Polymer Vesicles Research Articles

Rational design of elastin-like polypeptide fusion proteins to tune self-assembly and properties of protein vesicles.

Protein vesicles made from bioactive proteins have potential value in drug delivery, biocatalysis, and as artificial cells. As the proteins are produced recombinantly, the ability to precisely tune the protein sequence provides control not possible with polymeric vesicles. The tunability and biocompatibility motivated this work to develop protein vesicles using rationally designed protein building blocks to investigate how protein sequence influences vesicle self-assembly and properties. We have reported an elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper (ZR) and functional, globular proteins fused to a glutamate-rich leucine zipper (ZE) that self-assemble into protein vesicles when warmed from 4 to 25 °C due to the hydrophobic transition of ELP. Previously, we demonstrated the ability to tune vesicle properties by changing protein and salt concentration, ZE : ZR ratio, and warming rate. However, there is a limit to the properties that can be achieved via assembly conditions. In order to access a wider range of vesicle diameter and stability profiles, this work investigated how modifiying the hydrophobicity and length of the ELP sequence influenced self-assembly and the final properties of protein vesicles using mCherry as a model globular protein. The results showed that both transition temperature and diameter of protein vesicles were inversely correlated to the ELP guest residue hydrophobicity and the number of ELP pentapeptide repeats. Additionally, sequence manipulation enabled assembly of vesicles with properties not accessible by changes to assembly conditions. For example, introduction of tyrosine at 5 guest residue positions in ELP enabled formation of nanoscale vesicles stable at physiological salt concentration. This work yields design guidelines for modifying the ELP sequence to manipulate protein vesicle transition temperature, size and stability to achieve desired properties for particular biofunctional applications.

Preparation of polymeric vesicles via redox-initiated RAFT dispersion polymerization

2-(diisopropylamino) ethyl methacrylate (DIPEMA) and glycidyl methacrylate (GlyMA) were used for the investigation of the redox-initiated reversible addition-fragmentation chain transfer (RAFT) dispersion polymerization, by using poly (ethylene oxide)-4-(4-Cyanopentanoic acid) dithiobenzoate (mPEG-CPADB) as the macro-chain-transfer agent (macro-CTA). The particle growth process study indicated that the particles changed from spheres to worms, and then to vesicles during the polymerization process. The degree of polymerization (DP) of the hydrophobic block P(DIPEMA-co-GlyMA) affected the particle morphology, particle diameter and molecular weight significantly. Pure polymeric vesicles could be obtained when DP was equal to or higher than 60, and with increasing of the DP of the hydrophobic block, the diameter and the molecular weight of the particles both increased.

Generation of sub-5 nm AuNPs in the special space of the loop-cluster corona of a polymer vesicle: preparation and its unique catalytic performance in the reduction of 4-nitrophenol.

The hybrid vesicle AuNP@LCCV, in which a large number of AuNPs with an average size of about 2.8 nm were densely and uniformly distributed in an isolated state throughout the corona of the unusual polymer vesicle, was prepared via in situ reduction of Au3+ ions, which were encapsulated in advance in the unique polymer vesicle (LCCV) consisting of a hydrophobic membrane of poly(2-phenyl-2-oxazoline) and a hydrophilic loop-cluster corona of polyethyleneimine. The vesicle was formed via self-assembly from a comb-like block copolymer in which a polystyrenic main chain was grafted densely with diblock polyethyleneimine-b-poly(2-phenyl-2-oxazoline) and acted as a reactor for the reduction of Au3+. The hybrid vesicle AuNP@LCCV showed powerful catalytic ability in the reduction of nitrophenols (NPs). Interestingly, the reduction reactions of NPs showed a remarkably long induction time, which could be shortened dramatically from 60 min to 1-2 min by greatly increasing the concentration of NaBH4. It is revealed that the oxygen adsorbed on the AuNPs significantly inhibited the reduction, causing the induction time. Once the oxygen is chemically cleaned from the surface of the AuNPs, the reduction of 4-NP proceeds gradually for a while and then completes suddenly. The reduction mechanism accompanying the oxygen-dependent induction time is proposed from the view of the strong oxygen affinity of the catalyst AuNP@LCCV.

