Stretched or wrinkled? Looking into the polymer conformation within polymersome membranes.

Self-assembly of amphiphilic block-copolymers into polymersomes is a well-established concept. In this membrane, the hydrophilic part is considered to be loosely assembled towards the solvent, and the hydrophobic part on the inside of the membrane is considered to be more densely packed. Within the membrane, this hydrophobic part could now have a stretched conformation or be a random coil, depending on the available space and also on the chemical nature of the polymer. We now analysed the literature for works on polymersomes that determined the membrane thickness via cryo-TEM and analysed the hydrophobic part of their polymers for their conformation. Over all available block-copolymers, a variety of trends became obvious: the longer a hydrophobic block, the more coiled the conformation and the bulkier the side chains, the more stretched the polymer became. Polymers with less conformational freedom like semi-crystalline ones were present in a more stretched conformation. Both trends could be exemplified on various occasions in this cross-literature meta-study. This overview hence provides additional insight into the physical chemistry of block-copolymer membranes.

Surface Topology of Redox- and Thermoresponsive Nanogel Droplets.

Hydrogels are usually depicted as a homogenous polymer block with a distinct surface. While defects in the polymer structure are looked into frequently, structural irregularities on the hydrogel surface are often neglected. In this work, thin hydrogel layers of ≈100 nm thickness (nanogels) are synthesized and characterized for their structural irregularities, as they represent the surface of macrogels. The nanogels contain a main-chain responsiveness (thermo responsive) and a responsiveness in the cross-linking points (redox responsive). By combining data from ellipsometry using box-model and two-segment-model analysis, as well as atomic force microscopy, a more defined model of the nanogel surface can be developed. Starting with a more densely cross-linked network at the silica wafer surface, the density of cross-linking gradually decreases toward the hydrogel-solvent interface. Thermo-responsive behavior of the main chain affects the entire network equally as all chain segments change solubility. Cross-linker-based redox-responsiveness, on the other hand, is only governed by the inner, more cross-linked layers of the network. Such dual responsive nanogels hence allow for developing a more detailed model of a hydrogel surface from free radical polymerization. It provides a better understanding of structural defects in hydrogels and how they are affected by responsive functionalities.

Amphiphilic Block Copolymers PEG-b-PMTCs: Synthesis, Self-Assembly, Degradation Properties and Biocompatibility.

As a hydrophilic cyclic ketene acetal (CKA), 2-methylene-1,3,6-trioxocane (MTC) has recently attracted a lot of attention owing to its ability to promote a quicker (bio)degradation as compared to other heavily studied CKAs. Here, we prepared amphiphilic block copolymers based on poly-MTC with varying chain lengths by radical ring opening polymerization. Self-assemblies of these amphiphiles were performed in PBS buffer to generate nanoparticles with sizes from 40 to 105 nm, which were verified by dynamic light scattering, electron microscopy, and static light scattering (Zimm plots). Subsequently, fluorescence spectroscopy was applied to study the enzymatic degradation of Nile red-loaded nanoparticles. By performing a point-by-point comparison of fluorescence intensity decline patterns between nanoparticles, we demonstrated that lipase from Pseudomonas cepacia was very efficient in degrading the nanoparticles. Hydrolysis degradations under basic conditions were also carried out, and a complete degradation was achieved after 4 h. Additionally, cytotoxicity assays were carried out on HEK293 cells, and the results affirmed cell viabilities over 90% when incubated with up to 1 mg/mL nanoparticles for 24 h. These biodegradable and biocompatible nanoparticles hence hold great potential for future applications such as drug release.

A modular RNA delivery system comprising spherical nucleic acids built on endosome-escaping polymeric nanoparticles.

Nucleic acid therapeutics require delivery systems to reach their targets. Key challenges to be overcome include avoidance of accumulation in cells of the mononuclear phagocyte system and escape from the endosomal pathway. Spherical nucleic acids (SNAs), in which a gold nanoparticle supports a corona of oligonucleotides, are promising carriers for nucleic acids with valuable properties including nuclease resistance, sequence-specific loading and control of receptor-mediated endocytosis. However, SNAs accumulate in the endosomal pathway and are thus vulnerable to lysosomal degradation or recycling exocytosis. Here, an alternative SNA core based on diblock copolymer PMPC25-PDPA72 is investigated. This pH-sensitive polymer self-assembles into vesicles with an intrinsic ability to escape endosomes via osmotic shock triggered by acidification-induced disassembly. DNA oligos conjugated to PMPC25-PDPA72 molecules form vesicles, or polymersomes, with DNA coronae on luminal and external surfaces. Nucleic acid cargoes or nucleic acid-tagged targeting moieties can be attached by hybridization to the coronal DNA. These polymeric SNAs are used to deliver siRNA duplexes against C9orf72, a genetic target with therapeutic potential for amyotrophic lateral sclerosis, to motor neuron-like cells. By attaching a neuron-specific targeting peptide to the PSNA corona, effective knock-down is achieved at doses of 2 particles per cell.

