Ultrafast light-activated polymeric nanomotors.

Synthetic micro/nanomotors have been extensively exploited over the past decade to achieve active transportation. This interest is a result of their broad range of potential applications, from environmental remediation to nanomedicine. Nevertheless, it still remains a challenge to build a fast-moving biodegradable polymeric nanomotor. Here we present a light-propelled nanomotor by introducing gold nanoparticles (Au NP) onto biodegradable bowl-shaped polymersomes (stomatocytes) via electrostatic and hydrogen bond interactions. These biodegradable nanomotors show controllable motion and remarkable velocities of up to 125 µm s-1. This unique behavior is explained via a thorough three-dimensional characterization of the nanomotor, particularly the size and the spatial distribution of Au NP, with cryogenic transmission electron microscopy (cryo-TEM) and cryo-electron tomography (cryo-ET). Our in-depth quantitative 3D analysis reveals that the motile features of these nanomotors are caused by the nonuniform distribution of Au NPs on the outer surface of the stomatocyte along the z-axial direction. Their excellent motile features are exploited for active cargo delivery into living cells. This study provides a new approach to develop robust, biodegradable soft nanomotors with application potential in biomedicine.

Positively Charged Biodegradable Polymersomes with Structure Inherent Fluorescence as Artificial Organelles.

Polymersomes, nanosized polymeric vesicles, have attracted significant interest in the areas of artificial cells and nanomedicine. Given their size, their visualization via confocal microscopy techniques is often achieved through the physical incorporation of fluorescent dyes, which however present challenges due to potential leaching. A promising alternative is the incorporation of molecules with aggregation-induced emission (AIE) behavior that are capable of fluorescing exclusively in their assembled state. Here, we report on the use of AIE polymersomes as artificial organelles, which are capable of undertaking enzymatic reactions in vitro. The ability of our polymersome-based artificial organelles to provide additional functionality to living cells was evaluated by encapsulating catalytic enzymes such as a combination of glucose oxidase/horseradish peroxidase (GOx/HRP) or ß-galactosidase (ß-gal). Via the additional incorporation of a pyridinium functionality, not only the cellular uptake is improved at low concentrations but also our platform's potential to specifically target mitochondria expands.

Dipeptide coacervates as artificial membraneless organelles for bioorthogonal catalysis.

Artificial organelles can manipulate cellular functions and introduce non-biological processes into cells. Coacervate droplets have emerged as a close analog of membraneless cellular organelles. Their biomimetic properties, such as molecular crowding and selective partitioning, make them promising components for designing cell-like materials. However, their use as artificial organelles has been limited by their complex molecular structure, limited control over internal microenvironment properties, and inherent colloidal instability. Here we report the design of dipeptide coacervates that exhibit enhanced stability, biocompatibility, and a hydrophobic microenvironment. The hydrophobic character facilitates the encapsulation of hydrophobic species, including transition metal-based catalysts, enhancing their efficiency in aqueous environments. Dipeptide coacervates carrying a metal-based catalyst are incorporated as active artificial organelles in cells and trigger an internal non-biological chemical reaction. The development of coacervates with a hydrophobic microenvironment opens an alternative avenue in the field of biomimetic materials with applications in catalysis and synthetic biology.

Polymeric Microreactors with pH-Controlled Spatial Localization of Cascade Reactions.

Lipid and polymer vesicles provide versatile means of creating systems that mimic the architecture of cells. However, these constructs cannot mimic the adaptive compartmentalization observed in cells, where the assembly and disassembly of subcompartments are dynamically modulated by environmental cues. Here, we describe a fully polymeric microreactor with a coacervate-in-vesicle architecture that exhibits an adaptive response to pH. The system was fabricated by microfluidic generation of semipermeable biomimetic polymer vesicles within 1 min using oleyl alcohol as the oil phase. The polymersomes allowed for the diffusion of protons and substrates acting as external signals. Using this method, we were able to construct adaptive microreactors containing internal polyelectrolyte-based catalytic organelles capable of sequestering and localizing enzymes and reaction products in a dynamic process driven by an external stimulus. This approach provides a platform for the rapid and efficient construction of robust adaptive microreactors that can be used in catalysis, biosensing, and cell mimicry.

