Tannic Acid as a Degradable Mucoadhesive Compound - ACS

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES

Article

Tannic Acid as a Degradable Mucoadhesive Compound Mikyung Shin, Keumyeon Kim, Whuisu Shim, Jae Wook Yang, and Haeshin Lee ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00051 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 26, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Biomaterials Science & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Biomaterials Science & Engineering

Tannic Acid as a Degradable Mucoadhesive Compound Mikyung Shin,† Keumyeon Kim,†,§ Whuisu Shim, ‡ Jae Wook Yang,∥ and Haeshin Lee*,†,‡,§

†The Graduate School of Nanoscience and Technology, ‡Department of Chemistry, Korea Advanced Institute of Science and Technology (KAIST), 291 University Rd, Yuseong-gu, Daejeon, 34141, South Korea §InnoTherapy Inc., 97 Uisadang-daero, Yeongdeungpo-gu, Seoul, 07327, South Korea ∥Department of Ophthalmology, Inje University Pusan Paik Hospital, Inje University College of Medicine, 75 Bokgi-ro, Busanjin-gu, Busan, 47392, South Korea KEYWORDS mucoadhesive, intermolecular interaction, tannic acid, esophagus

ABSTRACT: To achieve site-specific delivery of pharmaceuticals, the development of effective mucoadhesive polymers is essential. Thus far, only a few polymers, such as thiolated ones and related variants, have been studied. However, their mucoadhesiveness varies depending on the type of polymer and the degree of chemical functionalization. Furthermore, the chemistry of tethering often requires harsh reaction conditions. Recently, pyrogallol-containing molecules have emerged as good tissue and hemostatic adhesives, but their in vivo mucoadhesive properties

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 39

have not been demonstrated. Herein, we found that pyrogallol-rich tannic acid (TA) formulated with poly(ethylene glycol) (PEG), named TAPE, exhibits superior mucoadhesive properties. TAPE is prepared by a simple physical mixture of TA and PEG. It remained on esophageal mucus layers for at least several hours (< 8 hours) after oral feeding. The mucoadhesion originated from interactions between the polyphenols of TA and mucin, exhibiting pH dependency. TAPE adhered strongly to mucin in neutral conditions but bound weakly in acidic conditions due to different hydrolysis rates of the ester linkages in TA. Thus, TAPE might be useful as a long-lasting esophageal mucoadhesive composite.

INTRODUCTION Bioadhesive materials have been considered to be promising materials for designing sitespecific drug delivery systems to the mucosal surfaces of the vaginal, buccal, or gastrointestinal regions.1, 2 There are various formulations, such as bioadhesive films and tablets, containing an antifungal agent or an anti-human immunodeficiency virus (HIV) drug for vaginal drug delivery, or bioadhesive hydrogel for ocular delivery.3-5 A major research direction to develop mucoadhesive polymer has been the chemical conjugation of adhesive moieties to polymeric backbones. A representative example is thiolation.6 It enhances intrinsic mucoadhesive properties of various polymers, such as chitosan, poly(acrylic acid) (PAA), alginate, or hyaluronic acid, via disulfide bonds with mucus layers.7, 8 The thiolated mucoadhesive polymers are stable up to 360 hours if the thiol groups are fully tethered to the polymer backbone.9 However, the disulfide bonds with the mucus layer are


2

Page 3 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reversible at neutral pH, and the modification is conducted under harsh reaction conditions and toxic solvents. Recently, the chemical tethering of catechol or its derivatives onto polymers has been an emerging study aim. Catechol is found in mussel adhesive proteins and is generally considered to be a key molecule that increases wet-resistant adhesion.10,

11

Thus, attempts to develop new

mucoadhesive polymers by catechol conjugation have been reported.12-15 For example, catecholconjugated hyaluronic acid has been shown to form a mechanically strong hydrogel by intermolecular interaction with mucin.12 Chitosan-catechol conjugates show in vivo intestinal mucoadhesion for 10 hours13 and are effective for buccal mucoadhesion.14 The introduction of catechol onto poly(ethylene glycol) (PEG) enhances the mucoadhesion of PEG comparably to intrinsic mucoadhesive polymers, chitosan, gantrez, or PAA.15 Furthermore, a similar catechol derivative called pyrogallol shows excellent wet-resistant adhesive properties.16, 17 In particular, a pyrogallol/catechol/PEG mixture called TAPE, a composite polymer resulting from hydrogen bonds between tannic acid (TA) and poly(ethylene glycol) (PEG), has exhibited good hemostatic and tissue adhesive capabilities.16 The mucoadhesive properties of catechol have been reported, but the mucoadhesion of pyrogallol has not been previously studied. Additionally, TA is already an FDA-approved compound, so studies of the mucoadhesion of TA will be very useful for the future development of mucoadhesive drug delivery systems. TA is a polyphenol with high affinity for various proteins, such as proline-rich salivary proteins.18 However, the in vivo mucoadhesive properties of TA have not been demonstrated despite the considerable knowledge of wet-resistant bioadhesion based on its intermolecular interaction with proteins. Herein, we demonstrate that a TA-containing adhesive, TAPE, shows excellent mucoadhesive properties to the esophagus and exhibits subsequent rapid degradation within the stomach due to


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 39

the acidic environment. In thirty minutes after oral feeding of TAPE, we found that TAPE attached strongly to the esophagus and did not rapidly flow down into the subsequent organ of the stomach, indicating strong esophageal mucoadhesion. 8 hours after TAPE feeding, most of the TAPE was found in the stomach, with a trace amount of residual TAPE in the esophageal mucus layer. Once the TAPE entered the stomach, the ester bonds in TA were rapidly degraded, and its mucoadhesive properties were considered to be lost. Therefore, TAPE can be appropriate for esophageal mucoadhesive formulation. Furthermore, we demonstrated a controlled release of 5-FU from TAPE, the most popular anti-cancer drug for treating esophageal cancer. Considering that the esophageal track is a challenging target for drug delivery due to the low permeability and the dynamic nature of the esophagus, our study shows the potential utility of TAPE as a new wet-resistant adhesive platforms for esophageal target drug delivery.

EXPERIMENTAL METHODS Materials Indocyanine green (ICG), tannic acid (TA; MW 1,701.20), poly(ethylene glycol) (PEG; Mn 4,400 - 4,800 (4.6 kDa), Mn 8,500 - 11,500 (10 kDa) or Mn 16,000 - 24,000 (20 kDa)), and catechin hydrate (MW 290.3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Artificial gastric juice (pH 1.2) consisted of 7 mL HCl (35 wt%) and 2 g NaCl dissolved in 1000 mL deionized water, and the neutral buffer (pH 7.0) consisted of 6.8 g NaH2PO4 and 21.5 mL of 1 M NaOH dissolved in 1000 mL deionized water. 19, 20 Mucin type II from porcine stomach was obtained from Sigma-Aldrich. In addition, glycerin was purchased from Duchefa Biochemie (Haarlem, Netherlands). For the ex vivo mucoadhesion test, the Krebs-Ringer bicarbonate buffer


4

Page 5 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


contained, on a g/L basis, D-glucose 1.8; MgCl2 0.0468; KCl 0.34; NaCl 7.0; NaH2PO4 0.18; Na2HPO4 0.1. In addition, the simulated saliva consisted of KCl (0.4 mg/mL), NaCl (0.4 mg/mL), Na2SO4 (0.013 mg/mL), MgCl2 (0.018 mg/mL), K2HPO4 (4.2 mg/mL) KH2PO4 (3.2 mg/mL), and KOH (0.19 mg/mL).

