Shape-Shifting Micro- and Nanopatterns Controlled by Temperature

Herein, features that alter their shape to form a different pattern upon an external trigger are described. Electron-beam lithography was used to fabricate micrometer- and nanometer-sized surface immobilized poly­(triethylene glycol methacrylate) (pTEGMA) that exhibits significant thermal responsivity; the resulting hydrogels collapsed by up to 95% of their height upon addition of heat. Multicomponent features composed of both the thermoresponsive polymer and nonresponsive poly­(ethylene glycol) (PEG) were then prepared. Upon increase in temperature, only the thermally responsive component of the pattern collapsed, causing a significant and predictable alteration in the overall pattern. Reversible micrometer- and nanometer-sized square-to-triangles, squares-to-checkerboards, smiles-to-neutral face, and zeros-to-ones shapes were shown

Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors

Herein, we report the synthesis of trehalose side chain polymers for stabilization of protein conjugates to environmental stressors. The glycomonomer 4,6-<i>O</i>-(4-vinylbenzylidene)-α,α-trehalose was synthesized in 40% yield over two steps without the use of protecting group chemistry. Polymers containing the trehalose pendent groups were prepared via reversible addition–fragmentation chain transfer (RAFT) polymerization using two different thiol-reactive chain transfer agents (CTAs) for subsequent conjugation to proteins through disulfide linkages. The resulting glycopolymers were well-defined, and a range of molecular weights from 4200 to 49 500 Da was obtained. The polymers were conjugated to thiolated hen egg white lysozyme and purified. The glycopolymers when added or covalently attached to protein significantly increased stability toward lyophilization and heat relative to wild-type protein. Up to 100% retention of activity was observed when lysozyme was stressed ten times with lyophilization and 81% activity when the protein was heated at 90 °C for 1 h; this is in contrast to 16% and 18% retention of activity, respectively, for the wild-type protein alone. The glycopolymers were compared to equivalent concentrations of trehalose and poly­(ethylene glycol) (PEG) and found to be superior at stabilizing the protein to lyophilization and heat. In addition, the protein–glycopolymer conjugates exhibited significant increases in lyophilization stability when compared to adding the same concentration of unconjugated polymer to the protein

Protein–Polymer Conjugation via Ligand Affinity and Photoactivation of Glutathione <i>S</i>‑Transferase

A photoactivated, site-selective conjugation of poly­(ethylene glycol) (PEG) to the glutathione (GSH) binding pocket of glutathione <i>S</i>-transferase (GST) is described. To achieve this, a GSH analogue (GSH-BP) was designed and chemically synthesized with three functionalities: (1) the binding affinity of GSH to GST, (2) a free thiol for polymer functionalization, and (3) a photoreactive benzophenone (BP) component. Different molecular weights (2 kDa, 5 kDa, and 20 kDa) of GSH-BP modified PEGs (GSBP-PEGs) were synthesized and showed conjugation efficiencies between 52% and 76% to GST. Diazirine (DA) PEG were also prepared but gave conjugation yields lower than for GSBP-PEGs. PEGs with different end-groups were also synthesized to validate the importance of each component in the end-group design. End-groups included glutathione (GS-PEG) and benzophenone (BP-PEG). Results showed that both GSH and BP were crucial for successful conjugation to GST. In addition, conjugations of 5 kDa GSBP-PEG to different proteins were investigated, including bovine serum albumin (BSA), lysozyme (Lyz), ubiquitin (Ubq), and GST-fused ubiquitin (GST-Ubq) to ensure specific binding to GST. By combining noncovalent and covalent interactions, we have developed a new phototriggered protein–polymer conjugation method that is generally applicable to GST-fusion proteins

Fluorous Comonomer Modulates the Reactivity of Cyclic Ketene Acetal and Degradation of Vinyl Polymers

