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Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Green Fabrication of Ovalbumin Nanoparticles as Natural Polyphenol Carriers for Ulcerative Colitis Therapy Shuangquan Gou,† Qiubing Chen,† Yan Liu,‡ Liang Zeng,‡ Heliang Song,⊥ Zhigang Xu,† Yuejun Kang,*,† Changming Li,† and Bo Xiao*,† †

ACS Sustainable Chem. Eng. Downloaded from pubs.acs.org by DURHAM UNIV on 08/27/18. For personal use only.

Institute for Clean Energy and Advanced Materials, Faculty of Materials and Energy, Southwest University, No. 2 Tiansheng Road, Beibei, Chongqing 400715, P. R. China ‡ College of Food Science, Southwest University, No. 2 Tiansheng Road, Beibei, Chongqing 400715, P. R. China ⊥ Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, 100 Piedmont Avenue SE, Atlanta, Georgia 30302, United States S Supporting Information *

ABSTRACT: Nanoparticles (NPs) have the capacity to improve the bioactivity and bioavailability of anti-inflammatory drugs. Here, a green drug nanocarrier based on ovalbumin (OVA) was facilely produced via a self-assembling process by adding epigallo-catechin 3-gallate (EGCG) aqueous solution to OVA aqueous solution at 80 °C. The obtained EGCG-NPs had an average hydrodynamic diameter (around 202.9 nm), negative surface charge (approximately −13.2 mV), high EGCG encapsulation efficiency (98.1%), and reduction-responsive EGCG release capacity. Additionally, they possessed excellent biocompatibility and achieved a much higher cellular uptake rate of EGCG than pristine EGCG. Furthermore, EGCG-NPs had remarkably stronger capacity to suppress the secretion of pro-inflammatory mediators (e.g., tumor necrosis factor α, interleukin-6, interleukin-12) and promote the production of anti-inflammatory factor (interleukin-10), in comparison with pristine EGCG. Finally, in vivo experiments demonstrated the excellent therapeutic efficacy of EGCG-NPs in alleviating ulcerative colitis (UC). The present study collectively suggests that these facilely fabricated OVA-based NPs could be exploited as an efficient EGCG carrier for UC therapy. KEYWORDS: Cellular internalization, Anti-inflammation, Epigallo-catechin 3-gallate, Ovalbumin, Nanoparticle, Ulcerative colitis

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INTRODUCTION Ulcerative colitis (UC) is a common inflammatory disease in the colon, which is characterized by diarrhea, mucosal ulceration, and rectal bleeding.1,2 This disease affects several million patients worldwide, and its morbidity continues to increase sharply in recent decades. The main objectives for UC therapy in the clinic are to reduce inflammation and achieve mucosal healing.3,4 Currently, the therapeutics for UC mainly relies on the utilization of anti-inflammatory agents, immunomodulatory drugs and biological products.5−7 Even though these medications are capable of temporarily alleviating UC, their long-term utilizations have been restricted by limited therapeutic outcomes and severe adverse effects.8 Epigallo-catechin 3-gallate (EGCG) takes up more than 40% of the total polyphenol amount in green tea, and it is a natural bioactive agent with good water solubility.9,10 A large number of investigations have confirmed its bioactivities in antioxidant, antiobesity, antibacterial, and protective functions against various diseases (e.g., inflammation, cancer, and Alzheimer’s).11−15 It was found that EGCG could reduce the symptoms of colonic oxidative stress and inflammation in IL© XXXX American Chemical Society

10 knockout mice, which would spontaneously develop colitis otherwise.16 A similar study demonstrated that EGCG could efficiently mitigate the loss of body weight in a mouse model of UC induced by dextran sulfate sodium (DSS).17 Notably, a recent investigation demonstrated that EGCG was able to maintain the integrative structure of colonic epithelial layer and reduce the intestinal permeability in vitro as well as in UC mouse models.18 In spite of these promising advantages of EGCG in UC therapy, its clinical translation has been constrained by several drawbacks, including poor stability, limited bioavailability, and low absorption efficiency.19 It is known that EGCG readily undergoes oxidation in solution, resulting in poor stability and low bioavailability.20 In addition, EGCG is taken up by cells via passive internalization accompanied by drainage by efflux transporters, leading to relatively low absorption efficiency.21 To address these issues, lots of drug carriers have been employed to deliver EGCG, Received: April 10, 2018 Revised: August 12, 2018 Published: August 17, 2018 A

