Aqueous two-phase systems (ATPSs) are liquid-liquid equilibria between two aqueous phases that usually contain over 70% water content each, which results in a nontoxic organic solvent-free environment for biological compounds and biomolecules. ATPSs have attracted significant interest in applications for formulating carriers (microparticles, nanoparticles, hydrogels, and polymersomes) which can be prepared using the spontaneous phase separation of ATPSs as a driving force, and loaded with a wide range of bioactive materials, including small molecule drugs, proteins, and cells, for delivery applications. This review provides a detailed analysis of various ATPSs, including strategies employed for particle formation, polymerization of droplets in ATPSs, phase-guided block copolymer assemblies, and stimulus-responsive carriers. Processes for loading various bioactive payloads are discussed, and applications of these systems for drug delivery are summarized and discussed.

Therapeutics based on proteins and peptides have profoundly transformed the landscape of treatment for diseases, from diabetes mellitus to cancers, yet the short half-life and low bioavailability of therapeutic proteins and peptides hinder their wide applications. To break through this bottleneck, biomolecules-loaded polymersomes with strong adjustability and versatility have attracted more and more attentions recently. Loading proteins or peptides into polymersomes is the first but extremely important step towards developing high-quality formulation products. However, increasing protein and peptide loading content is quite challenging due to the inherent nature of self-assembled vesicle formation mechanism and physiochemical characteristics of biomacromolecules. This review highlights the potential of polymersomes as the next-generation therapeutic proteins and peptides carrier and emphatically introduces novel approaches and recent progress to achieve satisfactory encapsulation capability of polymersomes for proteins and peptides. On the one hand, with the help of intermolecular interactions, such as electrostatic, lipid-protein, and hydrophobic interactions, the drug loading could be significantly improved. On the other hand, loading improvement could be attained through innovation of preparation methods, ranging from modified traditional film hydration techniques to the novel phase-guided assembly method.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Despite ongoing advancements in research, the inability of therapeutics to cross the blood-brain barrier (BBB) makes the treatment of neurological disorders (NDs) a challenging task, offering only partial symptomatic relief. Various adverse effects associated with existing approaches are another significant barrier that prompts the usage of structurally diverse phytochemicals as preventive/therapeutic lead against NDs in preclinical and clinical settings. Despite numerous beneficial properties, phytochemicals suffer from poor pharmacokinetic profile which limits their pharmacological activity and necessitates the utility of nanotechnology for efficient drug delivery. Nanocarriers have been shown to be proficient carriers that can enhance drug delivery, bioavailability, biocompatibility, and stability of phytochemicals. We, thus, conducted a meticulous literature survey using several electronic databases to gather relevant studies in order to provide a comprehensive summary about the use of nanocarriers in delivering phytochemicals as a treatment approach for NDs. Additionally, the review highlights the mechanisms of drug transport of nanocarriers across the BBB and explores their potential future applications in this emerging field.

Multicompartment structures have the potential for biomedical applications because they can act as multifunctional systems and provide simultaneous delivery of drugs and diagnostics agents of different types. Moreover, some of them mimic biological cells to some extent with organelles as separate sub-compartments. This article analyses multicompartment colloidal structures with smaller sub-units covered with lipid or polymer membranes that provide additional protection for the encapsulated substances. Vesosomes with small vesicles encapsulated in the inner pools of larger liposomes are the most studied systems to date. Dendrimer molecules are enclosed by a lipid bilayer shell in dendrosomes. Capsosomes, polymersomes-in-polymer capsules, and cubosomes-in-polymer capsules are composed of sub-compartments encapsulated within closed multilayer polymer membranes. Janus or Cerberus emulsions contain droplets composed of two or three phases: immiscible oils in O/W emulsions and aqueous polymer or salt solutions that are separated into two or three phases and form connected droplets in W/O emulsions. In more cases, the external surface of engulfed droplets in Janus or Cerberus emulsions is covered with a lipid or polymer monolayer. eLiposomes with emulsion droplets encapsulated into a bilayer shell have been given little attention so far, but they have very great prospects. In addition to nanoemulsion droplets, solid lipid nanoparticles, nanostructured lipid carriers and inorganic nanoparticles can be loaded into eLiposomes. Molecular engineering of the external membrane allows the creation of ligand-targeted and stimuli-responsive multifunctional systems. As a result, the efficacy of drug delivery can be significantly enhanced.

A bottom-up approach to fabricating monodisperse, two-component polymersomes that possess phase-separated ("patchy") chemical topology is presented. This approach is compared with already-existing top-down preparation methods for patchy polymer vesicles, such as film rehydration. These findings demonstrate a bottom-up, solvent-switch self-assembly approach that produces a high yield of nanoparticles of the target size, morphology, and surface topology for drug delivery applications, in this case patchy polymersomes of a diameter of ≈50 nm. In addition, an image processing algorithm to automatically calculate polymersome size distributions from transmission electron microscope images based on a series of pre-processing steps, image segmentation, and round object identification is presented.

