A self-deliverable H2O2-responsive tocopherol dimer for enhanced antioxidant and liposomal delivery

A self-deliverable H2O2-responsive tocopherol dimer for enhanced antioxidant and liposomal delivery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A self-deliverable H2O2-responsive tocopherol dimer for enhanced antioxidant and liposomal delivery Hanui Jo, Ayoung Kim, Changhee Park, soyoon Baek, Inki Hong, Mingi Kim, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7117698/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Oxidative stress caused by excessive hydrogen peroxide (H₂O₂) plays a central role in skin damage, inflammation, and premature aging, particularly through light-induced photosensitization. Tocopherol (TCP) is a widely used antioxidant in cosmetics, yet its potential in H₂O₂-responsive systems remains underexplored. Here, we report the design and characterization of ditocopheryl peroxalate (TOT), a novel tocopherol dimer linked via an H₂O₂-cleavable peroxalate bond. TOT remains stable under physiological conditions but selectively degrades in response to H₂O₂, simultaneously scavenging H₂O₂ and releasing two TCP molecules. TOT exhibited comparable radical scavenging activity to TCP but showed superior H₂O₂-scavenging efficiency, stronger antioxidant and anti-inflammatory effects in H₂O₂-stimulated cells, and excellent biocompatibility. Its rigid, linear structure promoted alignment within dipalmitoylphosphatidylcholine (DPPC) bilayers, enabling formulation of stable, H₂O₂-responsive liposomes with effective cellular uptake. These findings highlight TOT as a multifunctional, self-degradable antioxidant with strong potential as a cosmetic ingredient for protecting skin from oxidative and phototoxic damage. tocopherol hydrogen peroxide antioxidant liposome dimer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Cells continuously generate reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), superoxide anions, and nitric oxide, as byproducts of metabolic processes.(Forrester et al. 2018 ) While ROS play essential roles in cell proliferation, signaling, and immune responses, their excessive accumulation leads to oxidative stress, which damages biomolecules such as lipids, proteins, and DNA (E. Birben 2012; Jung et al. 2021 ). Among various ROS, H₂O₂ is the most abundant and relatively mild in reactivity; however, it serves as a precursor to hydroxyl radicals, one of the most potent and damaging oxidative species (Kwon et al. 2016 ). In the skin, oxidative stress is exacerbated by light-induced photosensitization of cosmetic ingredients,(Maddaleno et al. 2024 ) which generates free radicals via type-I and type-II photosensitization mechanisms (Baptista et al. 2017 ). These free radicals contribute to DNA damage, lipid peroxidation, and protein oxidation, leading to phototoxicity, skin irritation, and premature aging. Given the detrimental impact of excess ROS, particularly H₂O₂, developing effective antioxidant strategies is crucial for mitigating oxidative damage and improving skin health (Jaffri 2023 ). Tocopherol (TCP) is a family of natural phenols derived from shikimic acid, characterized by a polar chromanol head and isoprenic side chain (Burton and Ingold 1986 ). There are four different tocopherols (α, β, γ and δ) differing in the number and position of methyl groups on the chromanol head (Saladino et al. 2008 ). As a fat-soluble antioxidant essential for human health, TCP protects cell membranes from damage by ROS and plays essential roles in immune function, skin health, and vision (Latib et al. 2024 ; Verleyen et al. 2001 ). Its antioxidant activity is ascribed to the easy hydrogen transfer to peroxy radical, leading to the production of tocopheroxy radical, which combines with another lipid peroxy radical to subsequently generating various nonradical tocopherol oxidation products (Heo et al. 2025 ; Kumar et al. 2020 ; Saladino et al. 2008 ). TCP has been widely utilized in both pharmaceutical and cosmetic applications due to its ability to neutralize lipid peroxidation and enhance skin barrier function (Olbinska et al. 2023 ). However, the potential of tocopherol-based systems that leverage H₂O₂-scavenging mechanisms has yet to be fully realized. H₂O₂-activated prodrugs have been extensively studied in pharmaceutical and biomedical fields for their targeted therapeutic benefits (Jung et al. 2019 ; Jung et al. 2021 ). Peroxalate linkage has emerged as an effective H₂O₂-cleavable linker, facilitating the controlled activation of prodrugs through oxidative degradation (Lee et al. 2013 ; Lee et al. 2019 ). In contrast, their application in cosmetic formulations remains unexplored. To address this gap, we have developed ditocopheryl peroxalate (TOT), a novel antioxidant molecule, in which two tocopherol units are linked via a peroxalate bond. TOT exhibits stability under physiological conditions but undergoes degradation in the presence of H₂O₂, releasing TCP while simultaneously scavenging excess H₂O₂. Peroxalate linkage was exploited as a linker because of its H₂O₂-responsiveness in the development of drug delivery systems. Additionally, the H₂O₂-responsiveness of peroxalate can be finely tuned by adjusting the chemical structures. Electro-withdrawing substituents enhance its H₂O₂-responsiveness, while reducing the stability against hydrolytic degradation (Park et al. 2010 ). Because of these advantages and excellent biocompatibility, peroxalate has been widely utilized in biodegradable polymers that serve as antioxidant prodrugs and drug delivery systems. Unlike conventional antioxidants that function as payloads within delivery systems, TOT aligns within lipid bilayers, enabling its incorporation as a structural component in cosmetic formulations such as liposomes. Moreover, TOT effectively protects cells from H₂O₂-induced oxidative stress, making it a promising candidate for cosmetic applications aimed at mitigating phototoxicity and oxidative damage. In this study, we report the physicochemical properties of TOT and its potential as an active ingredient for cosmetic applications. 2. Materials and Methods 2.1 Synthesis and characterization of TOT TOT was synthesized from the reaction of TCP and oxalyl chloride. Briefly, oxalyl chloride (4.64 mmol) was dissolved in dichloromethane (DCM) and stirred in iced water bath for 30 min. TCP (9.28 mmol) dissolved in DCM was added dropwise while maintaining the temperature at 0°C. After 2 h reaction, the mixture was purified by silica gel chromatography using hexane/ethyl acetate (7:1, v/v) as the eluent. TOT was obtained as yellow wax (70% yield), and its chemical structure was confirmed by 500 MHz 1 H and 13 C NMR spectroscopy (JNM-EX500, JEOL) using CDCl 3 as the solvent. Transition temperature of TOT was determined using differential scanning calorimeter (TA Instruments, Discovery DSC2500). 2.2. Synthesis of α-Tocopherol-Derivative Containing Hexadecanol (TOH) TOH was synthesized by two steps. In the initial step of the synthesis, hexadecanol was dissolved in anhydrous diethyl ether and subsequently reacted with an excess amount of oxalyl chloride under ice-bath condition (0°C), leading to the formation of a monoester-acyl chloride intermediate. The crude reaction mixture was then subjected to liquid-liquid extraction using water and diethyl ether. During this work-up, the remaining acyl chloride moiety was selectively hydrolyzed to the corresponding carboxylic acid, yielding the monoester-monoacid product. In the subsequent step, the monoester-monoacid obtained from the previous reaction was dissolved in DCM, followed by the addition of α-tocopherol (1.5 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 4.0 eq.), and dimethylaminopyridine (DMAP, 0.4 eq.). The reaction mixture was stirred at room temperature overnight to allow esterification. Upon completion, the mixture was washed with distilled water (2–3 times) to remove water-soluble byproducts. The organic phase was dried over anhydrous sodium sulfate (Na 2 SO₄), filtered, and concentrated under reduced pressure. The crude product was then purified by column chromatography using HEX:EA (7:1, v/v) as the eluent, yielding the final ester compound (20% yield). Chemical structure was identified using 500 MHz ¹H, ¹³C NMR spectroscopy (JNM-EX500 JEOL) using CDCl₃ as a solvent. 2.3. H₂O₂-induced oxidation of TCP and TOT To investigate H₂O₂-induced oxidative modifications of TCP and TOT, each compound was dissolved in tetrahydrofuran (THF) at approximately 3 mM. Equimolar amounts of H₂O₂ were added, and the solvent was evaporated under reduced pressure. The residues were dissolved in deuterated CDCl₃ and analyzed by 1 H-NMR spectroscopy to confirm structural changes. Additionally, liquid chromatography-tandem mass spectrometry (LC/MS/MS) was performed to identify oxidation products and infer degradation pathways based on fragmentation patterns. 