Tuning of Cationic Polymer Functionality in Complex Coacervate Artificial Cells for Optimized Enzyme Activity.

Complex coacervates are a versatile platform to mimic the structure of living cells. In both living systems and artificial cells, a macromolecularly crowded condensate phase has been shown to be able to modulate enzyme activity. Yet, how enzyme activity is affected by interactions (particularly with cationic charges) inside coacervates is not well studied. Here, we synthesized a series of amino-functional polymers to investigate the effect of the type of amine and charge density on coacervate formation, stability, protein partitioning, and enzyme function. The polymers were prepared by RAFT polymerization using as monomers aminoethyl methacrylate (AEAM), 2-(dimethylamino)ethyl methacrylate (DMAEMA), imidazolepropyl methacrylamide (IPMAm), and [2-(methacryloyloxy)ethyl] trimethylammonium chloride (TMAEMA). Membranized complex coacervate artificial cells were formed with these polycations and an anionic amylose derivative. Results show that polycations with reduced charge density result in higher protein mobility in the condensates and also higher enzyme activity. Insights described here could help guide the use of coacervate artificial cells in applications such as sensing, catalysis, and therapeutic formulations.

Matrix metalloproteinase responsive hydrogel microplates for programmed killing of invasive tumour cells.

Interactive materials are an emerging class of systems that can offer control over response and adaptivity in polymer structures towards the meso- and macroscale. Here, we use enzyme regulated cleavage of peptide crosslinkers in polymer hydrogels to release a cytotoxic therapeutic nanoparticle with an adaptable mechanism. Hydrogel microplates were formed through polyethylene glycol/peptide photoinitiated thiol-ene chemistry in a soft-lithography process to give square plates of 20 by 20 µm with a height of 10 µm. The peptide was chosen to be degradable in the presence of matrix metalloproteinase 2/9 (MMP-2/9). The hydrogel material's mechanical properties, swelling, and protease degradation were characterised. The microfabricated hydrogels were loaded with docetaxel (DTXL) containing poly(dl-lactide-co-glycolide) (PLGA) nanoparticles, and characterised for enzyme responsivity, and toxicity to MMP-2/9 overexpressing brain cancer cell line U87-MG. A 5-fold decrease in EC50 was seen compared to free DTXL, and a 20-fold decrease was seen for the MMP responsive microplates versus a non-degradable control microplate. Potential applications of this system in post-resection glioblastoma treatment are envisioned.

Complex Coacervate Materials as Artificial Cells.

Cells have evolved to be self-sustaining compartmentalized systems that consist of many thousands of biomolecules and metabolites interacting in complex cycles and reaction networks. Numerous subtle intricacies of these self-assembled structures are still largely unknown. The importance of liquid-liquid phase separation (both membraneless and membrane bound) is, however, recognized as playing an important role in achieving biological function that is controlled in time and space. Reconstituting biochemical reactions in vitro has been a success of the last decades, for example, establishment of the minimal set of enzymes and nutrients able to replicate cellular activities like the in vitro transcription translation of genes to proteins. Further than this though, artificial cell research has the aim of combining synthetic materials and nonliving macromolecules into ordered assemblies with the ability to carry out more complex and ambitious cell-like functions. These activities can provide insights into fundamental cell processes in simplified and idealized systems but could also have an applied impact in synthetic biology and biotechnology in the future. To date, strategies for the bottom-up fabrication of micrometer scale life-like artificial cells have included stabilized water-in-oil droplets, giant unilamellar vesicles (GUV's), hydrogels, and complex coacervates. Water-in-oil droplets are a valuable and easy to produce model system for studying cell-like processes; however, the lack of a crowded interior can limit these artificial cells in mimicking life more closely. Similarly membrane stabilized vesicles, such as GUV's, have the additional membrane feature of cells but still lack a macromolecularly crowded cytoplasm. Hydrogel-based artificial cells have a macromolecularly dense interior (although cross-linked) that better mimics cells, in addition to mechanical properties more similar to the viscoelasticity seen in cells but could be seen as being not dynamic in nature and limiting to the diffusion of biomolecules. On the other hand, liquid-liquid phase separated complex coacervates are an ideal platform for artificial cells as they can most accurately mimic the crowded, viscous, highly charged nature of the eukaryotic cytoplasm. Other important key features that researchers in the field target include stabilizing semipermeable membranes, compartmentalization, information transfer/communication, motility, and metabolism/growth. In this Account, we will briefly cover aspects of coacervation theory and then outline key cases of synthetic coacervate materials used as artificial cells (ranging from polypeptides, modified polysaccharides, polyacrylates, and polymethacrylates, and allyl polymers), finishing with envisioned opportunities and potential applications for coacervate artificial cells moving forward.

Shape-specific microfabricated particles for biomedical applications: a review.

