Temperature-Controlled Masking/Unmasking of Cell-Adhesive Cues with Poly(ethylene glycol) Methacrylate Based Brushes

Thin, thermoresponsive polymer coatings that allow to reversibly modulate cell adhesion and detachment are attractive substrates for cell sheet engineering. Usually, this is accomplished by applying thin poly­(<i>N</i>-isopropylacrylamide) (PNIPAM) coatings, which allow cell adhesion via nonspecific interactions above the collapse temperature (<i>T</i>_T) of the surface-attached polymer and cell detachment upon cooling below <i>T</i>_T. This Article presents an alternative, thermoresponsive polymer platform that is based on 2-(2-methoxyethoxy)­ethyl methacrylate (MEO₂MA) containing copolymer brushes prepared via surface-initiated atom transfer radical polymerization (SI-ATRP). These brushes are interesting as they gradually collapse and dehydrate upon increasing the temperature from 10 to 40 °C, yet resist nonspecific adhesion of cells over this entire temperature window. The MEO₂MA based brushes presented here were modified via a two-step postpolymerization modification protocol to introduce cell-adhesive RGD containing peptide ligands. The possibility to reversibly control the swelling and collapse of these brush films by varying temperature allows to modulate the effectively available surface concentration of these cell-adhesive cues and thus provides a way to mask/unmask their biological activity. As a first proof of concept, this Article demonstrates that these MEO₂MA brush copolymer films enable integrin-mediated adhesion of 3T3 fibroblasts at 37 °C and allow release of these cells by cooling to 23 °C. The use of cell-adhesive ligands, which can be thermoreversibly masked/unmasked, is attractive as it enables the use of serum-free cell culture conditions. This is advantageous since it avoids possible concerns regarding eventual toxicity and immunological side effects of serum proteins and also provides opportunities to select for particular cell types and for enhanced control over cell stimulation and differentiation

Aqueous Fabrication of pH-Gated, Polymer-Brush-Modified Alumina Hybrid Membranes

In this Article, we studied the surface immobilization of five organic-acid-modified atom-transfer radical polymerization (ATRP) initiators based on salicylic acid, catechol, phthalic acid, and <i>m</i>- and <i>p</i>-benzoic acid on alumina, and we also investigated the growth of hydrophilic poly­(2-hydroxyethyl methacrylate) (PHEMA) and poly­(poly­(ethylene glycol)­methycrylate) (PPEGMA₆) brushes from the resulting initiator-modified substrates. Whereas the surface immobilization of phthalic acid- and benzoic acid-based initiators results in only very thin brushes or no brush growth at all, SI-ATRP of HEMA and PEGMA₆ from alumina surfaces modified with salicylate or catechol generates brushes with thicknesses comparable to those obtained using organosilane-based initiators. Most interestingly, the surface immobilization of the catechol- and salicylate based-initiators was found to be pH-dependent, which allowed facile variation of the ATRP initiator surface concentration and, concomitantly, the polymer brush grafting density by adjusting the pH of the aqueous solution that was used to immobilize the initiator. This is in contrast to organosilane-based initiators, where the variation of the grafting density is usually accomplished using mixtures of the ATRP initiator and an ATRP inactive “dummy”. Another difference between the organosilane-based initiators and the organic acid analogues is the stability of hydrophilic brushes grown from alumina. After a certain threshold thickness was exceeded, organosilane-tethered PPEGMA₆ brushes were observed to detach from the substrate, in contrast to brushes grown from catechol or salicylate initiators, which did not show signs of degradation. Finally, as a first proof-of-concept, the salicylate-based initiator was used to develop an all-aqueous protocol for the modification of alumina membranes with hydrophilic PHEMA and succinic anhydride post-modified polymer brushes. The water permeation properties of these hybrid membranes can be controlled by adjusting the brush thickness in the case of the neutral PHEMA brush coating or can be pH-gated after post-polymerization modification to introduce carboxylic acid groups