Protein-based therapeutics are an attractive alternative to established therapeutic approaches and represent one of the fastest growing families of drugs. While many of these proteins can be delivered using established formulations, the intrinsic sensitivity of proteins to denaturation sometimes calls for a protective carrier to allow administration. Historically, lipid-based self-assembled structures, notably liposomes, have performed this function. After the discovery of polymersome-based targeted drug-delivery systems, which offer manifold advantages over lipid-based structures, the scientific community expected that such systems would take the therapeutic world by storm. However, no polymersome formulations have been commercialised. In this review article, we discuss key obstacles for the sluggish translation of polymersome-based protein nanocarriers into approved pharmaceuticals, which include limitations imparted by the use of non-degradable polymers, the intricacies of polymersome production methods, and the complexity of the in vivo journey of polymersomes across various biological barriers. Considering this complex subject from a polymer chemist's point of view, we highlight key areas that are worthy to explore in order to advance polymersomes to a level at which clinical trials become worthwhile and translation into pharmaceutical and nanomedical applications is realistic.

Membrane proteins (MPs) play key roles in cellular signaling pathways and are responsible for intercellular and intracellular interactions. Dysfunctional MPs are directly related to the pathogenesis of various diseases, and they have been exploited as one of the most sought-after targets in the pharmaceutical industry. However, working with MPs is difficult given that their amphiphilic nature requires protection from biological membrane or membrane mimetics. Polymersomes are bilayered nano-vesicles made of self-assembled block copolymers that have been widely used as cell membrane mimetics for MP reconstitution and in engineering of artificial cells. This review highlights the prevailing trend in the application of polymersomes in MP study and drug discovery. We begin with a review on the techniques for synthesis and characterization of polymersomes as well as methods of MP insertion to form proteopolymersomes. Next, we review the structural and functional analysis of the different types of MPs reconstituted in polymersomes, including membrane transport proteins, MP complexes, and membrane receptors. We then summarize the factors affecting reconstitution efficiency and the quality of reconstituted MPs for structural and functional studies. Additionally, we discuss the potential in using proteopolymersomes as platforms for high-throughput screening (HTS) in drug discovery to identify modulators of MPs. We conclude by providing future perspectives and recommendations on advancing the study of MPs and drug development using proteopolymersomes.

We report a new aqueous polymerization-induced self-assembly (PISA) formulation that enables the hydrophobic block to be prepared first when targeting diblock copolymer nano-objects. This counter-intuitive reverse sequence approach uses an ionic reversible addition-fragmentation chain transfer (RAFT) agent for the RAFT aqueous dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA) to produce charge-stabilized latex particles. Chain extension using a water-soluble methacrylic, acrylic or acrylamide comonomer then produces sterically stabilized diblock copolymer nanoparticles in an aqueous one-pot formulation. In each case, the monomer diffuses into the PHPMA particles, which act as the locus for the polymerization. A remarkable change in morphology occurs as the ≈600 nm latex is converted into much smaller sterically stabilized diblock copolymer nanoparticles, which exhibit thermoresponsive behavior. Such reverse sequence PISA formulations enable the efficient synthesis of new functional diblock copolymer nanoparticles.

We compare the fusion of giant lipid and block-copolymer vesicles on glass and poly(dimethylsiloxane) substrates. Both types of vesicles are similar in their ability to fuse to hydrophilic substrates and form patches with distinct heart or circular shapes. We use epifluorescence/confocal microscopy and atomic force microscopy on membrane patches to (i) characterize bilayer fluidity and patch-edge stability and (ii) follow the intermediate stages in the formation of continuous supported bilayers. Polymer membranes show much lower membrane fluidity and, unlike lipids, an inability of adjacent patches to fuse spontaneously into continuous membranes. We ascribe this effect to hydration repulsion forces acting between the patch edges, which can be diminished by increasing the sample temperature. We show that large areas of supported polymer membranes can be created by fusing giant vesicles on glass or poly(dimethylsiloxane) substrates and annealing their edges.