2.4. UV-Vis spectroscopy of TOT UV-Vis absorption spectra were recorded using a spectrophotometer (S-3100, Scinco, Korea) with a 1 cm quartz cuvette. TOT was dissolved in THF, and spectra were acquired at room temperature over the wavelength range of 200∼800 nm in the presence or absence of H₂O₂. THF served as both the solvent and blank. 2.5. H₂O₂ scavenging assay The H₂O₂ scavenging ability of TOT was evaluated using a peroxalate-based chemiluminescence assay. A fluorescent working solution containing rubrene, diphenyloxalate (DPO), and THF was used as the detection system. Upon reaction with H₂O₂, DPO produces a high-energy intermediate that excites rubrene, emitting detectable light. TOT or TCP (used as a reference antioxidant) was dissolved in THF and added at varying concentrations (5, 10, 20, 50, and 100 µM) to a fixed 100 µM H₂O₂ solution. Residual H₂O₂ levels were quantified by measuring chemiluminescence intensity using a luminometer. For time-dependent evaluation, 100 µM of TOT was incubated with 100 µM H₂O₂, and luminescence was measured at 0, 1, 4, 8, 12, and 24 h. 2.6. Cell viability assay RAW264.7 (murine macrophage cell lines) cells were purchased from Korea Cell Line Bank (Korea) and cultured in the incubator with 5% CO 2 at 37°C. Cells were seeded into 24-well plates at a density of 2×10⁵ cells/well and cultured to ~ 80% confluency. Cells were treated with TCP or TOT at 10, 20, 50, and 100 µM for 24 h. After the treatment, 100 µL of MTT solution was added to each well, followed by 3 h incubation. One milliliter of dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystal and absorbance was measured at 570 nm using a microplate reader (Synergy MX, BioTek, Winooski, VT, USA). For oxidative stress protection, cells were pretreated with 100 µM H₂O₂ for 30 min, followed by antioxidant treatment. 2.7. TNF-α secretion by ELISA RAW264.7 cells were seeded at ~ 2 × 10⁵ cells/well and incubated with TCP or TOT (10 or 20 µM). After 24 h of incubation, culture mediums were collected, centrifuged at 10,000 × g for 10 min, and analyzed using a Mouse TNF-α ELISA kit (Thermo Fisher Scientific) per manufacturer’s instructions. Absorbance was read at 450 nm and cytokine levels calculated from a standard curve. 2.8. Formulation and characterization of TOT-containing liposomes Liposomes were prepared using DPPC, cholesterol, and TOT at a 6:2:2 (w/w/w) ratio (a total of 10 mg). DPPC and cholesterol were dissolved in ethanol, and TOT in ethyl acetate. The solutions were combined to 1 mL total volume and incubated at 40°C in a water bath sonicator for 30 min. One milliliter of distilled water was added gradually with stirring. The mixture underwent high-vacuum evaporation to remove solvents, yielding liposomes at a concentration of 10 mg/mL. A mini-extruder system was used to obtain uniformly sized liposomes. Cryo-TEM was performed to observe the morphology of the liposome under near-native conditions. Samples were first vitrified to prevent ice crystal formation by rapidly freezing them via plunge freezing in liquid nitrogen. The vitrified samples were then applied onto specialized cryo-TEM grids. Cryo-TEM analysis was performed at the Center for University Joint Research Facilities, Jeonbuk National University. The hydrodynamic size and polydispersity index (PDI) of TOT-containing liposomes were measured by dynamic light scattering using a particle size analyzer (Brookhaven Instrument Corp., Holtsville, NY. US). For fluorescence imaging and photoluminescence Nile Red (1.0 wt%) was loaded in TOT-containing liposomes. Nile Red was added in ethanol containing DPPC and cholesterol. The following procedure was the same as TOT-containing liposomes. The fluorescence emission of Nile Red-liposomes was obtained using spectrofluorometer (FP6500 Jasco, Japan) in the presence and absence of H 2 O 2 . 2.9. Fluorescence imaging To evaluate the ability of TOT and TOT-containing liposomes to suppress oxidative stress, cells were seeded in a confocal dish (SPL life Sciences, Korea) at a 6×10 4 cells per dish. Liposomes and TOT at various concentrations were added in each dish and incubated for 24 h. Afterward, gentle washing with PBS, cells were treated with 20 µM of 2’,7’-dichlorofluorescein-diacetate (DCFH-DA) dissolved in DMSO for 30 min. Cells were washed with PBS and observed under the confocal laser scanning microscope (CLSM, Carl Zeiss, Germany) to determine the level of intracellular ROS. To observe the internalization of TOT-containing liposomes, cells seeded in a confocal dish at a 6×10 4 cells were treated with Nile red-loaded liposomes for 0.5, 2 or 6 h and washed with PBS. 2.10. LDH Cytotoxicity Assay Cells seeded in 6-well plates were incubated with TOT-containing liposomes for 24 h. After treatment, cells were lysed and centrifuged at 600 × g for 5 min. Supernatants were analyzed using a WST-based LDH detection kit. A reaction mixture was prepared by mixing WST Substrate Mix with Assay Buffer, and 100 µL was added to each well in a 96-well plate. Plates were incubated for 30 min at room temperature in the dark. Absorbance was measured at 450 nm using a microplate reader. 3. Results and Discussion 3.1 Synthesis and characterization of TOT TOT was designed as a H 2 O 2 -responsive dimer of TCP, leveraging the unique chemistry of peroxalate. It has been well known that peroxalate readily reacts with nucleophilic H 2 O 2 to generate two alcohols. TOT was synthesized from the simple reaction of TCP with oxalyl chloride (Fig. 1 a). After purification, TOT was obtained as a waxy solid, unlike oily TCP. The synthesis of TCP was confirmed by NMR and mass analysis. The hydroxy proton signal (∼4.2 ppm) disappeared and the two equivalent methyl protons (∼2.2 ppm) shifted (Figure S1), indicating the successful synthesis of TOT. As TOT was synthesized as a H 2 O 2 -cleavable antioxidant, we examined whether TOT releases TCP in a H 2 O 2 -triggered manner. LC/MS MS chromatography revealed that H 2 O 2 -treatment led to TOT degradation, generating TCP (Fig. 1 b). In the aqueous environment containing H 2 O 2 , TCP would be readily oxidized by H 2 O 2 to form various oxidized TCP products including tocopherylquionine, as evidenced by the changes in chemical shifts at 2.2 ppm corresponding to methyl protons on the chromanol ring and the significant decrease in the proton signal corresponding to hydroxy proton (4.2 ppm) (Fig. 1 c). Formation of oxidized TCP was also confirmed by MS spectroscopy. TOT treated with H 2 O 2 exhibited the same proton signal as H 2 O 2 -treated TCP. These findings suggest that TOT degrades in a H 2 O 2 -triggered manner to release TCP. For comparison, we also synthesized TOH as a control that integrates TCP and hexadecanol through a peroxalate linker (Figure S2). 3.2. H 2 O 2 -Responsiveness of TOT H 2 O 2 itself is not highly reactive with biological molecules, but is a precursor of hydroxyl radical that is considered the most toxic to cells. We therefore assessed the H 2 O 2 -scavenging ability of TOT as a measure of antioxidant activity. After the addition of TOT to the H 2 O 2 solution, its H 2 O 2 scavenging ability was evaluated in direct comparison with TCP and TOH (Fig. 2 a). While TCP had negligible impact on the level of H 2 O 2 , TOT exhibited time-dependent H 2 O 2 scavenging, with 40% reduction at 30 min. To further elucidate the H 2 O 2 -responsiveness of peroxalate, TOH was also used as a control. TOT showed a significantly stronger H 2 O 2 -scavenging ability than TOH. TOH has a peroxalate linkage connected to both aryl and alkyl substitutes, unlike TOT possessing peroxalate with two aryl groups. It is well known that electron-withdrawing substituents increase the reactivity of peroxalate to H 2 O 2 , while electron-donating moiety diminishes its H 2 O 2 -reactivity (Lee et al. 2011 ). The higher H 2 O 2 -scavenging ability of TOT over TOH is attributed to its stronger electron-withdrawing power of two aromatic moieties attached to peroxalate. As shown in Fig. 2 b-c, TOT exhibited time- and concentration-dependent H 2 O 2 scavenging. The H 2 O 2 -responsiveness of TOT was further assessed by UV spectroscopy. As an oxygen donor H 2 O 2 oxidizes TCP to form oxidized products including tocopheryl quinone, tocopheryl hydroquinone, and dimers (Saladino et al. 2008 ). The H 2 O 2 -induced TCP oxidation was