Preparation of ICG-encapsulated tannic acid/poly(ethylene glycol) glue (ICG-TAPE) Tannic acid (TA) and poly(ethylene glycol) (PEG) were each dissolved in deionized water at a concentration of 1 g/mL. Three milligrams of ICG was liquefied in PEG solution (311 µL). TA solution (685 µL) was added to the ICG/PEG solution and mixed vigorously. The complex was prepared at a ratio of pyrogallol groups in TA to hydroxyl groups in PEG of 30:1. The resulting mixture was centrifuged for 5 minutes at 12,300 x g (Labogene mini with 12 holemicrorotor, Korea). In the course of TAPE(or /ICG) formulation, TAPE was formed at the bottom of bottles (or tubes), and the liquid byproduct was spontaneously formed, as the density of TAPE is larger than that of the byproduct. After the removal of the byproduct, the TAPE was incubated at 60 °C for 1 hour to remove all residual liquid.

Chemical analyses for the intermolecular interaction between TA and PEG in TAPE. For analyzing the intermolecular interaction between TA and PEG in TAPE, proton nuclear magnetic resonance (1H-NMR; Bruker Avance 300 MHz, UK) spectroscopy and fourier transform infrared (FT-IR; Agilent technologies, Cary 600 series, USA) spectroscopy were used. First, for 1H-NMR analysis, TAPE was solubilized in dimethyl sulfoxide-d6 (DMSO-d6). TA and


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 39

PEG (10 mg/mL) were also dissolved in the same solvent. Second, to obtain FT-IR spectra, TAPE, TA and PEG were freeze-dried, and the lyophilized powders were dispersed in potassium bromide (KBr) and compressed into disks. All FT-IR spectra were performed 20 scans over the range of 400 - 4000 cm-1.

Universal testing machine (UTM) studies for the adhesive force of TAPE The adhesion properties of TAPE were examined using UTM (Instron 5943, USA). To examine the adhesion properties dependent on the molecular ratio of catechol/pyrogallol groups in TA to the terminal hydroxyl groups in PEG and the molecular weight of PEG, the samples were prepared as five types, TAPEs with the ratio of 10 : 1, 20 : 1 or 30 : 1 using 4.6 kDa PEG and TAPEs using 10 kDa PEG or 20 kDa PEG at a ratio of 30 : 1. The samples were prepared on overhead projector (OHP) films (1 x 5 cm2). TAPE was compressed between OHP films with an area 1 x 1 cm2, and shear stress was applied using a 500 N cell at a rate of 10 mm/min. The adhesion force (kPa) was determined as the maximum load (kN) divided by the area (m2).

Absorbance and fluorescence emission of ICG in various solvents, deionized water, the artificial gastric juice (pH 1.2), TA, and PEG To measure the absorption and the fluorescence emission of ICG in different solvents, 10 µg/mL of ICG was dissolved in deionized water, artificial gastric juice (pH 1.2), TA solution (1 g/mL), and PEG solution (1 g/mL). The absorbance was measured by ultraviolet/visible (UV/vis)


6

Page 7 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


spectroscopy (HP8453, Hewlett Packard, USA). The fluorescence emission spectra were obtained with an Infinite M200 plate reader (Tecan, Austria).

Wet-adhesion properties of TAPE against water flow To confirm the wet-adhesive properties of TAPE and TA compared to PEG, TA solution (1 g/mL, 100 µL), PEG solution (1 g/mL, 100 µL) with commercial red food coloring dye with monoazo chromophores (Food Red No. 2, Bowon Food, Korea), and 100 µL of TAPE were prepared. Each material was put on a grid square dish tilted at an angle of ~ 60 degrees, and we measured the removal time of the materials under dropping water at a rate of ~ 2 mL/min

In vitro degradation kinetics of ICG-TAPE depending on pH To study the in vitro degradation kinetics of ICG-TAPE depending on pH, the weight change (%) of TAPE was measured. The adhesive (~100 mg) was soaked in artificial gastric juice (pH 1.2, 13 mL) or neutral buffer (pH 7.0, 13 mL). At predetermined time intervals (15 minutes, 1, 2, 4, and 6 hours), the weight of TAPE was measured after removing water drops on the surface of TAPE. In addition, rheological properties of TAPE degraded for 4 hours were investigated using a rotating rheometer (Bohlin Advanced Rheometer, Malvern Instruments, UK). The adhesives were loaded on the 20 mm parallel plate with a gap size 100 µm. The test range of frequency was varied from 0.1 to 10 Hz, and all measurements were conducted under a constant stress of 100 Pa at room temperature. Furthermore, to demonstrate the amount of the degradation products, the adhesive (~200 mg) was soaked in the same buffer (pH 1.2 or pH 7.0, 20 mL). Likewise, at a


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 39

pre-determined time interval, each released solution (500 µL) from ICG-TAPE was obtained. For relative quantitative analysis of the degraded products in the sampling solution, we measured the UV/vis absorption, and the absorption maximum wavelength (λmax) was compared at 260 nm for pH 1.2 and 272 nm for pH 7.0. In addition, the degradation of ICG-TAPE by pH alternation was tested. First, the ICG-TAPE was incubated in a neutral condition for 1 hour. Afterward, the adhesive was moved to the artificial gastric juice (pH 1.2) from 1 to 4 hours. Likewise, the UV/vis spectra for the released solutions were obtained. The amount of degradation products was calculated using the standard curves established in the concentration of pyrogallol from 0.02 to 0.13 mg/mL. Furthermore, the fluorescence emission of ICG encapsulated in TAPE depending on pH was measured by the IVIS 200 imaging system (Xenogen, CA, USA) at time intervals of 0, 10, 15, 30, and 45 minutes.

Intermolecular interaction between TA and mucin To demonstrate the intermolecular interaction between TA and mucin, we performed three experiments: turbidimetric titration, surface plasmon resonance (SPR) investigation, and imaging by scanning probe microscopy (SPM). First, for the turbidimetric titration, mucin was dissolved in phosphate buffered saline (PBS, pH 7.0 or pH 2.0) at a concentration of 1 mg/mL and dispersed using a high-power probe-type sonicator (Sonics Inc., USA) for 15 minutes with pulse (1 second sonication with 1 second interval). TA or PEG (10 mg/mL) was dissolved in PBS (pH 7.0 or pH 2.0). In addition, TAPE solution was prepared with the final concentration of 10 mg/mL, by mixing TA and PEG solutions (20 mg/mL respectively). TAPE, TA or PEG was dropped into mucin solution (500 µL) at ratios (g/g) of polymer to mucin of 0.2, 0.4, 2, 4, 8, and