Recent advances in permeable polymersomes: fabrication, responsiveness, and applications.

Polymersomes are vesicular nanostructures enclosed by a bilayer-membrane self-assembled from amphiphilic block copolymers, which exhibit higher stability compared with their biological analogues (e.g. liposomes). Due to their versatility, polymersomes have found various applications in different research fields such as drug delivery, nanomedicine, biological nanoreactors, and artificial cells. However, polymersomes prepared with high molecular weight components typically display low permeability to molecules and ions. It hence remains a major challenge to balance the opposing features of robustness and permeability of polymersomes. In this review, we focus on the design and strategies for fabricating permeable polymersomes, including polymersomes with intrinsic permeability, the formation of nanopores in the membrane bilayers by protein insertion, and the construction of stimuli-responsive polymersomes. Then, we highlight the applications of permeable polymersomes in the fields of biomimetic nanoreactors, artificial cells and organelles, and nanomedicine, to underline the challenges in the development of polymersomes as soft matter with biomedical utilities.

Inherently Fluorescent Peanut-Shaped Polymersomes for Active Cargo Transportation.

Nanomotors have been extensively explored for various applications in nanomedicine, especially in cargo transportation. Motile properties enable them to deliver pharmaceutical ingredients more efficiently to the targeted site. However, it still remains a challenge to design motor systems that are therapeutically active and can also be effectively traced when taken up by cells. Here, we designed a nanomotor with integrated fluorescence and therapeutic potential based on biodegradable polymersomes equipped with aggregation-induced emission (AIE) agents. The AIE segments provided the polymersomes with autofluorescence, facilitating the visualization of cell uptake. Furthermore, the membrane structure enabled the reshaping of the AIE polymersomes into asymmetric, peanut-shaped polymersomes. Upon laser irradiation, these peanut polymersomes not only displayed fluorescence, but also produced reactive oxygen species (ROS). Because of their specific shape, the ROS gradient induced motility in these particles. As ROS is also used for cancer cell treatment, the peanut polymersomes not only acted as delivery vehicles but also as therapeutic agents. As an integrated platform, these peanut polymersomes therefore represent an interesting delivery system with biomedical potential.

Assembly of biomimetic microreactors using caged-coacervate droplets.

Complex coacervates are liquid-like droplets that can be used to create adaptive cell-like compartments. These compartments offer a versatile platform for the construction of bioreactors inspired by living cells. However, the lack of a membrane significantly reduces the colloidal stability of coacervates in terms of fusion and surface wetting, which limits their suitability as compartments. Here, we describe the formation of caged-coacervates surrounded by a semipermeable shell of silica nanocapsules. We demonstrate that the silica nanocapsules create a protective shell that also regulates the molecular transport of water-soluble compounds as a function of nanocapasule size. The adjustable semipermeability and intrinsic affinity of enzymes for the interior of the caged-coacervates allowed us to assemble biomimetic microreactors with enhanced colloidal stability.

Controlled Membrane Transport in Polymeric Biomimetic Nanoreactors.

Polymersome-based biomimetic nanoreactors (PBNs) have generated great interest in nanomedicine and cell mimicry due to their robustness, tuneable chemistry, and broad applicability in biologically relevant fields. In this concept review, we mainly discuss the state of the art in functional polymersomes as biomimetic nanoreactors with membrane-controlled transport. PBNs that use environmental changes or external stimuli to adjust membrane permeability while maintaining structural integrity are highlighted. By encapsulating catalytic species, PBNs are able to convert inactive substrates into functional products in a controlled manner. In addition, special attention is paid to the use of PBNs as tailored artificial organelles with biomedical applications in vitro and in vivo, facilitating the fabrication of next-generation artificial organelles as therapeutic nanocompartments.

Responsive Peptide Nanofibers with Theranostic and Prognostic Capacity.

