyelectrolyte homopolymers with different terminal groups are designed and synthesized, which under acidic aqueous solution direct the self-assembly to form well-defined nanostructures. This assembly behavior was also observed for neutral polymers, and it was determined that the structure of the hydrophobic end groups (and thus choice of RAFT CTA) are very important in facilitating this unusual self-assembly behavior of hydrophilic homopolymers. It is proposed that the functionality of commonly used CTAs such as DDMAT, can affect the solution association of the resultant homopolymers and can in fact afford ABA' type polymers, which can undergo self-assembly to form higher-order nanostructures., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The design and synthesis of a tuneable and reversible morphology switching copolymer system is reported. The kinetics of the transition under a range of conditions has been explored, as has the stabilization of the resultant structures.

In highly selective solvents, block copolymers (BCPs) form association colloids, while in solvents with poor selectivity, they exhibit a temperature-controlled (de)mixing behavior. Herein, it is shown that a temperature-responsive self-assembly behavior emerges in solvent mixtures of intermediate selectivity. A biocompatible poly-ethylene(oxide)-block-poly-ε-caprolactone (PEO-PCL) BCP is used as a model system. The polymer is dissolved in solvent mixtures containing water (a strongly selective solvent for PEO) and ethanol (a poorly selective solvent for PEO) to tune the solvency conditions. Using synchrotron X-ray scattering, cryogenic transmission electron microscopy, and scanning probe microscopy, it is shown that a rich temperature-responsive behavior can be achieved in certain solvent mixtures. Crystallization of the PCL block enriches the phase behavior of the BCP by promoting sphere-to-cylinder morphology transitions at low temperatures. Increasing the water fraction in the solvent causes a suppression of the sphere-to-cylinder morphology transition. These results open up the possibility to induce temperature-responsive properties on demand in a wide range of BCP systems.

Direct polymerization of an oxaliplatin analogue was used to reproducibly generate amphiphiles in one pot, which consistently and spontaneously self-assemble into well-defined nanoparticles (NPs). Despite inefficient drug leakage in cell-free assays, the NPs were observed to be as cytotoxic as free oxaliplatin in cell culture experiments. We investigated this phenomenon by super-resolution fluorescence structured illumination microscopy (SIM) and nanoscale secondary ion mass spectrometry (NanoSIMS). In combination, these techniques revealed NPs are taken up via endocytic pathways before intracellular release of their cytotoxic cargo. As with other drug-carrying nanomaterials, these systems have potential as cellular delivery vehicles. However, high-resolution methods to track nanocarriers and their cargo at the micro- and nanoscale have been underutilized in general, limiting our understanding of their interactions with cells and tissues. We contend this type of combined optical and isotopic imaging strategy represents a powerful and potentially generalizable methodology for cellular tracking of nanocarriers and their cargo.

Bioreducible polymeric mRNA carriers are an emerging family of vectors for gene delivery and vaccine development. A few bioreducible systems have been generated through aqueous‐phase ring‐opening polymerization of lipoic acid derivatives, however this methodology limits hydrophobic group incorporation and functionality into resulting polymers. Herein, a poly(active ester)disulfide polymer is synthesized that can undergo facile aminolysis with amine‐containing substrates under stoichiometric control and mild reaction conditions to yield a library of multifunctional polydisulfide polymers. Functionalized polydisulfide polymer species form stable mRNA‐polymer nanoparticles for intracellular delivery of mRNAs in vitro. Alkyl‐functionalized polydisulfide‐RNA nanoparticles demonstrate rapid cellular uptake and excellent biodegradability when delivering EGFP and OVA mRNAs to cells in vitro. This streamlined polydisulfide synthesis provides a new facile methodology for accessing multifunctional bioreducible polymers as biomaterials for RNA delivery and other applications. [ABSTRACT FROM AUTHOR]

Review: 50 refs. [ABSTRACT FROM AUTHOR]

Amphiphilic small molecules and polymers form commonplace nanoscale macromolecular compartments and bilayers, and as such are truly essential components in all cells and in many cellular processes. The nature of these architectures, including their formation, phase changes, and stimuli-response behaviors, is necessary for the most basic functions of life, and over the past half-century, these natural micellar structures have inspired a vast diversity of industrial products, from biomedicines to detergents, lubricants, and coatings. The importance of these materials and their ubiquity have made them the subject of intense investigation regarding their nanoscale dynamics with increasing interest in obtaining sufficient temporal and spatial resolution to directly observe nanoscale processes. However, the vast majority of experimental methods involve either bulk-averaging techniques including light, neutron, and X-ray scattering, or are static in nature including even the most advanced cryogenic transmission electron microscopy techniques. Here, we employ in situ liquid-cell transmission electron microscopy (LCTEM) to directly observe the evolution of individual amphiphilic block copolymer micellar nanoparticles in solution, in real time with nanometer spatial resolution. These observations, made on a proof-of-concept bioconjugate polymer amphiphile, revealed growth and evolution occurring by unimer addition processes and by particle-particle collision-and-fusion events. The experimental approach, combining direct LCTEM observation, quantitative analysis of LCTEM data, and correlated in silico simulations, provides a unique view of solvated soft matter nanoassemblies as they morph and evolve in time and space, enabling us to capture these phenomena in solution. [ABSTRACT FROM AUTHOR]