Fluorine-containing polymers have potential for use in medicine and other applications, but the synthesis of degradable fluorous polymers is underexplored. In this report, we present a facile route to degradable fluorinated polymers and characterize the effect of fluorous comonomer identity on the polymerization as well as the degradation kinetics of the resulting polymer. Copolymers of poly­(ethylene glycol methyl ether methacrylate) (PEGMA), fluorous methacrylate (1<i>H</i>,1<i>H</i>,2<i>H</i>,2<i>H</i>-perfluorooctyl or 1<i>H</i>,1<i>H</i>,2<i>H</i>,2<i>H</i>,3<i>H</i>,3<i>H</i>-perfluoropentyl methacrylate), and cyclic ketene acetal 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) were synthesized via ruthenium-catalyzed living radical polymerization. It was observed that increasing the fluorous monomer content led to enhanced BMDO incorporation in the resulting polymer. Density functional theory calculations suggest that this is due to the decreased energy gap between the singly occupied molecular orbital (SOMO) of the methacrylate radical and the highest occupied molecular orbital (HOMO) of BMDO. Moreover, polymers with higher fluorous monomer content were more hydrolytically stable, with a degradation rate constant 100-fold smaller for the polymer with highest fluorous content compared to the nonfluorous polymer. This work provides easy access to degradable fluorous polymers using vinyl monomers. In addition, the insights gained into modulation of reactivity of cyclic ketene acetals and polymer degradation will be useful in applying fluorous polymers for a variety of biomedical applications

Aminooxy and Pyridyl Disulfide Telechelic Poly(poly(ethylene glycol) acrylate) by RAFT Polymerization

An efficient method to synthesize telechelic, bioreactive polymers is described. Homotelechelic polymers were synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization in one step by employing bifunctional chain transfer agents (CTAs). A bis-carboxylic acid CTA was coupled to <i>N</i>-Boc-aminooxy ethanol or pyridyl disulfide ethanol, resulting in a bis-<i>N</i>-Boc-aminooxy CTA and a bis-pyridyl disulfide CTA, respectively. RAFT polymerization of poly­(ethylene glycol) (PEG) acrylate in the presence of both CTAs resulted in a series of polymers over a range of molecular weights (∼8.4–35.2 kDa; polydispersity indices, PDIs, of 1.11–1.44) with retention of end-groups postpolymerization. The polymers were characterized by ¹H NMR spectroscopy and gel permeation chromatography (GPC). Conjugations of small molecules and peptides resulted in homotelechelic polymer conjugates

Imine Hydrogels with Tunable Degradability for Tissue Engineering

A shortage of available organ donors has created a need for engineered tissues. In this context, polymer-based hydrogels that break down inside the body are often used as constructs for growth factors and cells. Herein, we report imine cross-linked gels where degradation is controllable by the introduction of mixed imine cross-links. Specifically, hydrazide-functionalized poly­(ethylene glycol) (PEG) reacts with aldehyde-functionalized PEG (PEG-CHO) to form hydrazone linked hydrogels that degrade quickly in media. The time to degradation can be controlled by changing the structure of the hydrazide group or by introducing hydroxylamines to form nonreversible oxime linkages. Hydrogels containing adipohydrazide-functionalized PEG (PEG-ADH) and PEG-CHO were found to degrade more rapidly than gels formed from carbodihydrazide-functionalized PEG (PEG-CDH). Incorporating oxime linkages via aminooxy-functionalized PEG (PEG-AO) into the hydrazone cross-linked gels further stabilized the hydrogels. This imine cross-linking approach should be useful for modulating the degradation characteristics of 3D cell culture supports for controlled cell release

A Heparin-Mimicking Block Copolymer Both Stabilizes and Increases the Activity of Fibroblast Growth Factor 2 (FGF2)

