Dual Thermal- and Oxidation-Responsive Polymers Synthesized by a Sequential ROP-to-RAFT Procedure Inherently Temper Neuroinflammation.

This study is about multiple responsiveness in biomedical materials. This typically implies "orthogonality" (i.e., one response does not affect the other) or synergy (i.e., one increases efficacy or selectivity of the other), but an antagonist effect between responses may also occur. Here, we describe a family of very well-defined amphiphilic and micelle-forming block copolymers, which show both oxidative and temperature responses. They are produced via successive anionic ring-opening polymerization of episulfides and RAFT polymerization of dialkylacrylamides and differ only in the ratio between inert (N,N-dimethylacrylamide, DMA) and temperature-sensitive (N,N-diethylacrylamide, DEA) units. By scavenging Reactive Oxygen Species (ROS), these polymers are anti-inflammatory; through temperature responsiveness, they can macroscopically aggregate, which may allow them to form depots upon injection. The localization of the anti-inflammatory action is an example of synergy. An extensive evaluation of toxicity and anti-inflammatory effects on in vitro models, including BV2 microglia, C8D30 astrocytes and primary neurons, shows a link between capacity of aggregation and detrimental effects on viability which, albeit mild, can hinder the anti-inflammatory potential (antagonist action). Although limited in breadth (e.g., only in vitro models and only DEA as a temperature-responsive unit), this study suggests that single-responsive controls should be used to allow for a precise assessment of the (synergic or antagonist) potential of double-responsive systems.

A Reactive Oxygen Species-Scavenging 'Stealth' Polymer, Poly(thioglycidyl glycerol), Outperforms Poly(ethylene glycol) in Protein Conjugates and Nanocarriers and Enhances Protein Stability to Environmental and Biological Stressors.

This study addresses well-known shortcomings of poly(ethylene glycol) (PEG)-based conjugates. PEGylation is by far the most common method employed to overcome immunogenicity and suboptimal pharmacokinetics of, for example, therapeutic proteins but has significant drawbacks. First, PEG offers no protection from denaturation during lyophilization, storage, or oxidation (e.g., by biological oxidants, reactive oxygen species); second, PEG's inherent immunogenicity, leading to hypersensitivity and accelerated blood clearance (ABC), is a growing concern. We have here developed an 'active-stealth' polymer, poly(thioglycidyl glycerol)(PTGG), which in human plasma is less immunogenic than PEG (35% less complement activation) and features a reactive oxygen species-scavenging and anti-inflammatory action (∼50% less TNF-α in LPS-stimulated macrophages at only 0.1 mg/mL). PTGG was conjugated to proteins via a one-pot process; molar mass- and grafting density-matched PTGG-lysozyme conjugates were superior to their PEG analogues in terms of enzyme activity and stability against freeze-drying or oxidation; the latter is due to sacrificial oxidation of methionine-mimetic PTGG chains. Both in mice and rats, PTGG-ovalbumin displayed circulation half-lives up to twice as long as PEG-ovalbumin, but most importantly─and differently from PEG─without any associated ABC effect seen either in the time dependency of blood concentration, in the liver/splenic accumulation, or in antipolymer IgM/IgG titers. Furthermore, similar pharmacokinetic results were obtained with PTGGylated/PEGylated liposomal nanocarriers. PTGG's 'active-stealth' character therefore makes it a highly promising alternative to PEG for conjugation to biologics or nanocarriers.

Pectin as Rheology Modifier of a Gelatin-Based Biomaterial Ink.

Gelatin is a natural biopolymer extensively used for tissue engineering applications due to its similarities to the native extracellular matrix. However, the rheological properties of gelatin formulations are not ideal for extrusion-based bioprinting. In this work, we present an approach to improve gelatin bioprinting performances by using pectin as a rheology modifier of gelatin and (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as a gelatin-pectin crosslinking agent. The preparation of gelatin-pectin formulations is initially optimized to obtain homogenous gelatin-pectin gels. Since the use of GPTMS requires a drying step to induce the completion of the crosslinking reaction, microporous gelatin-pectin-GPTMS sponges are produced through freeze-drying, and the intrinsic properties of gelatin-pectin-GPTMS networks (e.g., porosity, pore size, degree of swelling, compressive modulus, and cell adhesion) are investigated. Subsequently, rheological investigations together with bioprinting assessments demonstrate the key role of pectin in increasing the viscosity and the yield stress of low viscous gelatin solutions. Water stable, three-dimensional, and self-supporting gelatin-pectin-GPTMS scaffolds with interconnected micro- and macroporosity are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The proposed biofabrication approach does not require any additional temperature controller to further modulate the rheological properties of gelatin solutions and it could furthermore be extended to improve the bioprintability of other biopolymers.