Current Standing on Radical Ring-Opening Polymerizations of Cyclic Ketene Acetals as Homopolymers and Copolymers with one another.

Radical Ring-opening polymerization (RROP) of cyclic ketene acetals (CKAs) emerges to be a valuable polymerization technique. In attracting more attention, RROP has seen a new spike in publications, which the authors will put into perspective. This review will hence address the progress made on the number of available CKAs and the synthetic strategies to get them. In grouping, the available monomers into distinct categories, the enormous variety of available CKAs will be highlighted. Polymerizations of CKAs without vinylenes have the potential to yield fully biodegradable polymers, which is why this kind of polymerization is the focus of this review. Detailing the current understanding of the mechanism, the various side reactions will be noted and also their effect on the overall properties of the final polymers. Current attempts to control the ring-retaining and branching reactions will be discussed as well. In addition to the polymerization itself, the available materials will be discussed as well as homopolymers, copolymers of CKAs, and block-copolymers with pure CKA-blocks have significantly widened the range of possible applications of materials from RROP. Altogether this review highlights the progress in the entire field of RROP just of CKAs to give a holistic overview of the field.

Reversible Molecular Capture and Release in Microfluidics by Host-Guest Interactions in Hydrogel Microdots.

The integration of microscopic hydrogels with high specific surface area and physically reactive groups into microfluidic systems for selective molecular interactions is attracting increasing attention. Herein, the reversible capture and release of molecules through host-guest interactions of hydrogel dots in a microfluidic device is reported, which translates the supramolecular chemistry to the microscale conditions under continuous flow. Polyacrylamide (PAAm) hydrogel arrays with grafted ß-cyclodextrin (ß-CD)  modified poly(2-methyl-2-oxazoline) (CD-PMOXA) chains are fabricated by photopolymerization and integrated into a polydimethylsiloxane (PDMS)-on-glass chip. The ß-CD/adamantane (ß-CD/Ada) host-guest complex is confirmed by two dimensional Nuclear Overhauser Effect Spectroscopy NMR (2D NOESY NMR) prior to transfer to microfluidics. Ada-modified molecules are successfully captured by host-guest interaction formed between the CD-PMOXA grafted chains in the hydrogel network and the guest molecule in the solution. Furthermore, the captured molecules are released by perfusing free ß-CD with higher binding affinity than those grafted in the hydrogel array. A small guest molecule adamantane-fluorescein-isothiocyanate (Ada-FITC) and a macromolecular guest molecule (Ada-PMOXA-Cyanine 5 (Cy5)) are separately captured and released for three times with a release ratio up to 46% and 92%, respectively. The reproducible capture and release of functional molecules with different sizes demonstrates the stability of this hydrogel system in microfluidics and will provide an opportunity for future applications.

Reversible Protein Capture and Release by Redox-Responsive Hydrogel in Microfluidics.

Stimuli-responsive hydrogels have a wide range of potential applications in microfluidics, which has drawn great attention. Double cross-linked hydrogels are very well suited for this application as they offer both stability and the required responsive behavior. Here, we report the integration of poly(N-isopropylacrylamide) (PNiPAAm) hydrogel with a permanent cross-linker (N,N'-methylenebisacrylamide, BIS) and a redox responsive reversible cross-linker (N,N'-bis(acryloyl)cystamine, BAC) into a microfluidic device through photopolymerization. Cleavage and re-formation of disulfide bonds introduced by BAC changed the cross-linking densities of the hydrogel dots, making them swell or shrink. Rheological measurements allowed for selecting hydrogels that withstand long-term shear forces present in microfluidic devices under continuous flow. Once implemented, the thiol-disulfide exchange allowed the hydrogel dots to successfully capture and release the protein bovine serum albumin (BSA). BSA was labeled with rhodamine B and functionalized with 2-(2-pyridyldithio)-ethylamine (PDA) to introduce disulfide bonds. The reversible capture and release of the protein reached an efficiency of 83.6% in release rate and could be repeated over 3 cycles within the microfluidic device. These results demonstrate that our redox-responsive hydrogel dots enable the dynamic capture and release of various different functionalized (macro)molecules (e.g., proteins and drugs) and have a great potential to be integrated into a lab-on-a-chip device for detection and/or delivery.

