Sodium butyrate | Perk signal

Polyvinyl Butyrate Nanoparticles as Butyrate Donors for Colitis Treatment

ImageYunmei Mu, Yusuke Kinashi, Jinting Li, Takuma Yoshikawa, Akihiro Kishimura, Mitsuru Tanaka, Toshiro Matsui, Takeshi Mori,* Koji Hase,* and Yoshiki Katayama*

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ABSTRACT: Butyrate has been attracting attention for the suppression of inﬂammatory bowel disease (IBD). However, clinical trials of butyrate for IBD treatment have resulted in controversial outcomes, likely owing to the adverse eﬀect of butyrate on the intestinal epithelium that was observed at high butyrate concentrations. Herein, we propose polyvinyl butyrate (PVBu) nanoparticles (NPs) as butyrate donors for delivery to the lower part of the intestine for the treatment of colitis. The PVBu NPs suppressed the inﬂammatory activation of macrophages in vitro, although sodium butyrate inversely further activated macrophages. Oral admin- istration of NPs did not change the luminal concentration of free butyrate; however, NPs showed a therapeutic eﬀect on a colitis mouse model. In addition, incorporation of vitamin D3 into the NPs enhanced the therapeutic eﬀect on colitis. Hence, PVBu NPs are a promising therapeutic for IBD treatment, not only as a butyrate donor but also as a carrier for hydrophobic drugs like vitamin D3.

KEYWORDS: butyrate, polyvinyl butyrate, nanoparticles, inﬂammatory bowel disease, vitamin D3

⦁ INTRODUCTION
Inﬂammatory bowel disease (IBD) is an autoimmune
diseaseincluding Crohn’s disease and ulcerative colitis that is characterized by long-term chronic inﬂammation in the gastrointestinal (GI) tract.1 Immune cells in the intestine that maintain the intestinal homeostasis are conversely activated in IBD.2 Alteration of gut microbiota and derived metabolites have been implicated in the pathogenesis of IBD.3 Despite the
eﬀorts of researchers, there is currently no eﬀective medicine for IBD.4
Butyrate, which is a metabolite of dietary ﬁber produced by intestinal bacteria, has attracted attention as a potential therapeutic for IBD. Butyrate exists in high concentration (11−25 mM) in human feces5−7 and works as an inhibitor of histone deacetylase (HDAc)8,9 or a ligand of G protein- coupled receptors10,11 in epithelium and immune cells including macrophagesto preserve gastrointestinal health,

Received: August 31, 2020
Accepted: January 25, 2021
Published: February 11, 2021
maintaining the intestinal barrier and the anti-inﬂammatory conditions in mucosal immunity.12 Patients with IBD have been reported to have a reduced intestinal concentration of butyrate.7,13 Oral supplementation of butyrate has been used for the treatment of an animal colitis model.12−16

The approaches used in these studies to avoid rapid absorption of butyrate in the upper part of the GI tract include feeding a Because of the unpleasant smell and taste of butyrate, rectal enema and oral application of capsules embedded with butyrate have been developed for use in clinical trials.18−22 The reported therapeutic eﬀects of butyrate in the clinical trials have been controversial.5 A possible reason for the controversial results for butyrate is its conﬂicting eﬀect on mucosal health. High concentrations of butyrate showed a negative eﬀect as a result of suppressing proliferation of intestinal epithelia23 and inducing production of inﬂammatory cytokines from immune cells.24 It is diﬃcult to control the luminal concentration of butyrate with the current rectal enema and oral capsule formulations, which results in the risk
of inducing adverse eﬀects of butyrate. The ideal formulation of butyrate would allow for oral delivery to the lower part of the intestine with controlled luminal concentration.

Here, we propose polyvinyl butyrate (PVBu) as a novel butyrate donor for IBD treatment (Figure 1). Although PVBu is water-insoluble, its water solubility increases gradually with the hydrolysis of butyrate. Because the glass transition butyrate-mixed diet14,15 and feeding starch modiﬁed with
butyrate through ester bonds.16,17

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https://dx.doi.org/10.1021/acsabm.0c01105

ACS Appl. Bio Mater. 2021, 4, 2335−2341
Proposed mechanism of action of PVBu NPs for IBD treatment.

