Modification of 3D-Printed PLA Structures Using Photo-Iniferter Polymerization: Toward On-Demand Antimicrobial Water Filters.

Water filtration is an important application to ensure the accessibility of clean drinking water. As requirements and contaminants vary on a local level, adjustable filter devices and their evaluation with contaminants are required. Within this work, modular filter devices are designed featuring an adjustable surface functionalization. For this purpose, 3D-printed structures are created consisting of bio-based poly(lactic acid) (PLA) that are manufactured by extrusion printing. The surface of PLA is activated with amino groups that are used to install xanthates as chain transfer agents. Subsequently, photo-iniferter (PI) polymerization is used to create cationic polymer brushes on the surface of PLA substrates. Multiple surface characterization techniques are employed to prove successful growth of polymer brushes on PLA. After initial optimization studies on flat surfaces, filter devices are printed, functionalized, and used to remove bacteria from contaminated water. Significant reduction of the number of microorganisms is detected after filtration (single filtration or cycling) and contaminating organism can also be removed from freshwater samples by simple incubation with a 3D-printed filter. The herein developed setup for producing functional filter devices and probing their performance in affinity filtration is a useful platform technology, enabling the rapid testing of polymer brushes for such applications.

Fabrication of Patchy Silica Microspheres with Tailor-Made Patch Functionality using Photo-Iniferter Reversible-Addition-Fragmentation Chain-Transfer (PI-RAFT) Polymerization.

Their inherent directional information renders patchy particles interesting building blocks for advanced applications in materials science. In this study, a feasible method to fabricate patchy silicon dioxide microspheres is demonstrated, which they are able to equip with tailor-made polymeric materials as patches. Their fabrication method relies on a solid-state supported microcontact printing (µCP) routine optimized for the transfer of functional groups to capillary-active substrates, which is used to introduce amino functionalities as patches to a monolayer of particles. Acting as anchor groups for polymerization, photo-iniferter reversible addition-fragmentation chain-transfer (RAFT) is used to graft polymer from the patch areas. Accordingly, particles with poly(N-acryloyl morpholine), poly(N-isopropyl acrylamide), and poly(n-butyl acrylate) are prepared as representative acrylic acid-derived functional patch materials. To facilitate their handling in water, a passivation strategy of the particles for aqueous systems is introduced. The protocol introduced here, therefore, promises a vast degree of freedom in engineering the surface properties of highly functional patchy particles. This feature is unmatched by other techniques to fabricate anisotropic colloids. The method, thus, can be considered a platform technology, culminating in the fabrication of particles that possess locally precisely formed patches on particles at a low µm scale with a high material functionality.

Light-Dependent RAFT Polymerization of Spiropyran Acrylates.

A light-assisted RAFT copolymerization protocol is established and investigated for the synthesis of 2-hydroxyethyl acrylate (HEA) / spiropyran acrylate (SPA) copolymers with enhanced SPA-contents. Radiation with visible light prevents the spiropyran (SP) motif from isomerizing into the open merocyanine (MC) form which can interfere with the polymerization process by abstracting a hydrogen atom from an active radical via its phenolic oxygen.

Toward Protein-Repellent Surface Coatings from Catechol-Containing Cationic Poly(2-ethyl-2-oxazoline).

Inspired by mussel proteins that enable surface binding in harsh marine environments, we envisioned a platform of protein-repellent macromolecules based on poly(2-ethyl-2-oxazoline) carrying catechol and cationic functional groups. To facilitate surface attachment, catechol units were installed by copolymerizing a functional comonomer, i.e., 2-(3,4-dimethoxyphenyl)-2-oxazoline, in a gradient fashion. Cationic units were introduced by partial acidic hydrolysis. The surface affinity of these polymers was probed using a quartz crystal microbalance with dissipation monitoring (QCM-D), and it was found that polymers with catechol units had a strong tendency to form surface-bound layers on different substrates, i.e., gold, iron, borosilicate, and polystyrene. While the neutral catechol-containing polymers showed strong, but uncontrolled binding, the ones with additional cationic units were able to form defined and durable polymer films. These coatings were able to prevent the attachment of different model proteins, i.e., bovine serum albumin (BSA), fibrinogen (FI), or lysozyme (LYZ). The herein-introduced platform offers straightforward access to nonfouling surface coatings using a biomimetic approach.

