Design of Synthetic Polymer Nanoparticles Specifically Capturing

Mar 8, 2019 - Synthetic polymers are of interest as stable and cost-effective biomolecule-affinity reagents, since these polymers interact with target...
0 downloads 0 Views 2MB Size
Download PDF
Article Cite This: Biomacromolecules XXXX, XXX, XXX−XXX

pubs.acs.org/Biomac

Design of Synthetic Polymer Nanoparticles Specifically Capturing Indole, a Small Toxic Molecule Anna Okishima,† Hiroyuki Koide,*,† Yu Hoshino,‡ Hiromichi Egami,§ Yoshitaka Hamashima,§ Naoto Oku,†,∥ and Tomohiro Asai†

Downloaded via MCMASTER UNIV on March 17, 2019 at 01:02:20 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

Department of Medical Biochemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan ‡ Department of Chemical Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan § Department of Synthetic Organic Chemistry, Graduate School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan ∥ Faculty of Pharma-Science, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan S Supporting Information *

ABSTRACT: Synthetic polymers are of interest as stable and cost-effective biomolecule-affinity reagents, since these polymers interact with target biomolecules both in vitro and in the bloodstream. However, little has been reported about orally administered polymers capable of capturing a target molecule and inhibiting its intestinal absorption. Here, we describe the design of synthetic polymer nanoparticles (NPs) specifically capturing indole, a major factor exacerbating chronic kidney disease, in the intestine. N-isopropylacrylamide-based NPs were prepared with various hydrophobic monomers. The amounts of indole captured by NPs depended on the structures and feed ratios of the hydrophobic monomers and the polymer density but not on the particle size. The combination of hydrophobic and quadrupole interaction was effective to enhance the affinity and specificity of NPs for indole. The optimized NPs specifically inhibited intestinal absorption of orally administered indole in mice. These results showed the potential of synthetic polymer NPs for inhibiting the intestinal absorption of a target molecule.

■

INTRODUCTION

effective reagent is of significant interest for the treatment and prevention of specific diseases and infections. Synthetic polymers such as linear polymers,18−20 dendrimers,21,22 and polymer nanoparticles (NPs)23−25 have emerged as stable and cost-effective affinity reagents and are expected to be alternatives to antibodies, since they are capable of binding to and neutralizing a target protein in vitro and/or in the bloodstream.26 For example, Schrader and co-workers have designed methacrylamide-based copolymers that have intrinsic affinity to target enzymes and bind to a specific epitope of the protein.27−29 Multiblock glycopolymers inhibit the lectin dendritic cell-specific intracellular adhesion molecule 3grabbing nonintegrin (DC-SIGN) by controlling the composition of glycomonomers.30,31 Dendrimers functionalized with many sulfate groups suppress the function of selectins expressed on the cell surface in vivo.32−34 Pieters and coworkers demonstrated that dendrimers functionalized with oligosaccharides neutralize toxins and inhibit bacterial

Oral intake of nutrients, drugs, and electrolytes is inevitable to sustain the normal life of human beings.1−3 These ingested substances are absorbed from the intestine into the body after the digestion by enzymes and/or metabolism by enteric bacteria. However, in the case of patients with kidney disease,4 hypertension5 or diabetes,6 the intestinal absorption of a certain specific molecule(s) must be inhibited. These molecules are contained in the daily foods or synthesized from essential nutrients by enteric bacteria in the intestine;7 therefore, restriction of their oral intake is not feasible. Capturing target molecules in the intestine and inhibiting their intestinal absorption is an attractive strategy for patients.8−12 Since nonspecific capture eventually causes malnutrition and reduced drug efficacy,13−16 specific inhibition of the targetmolecule absorption is important. A leading example of a reagent specifically binding to a target molecule is antibody or its fragment;17 however, they are easily digestible by digestive enzymes before/after capturing of the target molecule. Therefore, specific capture of the target molecule and inhibition of the intestinal absorption by a stable and cost© XXXX American Chemical Society

Received: December 26, 2018 Revised: March 6, 2019 Published: March 8, 2019 A

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules adhesins.35−37 More recently, Shea’s group have demonstrated that lightly cross-linked poly N-isopropylacrylamide (pNIPAm) nanoparticles (NPs) synthesized with functional monomers bind to and neutralize target peptides and proteins in the bloodstream of living animals.38−43 However, there are few examples of an abiotic polymer specifically capturing a small molecule that exists in the intestine of living animals and inhibits its intestinal absorption. Herein, we describe the design of synthetic NPs capable of specifically capturing a toxic molecular target in the intestine and inhibiting its intestinal absorption in vivo. In the present study, we selected indole as a model target molecule in the intestine, a major factor exacerbating chronic kidney disease (CKD).44,45 Indole is an aromatic lowmolecular compound, which is synthesized from diet-induced tryptophan by intestinal bacteria such as Escherichia coli.46 Indole is then metabolized to indoxyl sulfate (IS), a uremic toxin, in the liver after being absorbed from the intestine.47−49 IS is immediately excreted through the kidneys in healthy subjects; however, it accumulates in patients with CKD because of functionally reduced renal clearance.50 IS damages renal tubular cells by oxidative stresses and leads to exacerbation of CKD.51−54 Therefore, preventing the intestinal absorption of indole is the attractive strategy for avoiding deterioration of patients with CKD. To capture indole in intestines, we synthesized more than 40 kinds of NPs by changing their containing hydrophobic monomers, the degree of cross-linking, and resulting NP diameters. We used indole derivatives and cyanocobalamin (Vitamin B12, VB12) for demonstration of the NP specificity for indole. Caco-2 cell monolayers were selected as a model of intestinal epithelium in vitro for assessing the inhibitory effect of NP on the intestinal absorption of indole. For in vivo studies, we used radiolabeled NIPAm for demonstrating the biodistribution of NPs and radiolabeled indole for establishing the inhibitory effect of NPs on indole absorption.

■

n-hexane (Wako Pure Chemical Industries, Ltd., 96.0%) before polymerization. Cell Culture. Dulbecco’s Modified Eagle’s Medium (DMEM) high glucose was purchased from Wako Pure Chemical Industries, Ltd.; fetal bovine serum (FBS), from AusGeneX, Pty. Ltd. (Loganholme, QLD, Australia) and TPP tissue culture T-75 flasks, from Thermo Fisher Scientific, Inc. (Waltham, MA, USA); penicillin-streptomycin (10 000 IU Pen/mL, 10 000 μg Strep/mL), from MP Biomedicals, Inc. (Solon, OH, USA); collagen type I (for cell culture, from calf skin), from SIGMA-Aldrich, Inc.; Corning BioCoat intestinal epithelium differentiation environment including Seeding Basal Medium, Entero-STIM Differentiation Medium, and MITO+ serum extender (epidermal growth factor, transferrin, insulin, endothelial cell growth supplement, triiodothyronine, hydrocortisone, progesterone, testosterone, estradiol-17β, selenium and o-phosphorylethanolamine), and Transwell inserts (24 wells, 0.33 cm2, 1 μm pure size), from Corning, Inc. (Corning, NY, USA). Methods. Preparation of NPs. NPs were prepared by an aqueous dispersion copolymerization. NIPAm, TBAm, PAA, 5FPAA, Bis, and SDS (2 mg) were dissolved in nanopure water (10 mL). TBAm was dissolved in ethanol (600 μL) and PAA or 5FPAA was dissolved in acetone (1 mL) before the addition into the monomer solution. The total monomer concentration was 65 mM. Nitrogen gas was bubbled through the reaction mixtures for 30 min. Following the addition of APS aqueous solution (6 mg in 400 μL of water), the prepolymerization mixture was sealed under nitrogen gas. Polymerization was carried out under a nitrogen atmosphere with gentle stirring for 3 h at 65 °C. The polymerized polymer solutions were purified by dialysis against an excess amount of pure water (changed more than twice a day) for 4 days. Characterization of NPs. The particle sizes, ζ-potentials, and polydispersity index (PDI) of NPs in nanopure water were determined at 25 ± 0.1 °C by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK). The incorporated ratio of each monomer was determined as shown in the supplementary experimental section. Affinity of NPs for Indole. An ultraviolet−visible (UV−vis) spectrometry (SmartSpec3000, BIO-RAD Laboratories, CA, USA) was used to quantify the affinity of the NPs for indole. Indole was dissolved in a 25 mM phosphate buffer (PB, pH7.4) and mixed with NP (final concentrations of indole and NP of 85 μM and 2.5 mg/mL, respectively). NP and indole were ultracentrifuged (37 °C, 368,000g, 15 min) just after mixing each other. Then, the amount of free indole in the supernatant was determined by measuring the absorbance at 279 nm. The amounts of indole captured by NPs were calculated by using eq 1, where Cfree is the concentration of free indole in the supernatant and Ctotal is the initial concentration of indole (85 μM).

