Streamlined Formation and Manipulation of Charged Polymersomes.

Charged polymersomes are attractive for advanced material applications due to their versatile encapsulation capabilities and charge-induced functionality. Although desirable, the pH-sensitivity of charged block copolymers adds complexity to its self-assembly process, making it challenging to produce charged polymersomes in a reliable manner. In this work, a flow approach to control and strike a delicate balance between solvent composition and pH for self-assembly is used. This allows for the identification of a phase window to reliably produce of charged polymersomes. The utility of this approach to streamline downstream processes, such as morphological transformation or in-line purification is further demonstrated. As proof-of-concept, it is shown that the processed polymersomes can be used for surface modifications facilitated by charge complexation.

Mixtures of Acid- and Base-Functionalized Microparticles for One-Pot Cascade Reaction.

This study reports on the synthesis of surface-protected acid and base microparticles via surfactant-free emulsion polymerization. The protective layer allows to form particle mixtures with both colloidal catalysts that show excellent performance in a well-established model cascade reaction involving an acid-catalyzed deacetalization and a subsequent base-catalyzed Knoevenagel condensation. Compared to nanoreactors that contain both catalysts, the colloidal mixture shows comparable reaction kinetics despite the local separation of both catalysts onto separate particles. Since the synthesis of individual microparticles requires less steps, is scalable, and more versatile in terms of catalyst variation, colloidal mixtures are certainly an alternative to multifunctional nanoreactors, making them attractive for practical applications.

Scalable and Recyclable All-Organic Colloidal Cascade Catalysts.

We report on the synthesis of core-shell microparticles (CSMs) with an acid catalyst in the core and a base catalyst in the shell by surfactant-free emulsion polymerization (SFEP). The organocatalytic monomers were separately copolymerized in three synthetic steps allowing the spatial separation of incompatible acid and base catalysts within the CSMs. Importantly, a protected and thermo-decomposable sulfonate monomer was used as acid source to circumvent the neutralization of the base catalyst during shell formation, which was key to obtain stable, catalytically active CSMs. The catalysts showed excellent performance in an established one-pot model cascade reaction in various solvents (including water), which involved an acid-catalyzed deacetalization followed by a base-catalyzed Knoevenagel condensation. The CSMs are easily recycled, modified, and their synthesis is scalable, making them promising candidates for organocatalytic applications.

Size-Controlled Formation of Polymer Janus Discs.

A straightforward method is presented for the preparation of nano- to micrometer-sized Janus discs with controlled shape, size, and aspect ratio. The method relies on cross-linkable ABC triblock terpolymers and involves first the preparation of prolate ellipsoidal microparticles by combining Shirasu porous glass (SPG) membrane emulsification with evaporation-induced confinement assembly (EICA). By varying the pore diameter of the SPG membrane, we produce Janus discs with controlled size distributions centered around hundreds of nanometers to several microns. We further transferred the discs to water by mild sulfonation of PS to polystyrene sulfonic acid (PSS) and verified the Janus character by subsequent labelling with cationic nanoparticles. Finally, we show that the sulfonated Janus discs are amphiphilic and can be used as efficient colloidal stabilizers for oil-in-water (O/W) emulsions.

Vesicular Polymer Hexosomes Exhibit Topological Defects.

Polymer hexosomes are block copolymer solution morphologies that adopt an internal structure composed of an inverse hexagonal (HII) phase. To date, most polymer hexosomes are reportedly rotationally symmetric solid structures that possess a common feature where hexagonally ordered inverted cylinders rotate along a central axis of symmetry to form circular hoops. Here, we report on the formation of polymer hexosomes whose inverted cylinders orient in an unusual manner, forming hoops that are noncircular. For topological reasons, this led to the generation of four defects in the resulting hexosome structure. We find that these defect-bearing hexosomes are hollow, thereby resembling polymer vesicles or polymersomes with an inverse hexagonal cylindrical morphology in the shell. The topological defects of these so-called "vesicular hexosomes" are enticing as they could serve as a platform to spatially anchor targeting ligands or biomolecules on the surface, while the hollow cylindrical shell and the vesicular lumen could spatially accommodate cargoes within the different domains. We propose that these vesicular hexosomes do not form via a conventional nucleation-growth self-assembly pathway, but rather via a two-step process involving first liquid-liquid phase separation followed by polymer microphase separation.

pH-Controlled Hierarchical Assembly/Disassembly of Multicompartment Micelles in Water.