Xanthate-supported photo-iniferter (XPI)-RAFT polymerization: facile and rapid access to complex macromolecules.

Xanthate-supported photo-iniferter (XPI)-reversible addition-fragmentation chain-transfer (RAFT) polymerization is introduced as a fast and versatile photo-polymerization strategy. Small amounts of xanthate are added to conventional RAFT polymerizations to act as a photo-iniferter under light irradiation. Radical exchange is facilitated by the main CTA ensuring control over the molecular weight distribution, while xanthate enables an efficient photo-(re)activation. The photo-active moiety is thus introduced into the polymer as an end group, which makes chain extension of the produced polymers possible directly by irradiation. This is in sharp contrast to conventional photo-initiators, or photo electron transfer (PET)-RAFT polymerizations, where radical generation depends on the added small molecules. In contrast to regular photo-iniferter-RAFT polymerization, photo-activation is decoupled from polymerization control, rendering XPI-RAFT an elegant tool for the fabrication of defined and complex macromolecules. The method is oxygen tolerant and robust and was used to perform screenings in a well-plate format, and it was even possible to produce multiblock copolymers in a coffee mug under open-to-air conditions. XPI-RAFT does not rely on highly specialized equipment and qualifies as a universal tool for the straightforward synthesis of complex macromolecules. The method is user-friendly and broadens the scope of what can be achieved with photo-polymerization techniques.

Shape Matters: Highly Selective Antimicrobial Bottle Brush Copolymers via a One-Pot RAFT Polymerization Approach.

The one-pot synthesis of antimicrobial bottle brush copolymers is presented. Reversible addition-fragmentation chain-transfer (RAFT) polymerization is used for the production of the polymeric backbone, as well as for the grafts, which were installed using a grafting-from approach. A combination of N-isopropyl acrylamide and a Boc-protected primary amine-containing acrylamide was used in different compositions. After deprotection, polymers featuring different charge densities were obtained in both linear and bottle brush topologies. Antimicrobial activity was tested against three clinically relevant bacterial strains, and growth inhibition was significantly increased for bottle brush copolymers. Blood compatibility investigations revealed strong hemagglutination for linear copolymers and pronounced hemolysis for bottle brush copolymers. However, one bottle brush copolymer with a 50% charge density revealed strong antibacterial activity and negligible in vitro blood toxicity (regarding hemolysis and hemagglutination tests) resulting in selectivity values as high as 320. Membrane models were used to probe the mechanism of shown polymers that was found to be based on membrane disruption. The trends from bioassays are accurately reflected in model systems indicating that differences in lipid composition might be responsible for selectivity. However, bottle brush copolymers were found to possess increased cytotoxicity against human embryonic kidney (HEK) cells compared with linear analogues. The introduced synthetic platform enables screening of further, previously inaccessible parameters associated with the bottle brush topology, paving the way to further improve their activity profiles.

Probing the Effect of Rigidity on the Cellular Uptake of Core-Shell Nanoparticles: Stiffness Effects are Size Dependent.

Nanoparticles are well established vectors for the delivery of a wide range of biomedically relevant cargoes. Numerous studies have investigated the impact of size, shape, charge, and surface functionality of nanoparticles on mammalian cellular uptake. Rigidity has been studied to a far lesser extent, and its effects are still unclear. Here, the importance of this property, and its interplay with particle size, is systematically explored using a library of core-shell spherical PEGylated nanoparticles synthesized by RAFT emulsion polymerization. Rigidity of these particles is controlled by altering the intrinsic glass transition temperature of their constituting polymers. Three polymeric core rigidities are tested: hard, medium, and soft using two particle sizes, 50 and 100 nm diameters. Cellular uptake studies indicate that softer particles are taken up faster and threefold more than harder nanoparticles with the larger 100 nm particles. In addition, the study indicates major differences in the cellular uptake pathway, with harder particles being internalized through clathrin- and caveolae-mediated endocytosis as well as macropinocytosis, while softer particles are taken up bycaveolae- and non-receptormediated endocytosis. However, 50 nm derivatives do not show any appreciable differences in uptake efficiency, suggesting that rigidity as a parameter in the biological regime may be size dependent.

