Inflammation-Responsive Antioxidant Nanoparticles Based on a

Apr 16, 2013 - Polymer Fusion Research Center, Department of Polymer·Nano Science and Technology, Chonbuk National University, Jeonju,. 561-756 ...
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Inflammation-Responsive Antioxidant Nanoparticles Based on a Polymeric Prodrug of Vanillin Jeongil Kwon,† Jihye Kim,† Seunggyu Park,† Gilson Khang,‡ Peter M. Kang,†,§ and Dongwon Lee*,†,‡ †

Department of BIN Fusion Technology, Chonbuk National University, Jeonju, 561-756, Korea Polymer Fusion Research Center, Department of Polymer·Nano Science and Technology, Chonbuk National University, Jeonju, 561-756, Korea § Cardiovascular Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, United States ‡

S Supporting Information *

ABSTRACT: Oxidative stress is induced by accumulation of hydrogen peroxide (H2O2), and therefore, H2O2 could serve as a potential biomarker of various oxidative stress-associated inflammatory diseases. Vanillin is one of the major components of natural vanilla and has potent antioxidant and anti-inflammatory activities. In this work, we developed a novel inflammation-responsive antioxidant polymeric prodrug of vanillin, termed poly(vanillin oxalate) (PVO). In design, PVO incorporates H2O2-reacting peroxalate ester bonds and bioactive vanillin via acid-responsive acetal linkages in its backbone. Therefore, in cells undergoing damages by oxidative stress, PVO readily degrades into three nontoxic components, one of which is antioxidant and anti-inflammatory vanillin. PVO nanoparticles exhibit potent antioxidant activities by scavenging H2O2 and inhibiting the generation of ROS (reactive oxygen species) and also reduce the expression of pro-inflammatory cytokines in activated macrophages in vitro and in vivo. We, therefore, anticipate that PVO nanoparticles have great potential as novel antioxidant therapeutics and drug delivery systems for ROS-associated inflammatory diseases. GRAS (generally regarded as safe) status.11 It has recently been reported to exert potent antioxidant activity by scavenging superoxide and hydroxyl radical and protect membrane against protein oxidation and lipid peroxidation.12,13 Vanillin also has anti-inflammatory activity to suppress the expression of various pro-inflammatory cytokines such as IL (interleukin)-1β, IL-6, interferon-γ, and tumor necrosis factor-α (TNF-α), demonstrating tremendous potential for the treatment of inflammation-related diseases.10,14,15 However, vanillin shows minimal or insufficient therapeutic effects because orally administrated vanillin is rapidly decomposed in the upper digestive tract and intravenously injected vanillin is rapidly cleared from blood circulation.16 Therefore, its successful clinical applications require strategies to deliver vanillin to diseased cells or tissues and enhance its therapeutic efficacy. Drug delivery systems capable of a controlled and stimulusresponsive release have gained increasing attention in the field of pharmaceutics. In general, drug carriers encapsulate drugs physically and protect them from harsh in vivo conditions that lead to proteolytic degradation.17,18 A novel strategy for the delivery of chemotherapeutic drugs involves polymeric prodrugs, which incorporate drugs in the biodegradable polymer backbone, not side groups and release drugs during their degradation.5,19,20 Uhrich et al. reported a pioneering

1. INTRODUCTION ROS (reactive oxygen species) are chemically reactive and diffusible entities that are produced from as byproducts of normal oxygen metabolism and include H2O2, superoxide, and hydroxyl radicals.1,2 Despite their important role as a mediator in a variety of biological and pathological events, excessive and unregulated production of ROS is known to cause oxidative stress, leading to functional decline of organs and tissues.3,4 The accumulation of oxidative stress over time has been implicated in debilitating conditions such as cancer, cardiovascular diseases, neurodegenerative diseases, and acute and chronic inflammatory process.5,6 In particular, H2O2 is an essential oxygen metabolite in living organisms and plays fundamental roles in the cellular signaling pathway. However, it is also a major source of oxidative stress and a common marker of ROSassociated diseases.7 In addition, H2O2 is a precursor of highly reactive ROS such as hydroxyl radical, peroxynitrite, and hypochlorite, and the overexpression of H2O2 leads to oxidative damages and can be served as a biomarker of inflammation and aging-associated diseases.8,9 Therefore, there has been great interest in the development of antioxidants as therapeutic agents for various ROS-associated inflammatory diseases. Vanillin is an aromatic aldehyde (4-hydroxy-3-methoxybenzaldehyde) containing a hydroxyl group para to aldehyde and is the major component of natural vanilla, which has been widely used as a flavoring agent in food, beverage, and cosmetics.10 Vanillin is recognized as suitable for food use by the Food and Drug Administration in United States and has been given © XXXX American Chemical Society

