Bas G. P. van Ravensteijn

RNA therapeutics hold considerable promise for treating a wide range of diseases, but their clinical application remains limited by challenges in delivering intact RNA to target tissues. Lipid nanoparticles (LNPs) have emerged as leading RNA delivery vehicles; however, following systemic administration, their biodistribution and clearance are strongly influenced by the adsorption of plasma proteins onto the particle surface, forming the protein corona (PC). Because PC composition depends on LNP physicochemical properties, accurate characterization of the corona is essential for understanding LNP behavior in vivo. A major challenge in PC research is the isolation of LNPs from biological fluids without introducing methodological bias. Here, we systematically evaluated three approaches for LNP protein corona isolation: density gradient ultracentrifugation (DGU), magnetic capture of iron oxide nanoparticle (IONP)-loaded LNPs, and biotin-based capture (BIO) of biotinylated LNPs. Methods were optimized and compared with respect to LNP recovery, isolation selectivity, and resulting protein corona composition. Comparative analysis revealed distinct strengths and limitations for each workflow, highlighting trade-offs between capture efficiency, specificity, and experimental complexity. Despite methodological differences, six proteins—platelet factor 4, vitronectin, complement component 9, complement component 3, complement component 4B, and paraoxonase 1—were consistently enriched across all three approaches, identifying them as robust candidates for biologically relevant LNP–plasma protein interactions. Together, these findings provide a framework for selecting appropriate protein corona isolation strategies and represent an important step toward the standardization of LNP protein corona characterization in biological fluids.

(Macro)molecular self‐assembling systems are typically subjected to pathway complexity. The final structural outcome is not only determined by the system's composition but also depends on the details and kinetics of the pathway chosen to navigate the energetic landscape during the assembly process. In this study, a distinct manifestation of pathway complexity is presented in the formation of short double‐stranded DNA (dsDNA)‐loaded polyion complex (PIC) micelles. Besides the conventional assembly pathway of rapidly mixing dsDNA with preformed polycationic block copolymers, compositionally equivalent PIC micelles are prepared via polymerization‐induced electrostatic self‐assembly (PIESA). A combination of synchrotron time‐resolved small‐angle x‐ray scattering (TR‐SAXS), cryogenic transmission electron microscopy (cryo‐TEM), and fluorescence correlation spectroscopy (FCS) reveals the formation of spheroidal particles during direct mixing, whereas PIESA generates hexagonal disk‐shaped colloids with internal crystalline dsDNA ordering. A molecular assembly mechanism for PIESA is postulated, which relies on the continuous formation of charge‐neutralized complexes that act as primary building blocks for the colloidal hexagons. Mechanistic insights into pathway complexity during PIC micellization establish assembly pathway control as a strategy to tune the particle's physicochemical properties beyond conventional design parameters, such as polymer size and chemistry. This approache expands the opportunities for therapeutic oligonucleotide delivery through control over morphology and cargo loading.

(Macro)molecular self‐assembling systems are typically subjected to pathway complexity. The final structural outcome is not only determined by the system's composition but also depends on the details and kinetics of the pathway chosen to navigate the energetic landscape during the assembly process. In this study, a distinct manifestation of pathway complexity is presented in the formation of short double‐stranded DNA (dsDNA)‐loaded polyion complex (PIC) micelles. Besides the conventional assembly pathway of rapidly mixing dsDNA with preformed polycationic block copolymers, compositionally equivalent PIC micelles are prepared via polymerization‐induced electrostatic self‐assembly (PIESA). A combination of synchrotron time‐resolved small‐angle x‐ray scattering (TR‐SAXS), cryogenic transmission electron microscopy (cryo‐TEM), and fluorescence correlation spectroscopy (FCS) reveals the formation of spheroidal particles during direct mixing, whereas PIESA generates hexagonal disk‐shaped colloids with internal crystalline dsDNA ordering. A molecular assembly mechanism for PIESA is postulated, which relies on the continuous formation of charge‐neutralized complexes that act as primary building blocks for the colloidal hexagons. Mechanistic insights into pathway complexity during PIC micellization establish assembly pathway control as a strategy to tune the particle's physicochemical properties beyond conventional design parameters, such as polymer size and chemistry. This approache expands the opportunities for therapeutic oligonucleotide delivery through control over morphology and cargo loading.

