Oxidation Responsiveness of Polythioacetals and their Anti-Inflammatory Potential

The most commonly encountered strong oxidants in biology go under the collective name of Reactive Oxygen Species (ROS). The role of ROS is concentration-dependent: at low levels (e.g. µM for H2O2) they participate in survival pathways1, while at increasing concentration they become paracrine messengers, metabolic and inflammatory activators, inducers of apoptosis, eventually also acting as direct cytotoxic compounds (e.g. when H2O2 concentration is tens of mM)2, 3. What occurs in the presence of pathologically high levels of ROS is typically referred to as oxidative stress. In the central nervous system (CNS), oxidative stress is a key contributor to the onset and progression of neurodegenerative diseases, mental disorders and traumatic injuries4. Our group works on macromolecular ROS scavengers, often in the form of long-circulating nanoparticles. We specifically focus on sulfur(II)-containing macromolecular systems, whose responsiveness is based on the oxidation of sulfur to higher oxidation states. In poly(thioacetals) (PTAs) this oxidation triggers a cleavage of the whole group5; since the latter is incorporated in the macromolecular chain, these structures depolymerize during ROS scavenging; this may be beneficial in promoting a rapid clearance of the oxidation products, although care must be paid to also their potential toxicity. Polymer synthesis. We synthesized and characterized a ~20 kDa PTA (poly(1,3-dithio-6,9-dioxo-2-methylacetate-decane)), where each thioacetal group is flanked by a pendant methyl ester; hydrolysis of the latter to carboxylates increases the hydrophilicity of the macromolecule, and we have indeed produced materials that are fully water soluble (95% hydrolysis) or dispersable at pH>6/colloidal at pH<6 (45% hydrolysis). ROS responsiveness. We have evaluated the ROS scavenging capacity of the PTA parent (hydrophobic, non-hydrolyzed) polymer to that of similarly sized and hydrophobic poly(propylene sulfide) (PPS), a poly(thioether) extensively studied by our group6. PPS oxidation (thioethers to sulfoxides/sulfones) increases hydrophilicity without depolymerization, but this mechanistic difference did not play a major role in the ROS-scavenging kinetics of the dispersions of the two polymers (nanodroplets), suggesting the initial stage of sulfur oxidation to be the rate-determining step of the process. In vitro assessment. This phase was conducted on the hydrolyzed PTA, which are thermodynamically more stable in a water environment (either completely soluble or aggregated in micellar-like structures). Firstly, two model murine cell lines (BV2 microglia, C8D30 astrocytes) showed no decrease in viability with more than 100 µg/mL PTAs for 24 h, or up to 50 (amphiphilic PTA-COOH45) or 100 µg/mL (amphiphilic PTA-COOH95) for 48 h. The different morphology of the polymers in a water environment determined a different uptake mechanism in astrocytes, with the amphiphilic PTA-COOH45 able to permeate passively, and the hydrophilic PTA-COOH95 at least partially endocyted. No significant difference was seen in microglia, possibly due to their propensity towards phago- and macropinocytosis. The anti-inflammatory potential of hydrolyzed PTA (50 µg/mL) was then assessed in a CNS inflammation model using LPS-activated BV2 and C8D30 cells. Both polymers reduced the release of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in both cell cultures, with a more pronounced effect observed in BV2. PTA-COOH45 performed slightly better than PTA-COOH95, possibly due to a higher uptake. A similar trend was observed in the reduction of intracellular ROS - including hydrogen peroxide, superoxide, and nitric oxide - suggesting that the anti-inflammatory effects are at least partially mediated by the polymers’ antioxidant activity.