A nanoreactor containing an evolved mutant of Saccharolobus solfataricus phosphotriesterase (L72C/Y97F/Y99F/W263V/I280T) as a catalytic bioscavenger was made for detoxification of organophosphates. This nanoreactor intended for treatment of organophosphate poisoning was studied against paraoxon (POX). Nanoreactors were low polydispersity polymersomes containing a high concentration of enzyme (20 μM). The polyethylene glycol-polypropylene sulfide membrane allowed for penetration of POX and exit of hydrolysis products. In vitro simulations under second order conditions showed that 1 μM enzyme inactivates 5 μM POX in less than 10 s. LD₅₀-shift experiments of POX-challenged mice through intraperitoneal (i.p.) and subcutaneous (s.c.) injections showed that intravenous administration of nanoreactors (1.6 nmol enzyme) protected against 7 × LD₅₀ i.p. in prophylaxis and 3.3 × LD₅₀ i.p. in post-exposure treatment. For mice s.c.-challenged, LD₅₀ shifts were more pronounced: 16.6 × LD₅₀ in prophylaxis and 9.8 × LD₅₀ in post-exposure treatment. Rotarod tests showed that transitory impaired neuromuscular functions of challenged mice were restored the day of experiments. No deterioration was observed in the following days and weeks. The high therapeutic index provided by prophylactic administration of enzyme nanoreactors suggests that no other drugs are needed for protection against acute POX toxicity. For post-exposure treatment, co-administration of classical drugs would certainly have beneficial effects against transient incapacitation.

Polymer-based gene delivery relies on the binding, protection, and final release of nucleic acid cargo using polycations. Engineering polymeric vectors, by exploring novel topologies and cationic moieties, is a promising avenue to improve their performance, which hinges on the development of simple synthetic methods that allow facile preparation. In this work, we focus on cationic micelles formed from block polymers, which are examined as promising gene compaction agents and carriers. In this study, we report the synthesis and assembly of six amphiphilic poly(n-butyl acrylate)-b-poly(cationic acrylamide) diblock polymers with different types of cationic groups ((dialkyl)amine, morpholine, or imidazole) in their hydrophilic corona. The polycations were obtained through the parallel postpolymerization modification of a poly(n-butyl acrylate)-b-poly(pentafluorophenyl acrylate) reactive scaffold, which granted diblock polymers with equivalent degrees of polymerization and subsequent quantitative functionalization with cations of different pK_a. Ultrasound-assisted direct dissolution of the polycations in different aqueous buffers (pH = 1-7) afforded micellar structures with low size dispersities and hydrodynamic radii below 100 nm. The formation and properties of micelle-DNA complexes ("micelleplexes") were explored via DLS, zeta potential, and dye-exclusion assays revealing that binding is influenced by the cation type present in the micelle corona where bulkiness and pK_a are the drivers of micelleplex formation. Combining parallel synthesis strategies with simple direct dissolution formulation opens opportunities to optimize and expand the range of micelle delivery vehicles available by facile tuning of the composition of the cationic micelle corona.

Biomedical applications of polymersomes have been explored, including drug and gene delivery, insulin delivery, hemoglobin delivery, the delivery of anticancer agents, and various diagnostic purposes.

Polymersomes, which are self-assembled amphiphilic block copolymers, have received a lot of attention in drug delivery approaches. This review represents the methods of preparation of polymersomes including thin-film rehydration, electroformation, double emulsion, gel-assisted rehydration, PAPYRUS method, and solvent injection methods including various therapeutic applications of polymersomes.

Data we searched from PubMed, Google Scholar, and Science Direct through searching of keywords: Polymersomes, methods of preparation, amphiphilic block copolymers, anticancer drug delivery Results: Polymersomes provide both hydrophilic and hydrophobic drug delivery to a targeted site with an increase in the stability of the formulation and reduce the cytotoxic side effects of drugs.

A wide range of biological applications, including drug and gene delivery, insulin delivery, hemoglobin delivery, delivery of anticancer agents as well as in various diagnostic purposes. Recently, polymersomes have been used more frequently because of their stability, reducing the encapsulated drug's leakage, site-specific drug delivery, and increasing the bioavailability of the drugs and different diagnostic purposes. The liposomes encapsulate only hydrophilic drugs, but polymersomes encapsulate both hydrophilic and hydrophobic drugs in their cores.

Among non-viral vectors, cationic polymers, such as poly(propylene imine) (PPI), play a prominent role in nucleic acid delivery. However, limitations of polycationic polymer-based DNA delivery systems are (i) insufficient target specificity, (ii) unsatisfactory transgene expression, and (iii) undesired transfer of therapeutic DNA into non-target cells. We developed single-chain antibody fragment (scFv)-directed hybrid polyplexes for targeted gene therapy of prostate stem cell antigen (PSCA)-positive tumors. Besides mono-biotinylated PSCA-specific single-chain antibodies (scFv(AM1-P-BAP)) conjugated to neutravidin, the hybrid polyplexes comprise β-cyclodextrin-modified PPI as well as biotin/maltose-modified PPI as carriers for minicircle DNAs encoding for Sleeping Beauty transposase and a transposon encoding the gene of interest. The PSCA-specific hybrid polyplexes efficiently delivered a GFP gene in PSCA-positive tumor cells, whereas control hybrid polyplexes showed low gene transfer efficiency. In an experimental gene therapy approach, targeted transposition of a codon-optimized p53 into p53-deficient HCT116^p53-/-/PSCA cells demonstrated decreased clonogenic survival when compared to mock controls. Noteworthily, p53 transposition in PTEN-deficient H4^PSCA glioma cells caused nearly complete loss of clonogenic survival. These results demonstrate the feasibility of combining tumor-targeting hybrid polyplexes and Sleeping Beauty gene transposition, which, due to the modular design, can be extended to other target genes and tumor entities.

