Cationic Polysaccharides Bind to the Endothelial Cell Surface Extracellular Matrix Involving Heparan Sulfate.

Cationic polysaccharides have been extensively studied for drug delivery via the bloodstream, yet few have progressed to clinical use. Endothelial cells lining the blood vessel wall are coated in an anionic extracellular matrix called the glycocalyx. However, we do not fully comprehend the charged polysaccharide interactions with the glycocalyx. We reveal that the cationic polysaccharide poly(acetyl, arginyl) glucosamine (PAAG) exhibits the highest association with the endothelial glycocalyx, followed by dextran (neutral) and hyaluronan (anionic). Furthermore, we demonstrate that PAAG binds heparan sulfate (HS) within the glycocalyx, leading to intracellular accumulation. Using an in vitro glycocalyx model, we demonstrate a charge-based extent of association of polysaccharides with HS. Mechanistically, we observe that PAAG binding to HS occurs via a condensation reaction and functionally protects HS from degradation. Together, this study reveals the interplay between polysaccharide charge properties and interactions with the endothelial cell glycocalyx toward improved delivery system design and application.

Thermally Robust Solvent-Free Liquid Polyplexes for Heat-Shock Protection and Long-Term Room Temperature Storage of Therapeutic Nucleic Acids.

Nucleic acid therapeutics have attracted recent attention as promising preventative solutions for a broad range of diseases. Nonviral delivery vectors, such as cationic polymers, improve the cellular uptake of nucleic acids without suffering the drawbacks of viral delivery vectors. However, these delivery systems are faced with a major challenge for worldwide deployment, as their poor thermal stability elicits the need for cold chain transportation. Here, we demonstrate a biomaterial strategy to drastically improve the thermal stability of DNA polyplexes. Importantly, we demonstrate long-term room temperature storage with a transfection efficiency maintained for at least 9 months. Additionally, extreme heat shock studies show retained luciferase expression after heat treatment at 70 °C. We therefore provide a proof of concept for a platform biotechnology that could provide long-term room temperature storage for temperature-sensitive nucleic acid therapeutics, eliminating the need for the cold chain, which in turn would reduce the cost of distributing life-saving therapeutics worldwide.

Gradient Polymerization-Induced Self-Assembly: A One-Step Approach.

In this work, the authors report a novel single-step, one-pot process for the synthesis of self-assembled nanoparticles using a polymerization-induced self-assembly (PISA) mechanism. In contrast to conventional approaches employing a pre-formed macromolecular stabilizer, the disparate reactivities between two monomers, oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and diacetone acrylamide (DAAm), are exploited instead to synthesize a gradient copolymer directly in aqueous solution. Due to the hydrophobicity of poly(DAAm), these gradient copolymers can self-assemble in situ to form spheres and worms stabilized by the OEGMA residues. A surprisingly broad range of parameters are identified in which the worm morphology can be stabilized, which is highlighted by significant gelation of the reaction mixture in situ. This single-step gradient copolymerization approach to PISA is more efficient than conventional two-step syntheses. These results demonstrate improved reproducibility owing to the production of self-assembled nanoparticles directly in a one-pot and single-step synthesis.

Up in the air: oxygen tolerance in controlled/living radical polymerisation.

The requirement for deoxygenation in controlled/living radical polymerisation (CLRP) places significant limitations on its widespread implementation by necessitating the use of large reaction volumes, sealed reaction vessels as well as requiring access to specialised equipment such as a glove box and/or inert gas source. As a result, in recent years there has been intense interest in developing strategies for overcoming the effects of oxygen inhibition in CLRP and therefore remove the necessity for deoxygenation. In this review, we highlight several strategies for achieving oxygen tolerant CLRP including: "polymerising through" oxygen, enzyme mediated deoxygenation and the continuous regeneration of a redox-active catalyst. In order to provide further clarity to the field, we also establish some basic parameters for evaluating the degree of "oxygen tolerance" that can be achieved using a given oxygen scrubbing strategy. Finally, we propose some applications that could most benefit from the implementation of oxygen tolerant CLRP and provide a perspective on the future direction of this field.

Seeing the Light: Advancing Materials Chemistry through Photopolymerization.

