Polymer Brushes on Nanoparticles for Controlling the Interaction with Protein-Rich Physiological Media.

The interaction of nanoparticles (NPs) with biological environments triggers the formation of a protein corona (PC), which significantly influences their behavior in vivo. This review explores the evolving understanding of PC formation, focusing on the opportunity for decreasing or suppressing protein-NP interactions by macromolecular engineering of NP shells. The functionalization of NPs with a dense, hydrated polymer brush shell is a powerful strategy for imparting stealth properties in order to elude recognition by the immune system. While poly(ethylene glycol) (PEG) has been extensively used for this purpose, concerns regarding its stability and immunogenicity have prompted the exploration of alternative polymers. The stealth properties of brush shells can be enhanced by tailoring functionalities and structural parameters, including the molar mass, grafting density, and polymer topology. Determining correlations between these parameters and biopassivity has enabled us to obtain polymer-grafted NPs with high colloidal stability and prolonged circulation time in biological media.

Hydrogels Generated from Cyclic Poly(2-Oxazoline)s Display Unique Swelling and Mechanical Properties.

Cyclic macromolecules do not feature chain ends and are characterized by a higher effective intramolecular repulsion between polymer segments, leading to a higher excluded-volume effect and greater hydration with respect to their linear counterparts. As a result of these unique properties, hydrogels composed of cross-linked cyclic polymers feature enhanced mechanical strength while simultaneously incorporating more solvent with respect to networks formed from their linear analogues with identical molar mass and chemical composition. The translation of topology effects by cyclic polymers into the properties of polymer networks provides hydrogels that ideally do not include defects, such as dangling chain ends, and display unprecedented physicochemical characteristics.

Oxygen Tolerant and Cytocompatible Iron(0)-Mediated ATRP Enables the Controlled Growth of Polymer Brushes from Mammalian Cell Cultures.

The use of zerovalent iron (Fe0)-coated plates, which act both as a source of catalyst and as a reducing agent during surface-initiated atom transfer radical polymerization (SI-ATRP), enables the controlled growth of a wide range of polymer brushes under ambient conditions utilizing either organic or aqueous reaction media. Thanks to its cytocompatibility, Fe0 SI-ATRP can be applied within cell cultures, providing a tool that can broadly and dynamically modify the substrate's affinity toward cells, without influencing their viability. Upon systematically assessing the application of Fe-based catalytic systems in the controlled grafting of polymers, Fe0 SI-ATRP emerges as an extremely versatile technique that could be applied to tune the physicochemical properties of a cell's microenvironments on biomaterials or within tissue engineering constructs.

Robust and Biocompatible Functionalization of ZnS Nanoparticles by Catechol-Bearing Poly(2-methyl-2-oxazoline)s.

Zinc sulfide (ZnS) nanoparticles (NPs) are particularly interesting materials for their electronic and luminescent properties. Unfortunately, their robust and stable functionalization and stabilization, especially in aqueous media, has represented a challenging and not yet completely accomplished task. In this work, we report the synthesis of colloidally stable, photoluminescent and biocompatible core-polymer shell ZnS and ZnS:Tb NPs by employing a water-in-oil miniemulsion (ME) process combined with surface functionalization via catechol-bearing poly-2-methyl-2-oxazoline (PMOXA) of various molar masses. The strong binding of catechol anchors to the metal cations of the ZnS surface, coupled with the high stability of PMOXA against chemical degradation, enable the formation of suspensions presenting excellent colloidal stability. This feature, combined with the assessed photoluminescence and biocompatibility, make these hybrid NPs suitable for optical bioimaging.

Hairy and Slippery Polyoxazoline-Based Copolymers on Model and Cartilage Surfaces.

Comb-like polymers presenting a hydroxybenzaldehyde (HBA)-functionalized poly(glutamic acid) (PGA) backbone and poly(2-methyl-2-oxazoline) (PMOXA) side chains chemisorb on aminolized substrates, including cartilage surfaces, forming layers that reduce protein contamination and provide lubrication. The structure, physicochemical, biopassive, and tribological properties of PGA-PMOXA-HBA films are finely determined by the copolymer architecture, its reactivity toward the surface, i.e. PMOXA side-chain crowding and HBA density, and by the copolymer solution concentration during assembly. Highly reactive species with low PMOXA content form inhomogeneous layers due to the limited possibility of surface rearrangements by strongly anchored copolymers, just partially protecting the functionalized surface from protein contamination and providing a relatively weak lubrication on cartilage. Biopassivity and lubrication can be improved by increasing copolymer concentration during assembly, leading to a progressive saturation of surface defects across the films. In a different way, less reactive copolymers presenting high PMOXA side-chain densities form uniform, biopassive, and lubricious films, both on model aminolized silicon oxide surfaces, as well as on cartilage substrates. When assembled at low concentrations these copolymers adopt a "lying down" conformation, i.e. adhering via their backbones onto the substrates, while at high concentrations they undergo a conformational transition, assuming a more densely packed, "standing up" structure, where they stretch perpendicularly from the substrate. This specific arrangement reduces protein contamination and improves lubrication both on model as well as on cartilage surfaces.