Fully amorphous atactic and isotactic block copolymers and their self-assembly into nano- and microscopic vesicles.

The introduction of chirality into aqueous self-assemblies by employing isotactic block copolymers (BCPs) is an emerging field of interest as it promises special membrane properties of polymersomes not accessible by atactic BCPs. However, isotactic BCPs typically exhibit crystalline behaviour, inducing high membrane stiffness and limiting their applicability in systems involving membrane proteins or sensitive cargo. In this study, an isotactic yet fully amorphous BCP is introduced which overcomes these limitations. Three BCPs composed of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG), differing solely in their tacticities (R/S, R and S), were synthesised and characterised regarding their structural, optical and thermal properties. Their self-assembly into homogenous phases of nanoscopic polymersomes (referred to as small unilamellar vesicles, SUVs) was analysed, revealing stability differences between SUVs composed of the different BCPs. Additionally, microscopic giant unilamellar vesicles (GUVs) were prepared by double emulsion microfluidics. Only the atactic BCP formed GUVs which were stable over several hours, whereas GUVs composed of isotactic BCPs ruptured within several minutes after formation. The ability of atactic PBO-b-PG to form microreactors was elucidated by reconstituting the membrane protein OmpF in the GUV membrane by microfluidics and performing an enzyme reaction inside its lumen. The system presented here serves as platform to design versatile vesicles with flexible membranes composed of atactic or isotactic BCPs. Hence, they allow for the introduction of chirality into nano- or microreactors which is a yet unstudied field and could enable special biotechonological applications.

Double cross-linked supramolecular hydrogels with tunable properties based on host-guest interactions.

We report a novel double cross-linked hydrogel system based on polyacrylamide and poly(2-methyl-2-oxazoline) (PMOXA) network chains, as well as on supramolecular host-guest interactions with on-demand tailored mechanical properties. Well-defined vinyl-bearing PMOXA macromonomers, functionalized with either ß-cyclodextrin units (ß-CD-PMOXA) or adamantane units (Ada-PMOXA), were synthesized and confirmed using 1H NMR, MALDI-TOF-MS and GPC measurements. The complexation between adamantane and ß-CD modified macromonomers in solution towards bismacromonomers was confirmed by 2D NOESY NMR and DLS. After introducing these bismacromonomers into the polyacrylamide hydrogel, the supramolecular non-covalent Ada/ß-CD bond was responsible for the presence of PMOXA network chains to form a dense network. Once the interactions broke, the PMOXA chains no longer contributed to the network, but became dangling graft side chains in a predominated polyacrylamide network. Their dissociative nature influenced the physical properties, including the swelling behavior and mechanics of the final hydrogel. Rheological experiments proved that the E-modulus of the network was significantly increased by the supramolecular host-guest interactions. Tuning the lengths of PMOXA network chains even allowed the modification of the changes in mechanical strength, also through the addition of free ß-CD. The tunable properties of the double cross-linked supramolecular hydrogel proved their unique strength for future applications.

Deepening the insight into poly(butylene oxide)-block-poly(glycidol) synthesis and self-assemblies: micelles, worms and vesicles.

Aqueous self-assembly of amphiphilic block copolymers is studied extensively for biomedical applications like drug delivery and nanoreactors. The commonly used hydrophilic block poly(ethylene oxide) (PEO), however, suffers from several drawbacks. As a potent alternative, poly(glycidol) (PG) has gained increasing interest, benefiting from its easy synthesis, high biocompatibility and flexibility as well as enhanced functionality compared to PEO. In this study, we present a quick and well-controlled synthesis of poly(butylene oxide)-block-poly(glycidol) (PBO-b-PG) amphiphilic diblock copolymers together with a straight-forward self-assembly protocol. Depending on the hydrophilic mass fraction of the copolymer, nanoscopic micelles, worms and polymersomes were formed as well as microscopic giant unilamellar vesicles. The particles were analysed regarding their size and shape, using dynamic and static light scattering, TEM and Cryo-TEM imaging as well as confocal laser scanning microscopy. We have discovered a strong dependence of the formed morphology on the self-assembly method and show that only solvent exchange leads to the formation of homogenous phases. Thus, a variety of different structures can be obtained from a highly flexible copolymer, justifying a potential use in biomedical applications.

Directed Insertion of Light-Activated Proteorhodopsin into Asymmetric Polymersomes from an ABC Block Copolymer.