Received: February 18, 2013 Revised: April 5, 2013

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Scheme 1. Schematic Diagram of Fully Biodegradable and Dual Stimuli-Responsive PVO Nanoparticles

under a nitrogen atmosphere at room temperature for 6 h, and polymers were obtained through the extraction using dichloromethane and isolation by precipitating in cold hexane. The chemical structure of polymers was identified with a 400 MHz 1H NMR spectrometer (JNM-EX400 JEOL) CDCl3 as a solvent: 6.8−7.0 (m, 3H, Ar), 5.5 (s, 1H, CH(O)2), 4.5 (m, 2H, OCH2C), 3.8−4.2 (m, 4H, OCH2C), 3.9 (m, 3H, OCH3), 1.0 (s, 3H, CH3C). The molecular weight of PVO was determined by gel permeation chromatography (PL-GPC, Polymer Laboratories) equipped with a differential refractometer (2 × 10−7 RIU/mV sensitivity) using polystyrene standards at a flow rate of 1.0 mL/min (chloroform as a mobile phase). For the study of hydrolysis and vanillin release, PVO was incubated in D2O at 37 °C for 3 days. The supernatant was collected and analyzed with 1H NMR spectrometer. 2.4. Preparation and Characterization of PVO Nanoparticles. A total of 50 mg of PVO dissolved in 500 μL of dichloromethane was added to 5 mL of 5% PVA, poly(vinyl alcohol) solution. The mixture was sonicated using a sonicator (Fisher Scientific, Sonic Dismembrator 500) for 30 s and homogenized (PRO Scientific, PRO 200) with an output setting of 5 (rpm 17000−24000) for 1 min to form a fine oil/ water emulsion. The emulsion was added into 20 mL of PVA (1 w/v %) solution and homogenized for 1 min. The remaining solvent was removed using a rotary evaporator. Nanoparticles were obtained by centrifuging at 10000g for 5 min at 4 °C, washing with deionized water twice, and lyophilizing the recovered pellet. PVO nanoparticles were mounted on a metal stub and sputter-coated with Au−Pd for scanning electron microscopy (SEM, Hitachi). The morphology and size of PVO nanoparticles were observed by SEM with accelerating voltage of 10 kV. 2.5. Release of Vanillin from PVO Nanoparticles. To investigate the release behaviors of vanillin from the PVO nanoparticles, the prepared nanoparticles (100 mg) were placed in a test tube containing 5 mL of phosphate buffered saline (PBS) at 37 °C. At appropriate time intervals, 500 μL of the supernatant was removed and replaced with the same amount of fresh PBS. Vanillin released from PVO nanoparticles was analyzed using high performance liquid chromatography system (HPLC; Futecs, Korea), equipped with C-18 column (5 μm, 150 × 4.6 mm) and a P1000 solvent pump unit. HPLC was performed with a mobile phase consisting of acetonitrile (35%) and 0.1% formic acid solution (65%) at a flow rate of 1.0 mL/min. The effluents were monitored by UV−visible detector at 254 nm and quantified by comparing the peak areas with the standard curve. 2.6. Cytotoxicity Assay. The cytotoxicity of PVO nanoparticles was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells (RAW 264.7) were cultured using Dulbecco’s modified Eagle medium (Gibco, Gland Island, NY) containing 10% fetal bovine serum). Cells in a 24-well plate with ∼90% confluency were treated with various amounts of PVO nanoparticles for 4 h. The PVO nanoparticles-containing medium was replaced with fresh medium and cells were incubated for 24 h. Each well was given 100 μL of MTT solution and was incubated for 4 h. Dimethyl sulfoxide (1 mL) was added to cells to dissolve the resulting formazan crystals. After 10 min of incubation, the absorbance at 540 nm was measured using a microplate reader (Synergy MX, BioTek Instruments, Inc., Winooski, VT). 2.7. H2O2 Scavenging. The ability of PVO nanoparticles to scavenge H2O2 was evaluated by measuring the H2O2 concentration after incubation with 1 mg PVO nanoparticles. To 1 mL of H2O2

