Structure–Property Correlations of Reactive Oxygen Species

Sep 13, 2017 - To develop reactive oxygen species (ROS)-responsive anti-inflammatory materials and establish their structure–property correlations, ...
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Structure−Property Correlations of Reactive Oxygen SpeciesResponsive and Hydrogen Peroxide-Eliminating Materials with AntiOxidant and Anti-Inflammatory Activities Qixiong Zhang,†,‡ Fuzhong Zhang,† Yue Chen,† Yin Dou,† Hui Tao,† Dinglin Zhang,§ Ruibing Wang,∥ Xiaohui Li,‡ and Jianxiang Zhang*,† †

Department of Pharmaceutics, College of Pharmacy, Third Military Medical University, Chongqing 400038, China Institute of Materia Medica, College of Pharmacy, Third Military Medical University, Chongqing 400038, China § Department of Chemistry, College of Pharmacy, Third Military Medical University, Chongqing 400038, China ∥ State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau China ‡

S Supporting Information *

ABSTRACT: To develop reactive oxygen species (ROS)responsive anti-inflammatory materials and establish their structure−property correlations, a series of H2O2-eliminating materials (OxbCDs) were designed and synthesized by conjugating different phenylboronic acid pinacol ester (PBAP) groups onto a biocompatible scaffold compound βcyclodextrin via varied linker groups. Both the H2O2-triggered hydrolysis profiles and H2O2-eliminating capacities of these materials were dependent on the chemical structure of the PBAP moieties. Together with the elucidation of hydrolysis mechanisms, we established structure−property correlations of these OxbCD materials. Extensive in vitro experiments revealed nanoparticles (NPs) based on OxbCDs showed no adverse biological effects on normal cells. OxbCD NPs could effectively inhibit inflammatory responses and oxidative stress in stimulated macrophages. Consistently, OxbCD NPs efficaciously alleviated the symptoms of peritonitis in mice, with respect to reducing the counts of neutrophils and macrophages as well as inhibiting the secretion of pro-inflammatory cytokines, chemokines, and oxidative mediators. Similarly, OxbCD NPs loaded with anti-inflammatory drugs displayed superior efficacy in an acute inflammation model of peritonitis in mice. More importantly, OxbCD NPs showed good biocompatibility after administration via different routes. Consequently, besides serving as anti-inflammatory materials, the newly developed H2O2eliminating materials may be utilized as pharmacologically functional carriers for targeted therapy of many diseases associated with inflammation and oxidative stress. response to xenobiotics, cytokines, and bacterial invasion.13,14 Whereas normal levels of ROS are necessary for the regulation of oxygen hemostasis and cellular signaling,15 pathologically overproduced ROS may positively contribute to the development of different diseases, resulting from oxidation of biomolecules and exacerbation of oxidative injuries in different tissues, as well as amplification of inflammatory and immune responses.16−18 According to aberrantly increased ROS at diseased sites, a large number of ROS-triggerable compounds, materials, and drug delivery systems have been developed for imaging, diagnosis, and/or therapy of ROS-associated diseases.19−26 In

1. INTRODUCTION The rational design and development of responsive materials, according to abnormally enhanced molecular signals at diseased sites, has been an intensive focus of recent research in multidisciplinary fields of organic/polymer chemistry, materials science, pharmaceutics, and biomedical engineering.1−6 Among the diverse array of pathologically relevant biochemical signals in the intracellular and extracellular microenvironments, reactive oxygen species (ROS) have received much attention due to their important roles in the initiation and progression of numerous diseases including inflammatory disorders,7 infectious diseases,8 cardiovascular and neurodegenerative diseases,9,10 diabetes,11 and cancer.12 ROS, mainly containing hydrogen peroxide (H2O2), superoxide anion, and hydroxyl radical, are generated in a wide range of physiological processes, such as mitochondrial oxidative metabolism and cellular © 2017 American Chemical Society

Received: June 12, 2017 Revised: September 13, 2017 Published: September 13, 2017 8221

DOI: 10.1021/acs.chemmater.7b02412 Chem. Mater. 2017, 29, 8221−8238

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Chemistry of Materials

Figure 1. Design and synthetic routes of different H2O2-scavenging materials derived from β-CD as well as engineering of anti-inflammatory nanoparticles. (a) Schematic of different materials and nanoparticles. (b) Synthesis of OxbCD-1 and OxbCD-4. (c) Synthesis of OxbCD-2 and OxbCD-3. (d) Synthesis of OxbCD-5. BTC, bis(trichloromethyl) carbonate; DCM, dichloromethane; DMF, N,N-dimethylformamide; TEA, triethylamine; RT, room temperature; CDI, N,N′-carbonyldiimidazole.

inorganic−organic composite materials to synthesize ROSresponsive entities. Taking advantage of these ROS-sensitive materials or delivery vehicles, different imaging agents or therapeutics can be site-specifically delivered to target cells, tissues, or organs for detection or therapy applications. Of note,

this context, various ROS-labile moieties such as propylene sulfide,27,28 thioether ketal,29 thioketal,30−32 selenium- or tellurium-containing groups,33,34 peroxalates,35,36 oligoproline peptide,37 boronic acid esters,38−40 and Si−C covalent bonds41 are generally introduced in small molecules, polymers, or 8222

DOI: 10.1021/acs.chemmater.7b02412 Chem. Mater. 2017, 29, 8221−8238

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Chemistry of Materials

minopyridine (DMAP), phorbol 12-myristate 13-acetate (PMA), 2′,7′dichlorofluorescin diacetate (DCFH-DA), 3-amino,4-aminomethyl2′,7′-difluorescein diacetate (DAF-FM DA), and zymosan (from yeast cell) were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Anhydrous dichloromethane (DCM), anhydrous N,Ndimethylformamide (DMF), benzyl alcohol, and benzylamine were purchased from J&K (Beijing, China). Bis(trichloromethyl) carbonate (BTC), triethylamine (TEA), and lecithin were purchased from TCI (Tokyo, Japan). β-Cyclodextrin (β-CD) was supplied by Zhiyuan Biotechnology Co., Ltd. (Shandong Binzhou, China). 1,2-Distearoylsn-glycero-3-phosphoethanolamine-N-[(carbonyl-methoxy poly(ethylene glycol)-2000) (DSPE-PEG) was obtained from Avanti Polar Lipids Inc. (U.S.A.). Poly(lactide-co-glycolide) (PLGA, 75:25, with intrinsic viscosity of 0.50−0.65) was purchased from Polysciences Inc. (U.S.A.). RPMI-1640 medium, DMEM medium, fetal bovine serum (FBS), and trypsin were purchased from Gibco (Thermo Fisher Scientific, U.S.A.). TMRE-Mitochondrial Membrane Potential Assay Kit was obtained from Abcam (U.S.A.). Polycarbonate transwell inserts with 8-μm pore were purchased from Corning (U.S.A.). Dexamethasone (DXM) and indomethacin (IND) were supplied by MedChemExpress (MCE, U.S.A.). The APC-labeled antimouse Ly6G antibody and PE-labeled antimouse CD11b antibody were purchased from eBioscience (U.S.A.). FITC-labeled antimouse F4/80 antibody and FITC Annexin V Apoptosis Detection Kit with 7-AAD were purchased from Biolegend (U.S.A.). All ELISA kits were obtained from Neobioscience (Shenzhen, China), while kits for molecules related to oxidative stress were purchased from BioAssay Systems (U.S.A.). Reagents for Western blot analysis and dihydroethidium were supplied by Beyotime Biotechnology (China). All the other reagents are commercially available and were used as received. 2.2. Synthesis and Characterization of ROS-Responsive Materials. Three reaction routes were followed to synthesize various ROS-responsive materials based on β-CD (OxbCD, Figure 1b−d). OxbCD-1 and OxbCD-4 were obtained by Route 1, while Route 2 was adopted for the synthesis of OxbCD-2 and OxbCD-3. Meanwhile, OxbCD-5 was synthesized by Route 3. Route 1 (Figure 1b). Under nitrogen atmosphere, Compound 1 (0.338 g, 1.54 mmol) or Compound 4 (0.337 g, 1.54 mmol) and DMAP (0.564 g, 4.62 mmol) were dissolved in anhydrous DCM. Then BTC (0.457 g, 1.54 mmol) in DCM was added dropwise into the obtained solution. The resulting mixture was reacted for 3 h under magnetic stirring. The activated Compound 1 (BTC-1) or Compound 4 (BTC-4) was collected after the reaction mixture was filtered and concentrated in vacuum. Subsequently, β-CD (0.25 g, 0.22 mmol) and BTC-1 or BTC-4 were dissolved in anhydrous DMF, into which 0.25 mL of TEA was added. The mixture thus obtained was magnetically stirred overnight at −10 °C (OxbCD-1) or at room temperature (OxbCD-4). The final product OxbCD-1 or OxbCD-4 was obtained by precipitation from anhydrous ether and centrifugation at 5752g for 5 min. After thorough rinsing with anhydrous ether, the sample was dried to give light yellow powders. Route 2 (Figure 1c). Compound 2 (1.10 g, 4.68 mmol) or Compound 3 (1.22 g, 4.68 mmol) and CDI (1.51 g, 9.36 mmol) were dissolved in anhydrous DCM. The solution was magnetically stirred at room temperature for 60 min. Then 20 mL of DCM was added into the mixture, followed by washing with 30 mL of deionized water three times. The organic phase was further washed with saturated NaCl solution, dried over Na2SO4, and concentrated in vacuum to obtain CDI-activated Compound 2 (CDI-2) or Compound 3 (CDI-3). Subsequently, OxbCD-2 or OxbCD-3 was synthesized by conjugating CDI-2 or CDI-3 with β-CD, respectively. Specifically, under the protection of N2, β-CD (0.25 g, 0.22 mmol), TEA (0.25 mL, 1.80 mmol), and CDI-2 (0.50 g, 1.54 mmol) or CDI-3 (0.55 g, 1.54 mmol) were dissolved in anhydrous DMF. The mixture thus obtained was magnetically stirred overnight at room temperature. The final material OxbCD-2 was harvested by the above-mentioned method. To collect OxbCD-3, the final reaction solution was placed into dialysis tubing (with MWCO 1000 Da) for dialysis against deionized water at 25 °C. The outer aqueous solution was exchanged every 2 h. After 24 h of dialysis, the dialysate was lyophilized to give a white powder.