Fibroblast growth factor 2 (FGF2) is a protein involved in cellular functions in applications such as wound healing and tissue regeneration. Stabilization of this protein is important for its use as a therapeutic since the native protein is unstable during storage and delivery. Additionally, the ability to increase the activity of FGF2 is important for its application, particularly in chronic wound healing and the treatment of various ischemic conditions. Here we report a heparin mimicking block copolymer, poly­(styrenesulfonate-<i>co</i>-poly­(ethylene glycol) methyl ether methacrylate)-<i>b</i>-vinyl sulfonate) (p­(SS-<i>co</i>-PEGMA)-<i>b</i>-VS, that contains a segment that enhances the stability of FGF2 and one that binds to the FGF2 receptor. The FGF2 conjugate retained activity after exposure to refrigeration (4 °C) and room temperature (23 °C) for 7 days, while unmodified FGF2 was inactive after these standard storage conditions. A cell study performed with a cell line lacking native heparan sulfate proteoglycans indicated that the conjugated block copolymer facilitated binding of FGF2 to its receptor similar to the addition of heparin to FGF2. A receptor-based enzyme-linked immunosorbant assay (ELISA) confirmed the results. The conjugate also increased the migration of endothelial cells by 80% compared to FGF2 alone. Additionally, the FGF2-p­(SS-<i>co</i>-PEGMA)-<i>b</i>-VS stimulated endothelial cell sprouting 250% better than FGF2 at low concentration. These data verify that this rationally designed protein-block copolymer conjugate enhances receptor binding, cellular processes such as migration and tube-like formation, and stability, and suggest that it may be useful for applications in biomaterials, tissue regeneration, and wound healing

Poly(vinyl sulfonate) Facilitates bFGF-Induced Cell Proliferation

Heparin is a highly sulfated polysaccharide and is useful because of its diverse biological functions. However, because of batch-to-batch variability and other factors, there is significant interest in preparing biomimetics of heparin. To identify polymeric heparin mimetics, a cell-based screening assay was developed in cells that express fibroblast growth factor receptors (FGFRs) but not heparan sulfate proteoglycans. Various sulfated and sulfonated polymers were screened, and poly­(vinyl sulfonate) (pVS) was identified as the strongest heparin-mimicking polymer in its ability to enhance binding of basic fibroblast growth factor (bFGF) to FGFR. The results were confirmed by an ELISA-based receptor-binding assay. Different molecular weights of pVS polymer were synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization. The polymers were able to facilitate dimerization of FGFRs leading to cell proliferation in FGFR-expressing cells, and no size dependence was observed. The data showed that pVS is comparable to heparin in these assays. In addition, pVS was not cytotoxic to fibroblast cells up to at least 1 mg/mL. Together this data indicates that pVS should be explored further as a replacement for heparin

Direct Write Protein Patterns for Multiplexed Cytokine Detection from Live Cells Using Electron Beam Lithography

Simultaneous detection of multiple biomarkers, such as extracellular signaling molecules, is a critical aspect in disease profiling and diagnostics. Precise positioning of antibodies on surfaces, especially at the micro- and nanoscale, is important for the improvement of assays, biosensors, and diagnostics on the molecular level, and therefore, the pursuit of device miniaturization for parallel, fast, low-volume assays is a continuing challenge. Here, we describe a multiplexed cytokine immunoassay utilizing electron beam lithography and a trehalose glycopolymer as a resist for the direct writing of antibodies on silicon substrates, allowing for micro- and nanoscale precision of protein immobilization. Specifically, anti-interleukin 6 (IL-6) and antitumor necrosis factor alpha (TNFα) antibodies were directly patterned. Retention of the specific binding properties of the patterned antibodies was shown by the capture of secreted cytokines from stimulated RAW 264.7 macrophages. A sandwich immunoassay was employed using gold nanoparticles and enhancement with silver for the detection and visualization of bound cytokines to the patterns by localized surface plasmon resonance detected with dark-field microscopy. Multiplexing with both IL-6 and TNFα on a single chip was also successfully demonstrated with high specificity and in relevant cell culture conditions and at different times after cell stimulation. The direct fabrication of capture antibody patterns for cytokine detection described here could be useful for biosensing applications

Biocompatible Hydrogels by Oxime Click Chemistry

Oxime Click chemistry was used to form hydrogels that support cell adhesion. Eight-armed aminooxy poly­(ethylene glycol) (PEG) was mixed with glutaraldehyde to form oxime-linked hydrogels. The mechanical properties, gelation kinetics, and water swelling ratios were studied and found to be tunable. It was also shown that gels containing the integrin ligand arginine-glycine-aspartic acid (RGD) supported mesenchymal stem cell (MSC) incorporation. High cell viability and proliferation of the encapsulated cells demonstrated biocompatibility of the material