confirmed by the increase in UV absorption at ∼350 nm and ∼450 nm (Fig. 2 d). The absorption band at ∼350 nm is attributed to tocopherol quinone and an extended quinone systems. The distinct and strong absorption band at ∼450 nm suggests the formation of highly conjugated quinoid dimer. Both TCP and TOT showed H 2 O 2 concentration dependent oxidation, with higher UV absorption at a higher H 2 O 2 concentration (Fig. 2 e). The dimer formation is consistent with MS analysis (Fig. 1 b). The same pattern of UV absorption was observed with H 2 O 2 -treated TOT. Compared to TCP, TOT exhibited stronger absorption bands at ∼350 nm and ∼450 nm, which was also supported by the yellowish color (Figure S4). Because TOT contains two TCP units, it generates more quinone-like oxidation products, leading to stronger absorption bands at ∼350 and ∼450 nm. It can be also explained by the high reactivity of peroxalate that reacts with H 2 O 2 and likely generates reactive intermediates that accelerate oxidation. We also examined the time course of oxidation of TCP and TOT induced by 0.5 eq. H 2 O 2 . As shown in Fig. 2 d, TOT underwent faster H 2 O 2 -mediated oxidation than TCP. These findings suggest that TOT undergoes H 2 O 2 -triggered oxidation more effectively than TCP. 3.3 Cytoprotective and anti-inflammatory effects of TOT The toxicity of TOT against RAW264.7 cells was examined by MTT assay. Figure 3 a shows the viability of RAW264.7 cells treated with TCP and TOT for 24 h. TOT had negligible impact on the cell viability at concentrations up to 100 µM, suggesting its excellent biocompatibility. The antioxidant effect of TOT was assessed by measuring the level of intracellular ROS in H 2 O 2 -stimulated RAW264.7 cells using DCFH-DA which is a cell permeable fluorogenic probe commonly used to detect ROS. A large amount of ROS was generated in H 2 O 2 -stimulated cells, as evidenced by strong green fluorescence in the whole cytoplasm (Fig. 3 b). TCP inhibited H 2 O 2 -mediated ROS generation concentration dependently, suggesting its intrinsic antioxidant effects. However, TOT suppressed the ROS generation more effectively than TCP probably due to the combined effects of H 2 O 2 -scavenging peroxalate and the intrinsic antioxidant action of TCP. We also examined the cytoprotective effect of TOT from H 2 O 2 -mediated toxicity. Cell viability was markedly reduced after 24 h of incubation with 100 µM H 2 O 2 . TCP exerted cytoprotective effects at concentration of 100 µM (Fig. 3 c). TOT effectively protected cells from H 2 O 2 -mediated cytotoxicity at significantly lower concentrations. Notably, 10 µM TOT exhibited cytoprotective effects comparable to 100 µM TCP. We next examined the anti-inflammatory effects of TOT by measuring the level of TNF-α which is one of key pro-inflammatory cytokines and involves chronic inflammation. H 2 O 2 -stimulated cells showed a markedly elevated level of TNF-α (Fig. 3 d). TCP exhibited no or negligible effects on the level of TNF-α. However, TCP significantly suppressed the expression of TNF-α in H 2 O 2 -stimulated cells. These observations demonstrate that TOT exerts potent antioxidant and anti-inflammatory effects. 3.4 Liposomal formulation of TOT The therapeutic applications of TCP have been limited by its low water solubility and easy oxidation (Heo et al. 2025 ). Phospholipid liposomes have been widely used to improve its solubility and enhance delivery efficacy to cells and tissues. TCP is known to bind strongly with the lipids, possibly through hydrogen bond formation between the hydroxyl group of the former and one of the oxygen atoms of the latter (Srivastava et al. 1983 ). However, the low solubility of TCP in the aqueous core leads to uneven distribution within liposomal layer and make encapsulation inefficient. Additionally, oily TCP disrupts the liposomal membrane, leading to leakages or rupture of the liposomes during the long-term storage. In this regard, the composition and TCP content should be carefully adjusted to enhance the integrity of liposomes and reduce leakages. Based on the rationale that waxy TOT has a higher transition temperature than TCP (Figure S5) and therefore forms stable liposomes, we formulated liposomes composed of DPPC, cholesterol and TOT (or TCP) at a ratio, 6:2:2 (Fig. 4 a). The TOT-containing liposomes were spherical vesicles with a mean hydrodynamic diameter of ∼180 nm (Fig. 4 b-c). We also tested the stability of liposomes containing TCP and TOT. The colloidal stability of TOT-containing liposomes was assessed by monitoring their hydrodynamic diameter over 30 days of incubation in phosphate buffer (Fig. 4 d). The TOT-containing liposomes exhibited no discernable changes in diameter, while TCP-containing liposomes displayed abrupt increase in diameter from 6 days-post incubation, suggesting the enhanced colloidal stability of TOT-containing liposomes. It can be inferred that the linear structure and high transition temperature of TOT renders it aligns with lipid through the normal hydrophobic interaction. We also examined the H 2 O 2 -responsiveness of TOT-containing liposomes. After 18 h of H 2 O 2 treatment, the hydrodynamic diameter of TOT-containing liposomes markedly increased, indicating that H 2 O 2 -mediated cleavage of peroxalate ester in TOT induces the disruption of lipid bilayer. To further substantiate the H 2 O 2 -responsiveness of TOT-containing liposomes, we used Nile Red as a fluorescent probe which exhibits strong red fluorescence in lipid rich environments, while typically non fluorescent in hydrophilic environment. Nile Red-loaded liposomes exhibit distinct emission peak at 590 nm (Fig. 4 e), indicating that Nile Red is incorporated in the hydrophobic bilayer of liposomes. However, in the presence of H 2 O 2 , the fluorescence intensity gradually decreased with time (Fig. 4 f), indicating that Nile Red is released from the liposome and loses fluorescence. We also tested the stability and H 2 O 2 -responsiveness of TOH-liposomes for comparison purposes. TOH-containing liposomes exhibited a drastic change in hydrodynamic diameter from 7 day (Figure S6a). The hydrodynamic diameter increased rapidly increased after the addition of H 2 O 2 and gradually increased for 1 h of observation period (Figure S6b). The rapid H 2 O 2 -responsiveness of TOH-containing liposomes could be attributed to the inferior colloidal stability which induces rapid permeation of H 2 O 2 . 3.5 Cellular internalization and biological activity of TOT-containing liposomes TCP acts as a potent antioxidant primarily by scavenging ROS that can damage cells. This process occurs within the cellular environment, particularly in cell membranes, where TCP prevents oxidative damage to lipids and proteins. For bioactive substances like TOT to exert its antioxidant effects and modulate inflammatory pathways efficiently, they generally need to be internalized into cells. We therefore assessed the internalization of TOT-containing liposomes. Nile Red was co-loaded in TOT-containing liposomes as a fluorescent probe that exhibits strong red fluorescence in lipid rich environments, while typically non fluorescent in hydrophilic environments (Lee et al. 2022 ). Figure 5 a shows the fluorescence images of RAW264.7 cells incubated with fluorescent TOT-containing liposomes. From 30 min post-incubation, distinct red fluorescence was observed in cell membranes and whole cytoplasm. The fluorescence intensity increased with time, suggesting that the liposomes are taken up by cells through endocytosis and the liposomes remain intact. At 6 h post-incubation, the fluorescence intensity decreased, indicating the disruption of liposomes and subsequent release of Nile Red in a hydrophilic environment of cytosol. These observations suggest that TOT-containing liposomes are readily internalized into cells probably through endocytosis. It has been well accepted that liposomal formulations containing certain drugs can disrupt cell membrane, leading to the release of lactate dehydrogenase (LDH) as a sign of cell damage. We therefore assessed the LDH release from RAW264.7 cells incubated with TOT-containing liposomes for 12 h. As shown in Fig. 5 b, TOT-containing liposomes induced no or negligible LDH release, suggesting no discernable cell membrane damages. Additionally, TOT-containing liposomes exhibited negligible effects on the cell viability up to 200 µg/mL after 24 h of incubation (Fig. 5 c). As expected from Fig. 2 b, TOT-containing liposomes suppressed the ROS generation in H 2 O 2 -stimulated cells (Fig. 5 d). These results demonstrate that TOT-containing liposomes are biocompatible and exert antioxidant effects. 