■temperature of PVBu is below the room temperature, it exists as a melt at room temperature. Owing to the tunable hydrophobicity and ﬂexibility of the PVBu chain, nanoparticles (NPs) prepared from PVBu are expected to release butyrate at moderate speed by allowing access to hydrolysis enzymes. Accumulation in intestinal inﬂammatory lesions has been reported to be enhanced by tuning the NPs size to a few hundred nanometers.25,26 The small size allows the NPs to be engulfed by inﬂammatory cells such as resident macrophages, which are representative cells that respond to butyrate.8 In addition, we evaluate the suitability of PVBu NPs as a carrier for hydrophobic drug for delivery to the lower part of the intestine.

EXPERIMENTAL SECTION

Materials. Vinyl butyrate (VBu) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Azobisisobutyronitrile (AIBN), acetone, methanol, and toluene were purchased from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). Lipase from porcine pancreas and Pluronic F-127 were purchased from Sigma-Aldrich (St.Louis, USA). Calcitriol was obtained from Cayman chemical (≥97%, Michigan, USA). 3,3′-Dioctadecyloxacarbocyanine perchlorate (DiO) was purchased from Takara-Clontech (Shiga, Japan). 1,1′-Dioctade- cyltetramethyl indotricarbocyanine iodide (DiR) was purchased from
Caliper Life Sciences (Hopkinton, USA). Dextran sulfate sodium (DSS), colitis grade (36,000−50,000 Mw) was obtained from MP Biomedicals (Irvine, USA).
Synthesis of PVBu.

PVBu was synthesized by radical polymer- ization. To a 200 mL round-bottom ﬂask was added vinyl butyrate (20 g, 175 mmol), AIBN (0.28 g, 1.7 mmol), and acetone (80 g). Then, the solution was septa sealed and purged with nitrogen for 30 min. The ﬂask was then transferred to a preheated water bath at 50 °C to polymerize for 48 h. The polymerization solution was dialyzed against methanol for 48 h. Following dialysis, the methanol was removed via rotary evaporation and then lyophilized. The obtained polymer was conﬁrmed by 1H NMR (CDCl3, 400 MHz) spectros- copy.
Gel Permeation Chromatography Analysis. Weight average and number average molecular weights (Mw and Mn) of the polymer were determined on a multi detector SEC system ﬁtted with a Malvern Viscotek TDA 305 system and three Tosoh TSK-gel columns (TSKgel GMHHR-H × 2, G2000HHR). Tetrahydrofuran was used as the eluent with a ﬂow rate of 1.0 mL/min at 40 °C, and the system was calibrated to polystyrene standards from Agilent Technologies.
Preparation of PVBu NPs. 2 mL of toluene solution containing PVBu (0.4 g/mL) was prepared [in the cases of incorporation, vitamin D3 (1,25(OH)2D3) (0.63 μg/mL) or DiO or DiR (10 μg/ mL) was added to the toluene solution]. This solution was added to an aqueous solution (20 mL) containing Pluronic F-127 (5%) and homogenizer (T25 digital Ultra turax, IKA, Germany) at 12,000 rpm for 10 min and sonicated with a probe sonicator [UD-211 (TOMY) equipped with a TP-040 tip] for 5 min (20% power, 20 kHz, 20 W). Toluene was then evaporated from the solution by stirring (300 rpm) overnight. The NP dispersions were stored as prepared at 4 °C. Entrapment eﬃcacy of vitamin D3 in NPs was determined by HPLC following our previous report.27
Measurement of the NP Size and ζ-potential. The size, ζ- potential, and polydispersity index (PDI) of the obtained NPs were measured by a Zeta Sizer Nano Series (Malvern Instrument, UK) analyzer at 25 °C following our previous report.27

Butyrate Release from NPs. 100 mg of lipase was suspended in 40 mL of 2.5 mM Tris−HCl buﬀer (pH 7.4) containing NPs (34 mM butyrate conc.) or VBu (34 mM). The enzymatic reaction was carried out at 37 °C with the pH-stat method using an automatic titrator (DKK-TOA Co., Japan) as reported previously.28 Butyric acid released from the NPs was quantitatively titrated with 20 mM NaOH to maintain a constant pH (pH 7.4). During the enzymatic reaction, the dispersion was stirred vigorously with a magnetic stirrer. Cell Culture. RAW 264.7 macrophages and RAW 264.7 macrophages transfected with secreted alkaline phosphatase (SEAP) as a reporter gene under the transcriptional control of an NF-κB response element were cultured in complete Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 10% fetal bovine serum, 4.5 mg/mL glucose, 100 U/mL penicillin, and 100 U/mL streptomycin, supplemented with 500 μg/mL G418. The cells were maintained in a humidiﬁed environment containing 5% CO2 and 95% air at 37 °C.
Cellular Uptake of NPs.