EXPERIMENTAL SECTION

MATERIALS. Chemicals. The following materials were obtained from commercial sources: N-Isopropylacrylamide (NIPAm, ≥98.0% purity), N-phenyacrylamide (PAA, ≥98.0% purity), acrylic acid (AAc, ≥99.0% purity), acryroyl chloride (≥98.0% purity), hexadecyltrimethylammonium bromide (CTAB, ≥98.0% purity), 2,2,2-trifluoroethanol (≥99.0% purity), indole (≥99.0% purity), L-tryptophan (≥98.5% purity), 3-indoleacetic acid (≥98.0%, purity) and VB12 (≥95.0% purity), from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan); ammonium persulfate (APS, ≥98.0%, purity), tryptamine (≥97.0%, purity), and pepsin (3200−4500 units/mg, from porcine gastric mucosa), from SIGMA-Aldrich, Inc. (St Louis, MO, USA); N,N′methylenebis(acrylamide) (Bis, ≥97.0%, purity), N-tert-butylacrylamide (TBAm, ≥95.0%, purity), sodium dodecyl sulfate (SDS, ≥95.0%, purity), 2-propanol (≥99.7%, purity), hydrogen peroxide (analytical reagent 30.0−35.5%), pancreatin (26−46 protease activity units/mg of solid, from hog pancreas), and isoflurane (≥98.0% purity), from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); isopropylamine [2-3H] (209.3 mCi/mg) and indole [phenyl-14C] (1.2 mCi/mg), from American Radiolabeled Chemicals, Inc. (St Louis, MO, USA); N-(3-aminopropyl) methacrylamide (APM, >98% purity), from Polysciences, Inc. (Warrington, PA, USA); α,α′azobis(isobutyronitrile) (AIBN, ≥98.0% purity), from Nacalai Tesque, Inc. (Kyoto, Japan); and Hionic Fluor, from PerkinElmer Japan Co. Ltd. (Yokohama, Kanagawa, Japan). 2,3,4,5,6-Pentafluorophenyl acrylamide (5FPAA) was synthesized as described in the supplementary experimental section. NIPAm was recrystallized from

amount of indole captured by NPs (%) = [(C total − Cfree)/C total ] × 100

(1)

Specificity of NPs for Indole. VB12 and the indole derivatives (tryptophan, tryptamine, and indoleacetic acid) were dissolved in 25 mM PB (pH7.4) and mixed with NP to give final concentrations of each compound and NP of 10 μg/mL and 2.5 mg/mL, respectively. The solutions were ultracentrifuged (37 °C, 368,000g, 15 min) just after mixing each other. Then, the amounts of each free indole derivative and VB12 in the supernatants were determined by measuring the absorbance at 279 and 361 nm, respectively. The amounts of these molecules captured by NPs were calculated using eq 2, where Cfree X is the concentration of X (X: each indole derivative or VB12) in the supernatant and Ctotal X is the initial concentration of X. amount of X captured by NPs (%) = [(C total X − Cfree X)/C total X ] × 100

(2)

Preparation of Negatively Charged NPs. Negatively charged NPs were synthesized by an aqueous dispersion copolymerization as B

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules follows: NIPAm, Bis, TBAm, 5FPAA, AAc, and SDS (2 mg) were dissolved in nanopure water (10 mL). 5FPAA and TBAm were dissolved in acetone and ethanol, respectively, before the addition. The total monomer concentration was 65 mM. Following the addition of 200 μL APS aqueous solution (30 mg/mL), the reaction was carried out under a nitrogen atmosphere with gentle stirring for 3 h at 65 °C. The polymerized solutions were purified by dialysis against an excess amount of deionized water (changed more than twice a day) for 4 days. Preparation of Positively Charged NPs. Positively charged NPs were synthesized by an aqueous dispersion copolymerization as follows: NIPAm, Bis, TBAm, 5FPAA, APM, and CTAB (4 mg) were dissolved in nanopure water (10 mL). 5FPAA and TBAm were dissolved in acetone and ethanol, respectively, before the addition. The total monomer concentration was 65 mM. Following the addition of 200 μL AIBN solution (20 mg/mL acetone), the reaction was carried out under a nitrogen atmosphere with gentle stirring for 3 h at 65 °C. The polymerized solutions were purified by dialysis against an excess amount of deionized water (changed more than twice a day) for 4 days. Cell Culture and Differentiation. Human intestinal epithelial (Caco-2) cells were grown in a humidified atmosphere at 37 °C under 5% CO2. The cells were maintained in T-75 flasks with DMEM high glucose supplemented with 10% FBS, 100 units/mL penicillin, and 100 μg/mL streptomycin. Caco-2 cell monolayers were obtained by using a short-term Caco-2 cell culture system in accordance with manufacturer’s instructions. Briefly, Caco-2 cells were grown to a density of ≥250 000 cells/cm2 in T-75 flasks. Then, the cells were harvested and seeded onto collagen-coated Transwell inserts at a density of 2.0 × 105 cells/insert and grown in Basal Seeding Medium containing MITO+ Serum Extender for 24 h. The medium was changed to Entero-STIM Differentiation Medium supplemented with MITO+ Serum Extender. Then, the monolayers were used for experiments after measuring TEER values (trans-endothelial electrical resistance, ≥700 ohm × cm2). In Vitro Intestinal Absorption Assay. Caco-2 cell monolayers were incubated with 300 μL of Hanks’ balanced salt solution (HBSS) containing [14C]-labeled indole (43 μM, 370 Bq) and NPs (5.0 mg/ mL) in an apical compartment (inserts) and 1.0 mL of HBSS in a basolateral compartment (receivers) for 6 h. The amount of transepithelial transferred [14C]-indole from the apical to the basolateral compartment was determined by use of a liquid scintillation counter (LSC-7400, Hitachi Aloka Medical, Tokyo, Japan). Digestion of NPs. Artificial gastric juice (32 000 units/L pepsin, 8 g/L NaCl) was adjusted to pH 1.2 with 0.2 N HCl. Artificial intestinal juice (40 g/L pancreatin, 30 g/L KH2PO4) was adjusted to pH 6.8 with 0.2 N NaOH. [3H]-labeled NPs were prepared by adding a small amount of [3H]-labeled NIPAm (10 kBq/mg NP) synthesized as shown in the supporting experimental section to the initial solutions before each polymerization. [3H]-labeled TF-NP5, TF-NP5-p or TFNP5-n (1 mg) was incubated with 1 mL artificial gastric juice or artificial intestinal juice at 37 °C for 12 h. Then, they were ultracentrifuged (37 °C, 368,000g, 30 min) to divide the nondigested NPs from digested NPs (supernatant). The radioactivity in the supernatant was determined with a liquid scintillation counter. Experimental Animals. BALB/c male mice were purchased from Japan SLC Inc. (Shizuoka, Japan). The animals were cared for according to the Animal Facility Guidelines of the University of Shizuoka. All animal experiments were approved by the Animal and Ethics Review Committee of the University of Shizuoka (Approved No. 186332). Biodistribution of NPs. Five-week-old BALB/c male mice were given [3H]-labeled TF-NP5, TF-NP5-n or TF-NP5-p colloidal solutions (5 mg/50 kBq/mouse) via per os (p.o.) administration and housed in the metabolic cages at the University of Shizuoka Animal Care Facility and maintained at 23 ± 1 °C with a 12 h/12 h light/dark cycle (lights on at 8:00 a.m. and off at 8:00 p.m.) for 24 h. Then, these mice were sacrificed under deep anesthesia with isoflurane (1.0 mL/min, 3%; SurgiVet, Inc.) and their blood, heart, lungs, liver, spleen, and kidneys were removed and digested with