4. Conclusions To enhance antioxidant capacity of TCP, we developed ditocopherol peroxalate (TOT), a H 2 O 2 -responsive tocopherol dimer incorporating a peroxalate linkage. TOT undergoes selective degradation in the presence of H 2 O 2 , releasing two TCP molecules while simultaneously scavenging H 2 O 2 . Compared to TCP, TOT exhibited stronger antioxidant and anti-inflammatory effects in H 2 O 2 -stimulated cells. Its linear structure and enhanced rigidity promote alignment with DPPC, enabling the formation of H 2 O 2 -responsive liposomal formulation. These TOT-containing liposomes were efficiently internalized by cells and exhibited no cytotoxicity. Collectively, our results obviously demonstrate that TOT functions as a pro-antioxidant and H 2 O 2 -scavenger, offering superior protective effects over native TCP and holding great potential as an active cosmetic ingredient for oxidative stress mitigation. Declarations Conflicts of interest: The authors declare no conflict of interest. Acknowledgments This work was supported by a grant of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (RS-2024-00406625), Republic of Korea. The authors thank the Center for University Wide Research Facilities (CURF) at Jeonbuk National University for the valuable analysis of confocal laser scanning microscopy and electron microscopy. 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J Agric Food Chem 49:1508–1511. 10.1021/jf001142f Supplementary Files Supportinginfo.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7117698","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":503242927,"identity":"54caaf2c-24f1-4a9f-8c25-f0c0dd88934a","order_by":0,"name":"Hanui Jo","email":"","orcid":"","institution":"Jeonbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Hanui","middleName":"","lastName":"Jo","suffix":""},{"id":503242928,"identity":"1f26bae5-70c8-4317-aa4e-9aa988333b77","order_by":1,"name":"Ayoung Kim","email":"","orcid":"","institution":"Kolmar Korea","correspondingAuthor":false,"prefix":"","firstName":"Ayoung","middleName":"","lastName":"Kim","suffix":""},{"id":503242929,"identity":"18609ed3-3aa4-472e-9168-7e661a52fa77","order_by":2,"name":"Changhee Park","email":"","orcid":"","institution":"Kolmar Korea","correspondingAuthor":false,"prefix":"","firstName":"Changhee","middleName":"","lastName":"Park","suffix":""},{"id":503242930,"identity":"6049bc26-0979-4683-84e2-ffd589162ae5","order_by":3,"name":"soyoon Baek","email":"","orcid":"","institution":"Kolmar Korea","correspondingAuthor":false,"prefix":"","firstName":"soyoon","middleName":"","lastName":"Baek","suffix":""},{"id":503242931,"identity":"2a8432fe-e94c-4ffe-ac2a-9d9be3125697","order_by":4,"name":"Inki Hong","email":"","orcid":"","institution":"Kolmar Korea","correspondingAuthor":false,"prefix":"","firstName":"Inki","middleName":"","lastName":"Hong","suffix":""},{"id":503242932,"identity":"b20601fc-8995-4000-a20e-0804c332f753","order_by":5,"name":"Mingi Kim","email":"","orcid":"","institution":"ICBio","correspondingAuthor":false,"prefix":"","firstName":"Mingi","middleName":"","lastName":"Kim","suffix":""},{"id":503242933,"identity":"6b40397a-93a2-4878-86fd-644d0358056d","order_by":6,"name":"Dongwon Lee","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYDCC40D8geEAA4MEAzNYQIKglsMMDIwzSNbCzEOSFr7DzAcf29TckTOX7jE2YKixY5CcfQC/FsnDbMnGOceeGVvOOWOcwHAsmUGaLwG/FoPDPGbSuQ2HEzfcyDE+wMB2gEGOh4DDDA7zf/9tCdfyjygtPGzMjFAtCYxtBxikCWkB+sVYsufYYWODO8eKDRL7knkkewho4Tve/PDDj5rDcga3mzdLfPhmJydxhoAWVJDAwEDIWaNgFIyCUTAKiAEARYFA0mR5q9UAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0003-3035-6342","institution":"Jeonbuk National University","correspondingAuthor":true,"prefix":"","firstName":"Dongwon","middleName":"","lastName":"Lee","suffix":""}],"badges":[],"createdAt":"2025-07-14 06:39:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7117698/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7117698/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90177232,"identity":"832e330c-f357-4927-90ac-5712f27a59f3","added_by":"auto","created_at":"2025-08-29 12:43:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":197366,"visible":true,"origin":"","legend":"\u003cp\u003eSynthesis and characterization of TOT. (a) A synthetic route and degradation of TOT. (b) LC/MS MS chromatography of TCP, TOT and oxidation product of compounds. (c) \u003csup\u003e1\u003c/sup\u003eH-NMR spectroscopy of TCP, TOT and TCP and TOT oxidation products in CDCl\u003csub\u003e3\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7117698/v1/5e9a7005b85441e21af03ea4.png"},{"id":90177231,"identity":"175ca3a6-9d2b-4619-9a49-3f7c44592900","added_by":"auto","created_at":"2025-08-29 12:43:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":161800,"visible":true,"origin":"","legend":"\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-Responsiveness of TOT. (a) H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e reactivity assessment of TOT in comparison with TCP and TOH. (b) Time-dependent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging profile of TOT (100 μM). (c) Concentration-dependent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging profile of TOT over 24 h. Values are mean ± s.d. (n=3). (d) UV-vis spectroscopy spectrum of TCP and TOT oxidation products with different time of incubation with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (e) UV-vis spectroscopy spectrum of TCP and TOT oxidation products with different concentration of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7117698/v1/badb6d8809028c82187b8de0.png"},{"id":90177233,"identity":"53b57573-a859-4bab-9874-efc601053248","added_by":"auto","created_at":"2025-08-29 12:43:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":210954,"visible":true,"origin":"","legend":"\u003cp\u003eBiological activities of TOT. (a) Cytotoxicity of against RAW 264.7 cells. (b) Fluorescence images of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated RAW264.7 cells. The cells were stained with DCFH-DA as a ROS probe. Scale bar: 20 μm. (c) Cytoprotective effects of TOT on H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-simulated RAW 264.7 cells. (d) The level of TNF-α in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated RAW 264.7 cells. Values are mean ± s.d. (n=3). *p\u0026lt;0.05, ***p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7117698/v1/44e3a663cec455df298285d6.png"},{"id":90177236,"identity":"20103a6f-9d94-4879-83ae-df213c46b859","added_by":"auto","created_at":"2025-08-29 12:43:05","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":290364,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of TOT-containing liposomes. (a) Schematic illustration of TOT-containing liposomes with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsiveness. (b) Representative Cryo-TEM image of TOT-containing liposomes. (c) Representative dynamic light scattering of TOT-containing liposomes treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003efor 18 h. (d) Changes in diameter of TOT-containing liposomes over 30 days of observation period. (e) Changes in fluorescence emission spectra of Nile Red-loaded TOT-containing liposomes in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. (f) Photographs of Nile Red-loaded TOT-containing liposomes.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7117698/v1/5b4761149041eaed92df5e3f.png"},{"id":90177540,"identity":"b99c0207-d582-4f91-837f-329a1cf05ef9","added_by":"auto","created_at":"2025-08-29 12:51:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":410555,"visible":true,"origin":"","legend":"\u003cp\u003eBiological activity of TOT-containing liposomes. (a) Fluorescence images of RAW264.7 cells treated with Nile Red-loaded TOT liposomes. (b) The level of LDH released from cells treated with TOT liposomes (200 mg/mL). Values are mean ± s.d. (n=3). (c) Viability of RAW 264.7 cells treated with TOT liposomes. Values are mean ± s.d. (n=3). (d) Fluorescence images showing the intracellular level of ROS in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated RAW264.7 cells. Cells were stained with DCFH-DA.