DOI: 10.1021/acssuschemeng.8b01613 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering such as nanoparticles (NPs), nanoencapsulations, and liposomes.22−24 In recent years, natural protein-based NPs have attracted increasing attention for encapsulation and delivery of bioactive agents owing to their numerous benefits in biocompatibility, cost effectiveness and high drug encapsulation efficiency, as well as controlled drug release.25 For UC therapy, NPs could passively penetrate and accumulate in colitis tissues owing to the enhanced permeability and retention (EPR) effect, which is based on the loss of vascular endothelial integrity and poor lymphatic drainage in inflammatory tissues.26 Ovalbumin (OVA) is a globular glycoprotein, which can be easily extracted and purified from egg white.27 When heated in aqueous solution, the internal hydrophobic domains of OVA are exposed on their surfaces, resulting in the aggregation of OVA molecules encapsulating the drugs inside. A previous study reported that OVA had a very high affinity with hydrophilic drugs (e.g., caffeine, theophylline, and diprophylline) through physical interactions.28 Interestingly, Ognjenovic et al. reported that EGCG tended to bind to the IgE-binding domain in OVA, and they further speculated that such interaction would induce diverse biological effects.29 Nevertheless, to the best of our knowledge, there is no prior attempt to explore the therapeutic effects of EGCG-loaded OVA-based NPs (EGCG-NPs) for disease therapy. Thus, in the present study, we facilely fabricated EGCG-NPs via a selfassembling method without organic solvents and under mild conditions, and we further investigated their physicochemical characteristics. Finally, we described the first attempt to study the in vitro anti-inflammatory activities of EGCG-NPs and their in vivo therapeutic efficacies against UC.

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encapsulation efficiency weight of feeded drug − drug amount in supernatant = × 100% weight of feeded drug (1) drug loading weight of feeded drug − drug amount in supernatant = × 100% weight of dry NPs (2)

The average hydrodynamic particle size (nm), polydispersity index (PDI), and zeta potential (mV) of EGCG-NPs were studied using a dynamic light scattering (DLS) instrument (Malvan, Zetasizer NanoZS, U.K.). The average values were tested using three runs, and obtained on the basis of the measurements on different batches of NPs. The appearance of EGCG-NPs was examined using a JEOL JSM6510LV scanning electron microscope (SEM, Tokyo, Japan). Freezedried NPs were suspended in deionized water, and the NP suspension was dropped on a cleaned wafer and air-dried before SEM examination. The X-ray diffraction (XRD) spectra of pristine EGCG, OVA powders, and EGCG-NPs were measured (XRD-7000, Shimadzu, Japan) by scanning from 10° to 80° at a speed of 2 degree per min and operating at 40 kV and 30 mA. The FTIR spectra of pristine EGCG, OVA powders, and EGCGNPs were measured using a Bruker EQUINOX 55 FT-IR Spectrophotometer. The samples were blended with KBr, and the obtained mixtures were further pressed in pancake shape for measurements. Release Behavior of EGCG from EGCG-NPs. The release profiles of EGCG from EGCG-NPs were measured using a dialysis method. In brief, EGCG-NPs were suspended in various buffers (equivalent to 200 μg of EGCG), and the NP suspensions were added to dialysis bags (molecular weight cutoff = 10 000 Da). These bags were sealed in both sides and then put into 50 mL centrifuge tubes with various releasing buffers: PBS (pH 7.4) and sodium acetate/ acetic acid buffer (pH 6.2). The former buffer was chosen according to the pH in circulatory system or cytoplasm, and the latter one was selected on the basis of the pH in late endosome. These tubes were deposited in a thermostat shaker at 160 rpm and 37 °C. Thereafter, the releasing buffers were withdrawn at desired time points, and the released amounts of EGCG in releasing buffers were examined using HPLC. In Vitro Anti-Inflammatory Activity. Raw 264.7 macrophages were grown in 48-well plates, and their density was set as 1 × 105 cell per well. After culture for 12 h, the complete medium was replaced by a medium containing pristine EGCG, blank NPs or EGCG-NPs with various EGCG concentrations. The medium was subsequently discarded after 24 h of incubation. Thereafter, these cells were stimulated with LPS (0.5 μg/mL) for 3 h. The supernatant in each well was gathered, and the concentrations of TNF-α, interleukin-6 (IL-6), interleukin-12 (IL-12), and interleukin-10 (IL-10) were measured with their corresponding ELISA kits. LPS-stimulated cells and cells in the absence of LPS were employed as positive controls and negative controls, respectively. In Vivo Therapeutic Efficacy of NPs against UC. All animal care and protocols were carried out in accordance with the Southwest University Institutional Care and Use Committee. FVB male mice (8 weeks of age; Chongqing Tengxin Biotechnologies Company, Chongqing, P. R. China) were used in all animal experiments. UC mice model was established via the treatment of DSS (3.5%, w/v) in the drinking water. All mice were separated into four groups, namely, healthy control group, DSS control group, pristine EGCG-treated DSS group, and EGCG-NP-treated DSS group. The equivalent EGCG dosage of 5 mg/kg mice per day was applied for drug-treated DSS groups. Each formulation (200 μL) was administrated every other day by intravenous injection from the first day. Mice were weighed every day during the entire experiment. At day 9, all mice