Oxygen-generating polymer vesicles for enhanced sonodynamic tumor therapy.

The efficacy of sonodynamic therapy (SDT) is often limited by the insolubility of sonosensitizers and unfavorable hypoxia tumor microenvironment. To meet this challenge, an oxygen-generating polymer vesicle is developed to achieve enhanced SDT. The hydrophilic coronas and the hydrophobic membrane of polymer vesicles can function as different modules for the simultaneous delivery of manganese dioxide and chlorine e6 (designated as Ce6-MnO2-PVs). These Ce6-MnO2-PVs exhibited high catalase mimetic activity and could efficiently generate reactive oxygen species upon ultrasound activation. In vivo results showed that Ce6-MnO2-PVs almost completely eradicated the subcutaneous tumors (94% volume reduction) without any obvious systemic toxicity. Moreover, these Ce6-MnO2-PVs showed effective behavior for the attenuation of crucial tumor progression-releated factors. Specially, the expression levels of both hypoxia-inducible factor-1α and vascular endothelial growth factor at 4 h post-injection detected by immunofluorescence were reduced by 66% and 52%, respectively. These findings suggest that Ce6-MnO2-PVs may serve as an effective and safe platform for enahnced SDT in hypoxic tumors.

Oxidation-responsive polymer vesicles with order–disorder–order multiple-phase transitions

Oxidation-responsive PEG-b-PVBMS vesicles were prepared via RAFT-mediated PISA of VBMS in ethanol/water, and their sequential oxidation-induced order–disorder–order multiple-phase transitions were reported.

PH-Dependent disruption of giant polymer vesicles: a step towards biomimetic membranes

The spatiotemporal pH-triggered controlled release of a hydrophilic probe in a pH-responsive PGUV system demonstrates its potential as a biomimetic system for drug delivery, microreactors and artificial cell mimics.

Synthesis and Application of Responsive Polymersomes

Polymersomes as drug carriers make up for the shortcomings of traditional chemotherapeutic drugs, such as easy to synthesize, short circulation time, early release, and continuous cytotoxicity. Responsive polymersomes can alter their properties such as composition, size, shape, and surface functional groups according to different stimuli, enabling the delivery of cargo at hydrophilic, hydrophobic or other specific sites, and the control of drug release. This article provides an overview of what is known about different types of responsive polymeric vesicles and describes the main design choices. In this review, the mechanism and advantages of four responsive polymersomes are discussed. Furthermore, the article focuses on the synthesis methods and applications of representative examples of each stimulus. In particular, the latest progress of four type of responsive polymersomes are emphasized. Finally, based on the great progress in the current research on responsive polymersomes, this paper discusses the potential for future development and selection of more biosafety responsive polymersomes directions.

Tuning the morphology of block copolymer-based pH-triggered nanoplatforms as driven by changes in molecular weight and protocol of manufacturing

The ability to tune size and morphology of self-assemblies is particularly relevant in the development of delivery systems. By tailoring such structural parameters, one can provide larger cargo spaces or produce nanocarriers that can be loaded by hydrophilic and hydrophobic molecules starting ideally from the same polymer building unit. We herein demonstrate that the morphology of block copolymer-based pH-triggered nanoplatforms produced from poly(2-methyl-2-oxazoline)m-b-poly[2-(diisopropylamino)-ethyl methacrylate]n (PMeOxm-b-PDPAn) is remarkably influenced by the overall molecular weight of the block copolymer, and by the selected method used to produce the self-assemblies. Polymeric vesicles were produced by nanoprecipitation using a block copolymer of relatively low molecular weight (Mn ∼ 10 kg.mol−1). Very exciting though, despite the high hydrophobic weight ratio (wPDPA > 0.70), this method conducted to the formation of core–shell nanoparticles when block copolymers of higher molecular weight were used, thus suggesting that the fast (few seconds) self-assembly procedure is controlled by kinetics rather than thermodynamics. We further demonstrated the formation of vesicular structures using longer chains via the solvent-switch approach when the “switching” to the bad solvent is performed in a time scale of a few hours (approximately 3 hs). We accordingly demonstrate that using fairly simple methods one can easily tailor the morphology of such block copolymer self-assemblies, thereby producing a variety of structurally different pH-triggered nanoplatforms via a kinetic or thermodynamically-controlled process. This is certainly attractive towards the development of nanotechnology-based cargo delivery systems.