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7117698/v1/f651fc74fd3a2b0c4159c02b.png"},{"id":104779086,"identity":"ebb1a853-b4c6-4b89-aad7-105f0c0e802e","added_by":"auto","created_at":"2026-03-17 07:33:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1855142,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7117698/v1/6e73dcc5-da35-4563-94a2-5dd91c2592b8.pdf"},{"id":90177238,"identity":"ae806f51-5775-49f5-b4ab-90bca5d56e07","added_by":"auto","created_at":"2025-08-29 12:43:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":913190,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginfo.docx","url":"https://assets-eu.researchsquare.com/files/rs-7117698/v1/4ed6e8263488da10be9b4db8.docx"}],"financialInterests":"","formattedTitle":"A self-deliverable H2O2-responsive tocopherol dimer for enhanced antioxidant and liposomal delivery","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCells continuously generate reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), superoxide anions, and nitric oxide, as byproducts of metabolic processes.(Forrester et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) While ROS play essential roles in cell proliferation, signaling, and immune responses, their excessive accumulation leads to oxidative stress, which damages biomolecules such as lipids, proteins, and DNA (E. Birben 2012; Jung et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among various ROS, H₂O₂ is the most abundant and relatively mild in reactivity; however, it serves as a precursor to hydroxyl radicals, one of the most potent and damaging oxidative species (Kwon et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In the skin, oxidative stress is exacerbated by light-induced photosensitization of cosmetic ingredients,(Maddaleno et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) which generates free radicals via type-I and type-II photosensitization mechanisms (Baptista et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). These free radicals contribute to DNA damage, lipid peroxidation, and protein oxidation, leading to phototoxicity, skin irritation, and premature aging. Given the detrimental impact of excess ROS, particularly H₂O₂, developing effective antioxidant strategies is crucial for mitigating oxidative damage and improving skin health (Jaffri \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTocopherol (TCP) is a family of natural phenols derived from shikimic acid, characterized by a polar chromanol head and isoprenic side chain (Burton and Ingold \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). There are four different tocopherols (α, β, γ and δ) differing in the number and position of methyl groups on the chromanol head (Saladino et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). As a fat-soluble antioxidant essential for human health, TCP protects cell membranes from damage by ROS and plays essential roles in immune function, skin health, and vision (Latib et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Verleyen et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Its antioxidant activity is ascribed to the easy hydrogen transfer to peroxy radical, leading to the production of tocopheroxy radical, which combines with another lipid peroxy radical to subsequently generating various nonradical tocopherol oxidation products (Heo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Saladino et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). TCP has been widely utilized in both pharmaceutical and cosmetic applications due to its ability to neutralize lipid peroxidation and enhance skin barrier function (Olbinska et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, the potential of tocopherol-based systems that leverage H₂O₂-scavenging mechanisms has yet to be fully realized.\u003c/p\u003e\u003cp\u003eH₂O₂-activated prodrugs have been extensively studied in pharmaceutical and biomedical fields for their targeted therapeutic benefits (Jung et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jung et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Peroxalate linkage has emerged as an effective H₂O₂-cleavable linker, facilitating the controlled activation of prodrugs through oxidative degradation (Lee et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In contrast, their application in cosmetic formulations remains unexplored. To address this gap, we have developed ditocopheryl peroxalate (TOT), a novel antioxidant molecule, in which two tocopherol units are linked via a peroxalate bond. TOT exhibits stability under physiological conditions but undergoes degradation in the presence of H₂O₂, releasing TCP while simultaneously scavenging excess H₂O₂. Peroxalate linkage was exploited as a linker because of its H₂O₂-responsiveness in the development of drug delivery systems. Additionally, the H₂O₂-responsiveness of peroxalate can be finely tuned by adjusting the chemical structures. Electro-withdrawing substituents enhance its H₂O₂-responsiveness, while reducing the stability against hydrolytic degradation (Park et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Because of these advantages and excellent biocompatibility, peroxalate has been widely utilized in biodegradable polymers that serve as antioxidant prodrugs and drug delivery systems. Unlike conventional antioxidants that function as payloads within delivery systems, TOT aligns within lipid bilayers, enabling its incorporation as a structural component in cosmetic formulations such as liposomes. Moreover, TOT effectively protects cells from H₂O₂-induced oxidative stress, making it a promising candidate for cosmetic applications aimed at mitigating phototoxicity and oxidative damage. In this study, we report the physicochemical properties of TOT and its potential as an active ingredient for cosmetic applications.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Synthesis and characterization of TOT\u003c/h2\u003e\u003cp\u003eTOT was synthesized from the reaction of TCP and oxalyl chloride. Briefly, oxalyl chloride (4.64 mmol) was dissolved in dichloromethane (DCM) and stirred in iced water bath for 30 min. TCP (9.28 mmol) dissolved in DCM was added dropwise while maintaining the temperature at 0\u0026deg;C. After 2 h reaction, the mixture was purified by silica gel chromatography using hexane/ethyl acetate (7:1, v/v) as the eluent. TOT was obtained as yellow wax (70% yield), and its chemical structure was confirmed by 500 MHz \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectroscopy (JNM-EX500, JEOL) using CDCl\u003csub\u003e3\u003c/sub\u003e as the solvent. Transition temperature of TOT was determined using differential scanning calorimeter (TA Instruments, Discovery DSC2500).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Synthesis of α-Tocopherol-Derivative Containing Hexadecanol (TOH)\u003c/h2\u003e\u003cp\u003eTOH was synthesized by two steps. In the initial step of the synthesis, hexadecanol was dissolved in anhydrous diethyl ether and subsequently reacted with an excess amount of oxalyl chloride under ice-bath condition (0\u0026deg;C), leading to the formation of a monoester-acyl chloride intermediate. The crude reaction mixture was then subjected to liquid-liquid extraction using water and diethyl ether. During this work-up, the remaining acyl chloride moiety was selectively hydrolyzed to the corresponding carboxylic acid, yielding the monoester-monoacid product. In the subsequent step, the monoester-monoacid obtained from the previous reaction was dissolved in DCM, followed by the addition of α-tocopherol (1.5 eq.), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 4.0 eq.), and dimethylaminopyridine (DMAP, 0.4 eq.). The reaction mixture was stirred at room temperature overnight to allow esterification. Upon completion, the mixture was washed with distilled water (2\u0026ndash;3 times) to remove water-soluble byproducts. The organic phase was dried over anhydrous sodium sulfate (Na\u003csub\u003e2\u003c/sub\u003eSO₄), filtered, and concentrated under reduced pressure. The crude product was then purified by column chromatography using HEX:EA (7:1, v/v) as the eluent, yielding the final ester compound (20% yield). Chemical structure was identified using 500 MHz \u0026sup1;H, \u0026sup1;\u0026sup3;C NMR spectroscopy (JNM-EX500 JEOL) using CDCl₃ as a solvent.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. H₂O₂-induced oxidation of TCP and TOT\u003c/h2\u003e\u003cp\u003eTo investigate H₂O₂-induced oxidative modifications of TCP and TOT, each compound was dissolved in tetrahydrofuran (THF) at approximately 3 mM. Equimolar amounts of H₂O₂ were added, and the solvent was evaporated under reduced pressure. The residues were dissolved in deuterated CDCl₃ and analyzed by \u003csup\u003e1\u003c/sup\u003eH-NMR spectroscopy to confirm structural changes. Additionally, liquid chromatography-tandem mass spectrometry (LC/MS/MS) was performed to identify oxidation products and infer degradation pathways based on fragmentation patterns.