aryl boronic esters, with either ester or ether linkages, have been comprehensively investigated as ROS-labile groups.38−40,42−44 Compared to other ROS-responsive units, aryl boronic esters are selectively responsive to H2O2,38,45−48 showing superior in vitro degradation kinetics under physiologically relevant H2O2 levels. More importantly, materials functionalized with aryl boronic esters displayed good safety profiles in both in vitro tests and in vivo evaluations in different animal models.43,49−51 Consequently, these types of materials represent promising carriers to develop ROS-responsive drug delivery systems with great potential for clinical translation. Nevertheless, for different aryl boronic esters, particularly their linker groups, the structure−property correlations remain to be elucidated, with respect to sensitivity, hydrolysis kinetics, and in vitro/in vivo biocompatibility. On the other hand, as an important component of ROS, H2O2 is a strong oxidant from the decomposition of superoxide anion by superoxide dismutase.13 Overgenerated H2O2 actively participates in the pathogenesis of acute and chronic inflammation. Although it has been well documented that a stoichiometric amount of H2O2 will be consumed upon ROS-triggered hydrolysis of boronic esters,19,38,39,52 whether this capacity may afford antioxidant and anti-inflammatory activities is still an open question to be answered. Furthermore, both clinical studies and experiments in different animal models have demonstrated that small molecular compounds and polymeric micelles with the free radical-scavenging capability may function as effective therapeutics for the treatment of oxidative stress injuries.25,53,54 Based on the above issues, the first aim of this study was to establish structure−property correlations of aryl boronic esters, in order to develop ROS-responsive materials with wellcontrolled sensitivity, delicately tailored hydrolysis kinetics, and good biocompatibility. In addition, we attempt to demonstrate our hypothesis that H2O2-eliminating nanoparticles (NPs) may function as anti-oxidant and anti-inflammatory nanotherapies by efficiently consuming abnormally increased H2O2 in stimulated cells or diseased tissues. To this end, β-cyclodextrin (β-CD) was selected as a scaffold compound, in view of its low toxicity and low immunogenicity as well as its multiple hydroxyl groups that can be easily functionalized (Figure 1a).55−57 On the other hand, phenylboronic acid pinacol esters (PBAPs) with different linker groups were chosen as H2O2-labile model entities to synthesize ROS-responsive materials by covalently conjugating PBAPs onto β-CD. Besides studies on materials synthesis as well as hydrolysis kinetics and mechanisms, the H2O2-scavenging capability of various materials and NPs was interrogated. Moreover, in vitro cell culture experiments and in vivo evaluations were performed to examine anti-oxidant and anti-inflammatory activities of newly engineered H2O2-eliminating NPs. Also, we explored the potential of these new materials as pharmacologically active carriers for drug delivery applications. Finally, in vitro cytocompatibility and in vivo biocompatibility of these responsive materials derived NPs were separately studied in cells as well as in mice or rats after administration via different routes.

2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. 4-Hydroxylphenylboronic acid pinacol ester (Compound 1), 4-(hydroxymethyl)phenylboronic acid pinacol ester (Compound 2), 4-(carboxymethyl)phenylboronic pinacol ester (Compound 3), 4-aminophenylboronic acid pinacol ester (Compound 4), 4-(aminomethyl)phenylboronic acid pinacol ester (Compound 5), N,N′-carbonyldiimidazole (CDI), 4-dimethyla8223

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Chemistry of Materials Route 3 (Figure 1d). β-CD (0.25 g, 0.22 mmol) and CDI (0.249 g, 1.54 mmol) were dissolved in anhydrous DMF. The reaction was magnetically stirred at room temperature. After 90 min, the reaction mixture was added to anhydrous ether. Then the solution was filtered and thoroughly rinsed with anhydrous ether to obtain CDI-activated β-CD (CDI-bCD). Subsequently, Compound 5 (0.359 g, 1.54 mmol) and CDI-bCD were dissolved in anhydrous DMF, followed by addition of 0.25 mL of TEA. The mixture thus obtained was magnetically stirred overnight. The final product OxbCD-5 was precipitated from anhydrous ether and then collected by centrifugation at 5752g for 5 min. After thorough rinsing with anhydrous ether, the sample was dried to give a white powder. 2.3. Synthesis of Non-Responsive Control Materials. β-CD (0.25 g, 0.22 mmol) and CDI (0.249 g, 1.54 mmol) were dissolved in DMF. The reaction was magnetically stirred at room temperature. After 90 min, the reaction mixture was added to ether. Then the solution was filtered and thoroughly rinsed with ether to obtain CDIbCD. Subsequently, CDI-bCD and an excessive amount of benzyl alcohol or benzylamine were dissolved in DMF, followed by the addition of 0.25 mL TEA. The mixture thus obtained was magnetically stirred overnight. The final products of nonresponsive materials bCD2 and bCD-5 were precipitated from ether, and collected by centrifugation at 5752g for 5 min. After thorough rinsing with ether, the samples were dried to give white powders. 2.4. Characterization of Materials. 1H NMR spectra were acquired using an Agilent DD2 600 MHz NMR spectrometer. Fourier transform infrared spectroscopy (FT-IR) spectra were recorded on a PerkinElmer spectrometer (100S, U.S.A.). Thermogravimetric analysis (TGA) was run on a TGAQ50 instrument (TA Instruments). Matrix assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was performed on a MALDI-7090 TOF-TOF mass spectrometer (Shimadzu). Electrospray ionization mass spectrometry (ESI MS) was conducted using an ESI-Triple Quad mass spectrometer (Bruker). 2.5. Fabrication and Characterization of Nanoparticles by a Nanoprecipitation/Self-Assembly Method. All NPs based on different OxbCDs were prepared by a modified nanoprecipitation/selfassembly method.58 Briefly, 50 mg of carrier material (either OxbCD or PLGA) was dissolved in 2 mL of methanol. The obtained solution was added dropwise into 10 mL of deionized water containing 6 mg of DSPE-PEG and 4 mg of lecithin that was preheated at 65 °C. The suspension was stirred for 2 h at room temperature. Then the remaining organic solvent and water were removed by vacuum evaporation at 60 °C and concentrated to a volume of 8 mL. Finally, OxbCD NPs were obtained by freeze-drying. NPs containing different drugs were fabricated via a similar method. In this case, however, the residual free drug was removed by dialysis (MWCO: 3500 Da) against deionized water for 24 h, and drug-loaded NPs were collected after lyophilization. Dynamic light scattering and ξ-potential measurements were performed on a Malvern Zetasizer Nano ZS instrument at 25 °C. Transmission electron microscopy (TEM) observation was carried out using a JEM-1400 microscope (JEOL, Japan). Differential scanning calorimetry (DSC) was conducted on a DSC Q2000 instrument (TA, U.S.A.). 2.6. In Vitro Evaluation of the H2O2 Sensitivity of Different Materials or Nanoparticles. To examine the sensitivity of OxbCD NPs to different species of ROS, various ROS generators were prepared according to previously reported methods.59 Briefly, 50 μM H2O2 and 50 μM ferrous perchlorate were mixed in order to generate hydroxyl radicals (•OH). On the other hand, 50 μM H2O2 and 50 μM NaOCl were used to produce hypochlorite (OCl−), while peroxynitrite (ONOO−) was yielded by 50 μM H2O2 and 50 μM nitrite. To study the H2O2 sensitivity, OxbCD NPs at 1 mg/mL were incubated in 4 mL of PBS (pH 7.4) containing different species of ROS for 48 h. The hydrolysis degree was subsequently measured by UV−vis spectroscopy at 500 nm. 2.7. In Vitro H2O2-Eliminating Capability of Different Materials or Nanoparticles. Various concentrations of OxbCDs or OxbCD NPs were incubated in 4 mL of PBS containing 1.0 mM