AIE Polymer Micelle/Vesicle Photocatalysts Combined with Native Enzymes for Aerobic Photobiocatalysis

Biocatalytic transformation has attracted increasing attention in the green synthesis of chemicals due to the diversity of enzymes, their high catalytic activities and specificities, and environmentally benign conditions. Most redox enzymes in nature are dependent on nicotinamide cofactors like β-nicotinamide adenine dinucleotide (NAD+)/reduced nicotinamide adenine dinucleotide (NADH). The use of solar energy, especially visible light, in the regeneration of cofactors through the combination of photocatalysis and biocatalysis provides an extraordinary opportunity to make complete green processes. However, the combination of photocatalysts and enzymes has been challenged by the rapid degradation and deactivation of the enzymatic material by photogenerated reactive oxygen species (ROS). Here, we design core-shell structured polymer micelles and vesicles with aggregation-induced emission (AIE) as visible-light-mediated photocatalysts for highly stable and recyclable photobiocatalysis under aerobic conditions. NAD+ from NADH can be efficiently regenerated by the photoactive hydrophobic core of polymer micelles and the hydrophobic membrane of polymer vesicles, while the enzymatic material (glucose 1-dehydrogenase) is screened from the attack of photogenerated ROS by the hydrophilic surface layer of polymer colloids. After at least 10 regeneration cycles, the enzyme keeps its active state; meanwhile, polymer micelles and vesicles maintain their photocatalytic activity. These polymer colloids show the potential to be developed for the implementation of industrially relevant photobiocatalytic systems.

Microwave Irradiation-Assisted Reversible Addition-Fragmentation Chain Transfer Polymerization-Induced Self-Assembly of pH-Responsive Diblock Copolymer Nanoparticles.

Herein, we present a versatile platform for the synthesis of pH-responsive poly([N-(2-hydroxypropyl)]methacrylamide)-b-poly[2-(diisopropylamino)ethyl methacrylate] diblock copolymer (PHPMA-b-PDPA) nanoparticles (NPs) obtained via microwave-assisted reversible addition-fragmentation chain transfer polymerization-induced self-assembly (MWI-PISA). The N-(2-hydroxypropyl) methacrylamide (HPMA) monomer was first polymerized to obtain a macrochain transfer agent with polymerization degrees (DPs) of 23 and 51. Subsequently, using mCTA and 2-(diisopropylamino)ethyl methacrylate (DPA) as monomers, we successfully conducted MWI-PISA emulsion polymerization in aqueous solution with a solid content of 10 wt %. The NPs were obtained with high monomer conversion and polymerization rates. The resulting diblock copolymer NPs were analyzed by dynamic light scattering (DLS) and cryogenic-transmission electron microscopy (cryo-TEM). cryo-TEM studies reveal the presence of only NPs with spherical morphology such as micelles and polymer vesicles known as polymersomes. Under the selected conditions, we were able to fine-tune the morphology from micelles to polymersomes, which may attract considerable attention in the drug-delivery field. The capability for drug encapsulation using the obtained in situ pH-responsive NPs, the polymersomes based on PHPMA23-b-PDPA100, and the micelles based on PHPMA51-b-PDPA100 was demonstrated using the hydrophobic agent and fluorescent dye as Nile red (NR). In addition, the NP disassembly in slightly acidic environments enables fast NR release.

Overcoming the Dilemma of Permeability and Stability of Polymersomes through Traceless Cross-Linking.