The application of photochemistry to polymer and material science has led to the development of complex yet efficient systems for polymerization, polymer post-functionalization, and advanced materials production. Using light to activate chemical reaction pathways in these systems not only leads to exquisite control over reaction dynamics, but also allows complex synthetic protocols to be easily achieved. Compared to polymerization systems mediated by thermal, chemical, or electrochemical means, photoinduced polymerization systems can potentially offer more versatile methods for macromolecular synthesis. We highlight the utility of light as an energy source for mediating photopolymerization, and present some promising examples of systems which are advancing materials production through their exploitation of photochemistry.

A Self-Reporting Photocatalyst for Online Fluorescence Monitoring of High Throughput RAFT Polymerization.

Translating controlled/living radical polymerization (CLRP) from batch to the high throughput production of polymer libraries presents several challenges in terms of both polymer synthesis and characterization. Although recently there have been significant advances in the field of low volume, high throughput CLRP, techniques able to simultaneously monitor multiple polymerizations in an "online" manner have not yet been developed. Here, we report our discovery that 5,10,15,20-tetraphenyl-21H,23H-porphine zinc (ZnTPP) is a self-reporting photocatalyst that can mediate PET-RAFT polymerization as well as report on monomer conversion via changes in its fluorescence properties. This enables the use of a microplate reader to conduct high throughput "online" monitoring of PET-RAFT polymerizations performed directly in 384-well, low volume microtiter plates.

An Oxygen-Tolerant PET-RAFT Polymerization for Screening Structure-Activity Relationships.

The complexity of polymer-protein interactions makes rational design of the best polymer architecture for any given biointerface extremely challenging, and the high throughput synthesis and screening of polymers has emerged as an attractive alternative. A porphyrin-catalysed photoinduced electron/energy transfer-reversible addition-fragmentation chain-transfer (PET-RAFT) polymerisation was adapted to enable high throughput synthesis of complex polymer architectures in dimethyl sulfoxide (DMSO) on low-volume well plates in the presence of air. The polymerisation system shows remarkable oxygen tolerance, and excellent control of functional 3- and 4-arm star polymers. We then apply this method to investigate the effect of polymer structure on protein binding, in this case to the lectin concanavalin A (ConA). Such an approach could be applied to screen the structure-activity relationships for any number of polymer-protein interactions.

Macromol. Rapid Commun. 11/2016.

Back Cover: Polymers featuring photoresponsive groups have been exploited as promising drug delivery systems. Light as spatially and temporally controllable external stimulus can be readily tuned and focused to trigger changes in the structure of polymer chains to provide "on-command" drug delivery. A benign synthesis route for a novel controlled polymerization, known as photoinduced electron/energy transfer-reversible addition-fragmentation (PET-RAFT) polymerization, is presented. Further details can be found in the article by A. Bagheri, J. Yeow, H. Arandiyan, J. Xu, C. Boyer,* and M. Limon page 905.

Polymerization of a Photocleavable Monomer Using Visible Light.

The polymerization of the photocleavable monomer, o-nitrobenzyl methacrylate (NBMA), is investigated using photoinduced electron/energy transfer reversible addition-fragmentation chain transfer polymerization. The polymerizations under visible red (λ max = 635 nm, 0.7 mW cm(-2) ) and yellow (λ max = 560 nm, 9.7 mW cm(-2) ) light are performed and demonstrate rational evidence of a controlled/living radical polymerization process. Well-defined poly(o-nitrobenzyl methacrylate) (PNBMA) homopolymers with good control over the molecular weight and polymer dispersity are successfully synthesized by varying the irradiation time and/or targeted degree of polymerization. Chain extension of a poly(oligo(ethylene glycol) methyl ether methacrylate) macro-chain transfer agent with NBMA is carried out to fabricate photocleavable amphiphilic block copolymers (BCP). Finally, these self-assembled BCP rapidly dissemble under UV light suggesting the photoresponsive character of NBMA is not altered during the polymerization under yellow or red light. Such photoresponsive polymers can be potentially used for the remote-controlled delivery of therapeutic compounds.

An efficient and highly versatile synthetic route to prepare iron oxide nanoparticles/nanocomposites with tunable morphologies.