EXPERIMENTAL SECTION

Materials. EGCG was obtained from Taiyo Green Power Company (Wuxi, Jiangsu, P. R. China). OVA was purchased from Shanghai Yiji Industrial Company (Shanghai, P. R. China). DSS (36− 50 kDa) was obtained from MP Biomedical Inc. (Solon, OH, U.S.A.). Glutathione (GSH), phosphate-buffered saline (PBS), Triton X-100, potassium bromide (KBr), dimethyl sulfoxide (DMSO), lipopolysaccharide (LPS), and sodium dodecyl sulfate (SDS) were supplied by Sigma-Aldrich (St. Louis, MO, U.S.A.). 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) was from Invitrogen (Eugene, OR, U.S.A.). The mouse tumor necrosis factor α (TNF-α) ELISA kit and myeloperoxidase (MPO) kit were supplied by Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, P. R. China). Buffered formalin (10%) was from Thermo Fischer Scientific (Fair Lawn, NJ, U.S.A.). Hematoxylin and eosin (H&E) staining kit was purchased from Beyotime Institute of Biotechnology (Nanjing, Jiangsu, P. R. China). Fabrication of EGCG-NPs. Initially, OVA was dissolved in the phosphate buffer (pH 6.6), and its concentration was adjusted to 2.5 mg/mL. EGCG was also dissolved in phosphate buffer (pH 6.6) to obtain a fresh solution. After thermal denaturation of OVA at 80 °C for 20 min, EGCG solution was immediately added into thermal OVA solution to achieve various molar ratios of EGCG/OVA (1:1, 2:1, 4:1, and 10:1). The mixture was vortexed for 30 s, placed under running tap water for 3 min, and further sonicated for 1 min at an amplitude of 25%. The obtained EGCG-NPs were gathered by centrifugation, washed with deionized water, and freeze-dried for further applications. Physicochemical Characterization of EGCG-NPs. An indirect method was applied to determine the loading amount and encapsulation efficiency of EGCG in EGCG-NPs. Briefly, supernatants of EGCG-NP suspensions were obtained by centrifugation at 13 000g for 18 min and further analyzed using high-performance liquid chromatography (HPLC). The equations of drug loading amount and encapsulation efficiency were described as follows: B