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. UV-Vis spectroscopy of TOT\u003c/h2\u003e\u003cp\u003eUV-Vis absorption spectra were recorded using a spectrophotometer (S-3100, Scinco, Korea) with a 1 cm quartz cuvette. TOT was dissolved in THF, and spectra were acquired at room temperature over the wavelength range of 200\u0026sim;800 nm in the presence or absence of H₂O₂. THF served as both the solvent and blank.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. H₂O₂ scavenging assay\u003c/h2\u003e\u003cp\u003eThe H₂O₂ scavenging ability of TOT was evaluated using a peroxalate-based chemiluminescence assay. A fluorescent working solution containing rubrene, diphenyloxalate (DPO), and THF was used as the detection system. Upon reaction with H₂O₂, DPO produces a high-energy intermediate that excites rubrene, emitting detectable light. TOT or TCP (used as a reference antioxidant) was dissolved in THF and added at varying concentrations (5, 10, 20, 50, and 100 \u0026micro;M) to a fixed 100 \u0026micro;M H₂O₂ solution. Residual H₂O₂ levels were quantified by measuring chemiluminescence intensity using a luminometer. For time-dependent evaluation, 100 \u0026micro;M of TOT was incubated with 100 \u0026micro;M H₂O₂, and luminescence was measured at 0, 1, 4, 8, 12, and 24 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Cell viability assay\u003c/h2\u003e\u003cp\u003eRAW264.7 (murine macrophage cell lines) cells were purchased from Korea Cell Line Bank (Korea) and cultured in the incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. Cells were seeded into 24-well plates at a density of 2\u0026times;10⁵ cells/well and cultured to ~\u0026thinsp;80% confluency. Cells were treated with TCP or TOT at 10, 20, 50, and 100 \u0026micro;M for 24 h. After the treatment, 100 \u0026micro;L of MTT solution was added to each well, followed by 3 h incubation. One milliliter of dimethyl sulfoxide (DMSO) was added to each well to dissolve formazan crystal and absorbance was measured at 570 nm using a microplate reader (Synergy MX, BioTek, Winooski, VT, USA). For oxidative stress protection, cells were pretreated with 100 \u0026micro;M H₂O₂ for 30 min, followed by antioxidant treatment.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. TNF-α secretion by ELISA\u003c/h2\u003e\u003cp\u003eRAW264.7 cells were seeded at ~\u0026thinsp;2 \u0026times; 10⁵ cells/well and incubated with TCP or TOT (10 or 20 \u0026micro;M). After 24 h of incubation, culture mediums were collected, centrifuged at 10,000 \u0026times; g for 10 min, and analyzed using a Mouse TNF-α ELISA kit (Thermo Fisher Scientific) per manufacturer\u0026rsquo;s instructions. Absorbance was read at 450 nm and cytokine levels calculated from a standard curve.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Formulation and characterization of TOT-containing liposomes\u003c/h2\u003e\u003cp\u003eLiposomes were prepared using DPPC, cholesterol, and TOT at a 6:2:2 (w/w/w) ratio (a total of 10 mg). DPPC and cholesterol were dissolved in ethanol, and TOT in ethyl acetate. The solutions were combined to 1 mL total volume and incubated at 40\u0026deg;C in a water bath sonicator for 30 min. One milliliter of distilled water was added gradually with stirring. The mixture underwent high-vacuum evaporation to remove solvents, yielding liposomes at a concentration of 10 mg/mL. A mini-extruder system was used to obtain uniformly sized liposomes. Cryo-TEM was performed to observe the morphology of the liposome under near-native conditions. Samples were first vitrified to prevent ice crystal formation by rapidly freezing them via plunge freezing in liquid nitrogen. The vitrified samples were then applied onto specialized cryo-TEM grids. Cryo-TEM analysis was performed at the Center for University Joint Research Facilities, Jeonbuk National University. The hydrodynamic size and polydispersity index (PDI) of TOT-containing liposomes were measured by dynamic light scattering using a particle size analyzer (Brookhaven Instrument Corp., Holtsville, NY. US). For fluorescence imaging and photoluminescence Nile Red (1.0 wt%) was loaded in TOT-containing liposomes. Nile Red was added in ethanol containing DPPC and cholesterol. The following procedure was the same as TOT-containing liposomes. The fluorescence emission of Nile Red-liposomes was obtained using spectrofluorometer (FP6500 Jasco, Japan) in the presence and absence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. Fluorescence imaging\u003c/h2\u003e\u003cp\u003eTo evaluate the ability of TOT and TOT-containing liposomes to suppress oxidative stress, cells were seeded in a confocal dish (SPL life Sciences, Korea) at a 6\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells per dish. Liposomes and TOT at various concentrations were added in each dish and incubated for 24 h. Afterward, gentle washing with PBS, cells were treated with 20 \u0026micro;M of 2\u0026rsquo;,7\u0026rsquo;-dichlorofluorescein-diacetate (DCFH-DA) dissolved in DMSO for 30 min. Cells were washed with PBS and observed under the confocal laser scanning microscope (CLSM, Carl Zeiss, Germany) to determine the level of intracellular ROS. To observe the internalization of TOT-containing liposomes, cells seeded in a confocal dish at a 6\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells were treated with Nile red-loaded liposomes for 0.5, 2 or 6 h and washed with PBS.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. LDH Cytotoxicity Assay\u003c/h2\u003e\u003cp\u003eCells seeded in 6-well plates were incubated with TOT-containing liposomes for 24 h. After treatment, cells were lysed and centrifuged at 600 \u0026times; g for 5 min. Supernatants were analyzed using a WST-based LDH detection kit. A reaction mixture was prepared by mixing WST Substrate Mix with Assay Buffer, and 100 \u0026micro;L was added to each well in a 96-well plate. Plates were incubated for 30 min at room temperature in the dark. Absorbance was measured at 450 nm using a microplate reader.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results and Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Synthesis and characterization of TOT\u003c/h2\u003e\u003cp\u003eTOT was designed as a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsive dimer of TCP, leveraging the unique chemistry of peroxalate. It has been well known that peroxalate readily reacts with nucleophilic H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to generate two alcohols. TOT was synthesized from the simple reaction of TCP with oxalyl chloride (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). After purification, TOT was obtained as a waxy solid, unlike oily TCP. The synthesis of TCP was confirmed by NMR and mass analysis. The hydroxy proton signal (\u0026sim;4.2 ppm) disappeared and the two equivalent methyl protons (\u0026sim;2.2 ppm) shifted (Figure S1), indicating the successful synthesis of TOT. As TOT was synthesized as a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-cleavable antioxidant, we examined whether TOT releases TCP in a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-triggered manner.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLC/MS MS chromatography revealed that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treatment led to TOT degradation, generating TCP (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). In the aqueous environment containing H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, TCP would be readily oxidized by H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e to form various oxidized TCP products including tocopherylquionine, as evidenced by the changes in chemical shifts at 2.2 ppm corresponding to methyl protons on the chromanol ring and the significant decrease in the proton signal corresponding to hydroxy proton (4.2 ppm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec). Formation of oxidized TCP was also confirmed by MS spectroscopy. TOT treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e exhibited the same proton signal as H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated TCP. These findings suggest that TOT degrades in a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-triggered manner to release TCP. For comparison, we also synthesized TOH as a control that integrates TCP and hexadecanol through a peroxalate linker (Figure S2).