Cancer therapy is a significant challenge due to insufficient drug delivery to the cancer cells and non-selective killing of healthy cells by most chemotherapy agents. Nano-formulations have shown great promise for targeted drug delivery with improved efficiency. The shape and size of nanocarriers significantly affect their transport inside the body and internalization into the cancer cells. Non-spherical nanoparticles have shown prolonged blood circulation half-lives and higher cellular internalization frequency than spherical ones. Nanodiscs are desirable nano-formulations that demonstrate enhanced anisotropic character and versatile functionalization potential. Here, we review the recent development of theranostic nanodiscs for cancer mitigation ranging from traditional lipid nanodiscs encased by membrane scaffold proteins to newer nanodiscs where either the membrane scaffold proteins or the lipid bilayers themselves are replaced with their synthetic analogues. We first discuss early cancer detection enabled by nanodiscs. We then explain different strategies that have been explored to carry a wide range of payloads for chemotherapy, cancer gene therapy, and cancer vaccines. Finally, we discuss recent progress on organic-inorganic hybrid nanodiscs and polymer nanodiscs that have the potential to overcome the inherent instability problem of lipid nanodiscs.

Under certain cellular conditions, functional proteins undergo misfolding, leading to a transition into oligomers which precede the formation of amyloid fibrils. Misfolding proteins are associated with neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. While the importance of lipid membranes in misfolding and disease aetiology is broadly accepted, the influence of lipid membranes during therapeutic design has been largely overlooked. This study utilized a biophysical approach to provide mechanistic insights into the effects of two lipid membrane systems (anionic and zwitterionic) on the inhibition of amyloid-β 40 and α-synuclein amyloid formation at the monomer, oligomer and fibril level. Large unilamellar vesicles (LUVs) were shown to increase fibrillization and largely decrease the effectiveness of two well-known polyphenol fibril inhibitors, (-)-epigallocatechin gallate (EGCG) and resveratrol; however, use of immunoblotting and ion mobility mass spectrometry revealed this occurs through varying mechanisms. Oligomeric populations in particular were differentially affected by LUVs in the presence of resveratrol, an elongation phase inhibitor, compared to EGCG, a nucleation targeted inhibitor. Ion mobility mass spectrometry showed EGCG interacts with or induces more compact forms of monomeric protein typical of off-pathway structures; however, binding is reduced in the presence of LUVs, likely due to partitioning in the membrane environment. Competing effects of the lipids and inhibitor, along with reduced inhibitor binding in the presence of LUVs, provide a mechanistic understanding of decreased inhibitor efficacy in a lipid environment. Together, this study highlights that amyloid inhibitor design may be misguided if effects of lipid membrane composition and architecture are not considered during development.

Hierarchic self-assembly underpins much of the form and function seen in synthetic or biological soft materials. Lipids are paramount examples, building themselves in nature or synthetically in a variety of meso/nanostructures. Synthetic block copolymers capture many of lipid's structural and functional properties. Lipids are typically biocompatible and high molecular weight polymers are mechanically robust and chemically versatile. The development of new materials for applications like controlled drug/gene/protein delivery, biosensors, and artificial cells often requires the combination of lipids and polymers. The emergent composite material, a "polymer-lipid hybrid membrane", displays synergistic properties not seen in pure components. Specific examples include the observation that hybrid membranes undergo lateral phase separation that can correlate in registry across multiple layers into a three-dimensional phase-separated system with enhanced permeability of encapsulated drugs. It is timely to underpin these emergent properties in several categories of hybrid systems ranging from colloidal suspensions to supported hybrid films. In this review, we discuss the form and function of a vast number of polymer-lipid hybrid systems published to date. We rationalize the results to raise new fundamental understanding of hybrid self-assembling soft materials as well as to enable the design of new supramolecular systems and applications.