In nature, cells are highly compartmentalized into many organelles that are well separated from the rest of the cellular space by unique membrane structures, which are of crucial importance to allow cells to perform various physiological functions in such a small and crowded space. Learning from the ubiquitous membrane structures of cells and organelles has continuously inspired the development of artificial self-assembled nanostructures, with lipid vesicles (liposomes) and polymer vesicles (polymersomes) being the most representative examples. Similar to the membrane-bound structures of cells and organelles, both liposomes and polymersomes contain an aqueous interior enclosed by a bilayer membrane. Therefore, liposomes and polymersomes have been extensively investigated to mimic the fundamental structures and functions of living cells. For example, liposomes and polymersomes have been successfully engineered as nanocarriers, smart nanoreactors, artificial organelles, and so on. Notably, living cells can exchange both energy and materials with surrounding environments, benefiting from the selective permeability of lipid membranes. The permselectivity of cell membranes is thus an essential attribute of living organisms. Compared to liposomes, polymersomes have increased structural stability but low membrane permeability. Indeed, polymersomes are almost impermeable to small molecules, ions, and even water molecules. To improve the permeability of polymersomes, much effort has been devoted to the incorporation of channel proteins, the coassembly of oppositely charged block copolymers (BCPs), the development of stimuli-responsive BCPs, and so on. Despite great achievements, these approaches generally lead to decreased stability of polymersomes and, sometimes, polymersome disintegration. In this Account, we discuss our recent efforts to reconcile the stability and permeability of polymersomes via a traceless cross-linking approach. Although cross-linking reactions within bilayer membranes generally lead to decreased permeability, the traceless cross-linking approach can concurrently improve the stability and permeability of polymersomes. Specifically, stimuli-responsive polymersomes undergo either covalent cross-linking or noncovalent cross-linking reactions under specific stimuli to increase bilayer stability, while the cross-linking processes can concurrently permeabilize polymersome bilayers through cross-linking-driven hydrophobic-to-hydrophilic transitions. Notably, unlike conventional cross-linking processes requiring additional cross-linkers, the traceless cross-linking process does not involve extra cross-linking agents but takes full advantage of the in situ generated active moieties. By taking advantage of the simultaneous modulation of the stability and permeability of polymersomes via traceless cross-linking, these polymersomes can be further engineered as smart nanocarriers and nanoreactors. The robustness and generality of this approach have been validated by both extracellular and intracellular stimuli such as light irradiation, glutathione, and hydrogen peroxide. Moreover, many functional groups such as fluorescent dyes and contrast agents can be integrated into this versatile platform as well, enabling the construction of theranostic nanovectors capable of responding to pathological microenvironments. This Account provides a new approach to regulating the permeability of polymersomes while maintaining their structural stability.

Potential of Small‐Angle Neutron Scattering for Evaluating Protein Locus within a Polymersome

AbstractPost‐loading of polymersomes with different bio(macro)molecules has been successfully demonstrated, thus mimicking the diffusion processes through biological membranes. However, it is still an open issue the extent to which this diffusion process leads to transmembrane transportation, or rather encapsulation of cargo within the membrane. In this study, well‐established pH‐responsive and cross‐linked polymeric vesicles are studied. A pH‐controllable and stable membrane, as well as a hollow particle shape and membrane uniformity are confirmed using dynamic light scattering (DLS) and cryogenic‐transmission electron microscopy (Cryo‐TEM). Post‐loading with myoglobin (Mb) as a model enzyme is analyzed using multidetector asymmetrical flow field‐flow fractionation (AF4). Advanced analysis of conformational parameters allowed for the estimation of enzyme localization and the pH‐dependent loading efficiency thereof. Static light scattering coupled to AF4 is employed to successfully deliver information on the global size of the polymersomes (>50 nm). Furthermore, membrane structure and thickness, which are in the few nanometer range, can be successfully analyzed using small‐angle neutron scattering (SANS). Deuterated solvent as well as Mb deuteration for tuning the contrast are considered. Two different vesicle model fits, as well as Kratky–Porod interpretation confirm effective determination of vesicle core and membrane thickness and for evaluation of the membrane changes after post‐loading.

Synthetic Biology: Bottom-Up Assembly of Molecular Systems.

