Engineering of Materials Interfaces by Grafting of Designed Polymers

Macromolecular engineering of surfaces by polymer functionalization is the key strategy for regulating the physico-chemical properties of materials’ interfaces, their integration within a medium (such as in the case of nanoparticles suspensions), and the interaction of a material with the surrounding environment. In particular, we exploit the assemblies of chain-end-grafted polymers, which form polymer “brushes”, to functionalize a variety of substrates, including inorganic and organic materials. The chemical composition and molecular architecture of the polymer grafts determine the physical properties of the coatings, such as layer swelling, interfacial stiffness and lubricity, and regulate the biopassive/bioactive character of the films. Examples are reported in Scheme 1 and include linear polymer brushes (a), graft-copolymer adsorbates forming thin brush layers (b), macromolecules presenting controlled branching (c) or crosslinks (d) and cyclic polymer assemblies (e). The polymer compositions applied encompass, but are not restricted to, poly(ethylene glycol)s (PEG) and PEG-derivatives, poly(acrylamide)s (PAAm), poly(hydroxyethyl methacrylate) (PHEMA) and poly-2(alkyl-2-oxazoline)s (PAOXAs). These diverse formulations are applied to functionalize a variety of supports. In particular, we have been concentrating on modifying scaffolds for tissue engineering, in order to regulate the behavior of adhering cells; synthesizing polymer adsorbates for nanoparticles (NPs) forming shells with precise composition and physical properties; fabricating polymer nano-assemblies with tunable nanomechanical and nanotribological properties on flat, model surfaces and on natural cartilage surfaces, to prevent tissue degeneration; and establishing novel and robust synthetic and characterization methods to regulate interactions between polymer films and biological media, including proteins and cells.

In all these fabrications, fundamental importance is given to the characterization of the physico-chemical properties of the grafted polymer assemblies. These are studied by a combination of surface-sensitive techniques, such as variable-angle spectroscopic ellipsometry (VASE), X-ray photoelectron spectroscopy (XPS), quartz crystal microbalance with dissipation (QCM-D) and atomic force microscopy (AFM) methods.


Topological Polymer Chemistry Enters Surface Science

The application of distinctive polymer topologies, beyond linearity, to yield cyclic and “loops”-forming surface-grafted polymer assemblies, enables a broad modulation of highly relevant, interfacial, physicochemical properties that are difficult to be addressed by linear polymer brushes. On flat surfaces, the ultra-dense and highly compact character of cyclic poly-2-ethyl-2-oxazoline (PEOXA) brushes provide enhanced steric stabilization of the interface, improved biopassivity and highly lubricious behavior, if compared to linear brushes with similar molar mass. The application of random PEOXA-based copolymers with a variable concentration of surface-reactive co-monomers generates mixtures of grafted loops and “tails”. The precise tuning of the relative surface concentration of these two polymer topologies allows shifting of the interfacial properties either towards the ones showed by linear brushes, or near the characteristics of cyclic analogues. An increase in the loops fraction generates an increment of steric repulsion, a concomitant decrease of friction, and an improvement of the antifouling character of the polymer films. All these findings highlight how polymer topological effects, typically observed in bulk or in solution are amplified by adding an additional boundary such as a grafting surface. Their precise tuning translates into materials with unprecedented characteristics and extremely high applicability.

Concentrating on the applicability of polymer stabilizers for nanoparticles (NPs) beyond chain linearity, we demonstrate that cyclic PEOXA ligands, applied on superparamagnetic F3O4 NPs provide enhanced colloidal stability and bioinertness in physiological media. When linear PEOXA brush shells fail in providing colloidal stabilization to NPs, the cyclic ones assure long lasting dispersions. While the thermal-induced de-hydration of linear PEOXA shells cause irreversible aggregation of the NPs due to the insufficient screening of their inorganic cores, the collapse and subsequent re-hydration of similarly grafted cyclic brushes, allow the full recovery of individually dispersed NPs. Although linear PEOXA ligands are densely grafted on F3O4 cores, a small plasma protein like bovine serum albumin (BSA) still physisorbs within their shells. In contrast, the impenetrable entropic shield provided by cyclic brushes efficiently prevents nonspecific interaction with proteins. All the unique properties of cyclic polymer brush shells suggest the next-generation design for the development of bioimaging and drug-delivery systems based on inorganic NPs.


Relevant publications:

Angew. Chem., Int. Ed. 2016, 55, 15583; Angew. Chem., Int. Ed. 2017, 56, 4507.