Multicompartment micelles (MCMs) have become attractive drug delivery systems as they allow the separate storage of two or more incompatible cargos in their core compartments (e.g., drugs and dyes for imaging). A recent hierarchical self-assembly process for hydrophobic terpolymers in organic solvents showed the ability to form very homogeneous MCM populations, yet the transfer of this process into water requires a better understanding of the formation mechanism and influence of chain mobility during assembly. Here, the synthesis of a linear poly(oligo(ethylene glycol) methacrylate)-block-poly(benzyl acrylate)-block-poly(4-vinylpyridine) (POEGMA-b-PBzA-b-P4VP) triblock terpolymer by reversible addition-fragmentation chain transfer (RAFT) polymerization is reported as well as its step-wise assembly into MCMs in water with POEGMA corona, PBzA patches, and P4VP core. Reversible assembly/disassembly of the MCMs is investigated through protonation/deprotonation of the P4VP core. Interestingly, the low glass transition temperature (Tg ) of the hydrophobic PBzA middle block causes MCMs to directly disassemble into molecularly dissolved chains instead of patchy micelles due to mechanical stress from electrosteric repulsion of the protonated P4VP corona chains. In addition, pH resistant MCMs are created by core-crosslinking and fluorescent properties are added by covalent anchoring of fluorescent dyes via straightforward click chemistry.

Non-spherical polymersomes: formation and characterization.

Polymersomes are self-assembled hollow membrane sacs that are not only able to encapsulate hydrophobic and/or hydrophilic molecules, but also possess exceptional chemical and physical stability, structural versatility, and surface modifiability. For the above reasons, polymersomes have in recent years emerged as a powerful tool for a wide range of applications in the fields of biomimicry and drug delivery. The full potential of polymersomes, however, has yet to be harnessed due to a lack of appreciation of existing shape control methods. This very much contrasts the field of inorganic nanoparticle synthesis where non-spherical hollow metal nanoparticles are routinely prepared and used. Here, we summarize recent efforts over the past decade to study the morphological transformation of conventionally spherical polymersomes into non-spherical polymersomes.

Bioactive Patchy Nanoparticles with Compartmentalized Cargoes for Simultaneous and Trackable Delivery.

Recent years have seen an increased interest in the use of ABC triblock terpolymers to bottom-up assemble multicompartment patchy nanoparticles. Despite these experimental and theoretical efforts, the applications of polymer-based patchy nanoparticles remain rather limited. One of the major challenges that eclipses their potential is the lack of knowledge to selectively encapsulate cargoes within different compartments that are separated in the nanometer length scale. Here, strategies are reported to segregate two chemically distinct molecules in either the patches or core compartment of patchy nanoparticles that bear a (bioactive) sugar corona. The potential use of these bioactive patchy nanoparticles containing compartmentalized cargoes for simultaneous drug delivery with real-time release monitoring capabilities is further demonstrated.

Polymersomes prepared from thermoresponsive fluorescent protein-polymer bioconjugates: capture of and report on drug and protein payloads.

Polymersomes provide a good platform for targeted drug delivery and the creation of complex (bio)catalytically active systems for research in synthetic biology. To realize these applications requires both spatial control over the encapsulation components in these polymersomes and a means to report where the components are in the polymersomes. To address these twin challenges, we synthesized the protein-polymer bioconjugate PNIPAM-b-amilFP497 composed of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) and a green-fluorescent protein variant (amilFP497). Above 37 °C, this bioconjugate forms polymersomes that can (co-)encapsulate the fluorescent drug doxorubicin and the fluorescent light-harvesting protein phycoerythrin 545 (PE545). Using fluorescence lifetime imaging microscopy and Förster resonance energy transfer (FLIM-FRET), we can distinguish the co-encapsulated PE545 protein inside the polymersome membrane while doxorubicin is found both in the polymersome core and membrane.