Antimicrobial Polymers of Linear and Bottlebrush Architecture: Probing the Membrane Interaction and Physicochemical Properties.

Polymeric antimicrobial peptide mimics are a promising alternative for the future management of the daunting problems associated with antimicrobial resistance. However, the development of successful antimicrobial polymers (APs) requires careful control of factors such as amphiphilic balance, molecular weight, dispersity, sequence, and architecture. While most of the earlier developed APs focus on random linear copolymers, the development of APs with advanced architectures proves to be more potent. It is recently developed multivalent bottlebrush APs with improved antibacterial and hemocompatibility profiles, outperforming their linear counterparts. Understanding the rationale behind the outstanding biological activity of these newly developed antimicrobials is vital to further improving their performance. This work investigates the physicochemical properties governing the differences in activity between linear and bottlebrush architectures using various spectroscopic and microscopic techniques. Linear copolymers are more solvated, thermo-responsive, and possess facial amphiphilicity resulting in random aggregations when interacting with liposomes mimicking Escheria coli membranes. The bottlebrush copolymers adopt a more stable secondary conformation in aqueous solution in comparison to linear copolymers, conferring rapid and more specific binding mechanism to membranes. The advantageous physicochemical properties of the bottlebrush topology seem to be a determinant factor in the activity of these promising APs.

Bottlebrush copolymers for gene delivery: influence of architecture, charge density, and backbone length on transfection efficiency.

The influence of polymer architecture of polycations on their ability to transfect mammalian cells is probed. Polymer bottle brushes with grafts made from partially hydrolysed poly(2-ethyl-2-oxazoline) are used while varying the length of the polymer backbone as well as the degree of hydrolysis (cationic charge content). Polyplex formation is investigated via gel electrophoresis, dye-displacement and dynamic light scattering. Bottle brushes show a superior ability to complex pDNA when compared to linear copolymers. Also, nucleic acid release was found to be improved by a graft architecture. Polyplexes based on bottle brush copolymers showed an elongated shape in transmission electron microscopy images. The cytotoxicity against mammalian cells is drastically reduced when a graft architecture is used instead of linear copolymers. Moreover, the best-performing bottle brush copolymer showed a transfection ability comparable with that of linear poly(ethylenimine), the gold standard of polymeric transfection agents, which is used as positive control. In combination with their markedly lowered cytotoxicity, cationic bottle brush copolymers are therefore shown to be a highly promising class of gene delivery vectors.

A Dual pH- and Light-Responsive Spiropyran-Based Surfactant: Investigations on Its Switching Behavior and Remote Control over Emulsion Stability.

A cationic surfactant containing a spiropyran unit is prepared exhibiting a dual-responsive adjustability of its surface-active characteristics. The switching mechanism of the system relies on the reversible conversion of the non-ionic spiropyran (SP) to a zwitterionic merocyanine (MC) and can be controlled by adjusting the pH value and via light, resulting in a pH-dependent photoactivity: While the compound possesses a pronounced difference in surface activity between both forms under acidic conditions, this behavior is suppressed at a neutral pH level. The underlying switching processes are investigated in detail, and a thermodynamic explanation based on a combination of theoretical and experimental results is provided. This complex stimuli-responsive behavior enables remote-control of colloidal systems. To demonstrate its applicability, the surfactant is utilized for the pH-dependent manipulation of oil-in-water emulsions.

Photo-Iniferter RAFT Polymerization.

Light-mediated polymerization techniques offer distinct advantages over polymerization reactions fueled by thermal energy, such as high spatial and temporal control as well as the possibility to work under mild reaction conditions. Reversible addition-fragmentation chain-transfer (RAFT) polymerization is a highly versatile radical polymerization method that can be utilized to control a variety of monomers and produce a vast number of complex macromolecular structures. The use of light to drive a RAFT-polymerization is possible via multiple routes. Besides the use of photo-initiators, or photo-catalysts, the direct activation of the chain transfer agent controlling the RAFT process in a photo-iniferter (PI) process is an elegant way to initiate and control polymerization reactions. Within this review, PI-RAFT polymerization and its advantages over the conventional RAFT process are discussed in detail.