work in polymeric prodrugs, termed PolyAspirin, which incorporates salicylic acid in the backbone of biodegradable polyanhydride. PolyAspirin was designed to release active salicylic acid upon its hydration and degrade completely into biocompatible small molecules.21,22 This design allowed for a high percentage (∼62 wt %) of deliverable drugs that are available as the polymer degrades. Another elegant example is poly(trolox ester) which was synthesized from a carbodiimidebased polymerization of water-soluble analogue of vitamin E.6,23 Poly(trolox ester) nanoparticles showed little to no cytotoxicity and degraded enzymatically to release active antioxidant trolox to suppress oxidative stress injury in the cells. By taking cues from the previous polymeric prodrugs, we have developed poly(vanillin oxalate) (PVO) as a polymeric prodrug of vanillin, which covalently incorporates vanillin in its backbone and release them during its hydrolytic degradation, as shown Scheme 1.21 In addition, PVO was molecularly engineered to contain both H2O2-responsive peroxalate ester bonds and acid-cleavable acetal linkages in its backbone because inflammation is characterized by acidic pH and a large generation of ROS, such as H2O2.24 We therefore hypothesized that PVO releases vanillin during its H2O2- and acid-catalyzed backbone degradation and is capable of serving as therapeutic agents in ROS-associated inflammatory diseases. As a proof of concept, we report dual stimuli-responsive PVO nanoparticles as therapeutic agents for oxidative stress-associated diseases.

2. MATERIALS AND METHODS 2.1. Materials. Vanillin, hydroxymethyl-2-methylpropane-1,3-diol, oxalyl chloride, para-toluenesulfonic acid (pTSA), and poly(vinyl alcohol) were obtained from Sigma-Aldrich (St. Louis, MO). Dichloromethane and pyridine were obtained from Showa (Japan). pTSA was recrystallized from methanol and chloroform. Dichloromethane was distilled over calcium hydride. RAW 264.7 cells were purchased from Korea Cell Line Bank (Seoul, Korea). 2.2. Synthesis of 4-(5-(Hydroxymethyl)-5-methyl-1,3-dioxan-2-yl)-2-methoxyphenol. 2-(Hydroxymethyl)-2-methylpropane1,3-diol (7.896 g, 65.72 mmol) and vanillin (10 g, 65.72 mmol) were dissolved in dry tetrahydrofuran. A catalytic amount of ptoluenesulfonic acid, 43 mg (0.25 mmol), was then added to the mixture, and the reaction was allowed at 85 °C overnight. The resulting colorless liquid was purified via flash column chromatography with a mixture of ethylacetate/hexane (6:4). The chemical structure of 4-(5-(hydroxymethyl)-5-methyl-1,3-dioxan-2-yl)-2-methoxyphenol was identified with a 400 MHz 1H NMR spectrometer (JNM-EX400 JEOL) using CDCl3 as a solvent: 6.8−7.0 (m, 3H, Ar), 5.6 (s, 1H, CHO2), 5.3 (s, 1H, Ar−OH), 4.0 (m, 2H, OCH2C), 3.8 (m, 3H, OCH3), 3.6 (m, 2H, OCH2C), 0.9 (m, 3H, CCH3). 2.3. Synthesis of PVO. PVO was synthesized from a reaction of 4(5-(hydroxymethyl)-5-methyl-1,3-dioxan-2-yl)-2-methoxyphenol and oxalyl chloride. 4-(5-(Hydroxymethyl)-5-methyl-1,3-dioxan-2-yl)-2methoxyphenol (3.941 mmol) was dissolved in dry dichloromethane (DCM), under nitrogen, to which pyridine (9.852 mmol) was added dropwise at 4 °C. Oxalyl chloride (3.941 mmol) in 25 mL of dry DCM was added to the mixture dropwise at 4 °C. The reaction was kept B