H2O2 at 37 °C for 24 h. Nonresponsive materials bCD-2 and bCD-5 were used as negative controls. Using the QuantiChrom Peroxide Assay Kit (BioAssay Systems, U.S.A.), the concentration of remaining H2O2 was quantified by measuring the absorbance at 585 nm, and the H2O2-eliminating capacity was calculated. 2.8. In Vitro Hydrolysis of OxbCD NPs in Various Solutions. The hydrolysis of OxbCD NPs (1.0 mg/mL) was performed in 4 mL of PBS buffers (0.01 M, pH 7.4) containing various concentrations of H2O2 at 37 °C. Quantitative experiments were conducted by measuring the absorbance of NP-containing aqueous solution at 500 nm at various time points. Similarly, hydrolysis of OxbCD NPs in buffers with 1.0 mM H2O2 at different pH values of pH 1.2, 4.8, 7.4, 9, and 11 was also examined. In another set of in vitro hydrolysis tests, the hydrolysis solution was switched into the culture medium. In this context, 4 mL of culture medium was collected after mouse macrophage RAW264.7 cells were exposed to various stimulators. Subsequently, 4 mg of NPs was incubated with these different solutions at 37 °C for 48 h. The hydrolysis degree was calculated by measuring absorbance of NPcontaining solution at 500 nm. 2.9. In Vitro Drug Release Tests. Typically, 10 mg of newly prepared NPs containing either DXM or IND in 100 μL of deionized water was added into dialysis tubing (MWCO: 3500 Da), which was immersed into 40 mL of PBS or PBS containing 1.0 mM H2O2. At appropriate intervals, 4.0 mL of the supernatant was withdrawn, and the absorbance at 240 or 320 nm was measured by a UV−vis spectrophotometer (TU-1901, PERSEE Co., Ltd., Beijing) to quantify the DXM or IND concentrations, respectively. 2.10. Cytotoxicity Evaluation by MTT Assay. RAW264.7 cells and mouse aortic vascular smooth muscle cells (MOVAS-1) were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin in a 5% CO2 humidified environment at 37 °C. For the methyl thiazolyl tetrazolium (MTT) assay, cells were planted at 1 × 104 cells/well in 96-well plates for 24 h before different NPs were added. Subsequently, cells were treated with the medium containing NPs at various concentrations for 12 or 24 h. The cell viability was quantified by the MTT assay. 2.11. Measurement of Mitochondrial Membrane Potential. Tetramethylrhodamine (TMRE) was used to analyze mitochondrial membrane potential (ΔΨm). To this end, RAW264.7 cells were seeded into 6-well plates and treated with various NPs at 100 μg/mL) or at H2O2 100 or 200 μM for 24 h. Then, cells were trypsinized, washed, and incubated with 100 nM of TMRE in PBS at 37 °C for 30 min. The incubated cells were washed twice with PBS and subsequently assayed by flow cytometry. 2.12. Determination of Intracellular and Extracellular H2O2 Generation in RAW264.7 Cells. RAW264.7 cells (2 × 105 cells/ well) were cultured on glass coverslips in 12-well plates with growth culture medium (RPMI-1640, 10 wt % FBS, 1 wt % penicillin− streptomycin solution) for 12 h. Then fresh medium was changed and cultured in the presence of 100 ng/mL PMA for 1 h or 100 μg/mL NPs for 6 h. Subsequently, cells were washed thrice with Hank’s balanced salt solution (HBSS) and treated with 10 μM DCFH-DA in the dark at 37 °C for 40 min. Cells were washed thrice with HBSS and fixed by 4 wt % paraformaldehyde for 30 min at room temperature. The coverslips were fixed and mounted, and fluorescence microscopy images were acquired (BX51TRF, Olympus). Quantitative analyses were conducted by flow cytometry assay. To this end, RAW264.7 cells were cocultured with PMA or NPs in 12-well plates as aforementioned. Cells were then treated with 10 μM DCFHDA in the dark at 37 °C for 40 min. After the collected cells were washed thrice by HBSS, fluorescent intensities were measured by a laser flow cytometer (Accuri C6, BD). To determine extracellular H2O2, RAW264.7 cells (1 × 106 cells/ well) were seeded in 6-well plates. After incubation with 100 ng/mL PMA for 2 h or 100 μg/mL NPs for 6 h, the culture medium was collected. The concentration of H2O2 in the medium was measured by the QuantiChrom Peroxide Assay Kit. 2.13. Quantification of Inflammatory Cytokines and Chemokines in RAW264.7 Cells. RAW264.7 cells were seeded into 6-well 8224

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Chemistry of Materials plates (1 × 106 cells/well). Cells were treated with 100 ng/mL PMA for 1 h or 100 μg/mL NPs for 6 h. Cells in the control group were cultured with growth medium, while cells in the positive group were treated by 20 or 40 μM H2O2 for 6 h. Other groups were treated with 40 μM H2O2 and 100 μg/mL NPs for 6 h. Then the culture media were collected, and the levels of inflammatory cytokines and chemokines including tumor necrosis factor-α (TNF-α), interleukin1β (IL-1β), interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), and interleukin-8 (IL-8) were determined by commercial mouse ELISA kits (Neobioscience) according to the manufacturer’s instructions. On the other hand, cells were washed three times with cold HBSS and lysed in the ice-cold RIPA buffer containing 1.0 mM phenylmethylsulfonyl fluoride. Cellular lysates were centrifuged at 14 000g at 4 °C for 10 min to collect the supernatant. After quantification of total proteins by BCA protein assay (Beyotime Biotechnology, China), proteins were separated by 8 wt % SDS-polyacrylamide gel electrophoresis. The separated proteins were then transferred from the gel to polyvinylidene fluoride membranes, which were blocked for 2 h in TBS-T containing 5% w/v nonfat dry milk. Subsequently, each membrane was incubated with related antibodies at 4 °C overnight, followed by incubation with appropriate horseradish peroxidaselabeled antibodies. Finally, the proteins were detected by enhanced chemiluminescence using the ECL Western blot kit. 2.14. Determination of NO and NO Synthase in RAW264.7 Cells. For determination of NO generation, RAW264.7 cells were seeded into 6-well plates at 1 × 106 cells/well with DMEM growth medium without nitrate. Cells in the positive control group were treated by medium containing 20 or 40 μM H2O2 for 6 h, while the control group was treated with the same volume of medium. Other groups were treated by 40 μM H2O2 and 100 μg/mL NPs for 6 h. Then cells were washed thrice with HBSS and treated with 5 μM DAF-FM DA in the dark at 37 °C for 30 min. Prior to measurements, cells were washed thrice with HBSS. Observation by fluorescence microscopy and quantification by flow cytometry were conducted as mentioned above. To detect the level of NO synthase (NOS), RAW264.7 cells were seeded into 12-well plates at 1 × 106 cells/well. Cells were stimulated with different mediators as mentioned above. According to the manufacturer’s instructions of Nitric Oxide Synthase Assay Kit (Beyotime, China), DAF-FM DA was utilized to detect intracellular NO generated by NOS under adequate substrates of L-arginine and NADPH. After washing thrice with HBSS, flow cytometry analysis was conducted. The relative activity of NOS can be calculated according to the fluorescent intensity of DAF-FM DA. 2.15. Animals. All the animal care and experimental protocols were performed with review and approval by the Animal Ethical and Experimental Committee of the Third Military Medical University. Sprague−Dawley rats (200−220 g) and BALB/c mice (18−20 g) were obtained from the Animal Center at the Third Military Medical University. Animals were housed in standard mouse cages under conditions of optimum light (12:12 h light−dark cycle), temperature (22 ± 1 °C), and humidity (50−60%), with ad libitum access to water and food. Before further experiments were performed, all mice were acclimatized for at least 7 days. 2.16. Isolation of Mouse Peritoneal Neutrophils. To induce the production of neutrophils, 1 mL of aqueous solution of thioglycollate (3 wt %) was intraperitoneally (i.p.) administrated to BALB/c mice. After 4 h, mice were euthanized, and 5 mL of sterile HBSS was injected into the peritoneal cavity. Subsequently, 4 mL of peritoneal exudates containing neutrophils (>90%) was collected, and the suspension was centrifuged at 400g for 10 min. After resuspension in HBSS, total cell counts of neutrophils were determined with a hemocytometer. 2.17. In Vitro Migration of RAW264.7 Cells. A transwell assay was performed to investigate whether OxbCD NPs may cause migration of RAW264.7 cells. Specifically, cells were seeded in 24-well plates at 2 × 105 cells/well and allowed to adhere overnight. Cells were treated with various NPs at 100 μg/mL for 6 h. Then each well was washed, trypsinized, resuspended in 0.5 mL of RPMI-1640 medium,