RAW264.7 macrophages (1 × 105 cells/ mL) were seeded in a 96-well glass surface plate. The cells were washed with Dulbecco’s phosphate-buﬀered saline (PBS) after incubation for 24 h. NP-DiO (5 mM butyrate conc.) dispersed in DMEM was added to the wells. After incubation for 24 h at 37 °C, the cells were observed using ﬂuorescence microscopy (BZ-X700, Keyence Co., USA).
Suppressive Effect of NPs on Macrophage Activation. SEAP-transfected RAW 264.7 macrophages (1 × 106 cells/mL) were cultured with DMEM in a 96-well plate overnight. Then, DMEM was replaced with DMEM containing sodium butyrate (SB) (0.1−5 mM) or NPs (5 or 20 mM butyrate conc.). After incubation for 6 h, lipopolysaccharide (LPS) (ﬁnal conc. 20 ng/mL) was added, and the plate was incubated for 18 h. The supernatants were collected for measurement of the SEAP level as described in a previous study.29

Observation of Retention of NPs in the GI Tract. Five-week- old C57BL/6J male mice were purchased from Kyudo Co., Ltd (Saga, Japan). Mice were housed in a facility maintained at 25 °C with a 12 h light/dark cycle. All animal experiments were performed using protocols approved by the Animal Ethics Committees of Kyushu University. The mice were fed with AIN 76A puriﬁed diet (Research Diets, Inc., USA) for 1 week. The mice were administrated NP-DiR [0.27 g/mL PVBu (2.4 M butyrate conc.), 10 mL/kg] by oral gavage. The mice were euthanized after 1, 3, or 4 h, and the intestine was resected and soaked in PBS for ﬂuorescence measurement using an IVIS Lumina Imaging system (Xenogen Co., USA).

Measurement of Butyrate Concentration in Cecum. The butyrate concentration of the cecum contents was determined by gas chromatography (GC) according to the reported method.14 The 5- week-old C57BL/6J mice were fed with the AIN93G-formula diet (Oriental Yeast Co., Ltd, Japan) for 1 week. The mice were then orally administered the NPs [0.27 g/mL PVBu (2.4 M butyrate conc.), 10 mL/kg]. After 4 h, the mice were euthanized, and 100 mg of the cecum contents was disrupted and homogenized in 1 mL of water containing 5 ppm of isobutyric acid as an internal standard. The homogenates were mixed with extraction solution (mixture of 500 μL of 2.5 M hydrochloric acid and 10 mL of ether) followed by vigorous shaking and overnight storage at 4 °C. After centrifugation of the mixture, the ether layer was collected and dehydrated with anhydrous sodium sulfate. The samples were measured using a GC-18A (Shimadzu, Japan) chromatograph equipped with a DB-FFAP column (30 m × 0.32 mm × 0.25 μm) and a ﬂame ionization detector. The

GC experiment was run under the following conditions: carrier gas, helium at a ﬂow rate of 0.95 mL/min (50 kPa); split ratio, 1:20; temperature program (40−240 °C, 5 °C/min); and detector temperature 240 °C.
DSS-Induced Colitis Model Study. The 5-week-old C57BL/6J male mice were fed a low-ﬁber diet (AIN93G-formula diet). Five mice in each group received drinking water containing NPs (94 or 190 mM butyrate conc.), NP-D3 NPs (94 mM butyrate conc., 5.6 ng/mL vitamin D3 conc.), or SB (190 mM) for 10 days. On day 3, the mice were treated with 2% wt/v DSS in drinking water for 7 days, until day
9. On the last day, the mice received water without DSS. Body weights and clinical scores based on the severity of diarrhea and colonic hemorrhage30 were monitored daily. After euthanizing the mice on day 10, the colon was collected to measure its length.

■Statistical Data Analyses. Statistical data analyses were performed using analysis of variance followed by Dunnett’s test. A p value of <0.05 is the minimal level of statistical signiﬁcance.