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Figure 1. Antioxidant and anti-inflammatory nanoparticles based on a polymeric prodrug of vanillin (PVO). (A) A synthetic route of PVO precursor, 4-(5-(hydroxymethyl)-5-methyl-1,3-dioxan-2-yl)-2-methoxyphenol, (B) a synthetic route and degradation of PVO, (C) 1H NMR spectrum of PVO in CDCl3, and (D) 1H NMR spectrum of degradation products of PVO after 3 days of hydrolysis in D2O. PVO hydrolytically degrades into three components and its degradation is accelerated by acidic pH and H2O2. One of the degradation products is vanillin, which exerts antioxidant and antiinflammatory activity. solution (10 μM) was added 1 mg of PVO nanoparticles and the solution was left at 37 °C under gentle mechanical stirring. At appropriate time intervals, the solution was centrifuged at 1000g and the H2O2 concentration of the supernatant was measured using the Amplex Red assay (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. 2.8. Measurement of NO (Nitric Oxide). RAW 264.7 cells (4 × 105 cells/well in a 24 well plate) were pretreated with a various amount of PVO nanoparticles for 20 h and then treated with 3 μL of lipopolysaccharide (LPS, 1 mg/mL) for 4 h. Cell culture medium containing PVO nanoparticles was replaced with fresh medium and cells were incubated for 6 h. The concentration of NO was determined using a colorimetric assay based on the Griess reaction. A total of 50 μL of cell culture medium was collected and given 100 μL of Griess reagent (6 mg/mL) at room temperature for 10 min, and then the NO concentration was determined by measuring the absorbance at 540 nm using a microplate reader (Synergy MX, BioTek Instruments, Inc., Winooski, VT). The NO standard curve was constructed using known concentrations of sodium nitrite. Untreated cells were used as negative control. 2.9. Measurement of Intracellular ROS. RAW 264.7 cells (4 × 105 cells) were seeded in a glass bottom dish (MatTek Corp. Ashland, MA) and incubated for 24 h. Cells were treated with vanillin or PVO nanoparticles for 10 h and incubated with 1 μg of LPS for 20 h. DCFH-DA (dichlorofluorescein-diacetate) of 10 μM was added to each dish, and 0.5 h later, the fluorescence images were made with a confocal laser scanning microscope (LSM 510 Meta, Carl Zeiss, Germany). To quantify the fluorescent cells, flow cytometry was also performed with a Flow Cytometry Caliber (Becton Dickinson, U.S.A.). The percentage of cells in positive events was calculated as the events within the gate divided by total number of events, then subtracting the percentage of the control sample (untreated cells).

2.10. Reverse Transcription-Polymerase Chain Reaction. RAW 264.7 cells seeded at a density of 4 × 105 in a 24-well culture plate were pretreated with 0.25 mM vanillin and a various amount of PVO nanoparticles for 20 h and then treated with 1 μL of LPS (1 mg/ mL) for 10 h. Total cellular RNA was isolated using 1 mL of Trizol (Invitrogen, Life Technologies Co, Groningen, Netherlands) according to the manufacturer’s instructions. A total of 3 μg of total RNA was reverse-transcribed into cDNA using oligo (dT) primer (Invitrogen), 5× First Strand buffer (Invitrogen), dNTP (dGTP, dATP, dTTP, dCTP, Gibco), RNase inhibitor (Invitrogen), SuperScript II (Invitrogen), RNase H reverse transcriptase (Invitrogen), and DNase/RNase free water (Gibco,Gland Island, NY). PCR was performed on aliquots of the cDNA preparations to detect TNF-α and GAPDH (the internal standard) gene expressions by Authorized Thermal Cycler (TP 600, Takara Bio Inc., Japan). After amplification, portions of the PCR reactions were subjected to electrophoresis using 2% agarose gel and visualized under UV (365 nm) after ethidium bromide staining. 2.11. Tissue Compatibility. Balb/c mice (∼20 g, Orient Bio, Korea) were anesthetized by an intramuscular injection of a mixture (150 μL) of Zoletil 50 and Domitor (the ratio of 2:1). Mice were intramuscularly injected with 100 μL of PLGA or PVO (1 mg/mL) particles suspended in PBS. For histological examination, mice were euthanized and tissues surrounding injection sties were extracted at 7 days postinjection. The extracted tissues were fixed in 4% formalin (Sigma-Aldrich, St. Louis, MO) and embedded in paraffin blocks. The specimens were sectioned with to a 4 μm thickness. The sections were stained with H&E (hematoxylin and eosin) and ED-1. All experiment procedures were performed with the approval of Chonbuk National University Animal Care Committee, Jeonju, Korea. 2.12. Mouse Model of APAP-Induced Liver Injury. Mice (∼20 g) were fasted for 12 h prior to the experiments. Vanillin (0.75 mg/kg) C