and plated onto 8-μm pore polycarbonate inserts in a 24-well plate with 1.5 mL of medium. In the positive control group, cells were stimulated with either 10 nM MCP-1 or 40 μM H2O2 alone, while the negative control group was treated with saline. Cells were allowed to migrate for 12 h, and cells on the upper side of each insert were gently removed with a cotton swab. Subsequently, cells on the lower side of each insert were fixed and stained with 0.1 wt % crystal violet. After staining, 5 images of migrated cells per high-power filed (HPF, 10 × 10) were recorded and counted by optical microscopy. Also, the similar transwell assay was performed to study the antimigration activity of OxbCD NPs in RAW264.7 cells induced by H2O2. Specifically, cells were seeded in 24-well plates at 2 × 105 cells/well and allowed to adhere overnight. In this case, no NPs or H2O2 was added in the normal control group, while cells were stimulated with 40 μM H2O2 alone in the positive control group. An additional transwell assay was conducted to examine antimigration activity of OxbCD NPs in RAW264.7 cells induced by activated-neutrophils. In this case, RAW264.7 cells were seeded in 24well plates at 5 × 105 cells/well. To this end, activated neutrophils (2 × 104 cells) and 100 μg/mL of OxbCD NPs or 100 U/mL catalase were added into the lower compartment of the chamber. The normal control group was treated with fresh medium, while cells were stimulated with activated neutrophils alone in the positive control group. Subsequently, similar procedures as aforementioned were followed. 2.18. In Vitro Anti-Apoptotic Activity of OxbCD NPs in RAW264.7 Cells. Apoptosis analysis was conducted using FITC Annexin V Apoptosis Detection Kit with 7-AAD according to the manufacture’s protocol. Specifically, RAW264.7 cells were seeded in a 6-well plate at 4 × 105 cells/well and incubated overnight. The medium was then replaced with fresh growth medium containing 200 U/mL catalase or various NPs. After 2 h of incubation, cells were treated with 200 μM H2O2 for 8 h. Then, cells were washed with cold BioLegend’s cell staining buffer, digested with 0.25 wt % trypsin, and collected by centrifugation. After the cells were resuspended in 100 μL of Annexin V binding buffer with 2.5 μL of Annexin V and 5 μL of 7AAD viability staining solution at 1 × 105 cells/mL, they were vortexed gently and incubated in a dark room for 15 min. Finally, 400 μL of Annexin V binding buffer was added for analysis by flow cytometry (Accuri C6, BD). 2.19. In Vivo Irritation Effects of OxbCD NPs. Sprague−Dawley rats were used to examine possible irritation effects of NPs after intramuscular (i.m.) injection. For this purpose, 1.0 mL of aqueous solution containing OxbCD-2 NP, OxbCD-5 NP, or PLGA NP at 25 mg/mL was injected into the quadriceps femoris muscle of rats, while saline was injected in the control group. At day 7 post injection, rats were euthanized. Tissues at the injection sites were harvested and fixed in formalin, and pathological sections were prepared and stained with hematoxylin-eosin (H&E). In addition, the injected tissues were fixed in O.C.T immediately, and 7-μm cryosections were made and stained with dihydroethidium (DHE, 5 μM) that emit red fluorescence after oxidation by superoxide anion. Observation by fluorescence microscopy was then performed. In addition, BALB/c mice were used to evaluate the level of local oxidative stress after subcutaneous or i.p. injection of OxbCD NPs. Briefly, 100 μL of PBS (pH 7.4) containing 1 mg/mL of zymosan or 100 μg/mL of NPs was subcutaneously injected into the backs of mice. After 24 h, mice were anesthetized by isoflurane inhalation and then injected with 100 μL of saline containing 0.4 mg of luminol-Na. On the other hand, mice were i.p. injected with 1 mL of saline containing 1.0 mg of zymosan and 100 μg of NPs. After 6 h of stimulation, 500 μL of saline containing 0.8 mg/mL luminol-Na was i.p. administered. Animals were imaged using a living imaging system (IVIS Spectrum, PerkinElmer, U.S.A.) with exposure time of 5 min, and images were analyzed using Living Image 4.4 Software. 2.20. Hemolysis Tests. Blood samples were collected from female Sprague−Dawley rats, from which 2% erythrocytes suspension in saline was prepared. The erythrocytes suspension was then mixed with various NPs solutions at 2.0 mg/mL (total 2 mL). In the positive control group, deionized water was added, while saline solution was 8225

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Chemistry of Materials added in the negative control group. The mixed suspensions were incubated at 37 °C. One hour after incubation, all samples were centrifuged at 734g for 5 min, and the optical density (OD) of the supernatant was measured at 545 nm. The hemolytic degree was then calculated. 2.21. Acute Toxicity Evaluation. Male BALB/c mice were randomly divided into five groups (n = 10). OxbCD-2 NP or OxbCD5 NP in saline was intravenously (i.v.) administered via tail vein injection at doses of 500 or 1000 mg/kg. In the control group, mice were injected with 100 μL of saline solution. After administration, mice were weighed at specific time points, and their behaviors were monitored for any signs of illness each day. After 2 weeks, animals were euthanized. Blood samples were collected for hematological analysis (Sysmex KX-21, Sysmex Co., Japan) and quantification of biomarker molecules relevant to hepatic/kidney functions. Major organs including heart, liver, spleen, lung, and kidneys were harvested and weighed. The organ index was calculated. In addition, histopathological sections of the collected organs were prepared and stained with H&E. 2.22. Treatment of Peritonitis by OxbCD NPs in Mice. Male BALB/c mice were randomly allocated into 4 groups. The normal control group was treated by i.p. injection of 0.5 mL of saline. Mice in the model or zymosan group were i.p. injected with 1 mL of zymosan suspension in saline (1 mg/mL), and this was followed by i.p. administration of 0.5 mL of saline at 1 h after zymosan injection. For the positive control group, mice were i.p. injected with 0.5 mL of 200 U/mL catalase in PBS at 1 h after zymosan administration. In the case of the NPs group, 1 h after zymosan administration, mice were i.p. injected with 0.5 mL of saline containing OxbCD-2 NP, OxbCD-5 NP, or PLGA NP at 11 mg/kg. At 6 h after zymosan administration, mice were euthanized and i.p. injected with 4.5 mL of cold PBS. After gently shaking, peritoneal exudates were collected for the assay of inflammatory cells and cytokines. Specifically, 3 mL of peritoneal exudates were incubated with 2 mL of Red Blood Cell Lysis Buffer at 37 °C for 10 min. Then, 2 mL of RPMI-1640 containing 10 wt % FBS was added. Cells were collected by centrifugation at 400g for 10 min, washed 3 times with PBS, and resuspended in 100 μL of cold PBS. For each sample, 0.5 μL of antimouse FITC-F4/80 antibody, 0.625 μL of antimouse APCLy6G antibody, and 0.625 μL of antimouse PE-CD11b were added. After incubation in the dark at room temperature for 25 min, cells were washed 3 times with PBS. Subsequently, isolated cells were resuspended in 500 μL of PBS. Flow cytometric analysis was performed on an Accuri C6 flow cytometer, and data were analyzed using the FlowJo v10 software. The levels of TNF-α, IL-6, myeloperoxidase (MPO), IL-8, MCP-1, superoxide anion, and NO in peritoneal exudates were quantified using commercially available kits. In brief, peritoneal exudates were collected and incubated with Triton X-100 for 2 min. Then 100 μL of peritoneal exudates was added into 96-well plates with precoated antibodies, and the manufacturer’s instructions were followed for quantification. In addition, luminescence imaging was performed to evaluate the degree of inflammation. At 6 h after administration, mice were i.p. injected with 0.5 mL of saline containing 0.8 mg/mL of luminol-Na. Immediately, bioluminescence imaging was performed by a living imaging system as aforementioned. 2.23. Therapeutic Effects of Drug-Loaded NPs on Peritonitis in Mice. Male BALB/c mice were randomly assigned into 4 groups. The control group was treated with saline, while mice in the zymosan group were i.p. injected with 1 mL of saline containing 1 mg/mL zymosan and 0.5 mL of saline 1 h after zymosan administration. For the positive control group, mice were i.p. injected with 0.5 mL of saline containing DXM or IND at 11 mg/kg. For the drug-containing NPs group, mice were i.p. injected with 0.5 mL of saline containing DXMor IND-loaded NPs at 11 mg/kg, at 1 h after administration of zymosan. Subsequently, the aforementioned procedures were followed to examine therapeutic benefits of different formulations. 2.24. Statistical Analysis. Data are expressed as mean ± SEM. Statistical analysis was assessed using the One-way ANOVA test. A value of p < 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of OxbCDs. Since a stoichiometric amount of H2O2 may be consumed upon hydrolysis of PBAP, PBAPs were employed as H2O2-responsive moieties to synthesize ROS-responsive anti-inflammatory materials (OxbCDs) based on β-CD, a cyclic oligosaccharide with good in vitro and in vivo safety profiles.55 To establish the structure−property relationship of OxbCDs, a series of materials including OxbCD-1, OxbCD-2, OxbCD-3, OxbCD4, and OxbCD-5 were synthesized via the functionalization of β-CD with PBAPs containing different linking groups. Three different routes were chosen according to functional groups of different PBAPs (Figure 1b−d). The obtained products were first characterized by FT-IR spectroscopy. The presence of characteristic absorption bands corresponding to varied PBAPs in resulting materials indicated their successful conjugation onto β-CD (Figure S1). For OxbCD-1 and OxbCD-2, the newly generated linker between PBAP and β-CD is a carbonate ester, while it is an ester in OxbCD-3. In the case of OxbCD-4 and OxbCD-5, the linking bond is carbamate. This was further affirmed via 1H NMR spectroscopy (Figure S2a−e). Calculations based on the intensity ratio of proton signals due to functional groups and the parent compound β-CD suggested that approximately 4−5 PBAP units were conjugated. These results are consistent with MALDI-TOF MS spectra that showed peaks corresponding to β-CD derivatives conjugated with varied numbers of PBAP groups (Figure S3). Accordingly, different OxbCDs can be successfully and facilely synthesized by our established methods. Compared with previously reported ROS-responsive compounds or polymers (Table S1), the yields of various OxbCDs are relatively high (Figure 1). In addition, four materials with different linking groups were synthesized in the current study. Moreover, by changing the feeding ratio of PBAP to β-CD, OxbCDs with varied numbers of responsive groups can be conveniently synthesized. For instance, OxbCD-2 and OxbCD-5 with about 1, 3, 5, and 7 PBAP units were obtained, giving rise to OxbCD-2O/OxbCD-5O, OxbCD-2T/OxbCD5T, OxbCD-2F/OxbCD-5F, and OxbCD-2S/OxbCD-5S, respectively. Their structures were verified by 1H NMR and MALDI-TOF MS (Figure S4−5). Different from parent β-CD, these OxbCD materials can be dissolved in common organic solvents such as methanol, ethanol, acetonitrile, DMSO, and DMF. Also, they are slightly soluble in THF and acetone. TGA measurement revealed different thermal stabilities for different materials (Figure S6). While OxbCD-2, OxbCD-3, and OxbCD-5 showed TGA curves comparable to β-CD itself, the stability of OxbCD-1 and OxbCD-4 was decreased to a certain degree. This difference in stability is closely related to the chemical structure of the linker between PBAP and β-CD. To further demonstrate that the ROS sensitivity is due to phenylboronic pinacol ester, two control materials bCD-2 and bCD-5 were also synthesized and characterized by 1H NMR and MALDI-TOF MS, spectroscopy (Figure S7a−d), corresponding to OxbCD-2 and OxbCD-5, respectively. 3.2. Hydrolysis and the H2O2-Scavenging Capability of Different OxbCDs. In the presence of H2O2, all OxbCDs can be completely hydrolyzed as confirmed by 1H NMR spectroscopy and ESI MS (Figure S2f−j and Figure S8). For example, after OxbCD-2 was incubated with excess H2O2, characteristic proton signals corresponding to β-CD, pinacol, and 4-hydroxybenzyl alcohol were found in the 1H NMR 8226