Quasi-3D-Structured Interfaces by Polymer Brushes.

The fabrication of polymer brushes via surface-initiated controlled radical polymerizations has progressively developed beyond a simple surface functionalization technique, enabling the design of complex polymer interfaces with a quasi-3D molecular organization. The modulation of polymer brush structure has led to an extremely broad tuning potential for technologically relevant interfacial, physicochemical properties, allowing one to precisely tune swelling, nanomechanical, and nanotribological characteristics of polymer films. In addition, the synthesis of multilayer brush interfaces with hierarchical architecture has been exploited to control biological phenomena on modified platforms, such as cell adhesion and settlement, or to fully prevent biological contamination from bacteria. In this feature article, the most recent developments in the synthesis and application of quasi-3D structured polymer brushes are summarized, placing particular attention on how the tuning of grafted-polymer architecture could translate into a variation of interfacial characteristics.

Cyclic Polymer Grafts That Lubricate and Protect Damaged Cartilage.

Tissue-reactive graft copolymers were designed to protect the cartilage against enzymatic degradation and restore its lubrication properties during the early stages of osteoarthritis (OA). The copolymers feature a poly(glutamic acid) (PGA) backbone bearing hydroxybenzaldehyde (HBA) functions and cyclic poly(2-methyl-2-oxazoline) (PMOXA) side chains. PGA-PMOXA-HBA species chemisorb on the degraded tissue via Schiff bases and expose the biopassive and lubricious PMOXA cyclic grafts at the interface. The smaller hydrodynamic radius by cyclic PMOXA side chains coupled to the intrinsic absence of chain ends generate denser and more lubricious films on cartilage when compared to those produced by copolymers bearing linear PMOXA. Topology effects demonstrate how the introduction of cyclic polymers within tissue-reactive copolymers substantially improve their tribological and biopassive properties, suggesting a plethora of possible applications for cyclic macromolecules in biomaterials formulations.

Chemical Design of Non-Ionic Polymer Brushes as Biointerfaces: Poly(2-oxazine)s Outperform Both Poly(2-oxazoline)s and PEG.

The era of poly(ethylene glycol) (PEG) brushes as a universal panacea for preventing non-specific protein adsorption and providing lubrication to surfaces is coming to an end. In the functionalization of medical devices and implants, in addition to preventing non-specific protein adsorption and cell adhesion, polymer-brush formulations are often required to generate highly lubricious films. Poly(2-alkyl-2-oxazoline) (PAOXA) brushes meet these requirements, and depending on their side-group composition, they can form films that match, and in some cases surpass, the bioinert and lubricious properties of PEG analogues. Poly(2-methyl-2-oxazine) (PMOZI) provides an additional enhancement of brush hydration and main-chain flexibility, leading to complete bioinertness and a further reduction in friction. These data redefine the combination of structural parameters necessary to design polymer-brush-based biointerfaces, identifying a novel, superior polymer formulation.

Easy to Apply Polyoxazoline-Based Coating for Precise and Long-Term Control of Neural Patterns.