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ACS Sustainable Chemistry & Engineering Table 1. Basic Characteristics of EGCG-loaded NPs at Various Molar Ratios of EGCG/OVA (n = 3) EGCG/OVA molar ratio 1:1 2:1 4:1 10:1

particle size (nm) 170.6 202.9 315.7 479.7

± ± ± ±

0.7 2.8 1.4 6.4

zeta potential (mV) −10.2 −13.2 −14.6 −17.8

± ± ± ±

0.5 0.5 0.4 1.0

loading amount (μg/mg) 19.1 40.0 64.0 109.0

± ± ± ±

0.9 3.1 2.1 7.3

encapsulation efficiency (%) 99.7 98.1 91.2 80.7

± ± ± ±

0.3 0.1 1.0 0.5

Figure 1. (a) Representative SEM images of EGCG-NPs and (b) their corresponding size distributions. (c) XRD patterns of pristine EGCG, OVA powders, and EGCG-NPs. were sacrificed by CO2 inhalation. Colon tissues were excised and flushed gently with PBS to eliminate fecal matter. Their MPO activities were examined using a MPO kit. Major organs (heart, liver, spleen, lung, and kidney) were also excised, sectioned into slices (5 μm), and further stained with H&E. In Vivo Distribution of EGCG. Mice with colitis were intravenously injected with pristine EGCG or EGCG-NPs at an equal EGCG dose of 5 mg/kg mice. After 24 h of injection, all mice were sacrificed by CO2 inhalation. Major organs/tissues of mice were excised, and feces were collected. The obtained samples were homogenized, centrifuged, and further analyzed by measuring the amounts of EGCG by HPLC.

temperature due to the hydrophobic interaction compared with the conditions at 75 and 85 °C.34 Table 1 depicted that the hydrodynamic average diameters of EGCG-NPs obviously increased with the increase of molar ratios of EGCG/OVA. Typically, the particle size at a molar ratio of 10:1 (479.7 nm) was about 2.8-fold as large as that at 1:1. It was speculated that EGCG was encapsulated into OVA-based NPs through two approaches, which were based on the adsorption of EGCG to EGCG-binding sites in OVA molecules and the physical encapsulation of EGCG among OVA molecules, respectively. When the EGCG/OVA molar ratios were below 2:1, EGCG could be almost completely adsorbed to OVA molecules, resulting in high encapsulation efficiency. However, with further increasing their molar ratios, the adsorption of EGCG to OVA molecules was saturated. Meanwhile, hydrophilic EGCG would hinder the aggregation of OVA molecules with exposed hydrophobic cores, inducing the formation of loose assembly with large particle size and low drug encapsulation efficiency. In addition, all NPs showed slightly negative zeta potentials at approximately −14.0 mV. Furthermore, the encapsulation efficiencies of EGCG-NPs ranged from 80.7% to 99.7%, which exhibited a contrary tendency compared to that with regard to particle size. In spite of no notable difference in the EGCG encapsulation efficiency between 1:1 and 2:1, the drug loading amount of NPs at molar ratio of 1:1 was only half that at 2:1. Based on the overall analysis of particle size, encapsulation efficiency, and loading amount, EGCG-NPs at an EGCG/OVA molar ratio of 2:1 was selected for the subsequent studies.

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RESULTS AND DISCUSSION Preparation and Characterization of NPs. The selfassembling method is a well-established strategy for producing protein-based NPs.30 Hence, it was applied to fabricate EGCGNPs in this study. Theoretically, denaturation of OVA occurs under heating, so that the hydrophobic cores inside OVA molecules become exposed to aqueous phase.31 Subsequently, the hydrophobic interactions lead to the aggregation of NPs, which is eventually stiffened through the formation of disulfide bridges.32 As reported, particle size, size distribution, and surface charge are important parameters for NPs, which greatly impact their long-term stability, drug release behavior, cellular internalization profile and biodistribution.33 Here, the heating temperature of 80 °C was selected to fabricate EGCG-NPs with various molar ratios of EGCG and OVA. The reason is that OVA tends to form NPs with smallest particle size at this C

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Figure 2. (a) In vitro release profiles of EGCG molecules from EGCG-NPs in a buffer without GSH. The GSH-responsive EGCG release profiles in buffers with (b) pH 7.4 or (c) pH 6.8. Data are given as mean ± standard error of mean (SEM); n = 3.

