Models of Chemical Communication for Micro/Nanoparticles.

Engineering chemical communication between micro/nanosystems (via the exchange of chemical messengers) is receiving increasing attention from the scientific community. Although a number of micro- and nanodevices (e.g., drug carriers, sensors, and artificial cells) have been developed in the last decades, engineering communication at the micro/nanoscale is a recent emergent topic. In fact, most of the studies in this research area have been published within the last 10 years. Inspired by nature─where information is exchanged by means of molecules─the development of chemical communication strategies holds wide implications as it may provide breakthroughs in many areas including nanotechnology, artificial cell research, biomedicine, biotechnology, and ICT. Published examples rely on nanotechnology and synthetic biology for the creation of micro- and nanodevices that can communicate. Communication enables the construction of new complex systems capable of performing advanced coordinated tasks that go beyond those carried out by individual entities. In addition, the possibility to communicate between synthetic and living systems can further advance our understanding of biochemical processes and provide completely new tailored therapeutic and diagnostic strategies, ways to tune cellular behavior, and new biotechnological tools. In this Account, we summarize advances by our laboratories (and others) in the engineering of chemical communication of micro- and nanoparticles. This Account is structured to provide researchers from different fields with general strategies and common ground for the rational design of future communication networks at the micro/nanoscale. First, we cover the basis of and describe enabling technologies to engineer particles with communication capabilities. Next, we rationalize general models of chemical communication. These models vary from simple linear communication (transmission of information between two points) to more complex pathways such as interactive communication and multicomponent communication (involving several entities). Using illustrative experimental designs, we demonstrate the realization of these models which involve communication not only between engineered micro/nanoparticles but also between particles and living systems. Finally, we discuss the current state of the topic and the future challenges to be addressed.

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.

The average magnetic anisotropy of polystyrene in polymersomes self-assembled from poly(ethylene glycol)-b-polystyrene.

Using the diamagnetic anisotropy of polymers for the characterization of polymers and polymer aggregates is a relatively new approach in the field of soft-matter and polymer research. So far, a good and thorough quantitative description of these diamagnetic properties has been lacking. Using a simple equation that links the magnetic properties of an average polymer repeating unit to those of the polymer vesicle of any shape, we measured, using magnetic birefringence, the average diamagnetic anisotropy of a polystyrene (PS) repeating unit, ΔχPS, inside a poly(ethylene glycol)-polystyrene (PEG-PS) polymersome membrane as a function of the PS-length and as a function of the preparation method. All obtained values of ΔχPS have a negative sign which results in polymers tending to align perpendicular to an applied magnetic field. Combined, the same order of magnitude of ΔχPS (10-12 m3 mol-1) for all polymersome shapes proves that the individual polymers are organized similarly regardless of the PS length and polymersome shape. Furthermore, the value found is only a fraction (∼1%) of what it can maximally be due to the random coiling of the polymers. We, therefore, predict that further ordering of the polymers within the membrane could lead to similar responses at much lower magnetic fields, possibly obtainable with permanent magnets, which would be highly advantageous for practical applications.

Nonlinear Transient Permeability in pH-Responsive Bicontinuous Nanospheres.

We demonstrate the construction of pH-responsive bicontinuous nanospheres (BCNs) with nonlinear transient permeability and catalytic activity. The BCNs were assembled from amphiphilic block copolymers comprising pH-responsive groups and were loaded with the enzymes urease and horseradish peroxidase (HRP). A transient membrane permeability switch was introduced by employing the well-known pH-increasing effect of urease upon conversion of urea to ammonia. As expected, the coencapsulated HRP displayed a transiently regulated catalytic output profile upon addition of urea, with no significant product formation after the pH increase. This transient process displayed a nonlinear "dampening" behavior, induced by a decrease in membrane permeability as a result of significant local ammonia production. Furthermore, the catalytic output of HRP could be modulated by addition of different amounts of urea or by altering the buffer capacity of the system. Finally, this nonlinear dampening effect was not observed in spherical polymersomes, even though the membrane permeability could also be inhibited by addition of urea. The specific BCN morphology therefore allows to optimally control catalytic processes by pH changes in the nanoreactor microenvironment compared to bulk conditions due to its unique permeability profile.