Polymer Brush Gradients Grafted from Plasma-Polymerized Surfaces

A new method for generating a surface density gradient of polymer chains is presented. A substrate-independent polymer deposition technique was used to coat materials with a chemical gradient based on plasma copolymerization of 1,7-octadiene and allylamine. This provided a uniform chemical gradient to which initiators for atom transfer radical polymerization (ATRP) were immobilized. After surface-initiated atom transfer radical polymerization (SI-ATRP), poly­(2-hydroxyethyl methacrylate) (PHEMA) chains were grafted from the surface and the measured thickness profiles provided direct evidence for how surface crowding provides an entropic driving force resulting in chain extension away from the surface. Film thicknesses were found to increase with the position along the gradient surface, reflecting the gradual transition from collapsed to more extended surface-tethered polymer chains as the grafting density increased. The method described is novel in that the approach provides covalent linkages from the polymer coating to the substrate and is not limited to a particular surface chemistry of the starting material

Squaric Acid Mediated Synthesis and Biological Activity of a Library of Linear and Hyperbranched Poly(Glycerol)–Protein Conjugates

Polymer–protein conjugates generated from side chain functional synthetic polymers are attractive because they can be easily further modified with, for example, labeling groups or targeting ligands. The residue specific modification of proteins with side chain functional synthetic polymers using the traditional coupling strategies may be compromised due to the nonorthogonality of the side-chain and chain-end functional groups of the synthetic polymer, which may lead to side reactions. This study explores the feasibility of the squaric acid diethyl ester mediated coupling as an amine selective, hydroxyl tolerant, and hydrolysis insensitive route for the preparation of side-chain functional, hydroxyl-containing, polymer–protein conjugates. The hydroxyl side chain functional polymers selected for this study are a library of amine end-functional, linear, midfunctional, hyperbranched, and linear-block-hyperbranched polyglycerol (PG) copolymers. These synthetic polymers have been used to prepare a diverse library of BSA and lysozyme polymer conjugates. In addition to exploring the scope and limitations of the squaric acid diethylester-mediated coupling strategy, the use of the library of polyglycerol copolymers also allows to systematically study the influence of molecular weight and architecture of the synthetic polymer on the biological activity of the protein. Comparison of the activity of PG–lysozyme conjugates generated from relatively low molecular weight PG copolymers did not reveal any obvious structure–activity relationships. Evaluation of the activity of conjugates composed of PG copolymers with molecular weights of 10000 or 20000 g/mol, however, indicated significantly higher activities of conjugates prepared from midfunctional synthetic polymers as compared to linear polymers of similar molecular weight

pH-Sensitive Coiled-Coil Peptide-Cross-Linked Hyaluronic Acid Nanogels: Synthesis and Targeted Intracellular Protein Delivery to CD44 Positive Cancer Cells

The clinical translation of protein drugs that act intracellularly is limited by the absence of safe and efficient intracellular protein delivery vehicles. Here, pH-sensitive coiled-coil peptide-cross-linked hyaluronic acid nanogels (HA-cNGs) were designed and investigated for targeted intracellular protein delivery to CD44 overexpressing MCF-7 breast cancer cells. HA-cNGs were obtained with a small size of 176 nm from an equivalent mixture of hyaluronic acid conjugates with GY­(EIAALEK)₃GC (E3) and GY­(KIAALKE)₃GC (K3) peptides, respectively, at pH 7.4 by nanoprecipitation. Circular dichroism (CD) proved the formation of coiled-coil structures between E3 and K3 peptides at pH 7.4 while fast uncoiling at pH 5.0. HA-cNGs showed facile loading of cytochrome C (CC) and greatly accelerated CC release under mild acidic conditions (18.4%, 76.8%, and 91.4% protein release in 24 h at pH 7.4, 6.0, and 5.0, respectively). Confocal microscopy and flow cytometry displayed efficient internalization of CC-loaded HA-cNGs and effective endosomal escape of CC in MCF-7 cancer cells. Remarkably, HA-cNGs loaded with saporin, a ribosome inactivating protein, exhibited significantly enhanced apoptotic activity to MCF-7 cells with a low IC₅₀ of 12.2 nM. These coiled-coil peptide-cross-linked hyaluronic acid nanogels have appeared as a simple and multifunctional platform for efficient intracellular protein delivery