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Figure 2. Characterization of PVO nanoparticles. (A) A representative SEM image, (B) dynamic light scattering of PVO nanoparticles, and (C) release kinetics of vanillin from PVO nanoparticles at different pH. (D) H2O2 scavenging activity of PVO nanoparticles. Data presented are means ± SD (n = 4). or PVO nanoparticles (1.25 or 2.5 mg/kg) were injected in mice (n = 4) with various PVO nanoparticles through a tail vein. After 1 h, acute liver failure was induced by the intraperitoneal injection of 400 μL of APAP (22.5 mg/mL). Mice were sacrificed 24 h after APAP intoxication and whole blood and livers were collected. The activity of serum ALT was determined with an ALT enzymatic assay kit (Asan Pharma, Seoul, Korea) using a microplate reader (Synergy MX, BioTek Instruments, Inc., Winooski, VT). The liver tissues were fixed with 4% formalin (Sigma-Aldrich, St. Louis, MO) and embedded into paraffin. Histological sections were made and stained with hematoxylin and eosin (H&E). All the animal experiments were carried out according to the guidelines of the institution animal ethical committee. 2.13. In Vivo Biodistribution of PVO Nanoparticles. The biodistribution of PVO nanoparticles was determined using protoporphyrin-loaded PVO nanoparticles. Protoporphyrin-loaded PVO nanoparticles were formulated with the same procedure for empty PVO nanoparticles, as described above, except that protoporphyrin was added to dichloromethane containing PVO. Mice were injected with 100 μL of protoporphyrin-loaded PVO nanoparticles (10 mg/mL in PBS) via a tail vein injection and then sacrificed after 3 h. Immediately after the collection of organs, their fluorescence images were made using an IVIS imaging system (Xenogen, Alameda, CA).

environment of endosomes, facilitating the release of vanillin to cytosol after cellular uptake by inflammatory cells such as macrophages. We therefore reasoned that the H2O2- and acidresponsive PVO rapidly degrades in inflamed tissues to release vanillin which then exerts potent antioxidant and antiinflammatory activities. PVO is one analogue of biodegradable and biocompatible polyoxalates that has been used for medical devices such as absorbable sutures and controlled release.25,26 Polyoxalates have been prepared from a simple reaction of diols and oxalyl chloride or ester of oxalic acid. In this work, polyoxalate was chosen as a platform of PVO because of its easy synthesis, fast degradation kinetics, excellent biocompatibility, and H2O2responsiveness.5,20 To develop PVO as a polymeric prodrug of vanillin, we first synthesized 4-(5-(hydroxymethyl)-5-methyl1,3-dioxan-2-yl)-2-methoxyphenol from a reaction of 2(hydroxymethyl)-2-methylpropane-1,3-diol and vanillin, as shown in Figure 1. 4-(5-(Hydroxymethyl)-5-methyl-1,3-dioxan-2-yl)-2-methoxyphenol has an acetal linkage that can undergo acid-triggered cleavage. PVO was prepared from one-step growth polymerization using oxalyl chloride with 4-(5(hydroxymethyl)-5-methyl-1,3-dioxan-2-yl)-2-methoxyphenol in dichloromethane at room temperature. PVO was obtained as a pale yellow solid after drying under high vacuum. The chemical structure of PVO was confirmed by 1H NMR. The multiplet signals at ∼4.6 ppm correspond to methylene protons next to peroxalate ester bonds, and the acetal proton is observed at ∼5.5 ppm, demonstrating the condensation reaction between 4-(5-hydroxymethyl)-5-methyl-1,3-dioxan-2yl-2-methoxyphenol and oxalyl chloride to generate PVO

3. RESULTS AND DISCUSSION PVO was designed to contain H2O2-responsive peroxalate ester bonds as well as acid-labile acetal linkages in its backbone and release vanillin as it hydrolytically degrades. Therefore, in the inflamed tissues with a high concentration of H2O2, PVO nanoparticles are expected to scavenge H2O 2 because peroxalate ester bonds instantaneously react with H2O2 to be oxidized.5,20 PVO also possesses acid-sensitive acetal linkages which can be rapidly cleaved in the acidic (pH ∼5.5) D

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Figure 3. Antioxidant activities of PVO nanoparticles in LPS-stimulated RAW 264.7 cells. (A) Representative CLSM images of LPS-stimulated cells stained with DCFH-DA and (B) flow cytometric analysis of intracellular ROS generation in cells.

Figure 4. Anti-inflammatory activities of PVO nanoparticles. (A) Inhibition of NO production. Given amounts of PVO particles were added to each well containing 1 mL of culture medium. Data presented are means ± SD (n = 4), *p < 0.05, **p < 0.01, ***p < 0.001 relative to the LPS group. (B) Suppressed expression of iNOS and TNF-α in LPS-stimulated cells.