Polymer Vesicles with Integrated Photothermal Responsiveness.

Functionalized polymer vesicles have been proven to be highly promising in biomedical applications due to their good biocompatibility, easy processability, and multifunctional responsive capacities. However, photothermal-responsive polymer vesicles triggered by near-infrared (NIR) light have not been widely reported until now. Herein, we propose a new strategy for designing NIR light-mediated photothermal polymer vesicles. A small molecule (PTA) with NIR-triggered photothermal features was synthesized by combining a D-D'-A-D'-D configuration framework with a molecular rotor function (TPE). The feasibility of the design strategy was demonstrated through density functional theory calculations. PTA moieties were introduced in the hydrophobic segment of a poly(ethylene glycol)-poly(trimethylene carbonate) block copolymer, of which the carbonate monomers were modified in the side chain with an active ester group. The amphiphilic block copolymers (PEG44-PTA2) were then used as building blocks for the self-assembly of photothermal-responsive polymer vesicles. The new class of functionalized polymer vesicles inherited the NIR-mediated high photothermal performance of the photothermal agent (PTA). After NIR laser irradiation for 10 min, the temperature of the PTA-Ps aqueous solution was raised to 56 °C. The photothermal properties and bilayer structure of PTA-Ps after laser irradiation were still intact, which demonstrated that they could be applied as a robust platform in photothermal therapy. Besides their photothermal performance, the loading capacity of PTA-Ps was investigated as well. Hydrophobic cargo (Cy7) and hydrophilic cargo (Sulfo-Cy5) were successfully encapsulated in the PTA-Ps. These properties make this new class of functionalized polymer vesicles an interesting platform for synergistic therapy in anticancer treatment.

Biodegradable Grubbs-Loaded Artificial Organelles for Endosomal Ring-Closing Metathesis.

The application of transition-metal catalysts in living cells presents a promising approach to facilitate reactions that otherwise would not occur in nature. However, the usage of metal complexes is often restricted by their limited biocompatibility, toxicity, and susceptibility to inactivation and loss of activity by the cell's defensive mechanisms. This is especially relevant for ruthenium-mediated reactions, such as ring-closing metathesis. In order to address these issues, we have incorporated the second-generation Hoveyda-Grubbs catalyst (HGII) into polymeric vesicles (polymersomes), which were composed of biodegradable poly(ethylene glycol)-b-poly(caprolactone-g-trimethylene carbonate) [PEG-b-P(CL-g-TMC)] block copolymers. The catalyst was either covalently or non-covalently introduced into the polymersome membrane. These polymersomes were able to act as artificial organelles that promote endosomal ring-closing metathesis for the intracellular generation of a fluorescent dye. This is the first example of the use of a polymersome-based artificial organelle with an active ruthenium catalyst for carbon-carbon bond formation.

Compartmentalized Intracellular Click Chemistry with Biodegradable Polymersomes.

Polymersome nanoreactors that can be employed as artificial organelles have gained much interest over the past decades. Such systems often include biological catalysts (i.e., enzymes) so that they can undertake chemical reactions in cellulo. Examples of nanoreactor artificial organelles that acquire metal catalysts in their structure are limited, and their application in living cells remains fairly restricted. In part, this shortfall is due to difficulties associated with constructing systems that maintain their stability in vitro, let alone the toxicity they impose on cells. This study demonstrates a biodegradable and biocompatible polymersome nanoreactor platform, which can be applied as an artificial organelle in living cells. The ability of the artificial organelles to covalently and non-covalently incorporate tris(triazolylmethyl)amine-Cu(I) complexes in their membrane is shown. Such artificial organelles are capable of effectively catalyzing a copper-catalyzed azide-alkyne cycloaddition intracellularly, without compromising the cells' integrity. The platform represents a step forward in the application of polymersome-based nanoreactors as artificial organelles.