Nanoscopic artificial vesicles containing functional protein transporters are fundamental for synthetic biology. Energy-providing modules, such as proton pumps, are a basis for simple nanoreactors. We report on the first insertion of a functional transmembrane protein into asymmetric polymersomes from an ABC triblock copolymer. The polymer with the composition poly(ethylene glycol)-poly(diisopropylaminoethyl methacrylate)-poly(styrenesulfonate) (PEG-PDPA-PSS) was synthesized by sequential controlled radical polymerization. PEG and PSS are two distinctively different hydrophilic blocks, allowing for a specific orientation of our protein, the light-activated proton pump proteorhodopsin (PR), into the final proteopolymersome. A very interesting aspect of the PEG-PDPA-PSS triblock copolymers is that it allowed for simultaneous vesicle formation and oriented insertion of PR simply by adjusting the pH. The intrinsic positive charge of PR's intracellular surface was enhanced by a His-tag, which aligns readily with the negative charges of the PSS on the outside of the polymersomes. The directed insertion of PR was confirmed by a light-dependent pH change of the proteopolymersome solution, indicating the intended orientation. We have hereby demonstrated the first successful oriented insertion of a proton pump into an artificial asymmetric membrane.

Bottom-Up Evolution of Vesicles from Disks to High-Genus Polymersomes.

Polymersomes are vesicles formed by the self-assembly of amphiphilic copolymers in water. They represent one of the most promising alternatives of natural vesicles as they add new possibilities in the amphiphiles' molecular engineering of aqueous compartments. Here we report the design of polymersomes using a bottom-up approach wherein self-assembly of amphiphilic copolymers poly(2-(methacryloyloxy) ethyl phosphorylcholine)-poly(2-(diisopropylamino) ethyl methacrylate) (PMPC-PDPA) into membranes is tuned using pH and temperature. We report evolution from disk micelles, to vesicles, to high-genus vesicles (vesicles with many holes), where each passage is controlled by pH switch or temperature. We show that the process can be rationalized, adapting membrane physics theories to disclose scaling principles that allow the estimation of minimal radius of vesiculation as well as chain entanglement and coupling. This approach allows us to generate nanoscale vesicles with genus from 0 to 70, which have been very elusive and difficult to control so far.

Dynamic Docking and Undocking Processes Addressing Selectively the Outside and Inside of Polymersomes.

Increasing complexity and diversity of polymersomes and their compartments is a key issue for mimicking cellular functions and protocells. Thus, new challenges arise in terms of achieving tunable membrane permeability and combining it with control over the membrane diffusion process, and thus enabling a localized and dynamic control of functionality and docking possibilities within or on the surface of polymeric compartments. This study reports the concept of polymersomes with pH-tunable membrane permeability for controlling sequential docking and undocking processes of small molecules and nanometer-sized protein mimics selectively on the inside and outside of the polymersome membrane as a further step toward the design of intelligent multifunctional compartments for use in synthetic biology and as protocells. Host-guest interactions between adamantane and ß-cyclodextrin as well as noncovalent interactions between poly(ethylene glycol) tails and ß-cyclodextrin are used to achieve selective and dynamic functionalization of the inner and outer spheres of the polymersome membrane.

Chemotactic synthetic vesicles: Design and applications in blood-brain barrier crossing.

In recent years, scientists have created artificial microscopic and nanoscopic self-propelling particles, often referred to as nano- or microswimmers, capable of mimicking biological locomotion and taxis. This active diffusion enables the engineering of complex operations that so far have not been possible at the micro- and nanoscale. One of the most promising tasks is the ability to engineer nanocarriers that can autonomously navigate within tissues and organs, accessing nearly every site of the human body guided by endogenous chemical gradients. We report a fully synthetic, organic, nanoscopic system that exhibits attractive chemotaxis driven by enzymatic conversion of glucose. We achieve this by encapsulating glucose oxidase alone or in combination with catalase into nanoscopic and biocompatible asymmetric polymer vesicles (known as polymersomes). We show that these vesicles self-propel in response to an external gradient of glucose by inducing a slip velocity on their surface, which makes them move in an extremely sensitive way toward higher-concentration regions. We finally demonstrate that the chemotactic behavior of these nanoswimmers, in combination with LRP-1 (low-density lipoprotein receptor-related protein 1) targeting, enables a fourfold increase in penetration to the brain compared to nonchemotactic systems.

Vesicles in Multiple Shapes: Fine-Tuning Polymersomes' Shape and Stability by Setting Membrane Hydrophobicity.