As shown in Figure 2C, a majority (∼80%) of vanillin was released within 36 h at pH 5.4, while significantly less (40%) vanillin release occurred at pH 7.4. The pH-dependent release of vanillin is attributed to the acid-labile acetal linkage in the polymer backbone.27,28 It was also determined that 1 mg of PVO nanoparticles releases ∼300 μg of vanillin. We hypothesized that PVO nanoparticles are able to scavenge H2O2 and consequently reduce H2O2-mediated oxidative stress because PVO contains H2O2-reacting peroxalate ester bonds in its backbone, which are known to be oxidized by H2O2 spontaneously and instantaneously and decomposed into CO2.20,29 The ability of PVO nanoparticles to scavenge H2O2 was evaluated using the Amplex Red assay. A total of 1 μL of H2O2 solution (10 μM in pH 7.4 phosphate buffer) was incubated with 1 mM vanillin or 1 mg of PVO nanoparticles and the change of H2O2 concentration was monitored for 36 h (Figure 2D). Vanillin (1 mM) reduced the H2O2 concentration to some extent, with 20% reduction at 36 h. However, PVO nanoparticles showed noticeably higher H2O2 scavenging activity than free vanillin. After 36 h of incubation, a majority of H2O2 was removed by PVO nanoparticles, suggesting that H2O2 was destroyed through the oxidation of peroxalate ester bonds in the backbone of PVO. The potent H2O2 scavenging activity of PVO nanoparticles can be explained by the combined effects of vanillin and peroxalate ester bonds in the backbone of PVO. We investigated the ability of PVO nanoparticles to suppress lipopolysaccharide (LPS)-mediated oxidative stress in cells. RAW 264.7 cells were pretreated with free vanillin or PVO nanoparticles for 10 h and then stimulated with LPS (1 μg/ mL) as a stimulant for the generation of intracellular ROS. The generation of ROS in cells was observed by confocal laser

containing both peroxalate ester bonds and acetal linkages in the backbone (Figure 1B). Its weight average molecular weight (Mw) was determined to be ∼22000 Da with a polydispersity index of ∼1.6 by gel permeation chromatography. As aforementioned, PVO is designed to release vanillin during its hydrolytic degradation. In order to confirm the release of vanillin from PVO, PVO was hydrolyzed in D2O at 37 °C for 3 days and the degradation products were subjected to 1H NMR spectroscopy. Disappearance of the acetal proton signal and appearance of the aldehyde proton signal at ∼9.3 ppm were observed. In addition, methyl and methylene proton signals of 2-(hydroxymethyl)-2-methylpropane-1,3-diol appeared at ∼0.8 and ∼3.3 ppm, demonstrating that PVO undergoes hydrolytic degradation to release vanillin (Figure 1C). We also found that PVO undergoes hydrolytic degradation in a pH-dependent manner, with a half-life of hydrolysis of ∼24 h at pH 7.4 and ∼15 h at pH 5.5 due to the acid-cleavable acetal linkages. Hydrolytic degradation was also slightly accelerated by H2O2 due to the presence of peroxalate ester groups (Figures S3 and S4). DSC studies reveal that PVO is an amorphous polymer with a glass transition of ∼120 °C (Figure S5). PVO had a hydrophobic backbone and therefore could be formulated into nanoparticles by a conventional oil/water emulsion method. Figure 2 shows that PVO nanoparticles were round spheres with smooth surface and a mean hydrodynamic diameter of ∼260 nm (PDI = 1.50) in phosphate buffer (pH 7.4). PVO nanoparticles are expected to be phagocytosed by macrophages because phagocytes readily take up foreign matter with hydrophobic surface and size up to ∼3 μm.5 We next investigated the release kinetics of vanillin from PVO nanoparticles. E

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Figure 5. Biocompatibility and antioxidant activity of PVO nanoparticles. (A) Cytotoxicity of PVO nanoparticles determined by the MTT assay. Given amounts of PVO nanoparticles were added to each well containing 1 mL of culture medium. (B) H&E and ED-1 staining of tissues from PVO or PLGA nanoparticles injected mice.