Achieving Control in Micro-/Nanomotor Mobility.

Unprecedented opportunities exist for the generation of advanced nanotechnologies based on synthetic micro/nanomotors (MNMs), such as active transport of medical agents or the removal of pollutants. In this regard, great efforts have been dedicated toward controlling MNM motion (e.g., speed, directionality). This was generally performed by precise engineering and optimizing of the motors' chassis, engine, powering mode (i.e., chemical or physical), and mechanism of motion. Recently, new insights have emerged to control motors mobility, mainly by the inclusion of different modes that drive propulsion. With high degree of synchronization, these modes work providing the required level of control. In this Minireview, we discuss the diverse factors that impact motion; these include MNM morphology, modes of mobility, and how control over motion was achieved. Moreover, we highlight the main limitations that need to be overcome so that such motion control can be translated into real applications.

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.

Confined Motion: Motility of Active Microparticles in Cell-Sized Lipid Vesicles.

Active materials can transduce external energy into kinetic energy at the nano and micron length scales. This unique feature has sparked much research, which ranges from achieving fundamental understanding of their motility to the assessment of potential applications. Traditionally, motility is studied as a function of internal features such as particle topology, while external parameters such as energy source are assessed mainly in bulk. However, in real-life applications, confinement plays a crucial role in determining the type of motion active particles can adapt. This feature has been however surprisingly underexplored experimentally. Here, we showcase a tunable experimental platform to gain an insight into the dynamics of active particles in environments with restricted 3D topology. Particularly, we examined the autonomous motion of coacervate micromotors confined in giant unilamellar vesicles (GUVs) spanning 10-50 µm in diameter and varied parameters including fuel and micromotor concentration. We observed anomalous diffusion upon confinement, leading to decreased motility, which was more pronounced in smaller compartments. The results indicate that the theoretically predicted hydrodynamic effect dominates the motion mechanism within this platform. Our study provides a versatile approach to understand the behavior of active matter under controlled, compartmentalized conditions.

Hybrid Biodegradable Nanomotors through Compartmentalized Synthesis.

Designer particles that are embued with nanomachinery for autonomous motion have great potential for biomedical applications; however, their development is highly demanding with respect to biodegradability/compatibility. Previously, biodegradable propulsive machinery based on enzymes has been presented. However, enzymes are highly susceptible to proteolysis and deactivation in biological milieu. Biodegradable hybrid nanomotors powered by catalytic inorganic nanoparticles provide a proteolytically stable alternative to those based upon enzymes. Herein we describe the assembly of hybrid biodegradable nanomotors capable of transducing chemical energy into motion. Such nanomotors are constructed through a process of compartmentalized synthesis of inorganic MnO2 nanoparticles (MnPs) within the cavity of organic stomatocytes. We show that the nanomotors remain active in cellular environments and do not compromise cell viability. Effective tumor penetration of hybrid nanomotors is also demonstrated in proof-of-principle experiments. Overall, this work represents a new prospect for engineering of nanomotors that can retain their functionality within biological contexts.

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.

Biomimicry of Cellular Motility and Communication Based on Synthetic Soft-Architectures.

Cells, sophisticated membrane-bound units that contain the fundamental molecules of life, provide a precious library for inspiration and motivation for both society and academia. Scientists from various disciplines have made great endeavors toward the understanding of the cellular evolution by engineering artificial counterparts (protocells) that mimic or initiate structural or functional cellular aspects. In this regard, several works have discussed possible building blocks, designs, functions, or dynamics that can be applied to achieve this goal. Although great progress has been made, fundamental-yet complex-behaviors such as cellular communication, responsiveness to environmental cues, and motility remain a challenge, yet to be resolved. Herein, recent efforts toward utilizing soft systems for cellular mimicry are summarized-following the main outline of cellular evolution, from basic compartmentalization, and biological reactions for energy production, to motility and communicative behaviors between artificial cell communities or between artificial and natural cell communities. Finally, the current challenges and future perspectives in the field are discussed, hoping to inspire more future research and to help the further advancement of this field.