Solvable (PerkinElmer, MA, USA). The blood was heparinized and separated by centrifugation (700g, 15 min, 4 °C) to obtain the plasma. Urine and excreted feces were collected and weighted. The [3H]labeled NPs were extracted with 500 mL methanol from feces for 5 times. The radioactivities in plasma, each organ, urine, and feces were determined with a liquid scintillation counter (LSC-7400, Hitachi Aloka Medical, Tokyo, Japan) after the addition of Hionic Fluor (PerkinElmer). The total radioactivity in the plasma was calculated based on the average body weight of the mice, where the average plasma volume was assumed to be 4.27% of the body weight based on the data on total blood volume. The amounts of NPs in each organ, urine, and feces are expressed as percentage of injected dose. Inhibitory Effect of NPs on Indole-Intestinal Absorption in Vivo. Five-week-old BALB/c male mice were given [14C]-labeled indole (43 nmol/mouse) and NPs (5 mg/mouse) in water (400 μL) via p.o. administration after the preincubation of NPs and indole for 30 min. At 1 h after the administration, the mice were sacrificed under deep anesthesia with isoflurane (1.0 mL/min, 3%). After the mice had been bled from a carotid artery, their liver and kidneys were collected and digested with Solvable (PerkinElmer). Plasma samples were obtained by the heparinizing and centrifuging (700g, 15 min, 4 °C) the blood samples. The radioactivities in plasma and each organ were determined with a liquid scintillation counter (LSC-7400, Hitachi Aloka Medical).

■

RESULTS AND DISCUSSION Affinity of NPs for Indole, a Target Molecule. For the preparation of NPs that capture indole, we focused on the hydrophobicity and aromaticity of indole (Scheme 1a). TBAm,

Scheme 1. Synthesis of Indole-Capturing NPsa

a

(a) Structure of the target molecule, indole. (b) Preparation of NPs via a dispersion copolymerization and the structures of hydrophobic monomers adopted for capturing indole.

PAA, and 5FPAA were used as hydrophobic monomers that interact with indole via hydrophobic interactions and/or π−π stacking interaction. NIPAm-based NPs were synthesized with these hydrophobic monomers and Bis via an aqueous dispersion copolymerization as previously described55 and purified by repetitious dialyses (Scheme 1b). A summary of monomer feed ratios, sizes, polydispersity indexes (PDI), and ζ-potentials are shown in Table 1. Colloidal dispersions of monodispersed-NPs that ranged in particle size from 64 to 172 nm were obtained (Table 1). All NPs had a negatively charged surface because of the initiator, APS. Monomer-incorporation rates calculated by 1H NMR and 19F NMR spectra showed that the hydrophobic monomers were incorporated in proportion to the feed ratio. C

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Table 1. Characteristics of NPs Synthesized by Changing the Structures and Feed Ratios of Hydrophobic Monomersa Hydrophobic monomer feed (mol %) TBAm NP0 TB-NP1 TB-NP2 TB-NP3 TB-NP4 PA-NP1 PA-NP2 PA-NP3 PA-NP4 FP-NP1 FP-NP2 FP-NP3 FP-NP4

PAA

5FPAA

20 40 60 80 20 40 60 80 20 40 60 80

Incorporation ratio (mol %) Particle size (nm)

PdIb

± ± ± ± ± ± ± ± ± ± ± ± ±

0.030 0.009 0.033 0.033 0.022 0.066 0.056 0.053 0.064 0.078 0.062 0.019 0.021

172 152 137 122 102 115 100 88 96 102 89 83 64

18 18 17 27 11 17 24 9 10 7 4 6 3

ζ-potential (mV) −13 −24 −32 −37 −34 −26 −32 −36 −44 −25 −35 −45 −37

± ± ± ± ± ± ± ± ± ± ± ± ±

5 9 4 6 6 1 4 7 12 6 5 6 3

Yield (%) 92 82 80 92 85 89 89 96 94 76 77 81 87

TBAm

PAA

5FPAA

NIPAm

20.2 41.9 60.2 80.8

100 79.4 57.5 37.6 18.7 76.9 58.4 36.3 14.2 77.8 56.1 37.8 17.2

20.6 40.5 60.4 79.3 21.1 39.6 61.7 83.8

a

All NPs incorporated 2-mol % Bis. NIPAm was used to make up the remaining percentage. bPolydispersity index as a measure of the particle sizes.

Figure 1. Indole capture percentage by NPs. (a) Effect of hydrophobic monomer structures and feed ratios in NPs on capturing indole. (b) Influence of the NP diameters on capturing indole. (c) Effect of polymer density in TBAm NPs on capturing indole. TB-NP1, 2, 3, and 4 containing 2 mol % Bis; TB-NP5, 6, 7, and 8 containing 10 mol % Bis; TB-NP9, 10, 11, and 12 containing 20 mol % Bis; TB-NP13, 14, and 15 containing 40 mol % Bis; TB-NP1, 5, 9, and 13 containing 20 mol % TBAm; TB-NP2, 6, 10, and 14 containing 40 mol % TBAm;TB-NP3, 7, 11, and 15 containing 60 mol % TBAm; TB-NP4, 8, and 12 containing 80 mol % TBAm. (d) Effect of the hydrophobic monomer combination on capturing indole. The amounts of indole captured by NPs were calculated from the NP-capturing indole relative to added indole. Data represent the mean adsorption rates ± standard deviation (n = 3).

To establish that the synthesized NPs captured indole, we incubated the NPs (2.5 mg/mL) and indole (85 μM) at room temperature. Since NPs need to capture bacteria-induced indole before indole is absorbed into the body, it is important to design NPs capable of capturing the target within a minute. Thus, NP and indole were ultracentrifuged just after mixing each other in order to separate free indole from NP−indole complexes. Then, the concentration of free indole, which has not been associated with the NPs, was quantified with a UV− vis spectrometer (Figure 1a). Regardless of the monomer

structures, the affinity of NPs for indole was increased by increasing the feed ratio of hydrophobic monomers, suggesting that all hydrophobic interactions made a significant contribution to the capture of indole. For NPs with a high feed ratio of hydrophobic monomers, NPs containing fluorinated aromatic moieties (FP-NP4; 80-mol % 5FPAA) showed higher affinity for indole than the ones containing tert-butyl or phenyl groups (TB-NP4 or PA-NP4; 80-mol % TBAm or PAA, respectively), indicating that fluorinated aromatic moieties were important contributors to indole capture and that the enhanced affinity D

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Table 2. Monomer Composition and Characteristics of NPs Incorporating 2 Kinds of Hydrophobic Monomers, TBAm and 5FPAAa Hydrophobic monomer feed (mol %) TF-NP1 TF-NP2 TF-NP3 TF-NP4 TF-NP5 TF-NP6

TBAm

5FPAA

20 20 20 40 40 60

20 40 60 20 40 20

Incorporation ratio (mol %) Particle size (nm)

PdI

± ± ± ± ± ±

0.105 0.046 0.033 0.071 0.087 0.037

75 80 79 78 77 72

10 17 14 16 10 4

ζ-potential (mV) −39 −33 −40 −38 −40 −40

± ± ± ± ± ±

1 10 4 4 2 4

Yield (%)

TBAm

5FPAA

NIPAm

91 93 79 87 81 85

18.7 20.5 20.5 42.3 43.5 63.8

20.3 43.0 66.7 20.9 43.8 20.5

59.0 34.6 10.8 34.8 10.7 13.7

Particle sizes and ζ-potentials are given as mean ± SD (n = 3). All of NPs also incorporated 2-mol % Bis for cross-linking and NIPAm to make up the remaining percentages.