We report a versatile synthetic method for the in situ self-assembly of magnetic-nanoparticle-functionalized polymeric nanomorphologies, including spherical micelles and rod-like and worm-like micelles and vesicles. Poly(oligoethylene glycol methacrylate)-block-(methacrylic acid)-block-poly(styrene) (POEGMA-b-PMAA-b-PST) triblock copolymer chains were simultaneously propagated and self-assembled via a polymerization-induced self-assembly (PISA) approach. Subsequently, the carboxylic acid groups in the copolymers were used to complex an iron ion (Fe(II)/Fe(III)) mixture. Iron oxide nanoparticles were then formed in the central block, within the polymeric nanoparticles, via alkaline coprecipitation of the iron(II) and (III) salts. Nanoparticle morphologies, particle sizes, molecular weights, and chemical structures were then characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), size exclusion chromatography (SEC), and (1)H NMR measurements. TEM micrographs showed that the average size of the magnetic nanoparticles was ∼7 nm at the hydrophobic/hydrophilic nexus contained within the nanoparticles. In addition, XRD was used to confirm the formation of iron oxide nanoparticles. Importantly, the polymeric nanoparticle morphologies were not affected by the coprecipitation of the magnetic nanoparticles. The hybrid nanoparticles were then evaluated as negative MRI contrast agents, displaying remarkably high transverse relaxivities (r2, greater than 550 mM(-1) s(-1) at 9.4 T); a result, that we hypothesize, ensues from iron oxide nanoparticle clustering at the hydrophobic-hydrophilic interface. This simple synthetic procedure is highly versatile and produces nanocarriers of tunable size and shape with high efficacy as MRI contrast agents and potential utility as theranostic delivery vectors.

4D Multimaterial Printing of Soft Actuators with Spatial and Temporal Control.

Soft actuators (SAs) are devices which can interact with delicate objects in a manner not achievable with traditional robotics. While it is possible to design a SA whose actuation is triggered via an external stimulus, the use of a single stimulus creates challenges in the spatial and temporal control of the actuation. Herein, a 4D printed multimaterial soft actuator design (MMSA) whose actuation is only initiated by a combination of triggers (i.e., pH and temperature) is presented. Using 3D printing, a multilayered soft actuator with a hydrophilic pH-sensitive layer, and a hydrophobic magnetic and temperature-responsive shape-memory polymer layer, is designed. The hydrogel responds to environmental pH conditions by swelling or shrinking, while the shape-memory polymer can resist the shape deformation of the hydrogel until triggered by temperature or light. The combination of these stimuli-responsive layers allows for a high level of spatiotemporal control of the actuation. The utility of the 4D MMSA is demonstrated via a series of cargo capture and release experiments, validating its ability to demonstrate active spatiotemporal control. The MMSA concept provides a promising research direction to develop multifunctional soft devices with potential applications in biomedical engineering and environmental engineering.

Antimicrobial research of carbohydrate polymer- and protein-based hydrogels as reservoirs for the generation of reactive oxygen species: A review.

Reactive oxygen species (ROS) play an important role in biological milieu. Recently, the rapid growth in our understanding of ROS and their promise in antibacterial applications has generated tremendous interest in the combination of ROS generators with bulk hydrogels. Hydrogels represent promising supporters for ROS generators and can locally confine the nanoscale distribution of ROS generators whilst also promoting cellular integration via biomaterial-cell interactions. This review highlights recent efforts and progress in developing hydrogels derived from biological macromolecules with embedded ROS generators with a focus on antimicrobial applications. Initially, an overview of passive and active antibacterial hydrogels is provided to show the significance of proper hydrogel selection and design. These are followed by an in-depth discussion of the various approaches for ROS generation in hydrogels. The structural engineering and fabrication of ROS-laden hydrogels are given with a focus on their biomedical applications in therapeutics and diagnosis. Additionally, we discuss how a compromise needs to be sought between ROS generation and removal for maximizing the efficacy of therapeutic treatment. Finally, the current challenges and potential routes toward commercialization in this rapidly evolving field are discussed, focusing on the potential translation of laboratory research outcomes to real-world clinical outcomes.

Differences in Human Plasma Protein Interactions between Various Polymersomes and Stealth Liposomes as Observed by Fluorescence Correlation Spectroscopy.