The storied history of controlled the release systems has evolved over time; from degradable drug-loaded sutures to monolithic zero-ordered release devices and nano-sized drug delivery formulations. Scientists have tuned the physico-chemical properties of these drug carriers to optimize their performance in biomedical/pharmaceutical applications. In particular, particle drug delivery systems at the micron size regime have been used since the 1980s. Recent advances in micro and nanofabrication techniques have enabled precise control of particle size and geometry-here we review the utility of microplates and discoidal polymeric particles for a range of pharmaceutical applications. Microplates are defined as micrometer scale polymeric local depot devices in cuboid form, while discoidal polymeric nanoconstructs are disk-shaped polymeric particles having a cross-sectional diameter in the micrometer range and a thickness in the hundreds of nanometer range. These versatile particles can be used to treat several pathologies such as cancer, inflammatory diseases and vascular diseases, by leveraging their size, shape, physical properties (e.g., stiffness), and component materials, to tune their functionality. This review highlights design and fabrication strategies for these particles, discusses their applications, and elaborates on emerging trends for their use in formulations.

Harnessing Endogenous Stimuli for Responsive Materials in Theranostics.

Materials that respond to endogenous stimuli are being leveraged to enhance spatiotemporal control in a range of biomedical applications from drug delivery to diagnostic tools. The design of materials that undergo morphological or chemical changes in response to specific biological cues or pathologies will be an important area of research for improving efficacies of existing therapies and imaging agents, while also being promising for developing personalized theranostic systems. Internal stimuli-responsive systems can be engineered across length scales from nanometers to macroscopic and can respond to endogenous signals such as enzymes, pH, glucose, ATP, hypoxia, redox signals, and nucleic acids by incorporating synthetic bio-inspired moieties or natural building blocks. This Review will summarize response mechanisms and fabrication strategies used in internal stimuli-responsive materials with a focus on drug delivery and imaging for a broad range of pathologies, including cancer, diabetes, vascular disorders, inflammation, and microbial infections. We will also discuss observed challenges, future research directions, and clinical translation aspects of these responsive materials.

RAFT Emulsion Polymerization as a Platform to Generate Well-Defined Biocompatible Latex Nanoparticles.

Current approaches to generate core-shell nanoparticles for biomedical applications are limited by factors such as synthetic scalability and circulatory desorption of cytotoxic surfactants. Developments in controlled radical polymerization, particularly in dispersed states, represent a promising method of overcoming these challenges. In this work, well-defined PEGylated nanoparticles are synthesized using reversible addition fragmentation chain transfer emulsion polymerization to control particle size and surface composition and were further characterized with light scattering, electron microscopy, and size exclusion chromatography. Importantly, the nanoparticles are found to be tolerated both in vitro and in vivo, without the need for any purification after particle synthesis. Pharmacokinetic and biodistribution studies in mice, following intraperitoneal injection of the nanoparticles, reveal a long (>76 h) circulation time and accumulation in the liver.

Efficient Binding, Protection, and Self-Release of dsRNA in Soil by Linear and Star Cationic Polymers.

Double stranded RNA (dsRNA) exhibits severe degradation within 3 days in live soil, limiting its potential application in crop protection. Herein we report the efficient binding, protection, and self-release of dsRNA in live soil through the usage of a cationic polymer. Soil stability assays show that linear poly(2-(dimethylamino)ethyl acrylate) can delay the degradation of dsRNA by up to 1 week while the star shaped analogue showed an increased stabilization of dsRNA by up to 3 weeks. Thus, the architecture of the polymer can significantly affect the lifetime of dsRNA in soil. In addition, the hydrolysis and dsRNA binding and release profiles of these polymers were carefully evaluated and discussed. Importantly, hydrolysis could occur independently of environmental conditions (e.g., different pH, different temperature) showing the potential for many opportunities in agrochemicals where protection and subsequent self-release of dsRNA in live soil is required.

Branched poly (trimethylphosphonium ethylacrylate-co-PEGA) by RAFT: alternative to cationic polyammoniums for nucleic acid complexation.

Cationic and highly branched poly (trimethylphosphonium ethylacrylate-co-poly (ethylene glycol) acrylate) (p (TMPEA-co-PEGA)), and its ammonium equivalent, have been synthesised from post-polymerisation modification of a poly (bromo ethylacrylate-co-poly (ethylene glycol) acrylate) (p (BEA-co-PEGA)) precursor polymer produced using reversible addition fragmentation chain transfer (RAFT) polymerisation. The cationic polymers were evaluated for their ability to complex nucleic acids, their in vitro cytotoxicity and their GFP pDNA transfection efficiency. The results show RAFT copolymerisation of BEA and PEGA is a simple route to polyphosphoniums showing reduced cytotoxicities and higher transfection efficiencies than their polyammonium alternatives.

Ambient method for the production of an ionically gated carbon nanotube common cathode in tandem organic solar cells.

A method of fabricating organic photovoltaic (OPV) tandems that requires no vacuum processing is presented. These devices are comprised of two solution-processed polymeric cells connected in parallel by a transparent carbon nanotubes (CNT) interlayer. This structure includes improvements in fabrication techniques for tandem OPV devices. First the need for ambient-processed cathodes is considered. The CNT anode in the tandem device is tuned via ionic gating to become a common cathode. Ionic gating employs electric double layer charging to lower the work function of the CNT electrode. Secondly, the difficulty of sequentially stacking tandem layers by solution-processing is addressed. The devices are fabricated via solution and dry-lamination in ambient conditions with parallel processing steps. The method of fabricating the individual polymeric cells, the steps needed to laminate them together with a common CNT cathode, and then provide some representative results are described. These results demonstrate ionic gating of the CNT electrode to create a common cathode and addition of current and efficiency as a result of the lamination procedure.