Nano drug delivery systems have attracted researchers' growing attention and are gradually emerging into the public views. More and more nano-formulations are being approved for marketing or clinical use, representing the field's booming development. Copolymer self-assembly systems such as micelles, nanoparticles, polymersomes occupy a prominent position in the field of nano-drug delivery carriers. Among them, polymersomes, unlike micelles or nanoparticles, resemble liposomes' structure and possess large internal hollow hydrophilic reservoirs, allowing them to carry hydrophilic drugs. Nevertheless, their insufficient drug loading efficiency and unruly self-assembly morphology have somewhat constrained their applications. Especially for the delivery of biomacromolecule such as peptides, the encapsulation efficiency is always considered to be a formidable obstacle, even if the enormous hydrophilic core would render the polymersomes to have considerable potential in this regard. Reassuringly, the emergence of asymmetric polymersomes holds the prospect of solving this problem. With the development of synthetic technology and a deeper understanding of the self-assembly process, the asymmetric polymersomes which are with different inner and outer shell composition have been gradually recognized by researchers. It has made possible elevated drug loading, more controllable assembly processes and release performance. The internal hydrophilic blocks different from the outer shell could be engineered to have a more remarkable affinity to the cargos or could contain a non-watery aqueous phase to enable the thermodynamically preferred encapsulation of cargos, which would allow for a substantial improvement in drug encapsulation efficiency compared to the conventional approach. In this paper, we aim to deepen the understanding to asymmetric polymersomes and lay the foundation for the development of this field by describing four main elements: the mechanism of their preparation and asymmetric membrane formation process, the characterization of asymmetric membranes, the efficient drug loading, and the special stimulus-responsive release mechanism.

A variety of artificial cells springs from the functionalization of liposomes with proteins. However, these models suffer from low durability without repair and replenishment mechanisms, which can be partly addressed by replacing the lipids with polymers. Yet natural membranes are also dynamically remodeled in multiple cellular processes. Here, we show that synthetic amphiphile membranes also undergo fusion, mediated by the protein machinery for synaptic secretion. We integrated fusogenic SNAREs in polymer and hybrid vesicles and observed efficient membrane and content mixing. We determined bending rigidity and pore edge tension as key parameters for fusion and described its plausible progression through cryo-EM snapshots. These findings demonstrate that dynamic membrane phenomena can be reconstituted in synthetic materials, thereby providing new tools for the assembly of synthetic protocells.

Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293T^PSCA/ffLuc and PC3^PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3^PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition.

Polymersomes are a class of synthetic vesicles composed of a polymer membrane surrounding an aqueous inner cavity. In addition to their overall size, the thickness and composition of polymersome membranes determine the range of potential applications in which they can be employed. While synthetic polymer chemists have made great strides in controlling polymersome membrane parameters, measurement of their permeability to various analytes including gases, ions, organic molecules, and macromolecules remains a significant challenge. In this Outlook, we compare the general methods that have been developed to quantify polymersome membrane permeability, focusing in particular on their capability to accurately measure analyte flux. In addition, we briefly highlight strategies to control membrane permeability. Based on these learnings, we propose a set of criteria for designing future methods of quantifying membrane permeability such that the passage of a variety of molecules into and out of their lumens can be better understood.

Self-assembled polymer nanoparticles have tremendous potential in biomedical and environmental applications. For all applications, tailored polymer chemistries are critical. In this study, we demonstrate a precursor approach in which an activated, organic solvent-soluble block polymer precursor is modified through mild postpolymerization modifications to access new polymer structures. We synthesized and characterized poly(isoprene)-block-poly(di-Boc acrylamide) diblock polymers. This activated-acrylamide-based polymer was then reacted with amines or reductants in the absence of catalysts to yield the hydrophilic blocks polyacrylamide, poly(hydroxypropylene), and poly(N-ethyl acrylamide). The resulting amphiphilic block polymers self-assembled in water to form polymersomes, as confirmed by cryo-electron microscopy and confocal microscopy. The approach also enables simple functionalization with specialized ligands, which we demonstrated by tagging polymers with an amino-fluorophore and imaging by confocal microscopy. We expect that the methodologies established in this study will open doors to new and useful solution nanostructures with surface chemistries that can be optimized for various applications.

Compartmentalization and spatial organization of biochemical reactions are essential for the establishment of complex metabolic pathways inside synthetic cells. Phospholipid and fatty acid membranes are the most natural candidates for this purpose, but also polymers have shown great potential as enclosures of artificial cell mimics. Herein, we report on the formation of giant vesicles in a size range of 1 μm-100 μm using amphiphilic elastin-like polypeptides. The peptide vesicles can accommodate cell-free gene expression reactions, which is demonstrated by the transcription of a fluorescent RNA aptamer and the production of a fluorescent protein. Importantly, gene expression inside the vesicles leads to a strong growth of their size-up to an order of magnitude in volume in several cases-which is driven by changes in osmotic pressure, resulting in fusion events and uptake of membrane peptides from the environment.