A significant factor hindering the clinical translation of polymersomes as vesicular nanocarriers is the limited availability of comparative studies detailing their interaction with blood plasma proteins compared to liposomes. Here, polymersomes are self-assembled via film rehydration, solvent exchange, and polymerization-induced self-assembly using five different block copolymers. The hydrophilic blocks are composed of anti-fouling polymers, poly(ethylene glycol) (PEG) or poly(2-methyl-2-oxazoline) (PMOXA), and all the data is benchmarked to PEGylated "stealth" liposomes. High colloidal stability in human plasma (HP) is confirmed for all but two tested nanovesicles. In situ fluorescence correlation spectroscopy measurements are then performed after incubating unlabeled nanovesicles with fluorescently labeled HP or the specific labeled plasma proteins, human serum albumin, and clusterin (apolipoprotein J). The binding of HP to PMOXA-polymersomes could explain their relatively short circulation times found previously. In contrast, PEGylated liposomes also interact with HP but accumulate high levels of clusterin, providing them with their known prolonged circulation time. The absence of significant protein binding for most PEG-polymersomes indicates mechanistic differences in protein interactions and associated downstream effects, such as cell uptake and circulation time, compared to PEGylated liposomes. These are key observations for bringing polymersomes closer to clinical translation and highlighting the importance of such comparative studies.

Photoswitchable gating of non-equilibrium enzymatic feedback in chemically communicating polymersome nanoreactors.

The circadian rhythm generates out-of-equilibrium metabolite oscillations that are controlled by feedback loops under light/dark cycles. Here we describe a non-equilibrium nanosystem comprising a binary population of enzyme-containing polymersomes capable of light-gated chemical communication, controllable feedback and coupling to macroscopic oscillations. The populations consist of esterase-containing polymersomes functionalized with photo-responsive donor-acceptor Stenhouse adducts (DASA) and light-insensitive semipermeable urease-loaded polymersomes. The DASA-polymersome membrane becomes permeable under green light, switching on esterase activity and decreasing the pH, which in turn initiates the production of alkali in the urease-containing population. A pH-sensitive pigment that absorbs green light when protonated provides a negative feedback loop for deactivating the DASA-polymersomes. Simultaneously, increased alkali production deprotonates the pigment, reactivating esterase activity by opening the membrane gate. We utilize light-mediated fluctuations of pH to perform non-equilibrium communication between the nanoreactors and use the feedback loops to induce work as chemomechanical swelling/deswelling oscillations in a crosslinked hydrogel. We envision possible applications in artificial organelles, protocells and soft robotics.

Microliter Scale Synthesis of Luciferase-Encapsulated Polymersomes as Artificial Organelles for Optogenetic Modulation of Cardiomyocyte Beating.

Constructing artificial systems that effectively replace or supplement natural biological machinery within cells is one of the fundamental challenges underpinning bioengineering. At the sub-cellular scale, artificial organelles (AOs) have significant potential as long-acting biomedical implants, mimicking native organelles by conducting intracellularly compartmentalized enzymatic actions. The potency of these AOs can be heightened when judiciously combined with genetic engineering, producing highly tailorable biohybrid cellular systems. Here, the authors present a cost-effective, microliter scale (10 µL) polymersome (PSome) synthesis based on polymerization-induced self-assembly for the in situ encapsulation of Gaussia luciferase (GLuc), as a model luminescent enzyme. These GLuc-loaded PSomes present ideal features of AOs including enhanced enzymatic resistance to thermal, proteolytic, and intracellular stresses. To demonstrate their biomodulation potential, the intracellular luminescence of GLuc-loaded PSomes is coupled to optogenetically engineered cardiomyocytes, allowing modulation of cardiac beating frequency through treatment with coelenterazine (CTZ) as the substrate for GLuc. The long-term intracellular stability of the luminescent AOs allows this cardiostimulatory phenomenon to be reinitiated with fresh CTZ even after 7 days in culture. This synergistic combination of organelle-mimicking synthetic materials with genetic engineering is therefore envisioned as a highly universal strategy for the generation of new biohybrid cellular systems displaying unique triggerable properties.

Potent Virustatic Polymer-Lipid Nanomimics Block Viral Entry and Inhibit Malaria Parasites In Vivo.