Solid-Phase Microcontact Printing for Precise Patterning of Rough Surfaces: Using Polymer-Tethered Elastomeric Stamps for the Transfer of Reactive Silanes.

We present a microcontact printing (µCP) routine suitable to introduce defined (sub-) microscale patterns on surface substrates exhibiting a high capillary activity and receptive to a silane-based chemistry. This is achieved by transferring functional trivalent alkoxysilanes, such as (3-aminopropyl)-triethoxysilane (APTES) as a low-molecular weight ink via reversible covalent attachment to polymer brushes grafted from elastomeric polydimethylsiloxane (PDMS) stamps. The brushes consist of poly{N-[tris(hydroxymethyl)-methyl]acrylamide} (PTrisAAm) synthesized by reversible addition-fragmentation chain-transfer (RAFT)-polymerization and used for immobilization of the alkoxysilane-based ink by substituting the alkoxy moieties with polymer-bound hydroxyl groups. Upon physical contact of the silane-carrying polymers with surfaces, the conjugated silane transfers to the substrate, thus completely suppressing ink-flow and, in turn, maximizing printing accuracy even for otherwise not addressable substrate topographies. We provide a concisely conducted investigation on polymer brush formation using atomic force microscopy (AFM) and ellipsometry as well as ink immobilization utilizing two-dimensional proton nuclear Overhauser enhancement spectroscopy (1H-1H-NOESY-NMR). We analyze the µCP process by printing onto Si-wafers and show how even distinctively rough surfaces can be addressed, which otherwise represent particularly challenging substrates.

Impact of Multivalence and Self-Assembly in the Design of Polymeric Antimicrobial Peptide Mimics.

Antimicrobial resistance is an increasingly serious challenge for public health and could result in dramatic negative consequences for the health care sector during the next decades. To solve this problem, antibacterial materials that are unsusceptible toward the development of bacterial resistance are a promising branch of research. In this work, a new type of polymeric antimicrobial peptide mimic featuring a bottlebrush architecture is developed, using a combination of reversible addition-fragmentation chain transfer (RAFT) polymerization and ring-opening metathesis polymerization (ROMP). This approach enables multivalent presentation of antimicrobial subunits resulting in improved bioactivity and an increased hemocompatibility, boosting the selectivity of these materials for bacterial cells. Direct probing of membrane integrity of treated bacteria revealed highly potent membrane disruption caused by bottlebrush copolymers. Multivalent bottlebrush copolymers clearly outperformed their linear equivalents regarding bioactivity and selectivity. The effect of segmentation of cationic and hydrophobic subunits within bottle brushes was probed using heterograft copolymers. These materials were found to self-assemble under physiological conditions, which reduced their antibacterial activity, highlighting the importance of precise structural control for such applications. To the best of our knowledge, this is the first example to demonstrate the positive impact of multivalence, generated by a bottlebrush topology in polymeric antimicrobial peptide mimics, making these polymers a highly promising material platform for the design of new bactericidal systems.

Dual self-assembly of supramolecular peptide nanotubes to provide stabilisation in water.

Self-assembling peptides have the ability to spontaneously aggregate into large ordered structures. The reversibility of the peptide hydrogen bonded supramolecular assembly make them tunable to a host of different applications, although it leaves them highly dynamic and prone to disassembly at the low concentration needed for biological applications. Here we demonstrate that a secondary hydrophobic interaction, near the peptide core, can stabilise the highly dynamic peptide bonds, without losing the vital solubility of the systems in aqueous conditions. This hierarchical self-assembly process can be used to stabilise a range of different ß-sheet hydrogen bonded architectures.

Stimuli-responsive membrane activity of cyclic-peptide-polymer conjugates.

