Dual stimuli-responsive antioxidant polymer nanoparticles as therapeutics for oxidative stress-associated diseases

SUMMARY Vanillin is a naturally occurring molecule with potent antioxidant and anti-inflammatory activities. We synthesized a polymeric prodrug of vanillin, poly(vanillin oxalate)(PVO), in which vanillin is covalently incorporated in its backbone. PVO was also designed to have acid-cleavable acetal linkages and hydrogen peroxide (H2O2)-responsive peroxalate linkages in its backbone. The dual stimuliresponsive PVO nanoparticles administrated intravenously accumulate in liver and show therapeutic activities in mice suffering from acute liver failure and hepatic ischemia/reperfusion injury. INTRODUCTION Excessive and unregulated production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), superoxide and hydroxyl radicals is known to cause oxidative stress. Therefore, there has been great interest in the development of antioxidants as therapeutic agents for various ROS-associated inflammatory diseases. Vanillin is the major component of natural vanilla and has antiinflammatory activity to suppress the expression of various pro-inflammatory cytokines. However, orally administrated vanillin is rapidly decomposed in the upper digestive tract and intravenously injected vanillin is rapidly cleared from blood circulation. Therefore, its successful clinical applications need a strategy to deliver vanillin to diseased cells or tissues and enhance its therapeutic efficacy. In this study, we developed a polymeric prodrug of vanillin, poly(vanillin oxalate)(PVO), in which vanillin is covalently incorporated in its backbone and can be released during its hydrolytic degradation. Herein, we report the synthesis and physicochemical properties of PVO and the potential of PVO nanoparticles as therapeutic agents for oxidative stress-associated diseases. EXPERIMENTAL METHODS PVO was synthesized through two step. First, 4-(5hydroxymethyl)-5-mthyl-1,3-dioxan-2yl)-2methoxyphenol were made using vanillin (4hydroxy-3-methoxybenzaldehyde), 1,1,1-Tris (hydroxymethyl)-ethane, dry tetrahydrofuran (THF) and p-TSA as a catalyst. Second, PVO was develped using 4-(5-hydroxymethyl)-5-mthyl-1,3-dioxan2yl)-2-methoxy-phenol, oxalyl chloride and pyridine in 1:1:2.5 mixture. The chemical structure of 4-(5hydroxymethyl)-5-mthyl-1,3-dioxan-2yl)-2methoxyphenol and PVO was confirmed by 1 H NMR. Its average molecular weight was determined using gel permeation chromatography (GPC). PVO nanoparticles were prepared using an o/w single emulsion method. We investigated the release kinetics of vanillin from PVO nanoparticles. We assessed the H2O2 scavenging ability of PVO nanoparticles using Amplex Red assay and evaluated ability of PVO nanoparticles to reduce the generation of intracellular ROS through the confocal laser scanning microscopy (CLSM) and flow cytometry. Antioxidant and anti-inflammatory activities of PVO nanoparticles were assessed through detection of NO and RT-PCR. We also performed the MTT assay to evaluate the cytotoxic effects of PVO nanoparticles on RAW 264.7 cells. The potential of PVO nanoparticles as therapeutic agents was evaluated using mouse models of acute liver failure and ischemia/reperfusion injury. Fig. 1. Synthetic and degradation routes of PVO as a polymeric prodrug of vanillin. RESULTS AND DISCUSSION PVO contained both peroxalate ester bonds and acetal linkages in the backbone. Therefore PVO hydrolytically degrades into three components and its degradation is accelerated by low pH and H2O2. Its molecular weight was determined to be ~22,000 Da with polydispersity index of ~1.6 by GPC. PVO had hydrophobic backbone and therefore could be formulated into nanoparticles of round spheres and a mean hydrodynamic diameter of ~230nm in phosphate buffer (pH 7.4). Fig. 2. Characterization of PVO nanoparticles. (A) A SEM image and (B) dynamic light scattering, (C) H2O2 scavenging by PVO nanoparticles, (D) Release kinetics of vanillin from PVO nanoparticles. PVO nanoparticles showed higher H2O2 scavenging activity than free vanillin. This result can be explained by the combined effects of vanillin and H2O2-reacting peroxalate ester bonds in the backbone of PVO. PVO nanoparticles exhibited low cytotoxicity and excellent biocompatibility through MTT assay. Fig. 3. Antioxidant and anti-inflammatory activities of PVO nanoparticles in LPS-stimulated cells. (A) Inhibition of ROS production, (B) Inhibition of nitric oxide (NO), (C) Inhibitory effects on proinflammatory mediators. We investigated the ability of PVO nanoparticles to suppress LPS-mediated oxidative stress in RAW 264.7 cells, measured by CLSM and flow cytometry. As a result, the intracellular ROS generation was suppressed by PVO nanoparticles. PVO nanoparticles showed the stronger anti-inflammatory activity than vanillin may be due to their strong antioxidant activity. It can be explained by the PVO scavenges H2O2 generated during LPS-induced inflammatory responses and PVO releases vanillin during its degradation, which inhibits the further generation of other ROS and expression of TNF- and iNOS. PVO nanoparticles effectively reduced hepatic damages during ischemia/reperfusion. As shown in Fig. 4, PVO nanoparticles significantly reduced the activity of ALT (alanine transaminase) after hepatic ischemia/reperfusion and protected liver tissues from cell death. Fig. 4. Therapeutic effects of PVO nanoparticles on hepatic ischemia/reperfusion injury. (A) Reduction of ALT, (B) H&E staining of liver tissues. CONCLUSIONS We developed antioxidant and anti-inflammatory nanoparticles based on dual stimuli-responsive polymeric prodrug of vanillin (PVO). PVO showed pH-dependent hydrolytic degradation kinetics due to its acetal linkage. Also, PVO nanoparticles contained H2O2-responsive peroxalate ester bonds in their backbone and showed highly potent antioxidant activities by scavenging H2O2 and inhibiting the generation of oxidants in LPS-stimulated cells. In addition, PVO nanoparticles also exerted potent antiinflammatory activity by inhibiting the expression of TNF-α and iNOS. We anticipate that PVO nanoparticles as a prodrug of vanillin have tremendous potential as therapeutics and drug delivery systems for oxidative stress-associated inflammatory diseases. REFERENCES 1. J. Kwon. et al, Adv. Biomacromolecules, 2013, 14, 1618. 2. D. Lee, et al. Sci. Reports, 2013, 3, 2233. ACKNOWLEDGMENTS This work was supported by Brain Korea 21 plus program of National Research Foundation, Korea. (C) (D) (B) (A)