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Figure 2. Hydrolysis mechanisms of different OxbCDs and their H2O2-eliminating capability. (a) Schematic illustration of H2O2-triggered hydrolysis mechanisms of OxbCDs and the related hydrolysis products. (b) Elimination of H2O2 in a dose-dependent manner for different OxbCDs. (c) The calculated H2O2-eliminating efficiency of OxbCDs. (d, e) The regression curves showing dose-dependent elimination of H2O2 by OxbCD-2 (d) and elimination efficiency (e) of OxbCD-2 with varied PBAP numbers. (f, g) Dose-dependent elimination of H2O2 (f) and elimination efficiency (g) for OxbCD-5 with varied PBAP numbers. Data are mean ± SEM (n = 3).

spectrum (Figure S2g). Meanwhile, the ESI MS spectrum showed the presence of the molecular ion peak of β-CD ([M]−, found: 1135 m/z; calculated: 1134.98 m/z), the quasimolecular peak of 4-hydroxybenzyl alcohol ([M − 1]−, found: 123 m/z; calculated: 123.14 m/z), and the molecular ion peak of boric acid ([M]−, found: 62 m/z; calculated: 61.83 m/z). For PBAP groups, two major bonds can be broken once encountering H2O2. One is the C−B bond, and its fracture gives rise to phenol and boronic pinacol ester. It is well-known that pinacol ester is unstable and often breaks down in oxidative environments.38−40,42−44 Another labile bond lies in the newly generated linker, which may be broken via different electron transfer mechanisms depending on the PBAP structures (Figure 2a). Typically, the carbonate ester bond in OxbCD-1

or OxbCD-2 and the carbamate bond in OxbCD-4 or OxbCD5 may be cleaved, due to the quinone rearrangement-induced electron transfer to the electrophilic carbonyl carbon, generating CO or CO2, respectively. Of note, the carbon atom in the ester bond is more difficult to induce electron transfer than that in carbonate ester or carbamate. Consequently, there is a considerable obstacle for fracture of the ester bond in OxbCD-3, thereby impairing its ROS sensitivity and hydrolysis. These results are consistent with the previous finding that, in the presence of H2O2, PBAP may be oxidized to a phenolic compound and then undergo a quinone methide rearrangement (Figure 2a).38,39,48,60 The complete hydrolysis of OxbCDs led to the release of β-CD, pinacol, boric acid, and corresponding phenolic compounds. All of these small 8227

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Figure 3. Characterization and H2O2-responsive hydrolysis profiles of OxbCD NPs. (a) TEM images of various OxbCD NPs. (b) Hydrolytic curves of OxbCD NPs in pH 7.4 PBS containing various concentrations of H2O2. (c) Hydrolysis behaviors of different OxbCD NPs in buffers at varied pH values with 1.0 mM H2O2. (d) Comparison of hydrolysis profiles of various OxbCD NPs in pH 7.4 PBS with 1.0 mM H2O2. (e−h) Hydrolysis curves of NPs based on OxbCD-2 or OxbCD-5 conjugated with different numbers of PBAP in pH 7.4 PBS with 1.0 mM or 50 μM H2O2.

revealed that less OxbCD-3 was hydrolyzed at the examined time point (24 h). Therefore, OxbCD-3 was relatively stable against H2O2-triggered hydrolysis. On the other hand, the H2O2-scavenging capability was positively correlated to the number of PBAP units conjugated on β-CD (Figure 2d−g), as clearly shown for OxbCD-2 and OxbCD-5. For OxbCD-2, the consumption efficiency was 0.7, 1.0, 1.5, and 3.0 μmol H2O2 per mg of the material with approximately 1, 3, 5, and 7 PBAP units, respectively (Figure 2e). Comparatively, OxbCD-5 with approximately 1, 3, 5, and 7 PBAP units could eliminate H2O2 at 0.7, 1.2, 1.9, and 2.7 μmol/mg (Figure 2g), respectively. In line with their hydrolysis inert properties, both bCD-2 and bCD-5 were unable to scavenge H2O2 (Figure S7h). Together, these results demonstrated that OxbCDs may effectively scavenge H2O2 upon hydrolysis, and the H2O2eliminating capability is positively correlated with the number of PBAP groups conjugated on β-CD, which can be easily tailored by modulating the feeding ratio during synthesis. 3.3. Fabrication, Characterization, and Hydrolysis of OxbCD NPs. According to our previously established nano-

molecular metabolites can be easily excreted from the kidney by glomerular filtration and active tubular secretion. By contrast, direct observation and characterization by 1H NMR spectroscopy revealed that both bCD-2 and bCD-5 cannot be hydrolyzed in the presence of H2O2 (Figure S7e−g). This suggested that phenylboronic pinacol ester is essential to offer resulting materials with the ROS sensitivity. Concomitant with hydrolysis of OxbCDs, a stoichiometric amount of H2O2 may be scavenged. Consequently, we examined the H2O2-eliminating capability of different materials. Regardless of different OxbCDs, the amount of consumed H2O2 was increased with the dose of OxbCD (Figure 2b), showing a significant linear relationship. With the exception of OxbCD-3, a comparable H2O2-eliminating capacity was found for other OxbCDs. For OxbCDs bearing 4−5 PBAP units, the H2O2-consuming capacity was approximately 1.4, 1.5, 0.9, 1.6, and 1.9 μmol/mg for OxbCD-1, OxbCD-2, OxbCD-3, OxbCD4, and OxbCD-5, respectively (Figure 2c). Since the amount of H2O2 consumed by OxbCDs is mainly determined by the content of PBAP that can directly react with H2O2, this result 8228

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Chemistry of Materials precipitation/self-assembly method,43 OxbCDs could be processed into well-defined NPs in the presence of a small amount of lecithin and DSPE-PEG (both of them have been approved for clinical applications in liposomal formulations). Lecithin was employed to introduce an amphiphilic layer surrounding the hydrophobic core, while DSPE-PEG can be effectively anchored in the outer layer, thereby providing NPs with good colloidal stability. TEM images revealed spherical core−shell NPs could be prepared based on different OxbCDs (Figure 3a and Figure S9a). Independent of varied materials, OxbCD NPs produced under the similar formulation conditions displayed comparable size, with mean size varying from 92.5 ± 4.2 to 111.4 ± 5.7 nm (Figure S9b,c). The polydispersity index of OxbCD NPs varied from 0.061 to 0.176, indicating relatively narrow size distribution. The zeta-potential values of different OxbCD NPs were negative in either 0.01 M PBS at pH 7.4 or deionized water (Figure S9d). We also examined the hydrolysis profiles of OxbCD NPs to selective oxidative species. Among different reactive oxygen or nitrogen species, only slight hydrolysis occurred in PBS alone or PBS containing hydroxyl radical (•OH), hypochlorite ion (OCl−), or peroxonitrite (ONOO−), while significant hydrolysis was observed in the existence of H2O2 (Figure S9e). These results substantiated that hydrolysis of OxbCD NPs can be selectively triggered by H2O2, which is consistent with the properties of OxbCD materials. Of note, a control PLGA NP with a comparable size showed no responsive hydrolysis under all examined conditions (Figure S9e and Figure S10). Subsequently, hydrolysis behaviors of all OxbCD NPs were quantified in PBS (0.01 M, pH 7.4) containing various concentrations of H2O2 or at different pH values. Independent of varied OxbCD NPs, their hydrolysis rate was closely related to the concentration of H2O2. For NPs based on OxbCD-1, -2, -4, or -5, rapid and effective hydrolysis was observed at 0.25, 0.5, and 1.0 mM H2O2 (Figure 3b), while only limited hydrolysis occurred at 0.01 or 0.05 mM H2O2. For OxbCD-2 NP and OxbCD-5 NP, their hydrolysis behaviors were directly illuminated by digital photos (Figure S11). The bluish colloidal solutions could be clearly observed after 5 h of incubation in PBS (pH 7.4) alone, while they became nearly clear at 2 h in PBS containing 1.0 mM H2O2. By contrast, no significant signs of hydrolysis were observed for PLGA NP incubated in either PBS or PBS with 1.0 mM H2O2. In the case of OxbCD-3 NP, slow hydrolysis and low hydrolysis degrees were observed, which should be due to the more stable ester linker in OxbCD3. Even at 1.0 mM H2O2, the hydrolysis percentage was ∼45% after 5 h of incubation, which is dramatically lower than that of other OxbCD NPs. This slow hydrolysis profile of OxbCD-3 NP may account for the low H2O2-eliminating capability of both OxbCD-3 and OxbCD-3 NP. Further experiments were conducted in PBS containing 1.0 mM H2O2 at different pH values. All OxbCD NPs displayed negligible hydrolysis at pH 1.2 and slightly higher hydrolysis degrees at pH 4.8 (Figure 3c). This acid-attenuated hydrolysis may be attributed to suppressed cleavage of pinacol ester from the phenyl group at low pH, since hydroxyl ion is generally required for this reaction.60 By contrast, the hydrolysis rates and degrees of OxbCD NPs were considerably improved in alkaline buffers. Notably, the most sensitive hydrolysis profiles were found for OxbCD NPs at pH 7.4. Likewise, poor H2O2 sensitivity was found for OxbCD-3 NP at different pH values. These results revealed that a low pH may inhibit hydrolysis of OxbCD NPs, regardless of the presence of H2O2.