RESULTS AND DISCUSSION
NP Preparation. PVBu was prepared by radical polymer- ization using AIBN as an initiator. PVBu was puriﬁed by dialysis and obtained as a viscous liquid. 1H NMR of the obtained PVBu showed the successful removal of impurities (Figure S1). Mw and Mw/Mn were determined by gel permeation chromatography (GPC) analysis to be 1.6 × 104 and 1.8, respectively (Figure S2). PVBu NPs were prepared by the O/W emulsion method using Pluronic F-127 as a stabilizer, followed by evaporation of the organic solvent by stirring overnight. To incorporate vitamin D3 (dihydroxycho- lecalciferol; 1,25(OH)2D3) as a hydrophobic drug or ﬂuorescent molecules for labeling, these compounds were dissolved in the organic phase for emulsiﬁcation. The entrapment eﬃciency of vitamin D3 in the NPs (NP-D3) was determined to be 65.6 ± 4.0%. The characteristics of the NPs are summarized in Table 1. The NP sizes were tuned to

Table 1. NP Propertiesa

name size (nm) PDI ζ-potential (mv)
NP 193 ± 1.6 0.20 ± 0.01 −0.6 ± 0.14
NP-D3 203 ± 2.1 0.22 ± 0.01 −0.7 ± 0.32
NP-DiO 190 ± 0.2 0.21 ± 0.01 −0.5 ± 0.03
NP-DiR 191 ± 0.6 0.21 ± 0.01 −0.6 ± 0.09
aData are given as mean ± SD (n = 3).
around 200 nm, which is suitable for accumulation in inﬂammatory lesions in the intestine.25,31−33 The surface charge was almost neutral owing to the surface coating of Pluronic F-127. Because the glass transition temperature of PVBu is below the room temperature, the NPs were found to fuse with each other upon freeze drying. The stability of the aqueous dispersion of NPs during storage at 4 °C was therefore examined (Figure 2). The size and PDI of the NPs were almost constant for at least 56 days, indicating the high stability of the NP dispersion.
When the NPs are administered orally, there are several pathways for butyrate release: pancreatic enzymes in the lumen of the upper intestine, intestinal bacterial enzymes, and intracellular enzymes of intestinal cells. We examined the release of butyrate from NPs using pancreatic lipase, which is one of the release pathways. As shown in Figure 3, butyrate release from the NPs was much slower than that from the VBu monomer and rapidly saturated at a lower butyrate release. This release proﬁle is thought to be due to the limited access of Size and PDI of NPs during storage as an aqueous dispersion at 4 °C. 0.27 mg/mL NPs in 10 mM HEPES buﬀer (pH 7.4).

Release of butyrate from vinyl butyrate (VBu) and NPs by pancreatic lipase at 37 °C. 100 mg of lipase was suspended in 40 mL of 2.5 mM Tris−HCl buﬀer (pH 7.4) containing NPs (34 mM butyrate conc.) or VBu (34 mM). the lipase to the hydrophobic PVBu chains in aqueous media. The resistance of the NPs to lipase indicates that orally administered NPs may provide protection from the rapid hydrolysis and consumption of butyrate in the upper intestine. Cellular Uptake and Anti-Inﬂammatory Effect of NPs in Vitro. Macrophages play an important role in the progression of the inﬂammation in colitis.8 First, we assessed the cellular uptake of the NPs by the macrophage cell line RAW264.7. As shown in Figure 4A, NP-DiO was actively taken up by macrophages. Then, the anti-inﬂammatory eﬀect of the NPs was examined in terms of suppression of NF-κB signaling
in the macrophages induced by LPS. LPS is a ligand of toll-like receptor 4, which induces an inﬂammatory response mainly through the NF-κB pathway.34 Here, we used RAW264.7 transfected with SEAP, which secret SEAP as a reporter gene via NF-κB signaling. As shown in Figure 4B, low doses of SB (≤1 mM) had no eﬀect on NF-κB signaling in LPS-stimulated macrophages, whereas 5 mM SB activated NF-κB signaling. Activation of the inﬂammatory response of macrophages by high concentrations of SB has been reported previously.35 In contrast, the NPs showed signiﬁcant suppression of NF-κB signaling at high doses (5 and 20 mM).

The diﬀerent eﬀects of the NPs and SB on the NF-κB signaling are thought to result from the diﬀerence in their mode of action on the macrophages. Free butyrate binds both cell surface butyrate receptors and intracellular HDAc following uptake via endocytosis or transporters.8 In contrast, NPs do not stimulate cell surface butyrate receptors but slowly release butyrate when subjected to intracellular esterase or lipases after endocytotic uptake to inhibit HDAc. The selectivity of NPs for HDAc over the cell surface butyrate receptors may be the reason for their suppressive eﬀect on NF-κB signaling. This is an ideal characteristic for a butyrate donor intended for treating intestinal inﬂammation by targeting inﬂammatory cells such as macrophages.