Amphiphilic block-copolymers are known to self-assemble into micelles and vesicles. In this paper, we discuss the multiple options between and beyond these boundaries using amphiphilic AB diblock and ABC triblock copolymers. We adjust the final structure reached by the composition of the mixture, by the preparation temperature, and by varying the time-scale of formation. This leads to the formation of vesicles and micelles, but also internal micelles in larger sheets, lamellar vesicles, and closed tubes, thus broadening the amount of self-assembly structures available and deepening our understanding of them.

Self-Assembly of Amphiphilic Block Copolypeptoids - Micelles, Worms and Polymersomes.

Polypeptoids are an old but recently rediscovered polymer class with interesting synthetic, physico-chemical and biological characteristics. Here, we introduce new aromatic monomers, N-benzyl glycine N-carboxyanhydride and N-phenethyl glycine N-carboxyanhydride and their block copolymers with the hydrophilic polysarcosine. We compare their self-assembly in water and aqueous buffer with the self-assembly of amphiphilic block copolypeptoids with aliphatic side chains. The aggregates in water were investigated by dynamic light scattering and electron microscopy. We found a variety of morphologies, which were influenced by the polymer structure as well as by the preparation method. Overall, we found polymersomes, worm-like micelles and oligo-lamellar morphologies as well as some less defined aggregates of interconnected worms and vesicles. Such, this contribution may serve as a starting point for a more detailed investigation of the self-assembly behavior of the rich class of polypeptoids and for a better understanding between the differences in the aggregation behavior of non-uniform polypeptoids and uniform peptoids.

Biomimetic Hybrid Nanocontainers with Selective Permeability.

Chemistry plays a crucial role in creating synthetic analogues of biomacromolecular structures. Of particular scientific and technological interest are biomimetic vesicles that are inspired by natural membrane compartments and organelles but avoid their drawbacks, such as membrane instability and limited control over cargo transport across the boundaries. In this study, completely synthetic vesicles were developed from stable polymeric walls and easy-to-engineer membrane DNA nanopores. The hybrid nanocontainers feature selective permeability and permit the transport of organic molecules of 1.5 nm size. Larger enzymes (ca. 5 nm) can be encapsulated and retained within the vesicles yet remain catalytically active. The hybrid structures constitute a new type of enzymatic nanoreactor. The high tunability of the polymeric vesicles and DNA pores will be key in tailoring the nanocontainers for applications in drug delivery, bioimaging, biocatalysis, and cell mimicry.

iRGD peptide conjugation potentiates intraperitoneal tumor delivery of paclitaxel with polymersomes.

Polymersomes are versatile nanoscale vesicles that can be used for cytoplasmic delivery of payloads. Recently, we demonstrated that pH-sensitive polymersomes exhibit an intrinsic selectivity towards intraperitoneal tumor lesions. A tumor homing peptide, iRGD, harbors a cryptic C-end Rule (CendR) motif that is responsible for neuropilin-1 (NRP-1) binding and for triggering extravasation and tumor penetration of the peptide. iRGD functionalization increases tumor selectivity and therapeutic efficacy of systemic drug-loaded nanoparticles in many tumor models. Here we studied whether intraperitoneally administered paclitaxel-loaded iRGD-polymersomes show improved efficacy in the treatment of peritoneal carcinomatosis. First, we demonstrated that the pH-sensitive polymersomes functionalized with RPARPAR (a prototypic CendR peptide) or iRGD internalize in the cells that express NRP-1, and that internalized polymersomes release their cargo inside the cytosol. CendR-targeted polymersomes loaded with paclitaxel were more cytotoxic on NRP-1-positive cells than on NRP-1-negative cells. In mice bearing peritoneal tumors of gastric (MKN-45P) or colon (CT26) origin, intraperitoneally administered RPARPAR and iRGD-polymersomes showed higher tumor-selective accumulation and penetration than untargeted polymersomes. Finally, iRGD-polymersomes loaded with paclitaxel showed improved efficacy in peritoneal tumor growth inhibition and in suppression of local dissemination compared to the pristine paclitaxel-polymersomes or Abraxane. Our study demonstrates that iRGD-functionalization improves efficacy of paclitaxel-polymersomes for intraperitoneal treatment of peritoneal carcinomatosis.

Purification of Nanoparticles by Size and Shape.

Producing monodisperse nanoparticles is essential to ensure consistency in biological experiments and to enable a smooth translation into the clinic. Purification of samples into discrete sizes and shapes may not only improve sample quality, but also provide us with the tools to understand which physical properties of nanoparticles are beneficial for a drug delivery vector. In this study, using polymersomes as a model system, we explore four techniques for purifying pre-formed nanoparticles into discrete fractions based on their size, shape or density. We show that these techniques can successfully separate polymersomes into monodisperse fractions.