Photodynamic therapy (PDT) is a highly promising therapeutic modality for cancer treatment. The development of stimuli-responsive photosensitizer nanomaterials overcomes certain limitations in clinical PDT. Herein, we report the rational design of a highly sensitive PEGylated photosensitizer-peptide nanofiber (termed PHHPEG 6 NF) that selectively aggregates in the acidic tumor and lysosomal microenvironment. These nanofibers exhibit acid-induced enhanced singlet oxygen generation, cellular uptake, and PDT efficacy in vitro , as well as fast tumor accumulation, long-term tumor imaging capacity and effective PDT in vivo . Moreover, based on the prolonged presence of the fluorescent signal at the tumor site, we demonstrate that PHHPEG 6 NFs can also be applied for prognostic monitoring of the efficacy of PDT in vivo , which would potentially guide cancer treatment. Therefore, these multifunctional PHHPEG 6 NFs allow control over the entire PDT process, from visualization of photosensitizer accumulation, via actual PDT to the assessment of the efficacy of the treatment.

Twin-Engine Janus Supramolecular Nanomotors with Counterbalanced Motion.

Supramolecular nanomotors were created with two types of propelling forces that were able to counterbalance each other. The particles were based on bowl-shaped polymer vesicles, or stomatocytes, assembled from the amphiphilic block copolymer poly(ethylene glycol)-block-polystyrene. The first method of propulsion was installed by loading the nanocavity of the stomatocytes with the enzyme catalase, which enabled the decomposition of hydrogen peroxide into water and oxygen, leading to a chemically induced motion. The second method of propulsion was attained by applying a hemispherical gold coating on the stomatocytes, on the opposite side of the opening, making the particles susceptible to near-infrared laser light. By exposing these Janus-type twin engine nanomotors to both hydrogen peroxide (H2O2) and near-infrared light, two competing driving forces were synchronously generated, resulting in a counterbalanced, "seesaw effect" motion. By precisely manipulating the incident laser power and concentration of H2O2, the supramolecular nanomotors could be halted in a standby mode. Furthermore, the fact that these Janus stomatocytes were equipped with opposing motile forces also provided a proof of the direction of motion of the enzyme-activated stomatocytes. Finally, the modulation of the "seesaw effect", by tuning the net outcome of the two coexisting driving forces, was used to attain switchable control of the motile behavior of the twin-engine nanomotors. Supramolecular nanomotors that can be steered by two orthogonal propulsion mechanisms hold considerable potential for being used in complex tasks, including active transportation and environmental remediation.

Light-Activated Membrane Transport in Polymeric Cell-Mimics.

Giant polymersomes are versatile and stable biomimetic compartments that are ideal for building cell-like systems. However, the transport of hydrophilic molecules across the membrane, which controls the function of cell-like systems, is limited by the low permeability of polymeric bilayers. Therefore, mechanisms to control the permeability of polymersomes are necessary to create functional cell-like systems. Here, we describe the design of giant polymersomes equipped with spiropyran-based permeability modulators. Photo-isomerization of the modulators leads to perturbation of the polymer membrane, resulting in increased permeability. The photoactivated polymersomes were used to construct two cell-like systems controlled by light-activated transport of hydrophilic molecules. First, we designed an enzymatic micro-reactor activated by light irradiation. Second, we constructed a hybrid coacervate-in-polymersome system that mimics the adaptive formation of biological condensates in cells.

Imaging, quantitation and kinetic modelling of intravitreal nanomaterials.

In this study, the intravitreal pharmacokinetics of nanomaterials were investigated in vivo in rats and rabbits. Impact of particle size and shape (spherical, longitudinal) on ocular particle distribution and elimination was investigated with fundus camera, optical coherence tomography and ocular fluorophotometry. Differently sized particles showed prolonged ocular retention and remarkable differences in vitreal elimination, but size dependence was consistent, suggesting that other features have influence on their vitreal kinetics. We also demonstrate that liposomes are eliminated from the rabbit vitreous mainly via the anterior route. Simulation of drug concentrations after injection of intravitreal particles shows the importance of synchronized particle retention and drug release rate for efficient drug delivery. In conclusion, we provide kinetic insights in intravitreally administered nanoparticles to improve retinal drug delivery.