Arranging cultured cells in patterns via surface modification is a tool used by biologists to answer questions in a specific and controlled manner. In the past decade, bottom-up neuroscience emerged as a new application, which aims to get a better understanding of the brain via reverse engineering and analyzing elementary circuitry in vitro. Building well-defined neural networks is the ultimate goal. Antifouling coatings are often used to control neurite outgrowth. Because erroneous connectivity alters the entire topology and functionality of minicircuits, the requirements are demanding. Current state-of-the-art coating solutions such as widely used poly(l-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) fail to prevent primary neurons from making undesired connections in long-term cultures. In this study, a new copolymer with greatly enhanced antifouling properties is developed, characterized, and evaluated for its reliability, stability, and versatility. To this end, the following components are grafted to a poly(acrylamide) (PAcrAm) backbone: hexaneamine, to support spontaneous electrostatic adsorption in buffered aqueous solutions, and propyldimethylethoxysilane, to increase the durability via covalent bonding to hydroxylated culture surfaces and antifouling polymer poly(2-methyl-2-oxazoline) (PMOXA). In an assay for neural connectivity control, the new copolymer's ability to effectively prevent unwanted neurite outgrowth is compared to the gold standard, PLL-g-PEG. Additionally, its versatility is evaluated on polystyrene, glass, and poly(dimethylsiloxane) using primary hippocampal and cortical rat neurons as well as C2C12 myoblasts, and human fibroblasts. PAcrAm-g-(PMOXA, NH2, Si) consistently outperforms PLL-g-PEG with all tested culture surfaces and cell types, and it is the first surface coating which reliably prevents arranged nodes of primary neurons from forming undesired connections over the long term. Whereas the presented work focuses on the proof of concept for the new antifouling coating to successfully and sustainably prevent unwanted connectivity, it is an important milestone for in vitro neuroscience, enabling follow-up studies to engineer neurologically relevant networks. Furthermore, because PAcrAm-g-(PMOXA, NH2, Si) can be quickly applied and used with various surfaces and cell types, it is an attractive extension to the toolbox for in vitro biology and biomedical engineering.

Controlling Enzymatic Polymerization from Surfaces with Switchable Bioaffinity.

The affinity of surfaces toward proteins is found to be a key parameter to govern the synthesis of polymer brushes by surface-initiated biocatalytic atom transfer radical polymerization (SI-bioATRP). While the "ATRPase" hemoglobin (Hb) stimulates only a relatively slow growth of protein repellent brushes, the synthesis of thermoresponsive grafts can be regulated by switching the polymer's attraction toward proteins across its lower critical solution temperature (LCST). Poly(N-isopropylacrylamide) (PNIPAM) brushes are synthesized in discrete steps of thickness at temperatures above LCST, while the biocatalyst layer is refreshed at T < LCST. Multistep surface-initiated biocatalytic ATRP demonstrates a high degree of control, results in high chain end group fidelity and enables the synthesis of multiblock copolymer brushes under fully aqueous conditions. The activity of Hb can be further modulated by tuning the accessibility of the heme pocket within the protein. Hence, the multistep polymerization is accelerated at acid pH, where the enzyme undergoes a transition from its native to a molten globule conformation. The controlled synthesis of polymer brushes by multistep SI-bioATRP highlights how a biocatalytic synthesis of grafted polymer films can be precisely controlled through the modulation of the polymer's interfacial physicochemical properties, in particular of the affinity of the surface toward proteins. This is not only of importance to gain a predictive understanding of surface-confined enzymatic polymerizations, but also represents a new way to translate bioadhesion into a controlled functionalization of materials.

Thin polymer brush decouples biomaterial's micro-/nanotopology and stem cell adhesion.

Surface morphology and chemistry of polymers used as biomaterials, such as tissue engineering scaffolds, have a strong influence on the adhesion and behavior of human mesenchymal stem cells. Here we studied semicrystalline poly(ε-caprolactone) (PCL) substrate scaffolds, which exhibited a variation of surface morphologies and roughness originating from different spherulitic superstructures. Substrates were obtained by varying the parameters of the thermal processing, that is, crystallization conditions. The cells attached to these polymer substrates adopted different morphologies responding to variations in spherulite density and size. In order to decouple substrate topology effects on the cells, sub-100 nm bioadhesive polymer brush coatings of oligo(ethylene glycol) methacrylates were grafted from PCL and functionalized with fibronectin. On surfaces featuring different surface textures, dense and sub-100 nm thick brush coatings determined the response of cells, irrespective to the underlying topology. Thus, polymer brushes decouple substrate micro-/nanoscale surface topology and the adhesion of stem cells.

Oxygen Tolerance in Surface-Initiated Reversible Deactivation Radical Polymerizations: Are Polymer Brushes Turning into Technology?

Over the past three decades, the development of reversible deactivation radical polymerizations (RDRP), and advancements toward more user-friendly and accessible experimental setups have opened the door for nonexperts to design complex macromolecules with well-defined properties. External mediation, improved tolerance to oxygen, and increased reaction volumes for higher synthetic output are some of the many noteworthy technical improvements. The development of RDRPs in solution was paralleled by their application on solid substrates to synthesize surface-grafted "polymer brushes" via surface-initiated RDRP (SI-RDRP). This Viewpoint paper provides a current perspective on recent developments in SI-RDRP methods that are tolerant to oxygen, especially highlighting those that could potentially enable scaling up of the synthesis of brushes for the functionalization of technologically relevant materials.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties.

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.

The role of poly(2-alkyl-2-oxazoline)s in hydrogels and biofabrication.