Physicochemical Characterization of Pectin-Gelatin Biomaterial Formulations for 3D Bioprinting.

Developing biomaterial formulations with specific biochemical characteristics and physical properties suitable for bioprinting of 3D scaffolds is a pivotal challenge in tissue engineering. Therefore, the design of novel bioprintable formulations is a continuously evolving research field. In this work, the authors aim at expanding the library of biomaterial inks by blending two natural biopolymers: pectin and gelatin. Cytocompatible formulations are obtained by combining pectin and gelatin at different ratios and using (3-glycidyloxypropyl)trimethoxysilane (GPTMS) as single crosslinking agent. It is shown that the developed formulations are all suitable for extrusion-based 3D bioprinting. Self-supporting scaffolds with a designed macroporosity and micropores in the bioprinted struts are successfully obtained by combining extrusion-based bioprinting and freeze-drying. The presence of gelatin in these formulations allows for the modulation of porosity, of water uptake and of scaffold stiffness in respect to pure pectin scaffolds. Results demonstrate that these new biomaterial formulations, processed with this specific approach, are promising candidates for the fabrication of tissue-like scaffolds for tissue regeneration.

Orbital floor repair using patient specific osteoinductive implant made by stereolithography.

The orbital floor (OF) is an anatomical location in the craniomaxillofacial (CMF) region known to be highly variable in shape and size. When fractured, implants commonly consisting of titanium meshes are customized by plying and crude hand-shaping. Nevertheless, more precise customized synthetic grafts are needed to meticulously reconstruct the patients' OF anatomy with better fidelity. As alternative to titanium mesh implants dedicated to OF repair, we propose a flexible patient-specific implant (PSI) made by stereolithography (SLA), offering a high degree of control over its geometry and architecture. The PSI is made of biodegradable poly(trimethylene carbonate) (PTMC) loaded with 40 wt % of hydroxyapatite (called Osteo-PTMC). In this work, we developed a complete work-flow for the additive manufacturing of PSIs to be used to repair the fractured OF, which is clinically relevant for individualized medicine. This work-flow consists of (i) the surgical planning, (ii) the design of virtual PSIs and (iii) their fabrication by SLA, (iv) the monitoring and (v) the biological evaluation in a preclinical large-animal model. We have found that once implanted, titanium meshes resulted in fibrous tissue encapsulation, whereas Osteo-PMTC resulted in rapid neovascularization and bone morphogenesis, both ectopically and in the OF region, and without the need of additional biotherapeutics such as bone morphogenic proteins. Our study supports the hypothesis that the composite osteoinductive Osteo-PTMC brings advantages compared to standard titanium mesh, by stimulating bone neoformation in the OF defects. PSIs made of Osteo-PTMC represent a significant advancement for patients whereby the anatomical characteristics of the OF defect restrict the utilization of traditional hand-shaped titanium mesh.

Engineering 3D parallelized microfluidic droplet generators with equal flow profiles by computational fluid dynamics and stereolithographic printing.

Microfluidic droplet generators excel in generating monodisperse micrometer-sized droplets and particles. However, the low throughput of conventional droplet generators hinders their clinical and industrial translation. Current approaches to parallelize microdevices are challenged by the two-dimensional nature of the standard fabrication methods. Here, we report the facile production of three-dimensionally (3D) parallelized microfluidic droplet generators consisting of stacked and radially multiplexed channel designs. Computational fluid dynamics simulations form the design basis for a microflow distributor that ensures similar flow rates through all droplet generators. Stereolithography is the selected technique to fabricate microdevices, which enables the manufacturing of hollow channels with dimensions as small as 50 µm. The microdevices could be operated up to 4 bars without structural damage, including deformation of channels, or leakage of the on-chip printed Luer-Lok type connectors. The printed microdevices readily enable the production of water-in-oil emulsions, as well as polymer containing droplets that act as templates for both solid and core-shell hydrogel microparticles. The cytocompatibility of the 3D printed device is demonstrated by encapsulating mesenchymal stem cells in hydrogel microcapsules, which results in the controllable formation of stem cell spheroids that remain viable and metabolically active for at least 21 days. Thus, the unique features of stereolithography fabricated microfluidic devices allow for the parallelization of droplet generators in a simple yet effective manner by enabling the realization of (complex) 3D designs.

Disulfide-Mediated Bioconjugation: Disulfide Formation and Restructuring on the Surface of Nanomanufactured (Microfluidics) Nanoparticles.