Synthetic Cells: From Simple Bio-Inspired Modules to Sophisticated Integrated Systems.

Bottom-up synthetic biology is the science of building systems that mimic the structure and function of living cells from scratch. To do this, researchers combine tools from chemistry, materials science, and biochemistry to develop functional and structural building blocks to construct synthetic cell-like systems. The many strategies and materials that have been developed in recent decades have enabled scientists to engineer synthetic cells and organelles that mimic the essential functions and behaviors of natural cells. Examples include synthetic cells that can synthesize their own ATP using light, maintain metabolic reactions through enzymatic networks, perform gene replication, and even grow and divide. In this Review, we discuss recent developments in the design and construction of synthetic cells and organelles using the bottom-up approach. Our goal is to present representative synthetic cells of increasing complexity as well as strategies for solving distinct challenges in bottom-up synthetic biology.

Engineering transient dynamics of artificial cells by stochastic distribution of enzymes.

Random fluctuations are inherent to all complex molecular systems. Although nature has evolved mechanisms to control stochastic events to achieve the desired biological output, reproducing this in synthetic systems represents a significant challenge. Here we present an artificial platform that enables us to exploit stochasticity to direct motile behavior. We found that enzymes, when confined to the fluidic polymer membrane of a core-shell coacervate, were distributed stochastically in time and space. This resulted in a transient, asymmetric configuration of propulsive units, which imparted motility to such coacervates in presence of substrate. This mechanism was confirmed by stochastic modelling and simulations in silico. Furthermore, we showed that a deeper understanding of the mechanism of stochasticity could be utilized to modulate the motion output. Conceptually, this work represents a leap in design philosophy in the construction of synthetic systems with life-like behaviors.

Therapeutic Stomatocytes with Aggregation Induced Emission for Intracellular Delivery.

Bowl-shaped biodegradable polymersomes, or stomatocytes, have much potential as drug delivery systems, due to their intriguing properties, such as controllable size, programmable morphology, and versatile cargo encapsulation capability. In this contribution, we developed well-defined therapeutically active stomatocytes with aggregation-induced emission (AIE) features by self-assembly of biodegradable amphiphilic block copolymers, comprising poly(ethylene glycol) (PEG) and AIEgenic poly(trimethylene carbonate) (PTMC) moieties. The presence of the AIEgens endowed the as-prepared stomatocytes with intrinsic fluorescence, which was employed for imaging of cellular uptake of the particles. It simultaneously enabled the photo-mediated generation of reactive oxygen species (ROS) for photodynamic therapy. The potential of the therapeutic stomatocytes as cargo carriers was demonstrated by loading enzymes (catalase and glucose oxidase) in the nanocavity, followed by a cross-linking reaction to achieve stable encapsulation. This provided the particles with a robust motile function, which further strengthened their therapeutic effect. With these unique features, enzyme-loaded AIEgenic stomatocytes are an attractive platform to be exploited in the field of nanomedicine.

Cucurbit-Like Polymersomes with Aggregation-Induced Emission Properties Show Enzyme-Mediated Motility.

Polymersomes that incorporate aggregation-induced emission (AIE) moieties are attractive inherently fluorescent nanoparticles with biomedical application potential for cell/tissue imaging and tracking, as well as phototherapeutics. An intriguing feature that has not been explored yet is their ability to adopt a range of asymmetric morphologies. Structural asymmetry allows nanoparticles to be exploited as active (motile) systems. Here, we present the design and preparation of AIE fluorophore integrated (AIEgenic) cucurbit-shaped polymersome nanomotors with enzyme-powered motility. The cucurbit scaffold was constructed via morphology engineering of biodegradable fluorescent AIE-polymersomes, followed by functionalization with enzymatic machinery via a layer-by-layer (LBL) self-assembly process. Because of the enzyme-mediated decomposition of chemical fuel on the cucurbit-like nanomotor surface, enhanced directed motion was attained, when compared with the spherical counterparts. These cucurbit-shaped biodegradable AIE-nanomotors provide a promising platform for the development of active delivery systems with potential for biomedical applications.