8

Page 9 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16. The samples were mixed vigorously, and the turbidity was measured at 600 nm by UV/vis spectroscopy (HP8453, Hewlett Packard, USA). The relative absorbance (A600) of mucin solution without any polymers was calculated as follows: Relative absorbance (A600) = (A600 of polymer/mucin mixed solution)/(A600 of mucin solution) Second, the intermolecular interaction was analyzed using SPR (Biacore 3000, GE Healthcare, USA). A gold sensor chip was normalized using glycerol 70% (GE healthcare) prior to all measurements. The mucin (1 mg/mL, pH 7.0 or pH 2.0) was then adsorbed onto a sensor chip for 15 minutes at a flow rate of 10 µL/min. To remove mucin molecules adsorbed weakly on the chip, the washing process was continued for 10 minutes using PBS. TA or PEG solution in PBS (4 mg/mL, pH 7.0 or pH 2.0) was injected to the mucin-adsorbed chip for 5 minutes at a flow rate of 30 µL/min. Likewise, the washing process was continued for 10 minutes using PBS. Finally, the TA/mucin and the TAPE/mucin complexes in PBS (pH 7.0) were prepared at a weight ratio (g/g) of TA to mucin of 2:1, and the height of the complex was analyzed by scanning probe microscopy (Nanoman, Veeco, USA). As a control, PEG/mucin mixed solution was prepared at the same weight ratio as for TA. A solution comprised solely of mucin was used as a control. The mixture (50 µL) was dropped onto a mica substrate and adsorbed for 15 minutes. The complex-adsorbed surface was washed three times using deionized water and dried overnight.

Ex vivo and in vivo mucoadhesion of TAPE


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 39

To demonstrate the ex vivo esophageal mucoadhesion of TAPE, we designed an experimental setup using an everted rat esophagus model. Briefly, the rat esophagus was dissected into 2-cm segments (three Normal SD rats, 400-500 g, 10 weeks, Male) and incubated in Krebs-Ringer bicarbonate buffer. The esophagus segments were everted and placed onto a plastic rod with a 4.5 mm ball tip. TAPE was coated onto the mucosal layer of the everted esophagus. Glycerin was used as a control. Glycerin or TAPE-coated tissues were then rinsed in the simulated saliva (pH 7.0), and weight changes were measured at time intervals of 0, 2, 5, 10, 20, and 30 minutes. The retention weight (%) of the TAPE or glycerin was calculated as follows: Retention weight (%) = (Weight of TAPE or glycerin coated tissue after rinsing (mg)) × 100 / (Initial weight of TAPE or glycerin coated tissue (mg)) Furthermore, to study the in vivo mucoadhesion of ICG-TAPE, mice were starved overnight (BALBc mice, 23-25 g, 8-10 weeks, Male). Then, 0.02 cc of the adhesive was orally fed to the mice. Afterward, simulated saliva (pH 7.0, 0.1 cc) was orally injected into the mice. Likewise, the experiment was conducted in a control group using glycerin, PEG, or TA containing ICG (ICG-glycerin, ICG-PEG, or ICG-TA). In addition, to compare the in vivo mucoadhesion property of TAPE to that of other catechol-containing adhesives, catechin/PEG glue was prepared. In brief, PEG (200 mg/mL) was dissolved in deionized water, and catechin was added to the solution at a weight ratio of catechin to PEG, 1 : 2. The solution was vigorously mixed and centrifuged for 10 minutes at 12,300 x g. The mice were then sacrificed after 30 minutes or 8 hours. A fluorescence image map of the dissected gastrointestinal (GI) tract was obtained with an IVIS 200 imaging system (Xenogen, CA, USA). Three or more mice for each group (control or sample group at each time point) were used for the in vivo mucoadhesion test. The authors performed the animal experimental procedures and animal care with the approval of the Animal


10

Page 11 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Care Committee of KAIST, and followed the ethical protocol given by the Korean Ministry of Health and Welfare.

Statistical analysis Data are presented as the means ± standard deviations (SD). Statistical analysis was performed using one way analysis of variance (ANOVA) followed by the Tukey’s post hoc test by Origin Software (OriginLab 8.0). The probability value (P) < 0.05 was considered to indicate significant difference.

RESULTS AND DISCUSSION


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 39

Figure 1. (a) Schematic illustration of preparation of ICG-encapsulated TAPE mucoadhesive. (b) 1H-NMR spectrum of TAPE (red), TA (blue) and PEG (black) dissolved in DMSO-d6. The red square shows the downshift of the proton peak in -OH groups of TA. (c) FT-IR spectrum of TAPE (red), TA (blue) and PEG (black). (d and e) The adhesion force of TAPEs with (d) varying molar ratios of the -OH groups in TA to the terminal hydroxyl group of PEG and (e)


12

Page 13 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


different molecular weight of PEG at a molar ratio of 30 : 1 (n = 4). Data are means ± SD. * Significant difference from control (value for 10 : 1 (d) or 20 kDa PEG (e)) (*P < 0.05, one-way ANOVA). (f and g) Changes in absorption and fluorescent emission spectra of ICGs encapsulated in TAPE. (f) Absorption spectra of the ICG dissolved in deionized water (black), artificial gastric juice containing HCl (0.24 wt%) and NaCl (0.034 M) (pH 1.2) (blue), TA (red), and PEG (green). (g) The fluorescence emission spectra of the ICG in deionized water (black), TA (red), PEG (blue), and TAPE (green). (h) Photographs demonstrating the wet-resistant adhesion properties of PEG (pink, left), TA (light brown, middle), and ICG-encapsulated TAPE (dark green, right).

We prepared indocyanine green (ICG) encapsulated TAPE (ICG-TAPE) to visualize its in vivo mucoadhesion behavior (Figure 1a). TAPE was easily prepared by mixing TA (1 g/mL) and PEG (1 g/mL) solutions, and then the mixture was immediately centrifuged, and finally the supernatant was removed.16 Volume ratios of used TA and PEG solutions were described in the experimental methods. The preparation yield of TAPE was 60.1 ± 4.5%, which was determined by measuring the final weight of TAPE solution compared to initial weight. To investigate the intermolecular force existed in TAPE, proton nuclear magnetic resonance (1H-NMR) spectroscopy and fourier transform infrared (FT-IR) spectroscopy were used (Figure 1b and 1c). When 1H-NMR peaks of TAPE (red), TA (blue), and PEG (black) dissolved in dimethyl sulfoxide-d6 (DMSO-d6) were compared, the only proton peak of –OHs from catechol/pyrogallol in TA21 was slightly down-shifted from 10.0 to 10.2 ppm (‘a’ peak in red dashed square) (Figure 1b). This result indicates that the hydrogen bonds between PEGs and TA play an important role in TAPE formation. In addition, in FT-IR spectrum, catechol/pyrogallol -OH stretching vibration