Poly(2-alkyl-2-oxazoline)s (PAOXAs) have been rapidly emerging as starting materials in the design of tissue engineering supports and for the generation of platforms for cell cultures, especially in the form of hydrogels. Thanks to their biocompatibility, chemical versatility and robustness, PAOXAs now represent a valid alternative to poly(ethylene glycol)s (PEGs) and their derivatives in these applications, and in the formulation of bioinks for three-dimensional (3D) bioprinting. In this review, we summarize the recent literature where PAOXAs have been used as main components for hydrogels and biofabrication mixtures, especially highlighting how their easily tunable composition could be exploited to fabricate multifunctional biomaterials with an extremely broad spectrum of properties.

Biomaterials applications of cyclic polymers.

Cyclic polymers are an intriguing class of polymers due to their lack of chain ends. This unique architecture combined with steric constraints adorn cyclic polymers as well as nano-, micro- and macro-scale materials containing cyclic polymers with distinctive physicochemical properties which can have a profound effect on the performance of these materials in a wide range of applications. Within a biomedical context, biomaterials based on cyclic polymers have shown very distinct properties in terms of biodistribution, pharmacokinetics, drug/gene delivery efficiency and surface activity. This review summarizes the applications of cyclic polymers in the field of biomaterials and highlights their potential in the biomedical field as well as addressing future challenges in this area.

Topology and Molecular Architecture of Polyelectrolytes Determine Their pH-Responsiveness When Assembled on Surfaces.

Polymer composition and topology of surface-grafted polyacids determine the amplitude of their pH-induced swelling transition. The intrinsic steric constraints characterizing cyclic poly(2-carboxypropyl-2-oxazoline) (c-PCPOXA) and poly(2-carboxyethyl-2-oxazoline) (c-PCEOXA) forming brushes on Au surfaces induce an enhancement in repulsive interactions between charged polymer segments upon deprotonation, leading to an amplified expansion and a significant increment in swelling with respect to their linear analogues of similar molar mass. On the other hand, it is the composition of polyacid grafts that governs their hydration in both undissociated and ionized forms, determining the degree of swelling during their pH-induced transition.

Fabrication of Three-Dimensional Polymer-Brush Gradients within Elastomeric Supports by Cu0-Mediated Surface-Initiated ATRP.

Cu0-mediated surface-initiated ATRP (Cu0 SI-ATRP) emerges as a versatile, oxygen-tolerant process to functionalize three-dimensional (3D), microporous supports forming single and multiple polymer-brush gradients with a fully tunable composition. When polymerization mixtures are dispensed on a Cu0-coated plate, this acts as oxygen scavenger and source of active catalyst. In the presence of an ATRP initiator-bearing microporous elastomer placed in contact with the metallic plate, the reaction solution infiltrates by capillarity through the support, simultaneously triggering the controlled growth of polymer brushes. The polymer grafting process proceeds with kinetics that are determined by the progressive infiltration of the reaction solution within the microporous support and by the continuous diffusion of catalyst regenerated at the Cu0 surface. The combination of these effects enables the accessible generation of 3D polymer-brush gradients extending across the microporous scaffolds used as supports, finally providing materials with a continuous variation of interfacial composition and properties.

Enzyme-functionalized polymer brush films on the inner wall of silicon-glass microreactors with tunable biocatalytic activity.

The lipase from Candida Rugosa was immobilized to a poly(methacrylic acid) polymer brush layer, grown on the inner wall of silicon-glass microreactors. The hydrolysis of 4-nitrophenyl acetate was used as a model reaction to study the activity of this biocatalytic system. The amount of bound lipase could be tuned by changing the polymerization time of the brush formation. The Michaelis-Menten constants and V(max) values, determined for immobilized and free lipase, are similar, demonstrating that the lipase's substrate affinity and its activity remain unchanged upon immobilization to the microchannel wall.

Polymer Topology Determines the Formation of Protein Corona on Core-Shell Nanoparticles.

Linear and cyclic poly(2-ethyl-2-oxazoline) (PEOXA) adsorbates provide excellent colloidal stability to superparamagnetic iron oxide nanoparticles (FexOy NPs) within protein-rich media. However, dense shells of linear PEOXA brushes cannot prevent weak but significant attractive interactions with human serum albumin. In contrast, their cyclic PEOXA counterparts quantitatively hinder protein adsorption, as demonstrated by a combination of dynamic light scattering and isothermal titration calorimetry. The cyclic PEOXA brushes generate NP shells that are denser and more compact than their linear counterparts, entirely preventing the formation of a protein corona as well as aggregation, even when the lower critical solution temperature of PEOXA in a physiological buffer is reached.