Figure 3. (a) In vitro toxicity of pristine EGCG and EGCG-NPs at different EGCG concentrations against Raw 264.7 macrophages after coincubation for 24 h. Data are given as mean ± SEM (n = 5). (b) Amounts of cellular internalization of EGCG into macrophages after incubation with pristine EGCG or EGCG-NPs for various time periods. Data are given as mean ± SEM (n = 3).

Particle size and surface charge have been considered as critical factors to evaluate the stability of NPs,35,36 and thus, these two factors were investigated. As can be seen in Figure S1, no drastic change was found in their particle size and zeta potential during a 7-day incubation at room temperature, suggesting the hydrodynamic stability of EGCG-NPs in aqueous condition. Figure 1a showed the representative SEM images of EGCGNPs (molar ratio = 2:1), revealing that they possessed spherical shape and smooth surface. Moreover, these NPs in SEM images had an average particle size less than 125.7 nm and narrow size distribution (Figure 1b). The diameter determined by SEM was significantly smaller than those measured by DLS because EGCG-NPs were at swelling state in DLS measurement, while they were in the deswollen state in SEM test. These finding was consistent with our previous reports.25,37 To determine the crystalline phases of EGCG in EGCGNPs, we studied the corresponding XRD pattern of EGCGNPs, with pristine EGCG and OVA powders as controls. As presented in Figure 1c, the representative XRD diffractogram of pristine EGCG had numerous sharp peaks, suggesting that it was in crystalline nature. Conversely, OVA powders and EGCG-NPs showed a full absence of these representative peaks, revealing that no crystalline complex was formed between EGCG and its OVA matrix. Therefore, these results provided clear evidence that the crystal EGCG was converted into amorphous molecules inside OVA matrix. The FTIR spectra of pristine EGCG, OVA powder, and EGCG-NPs are depicted in Figure S2. In the context of pristine EGCG, the bands presented at 3400 and 1663 cm−1

were due to O−H (phenolic hydroxyl) and C−O group, respectively. The aromatic rings CC (skeleton structure) was registered to 1450 and 1600 cm−1. As to the FTIR spectra of OVA powders and EGCG-NPs, peaks at 1545 and 1650 cm−1 were attributed to their N−H (scissoring vibrations) and C−N (resonance vibrations), respectively. The wide peak around 3200−3500 cm−1 was ascribed to the polar bands in hydroxyl, amine, and carboxyl groups. Furthermore, we found that the shape of peak around 3200−3500 cm−1 in EGCG-NPs was relatively flat compared with that of OVA powder, which was indicative of the newly formed hydrogen bonds and the conjugation between EGCG molecule and OVA within NPs. In Vitro Drug Release Profile. Drug release behavior has been considered as a vital feature for drug-loaded systems, which determines their application prospect.38 Figure 2a revealed the in vitro release properties of EGCG from EGCG-NPs as a function of pH. It was obvious that EGCGNPs displayed similar EGCG release rates at different pH conditions, but a decrease at pH from 7.4 to 6.2 was associated with a slight increase in EGCG release rate, which was probably ascribed to the enhanced degradation of OVA in acidic environment. Moreover, approximately 40% of the trapped EGCG was released from EGCG-NPs after 24 h of incubation, and over 70% cumulative EGCG release was achieved within 96 h in different releasing buffers with various pH conditions. It can be clearly seen that EGCG-NPs produced a biphasic EGCG release behavior, which was manifested by a burst release of EGCG within the first 24 h of incubation and a subsequent slower release period. In this study, the burst release phenomenon could be contributed to the fast dispersion of EGCG absorbed in the superficial D

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Figure 4. (a) Comparative studies of in vitro anti-inflammatory activities between blank NPs and EGCG-NPs. Comparative studies of in vitro antiinflammatory activities between pristine EGCG and EGCG-NPs in terms of (b) TNF-α, (c) IL-6, and (d) IL-12. Data are given as mean ± SEM (n = 3).