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 39

in TA (3371 cm-1, blue) was shifted to a high wavenumber of 3382 cm-1 in TAPE (red), and the carbonyl group (C=O) stretching vibration in TA (blue) was shifted from 1715 to 1723 cm-1 in TAPE (red) (Figure 1c). The obvious peak in PEG spectrum (black) appeared at 2884 cm-1, which occurs by C-H stretching. In TAPE, the C-H stretching of PEG was higher-shifted (2884 2914 cm-1). All these changes show the intermolecular interaction between TA and PEG via hydrogen bonds.16, 22 Meanwhile, the adhesive force of TAPE was dependent on the molar ratio of pyrogallol in TA and the terminal –OH of PEGs in TAPE (Figure 1d). As the pyrogallol ratio to the hydroxyl group of PEG increased, enhancement of adhesive force was observed. The adhesive force was 1.7 ± 0.8 kPa for the ratio of 10 : 1 (left bar) and 3.1 ± 0.7 kPa for the ratio of 20 : 1 (middle bar). However, the adhesive force further increased up to 25.9 ± 1.5 kPa at a ratio of 30 : 1 (right bar). Thus, for the following experiments, TAPE was prepared at a ratio of catechol/pyrogallol groups in TA to hydroxyl groups in PEG of 30 : 1, and ICG was encapsulated at a stoichiometric ratio of one hundredth of TA. As the ratio of the catechol/pyrogallol groups in TAPE is high, the mucoadhesion property of TAPE can be increased.16 However, TAPE prepared at such a high molar ratio > 30:1 was too adhesive and viscous to handle. Thus, the ratio of 30 : 1 can be regarded as an optimal formulation for easy handling while still exhibiting strong mucoadhesion properties. We investigated the molecular weight effect of PEG on adhesiveness. The use of high molecular weight of PEGs resulted in a decrease in the adhesive force of TAPE (Figure 1e). It was 25.9 ± 1.5 kPa for PEG with low molecular weight (Mn 4,400 - 4,800; 4.6 kDa) (left bar), which was further decreased to 1.2 ± 0.8 kPa for 10 kDa PEG (Mn 8,500 - 11,500) (middle bar), and 1.1 ± 0.3 kPa was measured for 20 kDa PEG (Mn 16,000 - 24,000) (right bar). Therefore, 4.6 kDa PEG was used for the preparation of TAPE. The lowered adhesion force is closely related to the ‘flexibility’ of TAPE which means


14

Page 15 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


that the use of long chain PEGs typically results in flexible TAPE. The increased flexibility made an adverse effect on the mucoadhesion properties of TAPE. We hypothesized that TAPE might exhibit an extended residual time in the esophageal mucus layer and show pH dependency for the presence of ester linkages between catechol and pyrogallol. Thus, the rapid breakdown of the ester bonds in acidic environments (i.e., the stomach) might degrade TAPE, resulting in complete loss of the original mucoadhesive capability. First, the excitation and fluorescence emission of ICG were investigated in TA, PEG, deionized water (DDW) and an artificial gastric juice (pH 1.2) (Figure 1f and 1g). In Figure 1b, the maximum absorption wavelength of ICG (778 nm) was independent of the change in pH from neutral DDW (black) to an acidic artificial gastric juice (pH 1.2, blue). However, the absorption wavelength was red-shifted when TA or PEG was physically mixed: (778 803 nm, red for TA) or PEG (778 788 nm, green for PEG). The results indicated intermolecular interactions between ICG and TA as well as ICG and PEG.23, 24 These results were consistent with those of the previous study in which the absorption peak of ICG-containing phospholipid (PL)-PEG micelles self-assembled by interactions between amphiphilic ICG and PL-PEG also exhibited red-shift.24 This red-shifted trend of the maximal peak also appeared in the emission spectra (Figure 1g). The maximum emission peak of ICG in DDW (black) was at 813 nm but was at 819 nm for TA (red) and at 821 nm for PEG (blue) and TAPE (green). In particular, the emission of ICG was quenched by the addition of TA, but in contrast, the addition of PEG enhanced the emission of ICG. ICG emission was decreased by approximately forty-two percent upon TA addition (181,342 in DDW 76,297 in TA), but approximately 200% enhancement was observed with the addition of PEG (181,342 in DDW 367,607 in PEG). In TAPE, the emission was counterbalanced by the effect of TA and PEG (181,342 in DDW 230,374 in TAPE). The increase in fluorescent emission might be


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 39

attributed to the enhancement of ICG solubility by PEG, overcoming the negative effect of TA’s fluorescent quenching.24 Second, the wet-resistant adhesion of TAPE was evaluated on a grid dish tilted at an angle of ~ 60 degrees (Figure 1h). Strong in vivo adhesion resists the washing caused by the flow of body fluids. Thus, we designed an experiment to measure the materials’ surface adhesion-dependent resistance to a flow of DDW onto TAPE, TA, or PEG. The residual time of each material was measured. PEG slid down after 5 seconds (Figure 1h, 3rd photograph) of DDW flow, but TA and TAPE remained for 17 seconds (5th). The wet adhesion property of TAPE might result from the ten catechol/pyrogallol groups in TA.


16

Page 17 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (a) Schematic illustration of the mucosal adhesion and degradation of ICG-TAPE during esophagus-to-stomach transition (pH 7.0 pH 1.2). (b) The weight change (%) of TAPE depending on pH (black for pH 1.2 and red for pH 7.0) (n = 3). (c) The rheological properties of


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 39

TAPE after 4-hour degradation (green arrow in Figure 2b) (black: TAPE as prepared, red: TAPE degradation at pH 7.0 for 4 hours, blue: TAPE degradation at pH 1.2 for 4 hours. The filled symbol = elastic moduli (G’), the open symbol = viscous moduli (G’’)). (d) In vitro degradation profiles of TAPE as a function of pH values (λ260 for pH 1.2, black and λ272 for pH 7.0, red) (left panel). The blue arrow shown in the right panel indicates a sudden pH change from pH 7.0 to 1.2. The black dashed line is the same data showing the degradation kinetics at pH 1.2 from the left panel (black line) (n = 3). (e) The fluorescence image map of ICG-TAPE in neutral or acidic pH as a function of time (left panel) and the corresponding quantitative analysis (right panel) for the fluorescent image maps (n = 3). All data are means ± SD. * P < 0.05 at individual time points (one-way ANOVA).

In general, ester linkages are more rapidly hydrolyzed in an acidic condition than a neutral one. Thus, we hypothesized that the ester bonds in TAPE might remain chemically intact, exhibiting robust adhesion onto the mucus layer of the esophagus (pH 7.0), but degrade rapidly in the stomach (pH 1.2), resulting in significant loss of adhesive properties (Figure 2a). To study the degradation kinetics of TAPE as a function of pH, we first measured bulk weight change (%) of TAPE as a function of pH (pH 1.2 or 7.0) (Figure 2b). In an initial stage (< 30 minutes, blue arrow), the weight of TAPE slightly increased up to 111.3 ± 0.8 % for pH 7.0 (red) and 107.4 ± 3.8 % for pH 1.2 (black), which exhibited initial swelling of TAPE. The result showed that the early stage hydration of TAPE was similar independent of surrounding pH values of 7.0 and 1.2. However, after 30 minutes, the degradation rate of TAPE was largely distinguishable between pH 7.0 and pH 1.2. In acidic condition, the bulk weight of TAPE rapidly decreased down to 70.3 ± 6.1 % for 4 hours, which is originated from accelerated hydrolysis of the ester groups in TA by