This study is about (1) nanomanufacturing (focusing on microfluidic-assisted nanoprecipitation), (2) advanced colloid characterization (focusing on field flow fractionation), and (3) the possible restructuring of surface disulfides. Disulfides are dynamic and exchangeable groups, and here we specifically focus, first, on their use to introduce biofunctional groups and, second, on their re-organization, which may lead to variable surface chemistries and uncontrolled cell interactions. The particles were obtained via microfluidic-assisted (flow-focused) nanoprecipitation of poly(ethylene glycol)-b-poly(ε-caprolactone) bearing or not a 2-pyridyl disulfide (PDS) terminal group, which quantitatively exchanges with thiols in solution. In this study, we have paid specific attention to size characterization, thereby also demonstrating the limitations of dynamic light scattering (DLS) as a stand-alone technique. By using asymmetric flow field flow fractionation coupled with DLS, static light scattering (SLS), and refractive index detectors, we show that relatively small amounts of >100 nm aggregates (cryogenic transmission electron microscopy and SLS/DLS comparison suggesting them to be wormlike micelles) dominated the stand-alone DLS results, whereas the "real" size distributions picked <50 nm. Our key result is that the kinetics of the conjugation based on PDS-thiol exchange was controlled by the thiol pKa, and this also determined the rate of the exchange between the resulting disulfides and glutathione (GSH). In particular, more acidic thiols (e.g., peptides, where a cysteine is flanked by cationic residues) react faster with PDS, but their disulfides hardly exchange with GSH; the reverse applies to thiols with a higher pKa. Disulfides that resist against restructuring via thiol-disulfide exchange allow for a stable bioconjugation, although they may be bad news for payload release under reducing conditions. However, experiments of both thiol release and nanoparticles uptake in cells (HCT116) show that also the disulfides formed from less-acidic and, therefore, less-reactive, and more exchangeable thiols were stable for at least a few hours even in a GSH-rich (10 mM) environment; this suggests a sufficiently long stability of surface groups to achieve, for example, a cell-targeting effect.

A drug eluting poly(trimethylene carbonate)/poly(lactic acid)-reinforced nanocomposite for the functional delivery of osteogenic molecules.

BACKGROUND: Poly(trimethylene carbonate) (PTMC) has wide biomedical applications in the field of tissue engineering, due to its biocompatibility and biodegradability features. Its common manufacturing involves photofabrication, such as stereolithography (SLA), which allows the fabrication of complex and controlled structures. Despite the great potential of SLA-fabricated scaffolds, very few examples of PTMC-based drug delivery systems fabricated using photo-fabrication can be found ascribed to light-triggered therapeutics instability, degradation, side reaction, binding to the macromers, etc. These concerns severely restrict the development of SLA-fabricated PTMC structures for drug delivery purposes. METHODS: In this context, we propose here, as a proof of concept, to load a drug model (dexamethasone) into electrospun fibers of poly(lactic acid), and then to integrate these bioactive fibers into the photo-crosslinkable resin of PTMC to produce hybrid films. The hybrid films' properties and drug release profile were characterized; its biological activity was investigated via bone marrow mesenchymal stem cells culture and differentiation assays. RESULTS: The polymer/polymer hybrids exhibit improved properties compared with PTMC-only films, in terms of mechanical performance and drug protection from UV denaturation. We further validated that the dexamethasone preserved its biological activity even after photoreaction within the PTMC/poly(lactic acid) hybrid structures by investigating bone marrow mesenchymal stem cells proliferation and osteogenic differentiation. CONCLUSION: This study demonstrates the potential of polymer-polymer scaffolds to simultaneously reinforce the mechanical properties of soft matrices and to load sensitive drugs in scaffolds that can be fabricated via additive manufacturing.

Kinetically stable metal ligand charge transfer complexes as crosslinks in nanogels/hydrogels: Physical properties and cytotoxicity.