a

for indole by fluorination of the phenyl residue was attributable to quadrupole interactions of the pentafluoro rings.25,56,57 On the other hand, when the feed ratio of hydrophobic monomers was relatively small, NPs containing tert-butyl (TB-NP1; 20mol % TBAm) showed higher affinity for indole than the ones containing phenyl or fluorophenyl groups (PA-NP1 or FPNP1; 20-mol % TBAm or PAA, respectively). The pentafluoro residue in FP-NP1 binds to indole in a one-to-one fashion. This is further supported by Figure S1, which shows that the amount of FP-NP-captured indole increased in proportion to the 5FPAA incorporation ratio. TB-NPs contain a hydrophobicity-rich environment created by self-assembled tert-butyl groups. This “hydrophobic plaza” plays an important role in the capturing of indole by TB-NPs. The amounts of indole captured by NPs should be susceptible to the quadrupole interactions produced by the pentafluoro rings compared with the hydrophobic plaza created by TBAm. NPs containing 90mol % TBAm captured less indole (35 ± 1.4%) than those containing 80-mol % TBAm (37 ± 0.1%; SI Figure S2). The incorporation of more than 80-mol % hydrophobic monomers should thus not be necessarily essential for capturing indole. Effect of Size and Polymer Density of NPs on Affinity for Indole. To evaluate the implications of NP diameters for indole-capture, TB-, PA-, or FP-NP4 was synthesized by a precipitation polymerization without SDS (TB-NP4b, PANP4b, or FP-NP4b). Although the particle sizes of TB-NP4, PA-NP4, and FP-NP4 synthesized with SDS were 102, 96, and 64 nm, respectively, those of TB-NP4b, PA-NP4b, and FPNP4b (synthesized without SDS) were 323, 434, and 172 nm (SI Table S1). Surprisingly, the amounts of indole captured by TB-NP4b, PA-NP4b, and FP-NP4b did not significantly change (Figure 1b). These results indicate that the diameter (surface area) of the NPs was not a critical factor for capturing indole; rather the capture depended only on the monomer composition of NPs, especially the feed ratio of hydrophobic monomers that produced high affinity for indole. Moreover, these results strongly support the possibility that indole bound not only to the NP surface but also to the inside of the NPs. We next demonstrated the effect of the cross-linking degree of NPs on the indole capture. TB-NPs were synthesized by changing feed ratio of Bis (cross-linker) from 2 to 40 mol %. The NP sizes slightly increased by changing the cross-linker percentage from 2 to 40 mol % (SI Table S2). Regardless of the TBAm-feed ratios, the amount of captured indole was decreased by increasing the cross-linking percentage (polymer density) in the NPs, from 5% to 40% (Figure 1c). This result is direct evidence that the flexible polymer network in the NPs was particularly important for capturing indole.58,59 However,

the amount of indole captured by the NP containing 1-mol % Bis was less than that of the NP containing 2-mol % Bis (SI Figure S3). The too low cross-linking degree (1-mol %) might prevent effective formation of hydrophobic plaza because polymer density in the NPs was too low. Hence, 2-mol % cross-linked NPs was adapted for the subsequent experiments. Effect of Combining 2 Kinds of Interaction on Indole Affinity. Since TB- and FP-NPs should bind to indole by different binding modes, we hypothesized that incorporation of both TBAm and 5FPAA into NPs would increase the affinity for indole by multi modal interactions. To test this hypothesis, we synthesized a series of NPs containing both TBAm and 5FPAA (TF-NP1−6, Table 2). Monomer feed ratio, diameters, and ζ-potentials of the TF-NPs are shown in Table 2. TF-NP size and ζ-potentials were not significantly different from single hydrophobic monomer-containing NPs (TB or FP-NPs). TFNPs showed higher affinity for indole compared with NPs containing the same percentages of single hydrophobic monomer (TF-NP3, TF-NP5, and TF-NP6 vs TB-NP4, PANP4, and FP-NP4; Figure 1d). It is well-known that the binding affinity of a polymer for target (macro)molecules is increased by multimodal and multipoint interactions.60 We clearly showed that the incorporation of 2 kinds of hydrophobic monomer increased the affinity even for a small molecular target. Specificity of NPs for Indole. As most nutrients and orally taken drugs are absorbed from the intestine, nonspecific capture of these nutrients and drugs by the NPs could cause malnutrition and attenuation of the drug’s action. Therefore, the affinity between NPs and indole should be specific. To verify the binding specificity of NPs for indole, we used indole derivatives such as tryptophan (hydrophilic molecule), tryptamin (basic molecule, pKa = 10.2), and indoleacetic acid (acidic molecule, pKa = 4.75), and VB12 (large molecule; Figure 2a). TB-NP4, FP-NP4, TF-NP5, and TF-NP6, which possessed relatively high affinity for indole, were used in the assay (Figure 2b). The amounts of tryptophan, indoleacetic acid, tryptamine, and VB12 captured by TB-NP4, TF-NP5, or TF-NP6 were less than 10%, significantly lower than the amounts of captured indole. FP-NP4 showed high affinity for tryptamine due to the quadrupole and electrostatic interactions (high dipole moment). Interestingly, the amounts of indoleacetic acid and tryptamine captured by TF-NP5 and TF-NP6 were significantly lower than those captured by FP-NP4; and those of tryptophan and VB12 captured by TF-NP5 and TFNP6 were lower than those captured by TB-NP4. Nevertheless, TF-NP5 and TF-NP6 possessed higher affinity for indole than TB-NP4 or FP-NP4. These data indicate that the E

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure S4a,b). From these results, the incorporation of 5-mol % negatively or positively charged monomers into NPs did not have a dramatic influence on the affinity for indole. However, TF-NP5 containing 5-mol % AAc (TF-NP5-n) showed the highest affinity for indole. Anionic monomer (AAc)-incorporation cause better indole adsorption than cationic monomer (APM)-incorporation. The key reason of this phenomenon is hydrogen bond. The oxygen atoms in AAc-carboxylic acid groups are creating hydrogen bond to indole NH-protons. LCST of these NPs were not in the range of 4 and 37 °C because the NP size at 4 °C was not significantly different at 37 °C (data not shown), indicating hydrophobic behavior of these NPs should not change even in in vivo. On the examination of specificity, while the affinity of NPs for indole derivatives and VB12 was significantly changed by the incorporation of the charged monomers, that for tryptamine, a positively charged molecule (pKb = 10.2) was tremendously increased by AAc-incorporation (Figure 3c, SI Figure S2). NPs containing APM did not show affinity for indoleacetic acid, a negatively charged molecule (pKa = 4.75) that is supposed to electrostatically interact with APMincorporating NPs. We do not clearly understand why NPs containing APM did not interact with indoleacetic acid; however, a functional monomeric structure and the incorporation ratios are important for the design of polymer NPs specifically binding to a target small molecule. NPs and nutrients and/or drugs would be in contact while passing through the digestive tract, and the incubation we used could be far shorter than the actual condition. Therefore, a simple experiment was performed to confirm that NPs did not capture indole derivatives by a long-time incubation. Indole derivatives or VB12 and NPs were incubated for 3 h, and then the concentration of free indole derivatives and VB12 was measured (Figure 3d). In this experiment, AST-120 (spherical porous carbon particles) was used as a control for an oral absorbent. It is the medicine used for capturing uremic toxins in the intestine, the most important of which is indole. AST-120 captured 42 ± 5% of indole during the incubation (data not shown). This means that the NPs were equal in indole-capturing ability to this currently used drug. However, AST-120 captured all indoleacetic acid, tryptamine, and even tryptophan, an essential amino acid (Figure 3d), suggesting that nonspecific capture by AST-120 might cause malnutrition. However, the nonspecific capture was significantly decreased by using NPs. Especially, none of the NPs tested captured tryptophan or VB12. It has been generally recognized that NPs interacting with biomolecules are coated with a “protein corona” in the biological milieu.41,65 This phenomenon results in a remarkable reduction in NP abilities. Hence, we next examined whether the indole-capturing ability of NPs might change when indole was mixed with indole derivatives. TF-NP5, TFNP5-p, or TF-NP5-n was introduced to a mixture of [14C]labeled indole (85 μM), tryptophan (49 μM), indoleacetic acid (57 μM), tryptamine (62 μM), and VB12 (7.0 μM) and incubated for 37 °C for 3 h. Then, the amount of captured indole was calculated by measuring the radioactivity of free [14C]-labeled indole (Figure 3e). The amount of NP-captured indole did not change even in the presence of this mixture of indole derivatives and VB12. These data indicate that the NPs could capture indole without their capturing ability being impaired even in an environment where other molecules were present.