The self-assembly of amphiphilic diblock copolymers into polymeric vesicles, commonly known as polymersomes, results in a versatile system for a variety of applications including drug delivery and microreactors. In this study, we show that the incorporation of hydrophobic plasmonic nanoparticles within the polymersome membrane facilitates light-stimulated release of vesicle encapsulants. This work seeks to achieve tunable, triggered release with non-invasive, spatiotemporal control using single-pulse irradiation. Gold nanoparticles (AuNPs) are incorporated as photosensitizers into the hydrophobic membrane of micron-scale polymersomes and the cargo release profile is controlled by varying the pulse energy and nanoparticle concentration. We have demonstrated the ability to achieve immediate vesicle rupture as well as vesicle poration resulting in temporal cargo diffusion. Additionally, changing the pulse duration, from femtosecond to nanosecond, provides mechanistic insight into the photothermal and photomechanical contributors that govern membrane disruption in this polymer-nanoparticle hybrid system.

Over the past two decades, polymersomes have been widely investigated for the delivery of diagnostic and therapeutic agents in cancer therapy. Polymersomes are stable polymeric vesicles, which are prepared using amphiphilic block polymers of different molecular weights. The use of high molecular weight amphiphilic copolymers allows for possible manipulation of membrane characteristics, which in turn enhances the efficiency of drug delivery. Polymersomes are more stable in comparison with liposomes and show less toxicity in vivo. Furthermore, their ability to encapsulate both hydrophilic and hydrophobic drugs, significant biocompatibility, robustness, high colloidal stability, and simple methods for ligands conjugation make polymersomes a promising candidate for therapeutic drug delivery in cancer therapy. This review is focused on current development in the application of polymersomes for cancer therapy and diagnosis. Graphical abstract.

Linear homopolymers of norbornene (NBE) derivatives equipped with short-chain alcohol pendant groups were prepared by ring-opening metathesis polymerization (ROMP) and subsequently assembled into well-defined structures in alcohol solvents. The ratios of hydrophobic carbons and hydrophilic alcohol groups at the repeating monomeric unit in these short-chain amphiphilic polymers were found to play an important role in determining the size and distribution of the final globular structures. Unlike the assembly of other linear homo- and copolymers possessing long-chain amphiphilicity, NBE-based linear polymers were readily transformed into spherical particles with a layered conformation, whose sizes range from a few hundred nanometers to micrometers with narrow distributions, simply by controlling the concentration and molecular weights of the linear homopolymers without using any surfactants. In addition, the degree of the intermolecular forces with solvents (e.g., solvation) possessing different surface tensions and polarities highly affected the final diameter and distribution of the polymer particles, implying the importance of the selection of a proper solvent to regulate their structural features. As such, understanding the assembly of these types of short-chain homopolymers into uniform particles can allow for regulating the transformation of diverse linear amphiphilic polymers into precisely controlled structures for various applications.

Sterilisation and preservation of vesicle formulations are important considerations for their viable manufacture for industry applications, particular those intended for medicinal use. Here, we undertake an initial investigation of the stability of hybrid lipid-block copolymer vesicles to common sterilisation and preservation processes, with particular interest in how the block copolymer component might tune vesicle stability. We investigate two sizes of polybutadiene-block-poly(ethylene oxide) polymers (PBd₁₂-PEO₁₁ and PBd₂₂-PEO₁₄) mixed with the phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) considering the encapsulation stability of a fluorescent cargo and the colloidal stability of vesicle size distributions. We find that autoclaving and lyophilisation cause complete loss of encapsulation stability under the conditions studied here. Filtering through 200 nm pores appears to be viable for sterilisation for all vesicle compositions with comparatively low release of encapsulated cargo, even for vesicle size distributions which extend beyond the 200 nm filter pore size. Freeze-thaw of vesicles also shows promise for the preservation of hybrid vesicles with high block copolymer content. We discuss the process stability of hybrid vesicles in terms of the complex mechanical interplay between bending resistance, stretching elasticity and lysis strain of these membranes and propose strategies for future work to further enhance the process stability of these vesicle formulations.

The impact of protein therapeutics in healthcare is steadily increasing, due to advancements in the field of biotechnology and a deeper understanding of several pathologies. However, their safety and efficacy are often limited by instability, short half-life and immunogenicity. Nanodelivery systems are currently being investigated for overcoming these limitations and include covalent attachment of biocompatible polymers (PEG and other synthetic or naturally derived macromolecules) as well as protein nanoencapsulation in colloidal systems (liposomes and other lipid or polymeric nanocarriers). Such strategies have the potential to develop next-generation protein therapeutics. Herein, we review recent research progresses on these nanodelivery approaches, as well as future directions and challenges.