18

Page 19 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


acid. In contrast, 99.4 ± 2.6% of TAPE remained at pH 7.0 for 4 hours (green arrow). Furthermore, we measured the rheological properties of all TAPE sample obtained after 4-hour degradation (samples from the green arrow in Figure 2b). Decreases in elastic moduli (G’) and viscous moduli (G’’) can be attributed to the effective molecular weight changes by degradation.25 As expected, the extent of decreases in G’ and G’’ values was greater at pH 1.2 than those measured at pH 7.0 (Figure 2c). G’ at 1 Hz was changed from 14,110 (black filled square) to 1,300 for pH 7.0 (red filled triangle) and 333.4 for pH 1.2 (blue filled circle). G’’ at 1 Hz was also changed from 7,181 (black empty square) to 3,185 for pH 7.0 (red filled triangle) and 1,425 for pH 1.2 (blue filled circle). In particular, in an acidic condition, G’’ was larger than G’ over the entire range of frequency. It indicates that the rheological behavior of TAPE is similar with viscous solution. Second, we measured the amount of degraded products from TAPE. UV/Vis spectroscopy can collectively measure the degraded species originating from TA due to the presence of pyrogallol groups (i.e., 3,4,5-trihydroxybenzoic acid or its similar compounds such as ellagic acid).17 Figure 2d shows the degradation profile of TAPE. As expected, the hydrolysis rate of TAPE was enhanced in acidic conditions (pH 1.2, black, λmax = 260 nm, for detecting primarily protonated gallic acid and minor other compounds) compared to neutral conditions (pH 7.0, red, λmax = 272 nm, for detecting primarily deprotonated pyrogallol and others) (Figure 2d, left panel). After four hours, the overall absorption at pH 1.2 (black) was almost two-fold greater than the value measured at pH 7.0 (red). This pH sensitivity was further demonstrated by the pH-altering experiment from 7.0 to 1.2 (Figure 2d, right panel, blue arrow). In this pH-altering experiment, interestingly, we found different degradation rates even at the same pH value of 1.2. The TAPE degradation was greatly accelerated, up to nine-fold, when experiencing the aforementioned pH down-shift (Figure 2d, right panel, blue). Although we did


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 39

observe the degradation of the same TAPE when it was incubated at pH 1.2 (the black line at the left panel and the black line at the right panel are the same data), simple acidic incubation at pH 1.2 in the absence of pH alteration exhibited a much slower degradation rate. The reason might be differences in the solvent infiltration into TAPE. In pH 7.0, a fraction of the hydroxyl groups of catechol/pyrogallol are deprotonated and negatively charged. Thus, electrostatic repulsion among TA molecules might allow a significant degree of solvent infiltration, resulting in ‘swelling’ (i.e., loss of intermolecular interactions between TA/TA or TA/PEG) of TAPE. However, at pH 1.2, nearly all hydroxyl groups of catechol/pyrogallol are protonated and neutrally charged. Thus, the electrostatic repulsion shown in neutral conditions is not present, resulting in ‘little swelling’ of TAPE. Thus, the swelling caused by pre-incubation in a neutral condition followed by a pH shift-down to 1.2 inevitably caused rapid and substantial degradation of the ester bonds of TA. This observed sensitivity of TAPE degradation to the pH changes (i.e., 7.0 1.2) is important because the pH-altering experiment can be a good mimic of the esophagus-to-stomach transition in vivo. That is, TAPE can adhere tightly to the esophageal mucus layer (pH 7.0) and then can be rapidly hydrolyzed upon reaching the stomach. At pH 1.2, which is similar to that of stomach acid, the fluorescence intensity image map of ICG-TAPE shown in Figure 2c exhibited rapid changes from green to blue in only 45 minutes (red graph in the right panel, bottom images). In contrast, at pH 7.0, the image map was not significantly changed, retaining its initial green color (black, top images). This result means that the degradation rate of TAPE decreased at pH 7.0. The right panel data in Figure 2c shows a quantitative analysis of the fluorescent images explained above. For neutral pH (black), a decrease in fluorescence was observed only during the initial 15 minutes (quantitatively from 1.5 x 1010 to 1.2 x 1010), and no further decrease was detected. However, for acidic pH (red), the


20

Page 21 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


trend of fluorescence decrease was similar for the initial 15 minutes (quantitatively 1.4 x 1010 to 9.4 x 109), but its emission attenuation was significant (7.6 x 109 for red and 1.2 x 1010 for black). After the initial 15 minutes, the fluorescence gradually decreased in an acidic environment (down to 7.6 x 109).

Figure 3. The intermolecular interaction between mucin and TA. (a) Turbidimetric titration adding TA to the mucin solutions. The relative absorbance (A600) of adding TA to mucin (black), adding TAPE to mucin (blue), adding PEG to mucin (red), or adding PBS to mucin as a control


21


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 39

(green), indicative of esophageal (pH 7.0, left) or gastric (pH 2.0, right) conditions. The photographs show TA/mucin complexation with pre-determined weight ratios (0.4, 2, 8, and 16 = [TA]/[Mucin]). Data are means ± SD. * Significant difference of value for adding TA to mucin from controls (adding TAPE, PEG or PBS to mucin) (P < 0.05, one-way ANOVA). (b) SPR studies of TA adsorption on mucin-coated surfaces depending on pH values (red for pH 7.0 and blue for pH 2.0): 1st arrow for mucin (1 mg/mL) injection, 2nd arrow for PBS washing, 3rd arrow for TA (red and blue) or PEG (black lines) injection (4 mg/mL), and 4th arrow for PBS washing. (c) SPM images of only mucin (top, left) TA/mucin complexes (bottom, left), PEG/mucin mixture as a control (top, right) and TAPE/mucin complexes (top, right) in an esophageal neutral condition.

TA is a polyphenol molecule that binds strongly to various proteins, including thrombin,26 elastin27 and a proline-rich saliva protein.18 Thus, we hypothesized that TA would be mucoadhesive if the molecule has a strong affinity for mucins secreted onto the mucosal surfaces of the gastrointestinal (GI) tract. Recently, the TA-containing adhesive TAPE has also been shown to have good tissue adhesion and hemostatic ability.16 The intermolecular interaction between TA and mucin was verified by turbidimetric titration. Generally, micro-sized, randomly shaped particles are generated when two biomacromolecules are strongly entangled with each other. Because of this large entanglement, incident visible light is scattered, which can be observed as a turbid solution. As shown in Figure 3a (an esophageal condition), the turbidity increased from a weight ratio of TA to mucins of 4 and significantly further increased (more than 25-fold) by a ratio of 16 at pH 7.0 (photos and black line shown in the left panel). Furthermore, adding TAPE to mucin also formed a turbid solution in an esophageal, neutral condition. At pH