Photoactivated Polymersome Nanomotors: Traversing Biological Barriers.

Synthetic nanomotors are appealing delivery vehicles for the dynamic transport of functional cargo. Their translation toward biological applications is limited owing to the use of non-degradable components. Furthermore, size has been an impediment owing to the importance of achieving nanoscale (ca. 100 nm) dimensions, as opposed to microscale examples that are prevalent. Herein, we present a hybrid nanomotor that can be activated by near-infrared (NIR)-irradiation for the triggered delivery of internal cargo and facilitated transport of external agents to the cell. Utilizing biodegradable poly(ethylene glycol)-b-poly(d,l-lactide) (PEG-PDLLA) block copolymers, with the two blocks connected via a pH sensitive imine bond, we generate nanoscopic polymersomes that are then modified with a hemispherical gold nanocoat. This Janus morphology allows such hybrid polymersomes to undergoing photothermal motility in response to thermal gradients generated by plasmonic absorbance of NIR irradiation, with velocities ranging up to 6.2±1.10 µm s-1 . These polymersome nanomotors (PNMs) are capable of traversing cellular membranes allowing intracellular delivery of molecular and macromolecular cargo.

Spatial Organization in Proteinaceous Membrane-Stabilized Coacervate Protocells.

As a model protocell, the membrane-free coacervate microdroplet is widely utilized in functional studies to provide insights into the physicochemical properties of the cell and to engineer cytomimetic soft technologies; however, the lack of a discrete membrane contributes to its instability and limits further application. Herein, a strategy is developed to fabricate a hybrid protocell based on the self-assembly of a proteinaceous membrane at the surface of coacervate microdroplets driven by a combination of electrostatic adhesion and steric/hydrophilic surface buoyancy. The semipermeable proteinaceous membrane can enhance coacervate stability obviously without compromising sequestration behavior. Significantly, such hybrid protocells demonstrate spatial organization whereby various functional enzymes can be located in discrete regions, which facilitates an on/off modulation for a cascade enzymatic reaction along with enhanced chemical communication between subpopulations.

Molecular Programming of Biodegradable Nanoworms via Ionically Induced Morphology Switch toward Asymmetric Therapeutic Carriers.

Engineering biodegradable nanostructures with precise morphological characteristics is a key objective in nanomedicine. In particular, asymmetric (i.e., nonspherical) nanoparticles are desirable due to the advantageous effects of shape in a biomedical context. Using molecular engineering, it is possible to program unique morphological features into the self-assembly of block copolymers (BCPs). However, the criteria of biocompatibility and scalability limit progress due to the prevalence of nondegradable components and the use of toxic solvents during fabrication. To address this shortfall, a robust strategy for the fabrication of morphologically asymmetric nanoworms, comprising biodegradable BCPs, has been developed. Modular BCPs comprising poly (ethylene glycol)-block-poly(caprolactone-gradient-trimethylene carbonate) (PEG-PCLgTMC), with a terminal chain of quaternary ammonium-TMC (PTMC-Q), undergo self-assembly via direct hydration into well-defined nanostructures. By controlling the solution ionic strength during hydration, particle morphology switches from spherical micelles to nanoworms (of varying aspect ratio). This ionically-induced switch is driven by modulation of chain packing with salts screening interchain repulsions, leading to micelle elongation. Nanoworms can be loaded with cytotoxic cargo (e.g., doxorubicin) at high efficiency, preferentially interact with cancer cells, and increase tumor penetration. This work showcases the ability to program assembly of BCPs and the potential of asymmetric nanosystems in anticancer drug delivery.

Physicochemical Characterization of Polymer-Stabilized Coacervate Protocells.