Vanillin is also known to exert anti-inflammatory activity by reducing the expression of pro-inflammatory mediators such as TNF-α. In addition, TNF-α is known to be essential for optimal production of iNOS gene expression.33 We therefore investigated the mRNA level of TNF-α in LPS-stimulated RAW 264.7 cells. As shown in Figure 4B, LPS-treatment caused a large production of TNF-α, but LPS-induced TNF-α expression was reduced by vanillin and PVO nanoparticles. PVO nanoparticles exhibited the higher inhibitory effects on TNF-α production than vanillin. The stronger anti-inflammatory activity of PVO nanoparticles than vanillin may result from their highly potent antioxidant activity. The cytotoxicity of PVO nanoparticles was assessed using a (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assay. Metabotically active cells reduce MTT to formazan, which is soluble in dimethylsulfoxide and can be quantified by measuring the absorbance intensity as 570 nm. RAW 264.7 cells were treated with various amounts of PVO nanoparticles up to 500 μg/mL and the cell viability was assessed at 24 h after the treatment (Figure 5A). PVO nanoparticles exhibited a dose-dependent cytotoxicity. However, no or minimal cytotoxicity was observed with PVO nanoparticles at doses less than 500 μg/mL, demonstrating the excellent biocompatibility of PVO nanoparticles. We next studied the tissue biocompatibility of PVO nanoparticles because one of key issues involving the development of biodegradable polymers and drug carriers is the foreign-body response by the host immune system.34 Mice were subjected to an intramuscular injection of PVO nanoparticles and sacrificed at 7 days after injection. For comparison purposes, poly(lactic-co-glycolic acid) (PLGA, MW 33000 Da) was formulated into nanoparticles with a mean diameter of ∼250 nm and used as a control because the foreignbody response is severe with polymers such as PLGA, which generates acidic degradation products.35 Figure 5B shows the histological sections of tissues stained with H&E (hematoxylinEosin) and inflammatory cell-specific marker ED-1. Injection of PVO nanoparticles caused very little recruitment of immune cells and ED-1 positive cells. However, a large influx of immune cells and ED-1 positive cells was caused by PLGA nanoparticles. The results demonstrate that PVO nanoparticles induce minimal inflammatory responses and have great potential as drug carriers for the treatment of inflammatory diseases. To extrapolate our encouraging in vitro findings, we investigated therapeutic potential of PVO nanoparticles in clinically relevant settings using a mouse model of acetamino-

scanning microscopy (CLSM) using DCFH-DA as a probe for intracellular ROS because it diffuses into cells and becomes fluorescent dichlorofluorescein (DCF) via ROS-mediated oxidation.20,30 Figure 3A shows the representative CLSM images of LPSstimulated cells treated with vanillin or PVO nanoparticles. While untreated cells showed negligible DCF fluorescence, LPS-treatment induced the generation of a large amount of intracellular ROS, including H2O2, evidenced by strong DCF fluorescence. The intracellular ROS generation was suppressed by 0.25 mM of vanillin and 100 μg/mL of PVO nanoparticles. Relative quantification of intracellular ROS generation was performed by flow cytometry (Figure 3B). Both vanillin (0.25 mM) and PVO nanoparticles (100 μg) noticeably suppressed the intracellular ROS generation, evidenced by the significant leftward shift in DCF fluorescence. However, PVO nanoparticles showed more suppressive effects on ROS generation than free vanillin. We also studied the effects of PVO nanoparticles on the generation of nitric oxide (NO) in LPS-stimulated cells because NO is a well-known pro-inflammatory mediator and has been implicated in many inflammatory diseases.31 NO production is known to be catalyzed by inducible nitric oxide synthase (iNOS), which is produced in macrophages after activation by endotoxin such as LPS.32 A large amount of NO was produced in cells by LPS-treatment, but vanillin and PVO nanoparticles suppressed the LPS-induced NO generation in a dosedependent manner (Figure 4A). For example, 100 μg/mL of PVO nanoparticles significantly inhibited NO generation. In order to confirm whether inhibitory effects of PVO nanoparticles on NO production results from the suppression of iNOS expression, the mRNA level of iNOS was evaluated with GAPDH (glyceraldehyde 3-phosphate dehydrogenase) as an internal gene. As shown in Figure 4B, vanillin and PVO nanoparticles reduced the expression of iNOS mRNA, indicating that PVO nanoparticles inhibit the NO generation by suppressing iNOS expression. In these experiments, 100 μg/ mL of PVO nanoparticles is able to theoretically release ∼30 μg of vanillin, which corresponds to ∼0.21 mM. Interestingly, 100 μg/mL of PVO nanoparticles exhibited significantly higher suppressive effects on the generation of intracellular ROS and NO than 0.25 mM of vanillin. It can be explained by the dual antioxidant activities of PVO. First, PVO scavenges H2O2 generated during LPS-induced inflammatory responses and reduces H2O2-mediated oxidative stress. Second, PVO releases vanillin during its degradation, which inhibits the further generation of other ROS. F

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Figure 6. Therapeutic effects of PVO nanoparticles in APAP-intoxicated mice. (A) Serum ALT level in APAP-intoxicated mice. Given amounts of PVO nanoparticles were added to each well containing 1 mL of culture medium. Data presented are means ± SD (n = 4), **p < 0.01, ***p < 0.001 relative to the APAP group. (B) H&E staining of liver tissues of APAP-intoxicated mice. All images are ×200 magnification (scale bar = 100 μm). APAP challenge induced severe tissue destruction with large areas of damaged and necrotic hepatocytes and loss of membrane integrity.