The bottom-up assembly of biological and chemical components opens exciting opportunities to engineer artificial vesicular systems for applications with previously unmet requirements. The modular combination of scaffolds and functional building blocks enables the engineering of complex systems with biomimetic or new-to-nature functionalities. Inspired by the compartmentalized organization of cells and organelles, lipid or polymer vesicles are widely used as model membrane systems to investigate the translocation of solutes and the transduction of signals by membrane proteins. The bottom-up assembly and functionalization of such artificial compartments enables full control over their composition and can thus provide specifically optimized environments for synthetic biological processes. This review aims to inspire future endeavors by providing a diverse toolbox of molecular modules, engineering methodologies, and different approaches to assemble artificial vesicular systems. Important technical and practical aspects are addressed and selected applications are presented, highlighting particular achievements and limitations of the bottom-up approach. Complementing the cutting-edge technological achievements, fundamental aspects are also discussed to cater to the inherently diverse background of the target audience, which results from the interdisciplinary nature of synthetic biology. The engineering of proteins as functional modules and the use of lipids and block copolymers as scaffold modules for the assembly of functionalized vesicular systems are explored in detail. Particular emphasis is placed on ensuring the controlled assembly of these components into increasingly complex vesicular systems. Finally, all descriptions are presented in the greater context of engineering valuable synthetic biological systems for applications in biocatalysis, biosensing, bioremediation, or targeted drug delivery.

Sustainable colorimetric/luminescent sensors enabled by armored lipid nanoparticles

In this study, we developed a highly stable polymeric vesicle using a nanosilica-armor membrane to achieve a sustainable colorimetric/luminescent response. The silica armor can be grown directly as ~ 5 nm spherical nanoparticles on the surface of the diacetylene (DA) vesicle with liposomal structure. This can be accomplished via the modified Stöber reaction in pure water on a layer of amine linkers deposited on the vesicles. Once formed, the structural stability of the DA vesicles dramatically increased and remained so even in a dried powder form that could be stored for a period of approximately 6 months. Then, redispersed in water, the armored vesicles did not agglomerate because of the electric charge of the silica armor. After polymerization, the polydiacetylene (PDA) vesicles maintained an average of 87.4% their sensing capabilities compared to unstored vesicles. Furthermore, the silica membrane thickness can be controlled by reiteration of the electrostatic layer-by-layer approach and the direct hydrolysis of silica. As the number of silica armor membranes increases, the passage of the stimuli passing through the membranes becomes longer. Consequently, three layers of silica armor gave the PDA vesicles size-selective recognition to filter out external stimuli. These discoveries are expected to have large-scale effects in the chemo- and biosensor fields by applying protective layers to organic nanomaterials.Graphical

Fast and Reversible Photoresponsive Self-Assembly Behavior of Rosin-Based Amphiphilic Polymers.

Designing stimulus-responsive amphiphilic polymers with a fast photoresponsive self-assembly behavior remains a challenge. Two series of rosin-terminated and azobenzene-terminated amphiphilic polymers (PAMn and PMAn) with fast and reversible photoresponsive properties were prepared using rosin-based azobenzene groups and polyethylene glycol, respectively. Under 5-10 s of UV irradiation, the polymers showed trans-to-cis isomerization and reached a photosteady state. For the PAMn polymer, the absorbance of the absorption peak at 325 nm recovered to more than 95% of the initial value under visible light for 5-10 s, whereas that of the PMAn polymer recovered completely. Notably, the PAMn and PMAn polymers initially self-assembled to vesicles or spherical micelles, and various morphological changes were achieved by manipulating UV irradiation time, with the initial morphology again recovered under dark conditions or visible-light irradiation. Remarkably, vesicles of the PAM34 and PMA34 polymers presented an intermediate open-vesicle state before being completely deformed under UV irradiation because of the existence of a π-π interaction. Finally, the ability of PAM34 and PMA34 polymer vesicles to perform the controlled release and reversible loading of a fluorescent probe was evaluated.