When the hydrolyses of different OxbCD NPs were compared in the same cohort of tests, we found OxbCD-2 and OxbCD-5 NPs were more sensitive to H2O2 (Figure 3d). Additionally, the H2O2-sensitivity of these NPs was intimately associated with the number of PBAP units conjugated on β-CD (Figure 3e,f), with significantly enhanced hydrolysis for NPs prepared with OxbCD-2 and OxbCD-5 bearing less PBAP. Even at 50 μM H2O2, we observed efficient hydrolysis of NPs derived from OxbCD-2T or OxbCD-5T containing about 3 PBAP units (Figure 3g,h). Consequently, besides tailoring the chemical structure of the linking group in OxbCDs, the H2O2 sensitivity of their NPs can also be effectively modulated by adjusting the number of conjugated PBAP units. Consistent with their H2O2-triggered hydrolysis profiles, OxbCD NPs can quantitatively eliminate H2O2 in a dosedependent manner (Figure S9f). Among different OxbCD NPs, OxbCD-3 NP exhibited the lowest H2O2-eliminating efficiency (Figure S9g), while comparable performance was observed for other NPs. This is in line with the characters of different OxbCDs (Figure 2b,c). Accordingly, this finding demonstrated that OxbCD NPs are able to mimick the H2O2-scavenging capability of catalase, a natural anti-oxidant enzyme within the body.61 Additionally, these OxbCD NPs may be used as passive targeting nanocarriers for the treatment of inflammation-related diseases, in view of their capability of triggerable hydrolysis under ROS-enriched environments. 3.4. In Vitro Biological Effects of OxbCD NPs in Different Cells. The safety of nanocarriers and nanotherapies is one of the most important issues influencing their clinical applications. Initially, we examined the in vitro cytotoxicity of OxbCD NPs in RAW264.7 and MOVAS-1 cells. After 12 h of incubation, the viability of RAW264.7 cells was gradually decreased with the increased dose of OxbCD NPs (Figure S12a). Nevertheless, a relatively high cell viability of 80% could still be observed at a dose as high as 1000 μg/mL. Even after 24 h of incubation, the cell viability of RAW264.7 cells was still above 60% at 1000 μg/mL for different OxbCD NPs (Figure S12b). Of note, viability values of OxbCD NP-treated cells were comparable to those treated with identical doses of NP fabricated using a FDA-approved biodegradable material PLGA. Similar results were found in MOVAS-1 cells (Figure S12c). It should be emphasized that, at higher concentrations of different NPs, their deposition on attached cells may form a physical barrier that might influence cell growth, thereby leading to relatively low cell viability. This is different from cell death directly caused by endocytosed NPs. These data suggested that OxbCD NPs displayed low toxicity in normal cells of RAW264.7 and MOVAS-1. In view of the important role of macrophages in the pathogenesis of inflammatory diseases, RAW264.7 cells were chosen for additional in vitro evaluations. First the effect of OxbCD NP treatment on mitochondrial membrane potential (ΔΨm) was quantified by flow cytometry using TMRE as a probe. Treatment with 100 or 200 μM of H2O2 caused significant decrease of ΔΨm in RAW264.7 cells (Figure S12d,e), indicating that H2O2 may lead to mitochondrial damage to a certain degree. By contrast, incubation with either PLGA NP or different OxbCD NPs at 100 μg/mL only slightly decreased ΔΨm, agreeing with their low cytotoxicity (Figure S12a,b). As well documented, overexpression of ROS is seriously related to both acute and chronic inflammation as well as oxidative stress in diverse inflammatory diseases.7−11,17 There8229

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Figure 4. In vitro antioxidation activity of OxbCD NPs in RAW264.7 macrophages. (a) Fluorescence microscopy images showing the intracellular generation of ROS in macrophages stimulated with 100 ng/mL of PMA and different NPs at 100 μg/mL for 6 h. DCFH-DA was used as a fluorescent probe for intracellular ROS. (b) Typical flow cytometry profiles (left) and quantitative analysis (right) of relative ROS levels in macrophages. Before analysis, cells were treated under the same conditions as for fluorescence microscopy. (c, d) Flow cytometric profiles (left) and quantitative analysis (right) illustrating cell apoptosis (c) or disruption of mitochondrial membrane potential (d) of macrophages. Before exposure to 200 μM H2O2 for 8 h, macrophages were preincubated with NPs at 100 μg/mL for 2 h. (e, f) Fluorescence micrographs (e) and flow cytometry quantification (f) of the intracellular NO production in macrophages. Before analysis, cells were incubated with 40 μM H2O2 and 100 μg/mL NPs for 6 h. (g) Quantitative analysis of intracellular NOS levels after different treatments. Left panel, flow cytometric curves; right panel, quantitative data. Scale bars, 100 μm. Data are mean ± SEM (n = 5); *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.

RAW264.7 cells with 100 ng/mL PMA remarkably increased the extracellular level of H2O2 (Figure S13d), while treatment with different NPs led to the low levels of H2O2 in the culture medium. In line with this result, a high hydrolysis degree was observed for OxbCD NPs in the medium collected from the PMA stimulated group (Figure S13e), which was dramatically higher than that in the culture medium from the control group. In contrast, the culture medium from macrophages treated with different NPs at 100 μg/mL for 6 h did not accelerate hydrolysis of corresponding NPs (Figure S13f). These results are consistent with the H2O2-responsive hydrolysis profiles of OxbCD NPs (Figure 3b−d).

fore, we examined the possible effects of OxbCD NPs on ROS production. Observation by fluorescence microscopy revealed the generation of considerable intracellular ROS when macrophages were stimulated by PMA, as illustrated by strong green fluorescence resulting from the oxidized fluorescent probe DCFH-DA (Figure S13a). Treatment with different OxbCD NPs and PLGA NP at 100 μg/mL, however, only afforded notably weak fluorescent signals, which were comparable to that of the control group. This finding was further affirmed by quantitative analysis via flow cytometry (Figure S13b,c). Also, we quantified the extracellular production of ROS under different conditions. Stimulation of 8230

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Figure 5. Antimigratory and anti-inflammatory activities of OxbCD NPs in macrophages. (a) Optical microscopy images showing migrated RAW264.7 cells positively stained by crystal violet. (b) Quantitative analysis of migrated cells per high-power field (HPF, 200 × ). (c−e) Inhibition of the expression of typical inflammatory chemokines including MCP-1 (c), MIP-2 (d), and IL-8 (e) by OxbCD NPs. (f−h) The expression levels of inflammatory cytokines including TNF-α (f), IL-1β (g), and IL-6 (h) after different treatments. In all cases, macrophages were incubated with 40 μM H2O2 and 100 μg/mL NPs for 6 h, followed by different analyses. Both cytokines and chemokines in culture media were determined by ELISA. Scale bars represent 50 μm. Data are mean ± SEM (n = 5, b; n = 6, c−h); *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.

Further, we determined the expression of typical inflammatory cytokines in RAW264.7 cells after incubation with different NPs. Quantification by an ELISA assay indicated that treatment of macrophages with NPs based on OxbCD-1, OxbCD-3, OxbCD-4, and PLGA only slightly increased TNF-α expression levels in culture media (Figure S14a), which were significantly lower than that of PMA-treated cells. Notably, the TNF-α expression in the OxbCD-2 NP or OxbCD-5 NP group was remarkably lower than that of the PLGA NP group. Additionally, there was no significant difference in the TNF-α level between the OxbCD-2 NP or OxbCD-5 NP treated group and the normal control. On the other hand, the IL-6 levels

showed no significant increase after treatment with various NPs, and they were remarkably lower than that of the PMA group (Figure S14b). These results were also confirmed by Western blot analysis of intracellular TNF-α and IL-6 levels (Figure S14c). Consistent with the nonproinflammatory characteristics of OxbCD NPs in RAW264.7 cells, we found that incubation with different NPs did not induce macrophage migration, while treatment with either MCP-1 or H2O2 resulted in significant migration (Figure S14d-e). Taken together, we demonstrated that OxbCD NPs showed low cytotoxicity in normal cells. Furthermore, treatment of macrophages with OxbCD NPs did not notably change 8231

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Figure 6. In vivo anti-inflammatory efficacy of OxbCD NPs in an acute inflammation model of peritonitis in mice induced by zymosan. (a) Bioluminescence images (left) and quantitative analysis (right) indicating the ROS production in mouse abdominal cavity after various treatments. (b, c) Representative flow cytometric profiles (left) and quantitative analysis (right) illustrate the numbers of neutrophils (b) and macrophages (c) in peritoneal exudates. (d) Migration of RAW264.7 macrophages induced by neutrophils after various treatments. Except the normal group, macrophages in other groups were stimulated with 2 × 104 neutrophils. The left image showing microscopy images (HPF, 100×), while the right image indicates quantitative data. (e−g) The levels of MPO (e) as well as typical inflammatory chemokines (f) and cytokines (g) in peritoneal lavage fluid from mice with zymosan peritonitis. Data are mean ± SEM (n = 5, a−d; n = 6, e−g); *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.