Figure 2. Specificity of TB-NP4, FP-NP4, TF-NP5, and TF-NP6 for indole. (a) Structures of the indole derivatives and VB12. (b) Percentages of capture of indole and its derivatives. Data represent the mean captured rates ± standard deviation (n = 3). Significant differences between FP-NP4 and TF-NP5 or TF-NP6 are indicated by asterisks (***P < 0.001, Tukey’s test subsequent to ANOVA).

incorporation of 2 kinds of hydrophobic monomers into the NPs increased not only their affinity but also specificity for indole. On the other hand, incorporation of TBAm into the FP-NP (TF-NP) instead of 5FPAA decreased the affinity for tryptamine without reducing that for indole. These results indicate that the affinity and specificity for a certain molecule could be modulated by the combination of different types of hydrophobic monomer. Effect of Charged Monomer Inclusion into NPs on Capture of Indole and Its Derivatives. It is known that the π-electron cloud of the indole interacts with cationic molecules by cation−π interaction.61 In addition, inclusion of charged monomers into NPs increase the NP-dispersion stability by electric repulsion. 62,63 Therefore, N-(3-aminopropyl)methacrylamide (APM, positively charged monomer)- or acrylic acid (AAc, negatively charged monomer)-incorporated NPs were prepared to examine whether the incorporation of charged monomers have a positive impact on indole capturing by the NPs (Figure 3a). The peak derived from APM or AAc in the 1H NMR spectra was hardly detected owing to the overlap of another functional group or exchange of protons with methanol-d6, respectively. However, incorporation efficiency of these monomers by using the same polymerization protocol were found to be >50% (APM)64 or around 40% (AAc) of the feed amount.39 The NP libraries were constructed by inclusion of 5-mol % charged monomers into TB-NP4, FP-NP4, TF-NP5, and TFNP6, all of which showed relatively high affinity for indole (Table 3, SI Table S3). The affinity of TF-NP5 or TF-NP6 for indole was slightly increased by the incorporation of a negatively charged monomer but not changed by that of a positively charged one (Figure 3b, SI Figure S4c). The incorporation of a charged monomer into TB-NP4 did not change the affinity for indole, whereas that of a charged monomer into FP-NP4 slightly decreased the affinity for it (SI F

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

Figure 3. Effect of charged-monomer incorporation into NPs on capturing indole or the specificity for indole. 5-mol % charged monomers were incorporated into TF-NP5. (a) Structures of the charged monomers: APM, 3-aminopropyl methacrylamide (blue); AAc, acrylic acid (red). (b) Amount of indole captured by TF-NP5 and it synthesized with APM (blue, TF-NP5-p) or AAc (red, TF-NP5-n) by each following incubation with indole (85 μM). (c) Influence of charged-monomer incorporation into NPs for specificity. (d) Amounts of tryptophan, tryptamine, indoleacetic acid, and VB12 captured by NPs following a 3 h incubation. (e) Indole capture rate by NPs in the mixture of indole, its derivatives, and VB12. Data are shown as the mean rates ± standard deviation (n = 3).

Table 3. Characteristics of the TF-NP5 Incorporating Charged Monomera Monomer feed ratio (mol %) TF-NP5 TF-NP5-p TF-NP5-n

TBAm

5FPAA

40 40 40

40 40 40

APM

Incorporation ratio (%)

AAc

Particle size (nm)

PdI

ζ-potential (mV)

Yield (%)

TBAm

5FPAA

NIPAm

5

77 ± 10 81 ± 17 83 ± 4

0.087 0.11 0.017

−40 ± 2 +49 ± 2 −39 ± 1

81 75 86

43.5 39.1 41.2

43.8 43.2 40.9

10.7 10.6 10.9

5

Particle sizes and ζ-potentials are given as mean ± SD (n = 3). All NPs incorporated 2-mol % Bis for cross-linking and NIPAm to make up the remaining percentages.

a

Inhibition of Indole-Intestinal Absorption by NPs in Vitro. We next demonstrated the NP-inhibitory effect on indole-intestinal absorption in vitro by using a Caco-2 cell monolayer, a model of an intestinal barrier. Caco-2 cells were seeded on a semipermeable membrane (trans-well) at the density of 2 × 105 cells/insert. After checking the membrane monolayer integrity by measuring TEER, [14C]-labeled indole (43 μM) and TF-NP5, TF-NP5-p or TF-NP5-n (5 mg/mL) were added to the monolayer and incubated for 6 h at 37 °C (Figure 4a). Then, the amounts of [14C]-labeled indole in the apical and basolateral compartments were measured. The [14C]-labeled indole in the basolateral compartment indicated the amount of indole absorbed from the intestine. About 50% of the indole added to the cell monolayer was normally transferred to the basolateral compartment, with 20% remaining in the apical compartment without being absorbed (Figure 4b). However, the indole transferred to the basolateral

compartment was conspicuously diminished by the addition of NPs. Additionally, the amount of indole remaining in the apical compartment was doubled by adding the NPs compared with vehicle addition. These results suggest that NPs could inhibit the intestinal adsorption of indole. The enhanced efficacy resulting from AAc incorporation was due to not only the high affinity for indole but also the electrostatic repulsion between negatively changed NPs and the cell membrane. In fact, although TF-NP5 and TF-NP5-n were not taken up into the cells, TF-NP5-p was taken up slightly possibly due to the positive charge (SI Figure S5). Inhibition of Indole Absorption by NPs in Vivo. To capture the target molecule in the intestine after the oral administration of NPs, these NPs need to arrive at the intestine without being degraded by the digestive enzymes. In addition, NPs should be excreted in the feces without being absorbed into the body to avoid their accumulation in the body. G

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules

pH = 7.4) at 37 °C for 12 h. Then, the solutions were ultracentrifuged to remove degraded parts (monomer residues and/or oligomers) and measured for radioactivity of the insoluble fraction, which was the nondegraded polymer. The levels of radioactivity in the insoluble fractions of TF-NP5, TFNP5-p, and TF-NP5-n were not changed after the incubation with digestive enzymes (Figure 5a), suggesting that the NPs were not degraded even in the digestive enzyme solutions, unlike in the case of antibodies. We next demonstrated the biodistribution of TF-NP5, TFNP5-p, and TF-NP5-n after the per os (p.o.) injection. Mice were orally administered [3H]-labeled TF-NP5, TF-NP5-p, or TF-NP5-n. At 24 h after the p.o. administration, radioactivities in each organ (heart, ling, spleen, liver, and kidneys) and plasma were measured. More than 95% of NPs was excreted in the feces, with NPs of 1% or less being present in the organs and plasma (SI Table S4, Figure 5b). These results indicate that almost none of the NPs were absorbed in the intestine but excreted in the feces. The accumulation rates of NPs were significantly decreased by the incorporation of a charged monomer (Figure 5b), even if the ζ-potential was not significantly different from that of TF-NP5 (Table 2). TFNP5-p and TF-NP5-n showed lower accumulation in each organ than TF-NP5. This phenomenon was explained reduced intestinal absorption by incorporating charged monomer. Hydrophobic nanomaterials are generally a bit taken up by cells. However, incorporation of charged monomer inhibits their intracellular migration. Among them, anionic parts cause electrostatic repulsion against to cell membrane of the intestine. Therefore, TF-NP5-n showed lowest accumulation in the organs.66−68 In the case of positively charged TF-NP5-p, they were taken up into the cells to some extent in vitro; however, their absorption rate after the oral administration was lower than that of TF-NP5-n and TF-NP5. A possible explanation is that TF-NP5-p made a complex with intestinal contents by the electrostatic interactions in the intestine. Since positive charge of the surface of TF-NP5-p was neutralized and

Figure 4. NP-inhibitory effect on indole intestinal absorption in vitro. Caco-2 monolayers were used as a model of the intestinal barrier. (a) Illustrated procedure for intestinal absorption assay. (b) Radioactivity of [14C]-labeled indole in the apical or basolateral compartment at 24 h after indole (5.0 μg/mL) and NPs (5.0 mg/mL) addition into the apical compartment. Data represent the mean ± standard deviation (n = 3). Significant differences between vehicle and NP-injected groups are indicated by ***, apical compartment; or †††, basolateral compartment (P < 0.001, Dunnett’s test subsequent to ANOVA).