Comparative Study on the In Vitro Cytotoxicity of Linear, Dendritic, and Hyperbranched Polylysine Analogues

Lysine-based polycations are widely used as nonviral carriers for gene delivery. This manuscript reports the results of a comparative study on the in vitro cytotoxicity of a library of three structural polylysine variants, namely, linear polylysine (LPL), dendritic polylysine (DPL), and hyperbranched polylysine (HBPL). The aim of this study was to identify possible effects of polymer molecular weight and architecture on both immediate and delayed cytotoxicity and also to provide a mechanistic understanding for possible differences. Acute cytotoxicities were evaluated using cell viability assays with CHO DG44 cells. At comparable molecular weights, the EC₅₀ values for the LPL analogues were ∼5–250 times higher as compared to the DPL and HBPL samples. For low molecular weight polycations, osmotic shock was found to be an important contributor to immediate cell death, whereas for the higher molecular weight analogues, direct cell membrane disruption was identified to play a role. Delayed cytotoxicity (≥3 h) was assessed by identifying several of the hallmark events that characterize apoptosis, including phosphatidyl serine translocation, mitochondrial membrane depolarization, cytoplasmic cytochrome C release, and caspase 3 activation. At comparable molecular weights, apoptosis was found to be more pronounced for DPL and HBPL as compared to LPL. This difference was ascribed to the fact that LPL is completely enzymatically degradable, in contrast to DPL and HBPL, which also contain ε-peptidic bonds and are only partially degradable. Because their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA

Polymer-Brush-Templated Three-Dimensional Molybdenum Sulfide Catalyst for Hydrogen Evolution

Earth-abundant hydrogen evolution catalysts are essential for high-efficiency solar-driven water splitting. Although a significant amount of studies have been dedicated to the development of new catalytic materials, the microscopic assembly of these materials has not been widely investigated. Here, we describe an approach to control the three-dimensional (3D) assembly of amorphous molybdenum sulfide using polymer brushes as a template. To this end, poly­(dimethylaminoethyl methacrylate) brushes were grown from highly oriented pyrolytic graphite. These cationic polymer films bind anionic MoS₄^2– through an anion-exchange reaction. In a final oxidation step, the polymer-bound MoS₄^2– is converted into the amorphous MoS_<i>x</i> catalyst. The flexibility of the assembly design allowed systematic optimization of the 3D catalyst. The best system exhibited turnover frequencies up to 1.3 and 4.9 s^–1 at overpotentials of 200 and 250 mV, respectively. This turnover frequency stands out among various molybdenum sulfide catalysts. The work demonstrates a novel strategy to control the assembly of hydrogen evolution reaction catalysts

Ductile, High-Lignin-Content Thermoset Films and Coatings

In the context of transitioning toward a more sustainable use of natural resources, the application of lignin to substitute commonly utilized petroleum-based plastics can play a key role. Although lignin is highly available at low cost and presents interesting properties, such as antioxidant and UV barrier activities, its application is limited by its low reactivity, which is a consequence of harsh conditions normally used to extract lignin from biomass. In this work, the use of glyoxylic acid lignin (GA lignin), rich in carboxylic acid groups and hence highly reactive toward epoxy cross-linkers, is presented. GA lignin, which is directly extracted from biomass via a one-step aldehyde-assisted fractionation process, allowed the preparation of thermoset films and coatings via a simple reaction with sustainable poly(ethylene glycol) diglycidyl ether and glycerol diglycidyl ether cross-linkers. This allows one to prepare freestanding films containing up to 70 wt % lignin with tunable mechanical properties and covalently surface-attached coatings containing up to 90 wt % lignin with high solvent resistance. Both films and coatings display antioxidant properties and combine excellent UV barrier activity with high visible transparency, which is attractive for applications in sustainable food packaging