Synthesis of Thermoresponsive, Catechol-Rich Poly(ethylene glycol) Brush Polymers for Attenuating Cellular Oxidative Stress.

Herein, we report a platform to integrate customizable quantities of catechol units into polymers by reacting caffeic acid carbonic anhydride with polymers having pendant amine groups. Brush poly(ethylene glycol)-caffeamide (PEG-CAF) copolymers based on oligo(ethylene glycol)methyl ether methacrylate (OEGMA500) were obtained with a catechol content of approximately 30, 40, and 50 mol % (vs OEGMA content). Owing to the hydrophobicity of the introduced CAF groups, the catechol copolymers exhibited cloud points in the range of 23-46 °C and were used to fabricate thermoresponsive FeIII metal-phenolic network capsules. Polymers with the highest CAF content (50 mol %) proved most effective for attenuating reactive oxygen species levels in vitro, in co-cultured fibroblasts, and breast cancer cells, even in the presence of an exogenous oxidant source. The reported approach to synthesize customizable catechol materials could be generalized to other amine-functional polymers, with potential biomedical applications such as adhesives or stimuli-responsive drug delivery systems.

Nitroxide-functional PEGylated nanostars arrest cellular oxidative stress and exhibit preferential accumulation in co-cultured breast cancer cells.

The limited application of traditional antioxidants to reducing elevated levels of reactive oxygen species (ROS) is potentially due to their lack of stability and biocompatibility when tested in a biological milieu. For instance, the poor biological antioxidant performance of small molecular nitroxides arises from their limited diffusion across cell membranes and their significant side effects when applied at high doses. Herein, we describe the use of nanostructured carriers to improve the antioxidant activity of a typical nitroxide derivative, (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO). Polymers with star-shaped structures were synthesised and were further conjugated to TEMPO moieties via amide linkages. The TEMPO-loaded stars have small hydrodynamic sizes (<20 nm), and are better tolerated by cells than free TEMPO in a breast cancer-fibroblast co-culture, a system exhibiting elevated ROS levels. At a well-tolerated concentration, the polymer with the highest TEMPO-loading capacity successfully downregulated ROS production in co-cultured cells (a significant decrease of up to 50% vs. basal ROS levels), which was accompanied by a specific reduction in superoxide anion generation in the mitochondria. In contrast, the equivalent concentration of free TEMPO did not achieve the same outcome. Further investigation showed that the TEMPO-conjugated star polymers can be recycled inside the cells, thus providing longer term scavenging activity. Cell association studies demonstrated that the polymers can be taken up by both cell types in the co-culture, and are found to co-locate with the mitochondria. Interestingly the stars exhibited preferential mitochodria targeting in the co-cultured cancer cells compared to accompanying fibroblasts. The data suggest the potential of TEMPO-conjugated star polymers to arrest oxidative stress for various applications in cancer therapy.

Trisulfide linked cholesteryl PEG conjugate attenuates intracellular ROS and collagen-1 production in a breast cancer co-culture model.

The progression of cancer has been closely-linked with augmentation of cellular reactive oxygen species (ROS) levels and ROS-associated changes in the tumour microenvironment (TME), including alterations to the extracellular matrix and associated low drug uptake. Herein we report the application of a co-culture model to simulate the ROS based cell-cell interactions in the TME using fibroblasts and breast cancer cells, and describe how novel reactive polymers can be used to modulate those interactions. Under the co-culture conditions, both cell types exhibited modifications in behaviour, including significant overproduction of ROS in the cancer cells, and elevation of the collagen-1 secretion and stained actin filament intensity in the fibroblasts. To examine the potential of using reactive antioxidant polymers to intercept ROS communication and thereby manipulate the TME, we employed H2S-releasing macromolecular conjugates which have been previously demonstrated to mitigate ROS production in HEK cells. The specific conjugate used, mPEG-SSS-cholesteryl (T), significantly reduced ROS levels in co-cultured cancer cells by approximately 50%. This reduction was significantly greater than that observed with the other positive antioxidant controls. Exposure to T was also found to downregulate levels of collagen-1 in the co-cultured fibroblasts, while exhibiting less impact on cells in mono-culture. This would suggest a possible downstream effect of ROS-mitigation by T on stromal-tumour cell signalling. Since fibroblast-derived collagens modulate crucial steps in tumorigenesis, this ROS-associated effect could potentially be harnessed to slow cancer progression. The model may also be beneficial for interrogating the impact of antioxidants on naturally enhanced ROS levels, rather than relying on the application of exogenous oxidants to simulate elevated ROS levels.

Trisulfide-Bearing PEG Brush Polymers Donate Hydrogen Sulfide and Ameliorate Cellular Oxidative Stress.

A potential approach to combat cellular dysfunction is to manipulate cell communication and signaling pathways to restore physiological functions while protecting unaffected cells. For instance, delivering the signaling molecule H2S to certain cells has been shown to restore cell viability and re-normalize cell behavior. We have previously demonstrated the ability to incorporate a trisulfide-based H2S-donating moiety into linear polymers with good in vitro releasing profiles and demonstrated their potential for ameliorating oxidative stress. Herein, we report two novel series of brush polymers decorated with higher numbers of H2S-releasing segments. These materials contain two trisulfide-based monomers co-polymerized with oligo(ethylene glycol methyl ether methacrylate) via reversible addition-fragmentation chain-transfer polymerization. The macromolecules were characterized to have a range of trisulfide densities with similar, well-defined molecular weight distribution, good H2S-releasing profiles, and high cellular tolerance. Using an amperometric technique, the H2S liberated and total sulfide release were found to depend on concentrations and chemical nature of triggering molecules (glutathione and cysteine) and, importantly, the position of reactive groups within the brush structure. Notably, when introduced to cells at well-tolerated doses, two macromolecular donors which have the same proportion as of the H2S-donating monomer (30%) but differ in releasing moiety location show similar cellular H2S-releasing kinetics. These donors can restore reactive oxygen species levels to baseline values, when polymer pretreated cells are exposed to exogenous oxidants (H2O2). Our work opens up a new aspect in preparing H2S macromolecule donors and their application to arresting cellular oxidative cascades.

Polymers with Dithiobenzoate End Groups Constitutively Release Hydrogen Sulfide upon Exposure to Cysteine and Homocysteine.

Dithioesters are well-established as efficient reversible addition-fragmentation chain transfer (RAFT) agents. More recently, certain small molecule dithioesters have been reported to release the biological signaling molecule hydrogen sulfide (H2S) upon exposure to cysteine. Herein, we examine the propensity of polymers synthesized using RAFT with a dithioester chain transfer agent to release H2S via reaction of cysteine with constitutive dithioester end-groups. Homocysteine-triggered release of H2S from these materials is also observed, with evidence suggesting that the mechanism is analogous to the reaction with cysteine.