Fabrication of 3D Protein Gradients on Scaffolds for Tissue Engineering

This project concentrates on the fabrication of microporous scaffolds presenting three-dimensional (3D) protein and cell gradients for tissue engineering. 3D supports featuring regular geometry and microporosity were functionalized by PEG-based brush coatings coupled to cell-adhesive cues to spatially direct the settlement of adhering stem cells. 3D gradient scaffolds can be constructed with a combination of rapid prototyping of biodegradable thermoplastics and subsequent functionalization by surface-initiated atom transfer radical polymerization (SI-ATRP) of poly(oligoethylene glycol)methacrylate (POEGMA). The 3D environments that were created showed regular microporosity and high surface energy thanks to PEOEGMA brush coatings, and allowed the controlled diffusion of protein solutions in a multidirectional fashion. Subsequent incubation in stem-cell suspensions ultimately allowed the precise positioning of cell cultures within the scaffold with micrometer precision.

The formation of radial and axial POEGMA-brush-supported 3D gradients of fibronectin (FN) within the scaffolds allowed the spatially controlled culturing of stem cells. The advantages of this fabrication method, as compared to functional hydrogel supports, for example, were its ease of preparation and its ability to form multiaxial gradients of proteins with very high precision. These platforms represent a powerful tool for the design of chemically tailored platforms mimicking natural extracellular matrices and will be especially directed towards regenerative medicine, a field that has a consistent need for implantable functional scaffolds.

Relevant publications:

J. Mater. Chem. B, 2016, 4, 4244; Adv. Health. Mater., 2015, 4, 1169; Acta Biomater. 2014, 10, 2367.


AFM-Based, Advanced Characterization of Structured Polymer Films and Biointerfaces

Within our research activities, fundamental importance is given to the characterization of the interfacial physical properties of polymeric assemblies applied to engineered biomaterials interfaces. AFM-based methods are especially applied in conjunction with other surface-sensitive techniques to study the properties of polymer surfaces and their relationship to the behavior of adhering proteins and cells. As an example, the nanomechanical properties of responsive, layered polymer grafts featuring linear, thermoresponsive poly(N-isopropyl acrylamide) (PNIPAM) brushes, alternating with covalently crosslinked, poly(hydroxyethyl)methacrylate (PHEMA) gels are studied by a combination of AFM nanoindentation and in situ ellipsometry. PNIPAM brush/PHEMA-hydrogel films are synthesized by sequential surface-initiated atom transfer radical polymerization (SI-ATRP) to form multilayered architectures displaying a well-defined composition for each layer. The responsive properties of trilayered, PNIPAM/PHEMA-hydrogel/PNIPAM films are subsequently studied in aqueous media, across the lower critical solution temperature (LCST) of PNIPAM, and they are compared to the behavior of linear, homopolymer PNIPAM brushes. Below the LCST, brush/hydrogel multilayered films display swelling and nanomechanical properties similar to linear PNIPAM homopolymer brushes. In contrast, above the LCST, the PHEMA hydrogel interlayer acts as a stiffening element within the collapsed multilayered film, as measured by AFM nanoindentation. This translates into a tenfold increase in the effective Young’s modulus (E) for the collapsed, trilayered films compared to PNIPAM homopolymer analogues. The (macro)molecular continuity between the brush main chains and the hydrogel constituents enables a chemically robust layering to form graded, quasi-3D grafted polymer architectures, which display a concerted and amplified temperature-triggered transition. Multilayered brush/hydrogel films showed a composite-like mechanical behavior above the PNIPAM LCST. Their characteristics go beyond the simple rule of mixing (or layering) and are strongly influenced by the covalent continuity between the grafted components of each layer.

AFM techniques are additionally applied to probe the interfacial physical properties of synthetic extra-cellular matrices (ECMs) consisting of POEGMA brush-fibronectin (FN) conjugates presenting a gradual variation of tethered-chain length (gradient of brush thickness). Application of friction force microscopy (FFM) allowed me to estimate the lateral deformability and friction of POEGMA-FN. These parameters have been shown to regulate hMSCs adhesion, which adapt their morphology and form focal adhesions (FAs) responding to FN brush-tether characteristics. POEGMA brush-FNs presenting different grafted-chain lengths can modulate cell interactions at ECM cell-binding sites. Across a brush-thickness gradient presenting uniform FN exposure, thin brushes stimulate cell spreading and the development of FAs. Conversely, thick and more laterally deformable polymer grafts induce a decrease in cell spreading and FA formation. A correlation between lateral forces experienced at the (macro)molecular scale and the behavior of stem cells has been found. This interaction can be clarified by exploring novel aspects of FFM, a powerful tool for dynamically probing the ECM environment that indirectly suggests ways to structure ECM in order to trigger specific cell responses.

Relevant Publications:

Fabrication of Structured Brush Films:

Macromolecules, 2015, 48, 7106; Nanoscale, 2015, 7, 13017; Polymer 2016, 98, 470; Langmuir, 2016, 32, 10317;

Macromolecules 2017, 50, 2495; Macromolecules 2017, 50, 2436.

Advanced AFM Methods:

Adv. Mater. Interfaces, 2016, DOI: 10.1002/admi.201500456; Langmuir, 2017, 33, 4164; Macromolecules 2017, 50, 2932.