A terpyridine end-functionalized 8-arm poly(ethylene glycol) was prepared using the reaction of a 4'-aminopentanoxy substituted terpyridine with a p-nitrophenyl chloroformate activated PEG-(OH)8. Supramolecular complexation of the polymer terpyridine moieties by Fe(2+) ions was investigated using NMR, UV-Vis and dynamic light scattering experiments. At low concentrations addition of Fe(2+) ions to an aqueous solution of the polymer conjugate afforded nanogels with a single size distribution around 250 nm. At concentrations above 3 wt%, and at a 1:2 metal to ligand molar ratio, hydrogels were formed with increasing mechanical properties at increasing polymer concentrations. Using bovine chondrocytes, the biocompatibility and potential cytotoxicity of the polymer conjugate, nanogels and hydrogels were studied. The polymer conjugate with free ligands was toxic to the cells likely due to depletion of essential metal ions. When the terpyridine groups were complexed with Fe(2+) ions, both nanogel suspensions and hydrogels showed no cytotoxicity in direct contact with chondrocytes. Indirect contact of gels with chondrocytes using transwells revealed the absence of toxic components by leaching. A Live-Dead assay on chondrocytes encapsulated in the hydrogels indicated that the hydrogels are cytocompatible, revealing the potential use of these materials for biomedical and pharmaceutical applications. STATEMENT OF SIGNIFICANCE: The binding between transition metal ions and ligands with multiple binding sites can be almost as strong as covalent bonds. This metal-ligand charge transfer (MLCT) complexation was used to crosslink water soluble polymers into hydrogels. This approach to novel materials may find applications in the biomedical and pharmaceutical fields. Transition metal ions are essential trace elements present in tissue but up to now no cytotoxicity data of free ligands are available. Data presented show that free ligands are toxic to cells likely by depletion of trace metal ions, whereas kinetically stable complexes are not cytotoxic even when embedded in hydrogels. These results provide fundamental issues to be considered in the design of hydrogels crosslinked through metal ligand complexation.

Preparation and mechanical properties of photo-crosslinked poly(trimethylene carbonate) and nano-hydroxyapatite composites.

Composite materials of photo-crosslinked poly(trimethylene carbonate) and nanoscale hydroxyapatite were prepared and their mechanical characteristics for application as orbital floor implants were assessed. The composites were prepared by solvent casting poly(trimethylene carbonate) macromers with varying amounts of nano-hydroxyapatite and subsequent photo-crosslinking. The incorporation of the nano-hydroxyapatite into the composites was examined by thermogravimetric analysis, scanning electron microscopy and gel content measurements. The mechanical properties were investigated by tensile testing and trouser tearing experiments. Our results show that nano-hydroxyapatite particles can readily be incorporated into photo-crosslinked poly(trimethylene carbonate) networks. Compared to the networks without nano-hydroxyapatite, incorporation of 36.3 wt.% of the apatite resulted in an increase of the E modulus, yield strength and tensile strength from 2.2 MPa to 51 MPa, 0.5 to 1.4 N/mm2 and from 1.3 to 3.9 N/mm2, respectively. We found that composites containing 12.4 wt.% nano-hydroxyapatite had the highest values of strain at break, toughness and average tear propagation strength (376% , 777 N/mm2 and 3.1 N/mm2, respectively).

Hydrogels by supramolecular crosslinking of terpyridine end group functionalized 8-arm poly(ethylene glycol).

Metallo supramolecular assemblies of an 8-arm poly(ethylene glycol) partially substituted with terpyridyl end-groups and the transition metal ions Ni(2+), Fe(2+), Co(2+) and Zn(2+) were studied for their nano-particle formation at dilute conditions and gelation at higher concentrations. The large differences in dissociation rate constants of the metal ligand complexes largely determine the assembly behavior. Thermodynamically stable complexes are generated with Ni(2+) and Fe(2+) chlorides, which lead to distinct particle sizes of ∼200 nm in dilute conditions. The Co(2+) and Zn(2+) chlorides provide multiple size distributions revealing that mono and bis-complexes are present at equilibrium. Upon complexation, terpyridyl groups move to the outer sphere giving aggregates with a charged surface. At polymer concentrations above 5 wt%, crosslinking upon addition of transition metal ions provides hydrogels. Elastic hydrogels were obtained with Ni(2+), Fe(2+) and Co(2+) having storage moduli in excess of 20 kPa, whereas Zn(2+) gels are relatively viscous. Only Zn(2+) gels show a thermoreversible sol to gel transition at a temperature of 25 °C independent of polymer concentration.

Thermoresponsive copolymer brushes possessing quaternary amine groups for strong anion-exchange chromatographic matrices.

A thermoresponsive copolymer incorporating a quaternary amine group, poly(N-isopropylacrylamide-co-3-acrylamidopropyl trimethylammonium chloride (APTAC)-co-tert-butylacrylamide), was conjugated to the surface of silica beads through surface-initiated atom transfer radical polymerization. Prepared copolymer- and copolymer brush-modified beads were characterized by CHN elemental analysis, X-ray photoelectron spectroscopy, gel permeation chromatography, and observation of phase transition profiles. Phase transition profiles of the prepared copolymer indicated that 5 mol % APTAC is suitable for enabling thermally modulated property changes in the copolymer. Chromatographic elution behaviors of adenosine nucleotides and proteins were observed using prepared beads as chromatography matrices. Higher retention time of adenosine nucleotides and strong protein adsorption behavior were observed compared with those on beads with tertiary amine groups, because of the strong basic properties. Therefore, copolymer brush modified beads will be useful as thermoresponsive ion-exchange chromatographic matrices.