Cell membranes control mass, energy, and information flow to and from the cell. In the cell membrane a lipid bilayer serves as the barrier layer, with highly efficient molecular machines, membrane proteins, serving as the transport elements. In this way, highly specialized transport properties are achieved by these composite materials by segregating the matrix function from the transport function using different components. For example, cell membranes containing aquaporin proteins can transport ∼4 billion water molecules per second per aquaporin while rejecting all other molecules including salts, a feat unmatched by any synthetic system, while the impermeable lipid bilayer provides the barrier and matrix properties. True separation of functions between the matrix and the transport elements has been difficult to achieve in conventional solute separation synthetic membranes. In this study, we created membranes with distinct matrix and transport elements through designed coassembly of solvent-stable artificial (peptide-appended pillar[5]arene, PAP5) or natural (gramicidin A) model channels with block copolymers into lamellar multilayered membranes. Self-assembly of a lamellar structure from cross-linkable triblock copolymers was used as a scalable replacement for lipid bilayers, offering better stability and mechanical properties. By coassembly of channel molecules with block copolymers, we were able to synthesize nanofiltration membranes with sharp selectivity profiles as well as uncharged ion exchange membranes exhibiting ion selectivity. The developed method can be used for incorporation of different artificial and biological ion and water channels into synthetic polymer membranes. The strategy reported here could promote the construction of a range of channel-based membranes and sensors with desired properties, such as ion separations, stimuli responsiveness, and high sensitivity.

The extracellular and subcellular compartments are characterized by specific pH levels that can be modified by pathophysiological states. This scenario encourages the use of environmentally responsive nanomedicines for the treatment of damaged cells. We have engineered doxorubicin (DOX)-loaded pH-responsive polymersomes using poly([ N-(2-hydroxypropyl)]methacrylamide)- b-poly[2-(diisopropylamino)ethyl methacrylate] block copolymers (PHPMA _m- b-PDPA _n). We demonstrate that, by taking advantage of the microfluidic technology, quasi-monodisperse assemblies can be created. This feature is of due relevance because highly uniform nanoparticles commonly exhibit more consistent biodistribution and cellular uptake. We also report that the size of the polymer vesicles can be tuned by playing with the inherent mechanical parameters of the microfluidic protocol. This new knowledge can be used to engineer size-specific nanomedicines for enhanced tumor accumulation if the manufacturing is performed with previous knowledge of tumor characteristics (particularly the degree of vascularity and porosity). The pH-dependent DOX release was further investigated evidencing the ability of polymersome to sustain encapsulated hydrophilic molecules when circulating in physiological environment (pH 7.4). This suggests nonrelevant drug leakage during systemic circulation. On the other hand, polymersome disassembly in slightly acid environments takes place enabling fast DOX release, thereby making the colloidal carriers highly cytotoxic. These features encourage the use of such advanced pH-responsive platforms to target damaged cells while preserving healthy environments during systemic circulation.

The timing and rate of release of pharmaceuticals from advanced drug delivery systems is an important property that has received considerable attention in the scientific literature. Broadly, these mostly fall into two classes: controlled release with a prolonged release rate or triggered release where the drug is rapidly released in response to an environmental stimulus. This review aims to highlight the potential for developing adaptive release systems that more subtlety modulate the drug release profile through continuous communication with its environment facilitated through feedback control. By reviewing the key elements of this approach in one place (fundamental principles of nanomedicine, enzymatic nanoreactors for medical therapies and feedback-controlled chemical systems) and providing additional motivating case studies in the context of chronobiology, we hope to inspire innovative development of novel "chrononanomedicines."

A synthetic cell mimic in the form of giant glycosylated polymersomes (GGPs) comprised of a novel amphiphilic diblock copolymer is reported. A synthetic approach involving a poly(dimethylsiloxane) (PDMS) macro-chain transfer agent (macroCTA) and postpolymerization modification was used to marry the hydrophobic and highly flexible properties of PDMS with the biological activity of glycopolymers. 2-Bromoethyl acrylate (BEA) was first polymerized using a PDMS macroCTA ( M_n,th ≈ 4900 g·mol^-1, Đ = 1.1) to prepare well-defined PDMS- b-pBEA diblock copolymers ( Đ = 1.1) that were then substituted with 1-thio-β-d-glucose or 1-thio-β-d-galactose under facile conditions to yield PDMS- b-glycopolymers. Compositions possessing ≈25% of the glycopolymer block (by mass) were able to adopt a vesicular morphology in aqueous solution (≈210 nm in diameter), as indicated by TEM and light scattering techniques. The resulting carbohydrate-decorated polymersomes exhibited selective binding with the lectin concanavalin A (Con A), as demonstrated by turbidimetric experiments. Self-assembly of the same diblock copolymer compositions using an electroformation method yielded GGPs (ranging from 2-20 μm in diameter). Interaction of these cell-sized polymersomes with fimH positive E. coli was then studied via confocal microscopy. The glucose-decorated GGPs were found to cluster upon addition of the bacteria, while galactose-decorated GGPs could successfully interact with (and possibly immobilize) the bacteria without the onset of clustering. This demonstrates an opportunity to modulate the response of these synthetic cell mimics (protocells) toward biological entities through exploitation of selective ligand-receptor interactions, which may be readily tuned through a considered choice of carbohydrate functionality.