Cellular uptake and anti-inﬂammatory eﬀect of NPs in RAW264.7. (A) Cellular uptake of NP-DiO by RAW264.7 after incubation for 24
Imageh. (B) Suppressive eﬀect of SB and NPs on the LPS-induced NF-κB activation in RAW264.7. 6 h after the addition of SB or NPs, LPS (20 ng/mL) was added, and the samples were incubated for a further 18 h. The SEAP level in the supernatant was measured using a substrate. Data are given as mean ± SD (n = 3). ***p < 0.001 vs control; ###p < 0.001 and ns denotes not signiﬁcant vs non.

NP retention in the GI tract and butyrate concentration in cecum. (A) Following oral gavage administration of NP-DiR [0.27 g/mL PVBu (2.4 M butyrate conc.), 10 mL/kg body weight], intestines were collected from euthanized mice at each time point and observed by IVIS.
(B) Butyrate concentration in the cecum contents 4 h after oral gavage of NPs (same dose as panel A). Data are given as mean ± SD (n = 5). ns denotes not signiﬁcant.

Retention of NPs in the GI Tract and Butyrate Concentration in the Cecum. We examined the retention of NP-DiR in the GI tract after oral gavage administration in mice. As shown in Figure 5A, at 1 h, most of the DiR ﬂuorescence was observed in the stomach and intestine; then at 3 h, the ﬂuorescence had moved to the lower part of the intestine, cecum, and large intestine. At 4 h, most of the ﬂuorescence could no longer be detected. The retention time of NP-DiR in the GI tract is similar to those of particles with a similar diameter.36,37
We measured the increment of butyrate concentration in theGI tract by oral administration of NPs. We followed a reported protocol in which the increment of butyrate concentration was detected for butyrate-modiﬁed starch fed mice. Mice were fed a low-ﬁber diet to minimize diet-originating butyrate.14 4 h after oral gavage administration of NPs at a relatively high dose (∼0.5 mmol/mouse), the amount of free butyrate in the cecum was measured.

As shown in Figure 5B, the NPs showed no signiﬁcant diﬀerence in butyrate concentration compared with the nontreated group. Compared with butyrate-modiﬁed starch, the main chain of PVBu is not biodegradable, which may be the reason for the slow release of butyrate in the GI
tact. It is notable that the NPs did not increase the free butyrate concentration in the cecum even at such a high dose. NP-Ameliorated DSS-Induced Colitis. We assessed the therapeutic eﬀect of NPs on a colitis mouse model induced by DSS. The treatment scheme is shown in Figure 6A. NPs or SB was applied in drinking water, and two butyrate concentrations were examined; low (94 mM) and high (190 mM). DSS was added to the drinking water from day 3 to induce colitis. During the experiment, the daily intakes of water and feed were almost constant among all of the treatment groups (Figure S3). Figure 6B shows a representative colon of each group after 10 days of treatment, and Figure 6C summarizes the average colon length of ﬁve mice for each group, which reﬂects the severity of the colon inﬂammation. The colon length of healthy mice was 69.6 ± 4.0 mm. The NPs suppressed the shortening of the colon length more than SB at the same high dose, showing the advantage of the NPs in enhancing the eﬃcacy of butyrate delivery to the lower
intestine.

It is notable that the NPs did not raise the butyrate concentration in the cecum (Figure 5B) but showed the suppression of the inﬂammatory response both in vitro (Figure 4B) and in vivo (Figure 6). These results showed that the NPs Therapeutic eﬀect of NPs on a colitis mouse model induced by DSS. (A) Mouse treatment scheme. The application of samples (NP, NP-D3, SB) as drinking water was commenced 2 days before the application of 2% DSS. The three samples in drinking water contained the same concentration of butyrate (94 and 190 mM for low and high dose, respectively). Representative colon image (B) and average colon length (C) of mice from each treatment group following treatment for 10 days. Changes in body weight (D) and clinical score (E) during the treatment. Data are given as mean ± SD (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 vs the vehicle control.
††p < 0.01 for comparison as indicated. have moderate tolerance to hydrolysis enzymes in the intestinal lumen, while they provided enough intracellular concentration of butyrate in intestinal cells to induce anti-inﬂammatory eﬀect. Because a rapid increase of luminal concentration of butyrate induced toxicity,23,24 these characteristics of the NPs would be ideal as the butyrate donor.