Therefore, before performing the assay for the inhibitory effect of NPs on intestinal adsorption in vivo, we evaluated the degradation-resistance of TF-NP 5s to digestive enzymes. In this assay, [3H]-labeled TF-NP5, TF-NP5-p, and TF-NP5-n were prepared by the inclusion of [3H]-labeled NIPAm (10 kBq/mg NP) during NP-synthesis. The [3H]-labeled TF-NP5, TF-NP5-p, or TF-NP5-n was incubated in artificial gastric juice including pepsin (pH = 1.2), artificial intestinal juice including pancreatin (pH = 6.8), or phosphate buffer (control,

Figure 5. NP-inhibitory effect on indole intestinal absorption in vivo. (a) Stability of NPs in the presence of digestive enzyme (pepsin or pancreatin). (b) Biodistribution of TF-NP5, TF-NP5-p, and TF-NP5-n (5 mg/kg) at 24 h after oral administration. (c) Accumulation rates of [14C]-labeled indole at 1 h after the p.o. injection with or without the NPs. Data represent the mean ± standard deviation (n = 5). Significant differences in plasma-accumulation rates of vehicle-injected group versus NP-injected group: *P < 0.05 and **P < 0.01. Liver-accumulation rates: †P < 0.05, ††P < 0.01, and †††P < 0.001. Kidney-accumulation rates: ‡‡ P < 0.01 (Dunnett’s test subsequent to ANOVA). H

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules covered by making the complex with the intestinal contents, the absorption rate of TF-NP5-p could be decreased after the oral administration compared with that of TF-NP5-n and TFNP5. To demonstrate the safety of NPs, we next measured cell-released lactate dehydrogenase (LDH) after NP addition. 2H-11 cells were incubated with NPs (300 μg/mL) for 24 h at 37 °C. Then, the LDH concentration in the medium was measured. LDH release was not significantly increased by NP addition compared with that of the vehicle (PBS) (SI Figure S6), indicating that NPs had little toxicity, at least at the concentration of 300 μg/mL. Finally, to demonstrate inhibitory effect on indole-intestinal absorption in vivo, [14C]-labeled indole (43 nmol/mouse) and TF-NP5, TF-NP5-p, or TF-NP5-p (5 mg/mouse) were p.o. administered to mice. One hour after administration, radioactivities in the plasma, liver, and kidneys were measured. The accumulation rates of [14C]-labeled indole in the plasma and each organ were dramatically decreased by NP injection compared with those obtained with vehicle injection (Figure 5c). In addition, the accumulation of [14C]-labeled indole in the plasma and each organ was slightly decreased by TF-NP5-n injection compared with that found with TF-NP5 or TF-NP5p injection because of decreased intestinal absorption. These results indicate that NPs inhibited the intestinal absorption of indole in vivo by capturing indole.

■

AUTHOR INFORMATION

Corresponding Author

*H. Koide. E-mail: [email protected]. ORCID

Hiroyuki Koide: 0000-0003-1763-6593 Yu Hoshino: 0000-0001-9628-6979 Hiromichi Egami: 0000-0002-7784-4987 Yoshitaka Hamashima: 0000-0002-6509-8956 Naoto Oku: 0000-0002-1933-2545 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank Kae Misumi for technical advice. This work was supported by JSPS KAKENHI Grant Number 17K14519 and The Mochida Memorial Foundation for Medical and Pharmaceutical Research.

■

CONCLUSIONS In conclusion, we developed synthetic polymer nanoparticles (NPs) that captured indole, a target small molecule. The amounts of indole captured by the NPs depended on the hydrophobic monomer-structures, their feed ratios, and polymer densities, but not on particle size. The amount of indole captured by NPs incorporating 80-mol % 5FPAA was higher than that captured by those incorporating 80%-TBAm. The combination of 2 kinds of hydrophobic monomers (TBAm and 5FPAA, i.e., TF-NP) showed greater affinity for indole compared with single hydrophobic monomer-incorporated NPs (TB or FP-NP). A flexible polymer network was important for capturing indole. Furthermore, NPs specifically captured indole, unlike AST-120. The specificity for indole was improved by combining the 2 kinds of hydrophobic monomers without impairing indole-affinity. NPs were not degraded by digestive enzymes. TF-NPs significantly inhibited indoleintestinal absorption both in vitro and in vivo without being gastro-intestinally absorbed into the body. Until now, no drugs that specifically capture target molecules in the intestine have been developed. Most drugs capture not only the target molecules but also other nutrients and drugs. Since many nutrients and drugs are taken orally and absorbed in the intestine, nonspecific adsorption of these nutrient and drug result in malnutrition and reduced drug action. The present strategy of capturing a target molecule by synthetic polymers is applicable to several molecules by optimizing the functional monomer structure and composition. We believe that these results will prompt the development of novel strategies for the preparation of drugs that capture and inhibit the uptake of target molecules in the intestine.

■

Abundance parts per million analyses (PDF) Experimental procedures for the synthesis of 5FPAA and [3 H]-labeled NIPAm, procedure for LDH assay, procedures for evaluating monomer feed ratios of NPs, the characterization data (NMR, DLS) of NPs, and the data for the indole capture and cytotoxicity of NPs (PDF)

■

ABBREVIATIONS NIPAm, N-isopropylacrylamide; TBAm, N-tert-butylacrylamide; PAA, N-phenyacrylamide; 5FPAA, 2,3,4,5,6-pentafluorophenylacrylamide; Bis, N,N′-methylenebis(acrylamide); AAc, acrylic acid; APM, 2-N-(3-aminopropyl) methacrylamide; APS, ammonium persulfate; SDS, sodium dodecyl sulfate; AIBN, 2,2′-azodiisobutyronitrile; CTAB, hexadecyltrimethylammonium bromide; PBS, phosphate-buffered saline; DMEM, Dulbecco’s Modified Eagle’s Medium; FBS, fetal bovine serum; HBSS, Hank’s balanced salt solution

■

REFERENCES

(1) Trumbo, P.; Yates, A. A.; Schlicker, S.; Poos, M. Dietary reference intakes: vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. J. Am. Diet. Assoc. 2001, 101 (3), 294−301. (2) Tsuboyama-Kasaoka, N.; Takizawa, A.; Tsubota-Utsugi, M.; Nakade, M.; Imai, E.; Kondo, A.; Yoshida, K.; Okuda, N.; Nishi, N.; Takimoto, H. Dietary intake of nutrients with adequate intake values in the dietary reference intakes for Japanese. J. Nutr. Sci. Vitaminol. 2013, 59 (6), 584−95. (3) Yates, A. A. Dietary reference intakes: a new approach to setting nutrition standards in the USA and Canada. Bibl. Nutr. Dieta 2000, 55 (55), 4−13. (4) Schepers, E.; Glorieux, G.; Vanholder, R. The gut: the forgotten organ in uremia? Blood Purif. 2010, 29 (2), 130−6. (5) Ndanuko, R. N.; Tapsell, L. C.; Charlton, K. E.; Neale, E. P.; O’Donnell, K. M.; Batterham, M. J. Relationship between sodium and potassium intake and blood pressure in a sample of overweight adults. Nutrition 2017, 33, 285−290. (6) Bhartia, M.; Tahrani, A. A.; Barnett, A. H. SGLT-2 inhibitors in development for type 2 diabetes treatment. Rev. Diabet. Stud. 2011, 8 (3), 348−54. (7) Fernandez-Prado, R.; Esteras, R.; Perez-Gomez, M. V.; GraciaIguacel, C.; Gonzalez-Parra, E.; Sanz, A. B.; Ortiz, A.; Sanchez-Nino, M. D. Nutrients Turned into Toxins: Microbiota Modulation of Nutrient Properties in Chronic Kidney Disease. Nutrients 2017, 9 (5).