22

Page 23 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


7.0, the turbidity increased to > 5-fold at a ratio of 16 (blue line shown in the left panel). However, the transparency of the same solutions was largely maintained when adding PEGs or PBS to the mucin solutions (red line and green line shown in the left graph). Large changes in the results of turbidimetric titration were observed simply by changing pH from 7.0 to 2.0 (esophageal to gastric conditions). The turbidity due to the formation of TA/mucin or TAPE/mucin complexes was only slightly increased in acidic conditions (photos and black/blue lines shown in the right panel). Even when TA was added to the mucin solution at the weight ratio of 16, the turbidity only increased up to 2.5 times. Likewise, when TAPE was added to the mucin solution at the same ratio, the degree of turbidity was similar with the level of TA only (blue line shown in the right panel). For the case of pH 7.0, a twenty-five-fold increase in turbidity was measured by comparison. The result was interpreted to show that the affinity of TA toward mucin was decreased by fast ester hydrolysis at pH 2.0. In addition, surface plasmon resonance (SPR) spectroscopy was used to study the intermolecular interaction between TA and mucin. Previous studies have also used SPR spectroscopy to demonstrate the non-covalent interactions of biomolecules, for example, antigen-antibody binding or protein-polysaccharide interactions.28 For quantitative measurement of the interaction between TA and mucin depending on pH values, mucin was first adsorbed on the surface of gold sensor chips at pH 7.0 (Figure 3b, red and black) or pH 2.0 (blue and dashed black). The adsorption of mucin (1st arrow) was approximately 836 ± 61 pg/mm2 at pH 7.0 and 1,540 ± 291 pg/mm2 at pH 2.0, based on the change of 1 RU corresponding to 1 pg/mm2.29 To remove the weakly adsorbed mucin, the coated channel was washed using PBS (2nd arrow) for 10 minutes after the injection of mucin (1 mg/mL). TA (red and blue lines) or PEG (black lines) solution (4 mg/mL) was then injected onto the mucin-coated channel (3rd arrow). As expected, changes in RU values upon the injection of


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 39

PEG solutions at pH 7.0 and pH 2.0 remained nearly unchanged (∆ = -87 for pH 7.0 and 0 for pH 2.0). In contrast, the RU value significantly increased to (∆ =) 3,100 at pH 7.0 upon the adsorption of TA, showing the strong interaction between TA and mucin. At pH 2.0, the RU change was (∆ =) 499. The SPR result at pH 2.0 was less than at pH 7.4 despite a large amount of mucin adsorption, which is correlated with the turbidimetric titration results. The turbidity caused by mucin and TA at pH 7.0 was large compared to observations at pH 2.0. The TA/mucin complex at pH 7.4 was also confirmed by atomic force microscopy (AFM) (Figure 3c). We chose ratios of TAPE, TA, or PEG to mucin (g/g) of 2 : 1. The height of PEG/mucin mixture was 4.5 ± 1.6 nm, which is similar to the topology of mucin itself with the height of 4.1 ± 1.4 nm.30 However, the TA/mucin complex showed granule-like morphologies. The result indicates that the granule-like aggregates by the interaction between mucin and TA are formed at random. The height of TA/mucin complex was 26.3 ± 10.3 nm, which was 5 times higher than that of the PEG/mucin mixture. Likewise, the TAPE/mucin complex exhibited particle-like morphologies with the height of 15.5 ± 1.8 nm. The results indicate that TA is an important morphological determinant to form large, granule-like structures when complexed with mucin. Based on the aforementioned results, Figure 4 provides a schematic illustration of the role of TA (i.e. TAPE) in esophagus and stomach environments.


24

Page 25 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4 Schematic description explaining the esophageal mucoadhesion of TA by the intermolecular interaction between TA and mucin (pH 7.0) and subsequent acid-catalytic hydrolysis of TA in the stomach (pH 2.0).


25


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 39

Figure 5. Mucoadhesion of TAPE in GI tract. (a) Experimental setup for demonstrating TAPE ex vivo mucoadhesion to the esophageal mucus layer. (b) Ex vivo esophageal mucoadhesive properties of TAPE (red), TA (blue), PEG (green) or glycerin (black). Data are means ± SD. * P < 0.05 for the value of controls (glycerin, TA, and PEG) at individual time points (one-way ANOVA). (c) ICG-fluorescence image map for in vivo mucoadhesion of glycerin (1st panel), PEG (2nd panel), TA (3rd panel) or ICG-TAPE (4th panel) in GI tract 30 minutes (top) and 8 hours (bottom) after mouse oral feedings.


26

Page 27 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Finally, we tested the mucoadhesion of TAPE both ex vivo and in vivo. First, the ex vivo mucoadhesion of TAPE was evaluated on the mucus layer of the esophagus, which is the entrance to the GI tract (areas of neutral pH). Figure 5a shows the experimental design for the ex vivo mucoadhesion, which consisted of an everted rat esophagus mucus layer, TAPE, and the supporting bar.31 As a control, glycerin, a representative non-mucoadhesive material, TA and PEG were applied. As expected, TAPE showed excellent mucoadhesion characteristics on the esophageal mucus layer (Figure 5b, red). Almost no desorption of TAPE from the layer was observed (relative weight was nearly 100% compared to the weight measured at t = 0). However, glycerin was rapidly rinsed away after 2 minutes of soaking with the simulated saliva (pH 7.0). Only 16.9 ± 3.8% weight remained after 2 minutes and 14.8 ± 3.5% after 5 minutes (Figure 5b, black). TA and PEG were also rinsed away after 2 minutes of soaking, and the residual weight of each sample was 21.6 ± 2.1 % for TA (Figure 5b, blue) and 15.7 ± 5.0 % for PEG (Figure 5b, green). However, the weight of TAPE was nearly unchanged even after 30 minutes (101.0 ± 9.8 %). In addition, the initial weight of TAPE increased slightly to 107.0 ± 3.6 % in 5 minutes, which might be due to the water-retaining capability of the highly hydrophilic PEG. Second, we performed experiments focusing on TAPE’s behavior during esophagus-to-stomach movement. We found that TAPE was present on the esophageal mucus layers 30 minutes after mouse oral feedings (up image in 4th image of Figure 5c) and remained for at least several hours without rapid flow into the subsequent organ, the stomach. In contrast, glycerin, PEG, and TA were not found in the esophagus at similar timeframe (30 minutes) and moved rapidly into the small intestine, even passing through the stomach (up images in the 1st to 3rd images of Figure 5c). The result indicates the strong esophageal retention capability of TAPE. Afterward, most TAPE was observed in the stomach after 8 hours with trace amounts of residual TAPE in the esophagus


27


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 39

(bottom image in the 4th image of Figure 5c); however, most glycerin and PEG were found in the terminal of the large intestine (bottom images in the 1st and 2nd images of Figure 5c). Interestingly, almost the entire amount of TA remained in both small and large intestines, which exhibited superior mucoadhesion properties of TA than PEG or glycerin in a molecular level. In addition, we compared the mucoadhesion property of TAPE to that of catechin, one of other catechol-containing molecules, which can interact with PEG (Figure S1 in supporting information). The catechin-PEG adhesive (similar to tannic acid-PEG, TAPE) showed little esophageal-mucoadhesion, which was similar with the results of non-mucoadhesive materials, glycerin or PEG. The results indicate that the mucoadhesion of TA was considerably distinguishable compared to other controls and a catechol-derivative chemical. When TAPE reaches the stomach, the ester bonds in TA are rapidly hydrolyzed, resulting in the loss of its bulk mucoadhesive characteristics. Finally, we utilized the unique wet-resistant adhesive property of TAPE for local delivery of anti-cancer drugs. Due to the aforementioned excellent esophageal wet adhesion of TAPE, we chose and encapsulated 5-fluorouracil (5-FU), the most popular anti-cancer drug, for potential treatment of esophageal cancer, in which TAPE/5-FU would be an effective drug depot and subsequent controlled release of the drug (supporting Figure S2). The 5-FU release profile at pH 7.0 was analyzed by reverse-phase liquid chromatography (RP-LC). The peak appeared at 5.5 minutes was the released 5-FU followed by the degradation products of TA (~ 7.1 minutes). The peak area of 5-FU as a function of time was dramatically increased from 6,958 AUC (30 minutes) to 17,845 AUC (6 hours). This result demonstrates the practical utility of TAPE as a wet-resistant anti-cancer drug depot on wet tissues.