The NPs containing vitamin D3 (NP-D3) showed a superior eﬀect to that of the NPs when compared at the same low dose, indicating the synergetic eﬀect of the NPs and vitamin D3. The reduction in body weight due to the progression of DSS was suppressed at a high NP dose and a low NP-D3 dose (Figure 6D). The superior eﬀects for these two treatment groups were also observed in the clinical scores evaluated based on the severity of diarrhea and colonic hemorrhage (Figure 6E). Vitamin D3 is the ligand for the nuclear receptor (vitamin D3 receptor), which regulates the expression of various genes in many cell types.38 Recent experiments based on in vitro and IBD model animal experiments have revealed that vitamin D3 contributes to suppressing the progression of IBD through multiple pathways including the suppression of activation of immune cells.39 However, without using an eﬀective carrier to the lower part of the intestine, oral application of free vitamin D3 shows no therapeutic eﬀect on DSS-induced colitis mice.29

Thus, the synergy of NP-D3 showed that NPs work not only as a donor of butyrate but also as an eﬀective carrier for vitamin D3 delivery to the lower intestine.In this study, we examined PVBu NPs as a butyrate donor for delivery to the lower part of the intestine for the treatment of colitis induced by DSS. The NPs showed a superior suppressive eﬀect on the inﬂammatory response of macro- phages in vitro compared with SB. The superior eﬀect of the NPs is attributed to their selectivity in working more as an HDAc inhibitor than as a ligand for cell surface receptors of butyrate. Although oral administration of the NPs did not increase the luminal concentration of butyrate in the cecum, the NPs showed a therapeutic eﬀect on the colitis model mice, indicating that the NPs may target inﬂammatory cells such as resident macrophages by increasing the intracellular concen- tration of butyrate to suppress their activation. These characteristics of PVBu NPs make them suitable for avoiding the adverse eﬀects associated with high concentrations of butyrate. We also demonstrated that PVBu NPs are an eﬀective carrier for vitamin D3 which showed a synergistic eﬀect on the therapeutic eﬀect of butyrate. In conclusion, PVBu NPs are a promising therapeutic for IBD treatment, having both a suitable butyrate release proﬁle and the ability to accommodate hydrophobic drugs such as vitamin D3.

⦁ CONCLUSIONS

■ASSOCIATED CONTENT
*sı Supporting Information
The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acsabm.0c01105.

1H NMR spectrum of PVBu; GPC proﬁle of PVBu; and daily intake of diet and water (PDF)

⦁ AUTHOR INFORMATION

Corresponding Authors
Takeshi Mori − Graduate School of Systems Life Sciences, Department of Applied Chemistry, Faculty of Engineering, and Center for Future Chemistry, Kyushu University, Fukuoka 819-0395, Japan; orcid.org/0000-0002-1821- 5427; Email: [email protected]
Koji Hase − Division of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan; Division of Mucosal Barrierology, International
Research and Development Center for Mucosal Vaccines, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan; Email: [email protected]

Yoshiki Katayama − Graduate School of Systems Life Sciences, Department of Applied Chemistry, Faculty of Engineering,
ImageCenter for Future Chemistry, and International Research Center for Molecular System, Kyushu University, Fukuoka 819-0395, Japan; Department of Biomedical Engineering, Chung Yuan Christian University, Taoyuan 32023, Taiwan; orcid.org/0000-0002-4957-6241; Email: ykatatcm@
mail.cstm.kyushu-u.ac.jp

Authors
Yunmei Mu − Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 819-0395, Japan
Yusuke Kinashi − Division of Biochemistry, Faculty of Pharmacy, Keio University, Tokyo 105-8512, Japan
Jinting Li − Graduate School of Systems Life Sciences, Kyushu University, Fukuoka 819-0395, Japan
Takuma Yoshikawa − Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Fukuoka 819- 0395, Japan

Akihiro Kishimura − Graduate School of Systems Life Sciences, Department of Applied Chemistry, Faculty of Engineering, Center for Future Chemistry, and International Research Center for Molecular System, Kyushu University, Fukuoka 819-0395, Japan; orcid.org/0000-0002-0503- 1418
Mitsuru Tanaka − Research and Development Center for Five- Sense Devices, Kyushu University, Fukuoka 819-0395, Japan
ImageToshiro Matsui − Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School of Kyushu University, Fukuoka 819-0395, Japan; orcid.org/ 0000-0002-9137-8417
Complete contact information is available at: https://pubs.acs.org/10.1021/acsabm.0c01105

Notes
The authors declare no competing ﬁnancial interest.

⦁ ACKNOWLEDGMENTS
We thank Dr. Masaru Tanaka and Dr. Shingo Kobayashi for
the GPC analysis of PVBu. We thank Dr. Yukio Nagasaki for critical suggestions for animal experiment protocol.

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