caused no obvious cytotoxicity against Raw 264.7 macrophages in the range of applied dosages, indicating their excellent biocompatibility. Intracellular Uptake. Efficient cellular internalization is a prerequisite to improve the therapeutic efficacy of EGCG. To quantitatively compare the cellular internalization efficiencies of pristine EGCG and EGCG-NPs, we conducted the cell lysis and the dissociation of EGCG and OVA in EGCG-NPs, and we further evaluated the amount of intracellular EGCG over time for up to 6 h (Figure 3b). The assimilated amount of EGCG increased with the incubation time for both pristine EGCG and EGCG-NPs. Moreover, the final assimilated amount of pristine EGCG was only 7.0 μg after 6 h of incubation, which was 2.1-fold lower than that in the EGCGNP-treated cells. These results clearly suggested that endocytosis of NPs could mediate much higher efficiency in the cellular internalization of EGCG, in comparison with pristine EGCG. Although NPs remarkably enhanced the cellular uptake amounts of EGCG compared with pristine EGCG, they caused negligible cytotoxicities (Figure 3a). The possible reason is that EGCG-NPs have the capacity to constant release drug in the intracellular space and thus maintain the EGCG concentration within the optimal ranges. However, with the treatment of pristine EGCG, cells would be rapidly exposed to EGCG with relatively high concentration, resulting in strong toxicity. In Vitro Anti-Inflammation Test. TNF-α, one of the major pro-inflammatory factors, is mainly synthesized and excreted by macrophages in the pathological process of UC.43 Therefore, we investigated whether EGCG-NPs could suppress the secretion of TNF-α in activated macrophages. Figure 4a indicated that LPS-treated cells (positive control) significantly

domain of EGCG-NPs into the releasing buffers, and the following moderate release rate was due to the migration of EGCG from the internal domain to the NP surface. Collectively, we could conclude that EGCG-NPs had the capacity to realize a controlled EGCG release for more than 5 days. Although the extracellular environment is oxidizing, the intracellular compartment is in a reductive condition, and the amount of intracellular soluble GSH was significantly elevated during the activation of macrophages.39−41 Thus, the bioreducible property of EGCG-NPs was also examined in the present study. Figure 2b,c showed the release behaviors of EGCG from EGCG-NPs with or without GSH. The EGCG release rates in the presence of GSH were much faster than that in buffers without GSH. As reported, an OVA molecule has four interior sulfhydryl groups and one disulfide bond.34 The mechanism involved in the GSH-responsive release of EGCG might be ascribed to the breakage of disulfide bridges and the further decomposition of EGCG-NPs induced by GSH. The above results also suggested that EGCG-NPs were pretty stable in extracellular environment. Conversely, they became unstable in intracellular condition, which would facilitate the EGCG release within cells. In Vitro Biocompatibility Test. Cytotoxicity is a significant concern for drug-loaded systems, which are designed for UC therapy. Thus, the MTT test was utilized to determine the cytotoxicity of pristine EGCG and EGCGNPs at different EGCG concentrations against Raw 264.7 macrophages. As seen in Figure 3a, pristine EGCG exhibited very strong cytotoxicity, and inhibited the cell proliferation by over 60% at the concentration of 100 μg/mL, which was consistent with a previous study.42 In contrast, EGCG-NPs E

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Figure 5. Immunological responses to NPs in mice. (a) Pro-inflammatory cytokines and (b) immunological cells in the blood of mice injected with PBS (control), blank NPs, or EGCG-NPs. Data are given as mean ± SEM (n = 3).