28

Page 29 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


CONCLUSIONS In conclusion, we demonstrated for the first time the in vivo mucoadhesion behavior of the pyrogallol-containing adhesive, TAPE. This behavior was observed on an esophageal mucus layer 30 minutes after the TAPE oral feeding and remained there for several hours. Its esophageal retention was completed after 8 hours, at which point most of TAPE was found in the stomach. Once TAPE reached the stomach, its mucoadhesion was rapidly lost due to the loss of intermolecular interactions between TA and mucin, followed by rapid hydrolysis of the ester groups in TA at low pH. Therefore, TAPE is a promising, long-lasting esophageal mucoadhesive material.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ACKNOWLEDGMENT This study was financially supported from National Research Foundation of South Korea: Midcareer scientist grant (2014002855), Molecular-level Interface Research Center (20090083525). Also, this study was supported in part by Korea Healthcare Technology R&D Project from the Ministry of Health and Welfare (HI12C0005).

ASSOCIATED CONTENT *Supporting Information


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 39

The Supporting Information is available free of charge on the ACS Publications website at DOI:xx. In vivo fluorescence maps for mucoadhesion property of catechin/PEG complex and in vitro 5FU release profile from TAPE (PDF)

REFERENCES (1) Peppas, N. A.; Sahlin, J. J. Hydrogels as mucoadhesive and bioadhesive materials: a review. Biomaterials 1996, 17, 1553-1561. (2) Vasir, J. K.; Tambwekar, K.; Garg, S. Bioadhesive microspheres as a controlled drug delivery system. Int. J. Pharmaceut. 2003, 255, 13-32. (3) Ghosal, K.; Ranjan, A.; Bhowmik, B. B. A novel vaginal drug delivery system: anti-HIV bioadhesive film containing abacavir. J. Mater. Sci:. Mater. Med. 2014, 25, 1679-1689. (4) Cevher, E.; Açma, A.; Sinani, G.; Aksu, B.; Zloh, M.; Mülazımoğlu, L. Bioadhesive tablets containing cyclodextrin complex of itraconazole for the treatment of vaginal candidiasis. Int. J. Biol. Macromol. 2014, 69, 124–136. (5) Mayol, L.; Quaglia, F.; Borzacchiello, A.; Ambrosio, L.; La Rotonda, M. I. A novel poloxamers/hyaluronic acid in situ forming hydrogel for drug delivery: Rheological, mucoadhesive and in vitro release properties. Eur. J. Pharm. Biopharm. 2008, 70, 199-206. (6) Bernkop-Schnürch, A.; Schwarz, V.; Steininger, S. Polymers with thiol groups: A new generation of mucoadhesive polymers. Pharmaceut. Res. 1999, 16, 876-881.


30

Page 31 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) Bernkop-Schnürch, A. Thiomers: A new generation of mucoadhesive polymers. Adv. Drug. Deliv. Rev. 2005, 57, 1569-1582. (8) Ding, J.; He, R.; Zhou, G.; Tang, C.; Yin, C. Multilayered mucoadhesive hydrogel films based on thiolated hyaluronic acid and polyvinylalcohol for insulin delivery. Acta Biomater. 2012, 8, 3643-3651. (9) Roldo, M.; Hornof, M.; Caliceti, P.; Bernkop-Schnürch, A. Mucoadhesive thiolated chitosans as platforms for oral controlled drug delivery: synthesis and in vitro evaluation. Eur. J. Pharm. Biopharm. 2004, 57, 115-121. (10) Waite, J. H. Nature's underwater adhesive specialistInt. J. Adhes. Adhes. 1987, 7, 9-14. (11) Waite, J. H.; Ann, N. Y.; Reverse Engineering of Bioadhesion in Marine Mussels. Acad. Sci. 1999, 875, 301-309. (12) Lee, Y.; Chung, H. J.; Yeo, S.; Ahn, C. H.; Lee, H.; Messersmith, P. B.; Park, T. G. Thermo-sensitive, injectable, and tissue adhesive sol–gel transition hyaluronic acid/pluronic composite hydrogels prepared from bio-inspired catechol-thiol reaction. Soft Matter 2010, 6, 977-983. (13) Kim, K.; Kim, K.; Ryu, J. H.; Lee, H. Chitosan-catechol: A polymer with long-lasting mucoadhesive properties. Biomaterials 2015, 52, 161-170. (14) Xu, J.; Strandman, S.; Zhu, J. X.X.; Barralet, J.; Cerruti, M. Genipin-crosslinked catecholchitosan mucoadhesive hydrogels for buccal drug delivery. Biomaterials 2015, 37, 395-404. (15) Catron, N. D.; Lee, H.; Messersmith, P. B. Enhancement of poly(ethylene glycol) mucoadsorption by biomimetic end group functionalization. Biointerphases 2006, 1, 134-141.


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 39

(16) Kim, K.; Shin, M.; Koh, M.; Ryu, J. H.; Lee. M. S.; Hong, S.; Lee, H. TAPE: A medical adhesive inspired by a ubiquitous compound in plants. Adv. Funct. Mater. 2015, 25, 2402-2410. (17) Shin, M.: Ryu, J. H.; Park, J. P.; Kim, K.; Yang, J. W.; Lee, H. DNA/Tannic acid hybrid gel exhibiting biodegradability, extensibility, tissue adhesiveness, and hemostatic ability. Adv. Funct. Mater. 2015, 25, 1270–1278. (18) McRae, J. M.; Kennedy, J. A. Wine and grape tannin interactions with salivary proteins and their impact on astringency: A review of current research. Molecules 2011, 16, 2348-2364. (19) Zhang, Z.; Chen, L.; Zhao, C.; Bai, Y.; Deng, M.; Shan, H.; Zhuang, X.; Chen, X.; Jing, X. Thermo- and pH-responsive HPC-g-AA/AA hydrogels for controlled drug delivery applications. Polymer 2011, 52, 676-682. (20) Rao, A. V.; Shiwnarain, N.; Maharaj, I. Survival of microencapsulated bifidobacterium pseudolongum in simulated gastric and intestinal juices. J. lnst. Can. Sei. Technal. Aliment. 1989, 22, 345-349. (21) Peng, Y.; Zheng, Z.; Sun, P.; Wang, X.; Zhang, T. Synthesis and characterization of polyphenol-based polyurethane. NewJ. Chem. 2013, 37, 729-734. (22) Khoultchaev, Kh. Kh.; Pang, P.; Kerekes, R. J.; Englezos, P. Enhancement of the retention performance of the poly(ethy1ene oxide) - Tannic acid system by poly(diallyldimethyl ammonium chloride). Can. J. Chem. Eng. 1998, 76, 261-266. (23) Philip, R.; Penzkofer, A.; Bäumler, W.; Szeimies, R. M.; Abels, C. Absorption and fluorescence spectroscopic investigation of indocyanine green. J. Photoch. Photobio. A. 1996, 96, 137-148.