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biomac.8b01820. I

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules (8) Taniguchi, K.; Kakuta, H. Bixalomer, a novel phosphate binder with a small swelling index, improves hyperphosphatemia in chronic kidney disease rat. Eur. J. Pharmacol. 2015, 766, 129−34. (9) Schulman, G.; Vanholder, R.; Niwa, T. AST-120 for the management of progression of chronic kidney disease. Int. J. Nephrol. Renovasc. Dis. 2014, 7, 49−56. (10) Alswat, K. A. Role of Cholestyramine in Refractory Hyperthyroidism: A Case Report and Literature Review. Am. J. Case Rep. 2015, 16, 486−90. (11) Akizawa, T.; Origasa, H.; Kameoka, C.; Tsukada, J.; Kuroishi, K.; Yamaguchi, Y. Bixalomer in Hyperphosphatemic Patients With Chronic Kidney Disease Not on Dialysis: Phase 3 Randomized Trial. Ther. Apheresis Dial. 2016, 20 (6), 588−597. (12) Levy, R. I.; Fredrickson, D. S.; Stone, N. J.; et al. Cholestyramine in type ii hyperlipoproteinemia: A double-blind trial. Ann. Intern. Med. 1973, 79 (1), 51−58. (13) Muck, W.; Ritter, W.; Frey, R.; Wetzelsberger, N.; Lucker, P. W.; Kuhlmann, J. Influence of cholestyramine on the pharmacokinetics of cerivastatin. Int. J. Clin. Pharmacol. Ther. 1997, 35 (6), 250− 254. (14) Matsui, M. S.; Rozovski, S. J. Drug-nutrient interaction. Clin. Ther. 1982, 4 (6), 423−440. (15) Koya, Y.; Uchida, S.; Machi, Y.; Shobu, Y.; Namiki, N.; Kotegawa, T. Prediction of drug interaction between oral adsorbent AST-120 and concomitant drugs based on the in vitro dissolution and in vivo absorption behavior of the drugs. Eur. J. Clin. Pharmacol. 2016, 72 (11), 1353−1361. (16) Honda, Y.; Nakano, M. Studies on the Adsorption Characteristics of Spherical Charcoal (Kremezin). Byoin Yakugaku 1997, 23, 219−224. (17) Chiu, M. L.; Gilliland, G. L. Engineering antibody therapeutics. Curr. Opin. Struct. Biol. 2016, 38, 163−173. (18) You, C. C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I. B.; Erdogan, B.; Krovi, S. A.; Bunz, U. H.; Rotello, V. M. Detection and identification of proteins using nanoparticle-fluorescent polymer ’chemical nose’ sensors. Nat. Nanotechnol. 2007, 2 (5), 318−23. (19) Sandanaraj, B. S.; Demont, R.; Aathimanikandan, S. V.; Savariar, E. N.; Thayumanavan, S. Selective sensing of metalloproteins from nonselective binding using a fluorogenic amphiphilic polymer. J. Am. Chem. Soc. 2006, 128 (33), 10686−7. (20) Wada, Y.; Lee, H.; Hoshino, Y.; Kotani, S.; Shea, K. J.; Miura, Y. Design of multi-functional linear polymers that capture and neutralize a toxic peptide: a comparison with cross-linked nanoparticles. J. Mater. Chem. B 2015, 3 (8), 1706−1711. (21) Mignani, S.; El Kazzouli, S.; Bousmina, M. M.; Majoral, J. P. Dendrimer space exploration: an assessment of dendrimers/dendritic scaffolding as inhibitors of protein-protein interactions, a potential new area of pharmaceutical development. Chem. Rev. (Washington, DC, U. S.) 2014, 114 (2), 1327−42. (22) Imaoka, T.; Kawana, Y.; Kurokawa, T.; Yamamoto, K. Macromolecular semi-rigid nanocavities for cooperative recognition of specific large molecular shapes. Nat. Commun. 2013, 4, 2581. (23) Cutivet, A.; Schembri, C.; Kovensky, J.; Haupt, K. Molecularly imprinted microgels as enzyme inhibitors. J. Am. Chem. Soc. 2009, 131 (41), 14699−702. (24) Haupt, K.; Linares, A. V.; Bompart, M.; Bui, B. T. Molecularly imprinted polymers. Top. Curr. Chem. 2011, 325, 1−28. (25) Weisman, A.; Chen, Y. A.; Hoshino, Y.; Zhang, H.; Shea, K. Engineering Nanoparticle Antitoxins Utilizing Aromatic Interactions. Biomacromolecules 2014, 15 (9), 3290−3295. (26) Weisman, A.; Chou, B.; O’Brien, J.; Shea, K. J. Polymer antidotes for toxin sequestration. Adv. Drug Delivery Rev. 2015, 90, 81−100. (27) Latza, P.; Gilles, P.; Schaller, T.; Schrader, T. Affinity polymers tailored for the protein A binding site of immunoglobulin G proteins. Chem. - Eur. J. 2014, 20 (36), 11479−87. (28) Gilles, P.; Wenck, K.; Stratmann, I.; Kirsch, M.; Smolin, D. A.; Schaller, T.; de Groot, H.; Kraft, A.; Schrader, T. High-Affinity

Copolymers Inhibit Digestive Enzymes by Surface Recognition. Biomacromolecules 2017, 18 (6), 1772−1784. (29) Renner, C.; Piehler, J.; Schrader, T. Arginine- and lysinespecific polymers for protein recognition and immobilization. J. Am. Chem. Soc. 2006, 128 (2), 620−8. (30) Zhang, Q.; Collins, J.; Anastasaki, A.; Wallis, R.; Mitchell, D. A.; Becer, C. R.; Haddleton, D. M. Sequence-controlled multi-block glycopolymers to inhibit DC-SIGN-gp120 binding. Angew. Chem., Int. Ed. 2013, 52 (16), 4435−9. (31) Mitchell, D. A.; Zhang, Q.; Voorhaar, L.; Haddleton, D. M.; Herath, S.; Gleinich, A. S.; Randeva, H. S.; Crispin, M.; Lehnert, H.; Wallis, R.; Patterson, S.; Becer, C. R. Manipulation of cytokine secretion in human dendritic cells using glycopolymers with picomolar affinity for DC-SIGN. Chem. Sci. 2017, 8 (10), 6974−6980. (32) Schneider, T.; Welker, P.; Licha, K.; Haag, R.; Schulze-Tanzil, G. Influence of dendritic polyglycerol sulfates on knee osteoarthritis: an experimental study in the rat osteoarthritis model. BMC Musculoskeletal Disord. 2015, 16, 387. (33) Maysinger, D.; Ji, J.; Moquin, A.; Hossain, S.; Hancock, M. A.; Zhang, I.; Chang, P. K. Y.; Rigby, M.; Anthonisen, M.; Grutter, P.; Breitner, J.; McKinney, R. A.; Reimann, S.; Haag, R.; Multhaup, G. Dendritic Polyglycerol Sulfates in the Prevention of Synaptic Loss and Mechanism of Action on Glia. ACS Chem. Neurosci. 2018, 9 (2), 260− 271. (34) Dernedde, J.; Rausch, A.; Weinhart, M.; Enders, S.; Tauber, R.; Licha, K.; Schirner, M.; Zugel, U.; von Bonin, A.; Haag, R. Dendritic polyglycerol sulfates as multivalent inhibitors of inflammation. Proc. Natl. Acad. Sci. U. S. A. 2010, 107 (46), 19679−84. (35) Branderhorst, H. M.; Liskamp, R. M.; Visser, G. M.; Pieters, R. J. Strong inhibition of cholera toxin binding by galactose dendrimers. Chem. Commun. (Cambridge, U. K.) 2007, No. 47, 5043−5. (36) Haataja, S.; Verma, P.; Fu, O.; Papageorgiou, A. C.; Poysti, S.; Pieters, R. J.; Nilsson, U. J.; Finne, J. Rationally Designed Chemically Modified Glycodendrimer Inhibits Streptococcus suis Adhesin SadP at Picomolar Concentrations. Chem. - Eur. J. 2018, 24 (8), 1905− 1912. (37) Visini, R.; Jin, X.; Bergmann, M.; Michaud, G.; Pertici, F.; Fu, O.; Pukin, A.; Branson, T. R.; Thies-Weesie, D. M.; Kemmink, J.; Gillon, E.; Imberty, A.; Stocker, A.; Darbre, T.; Pieters, R. J.; Reymond, J. L. Structural Insight into Multivalent Galactoside Binding to Pseudomonas aeruginosa Lectin LecA. ACS Chem. Biol. 2015, 10 (11), 2455−62. (38) Hoshino, Y.; Kodama, T.; Okahata, Y.; Shea, K. J. Peptide imprinted polymer nanoparticles: a plastic antibody. J. Am. Chem. Soc. 2008, 130 (46), 15242−3. (39) Hoshino, Y.; Koide, H.; Furuya, K.; Haberaecker, W. W., 3rd; Lee, S. H.; Kodama, T.; Kanazawa, H.; Oku, N.; Shea, K. J. The rational design of a synthetic polymer nanoparticle that neutralizes a toxic peptide in vivo. Proc. Natl. Acad. Sci. U. S. A. 2012, 109 (1), 33− 8. (40) Lee, S. H.; Hoshino, Y.; Randall, A.; Zeng, Z.; Baldi, P.; Doong, R. A.; Shea, K. J. Engineered synthetic polymer nanoparticles as IgG affinity ligands. J. Am. Chem. Soc. 2012, 134 (38), 15765−72. (41) O’Brien, J.; Lee, S. H.; Onogi, S.; Shea, K. J. Engineering the Protein Corona of a Synthetic Polymer Nanoparticle for BroadSpectrum Sequestration and Neutralization of Venomous Biomacromolecules. J. Am. Chem. Soc. 2016, 138 (51), 16604−16607. (42) O’Brien, J.; Shea, K. J. Tuning the Protein Corona of Hydrogel Nanoparticles: The Synthesis of Abiotic Protein and Peptide Affinity Reagents. Acc. Chem. Res. 2016, 49 (6), 1200−10. (43) Koide, H.; Yoshimatsu, K.; Hoshino, Y.; Lee, S. H.; Okajima, A.; Ariizumi, S.; Narita, Y.; Yonamine, Y.; Weisman, A. C.; Nishimura, Y.; Oku, N.; Miura, Y.; Shea, K. J. A polymer nanoparticle with engineered affinity for a vascular endothelial growth factor (VEGF165). Nat. Chem. 2017, 9 (7), 715−722. (44) Niwa, T.; Miyazaki, T.; Hashimoto, N.; Hayashi, H.; Ise, M.; Uehara, Y.; Maeda, K. Suppressed serum and urine levels of indoxyl sulfate by oral sorbent in experimental uremic rats. Am. J. Nephrol. 1992, 12 (4), 201−6. J