Polymersomes with micellar patches.

Hollow block copolymer particles called polymer vesicles (polymersomes) serve as versatile containers for compartmentalization in synthetic biology and drug delivery. Recently, there has been growing interest in using polymersomes as colloidal building blocks for creating higher-order clustered structures. Most reports thus far rely on the use of DNA base-pairing interactions to "glue" polymersomes with other colloidal components. In this study, we present two alternative electrostatically driven approaches to assemble polymersomes and model colloids (micelles) into hybrid clusters. The first approach uses pH to manipulate electrostatic interactions and effectively control the clustering extent of micellar subunits on polymersomes, while the second approach relies on the hydrolysis of an acid trigger, glucono delta-lactone (GDL), to introduce temporal control over clustering. We envisage our approaches and structures reported herein will help inspire the creation of new prospects for materials science and biomedical applications.

Dynamic metastable polymersomes enable continuous flow manufacturing.

Polymersomes are polymeric analogues of liposomes with exceptional physical and chemical properties. Despite being dubbed as next-generation vesicles since their inception nearly three decades ago, polymersomes have yet to experience translation into the clinical or industrial settings. This is due to a lack of reliable methods to upscale production without compromising control over polymersome properties. Herein we report a continuous flow methodology capable of producing near-monodisperse polymersomes at scale (≥3 g/h) with the possibility of performing downstream polymersome manipulation. Unlike conventional polymersomes, our polymersomes exhibit metastability under ambient conditions, persisting for a lifetime of ca. 7 days, during which polymersome growth occurs until a dynamic equilibrium state is reached. We demonstrate how this metastable state is key to the implementation of downstream processes to manipulate polymersome size and/or shape in the same continuous stream. The methodology operates in a plug-and-play fashion and is applicable to various block copolymers.

Macrophage-Targeting and Complete Lysosomal Degradation of Self-assembled Two-Dimensional Poly(ε-caprolactone) Platelet Particles.

Understanding cellular uptake and particle trafficking within the cells is essential for targeted drug delivery applications. Existing studies reveal that the geometrical aspects of nanocarriers, for example, shape and size, determine their cell uptake and sub-cellular transport pathways. However, considerable efforts have been directed toward understanding the cell uptake mechanism and trafficking of spherical particles. Detailed analysis on the uptake mechanism and downstream intracellular processing of non-spherical particles remains elusive. Here, we used polymeric two-dimensional platelets based on poly(ε-caprolactone) (PCL) prepared by living crystallization-driven self-assembly as a platform to investigate the cell uptake and intracellular transport of non-spherical particles in vitro. PCL is known to degrade only slowly, and these platelets were still stable after 2 days of incubation in artificial lysosomal media. Upon cell uptake, the platelets were transported through an endo/lysosomal pathway and were found to degrade completely in the lysosome at the end of the cell uptake cycle. We observed a morphological transformation of the lysosomes, which correlates with the stages of platelet degradation in the lysosome. Overall, we found an accelerated degradation of PCL, which was likely caused by mechanical forces inside the highly stretched endosomes.

Direct Observation of Topological Defects in Striped Block Copolymer Discs and Polymersomes.