Polymersomes, also called polymeric vesicles, are self-assembled by amphiphilic copolymers. Due to their unique characters, polymersomes are attracting more and more interest as an important class of vehicles for nanopharmaceuticals. In this chapter, various methods to prepare and characterize polymersomes are introduced systematically with several applicable examples. In addition, the advantages and disadvantages of each method were compared and analyzed with the aim to help readers choose the appropriate method in the process of experiments. Although some methods we introduced here are effective in preparing and characterizing polymersomes, the remaining challenge in this filed is to develop new tools. The reason is that polymersome is a kind of complex nanostructure, and some minor factors can affect the formation of polymersome. Meanwhile, more advanced technology should be developed to precisely determine the structure of some complex polymersomes such as multilayer polymersomes.

Drug-delivery systems (DDSs), in which drug encapsulation in nanoparticles enables targeted delivery of therapeutic agents and their release at specific disease sites, are important because they improve drug efficacy and help to decrease side effects. Although significant progress has been made in the development of DDSs for the treatment of a wide range of diseases, new approaches that increase the scope and effectiveness of such systems are still needed. Concepts such as nanoreactors and nanofactories are therefore attracting much attention. Nanoreactors, which basically consist of vesicle-encapsulated enzymes, provide prodrug conversion to therapeutic agents rather than simple drug delivery. Nanofactories are an extension of this concept and combine the features of nanoreactors and delivery carriers. Here, the required features of nanofactories are discussed and an overview of current strategies for the design and fabrication of different types of nanoreactors, i.e., systems based on lipid or polymer vesicles, capsules, mesoporous silica, viral capsids, and hydrogels, and their respective advantages and shortcomings, is provided. In vivo applications of biocatalytic reactors in the treatment of cancer, glaucoma, neuropathic pain, and alcohol intoxication are also discussed. Finally, the prospects for further progress in this important and promising field are outlined.

Vesicles composed of self-assembled lipids or amphiphilic polymers have significant potential in applications such as delivery of cargo for therapeutics. However, they are fragile under physiological conditions such as inside living cells or the bloodstream, in which a vast number of other molecules are present in high concentrations. This is because vesicles are in dynamic equilibrium between unimers and vesicles. Therefore, the development of more robust vesicles by covalent cross-linking of the shell was focused on. Cross-linked polymer vesicles were prepared by the self-assembly of maltopentaose-b-poly(propylene glycol) followed by the reaction between divinyl sulfone and the hydroxyl group in a maltopentaose unit. It was found that two equivalents of DVS to the polymer is an optimal condition for the cross-linking without changing in size. The bilayer structures were retained after the cross-linking reactions. Importantly, the cross-linked polymer vesicles retained their size and polydispersity even in 50:50 v/v methanol/water solution. This work highlights the potential of the divinyl sulfone shell cross-link as a promising tool for stabilization of glycopolymer vesicles.

Sorafenib (SFN), a hydrophobic anticancer drug, has several limitations predominantly poor aqueous solubility and hepatic first-pass effect, limiting its oral delivery that results into several other complications. Present study aims to develop Sorafenib loaded polymersomes using poly butadiene block poly ethylene oxide (PB-b-PEO), an amphiphilic co-block polymer. Prior to drug loading, critical aggregate concentration (CAC) of polymer was calculated for stable formulation synthesis. The developed SFN loaded PB-b-PEO polymersomes (SFN-PB-b-PEO, test formulation) characterized by DLS and cryo-TEM showed particle size 282 nm, polydispersity (PDI) of less than 0.29 and membrane thickness of about 20 nm. SFN-PB-b-PEO polymersomes demonstrated encapsulation efficiency of 71% and showed sustained drug release up to 144 h. Formulation remained stable for 3 months in suspension form. In vitro cytotoxicity against HepG2 cells showed 1.7 folds improved toxicity compared to SFN suspension. In addition, oral administration of SFN-PB-b-PEO polymersomes in BALB/c mice showed increased C_max and AUC_0-96 by 1.7 and 2.77-fold respectively (p < 0.05) compared to those of SFN suspension (reference formulation). Findings suggest that the SFN-PB-b-PEO polymersomes can be a potential candidate for oral delivery of SFN.