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX

Article

Biomacromolecules (45) Niwa, T.; Ise, M.; Miyazaki, T. Progression of glomerular sclerosis in experimental uremic rats by administration of indole, a precursor of indoxyl sulfate. Am. J. Nephrol. 1994, 14 (3), 207−12. (46) Smith, T. A modification of the method for determining the production of indole by bacteria. J. Exp. Med. 1897, 2 (5), 543−547. (47) Gillam, E. M. J.; Notley, L. M.; Cai, H.; De Voss, J. J.; Guengerich, F. P. Oxidation of Indole by Cytochrome P450 Enzymes. Biochemistry 2000, 39 (45), 13817−13824. (48) Banoglu, E.; King, R. S. Sulfation of indoxyl by human and rat aryl (phenol) sulfotransferases to form indoxyl sulfate. Eur. J. Drug Metab. Pharmacokinet. 2002, 27 (2), 135−40. (49) Vanholder, R.; Schepers, E.; Pletinck, A.; Nagler, E. V.; Glorieux, G. The uremic toxicity of indoxyl sulfate and p-cresyl sulfate: a systematic review. J. Am. Soc. Nephrol. 2014, 25 (9), 1897− 907. (50) Duranton, F.; Cohen, G.; De Smet, R.; Rodriguez, M.; Jankowski, J.; Vanholder, R.; Argiles, A.; European Uremic Toxin Work Group. Normal and pathologic concentrations of uremic toxins. J. Am. Soc. Nephrol. 2012, 23 (7), 1258−1270. (51) Shimizu, H.; Yisireyili, M.; Nishijima, F.; Niwa, T. Indoxyl sulfate enhances p53-TGF-beta1-Smad3 pathway in proximal tubular cells. Am. J. Nephrol. 2013, 37 (2), 97−103. (52) Shimizu, H.; Bolati, D.; Adijiang, A.; Muteliefu, G.; Enomoto, A.; Nishijima, F.; Dateki, M.; Niwa, T. NF-kappaB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. Am. J. Physiol.: Cell Physiol. 2011, 301 (5), C1201−12. (53) Shimizu, H.; Bolati, D.; Adijiang, A.; Enomoto, A.; Nishijima, F.; Dateki, M.; Niwa, T. Senescence and dysfunction of proximal tubular cells are associated with activated p53 expression by indoxyl sulfate. Am. J. Physiol.: Cell Physiol. 2010, 299 (5), C1110−7. (54) Shimizu, H.; Bolati, D.; Adijiang, A.; Adelibieke, Y.; Muteliefu, G.; Enomoto, A.; Higashiyama, Y.; Higuchi, Y.; Nishijima, F.; Niwa, T. Indoxyl sulfate downregulates renal expression of Klotho through production of ROS and activation of nuclear factor-kB. Am. J. Nephrol. 2011, 33 (4), 319−24. (55) Yoshimatsu, K.; Koide, H.; Hoshino, Y.; Shea, K. J. Preparation of abiotic polymer nanoparticles for sequestration and neutralization of a target peptide toxin. Nat. Protoc. 2015, 10, 595. (56) Robson Marsden, H.; Fraaije, J. G.; Kros, A. Introducing quadrupole interactions into the peptide design toolkit. Angew. Chem., Int. Ed. 2010, 49 (46), 8570−2. (57) Collings, J. C.; Roscoe, K. P.; Thomas, R. L.; Batsanov, A. S.; Stimson, L. M.; Howard, J. A. K.; Marder, T. B. Arene-perfluoroarene interactions in crystal engineering. Part 3. Single-crystal structures of 1:1 complexes of octafluoronaphthalene with fused-ring polyaromatic hydrocarbons. New J. Chem. 2001, 25 (11), 1410−1417. (58) Nakamoto, M.; Hoshino, Y.; Miura, Y. Effect of Physical Properties of Nanogel Particles on the Kinetic Constants of Multipoint Protein Recognition Process. Biomacromolecules 2014, 15, 541−547. (59) Hoshino, Y.; Nakamoto, M.; Miura, Y. Control of ProteinBinding Kinetics on Synthetic Polymer Nanoparticles by Tuning Flexibility and Inducing Conformation Changes of Polymer Chains. J. Am. Chem. Soc. 2012, 134, 15209−15212. (60) Ofran, Y.; Rost, B. Analysing Six Types of Protein−Protein Interfaces. J. Mol. Biol. 2003, 325 (2), 377−387. (61) Ma, J. C.; Dougherty, D. A. The Cation−π Interaction. Chem. Rev. (Washington, DC, U. S.) 1997, 97 (5), 1303−1324. (62) Zhang, W. Nanoparticle Aggregation: Principles and Modeling. Adv. Exp. Med. Biol. 2014, 811, 19−43. (63) Boström, M.; Williams, D. R. M.; Ninham, B. W. Specific Ion Effects: Why DLVO Theory Fails for Biology and Colloid Systems. Phys. Rev. Lett. 2001, 87 (16), 168103. (64) Yonamine, Y.; Yoshimatsu, K.; Lee, S. H.; Hoshino, Y.; Okahata, Y.; Shea, K. J. Polymer nanoparticle-protein interface. Evaluation of the contribution of positively charged functional groups to protein affinity. ACS Appl. Mater. Interfaces 2013, 5 (2), 374−9.

(65) Cedervall, T.; Lynch, I.; Lindman, S.; Berggard, T.; Thulin, E.; Nilsson, H.; Dawson, K. A.; Linse, S. Understanding the nanoparticleprotein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (7), 2050−5. (66) Kettler, K.; Veltman, K.; van de Meent, D.; van Wezel, A.; Hendriks, A. J. Cellular uptake of nanoparticles as determined by particle properties, experimental conditions, and cell type. Environ. Toxicol. Chem. 2014, 33 (3), 481−492. (67) Abou Matar, T.; Karam, P. The Role of Hydrophobicity in the Cellular Uptake of Negatively Charged Macromolecules. Macromol. Biosci. 2018, 18, 1700309. (68) Alama, T.; Kusamori, K.; Katsumi, H.; Sakane, H.; Yamamoto, A. Absorption-enhancing effects of gemini surfactant on the intestinal absorption of poorly absorbed hydrophilic drugs including peptide and protein drugs in rats. Int. J. Pharm. (Amsterdam, Neth.) 2016, 499, 58−66.

K

DOI: 10.1021/acs.biomac.8b01820 Biomacromolecules XXXX, XXX, XXX−XXX