The bottom-up construction of cell mimics has produced a range of membrane-bound protocells that have been endowed with functionality and biochemical processes reminiscent of living systems. The contents of these compartments, however, experience semidilute conditions, whereas macromolecules in the cytosol exist in protein-rich, crowded environments that affect their physicochemical properties, such as diffusion and catalytic activity. Recently, complex coacervates have emerged as attractive protocellular models because their condensed interiors would be expected to mimic this crowding better. Here we explore some relevant physicochemical properties of a recently developed polymer-stabilized coacervate system, such as the diffusion of macromolecules in the condensed coacervate phase, relative to in dilute solutions, the buffering capacity of the core, the molecular organization of the polymer membrane, the permeability characteristics of this membrane towards a wide range of compounds, and the behavior of a simple enzymatic reaction. In addition, either the coacervate charge or the cargo charge is engineered to allow the selective loading of protein cargo into the coacervate protocells. Our in-depth characterization has revealed that these polymer-stabilized coacervate protocells have many desirable properties, thus making them attractive candidates for the investigation of biochemical processes in stable, controlled, tunable, and increasingly cell-like environments.

Adaptive Polymeric Assemblies for Applications in Biomimicry and Nanomedicine.

Dynamic and adaptive self-assembly systems are able to sense an external or internal (energy or matter) input and respond via chemical or physical property changes. Nanomaterials that show such transient behavior have received increasing interest in the field of nanomedicine due to improved spatiotemporal control of the nanocarrier function. In this regard, much can be learned from the field of systems chemistry and bottom-up synthetic biology, in which complex and intelligent networks of nanomaterials are designed that show transient behavior and function to advance our understanding of the complexity of living systems. In this Perspective, we highlight the recent advancements in adaptive nanomaterials used for nanomedicine and trends in transient responsive self-assembly systems to envisage how these fields can be integrated for the formation of next-generation adaptive stimuli-responsive nanocarriers in nanomedicine.

Development of Morphologically Discrete PEG-PDLLA Nanotubes for Precision Nanomedicine.

Precise control over the morphological features of nanoparticles is an important requisite for their application in nanomedical research. Parameters such as size and shape have been identified as critical features for effective nanotherapeutic technologies due to their role in circulation, distribution, and internalization in vivo. Tubular PEG-PDLLA polymersomes (nanotubes) exhibit an interesting morphology with potential for immunotherapeutics, as the elongated shape can affect cell-particle interactions. Developing methodologies that permit control over the precise form of such nanotubes is important for their biomedical implementation due to the stringent physicochemical constraints for efficacious performance. Through careful control over the engineering process, we demonstrate the generation of well-defined nanotubes based on polymersomes as small as 250 and 100 nm, which can be successfully shape transformed. The quality of the resulting nanostructures was established by physical characterization using AF4-MALS and cryo-TEM. Moreover, we show the successful loading of such nanotubes with model payloads (proteins and drugs). These findings provide a promising platform for implementation in biomedical applications in which discrete structure and functionality are essential features.

ATP-Mediated Transient Behavior of Stomatocyte Nanosystems.

In nature, dynamic processes are ubiquitous and often characterized by adaptive, transient behavior. Herein, we present the development of a transient bowl-shaped nanoreactor system, or stomatocyte, the properties of which are mediated by molecular interactions. In a stepwise fashion, we couple motility to a dynamic process, which is maintained by transient events; namely, binding and unbinding of adenosine triphosphate (ATP). The surface of the nanosystem is decorated with polylysine (PLL), and regulation is achieved by addition of ATP. The dynamic interaction between PLL and ATP leads to an increase in the hydrophobicity of the PLL-ATP complex and subsequently to a collapse of the polymer; this causes a narrowing of the opening of the stomatocytes. The presence of the apyrase, which hydrolyzes ATP, leads to a decrease of the ATP concentration, decomplexation of PLL, and reopening of the stomatocyte. The competition between ATP input and consumption gives rise to a transient state that is controlled by the out-of-equilibrium process.