Figure 7. Biodistribution of free PpIX and PpIX-loaded PVO nanoparticles in mice. A representative image of three independent experiments is presented.

phen (APAP)-induced liver injury. APAP overdose is known to induce a large generation of ROS such as H2O2, hydroxyl

radical, or nitric oxide, leading to oxidative stress, acute hepatic inflammation, and severe liver injury.36,37 Mice were treated G

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inflammatory diseases. Ongoing studies are dedicated to investigating the potential of PVO nanoparticles as drug delivery systems to tissues undergoing oxidative stress, using a clinically relevant animal model.

with free vanillin (0.75 mg/kg) or PVO nanoparticles (1.25 or 2.5 mg/kg) 1 h before APAP injection. We evaluated the liver injury at 24 h by measuring the level of serum alanine transaminase (ALT), which is a surrogate clinical marker for liver injury. PVO nanoparticles themselves showed no effects on ALT activity, but 24 h of APAP-intoxication dramatically increased the ALT level. However, as shown in Figure 6A, the elevated ALT level was significantly reduced by the treatment of PVO nanoparticles, in a dose-dependent manner. In contrast, vanillin alone (0.75 mg/kg) at the equivalent amount contained in PVO nanoparticles (2.5 mg/kg) showed modest, not significant, effects on ALT activity. We also performed the histological studies to further confirm the therapeutic efficacy of PVO nanoparticles in APAP-induced liver injury (Figure 6B). PVO nanoparticles themselves had no effects on liver histology. However, APAP overdose induced extensive liver damages and disruption of tissue architecture, evidenced by destruction of hepatocytes. PVO nanoparticles at a dose of 2.5 mg/kg remarkably reduced the liver tissue damage and histopathological alterations. These findings demonstrate that PVO nanoparticles exhibit highly potent antioxidant effects and have tremendous potential as therapeutic agents for liver injury associated with oxidative stress. The biodistribution of PVO nanoparticles was investigated using protoporphyrin (PpIX) as a fluorophore. As shown in Figure 7, free PpIX was accumulated in lung, liver and kidney. In contrast, a majority of PpIX-loaded PVO nanoparticles were accumulated in liver more than other organs, like particulate drug delivery systems, which have natural propensity to target liver, passively, but specifically.37 It is not surprising because of the rationale that the largest population of macrophages in contact with blood is located in liver sinuses (Kupffer cells) and particulates are localized to liver macrophages that have a primary scavenging role for foreign invaders.38 In addition, it is known that during acute liver injury, macrophages from circulation are activated and enter the injured liver, which enhances macrophage-mediated phagocytosis in liver.37 Thus, the ability of PVO nanoparticles to passively target liver may provide a rational strategy for effective treatment of acute liver injury using PVO. Given their targeting ability, we also anticipate that PVO nanoparticles have great potential as drug delivery systems for liver injury.

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ASSOCIATED CONTENT

S Supporting Information *

NMR, FT-IR and GPC data, thermal analysis, and flow cytometry data of PVO. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: 82-63-270-2344. Fax: 82-270-2341. E-mail: dlee@ chonbuk.ac.kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by the grant of National Research Foundation of Korea (2010-0021903, 2-11-0030912) and the World Class University program (R31-20029) funded by the Ministry of Education, Science and Technology.

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REFERENCES

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4. CONCLUSIONS In summary, we developed antioxidant and anti-inflammatory polymeric nanoparticles based on dual stimuli-responsive polymeric prodrugs of vanillin (PVO). PVO was designed to incorporate vanillin in its backbone and release vanillin during its H2O2- and acid-mediated hydrolytic degradation. PVO showed pH-dependent hydrolytic degradation kinetics and vanillin release behavior due to acid-cleavable acetal linkages in its backbone. PVO nanoparticles were able to scavenge H2O2 because they contain H2O2-responsive peroxalate ester bonds in their backbone. PVO nanoparticles showed highly potent antioxidant activities by scavenging H2O2 and inhibiting the generation of oxidants in LPS-stimulated cells. PVO nanoparticles also exerted highly potent anti-inflammatory activity by inhibiting the expression of TNF-α and iNOS. In addition, intravenous administration of PVO nanoparticles significantly reduced the APAP-induced acute hepatic injury. Based on their excellent biocompatibility, antioxidant, and anti-inflammatory activity, we anticipate that PVO nanoparticles have tremendous potential as therapeutics for oxidative stress-associated H

dx.doi.org/10.1021/bm400256h | Biomacromolecules XXXX, XXX, XXX−XXX

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dx.doi.org/10.1021/bm400256h | Biomacromolecules XXXX, XXX, XXX−XXX