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-Responsiveness of TOT\u003c/h2\u003e\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e itself is not highly reactive with biological molecules, but is a precursor of hydroxyl radical that is considered the most toxic to cells. We therefore assessed the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging ability of TOT as a measure of antioxidant activity. After the addition of TOT to the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e solution, its H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging ability was evaluated in direct comparison with TCP and TOH (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). While TCP had negligible impact on the level of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, TOT exhibited time-dependent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging, with 40% reduction at 30 min. To further elucidate the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsiveness of peroxalate, TOH was also used as a control. TOT showed a significantly stronger H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging ability than TOH. TOH has a peroxalate linkage connected to both aryl and alkyl substitutes, unlike TOT possessing peroxalate with two aryl groups. It is well known that electron-withdrawing substituents increase the reactivity of peroxalate to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, while electron-donating moiety diminishes its H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-reactivity (Lee et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The higher H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging ability of TOT over TOH is attributed to its stronger electron-withdrawing power of two aromatic moieties attached to peroxalate. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb-c, TOT exhibited time- and concentration-dependent H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e scavenging.\u003c/p\u003e\u003cp\u003eThe H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsiveness of TOT was further assessed by UV spectroscopy. As an oxygen donor H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e oxidizes TCP to form oxidized products including tocopheryl quinone, tocopheryl hydroquinone, and dimers (Saladino et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-induced TCP oxidation was confirmed by the increase in UV absorption at \u0026sim;350 nm and \u0026sim;450 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The absorption band at \u0026sim;350 nm is attributed to tocopherol quinone and an extended quinone systems. The distinct and strong absorption band at \u0026sim;450 nm suggests the formation of highly conjugated quinoid dimer. Both TCP and TOT showed H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration dependent oxidation, with higher UV absorption at a higher H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe dimer formation is consistent with MS analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). The same pattern of UV absorption was observed with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-treated TOT. Compared to TCP, TOT exhibited stronger absorption bands at \u0026sim;350 nm and \u0026sim;450 nm, which was also supported by the yellowish color (Figure S4). Because TOT contains two TCP units, it generates more quinone-like oxidation products, leading to stronger absorption bands at \u0026sim;350 and \u0026sim;450 nm. It can be also explained by the high reactivity of peroxalate that reacts with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and likely generates reactive intermediates that accelerate oxidation. We also examined the time course of oxidation of TCP and TOT induced by 0.5 eq.\u0026nbsp;H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, TOT underwent faster H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated oxidation than TCP. These findings suggest that TOT undergoes H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-triggered oxidation more effectively than TCP.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Cytoprotective and anti-inflammatory effects of TOT\u003c/h2\u003e\u003cp\u003eThe toxicity of TOT against RAW264.7 cells was examined by MTT assay. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea shows the viability of RAW264.7 cells treated with TCP and TOT for 24 h. TOT had negligible impact on the cell viability at concentrations up to 100 \u0026micro;M, suggesting its excellent biocompatibility. The antioxidant effect of TOT was assessed by measuring the level of intracellular ROS in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated RAW264.7 cells using DCFH-DA which is a cell permeable fluorogenic probe commonly used to detect ROS. A large amount of ROS was generated in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated cells, as evidenced by strong green fluorescence in the whole cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). TCP inhibited H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated ROS generation concentration dependently, suggesting its intrinsic antioxidant effects. However, TOT suppressed the ROS generation more effectively than TCP probably due to the combined effects of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenging peroxalate and the intrinsic antioxidant action of TCP.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eWe also examined the cytoprotective effect of TOT from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated toxicity. Cell viability was markedly reduced after 24 h of incubation with 100 \u0026micro;M H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. TCP exerted cytoprotective effects at concentration of 100 \u0026micro;M (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). TOT effectively protected cells from H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated cytotoxicity at significantly lower concentrations. Notably, 10 \u0026micro;M TOT exhibited cytoprotective effects comparable to 100 \u0026micro;M TCP. We next examined the anti-inflammatory effects of TOT by measuring the level of TNF-α which is one of key pro-inflammatory cytokines and involves chronic inflammation. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated cells showed a markedly elevated level of TNF-α (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). TCP exhibited no or negligible effects on the level of TNF-α. However, TCP significantly suppressed the expression of TNF-α in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated cells. These observations demonstrate that TOT exerts potent antioxidant and anti-inflammatory effects.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Liposomal formulation of TOT\u003c/h2\u003e\u003cp\u003eThe therapeutic applications of TCP have been limited by its low water solubility and easy oxidation (Heo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Phospholipid liposomes have been widely used to improve its solubility and enhance delivery efficacy to cells and tissues. TCP is known to bind strongly with the lipids, possibly through hydrogen bond formation between the hydroxyl group of the former and one of the oxygen atoms of the latter (Srivastava et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e1983\u003c/span\u003e). However, the low solubility of TCP in the aqueous core leads to uneven distribution within liposomal layer and make encapsulation inefficient. Additionally, oily TCP disrupts the liposomal membrane, leading to leakages or rupture of the liposomes during the long-term storage. In this regard, the composition and TCP content should be carefully adjusted to enhance the integrity of liposomes and reduce leakages. Based on the rationale that waxy TOT has a higher transition temperature than TCP (Figure S5) and therefore forms stable liposomes, we formulated liposomes composed of DPPC, cholesterol and TOT (or TCP) at a ratio, 6:2:2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The TOT-containing liposomes were spherical vesicles with a mean hydrodynamic diameter of \u0026sim;180 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb-c). We also tested the stability of liposomes containing TCP and TOT. The colloidal stability of TOT-containing liposomes was assessed by monitoring their hydrodynamic diameter over 30 days of incubation in phosphate buffer (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). The TOT-containing liposomes exhibited no discernable changes in diameter, while TCP-containing liposomes displayed abrupt increase in diameter from 6 days-post incubation, suggesting the enhanced colloidal stability of TOT-containing liposomes. It can be inferred that the linear structure and high transition temperature of TOT renders it aligns with lipid through the normal hydrophobic interaction. We also examined the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsiveness of TOT-containing liposomes. After 18 h of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment, the hydrodynamic diameter of TOT-containing liposomes markedly increased, indicating that H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-mediated cleavage of peroxalate ester in TOT induces the disruption of lipid bilayer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further substantiate the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsiveness of TOT-containing liposomes, we used Nile Red as a fluorescent probe which exhibits strong red fluorescence in lipid rich environments, while typically non fluorescent in hydrophilic environment. Nile Red-loaded liposomes exhibit distinct emission peak at 590 nm (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee), indicating that Nile Red is incorporated in the hydrophobic bilayer of liposomes. However, in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, the fluorescence intensity gradually decreased with time (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ef), indicating that Nile Red is released from the liposome and loses fluorescence. We also tested the stability and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsiveness of TOH-liposomes for comparison purposes. TOH-containing liposomes exhibited a drastic change in hydrodynamic diameter from 7 day (Figure S6a). The hydrodynamic diameter increased rapidly increased after the addition of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and gradually increased for 1 h of observation period (Figure S6b). The rapid H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsiveness of TOH-containing liposomes could be attributed to the inferior colloidal stability which induces rapid permeation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Cellular internalization and biological activity of TOT-containing liposomes\u003c/h2\u003e\u003cp\u003eTCP acts as a potent antioxidant primarily by scavenging ROS that can damage cells. This process occurs within the cellular environment, particularly in cell membranes, where TCP prevents oxidative damage to lipids and proteins. For bioactive substances like TOT to exert its antioxidant effects and modulate inflammatory pathways efficiently, they generally need to be internalized into cells. We therefore assessed the internalization of TOT-containing liposomes. Nile Red was co-loaded in TOT-containing liposomes as a fluorescent probe that exhibits strong red fluorescence in lipid rich environments, while typically non fluorescent in hydrophilic environments (Lee et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea shows the fluorescence images of RAW264.7 cells incubated with fluorescent TOT-containing liposomes. From 30 min post-incubation, distinct red fluorescence was observed in cell membranes and whole cytoplasm. The fluorescence intensity increased with time, suggesting that the liposomes are taken up by cells through endocytosis and the liposomes remain intact. At 6 h post-incubation, the fluorescence intensity decreased, indicating the disruption of liposomes and subsequent release of Nile Red in a hydrophilic environment of cytosol. These observations suggest that TOT-containing liposomes are readily internalized into cells probably through endocytosis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIt has been well accepted that liposomal formulations containing certain drugs can disrupt cell membrane, leading to the release of lactate dehydrogenase (LDH) as a sign of cell damage. We therefore assessed the LDH release from RAW264.7 cells incubated with TOT-containing liposomes for 12 h. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, TOT-containing liposomes induced no or negligible LDH release, suggesting no discernable cell membrane damages. Additionally, TOT-containing liposomes exhibited negligible effects on the cell viability up to 200 \u0026micro;g/mL after 24 h of incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). As expected from Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, TOT-containing liposomes suppressed the ROS generation in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). These results demonstrate that TOT-containing liposomes are biocompatible and exert antioxidant effects.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eTo enhance antioxidant capacity of TCP, we developed ditocopherol peroxalate (TOT), a H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsive tocopherol dimer incorporating a peroxalate linkage. TOT undergoes selective degradation in the presence of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, releasing two TCP molecules while simultaneously scavenging H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Compared to TCP, TOT exhibited stronger antioxidant and anti-inflammatory effects in H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-stimulated cells. Its linear structure and enhanced rigidity promote alignment with DPPC, enabling the formation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-responsive liposomal formulation. These TOT-containing liposomes were efficiently internalized by cells and exhibited no cytotoxicity. Collectively, our results obviously demonstrate that TOT functions as a pro-antioxidant and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e-scavenger, offering superior protective effects over native TCP and holding great potential as an active cosmetic ingredient for oxidative stress mitigation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflicts of interest:\u003c/strong\u003e\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e\u003cp\u003eThis work was supported by a grant of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (RS-2024-00406625), Republic of Korea. The authors thank the Center for University Wide Research Facilities (CURF) at Jeonbuk National University for the valuable analysis of confocal laser scanning microscopy and electron microscopy.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eData is contained within the article and the supplementary material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaptista MS, Cadet J, Di Mascio P et al (2017) Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways. 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J Agric Food Chem 49:1508\u0026ndash;1511. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1021/jf001142f\u003c/span\u003e\u003cspan address=\"10.1021/jf001142f\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"tocopherol, hydrogen peroxide, antioxidant, liposome, dimer","lastPublishedDoi":"10.21203/rs.3.rs-7117698/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7117698/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOxidative stress caused by excessive hydrogen peroxide (H₂O₂) plays a central role in skin damage, inflammation, and premature aging, particularly through light-induced photosensitization. Tocopherol (TCP) is a widely used antioxidant in cosmetics, yet its potential in H₂O₂-responsive systems remains underexplored. Here, we report the design and characterization of ditocopheryl peroxalate (TOT), a novel tocopherol dimer linked via an H₂O₂-cleavable peroxalate bond. TOT remains stable under physiological conditions but selectively degrades in response to H₂O₂, simultaneously scavenging H₂O₂ and releasing two TCP molecules. TOT exhibited comparable radical scavenging activity to TCP but showed superior H₂O₂-scavenging efficiency, stronger antioxidant and anti-inflammatory effects in H₂O₂-stimulated cells, and excellent biocompatibility. Its rigid, linear structure promoted alignment within dipalmitoylphosphatidylcholine (DPPC) bilayers, enabling formulation of stable, H₂O₂-responsive liposomes with effective cellular uptake. These findings highlight TOT as a multifunctional, self-degradable antioxidant with strong potential as a cosmetic ingredient for protecting skin from oxidative and phototoxic damage.\u003c/p\u003e","manuscriptTitle":"A self-deliverable H2O2-responsive tocopherol dimer for enhanced antioxidant and liposomal delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-29 12:43:00","doi":"10.21203/rs.3.rs-7117698/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2b0cfc4b-9014-4bee-b8e7-2d9e343df85e","owner":[],"postedDate":"August 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T19:18:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-29 12:43:00","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7117698","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7117698","identity":"rs-7117698","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}