Infectious diseases continue to pose a substantial burden on global populations, requiring innovative broad-spectrum prophylactic and treatment alternatives. Here, we have designed modular synthetic polymer nanoparticles that mimic functional components of host cell membranes, yielding multivalent nanomimics that act by directly binding to varied pathogens. Nanomimic blood circulation time was prolonged by reformulating polymer-lipid hybrids. Femtomolar concentrations of the polymer nanomimics were sufficient to inhibit herpes simplex virus type 2 (HSV-2) entry into epithelial cells, while higher doses were needed against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given their observed virustatic mode of action, the nanomimics were also tested with malaria parasite blood-stage merozoites, which lose their invasive capacity after a few minutes. Efficient inhibition of merozoite invasion of red blood cells was demonstrated both in vitro and in vivo using a preclinical rodent malaria model. We envision these nanomimics forming an adaptable platform for developing pathogen entry inhibitors and as immunomodulators, wherein nanomimic-inhibited pathogens can be secondarily targeted to sites of immune recognition.

Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines.

Self-amplifying RNA (saRNA) is a next-generation vaccine platform, but like all nucleic acids, requires a delivery vehicle to promote cellular uptake and protect the saRNA from degradation. To date, delivery platforms for saRNA have included lipid nanoparticles (LNP), polyplexes and cationic nanoemulsions; of these LNP are the most clinically advanced with the recent FDA approval of COVID-19 based-modified mRNA vaccines. While the effect of RNA on vaccine immunogenicity is well studied, the role of biomaterials in saRNA vaccine effectiveness is under investigated. Here, we tested saRNA formulated with either pABOL, a bioreducible polymer, or LNP, and characterized the protein expression and vaccine immunogenicity of both platforms. We observed that pABOL-formulated saRNA resulted in a higher magnitude of protein expression, but that the LNP formulations were overall more immunogenic. Furthermore, we observed that both the helper phospholipid and route of administration (intramuscular versus intranasal) of LNP impacted the vaccine immunogenicity of two model antigens (influenza hemagglutinin and SARS-CoV-2 spike protein). We observed that LNP administered intramuscularly, but not pABOL or LNP administered intranasally, resulted in increased acute interleukin-6 expression after vaccination. Overall, these results indicate that delivery systems and routes of administration may fulfill different delivery niches within the field of saRNA genetic medicines.

Benchtop Preparation of Polymer Brushes by SI-PET-RAFT: The Effect of the Polymer Composition and Structure on Inhibition of a Pseudomonas Biofilm.

We report a high-throughput method for producing surface-tethered polymeric brushes on glass substrates via surface-initiated photoinduced electron transfer-reversible addition-fragmentation chain transfer polymerization (SI-PET-RAFT). Due to its excellent oxygen tolerance, SI-PET-RAFT allows brush growth using low reagent volumes (30 µL) without prior degassing. An initial 28 homopolymer brush library was successfully prepared and screened with respect to their antifouling performance. The high-throughput approach was further exploited to expand the library to encompass statistical, gradient, and block architectures to investigate the effect of monomer composition and distribution using two monomers of disparate performance. In this manner, the degree of attachment from Gram-negative Pseudomonas aeruginosa (PA) bacterial biofilms could be tuned between the bounds set by the homopolymer brushes.

Big Is Beautiful: Enhanced saRNA Delivery and Immunogenicity by a Higher Molecular Weight, Bioreducible, Cationic Polymer.

Self-amplifying RNA (saRNA) vaccines are highly advantageous, as they result in enhanced protein expression compared to mRNA (mRNA), thus minimizing the required dose. However, previous delivery strategies were optimized for siRNA or mRNA and do not necessarily deliver saRNA efficiently due to structural differences of these RNAs, thus motivating the development of saRNA delivery platforms. Here, we engineer a bioreducible, linear, cationic polymer called "pABOL" for saRNA delivery and show that increasing its molecular weight enhances delivery both in vitro and in vivo. We demonstrate that pABOL enhances protein expression and cellular uptake via both intramuscular and intradermal injection compared to commercially available polymers in vivo and that intramuscular injection confers complete protection against influenza challenge. Due to the scalability of polymer synthesis and ease of formulation preparation, we anticipate that this polymer is highly clinically translatable as a delivery vehicle for saRNA for both vaccines and therapeutics.