32

Page 33 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(24) Zheng, X.; Xing, D.; Zhou, F.; Wu, B.; Chen, W. R. Indocyanine green-containing nanostructure as near infrared dual-functional targeting probes for optical imaging and photothermal therapy. Mol. Pharmaceutics 2011, 8, 447–456. (25) Tayal, A.; Pai, V. B.; Khan, S. A. Rheology and microstructural changes during enzymatic degradation of a guar-borax hydrogel. Macromolecules 1999, 32, 5567-5574. (26) Shukla, A.; Fang, J. C.; Puranam, S.; Jensen, F. R.; Hammond, P. T. Hemostatic multilayer coatings. Adv. Mater. 2012, 24, 492-496. (27) Isenburg, J. C.; Simionescu, D. T.; Vyavahare, N. R. Elastin stabilization in cardiovascular implants: improved resistance to enzymatic degradation by treatment with tannic acid. Biomaterials 2004, 25, 3293-3302. (28) Green, R. J.; Frazier, R. A.; Shakesheff, K. M.; Davies, M. C.; Roberts, C. J.; Tendler, S. J. B. Surface plasmon resonance analysis of dynamic biological interactions with biomaterials. Biomaterials 2000, 21, 1823-1835. (29) Stenberg, E.; Persson, B.; Roos, H.; Urbaniczky, C. Quantitative determination of surface concentration of protein with surface plasmon resonance using radiolabeled proteins. J. Colloid Interface Sci. 1991, 43, 513-526. (30) Yakubov, G. E.; Papagiannopoulos, A.; Rat, E.; Waigh, T. A. Charge and interfacial behavior of short side-chain heavily glycosylated porcine stomach mucin. Biomacromolecules 2007, 8, 3791-3799. (31) Dobrozsi, D. J.; Smith, R. L.; Sakr, A. A. Comparative mucoretention of sucralfate suspensions in an everted rat esophagus model. Int. J. Pharmaceut. 1999, 189, 81-89.


33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 39

*** For Table of Contents Use only

Tannic Acid as a Degradable Mucoadhesive Compound Mikyung Shin,† Keumyeon Kim,†,§ Whuisu Shim, ‡Jae Wook Yang,∥ and Haeshin Lee*,†,‡,§


34

Page 35 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. (a) Schematic illustration of preparation of ICG-encapsulated TAPE mucoadhesive. (b) 1H-NMR spectrum of TAPE (red), TA (blue) and PEG (black) dissolved in DMSO-d6. The red square shows the downshift of the proton peak in -OH groups of TA. (c) FT-IR spectrum of TAPE (red), TA (blue) and PEG (black). (d and e) The adhesion force of TAPEs with (d) varying molar ratios of the -OH groups in TA to the terminal hydroxyl group of PEG and (e) different molecular weight of PEG at a molar ratio of 30 : 1 (n = 4). Data are means ± SD. * Significant difference from control (value for 10 : 1 (d) or 20 kDa PEG (e)) (*P < 0.05, one-way ANOVA). (f and g) Changes in absorption and fluorescent emission spectra of ICGs encapsulated in TAPE. (f) Absorption spectra of the ICG dissolved in deionized water (black), artificial gastric juice containing HCl (0.24 wt%) and NaCl (0.034 M) (pH 1.2) (blue), TA (red), and PEG (green). (g) The fluorescence emission spectra of the ICG in deionized water (black), TA (red), PEG (blue), and TAPE (green). (h) Photographs demonstrating the wet-resistant adhesion properties of PEG (pink, left), TA (light brown, middle), and ICG-encapsulated TAPE (dark green, right). 205x242mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Page 36 of 39

Page 37 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. (a) Schematic illustration of the mucosal adhesion and degradation of ICG-TAPE during esophagusto-stomach transition (pH 7.0 ◊ pH 1.2). (b) The weight change (%) of TAPE depending on pH (black for pH 1.2 and red for pH 7.0) (n = 3). (c) The rheological properties of TAPE after 4-hour degradation (green arrow in Figure 2b) (black: TAPE as prepared, red: TAPE degradation at pH 7.0 for 4 hours, blue: TAPE degradation at pH 1.2 for 4 hours. The filled symbol = elastic moduli (G’), the open symbol = viscous moduli (G’’)). (d) In vitro degradation profiles of TAPE as a function of pH values (λ260 for pH 1.2, black and λ272 for pH 7.0, red) (left panel). The blue arrow shown in the right panel indicates a sudden pH change from pH 7.0 to 1.2. The black dashed line is the same data showing the degradation kinetics at pH 1.2 from the left panel (black line) (n = 3). (e) The fluorescence image map of ICG-TAPE in neutral or acidic pH as a function of time (left panel) and the corresponding quantitative analysis (right panel) for the fluorescent image maps (n = 3). All data are means ± SD. * P < 0.05 at individual time points (one-way ANOVA). 208x328mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

Page 38 of 39

Page 39 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. The intermolecular interaction between mucin and TA. (a) Turbidimetric titration adding TA to the mucin solutions. The relative absorbance (A600) of adding TA to mucin (black), adding TAPE to mucin (blue), adding PEG to mucin (red), or adding PBS to mucin as a control (green), indicative of esophageal (pH 7.0, left) or gastric (pH 2.0, right) conditions. The photographs show TA/mucin complexation with predetermined weight ratios (0.4, 2, 8, and 16 = [TA]/[Mucin]). Data are means ± SD. * Significant difference of value for adding TA to mucin from controls (adding TAPE, PEG or PBS to mucin) (P < 0.05, one-way ANOVA). (b) SPR studies of TA adsorption on mucin-coated surfaces depending on pH values (red for pH 7.0 and blue for pH 2.0): 1st arrow for mucin (1 mg/mL) injection, 2nd arrow for PBS washing, 3rd arrow for TA (red and blue) or PEG (black lines) injection (4 mg/mL), and 4th arrow for PBS washing. (c) SPM images of only mucin (top, left) TA/mucin complexes (bottom, left), PEG/mucin mixture as a control (top, right) and TAPE/mucin complexes (top, right) in an esophageal neutral condition. 202x205mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4 Schematic description explaining the esophageal mucoadhesion of TA by the intermolecular interaction between TA and mucin (pH 7.0) and subsequent acid-catalytic hydrolysis of TA in the stomach (pH 2.0). 168x135mm (150 x 150 DPI)


Page 40 of 39

Page 41 of 39

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Mucoadhesion of TAPE in GI tract. (a) Experimental setup for demonstrating TAPE ex vivo mucoadhesion to the esophageal mucus layer. (b) Ex vivo esophageal mucoadhesive properties of TAPE (red), TA (blue), PEG (green) or glycerin (black). Data are means ± SD. * P < 0.05 for the value of controls (glycerin, TA, and PEG) at individual time points (one-way ANOVA). (c) ICG-fluorescence image map for in vivo mucoadhesion of glycerin (1st panel), PEG (2nd panel), TA (3rd panel) or ICG-TAPE (4th panel) in GI tract 30 minutes (top) and 8 hours (bottom) after mouse oral feedings. 205x174mm (150 x 150 DPI)


Tannic Acid as a Degradable Mucoadhesive Compound - ACS

Recommend Documents