Topology and defects are of fundamental importance for ordered structures on all length scales. Despite extensive research on block copolymer self-assembly in solution, knowledge about topological defects and their effect on nanostructure formation has remained limited. Here, we report on the self-assembly of block copolymer discs and polymersomes with a cylinder line pattern on the surface that develops specific combinations of topological defects to satisfy the Euler characteristics for closed spheres as described by Gauss-Bonnet theorem. The dimension of the line pattern allows the direct visualization of defect emergence, evolution, and annihilation. On discs, cylinders either form end-caps that coincide with λ+1/2 disclinations or they bend around τ+1/2 disclinations in 180° turns (hairpin loops). On polymersomes, two λ+1/2 defects connect into three-dimensional (3D) Archimedean spirals, while two τ+1/2 defects form 3D Fermat spirals. Electron tomography reveals two complementary line patterns on the inside and outside of the polymersome membrane, where λ+1/2 and τ+1/2 disclinations always eclipse on opposing sides ("defect communication"). Attractive defects are able to annihilate with each other into +1 disclinations and stabilize anisotropic polymersomes with sharp tips through screening of high-energy curvature. This study fosters our understanding of the behavior of topological defects in self-assembled polymer materials and aids in the design of polymersomes with preprogrammed shapes governed by synthetic block length and topological rules.

Modulating the Selectivity and Stealth Properties of Ellipsoidal Polymersomes through a Multivalent Peptide Ligand Display.

There is a need for improved nanomaterials to simultaneously target cancer cells and avoid non-specific clearance by phagocytes. An ellipsoidal polymersome system is developed with a unique tunable size and shape property. These particles are functionalized with in-house phage-display cell-targeting peptide to target a medulloblastoma cell line in vitro. Particle association with medulloblastoma cells is modulated by tuning the peptide ligand density on the particles. These polymersomes has low levels of association with primary human blood phagocytes. The stealth properties of the polymersomes are further improved by including the peptide targeting moiety, an effect that is likely driven by the peptide protecting the particles from binding blood plasma proteins. Overall, this ellipsoidal polymersome system provides a promising platform to explore tumor cell targeting in vivo.

Surface roughness influences the protein corona formation of glycosylated nanoparticles and alter their cellular uptake.

Recently the role of protein absorption in nanoparticle drug delivery has gathered significant attention as the protein corona can significantly decide on the fate of nanoparticles in the body. Although it is known that the surface chemistry will significantly influence the amount and type of bound protein, there is little known about the effect of surface roughness and surface topography on the interaction. In this work, we show how patchy nanoparticles can noticeably reduce the adsorption of proteins compared to spherical nanoparticles with a smooth surface as demonstrated using six ABC triblock terpolymers based on glucose, mannose and galactose. To obtain patchy nanoparticles, poly(2-d-sugar ethyl acrylate)-b-poly (n-butyl acrylate)-b-poly(4-vinyl pyridine) (PSugEA-b-PBuA-b-P4VP) was prepared by reversible addition-fragmentation chain-transfer (RAFT) polymerization and assembled into nanoparticles with a patch-like appearance and a hydrodynamic diameter of around 130-160 nm. As control, smooth nanoparticles were prepared from poly(2-d-sugar ethyl acrylate)-b-poly (n-butyl acrylate)-b-polystyrene (PSugEA-b-PBuA-b-PS). The patchy nanoparticles displayed significantly reduced protein absorption when exposed to serum-supplemented cell culture media, as observed using dynamic light scattering. The smooth particles, however, supported the formation of a large protein corona. Additionally, an enrichment of haemoglobin was observed in the corona compared to the serum protein in solution. The amount of albumin on the surface was observed to be dependent on the type of sugar with glucose resulting in the highest absorption. The protein corona led to cellular uptake that was unrelated to the underlying sugar, which was supposed to help targeting specific cell lines. This example demonstrated how the protein corona can override any attempts to target receptor expressing cells.

Faceted polymersomes: a sphere-to-polyhedron shape transformation.

The creation of "soft" deformable hollow polymeric nanoparticles with complex non-spherical shapes via block copolymer self-assembly remains a challenge. In this work, we show that a perylene-bearing block copolymer can self-assemble into polymeric membrane sacs (polymersomes) that not only possess uncommonly faceted polyhedral shapes but are also intrinsically fluorescent. Here, we further reveal for the first time an experimental visualization of the entire polymersome faceting process. We uncover how our polymersomes facet through a sphere-to-polyhedron shape transformation pathway that is driven by perylene aggregation confined within a topologically spherical polymersome shell. Finally, we illustrate the importance in understanding this shape transformation process by demonstrating our ability to controllably isolate different intermediate polymersome morphologies. The findings presented herein should provide opportunities for those who utilize non-spherical polymersomes for drug delivery, nanoreactor or templating applications, and those who are interested in the fundamental aspects of polymersome self-assembly.