boosted the production of TNF-α compared with the negative control. The blank NPs without EGCG slightly decreased TNF-α secretion, whereas no statistical significance was found between blank NP-treated cells and positive control cells. Importantly, TNF-α secretion was remarkably decreased after the treatment of EGCG-NPs. We further found that EGCGNPs exhibited much stronger anti-inflammatory ability than pristine EGCG (Figure 4b), which could be attributable to the enhanced cellular internalization of EGCG mediated by NPs. Additionally, the secretion of other major pro-inflammatory mediators (IL-6 and IL-12) showed similar decrease patterns as that of TNF-α (Figure 4c,d). Interestingly, Figure S3 suggested that treatment with pristine EGCG or EGCG-NPs significantly promoted the amounts of the anti-inflammatory factor (IL-10). Notably, the amount of IL-10 secreted in the supernatant of EGCG-NP-treated cells was obviously higher than that of cells with the treatment of pristine EGCG. Hemocompatibility and Immunogenicity Tests. Hemocompatibility of the therapeutics needs to be evaluated prior to intravenous administration of NPs.44 Figure S4a,b showed that the positive control (1% Triton X-100) induced 100% hemolysis. However, there was no evident hemolysis observed in the tubes treated with EGCG-NPs even under the concentration up to 10 mg/mL. To further investigate the hemocompatibility of NPs in vivo, mice were injected with EGCG-NPs. After 6 or 24 h of injection, blood was collected for routine hematological analysis, and numerous hematological parameters were examined, including white blood cells (HBC), red blood cells (RBC), mean corpuscular hemoglobin (MCH), hemoglobin (HGB), and mean corpuscular hemoglobin concentration (MCHC). Figure S4c clearly indicated that there were no significant differences in these key hematological parameters between EGCG-NP-treated group

and normal control group, which were in accordance with the in vitro hemocompatibility results (Figure S4a,b). The immunological response to NPs was a critical issue in their application in disease treatment. Thus, we intravenously injected different NPs in mice, and further evaluated the outcomes of immune responses. As illustrated in Figure 5a, there was no difference in the amounts of the main proinflammatory mediators, including TNF-α, IL-6, and IL-12, between blank NP- or EGCG-NP-treated mice and control mice. We further found that the percentages and numbers of the main immunological cells in blood were within the normal ranges (Figure 5b). In summary, these results clearly suggest that these OVA-based NPs have excellent blood compatibility and no obvious immunological responses of a living organism. In Vivo Therapeutic Efficacy of NPs against UC. The DSS-induced UC mouse model has the main histological features (e.g., body weight loss, disruption of the colonic epithelium barrier and reduction in colon length, as well as the accumulation of immune cells), which are very close to the symptom of human UC.45,46 Hence, we applied this mouse model to investigate the therapeutic efficacy of EGCG-NPs against UC. As the drug distribution in disease sites is extremely important for UC therapy, we quantitatively compared the in vivo distribution of pristine EGCG and EGCG-NPs. As presented in Figure S5, EGCG-NP-treated mice with UC had remarkably higher EGCG amounts in colitis tissue sites, in comparison with pristine EGCG-treated mice. Moreover, we investigated the therapeutic efficacies of NPs against UC. Figure 6a showed that the mice body weight slightly increased over time in the healthy control group, whereas DSS induced body weight loss by approximately 20% after 10 days. We also noticed that there were significantly different therapeutic effects in various treatment groups. Importantly, the EGCGF

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Figure 6. (a) Body weights, (b) MPO activities in colon, (c) spleen weights, (d) colon images, (d) colon lengths, and (e) colon weights of mice from various treatment groups. Data are given as mean ± SEM (n = 5).

NP-treated group exhibited no obvious body weight loss, whereas the group treated with pristine EGCG showed body weight loss by approximately 5.8%. MPO is mainly produced by macrophages and granulocytes, and its concentration in colon tissue is taken as an important indicator of UC severity. As can be seen in Figure 6b, MPO concentrations in healthy control group and EGCG-NPtreated DSS group were much higher than that in the other three groups. More importantly, EGCG-NP-treated DSS group produced no statistically significant differences in the MPO activities, when compared with the healthy control group. As expected, we found a positive correlation between the increase of MPO activity and the body weight loss. Figure 6c showed that healthy control group possessed the lowest value in spleen weight. DSS control group and blank NP-treated DSS group developed considerably heavier spleen (up to 250 mg), in comparison with the other three groups. Furthermore, spleen weights were slightly increased in both EGCG-NP-treated DSS group and pristine EGCG-treated DSS group, which were not significantly different from those recorded in healthy control group. Meanwhile, an opposite trend was found in colon length (Figure 6d,e) and colon