Polyoxazoline-based surface modifiers

Increasingly sophisticated formulations for the development of bulk biomaterials have not been accompanied by the same advances in coating compositions, especially when biological inertness is required. The latter is mostly attained by relying on linear PEG derivatives expanding from flat interfaces or from the cores of NPs. In these morphological arrangements and at high grafting densities, hydrophilic PEGs adopt a brush configuration, which provide a steric and entropic barrier against particle aggregation, and hinder the interaction with biomolecules by the functionalized materials.

Despite its widespread use in biomedicine, PEG shows chemical instability in the presence of oxygen, transition metal ions or specific enzymes that can trigger oxidation and the subsequent formation of toxic compounds. In order to overcome these limitations, poly-2-alkyl-2-oxazolines (PAOXAs) have been emerging as convenient alternatives. PAOXAs show resistance towards enzymatic degradation, low toxicity, high biocompatibility and higher stability, compared to PEG analogues. When PAOXAs are applied as surface-modifiers, on both flat surfaces and NPs, their intrinsic chemical stability yields an outstanding resistance to biological contamination and leads to a lasting stabilization of functionalized nano-colloids. In particular, poly-2-methyl- and poly-2-ethyl-2-oxazoline (PMOXA and PEOXA) brush coatings, have shown outstanding performance as biopassive surfaces when compared to PEG. Molecularly designed PMOXA and PEOXA adsorbates are prepared by exploiting the versatility of cationic ring-opening polymerization (CROP). Controlled termination strategies and precise tuning of monomer composition enable the synthesis of polymers with well-defined architectures and chain-end chemistries. This allowed us to synthesize PAOXA-based adsorbates for the functionalization of different materials, including inorganic NPs for bioimaging applications; and natural tissue surfaces to develop injectable formulations for osteoarthritis treatment (OA).

In the first case, we synthesized and applied PMOXA surface modifiers, carrying substituted catechol ligands that strongly and irreversibly anchor to the surface of metal oxide colloids. Specifically ZnO NPs, prepared by a mini-emulsion process, are functionalized by PMOXA-nitrodopamine (PMOXA-ND) ligands, to form ultra-stable NP suspensions in aqueous media. The chain-length of PMOXA ligands and the coverage on ZnO NPs was varied in order to achieve the complete stabilization of the inorganic colloids, and the formation of clear dispersions, presenting monodispersed, platelet-like crystals. Thanks to the chemical stability of PMOXA and the strong and stable anchoring by ND, dispersions of single ZnO NPs showed excellent stability over long incubation times in water (up to 9 months). Similarly to the functionalization of ZnO NPs, grafting-to methods employing different catechol-binding chemistries are applied for the functionalization and stabilization of Fe3O4 and ZnS NPs. Fe3O4 NPs are particularly applied in magnetic resonance imaging (MRI) for biomedicine, while biocompatible, ZnS NPs can be employed as bioimaging probes via doping with transition metal or lanthanide ions. In addition, application of silane-based anchors within PAOXA surface modifiers, enabled the fabrication of core-shell, fluorescent, organically-modified silica NPs (ORMOSIL) of variable size.

Our group has further been concentrating on the development of a PMOXA-based copolymer formulation to be directly applied onto the natural cartilage surface for the treatment of the early stages of OA. This widespread disease causes a progressive loss of glycosaminoglycans (GAGs) from the articular surface of cartilage, leaving the underlying collagen network exposed and fibrillated. The increase in friction and wear of the once-smooth articular surfaces initiates a downward spiral of tissue destruction in which, ultimately, the function of the joint is compromised. Importantly, there are currently no clinically accepted OA therapies that prevent disease progression or restore the natural lubrication of articular joints. My strategy relies on an injectable formulation based on poly(glutamic acid)-g-PMOXA (PGA-g-PMOXA) graft-copolymers that can selectively bind on the osteoarthritic tissue and restore the lubricity of the fibrillated tissue surface through the formation of a highly hydrated, tissue-grafted polymer brush layer. These “bottle-brush” copolymers, inspired by the natural, lubricating glycoproteins of articular cartilage, offer tissue-adhesive benzaldehyde (HBA) functions capable of forming stable binding onto degenerated cartilage via Schiff-base formation. The PMOXA side chains form a dense brush layer at the tissue interface, reducing the adsorption of proteins from the synovial fluid and restoring lubricating properties of the native tissue (NC) on the degraded cartilage (DC)), within a cartilage-vs-cartilage tribological system studied in synovial fluid. The reactivity of the copolymers and the architecture of the formed assemblies can be modulated by varying copolymer composition. Namely, the concentration β of tissue reactive HBA groups determines copolymer reactivity towards the cartilage, while PMOXA side-chain length x and PMOXA grafting density α, influence the morphology of the copolymer film and its properties.

Relevant Own Publications:

Biomaterials, 2010, 31, 9462; Chem. Mater., 2015, 27, 2957; Angew. Chem., Int. Ed. 2016, 128, 8684; ACS Nano 2017, 11, 2794.