Just add sugar for carbohydrate induced self-assembly of curcumin.

In nature, self-assembly processes based on amphiphilic molecules play an integral part in the design of structures of higher order such as cells. Among them, amphiphilic glycoproteins or glycolipids take on a pivotal role due to their bioactivity. Here we show that sugars, in particular, fructose, are capable of directing the self-assembly of highly insoluble curcumin resulting in the formation of well-defined capsules based on non-covalent forces. Simply by mixing an aqueous solution of fructose and curcumin in an open vessel leads to the generation of capsules with sizes ranging between 100 and 150 nm independent of the initial concentrations used. Our results demonstrate that hydrogen bonding displayed by fructose can induce the self-assembly of hydrophobic molecules such as curcumin into well-ordered structures, and serving as a simple and virtually instantaneous way of making nanoparticles from curcumin in water with the potential for template polymerization and nanocarriers.

Formation of non-spherical polymersomes driven by hydrophobic directional aromatic perylene interactions.

Polymersomes, made up of amphiphilic block copolymers, are emerging as a powerful tool in drug delivery and synthetic biology due to their high stability, chemical versatility, and surface modifiability. The full potential of polymersomes, however, has been hindered by a lack of versatile methods for shape control. Here we show that a range of non-spherical polymersome morphologies with anisotropic membranes can be obtained by exploiting hydrophobic directional aromatic interactions between perylene polymer units within the membrane structure. By controlling the extent of solvation/desolvation of the aromatic side chains through changes in solvent quality, we demonstrate facile access to polymersomes that are either ellipsoidal or tubular-shaped. Our results indicate that perylene aromatic interactions have a great potential in the design of non-spherical polymersomes and other structurally complex self-assembled polymer structures.

An Oligonucleotide Transfection Vector Based on HSA and PDMAEMA Conjugation: Effect of Polymer Molecular Weight on Cell Proliferation and on Multicellular Tumor Spheroids.

A novel gene transfection vector was fabricated based on the conjugation of human serum albumin (HSA) and maleimide end functionalized poly[(N,N-dimethylamino) ethyl methacrylate] (PDMAEMA). The bioconjugation was achieved in a site-specific manner to yield well-defined polymer-protein conjugates. The biohybrid was able to bind DNA with high affinity resulting in nanoparticles with a HSA shell. This paper mainly focuses on the influence of polymeric chain length on the particle properties and their drug-carrying ability to deliver oligonucleotides into breast cancer cells. The cytotoxic agent of interest, ISIS5132, is an oligonucleotide which disrupts DNA function within the cell. There was no evidence that the polymeric chain length had any effects on the conjugation efficiency and the subsequent condensation ability of the conjugates to oligonucleotide. However, the polymeric chain length had an obvious effect on the size of the complex micelles. Low molecular weights only led to loosely compacted complexes with the oligonucleotide, while large molecular weight led to well-defined nanoparticle structures. More importantly, it was found that the variation in the length of the PDMAEMA block resulted in a change in cytotoxicity of the drug loaded complex micelle. That is, the concentration of 50% inhibition (IC50 ) of the complex micelle on MDA-MB-231 and MCF-7 cells reached the lowest value at a chain length of around 21 000 g mol(-1) . The IC50 value increased when the polymeric chain length was shorter (8000 g mol(-1) and 10 000 g mol(-1) ) while it increased again when PDMAMEA of M¯n = 47 000 g mol(-1) , probably due to insufficient release of the drug. These result were reflected when investigating the performance of the polyplex using MCF-7 multicellular tumor spheroids, where again the medium PDMAEMA chain length led to the best delivery vehicle for ISIS5132.