Biodegradable Polymersomes with Structure Inherent Fluorescence and Targeting Capacity for Enhanced Photo-Dynamic Therapy.

Biodegradable nanostructures displaying aggregation-induced emission (AIE) are desirable from a biomedical point of view, due to the advantageous features of loading capacity, emission brightness, and fluorescence stability. Herein, biodegradable polymers comprising poly (ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-P(CLgTMC)), with tetraphenylethylene pyridinium-TMC (PAIE) side chains have been developed, which self-assembled into well-defined polymersomes. The resultant AIEgenic polymersomes are intrinsically fluorescent delivery vehicles. The presence of the pyridinium moiety endows the polymersomes with mitochondrial targeting ability, which improves the efficiency of co-encapsulated photosensitizers and improves therapeutic index against cancer cells both in vitro and in vivo. This contribution showcases the ability to engineer AIEgenic polymersomes with structure inherent fluorescence and targeting capacity for enhanced photodynamic therapy.

Photoactivated nanomotors via aggregation induced emission for enhanced phototherapy.

Aggregation-induced emission (AIE) has, since its discovery, become a valuable tool in the field of nanoscience. AIEgenic molecules, which display highly stable fluorescence in an assembled state, have applications in various biomedical fields-including photodynamic therapy. Engineering structure-inherent, AIEgenic nanomaterials with motile properties is, however, still an unexplored frontier in the evolution of this potent technology. Here, we present phototactic/phototherapeutic nanomotors where biodegradable block copolymers decorated with AIE motifs can transduce radiant energy into motion and enhance thermophoretic motility driven by an asymmetric Au nanoshell. The hybrid nanomotors can harness two photon near-infrared radiation, triggering autonomous propulsion and simultaneous phototherapeutic generation of reactive oxygen species. The potential of these nanomotors to be applied in photodynamic therapy is demonstrated in vitro, where near-infrared light directed motion and reactive oxygen species induction synergistically enhance efficacy with a high level of spatial control.

Intravitreal Polymeric Nanocarriers with Long Ocular Retention and Targeted Delivery to the Retina and Optic Nerve Head Region.

Posterior eye tissues, such as retina, are affected in many serious eye diseases, but drug delivery to these targets is challenging due to various anatomical eye barriers. Intravitreal injections are widely used, but the intervals between invasive injections should be prolonged. We synthesized and characterized (1H NMR, gel permeation chromatography) block copolymers of poly(ethylene glycol), poly(caprolactone), and trimethylene carbonate. These polymers self-assembled to polymersomes and polymeric micelles. The mean diameters of polymersomes and polymeric micelles, about 100 nm and 30-50 nm, respectively, were obtained with dynamic light scattering. Based on single particle tracking and asymmetric flow field-flow fractionation, the polymeric micelles and polymersomes were stable and diffusible in the vitreous. The materials did not show cellular toxicity in cultured human umbilical vein endothelial cells in the Alamar Blue Assay. Pharmacokinetics of the intravitreal nanocarriers in the rabbits were evaluated using in vivo fluorophotometry. The half-lives of the polymersomes (100 nm) and the micelles (30 nm) were 11.4-32.7 days and 4.3-9.5 days. The intravitreal clearance values were 1.7-8.7 µL/h and 3.6-5.4 µL/h for polymersomes and polymeric micelles, respectively. Apparent volumes of distribution of the particles in the rabbit vitreous were 0.6-1.3 mL for polymeric micelles and 1.9-3.4 mL for polymersomes. Polymersomes were found in the vitreous for at least 92 days post-dosing. Furthermore, fundus imaging revealed that the polymersomes accumulated near the optic nerve and retained there even at 111 days post-injection. Polymersomes represent a promising technology for controlled and site-specific drug delivery in the posterior eye segment.