found that OxbCD NPs were able to effectively decrease the H2O2-induced intracellular production of nitric oxide (NO) (Figure 4e,f), an important component of reactive nitrogen species that may also cause oxidative stress.16,18 We also quantified the intracellular level of NO synthase (NOS). It was observed that the H2O2-induced high expression of NOS could be significantly inhibited by different OxbCD NPs (Figure 4g). Of note, no positive effects were observed after treatment with the same dose of PLGA NP in all cases. Therefore, the antiapoptotic activity of OxbCD NPs should be largely attributed to their capability of mitigating oxidative stress-induced mitochondrial dysfunction by eliminating H2O2. It has been demonstrated that ROS may stimulate the production of chemotactic factors by monocytes and macrophages, resulting in enhanced cell migration to inflammatory sites.63 The transwell migration assay showed that H2O2 can induce the migration of RAW264.7 macrophages (Figure S14d,e and Figure 5a,b). Treatment by OxbCD NPs significantly suppressed migration of RAW264.7 cells induced by H2O2. Correspondingly, OxbCD NPs remarkably inhibited the H2O2-induced expression of chemokines in macrophages, including MCP-1, MIP-2, and IL-8 (Figure 5c−e). After stimulation with H2O2, high levels of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6, were excreted by RAW264.7 cells (Figure 5f−h), which were dramatically

mitochondrial membrane potential, cause overproduction of intracellular/extracellular ROS, stimulate significant expression of inflammatory cytokines, or induce cell migration. These findings suggested that OxbCD NPs themselves may not induce oxidative stress, mitochondrial damage, and inflammatory responses in macrophages. It is worth noting that OxbCD NPs exhibited cellular compatibility comparable to or even better than that of PLGA NP. 3.5. In Vitro Anti-Oxidative Stress and Anti-Inflammatory Effects of OxbCD NPs in Macrophages. Based on the above findings, we examined in vitro antioxidative stress and anti-inflammatory effects of OxbCD NPs in macrophages. In accordance with their H2O2-scavenging capability, different OxbCD NPs can effectively reduce intracellular H 2 O 2 generated by PMA stimulation (Figure 4a,b). By eliminating H2O2, OxbCD NPs significantly inhibited cell apoptosis induced by H2O2 in RAW264.7 cells (Figure 4c). Notably, OxbCD-2 NP and OxbCD-5 NP showed an activity comparable to that of catalase, a common enzyme found in nearly all living organisms that can catalyze the decomposition of H2O2.16 As well documented, loss of ΔΨm is an early event in the apoptotic process of many cells.62 Our experiments demonstrated that different H2O2-eliminating OxbCD NPs could reverse depolarization of ΔΨm induced by H2O2 (Figure 4d). Using an intracellular fluorescent probe DAF-FM DA, we 8232

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note, OxbCD-2 and OxbCD-5 NPs showed efficacy comparative to catalase, while PLGA NP had no anti-inflammatory activity. Together, the above results demonstrated that OxbCD NPs alone can efficiently alleviate the symptoms of peritonitis, by reducing ROS production, inhibiting recruitment of neutrophils and macrophages to inflamed sites, and down-regulating the expression of inflammatory cytokines and chemokines. Therefore, we may conclude that OxbCD NPs display in vivo antiinflammatory efficacy. In combination with in vitro and in vivo results, we proposed the mechanisms underlying the antiinflammatory activity of OxbCD NPs (Figure 7). By

decreased by OxbCD NPs. In particular, both OxbCD-2 NP and OxbCD-5 NP groups showed no significant differences in the levels of TNF-α and IL-1β as compared to those of the normal control group. These results demonstrated that OxbCD NPs may attenuate inflammatory responses mediated by ROS. Taken together, OxbCD NPs can significantly inhibit cell apoptosis, notably reverse depolarization of ΔΨm, effectively attenuate overproduction of NO and NOS, and remarkably suppress cell migration, as well as dramatically decrease the high expression of pro-inflammatory cytokines and chemokines in macrophages induced by H2O2. The beneficial effects were mainly realized by efficiently eliminating H2O2, thereby reducing oxidative stress. These pharmacological activities make OxbCD NPs desirable anti-oxidant and anti-inflammatory nanotherapies. 3.6. In Vivo Anti-Inflammatory Efficacy of OxbCD NPs. We further examined in vivo anti-inflammatory efficacy of OxbCD NPs in mice with zymosan-induced peritonitis, a wellrecognized model of acute inflammation.64 OxbCD-2 and OxbCD-5 NPs were selected for subsequent experiments due to their better in vitro performance. At 6 h after i.p. injection of zymosan in mice, in vivo imaging showed significant luminescent signals post-local administration of luminol (Figure 6a), a bioluminescent probe generally utilized for inflammation imaging.65 By contrast, either the luminol group without zymosan stimulation or the saline group with zymosan showed negligible luminescent signals. This result demonstrated that the mouse peritonitis model was successfully established. The luminescent signals were notably decreased when mice with peritonitis were treated by i.p. injection of catalase, OxbCD-2 NP, or OxbCD-5 NP at 1 h after zymosan challenge, although the PLGA NP group showed luminescence comparable to that of the model group. These luminescence imaging results suggested that OxbCD-2 and OxbCD-5 NPs can efficaciously attenuate zymosan-induced peritonitis in mice. In support of this finding, flow cytometry analyses revealed significantly decreased inflammatory cell counts of neutrophils and macrophages in peritoneal exudates from mice with peritonitis, after treatment by OxbCD-2 NP or OxbCD-5 NP (Figure 6b,c). It has been demonstrated that neutrophils and macrophages may promote the development and progression of peritonitis, partly through the generation of ROS.66 While neutrophil extravasation to inflamed sites occurs at the early stages of peritonitis, macrophages can be recruited by neutrophilmediated cell migration. In vitro transwell assay substantiated that either OxbCD-2 NP or OxbCD-5 NP notably inhibited macrophage migration induced by neutrophils (Figure 6d). In addition, the expression of MPO, an indicator of neutrophil infiltration, was significantly reduced in peritoneal lavage fluid, after intervention by OxbCD-2 NP or OxbCD-5 NP (Figure 6e). Furthermore, the levels of MCP-1 and IL-8 were strikingly reduced in the OxbCD-2 NP and OxbCD-5 NP groups (Figure 6f). Since MCP-1 and IL-8 play an important role in selectively recruiting monocytes or neutrophils to inflamed sites, as well as in regulating the migration and infiltration of monocytes/ macrophages,67 our results demonstrated that both OxbCD-2 NP and OxbCD-5 NP can efficaciously suppress infiltration of neutrophils and neutrophil-mediated migration of macrophages by inhibiting the expression of chemokines. Moreover, treatment with either OxbCD-2 NP or OxbCD-5 NP dramatically decreased the levels of TNF-α and IL-6 (Figure 6g), indicating that inflammatory responses were mitigated. Of

Figure 7. Schematic illustration of anti-inflammatory mechanisms of H2O2-eliminating OxbCD NPs.

eliminating H2O2, OxbCD NPs inhibit the infiltration of neutrophils to inflamed sites, thereby attenuating macrophage recruitment induced by activated neutrophils. On the one hand, decreased neutrophils and macrophages may mitigate oxidative stress; while on the other hand, the reduced macrophage count decreases the expression of both pro-inflammatory cytokines and chemokines. Collectively, reduced oxidative stress and inflammatory responses further decrease the generation of ROS. 3.7. Preparation, Characterization, and in Vivo Evaluation of Drug-Loaded OxbCD NPs. To interrogate whether OxbCD materials can serve as ROS-responsive vehicles to potentiate efficacy of anti-inflammatory therapeutics, DXM and IND were selected as candidate drugs. Among them, DXM is a typical corticosteroid, while IND is a nonsteroidal anti-inflammatory drug. Different NPs loaded with DXM or IND were also prepared by the nanoprecipitation/self-assembly method. TEM observation indicated that core−shell structured spherical NPs, with relatively narrow size distribution profiles, were obtained based on different materials OxbCD-2, OxbCD5, or PLGA (Figure 8a,b). The mean diameters were 208.0 ± 0.4, 195.2 ± 1.0, 201.1 ± 1.0, 210.4 ± 0.6, 240.0 ± 1.0, and 220.6 ± 1.1 nm for DXM/OxbCD-2 NP, DXM/OxbCD-5 NP, DXM/PLGA NP, IND/OxbCD-2 NP, IND/OxbCD-5 NP, and IND/PLGA NP, respectively. All drug-loaded NPs had negative zeta-potential values (Figure S15a). OxbCD-2 and OxbCD-5 NPs exhibited significantly higher drug loading contents and entrapment efficiencies as compared to PLGA NP 8233

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Figure 8. Characterization and in vitro release profiles of different NPs containing anti-inflammatory drugs DXM or IND. (a) TEM images of drugloaded NPs. (b) Size distribution profiles of different NPs. (c) In vitro drug release curves of various NPs in pH 7.4 PBS containing 1.0 mM H2O2. Data in (c) are mean ± SEM (n = 3).