High‐aspect‐ratio polymeric micelles (filomicelles, FMs) are attractive anisotropic nanoparticles for a wide range of applications, however, their formation is typically confined to narrow hydrophilic/hydrophobic polymer block ratios, limiting systematic structure–property investigations. Consequently, independent control over micelle width, length, and stiffness remains challenging. A supramolecular polymerization‐induced self‐assembly (PISA) strategy using a bis‐urea core template was recently shown to robustly yield wormlike micelles across a wide range of core block lengths; however, the resulting FM lengths remained highly polydisperse. Herein, we investigate two strategies to tune the contour length: ( i ) Blending in polymer without the bis‐urea sticker and ( ii ) post‐micellization sonication. Unexpectedly, incorporating non‐sticker polymer can increase the contour length. Transmission electron microscopy (TEM), fluorescence cross‐correlation spectroscopy (FCCS), and high‐resolution atomic force microscopy (AFM) were used to elucidate the key factors contributing to this peculiar self‐assembly mechanism. Sonication‐induced cleavage led to shortening of the particles and narrower polydispersity in length. Combined, the two strategies provide access to a wide range of anisotropic micelles with accessible aspect ratios ranging from 1 to >150. Considering their long‐term (colloidal) stability, the presented system is an ideal model to study the effect of micellar anisotropy, for example, in drug delivery relevant scenarios.

A recently introduced one-pot strategy for polyelectrolyte self-assembly in aqueous solution, namely polymerization-induced electrostatic self-assembly (PIESA), enables in-situ polymerization and complexation of a polyelectrolyte block of a double hydrophilic block copolymer with an oppositely charged polymeric template. Unlike the conventional preparation of complex coacervate core micelles (C3Ms) by direct mixing of aqueous solutions of premade oppositely-charged polyelectrolytes, PIESA intrinsically couples polymerization and electrostatically-driven self-assembly within a single process. This coupling facilitates formation and equilibration of nanometer-sized C3M-like nanostructures. PIESA offers promising control over nanostructure formation, but the mechanistic pathways underlying structure formation and evolution in such systems remain poorly understood. To fill this knowledge gap, the structural evolution of nanoscale assemblies was tracked via time-variable small-angle X-ray scattering (SAXS) and light scattering measurements on quenched aliquots taken at different time points during a PIESA process. A detailed analysis of the SAXS profiles on an absolute intensity scale with molecular constraints, in conjunction with monomer conversions determined by 1H-NMR, reveals proceeding of nanostructure evolution in PIESA through three distinct stages: (I) Complexation and Particle Nucleation: a non-particulate regime marking the onset of nucleation, (II) Maturation and Growth: a particulate regime wherein particles grow via a diffusion-driven ripening mechanism, and (III) Polymerization Termination and Structural Stabilization: a particulate regime wherein particle coarsening and structural maturation cease. These findings provide fundamental insights into coupled polymerization and assembly kinetics in PIESA systems, offering a mechanistic framework that can guide the rational design and equilibration of next-generation polyelectrolyte nanostructures with tunable size and functionality.

Light-tunable DNA brushes enable isothermal spatiotemporal control of colloidal interactions and relaxation-driven crystallization.

There has been significant interest in the engineering of polymer topology to control rheology and mechanical stability in dilute solutions for applications involving extreme shear rate flows. However, methods to experimentally probe properties at relevant shear rates (≈ 10⁴–10⁶ s^–1) have remained ex situ, obscuring access to measures of polymer deformation and rheology that would otherwise provide mechanistic insight into the topology-dependent properties that control their behavior in extreme shear flows. In this study, we used novel in situ small angle neutron scattering measurements in a capillary rheometer (capillary rheo-SANS) to simultaneously measure solution viscosities and polymer deformations in high shear on a series of chemically homologous topology-defined polymers including linear, randomly branched, and star-shaped molecules. We demonstrate that differences in the onset of chain stretching and shear thinning of these polymers in dilute solution are controlled primarily by differences in their molecular relaxation time. These differences correlate with differences in chain scission inferred from ex situ measurements at more extreme shear rates. Together, the results demonstrate a direct coupling between chain deformation and scission, and suggest that the dominant effect of branching as a means to impart resilience against mechanical degradation is through differences in relaxation dynamics due to branching. We anticipate that these results will provide key insights to engineer topology-controlled polymers for rheological modification, mechanical stability, and controlled mechano-chemistry.

In biology, ligand-mediated transitions (LMT), where the binding of a molecular ligand onto the binding site of a receptor molecule leads to a well-defined change in the conformation of the receptor, are often referred to as "the second secret of life." Sharp, cooperative transitions arise in many biological cases, while examples of synthetic cooperative systems are rare. This is because well-defined conformational states are hard to "program" into a molecular design. Here, we impose an external constraint in the form of two immiscible liquids that effectively define and limit the available conformational states of two different synthetic and relatively simple macromolecules. We show that the mechanism of the observed cooperative transitions with ligand concentration is the coupling of ligand binding and conformation, similar to more complex biological systems. The systems studied are (1) hydrophobic polyelectrolytes (HPE) which are (bio) polymers that consist of hydrophobic as well as ionizable (proton and hydroxyl ligand binding) functional groups. (2) Oligomeric metal chelators (OMC), which are oligomers composed of metal ion chelating repeating groups that are able to bind metal ions (considered as the "ligands"), resulting in gel-like networks of oligomers cross-linked by coordinated metal ions. We find that in HPE, interactions between ligands and individual macromolecules explain the observed cooperative transitions. For OMC, coordinated bonds significantly enhance the degree of cooperativity, compared to HPE.