Evaluating the integrity of polymersomes by FRET for optimization of the lyophilization parameters

Polymersomes (polymeric vesicles) are non-viral delivery vehicles self-assembled from block copolymers (BCPs), holding promising potential in biomedical applications. Despite the higher chemical stabilities of the BCPs, lyophilization is still required for the long-term preservation of the polymersomes and their encapsulated drugs. However, formulation and processing parameters can affect the efficiency of the lyophilization. In this work, we present an approach to evaluate the integrity of polymersomes based on Fluorescence Resonance Energy Transfer (FRET) for rapid optimization of the lyophilization parameters. Specifically, Coumarin 6 (C6) and Nile red (NR)-encapsulated polymersomes were fabricated separately and mixed together for lyophilization. Upon reconstitution, FRET was detected for polymersomes which lost integrity during the lyophilization or reconstitution. The evaluation was carried out for poly(ethylene glycol)-b-poly(lactic acid) (PEG-b-PLA) polymersomes lyophilized with eight different excipients, and the results showed that the best polymersomes integrity could be achieved with the addition of hydroxypropyl-beta-cyclodextrin (HP-β-CD). We believe the approach described here would greatly facilitate the optimization of the polymersomes lyophilization, accelerating the translation of polymersomes to practical applications.

Fabrication of Polymersomes: A Macromolecular Architecture in Nanotherapeutics

In consideration of the issues of drug delivery systems, the artificial vesicle structures composed of block copolymers called polymersomes recently gained considerable attention. The possibility of tuning the mechanical parameter and increasing the scale-up production of polymersomes led to its wide application in healthcare. Bearing in mind the disease condition, the structure and properties of the polymersomes could be tuned to serve the purpose. Furthermore, specific ligands can be incorporated on the vesicular surface to induce smart polymersomes, thus improving targeted delivery. The synthesis method and surface functionalization are the two key aspects that determine the versatility of biological applications as they account for stability, specific targeting, degradability, biocompatibility, and bioavailability. A perfectly aligned polymer vesicle can mimic the cells/organelles and function by avoiding cytotoxicity. This supramolecular structure can carry and deliver payloads of a wide range, including drugs, proteins, and genes, contributing to the construction of next-generation therapeutics. These aspects promote the potential use of such components as a framework to approach damaged tissue while maintaining healthy environments during circulation. Herein, this article concentrates specifically on the drug delivery applications of polymersomes.

Mean-field models of size selection in polymer vesicles with and without interdigitation

ABSTRACT Block copolymers in solution can form vesicles with a preferred size when the degree of polymerisation of the hydrophobic block is significantly greater than that of the hydrophilic block. In an earlier publication, we introduced a mean-field theory that treats the vesicle membrane as a bilayer. Here, a model is presented in which the hydrophobic blocks form a single interdigitated layer. In the model with interdigitation, the vesicles still have a preferred radius, but this increases more slowly as a function of than in the bilayer model, in better agreement with experiment. However, the predictions of the bilayer model for the relationship between and the vesicle membrane thickness are consistent with the experimental result over a wide range of parameters, while the model with interdigitation predicts an exponent of around . This suggests that the structure of the vesicles may lie between these two cases.

Membrane Manipulation of Giant Unilamellar Polymer Vesicles with a Temperature-Responsive Polymer.

Understanding the complex behavior and dynamics of cellular membranes is integral to gain insight into cellular division and fusion processes. Bottom-up synthetic cells are as a platform for replicating and probing cellular behavior. Giant polymer vesicles are more robust than liposomal counterparts, as well as having a broad range of chemical functionalities. However, the stability of the membrane can prohibit dynamic processes such as membrane phase separation and division. Here, we present a method for manipulating the membrane of giant polymersomes using a temperature responsive polymer. Upon elevation of temperature deformation and phase separation of the membrane was observed. Upon cooling, the membrane relaxed and became homogeneous again, with infrequent division of the synthetic cells.

Membrane Manipulation of Giant Unilamellar Polymer Vesicles with a Temperature‐Responsive Polymer

AbstractUnderstanding the complex behavior and dynamics of cellular membranes is integral to gain insight into cellular division and fusion processes. Bottom‐up synthetic cells are as a platform for replicating and probing cellular behavior. Giant polymer vesicles are more robust than liposomal counterparts, as well as having a broad range of chemical functionalities. However, the stability of the membrane can prohibit dynamic processes such as membrane phase separation and division. Here, we present a method for manipulating the membrane of giant polymersomes using a temperature responsive polymer. Upon elevation of temperature deformation and phase separation of the membrane was observed. Upon cooling, the membrane relaxed and became homogeneous again, with infrequent division of the synthetic cells.