PLGA NP groups (Figure 9d−h and Figure S16). For drugcontaining OxbCD-2 and OxbCD-5 NPs, the synergistic effects of ROS-triggered rapid drug release and effective H2O2eliminating activity should have contributed to their best efficacy among different formulations. As for DXM/PLGA and IND/PLGA NPs, limited drug release may account for their poor therapeutic effect. Consequently, these results demonstrated that OxbCD NPs can serve as effective ROS-responsive nanocarriers to amply the efficacy of anti-inflammatory therapeutics. 3.8. In Vivo Safety Studies of OxbCD NPs. Based on the desirable therapeutic results and preliminary cell culture data, we performed additional animal studies to interrogate the in vivo biocompatibility of OxbCD NPs. At 24 h after subcutaneous (0.5 mg/kg) or i.p. (5 mg/kg) injection of different OxbCD NPs in mice, in vivo imaging using luminol as the probe revealed negligible luminescent signals, whereas mice induced with zymosan showed highly strong luminescence (Figure 10a, b). The luminescent signal of luminol is closely related to the ROS level (Figure S17), which may reflect the degree of both acute and chronic phases of the foreign body reaction.69 Therefore, our results suggested that subcutaneously or i.p. administered OxbCD NPs did not cause significant oxidative stress and inflammatory responses. Furthermore, at day 7 after intramuscular (i.m.) injection of OxbCD NPs at 25 mg in each rat, histological analysis of H&Estained sections revealed normal texture and alignment of muscle fibers without significant infiltration of inflammatory cells (Figure 10c), which were similar to that of the saline group. Meanwhile, fluorescence observation of muscular sections stained with a fluorescent probe for superoxide anion revealed considerable red fluorescence in the positive control group treated with lipopolysaccharide (LPS) (Figure 10d), while remarkably weak signals were found for other groups administered with NPs. These findings substantiated that i.m. administration of OxbCD NPs did not cause discernible tissue injuries or significant oxidative stress.

(Figure S15b). This implied that host−guest interactions between the β-CD cavity and the lipophilic group in DXM or IND may facilitate their packaging into NPs.68 Measurement by DSC suggested that loaded drug molecules were molecularly dispersed in the nanoscaled OxbCD matrix other than in a crystal form (Figure S15c), which is beneficial for rapid drug release upon triggering via ROS. In vitro release tests showed dramatically accelerated drug release from responsive OxbCD-2 NP or OxbCD-5 NP in the presence of 1.0 mM H2O2, which was significantly higher than that from PLGA NP (Figure 8c). For OxbCD-2 and OxbCD-5 NPs, more than 80% cumulative release was observed within 6 h for both DXM and IND, while less than 20% of the loaded drug was released from PLGA NP. Subsequently, we examined therapeutic benefits of different drug-loaded NPs in mice with peritonitis. At 1 h after initiation of peritonitis by i.p. injection of zymosan, different treatments were conducted by local administration. After 5 h, quantification by flow cytometry implicated that the neutrophil count in peritoneal exudates was notably decreased after treatment with free DXM or IND (Figure 9a). This effect was additionally potentiated by packaging them into responsive nanovehicles of OxbCD-2 or OxbCD-5. In contrast, drug-loaded PLGA NPs showed no significant efficacy as compared to the model group. Also, the numbers of peritoneal exudate macrophages were affected in similar manners after treatment with different DXMor IND-loaded NPs (Figure 9b), with the lowest level observed for responsive NPs. Consistent with this, the levels of MPO in peritoneal lavage fluid were most significantly decreased for mice treated with drug-loaded responsive NPs (Figure 9c), which even exhibited no significant difference as compared to the normal control. PLGA NPs containing DXM or IND also lowered the expression of MPO, indicating that a small quantity of released drug molecules were able to inhibit activation of neutrophils. Likewise, typical oxidative species (such as superoxide anion and NO) as well as pro-inflammatory cytokines and chemokines (including TNF-α, IL-6, MCP-1, and IL-8) were more significantly decreased in groups treated with drug-loaded responsive NPs, as compared to free drug and 8234

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Figure 9. In vivo anti-inflammatory effects of drug-loaded OxbCD NPs in peritonitis mice. (a, b) Representative flow cytometric profiles (left) and quantitative data (right) indicating the numbers of neutrophils (a) and macrophages (b) in peritoneal lavage fluid. (c−e) The levels of typical mediators associated with oxidative stress, including MPO (c), superoxide anion (d), and NO (e) in peritoneal exudates. (f−h) The concentrations of inflammatory cytokines and chemokines including TNF-α (f), IL-6 (g), and MCP-1 (h) in peritoneal lavage fluid. Data are mean ± SEM (n = 6); *p < 0.05, **p < 0.01, ***p < 0.001; ns, no significance.

OxbCD-2 and OxbCD-5 NPs (Figure S18). After a single i.v. injection at 500 or 1000 mg/kg in mice, the body weights of all animals were gradually increased (Figure S19a), without significant differences between NP-treated groups and the saline group. Mice administered with different NPs displayed

Subsequently, we examined the safety performance of OxbCD NPs for intravenous (i.v.) administration, using OxbCD-2 and OxbCD-5 NPs as typical examples. First, an in vitro hemolysis test was conducted, and both direct observation and quantification revealed no significant hemolysis for 8235

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Figure 10. In vivo safety studies of OxbCD NPs after local administration. (a, b) Luminescence images (left) and quantitative data (right) reveal the degree of oxidative stress after subcutaneous (a) or intraperitoneal (b) injection of different NPs in mice. (c, d) Evaluation of muscular irritation effects of OxbCD NPs after intramuscular injection in the right posterior thigh muscles in rats. Optical microscopy images of H&E stained sections (c) and fluorescence images of DHE-stained sections (d) of muscular tissues at day 7 after administration. Data are mean ± SEM (n = 3); ***p < 0.001.

onto β-CD through different covalent linkers. Regardless of their different structures, these OxbCD materials and their NPs can effectively eliminate H2O2 in a stoichiometry-dependent manner. This H2O2-scavenging capability is closely associated with the hydrolytic lability of different linker groups in the presence of H2O2. The slowest hydrolysis and lowest H2O2scavenging efficiency were found for OxbCDs bearing ester groups. Also, the ROS-sensitive hydrolysis profiles and the H2O2-eliminating capacities of OxbCDs can be modulated by adjusting the number of conjugated PBAP groups. The complete hydrolysis of OxbCDs gives rise to parent β-CD and other small molecules that can be easily excreted. By eliminating H2O2, OxbCD NPs effectively protected macrophages from oxidative stress and inflammatory responses induced by H2O2. Consistently, OxbCD NPs were able to efficaciously treat acute inflammation in mice, by inhibiting neutrophil infiltration and neutrophil-induced macrophage recruitment as well as suppressing the expression of proinflammatory cytokines and chemokines. Additionally, OxbCD NPs can serve as functional nanovehicles of anti-inflammatory drugs. By synergistic effects of drug molecules and carrier materials, drug-loaded OxbCD NPs more effectively promoted the resolution of acute inflammation in mice, when compared with corresponding free drug and drug-loaded PLGA NPs. More importantly, in vitro cell culture studies and in vivo evaluations after delivery via different routes demonstrated that OxbCD NPs displayed a very good safety profile. Consequently, these findings may provide new insight into the design of novel ROS-responsive and H 2O2 -eliminating materials. OxbCDs and their NPs may be used as pharmacologically active platforms for the treatment of acute

normal food and water intake, and no abnormal behaviors appeared. At day 14, the mice were euthanized. All the examined groups showed comparable organ index values for major organs including heart, liver, spleen, lung, and kidney (Figure S19b). Quantification of typical hematological parameters, such as white blood cell (WBC), red blood cell (RBC), platelet (PLT), and hemoglobin (HGB), indicated no significant differences between the saline group and OxbCD NP-treated group (Figure S19c). Moreover, clinical biochemistry quantification of biomarkers relevant to liver (ALT and AST) and kidney functions (BUN and CREA) revealed no abnormal variations in mice administered with OxbCD-2 or OxbCD-5 NP (Figure S19d). Further inspection on H&E stained histological sections indicated that there were no distinguishable injuries or pathological changes in the major organs of OxbCD NP-treated mice (Figure S20). Taken together, the above results demonstrated that OxbCD NPs, particularly OxbCD-2 and OxbCD-5 NPs, exhibited a good safety profile after administration by different routes, including subcutaneous, i.p., i.m., and i.v. injection. Although the hydrolysis of OxbCD-1 and OxbCD-4 may generate CO, the trace amount of CO does not elicit significant adverse effects. Therefore, OxbCD NPs themselves can be further developed as safe nanotherapies. Alternatively, they may serve as biocompatible ROS-responsive nanocarriers for the targeted delivery of a large array of therapeutics.

4. CONCLUSIONS In summary, we have successfully synthesized a series of ROSresponsive materials by conjugating responsive PBAP groups 8236

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and chronic inflammatory diseases as well as other diseases associated with oxidative stress.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b02412. Table S1 and Figures S1−S20 (PDF)



AUTHOR INFORMATION

Corresponding Author

*Jianxiang Zhang. E-mail: [email protected]; jxzhang@ tmmu.edu.cn. ORCID

Qixiong Zhang: 0000-0003-0330-1389 Ruibing Wang: 0000-0001-9489-4241 Jianxiang Zhang: 0000-0002-0984-2947 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This study was supported by the National Natural Science Foundation of China (No. 81471774), the Research Foundation of the Third Military Medical University (No. 2014XJY04), and the Graduate Student Research Innovation Project of Chongqing.



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DOI: 10.1021/acs.chemmater.7b02412 Chem. Mater. 2017, 29, 8221−8238