Cyclic peptide nanotubes (CPNT) consisting of an even number of amino acids with an alternating chirality are highly interesting materials in a biomedical context due to their ability to insert themselves into cellular membranes. However, unwanted unspecific interactions between CPNT and non-targeted cell membranes are a major drawback. To solve this issue we have synthetized a series of CPNT-polymer conjugates with a cleavable covalent connection between macromolecule and peptide. As a result, the polymers form a stabilizing and shielding shell around the nanotube that can be cleaved on demand to generate membrane active CPNT from non-active conjugates. This approach enables us to control the stacking and lateral aggregation of these materials, thus leading to stimuli responsive membrane activity. Moreover, upon activation, the systems can be adjusted to form nanotubes with an increased length instead of aggregates. We were able to study the dynamics of these systems in detail and prove the concept of stimuli responsive membrane interaction using CPNT-polymer conjugates to permeabilize liposomes as well as mammalian cell membranes.

Targeting intracellular, multi-drug resistant Staphylococcus aureus with guanidinium polymers by elucidating the structure-activity relationship.

Intracellular persistence of bacteria represents a clinical challenge as bacteria can thrive in an environment protected from antibiotics and immune responses. Novel targeting strategies are critical in tackling antibiotic resistant infections. Synthetic antimicrobial peptides (SAMPs) are interesting candidates as they exhibit a very high antimicrobial activity. We first compared the activity of a library of ammonium and guanidinium polymers with different sequences (statistical, tetrablock and diblock) synthesized by RAFT polymerization against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive strains (MSSA). As the guanidinium SAMPs were the most potent, they were used to treat intracellular S. aureus in keratinocytes. The diblock structure was the most active, reducing the amount of intracellular MSSA and MRSA by two-fold. We present here a potential treatment for intracellular, multi-drug resistant bacteria, using a simple and scalable strategy.

Polydimethylsiloxane-Based Giant Glycosylated Polymersomes with Tunable Bacterial Affinity.

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 ( Mn,th ≈ 4900 g·mol-1, D = 1.1) to prepare well-defined PDMS- b-pBEA diblock copolymers ( D = 1.1) that were then substituted with 1-thio-ß-d-glucose or 1-thio-ß-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.

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.

Systematic study of the structural parameters affecting the self-assembly of cyclic peptide-poly(ethylene glycol) conjugates.

Self-assembling cyclic peptides (CP) consisting of amino acids with alternating d- and l-chirality form nanotubes by hydrogen bonding, hydrophobic interactions, and π-π stacking in solution. These highly dynamic materials are emerging as promising supramolecular systems for a wide range of biomedical applications. Herein, we discuss how varying the polymer conformation (linear vs. brush), as well as the number of polymer arms per peptide unimer affects the self-assembly of PEGylated cyclic peptides in different solvents, using small angle neutron scattering. Using the derived information, strong correlations were drawn between the size of the aggregates, solvent polarity, and its ability to compete for hydrogen bonding interactions between the peptide unimers. Using these data, it could be possible to engineer cyclic peptide nanotubes of a controlled length.

Tumor targeting with pH-responsive poly(2-oxazoline)-based nanogels for metronomic doxorubicin treatment.

The synthesis of a new nanogel drug carrier system loaded with the anti-cancer drug doxorubicin (DOX) is presented. Poly(2-oxazoline) (POx) based nanogels from block copolymer micelles were cross-linked and covalently loaded with DOX using pH-sensitive Schiff' base chemistry. DOX loaded POx based nanogels showed a toxicity profile comparable to the free drug, while unloaded drug carriers showed no toxicity. Hemolytic activity and erythrocyte aggregation of the drug delivery system was found to be low and cellular uptake was investigated by flow cytometry and fluorescence microscopy. While the amount of internalized drug was enhanced when incorporated into a nanogel, the release of the drug into the nucleus was delayed. For in vivo investigations the nanogel drug delivery system was combined with a metronomic treatment of DOX. Low doses of free DOX were compared to equivalent DOX loaded nanogels in a xenograft mouse model. Treatment with POx based nanogels revealed a significant tumor growth inhibition and increase in survival time, while pure DOX alone had no effect on tumor progression. The biodistribution was investigated by microscopy of organs of mice and revealed a predominant localization of DOX within tumorous tissue. Thus, the POx based nanogel system revealed a therapeutic efficiency despite the low DOX concentrations and could be a promising strategy to control tumor growth with fewer side effects.