weight (Figure 6f) for all the examined groups compared with the results of spleen weight (Figure 6c). H&E staining was further carried out to verify the therapeutic outcomes of EGCG-NPs against UC. As expected, there is no obvious inflammatory region in the colonic section from healthy control group (Figure 7a).47 However, the colonic sections from DSS control group (Figure 7b) and blank NP-treated DSS group (Figure 7c) displayed an obvious evidence of inflammation, including disruption of colonic epithelia layer and accumulation of inflammatory cells. With respect to pristine EGCG-treated DSS group, the histological analysis showed a clear decrease in the severity of inflammation (Figure 7d) compared with that of DSS control group. Meanwhile, colon tissues from EGCG-NP-treated DSS group merely showed a minor injury in mucosa, while almost no accumulation of inflammatory cells was detected in the colonic mucosa that maintained a similar morphology as the healthy control (Figure 7e). It was important to note that the sections of major organs from all the mouse groups after treatments exhibited no histopathological evidence of damages (Figure 8), demonstrating the excellent biocompatibility of EGCG-NPs. G

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Figure 7. H&E staining of colonic sections from various mice groups. (a) Healthy control group, (b) DSS control group, (c) blank NP-treated DSS group, (d) pristine EGCG-treated DSS group, and (e) EGCG-NP-treated DSS group (scale bar = 200 μm).

Figure 8. H&E staining of the sections of heart, liver, spleen, lung, and kidney from various mice groups (scale bar = 100 μm).

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CONCLUSIONS In the present study, epigallo-catechin 3-gallate (EGCG)loaded ovalbumin (OVA)-based nanoparticles (EGCG-NPs) were successfully produced via self-assembly between OVA and EGCG. The synthetic process was simple and facile without organic solvents or hostile environments. The obtained NPs had a mean diameter of around 202.9 nm, monodispersity and slightly negative surface charge. In addition, they displayed a glutathione-responsive EGCG

release behavior in the reductive microenvironment. Benefited from their unique physicochemical properties, EGCG-NPs were able to be efficiently internalized into Raw 264.7 macrophages, suppress the production of the main proinflammatory mediators, and increase the secretion of antiinflammatory factor (IL-10). The in vivo experiments clearly indicated that intravenously administered EGCG-NPs produced significantly better therapeutic outcomes against ulcerative colitis (UC) than pristine EGCG. Collectively, the H

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ACS Sustainable Chemistry & Engineering

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obtained EGCG-NPs may serve as a promising therapeutic nanoplatform for efficient UC therapy with minimal systemic toxicity.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.8b01613.

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Experimental description, variations in diameters and zeta potentials of EGCG-NPs, FTIR spectra of samples, in vitro IL-10 secretion amounts, hemocompatibility of NPs, and in vivo distribution of EGCG (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail for Y.K.: [email protected]. Tel.: +86-6825-3204. Fax: +86-6825-4056. *E-mail for B.X.: [email protected]. Tel.: +86-68254762. Fax: +86-6825-4056. ORCID

Yan Liu: 0000-0003-3677-9290 Liang Zeng: 0000-0003-3860-7724 Zhigang Xu: 0000-0003-1805-5061 Yuejun Kang: 0000-0002-1021-0349 Changming Li: 0000-0002-4041-2574 Bo Xiao: 0000-0002-2992-6435 Author Contributions

S.G. and B.X. designed experiments, supervised the project, and wrote the draft of manuscript. S.G., Q.C., Y.L., L.Z., H.S., Z.X., Y.K., and C.L. performed the experiments. Y.K. and B.X. edited and revised the manuscript. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from the National Natural Science Foundation of China (51503172 and 81571807), the Fundamental Research Funds for the Central Universities (XDJK2017B058), the Young Core Teacher Program of the Municipal Higher Educational of Chongqing and the State Key Laboratory of Silkworm Genome Biology.

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REFERENCES

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DOI: 10.1021/acssuschemeng.8b01613 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acssuschemeng.8b01613 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX