Article pubs.acs.org/Biomac
Acrylate-Tethering Drug Carrier: Covalently Linking Carrier to Biological Surface and Application in the Treatment of Helicobacter pylori Infection Amornset Tachaprutinun,† Porntip Pan-In,† Pawatsanai Samutprasert,† Wijit Banlunara,‡ Nuntaree Chaichanawongsaroj,§ and Supason Wanichwecharungruang*,†,∥,⊥ †
Department of Chemistry, Faculty of Science, ‡Department of Pathology, Faculty of Veterinary Science, §Department of Transfusion Medicine, Faculty of Allied Health Sciences, and ∥The Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University, Bangkok, Thailand ⊥ Nanotec-CU Center of Excellence on Food and Agriculture, Bangkok, Thailand S Supporting Information *
ABSTRACT: The development of carriers to sustain drugs at stomach surface is an attractive strategy to increase drug bioavailability locally and systematically. So far, the only reported carrier that can form a covalent bond with mucus, the thiolated carrier, relies on a reversible disulfide exchange reaction between thiols on the carrier and disulfide bridges on the mucus. Here we show the design and fabrication of a cellulose carrier with tethering acrylate groups (denoted here as clickable carrier) that, under a nontoxic condition, can efficiently react with thiols on biomaterials in situ through the thermodynamically driven and kinetically probable Michael thiol−ene click reaction. Here we show the attachments of the clickable carriers to a mucin protein, a surface of human laryngeal carcinoma cells, and a surface of a fresh porcine stomach. We also show that the required thiol moieties can be generated in situ by reducing existing cystine disulfide bridges with either the edible vitamin C or the relatively nontoxic tris(2carboxyethyl) phosphine. Comparing to a control carrier, the clickable carrier can increase some drug concentrations in an ex vivo stomach tissue, and improve the Helicobacter pylori treatment in infected C57BL/6 mice.
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INTRODUCTION Ability to localize drug depots at biological surfaces such as stomach, buccal, vaginal, respiratory track, in a simple and nontoxic manner, will help prolonging systemic drug concentrations and localizing drug molecules at specific area. Up until now, the only reported strategy to covalently link drug depots onto biological surfaces is based on the disulfide exchange reaction between thiolated carriers, carriers with tethering thiol groups, and cystine moieties on biomaterials.1−5 Nevertheless, not only that the thiol−disulfide exchange reaction is equilibrium driven and reversible, but also the rate and likelihood of this reaction on biological surfaces still await more investigation. As a gentle and metal-free reaction, the Michael thiol−ene process has been extensively employed in various biological fields.6−8 The reaction can be performed under a mild base,9,10 a nucleophilic catalyst,11 or even in an absence of a catalyst in strongly polar solvents, such as water.12 The reaction is thermodynamically favored, kinetically probable, and very versatile so that it has been called click reaction. Therefore, here we have designed a new carrier that will covalently bond with biological surfaces through this Michael thiol−ene click © XXXX American Chemical Society
reaction. The design involves a carrier with tethering acrylate moieties that can efficiently form covalent bond with a thiol group on various biological surfaces, resulting in a secure retention of the carrier on the surface. Nevertheless, biological surfaces, including a mucus layer on a stomach surface, normally possess cystine disulfide bridges instead of free thiol groups. Thus, for this concept to work, another challenge besides the designing of the carrier with tethering terminal alkene moieties, is the in situ generating of free thiols on biomaterials under a nontoxic condition. This paper, therefore, also includes the study on the reduction reaction of cystine disulfide bridges in a mucin protein into thiol moieties by either vitamin C (vit C, an edible nontoxic reducing agent) or tris (2carboxyethyl) phosphine (TCEP, a relatively nontoxic reducing agent13,14). The abilities of the carriers with tethering acrylate moieties, so-called clickable carrier, to retain themselves on epithelial cells, a mucosal surface, and a porcine stomach surface (ex vivo) under a biologically friendly condition were Received: August 25, 2014 Revised: October 7, 2014
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demonstrated. The ability of the clickable carrier to improve some drug localizations in ex vivo stomach tissues was also tested and is reported here. To further proof an effectiveness of this simple yet novel carrier, an application of this clickable carrier to reduce Helicobacter pylori infection in mice was also experimented. The facts that H. pylori possesses one of the most fluid genomes within the prokaryotic kingdom15,16 can survive under stressed conditions by entering the coccoid form17 and colonizes in the hard to reach microaerophilic environment, on stomach epithelial cells, underneath the mucus layer, have made the treatment of this bacterial infection a big challenge. The failure in effectively sustaining antibiotics at the bacterial residence has caused drug resistant H. pylori.18−21 It is believed that antibiotics locally delivered through a mucus layer will be more effective for the treatment than drugs systemically delivered through a basolateral membrane. Reported delivery systems designed to cope with the H. pylori infection by increasing a drug residence time in a stomach can be divided into those using physical mechanisms such as floating, swelling, expanding, and magnetic force,22−26 and those using adhesion forces between mucoadhesive carriers and stomach surfaces.27−30 Adhesion forces employed by reported mucoadhesive carriers are usually noncovalent bindings, such as a hydrogen bond or a charge interaction; thus, a secure attachment cannot be obtained. The treatment of H. pylori infection is a perfect choice to demonstrate an effective attachment of this proposed clickable carrier on a stomach lining. This paper, therefore, includes a preliminary in vivo application of the clickable carrier to combat H. pylori in infected C57BL/6 mice.
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Scheme 1. Synthesis of Poly(ethylene oxide)monoacrylategraf ted-Ethylcellulose (Acrylate-EC) and TAMRA Labeling
MATERIALS AND METHODS
Materials. Ethylcellulose (EC) with 48% ethoxylation (viscosity 100 cps) and mucin from porcine stomach type II were from Aldrich (Steinheim, Germany) and polyethylene glycol 4000 monoacrylate (HO-PEO-acrylate) was from monomer-polymer and Dajac Laboratories (Pennsylvania, U.S.A.). Pyridine was from CARLO ERBA reagents (Val de Reuil, France). 1-Ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDCI·HCl) and tris (2-carboxyethyl) phosphine hydrochloride (TCEP) were from Acros Organics (Geel, Belgium). L-(+)-Ascorbic acid (Vit C) was from Wako Pure Chemical Industries, Ltd., Japan. 5,6-Carboxytetramethylrhodamine (TAMRA) was from Life Technology (Carlsbad, Ca, U.S.A.). All other chemicals were analytical grade reagent. Synthesis of Carboxy Terminated Poly(ethylene oxide)monoacrylate (COOH-PEO-Acrylate) (Scheme 1). Under a N2 atmosphere, succinic anhydride solution (250 mg in 20 mL DMF) was slowly added dropwise to the HO-PEO-acrylate solution (1.0 g in 50 mL DMF) with stirring. Then pyridine (2 drops) was added and the mixture was stirred overnight at 60 °C. The solution was then dialyzed against water using benzoylated membrane tube (Mw cutoff 4000, Sigma-Aldrich, U.S.A.), before being subjected to freeze-drying. Polyethylene Glycol Monoacrylate (HO-PEO-Acrylate), White Powder. 1H NMR (D2O, 400 MHz, δ, ppm): 6.28 (d, J = 17.6 Hz, HcHbCCHa−C(O)−), 6.03 (dd, J = 17.2, 10.4 Hz, HcHbCCHa− C(O)−), 5.81 (d, J = 10.4 Hz, HcHbCCHa−C(O)−), 3.30−3.75 (−CH2CH2O−), 3.0 (q, −CH2CH2OH). ATR-FTIR (cm−1): 2957 (CC−H str), 2874 (C−H str), 1719 (CO str), 1462 (CC str), and 1100 (C−O−C str). Succinylated Poly(ethylene oxide) Acrylate (COOH-PEO-acrylate), White Powder. 1H NMR (DMSO, 400 MHz, δ, ppm): 6.31 (d, J = 17.2 Hz, HcHbCCHa−C(O)−), 6.17 (dd, J = 17.2, 10.4 Hz, HcHbCCHa−C(O)−), 5.93 (d, J = 10.0 Hz, HcHbCCHa− C(O)−), 4.59 (t, −CH2CH2C(O)−), 3.10−3.80 (−CH2CH2O−). ATR-FTIR (cm−1): 2957 (CC−H str), 2883 (C−H str), 1719 (CO str), 1462 (CC str), and 1100 (C−O−C str).
Grafting of COOH-PEO-Acrylate onto EC (Scheme 1). The reaction was carried out under a N2 atmosphere. EDCI·HCl (63 mg) was added to the cold COOH-PEO-acrylate solution (652 mg in 40 mL DMF) and stirred for 30 min at 0 °C. Then EC solution (1.0 g in 100 mL DMF) was dropped into the mixture, followed with the addition of 1-hydroxybenzotriazole (HOBt, 44 mg, freshly recrystallized with methanol prior to use), and the mixture was stirred overnight at room temperature. The mixture was then dialyzed against water using a cellulose membrane tube (MW cutoff 12000−14000, Sigma-Aldrich, U.S.A.), and the obtained poly(ethylene oxide)monoacrylate-graf ted-ethylcellulose (acrylate-EC) in the dialysis bag was freeze-dried. TAMRA was grafted onto the acrylate-EC using EDCI/HOBt coupling method (Scheme 1). EDCI·HCl (12.4 mg) was added to the cold TAMRA solution (15.3 mg in 10 mL DMF) and the mixture was stirred under a N2 atmosphere for 30 min at 0 °C. Then the solution of PEG-acrylate-EC (1.0 g in 100 mL DMF) was added, followed with HOBt (8.8 mg in 10 mL DMF), and the stirring was continued overnight, then the product was purified by dialysis against water using cellulose membrane with 12000−14000 MW cutoff before being freeze-dried. EC was labeled with TAMRA using the same procedure as described for the acrylate-EC. Poly(ethylene oxide)monoacrylate-graf ted-Ethylcellulose (Acrylate-EC), White Powder. 1H NMR (DMSO, 400 MHz, δ, ppm): 6.34 B
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(d, J = 16.8 Hz, HcHbCCHa−C(O)− of acrylate), 6.20 (dd, J = 17.2, 10.0 Hz, HcHbCCHa−C(O)− of acrylate), 5.96 (d, J = 10.0 Hz, HcHbCCHa−C(O)− of acrylate), 2.90−4.00 (−CH2CH2O− of PEO and pyranose protons of EC), 1.15 (−OCH2CH3 of EC). ATRFTIR (cm−1): 3463 (O−H str), 2872 (C−H str), 1728 (CO str), and 1100 (C−O−C str). TAMRA-Poly(ethylene oxide)monoacylate-graf ted-Ethylcellulose (Acrylate-ECTAMRA): Pink Powder. 1H NMR (DMSO, 400 MHz, δ, ppm): 7.00−8.00 (ArH), 5.70−6.40 (−CH2CH−C(O)−), 2.75− 4.75 (−CH2CH2O− and pyranose protons of ethyl cellulose), 2.50 (−(O)C−CH2CH2C(O)−), 1.15 (−OCH2CH3). TAMRA-Ethylcellulose (ECTAMRA). Pink Powder. 1H NMR (D2O, 400 MHz, δ, ppm): 2.75−4.25 (pyranose protons on cellulose) and 6.50−8.00 (aromatic protons of TAMRA). Particle Formation. Particles from the four polymers (EC, acrylate-EC, ECTAMRA, and acrylate-ECTAMRA) were all prepared by the solvent displacement method, as previously described.31 The polymer solution (100 mg in 20 mL ethanol) was dialyzed against water using the cellulose membrane dialysis tube (MW cutoff 12000− 14000, Sigma-Aldrich, U.S.A.), and the obtained aqueous suspension of the particles in the dialysis bag was collected and subjected to scanning electron microscopic (SEM, JEOL JSM-6400), transmission electron microscopic (TEM, JEOL JEM-2100), dynamic light scattering (DLS, S4700 Malvern Zetasizer), and X-ray photoelectron spectroscopic (XPS, a Kratos AXIS Ultra DLD) analyses. Particles were dried by freeze-drying. Capsaicin Encapsulation. Nanoencapsulation of capsaicin was carried out by the solvent displacement method. The solution containing polymer (EC or acrylate-EC, 100 mg) and capsaicin (100 mg) in ethanol (20 mL) was dialyzed against water as described above. The obtained aqueous suspensions of capsaicin-loaded EC and capsaicin-loaded acrylate-EC spheres were subjected to SEM, TEM, and DLS analyses. Particles were freeze-dried. Capsaicin loading was determined by extracting the dry spheres with ethanol and then centrifugally filtering out the EC polymer from the extract using a membrane filter (MW cutoff of 10000 Da, Amicon Ultra-15, Millipore, Billerica, MA, U.S.A.) and quantifying capsaicin in the obtained liquid with HPLC. The loading was determined as follows:
%loading =
(0.5 mg) in D2O (1 mL), were also carried out at various post-mixing times. Reduction of Mucin by Vit C and TCEP. The reductions of disulfide moieties in mucin into thiol groups by TCEP and vit C, were quantified with Elmann reaction. First, 500 μL of mucin solution (10 mg mL−1) were incubated with 500 μL of reducing agent solution (5 mg mL−1 of TCEP solution or 20 mg mL−1 of vit C solution) for 4 h. The mixture was centrifuged (13500 rpm for 20 min) and the mucin pellet was collected and redispersed in 1000 μL of water. The centrifugation and redispersion cycles were repeated three times to clean up the reducing agent. The obtained reduced mucin sample was redispersed in 0.1 M phosphate buffer containing 1.0 mM EDTA and then added with 50 μL of Elmann reagent (10.0 mM) to give a final mucin concentration of 2.5 mg mL−1. The mixture was incubated for 2 h at room temperature before being subjected to a UV−visible absorption measurement at 412 nm (Optizen Pop QX UV−vis absorption spectrometer, Mecasys, South Korea). Amounts of thiol groups in the samples were then estimated with the aid of the calibration curve constructed using dithiothreitol standards. Adhesion of Carriers to the Mucosal Layer. Mucosa plates or glass slides covalently covered with mucin were prepared as previously described.32 The 1.8 × 1.8 cm mucosa plate was incubated with the aqueous suspension (2.0 mL) containing the fluorescence labeled carriers (acrylate-ECTAMRA or ECTAMRA, 0.3 mg mL−1) with or without a reducing agent (0.6 mg mL−1 vit C or 0.03 mg mL−1 TCEP) for 4 h. Then the slide was subjected to fluorescence intensity detection (Synergy MX monochromator-based multimode microplate reader, Biotek, U.S.A.) at the excitation and emission wavelengths of 540 and 580 nm, respectively. The obtained fluorescence intensities of different samples, although were roughly in the same range, were normalized to exactly the same level. The plate was then washed excessively with water, submerged in water, and subjected to fluorescence intensity detection. Fluorescence intensity values obtained from the washed plates were then normalized with the corresponding factors used previously for the unwashed plates. Fluorescence images of the washed slides were also acquired using a fluorescence microscope (Eclipse Ti Series, Nikon, Japan). Adhesion of Carriers to HEp-2 Cells. The HEp-2 (Human laryngeal carcinoma) cells (kindly provided by Dr. Pornthep Tiensiwakul, Faculty of Allied Health Sciences, Chulalongkorn University) were grown on tissue culture plasticware in RPMI 1640 media with 2.05 mM L-glutamine (Hyclone Laboratory, Inc., Logan, UT, U.S.A.) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic-antimycotic solution (Gibco BRL Laboratories, Grand Island, NY, U.S.A.) at 37 °C in 5% CO2 with 80% humidity. HEp-2 cells were washed twice with phosphate buffer saline (PBS), treated with trypsin, and counted. Approximately 1 × 106 cells were seeded on six-well tissue culture plates and incubated overnight to obtain 80% confluence. Each well was washed twice with PBS and incubated with RPMI medium (0.8 mL) and aqueous suspension (0.2 mL) containing the fluorescence-labeled carriers (acrylate-ECTAMRA or ECTAMRA, 1.5 g mL−1) with or without TCEP (0.15 mg mL−1) for 2 h before being subjected to fluorescence intensity measurement, as described above. Then the cells were washed twice with PBS, RPMI medium was added, and fluorescence intensity of the mixture was acquired again. Finally, the cells were treated with Acridine orange by adding 10 μL of 0.1 mg mL−1 Acridine orange solution into each well, incubating for 1 h, washing twice with PBS, and adding RPMI medium. The Acridine orange treated cells were then observed under a confocal fluorescent microscope (LSM 700, Carl Zeiss Micro Imaging GmbH, Germany). Adhesion of Carriers to Porcine Stomach. A total of 100 μL of aqueous suspension containing capsaicin-loaded carriers (1.0 mg mL−1 carrier, 1.0 mg mL−1 capsaicin, the carriers tested included ECTAMRA and acrylate-ECTAMRA) with or without TCEP (0.1 mg/mL) were dropped onto fresh porcine stomach pieces (1 × 2 cm size) and left for 2 h. After 5× washing with excess PBS, fluorescence images of the stomach pieces were then acquired using a confocal fluorescent microscope (Eclipse Ti Series, Nikon, Japan). The amount of capsaicin
wt of capsaicin found in the spheres × 100 wt of capsaicin−loaded spheres
Clarithromycin Encapsulation. Clarithromycin encapsulation was carried out as previously described.29 The solution of polymer (240 mg, acrylate-EC or unmodified EC) and clarithromycin (60 mg) in acetone (40 mL) was prepared. Then water (200 mL) was slowly dropped into the obtained solution under continuous stirring. Acetone was removed from the suspension by vacuum suction overnight and the suspension was freeze-dried. The dry particles were redispersed in water by stirring and the obtained suspension was subjected to SEM and DLS analyses. The suspension was also subjected to microscopic analysis to confirm an absent of any clarithromycin crystal/precipitate. Amount of free clarithromycin left in the system was determined by subjecting the particle suspension to centrifugal filtering using a membrane with MW cutoff of 100000 Da (Amicon Ultra-15, Millipore, Billerica, MA, U.S.A.), and the amount of clarithromycin in the filtrate liquid was determined using UV−visible spectrometry with the aid of a calibration curve. Thiol−Ene Reaction between Acrylate-EC and Mucin. The thiol−ene click reactions between mucin and acrylate moieties under different conditions were monitored with a 1H NMR technique. Here three samples, (i) solution of acrylate-EC (8 mg) and mucin (5 mg) in DMSO-d6 (1 mL), (ii) solution of acrylate-EC (8 mg), mucin (5 mg) and TCEP (0.5 mg) in DMSO-d6 (1 mL), and (iii) solution of acrylate-EC (8 mg), mucin (5 mg), and vit C (16 mg) in DMSO-d6 (1 mL), were prepared and subjected to 1H NMR analysis at 0, 1, 3, and 8 h post-mixing times at room temperature. In addition, the 1H NMR analysis of the two mixtures, (i) HO-PEO-acrylate (8 mg) and vit C (16 mg) in D2O (1 mL) and (ii) HO-PEO-acrylate (8 mg) and TCEP C
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in each stomach piece was evaluated by performing capsaicin extraction and quantification. Capsaicin was extracted from the stomach pieces with ethyl acetate. The stomach tissue was homogenized in ethyl acetate and the ethyl acetate extract was collected, dried, reconstituted in methanol, filtered, and subjected to capsaicin quantification with HPLC. Capsaicin quantitation by HPLC was carried out with the aid of capsaicin calibration standards, on C18-AR 100 × 4.6 mm column, with the mixture of acetonitrile and 1% aqueous acetic acid (at 42% v/ v acetonitrile) as mobile phase. Analysis was carried out by isocratic elution at a flow rate of 0.8 mL/min using UV detection at 280 nm and 20 μL injection volume. The retention time of capsaicin was approximately 10 min. Treatment of H. pylori Infection in Mice. Tested samples included distilled water (positive control), free clarithromycin, unloaded EC particles, clarithromycin-loaded EC particles, unloaded acrylate-EC particles, and clarithromycin-loaded acrylate-EC particles. An aqueous suspension of free clarithromycin was prepared at a concentration of 1.5 mg mL−1 by dispersing clarithromycin in water. Aqueous suspensions of the encapsulated clarithromycin were also prepared at the final clarithromycin concentration of 1.5 mg mL−1 (concentration of the polymer was 4.5 mg mL−1) by dispersing the freeze-dried particles in water. Control blank acrylate-EC particles were prepared as a 4.5 mg mL−1 aqueous suspension. Vit C solution was freshly prepared in water at a concentration of 30 mg mL−1. A total of 18 C57BL/6 mice and ATCC 43504 H. pylori strain were used. The procedures were approved by both the ethics committee and the institutional animal care and use committee (IACUC) of Chulalongkorn University (No. 11310079). Each mouse was infected with 109 CFU of H. pylori for 3 consecutive days. After 3 weeks, 18 H. pylori infected mice of both sexes were randomly divided into 6 groups of 3 mice each. The mice were fed once a day for 3 consecutive days with appropriate samples by direct oral administration using a feeding needle. Clarithromycin dose of 30 mg kg−1 body weight, carrier material dose of 90 mg kg−1 body weight, and vit C dose of 250 mg kg−1 body weight were used. Two days after administration of the final dose, the mice were sacrificed and the stomach samples were collected and fixed in 10% neutral buffered formalin. The samples were histologically processed and embedded in paraffin. The sections were cut with ultramicrotome at 4 μm thickness and stained with hematoxylin and eosin (H&E) using the Warthin-starry method. The immunohistochemical staining was performed using a specific H. pylori antibody. Briefly, the 4 μm thick tissue sections were pretreated by 0.1% trypsin for 25 min at 37 °C and then treated with 3% H2O2 for 15 min at room temperature to inactivate endogeneous peroxidase and then blocked with 1% bovine serum albumin for 45 min at 37 °C. The sections were incubated with polyclonal rabbit anti-H. pylori antibody (Dako, Glotrup, Denmark) overnight at 4 °C, followed by incubation with a chain polymer kit (Dako EnVision+ System-HRP, antirabbit, Dako, Tokyo, Japan) for 45 min at 37 °C. Finally, the presence of antigens was visualized by 3,3′diaminobenzidine-4·HCl (DAB) reaction and hematoxylin counterstain using light microscopy. The semiquantitative evaluation was blindly observed and the immune-reativity of each sample was graded into 4 levels: − (negative), + (low), ++ (moderate), and +++ (numerous) of the positive immune-staining.
the surface of stomach epithelial cells, resulting in some covalent bindings of the carriers at the residence of H. pylori. EC polymer grafted with poly(ethylene oxide) (PEO) acrylate chains, was prepared according to Scheme 1. The COOH functionality was first introduced onto the hydroxyl end of the PEG-acrylate through the esterification reaction between succinic anhydride and the hydroxyl end of the PEGacrylate chain. 1H NMR of the purified COOH-PEO-acrylate, showed resonance peak of the methylene protons from the succinyl group at 2.45 ppm (see Figures S1 and S2 in Supporting Information, SI), confirming the presence of succinyl moieties on the chain. The obtained COOH-PEOacrylate was then covalently bound to the EC chain through the esterification reaction between the COOH of the COOH-PEOacrylate and the hydroxyl group on the EC chain. 1H NMR spectrum of the grafted product (PEO-acrylate-grafted EC or denoted here as acrylate-EC) revealed the resonance peaks at 5.9−6.4 ppm from the acrylate protons, and analysis of the peak area indicated the substitution degree of 0.13 of acrylate group on the EC (see Figures S3 and S4 in SI). IR spectra of the functionalized EC also confirmed successful grafting through an appearance of small absorption peaks at 1716 cm−1 (−CO of acryl group at the terminus of each grafted PEO chain) and 1100 cm−1 (−C−O−C− of poly(ethylene oxide) group of PEO chains; Figure S6 in SI). Acrylate-EC (and EC) covalently labeled with fluorophore TAMRA was prepared via the carbodiimide assisted esterification reaction between a carboxylic group of the TAMRA and a hydroxyl group of the polymer. 1H NMR spectra of the acrylate-ECTAMRA and ECTAMRA revealed resonance peaks of TAMRA moieties (7.00−8.00 ppm; Figure S5 in SI), indicating successful labeling. The obtained acrylate-EC, acrylate-ECTAMRA, ECTAMRA, and the unmodified EC were then prepared into nanoparticles through self-assemble process using solvent displacement method (displacing ethanol with water). Dynamic light scattering analysis revealed the size distribution of 425 ± 25, 412 ± 10, 340 ± 169, and 324 ± 13 nm for the acrylate-EC, EC, acrylate-ECTAMRA, and ECTAMRA particles, respectively. Scanning and transmission electron microscopic (SEM and TEM) images show spherical morphology of the four particles (Figure 1). It was speculated that during the self-assembling, the hydrophilic moieties of the polymer chains arranged themselves at the outer surface of the spheres, with maximum contact with water, while the hydrophobic methylene groups positioned themselves inward with minimal contact with water. Since acrylate moieties were connected to EC via hydrophilic PEO chain, thus it was likely that the acrylate groups arranged themselves at the outside of the spheres. According to our design, this is an important feature for a successful linking of the particles to biomaterials. We confirmed the present of PEOacrylate moieties at the outer surface of the particles by subjecting the particles to X-ray photoelectron spectroscopic (XPS) analysis. The C 1s spectrum of the EC particles revealed peaks at 283.3, 285.1, and 288.4 eV, which corresponded to the binding energy of the C−C, C−OH, and OC−O bonds, respectively. However, as expected, the C 1s spectrum of the acrylate-EC particles showed peaks at 283.3, 285.1, 288.4, 284.2, and 286.7 eV, which corresponded to the binding energy of C−C, C−OH, OC−O, CC, and C−O−C bonds, respectively (Figure S7 in SI). Therefore, we concluded that the CC functionality (from acrylate moieties) and the PEO
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RESULTS AND DISCUSSIONS Carrier Design, Fabrication, and Characterization. Although we previously showed that EC particles possessed good attachment to a stomach surface in vivo,29−31 here we want to further improve the attachment in order to more efficiently retain drug carriers at the residence of H. pylori. Since the acrylate functionality can react instantly with thiol groups via Michael thiol−ene click reaction, here EC carriers with tethering acrylate moieties on their surfaces were designed and fabricated. We expect that the tethering acrylate moieties will efficiently react with thiol groups on both the mucus layer and D
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Scheme 2. Proposed Reactions between Mucin and AcrylateEC in the Presence of Different Reducing Agentsa
Figure 1. SEM images of EC (A), acrylate-EC (B), ECTAMRA(C), acrylate-ECTAMRA (D), clarithromycin-loaded EC (E), and clarithromycin-loaded acrylate-EC (F) particles. Corresponding TEM images are shown as insets.
chains were present at the surface of the self-assembled particles. Thiol−Ene Click Reaction between Clickable Carrier and Mucin. Our design is that the tethering acrylate moieties on the particles will react with thiol groups on either the gastric epithelial cell’s surface or on mucus layer through the Michael thiol−ene click reaction, so that the particles would be covalently bound to those surfaces through thioether bonds. Nevertheless, normally cysteine moieties in biomaterials, including mucin, are in the cystine disulfide form; it is, therefore, necessary to reduce the disulfides into thiol moieties. According to our biomedical purpose, the nontoxic nature of the catalyst-free Michael thiol−ene reaction in our design will be useless if the thiol generation step is toxic. Therefore, here we investigated the reduction of the cystine disulfide bridge with two reducing agents, the edible vit C and the relatively mild and nontoxic TCEP.13,14 We monitored the reaction between the acrylate-EC and the mucin protein under three conditions, reaction with no reducing agent, reaction in the presence of TCEP, and reaction in the presence of vit C. Progress of the reaction was monitored through the reduction of resonance peaks of the acrylate protons (6.34−5.96 ppm) in the 1H NMR spectra. In an absence of a reducing agent, the change in the intensity of acrylate proton peaks (ratio of acrylate proton peaks at 6.34−5.98 ppm and the methyl proton peaks at 1.1 ppm from the ethoxyl groups on ethylcellulose) was insignificant, indicating no reaction. In contrast, in the presence of TCEP, acrylate protons disappeared quickly (peaks were not observable after mixing), indicating instant reaction (Figures S8 and S9 in SI). Here two possibilities were possible (Scheme 2): (i) TCEP reduced disulfide bridges into thiols and the generated thiols underwent thiol−ene click reaction with acrylate moieties, and (ii) TCEP reacted directly with acrylate moieties under an acidic condition.33 To clarify this, the reaction between TCEP and acrylate-EC in an absence of
a
Two possible pathways for the reaction between mucin and acrylateEC in the presence of TCEP: (A) the reduction of disulfide bridges into thiols, followed by the thiol−ene reaction between the generated thiols and acrylate moieties on the EC, and (B) a direct reaction between TCEP and acrylate moieties on the EC. Only one pathway for the reaction between mucin and acrylate-EC in the presence of vit C: the reduction of disulfide bridges into thiols by vit C, followed by the thiol−ene reaction between the generated thiols and acrylate moieties on the EC (C).
mucin (thiol source) was monitored. 1H NMR spectra of the reaction mixture showed slow reduction of acrylate proton peaks, i.e., 8 h incubation at room temperature was needed to diminish all the acrylate protons (Figure S10 in SI). This indicated that the direct reaction between TCEP and acrylate moieties proceeded slowly. Thus, we concluded that both pathways, the reduction of disulfide bridges by TCEP into thiols followed with Michael thiol−ene click reaction, and the direct reaction between TCEP and acrylate, did take place during the incubation of acrylate-EC, mucin, and TCEP. Nevertheless, the rate of the former was tremendously higher than the latter, so our expected reaction (the thiol−ene reaction) did occur more predominately than the side reaction (acrylate and TCEP). It should be noted here that, for the thiol−ene pathway, TCEP not only reduced the disulfide bridges into thiols, but also acted as a catalyst for the subsequent thiol−ene reaction.33 When vit C was present in the mixture of acrylate-EC and mucin, the disappearance of acrylate protons occurred less quickly than that in the presence of TCEP, that is, complete disappearance was observed at 4 h post mixing (Figure S11 in E
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Figure 2. Attachments of various carriers on mucosa plates. Fluorescence images (A−D) and fluorescence intensities (E) of the mucosa plates that were previously incubated with particle samples and then thoroughly washed. The particle samples include (A) ECTAMRA, (B) acrylate-ECTAMRA, (C) TCEP + acrylate-ECTAMRA, and (D) vit C + acrylate-ECTAMRA. Fluorescence intensities in (E) are shown as the mean ± 1 SD, and statistical significant difference (P < 0.05) was determined by one-way ANOVA Duncan in which different lower case letters indicate significant difference. Representative model showing reaction between acrylate-EC particles and the mucosa plate in the presence of a reducing agent is shown in (F).
SI). To make sure that the disappearance of the acrylate protons was not from the direct reaction between acrylates and vit C, 1H NMR of the mixture of acrylate-EC and vit C (with no mucin) was monitored. The result showed no change in the intensity of acrylate proton peaks (Figure S12 in SI). This indicated that vit C did not react with the acrylate functionality and acrylate moieties were stable in the presence of vit C. Therefore, it was likely that in a presence of mucin, vit C reduced the disulfide bridges in the mucin into thiols and the generated thiols then reacted with acrylate moieties via the Michael thiol−ene click reaction (Scheme 2). However, without TCEP as a catalyst, the thiol−ene reaction proceeded more slowly than that observed previously with TCEP. The reduction of cystine disulfide bridges into thiol moieties in the mucin was confirmed with Elmann reaction, using dithiothreitol as a thiol standard. Mucin incubated with 5 mg mL−1 TCEP and 20 mg mL−1 vit C for 20 min gave approximately 3.33 ± 0.14 and 3.09 ± 0.33 mol of thiol/mol of mucin. This result confirmed that vit C could reduce the disulfide bridges in mucin into free thiols. It should be noted here that, vit C was easily and quickly degraded in water, thus, the reaction with mucin, a steric macromolecules, required a high concentration of the compound. After verifying the reaction between acrylate-EC and mucin in the presence of either vit C or TCEP, here we acquired the
degree of particle attachment on the mucosal layer, HEp-2 cells, and fresh porcine stomach, using ECTAMRA and acrylateECTAMRA particles. Attachment of Clickable Carriers on the Mucosal Layer. The mucosal layer used here was a mucin-grafted glass slide or a mucosa plate prepared by covalently coating a glass slide with some mucin protein.32 The mucosa plates were incubated with the fluorescent particles (acrylate-ECTAMRA or ECTAMRA), and both the fluorescence images and the fluorescence intensity of TAMRA on the slides after washing were acquired. The particle attachments were tested both in a presence and an absence of a reducing agent (vit C or TCEP). As expected, fluorescence images of the washed slides indicated higher level of particle attachment for acrylate-ECTAMRA particles when used with either vit C or TCEP, as compared to ECTAMRA particles, or reducing agent-free acrylate-ECTAMRA particles (Figure 2A−D). In a presence of either vit C or TCEP, fluorescence intensity of the plates treated with acrylateECTAMRA spheres was more than 10× higher than those treated with ECTAMRA spheres (Figure 2E). These results conformed to our design that the two reducing agents would generate thiols on the mucosa plates and the Michael thiol−ene reaction between acrylate moieties on the acrylate-ECTAMRA particles and the generated thiols on the mucosa plate then proceeded, forming thioether linkages between the plate and the particles. F
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This resulted in covalent attachment of the particles onto the plates in which thoroughly rinsing the plates with water could not rid the particles off (Figure 2F). It should be noted here that without the presence of any reducing agent, noncovalent attachment of ECTAMRA and acrylate-ECTAMRA particles to the mucosa plates could still take place (Figure 2A,B), which resulted in some attachment of the particles to the mucosa plates (Figure 2C,D) but at a much lower level compared to the attachment via covalent linkages. Therefore, we concluded that the two reducing agents, vit C and TCEP, were capable of generating thiol moieties on the mucosa plate and the thiol− ene reaction between the generated thiols and acrylate moieties of the acrylate-ECTAMRA particles increased the particle retention on the plates. Attachment of Clickable Carriers on Cells. Next, we tested for the ability of the acrylate-ECTAMRA particles to attach to human laryngeal carcinoma (HEp-2) cells. A previous report has indicated a similarity between the HEp-2 cells and porcine gastric epithelial cells.34 Therefore, an ability of the particles to attach to HEp-2 cells could roughly reflect their ability to attach to epithelial cells of a stomach surface. Attachment levels of ECTAMRA and acrylate-ECTAMRA particles to the HEp-2 cells were determined in the presence and absence of TCEP. In the absence of a reducing agent, the fluorescence intensity observed on the Hep-2 cells was roughly at a similar level to that observed on the mucosa plates. Among the four samples (ECTAMRA, ECTAMRA + TCEP, acrylate-ECTAMRA, and acrylateECTAMRA + TCEP), the acrylate-ECTAMRA + TCEP showed the best attachment level (Figure 3). Nevertheless, the presence of TCEP could increase the particle attachment level for only approximately 20% comparing to the attachment level obtained without a reducing agent. This small increase, comparing to the increase observed in the mucosa plate, implied that the surface of HEp-2 cells possessed limited disulfide bridges. Drug Sustainment on Porcine Stomach. Results above clearly indicated that acrylate-EC particles could attach to a mucus material and a surface of the HEp-2 cell in a presence of a reducing agent, thus, it was likely that the drug-loaded acrylate-EC carriers would increase the drug retention on a stomach lining. Here we tested the ability of the acrylate-EC particles to sustain some drug concentration on the ex vivo stomach tissue. We loaded capsaicin, a heat and light stable hydrophobic compound used as a model drug in this experiment (the compound could be extracted from the tissue and quantified by HPLC easily), into both the EC and the acrylate-EC carriers by allowing the polymers to self-assemble in the presence of capsaicin. The loading contents were 46 and 35% for the capsaicin-loaded EC and the capsaicin-loaded acrylate-EC particles, respectively. We compared five samples, unencapsulated capsaicin, the two encapsulated capsaicin (in EC and in acrylate-EC carriers) in the presence and absence of TCEP. The results (Figure 4A) showed the maximum capsaicin content in the stomach tissue exposed to capsaicin-loaded acrylate-EC particles with TCEP. It was likely that the TCEPreduced cystine moieties on the stomach surface into thiol groups and the generated thiols then added to the double bond in the acrylate moieties on the particles, forming thioether linkages between the particles and the stomach surface. The covalently linked particles on the stomach could withstand the washing. Capsaicin then diffused from the attached particles into stomach tissue via simple diffusion, driven by the concentration gradient between the inside and the outside of the particles. The amount of capsaicin obtained from stomach
Figure 3. Attachment of acrylate-EC particles on HEp-2 cells: (A) a graph showing the fluorescence intensity remained on the cells, that were previously incubated with ECTAMRA or acrylate-ECTAMRA particles in the presence or absence of TCEP and then washed twice (shown as the mean ± 1 SD, statistical significant difference (P < 0.05) was determined by one-way ANOVA Duncan, in which different lower case letters on the columns indicate significant difference), and (B) confocal fluorescence microscopic image of the HEp-2 cells that were previously treated with acrylate-ECTAMRA particles + TCEP and thoroughly washed.
Figure 4. Particle attachment and drug retention on ex vivo stomach surface. (A) Amounts of capsaicin in porcine stomach tissues which were previously incubated for 2 h with free capsaicin, capsaicin-loaded EC (with and without TCEP), and capsaicin-loaded acrylate-EC (with and without TCEP) at the stomach surface coverage of 0.05 mg capsaicin cm−2 and washed 5× (shown as capsaicin percentages relative to the amount of total capsaicin applied to the stomach, presented as mean ± 1 SD derived from three repeats, statistical significant difference (P < 0.05) was analyzed by one-way ANOVA Duncan and means with a different lower case letter are significantly different). (B) The fluorescence image of the stomach surface that was previously incubated with acrylate-ECTAMRA particle suspension, at the stomach surface coverage of 0.05 mg capsaicin cm−2, for 2 h and washed 5×. G
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Figure 5. In vivo combating H. pylori in the stomach. Immunohistochemical demonstration of H. pylori in the pylorus: a high numbers of short curve bacilli and coccoid H. pylori-positive immunostaining on top and within the epithelial lining of the mucosal layer can be observed in the positive control mice group (A); moderate to high numbers of short curve bacilli and coccoid H. pylori-positive immunostaining on top of the epithelial lining of the mucosal layer can be observed in the unloaded acrylate-EC particles + vit C group (B); moderate numbers of positive staining bacteria on top of the epithelial lining of the mucosal layer can be observed in the clarithromycin-loaded EC particle group (C); the clarithromycin-loaded acrylateEC + vit C treated group shows low numbers of crumble positive staining bacteria spaced out from the epithelial surface of the mucosal layer (D). Each image (derived from the Envision + system-HRP (Dako, Tokyo, Japan), 3,3′-diaminobenzidine-HCl method) shown is a representative of those seen from more than 20 pyloric samples. Model showing how the clickable carriers work (E).
tissue analysis included both the released capsaicin in the tissue and the capsaicin inside the attached particles. The particle attachment on a stomach surface was also verified by exposing the stomach surface to an aqueous suspension of the fluorescence labeled particles (ECTAMRA and acrylate-ECTAMRA particles), in the presence and absence of TCEP, washing the exposed surface, and then taking the fluorescence images of the washed stomach surface. The result clearly showed high numbers of the fluorescence particles on the stomach surface when acrylate-ECTAMRA particles were used in the presence of TCEP (Figure 4B). Treatment of H. pylori Infection. The results above encouraged us to combat H. pylori using the acrylate-EC carriers to deliver antibiotics to their residence. So we encapsulated clarithromycin into the EC (a control carrier) and the acrylate-EC carriers, using the solvent precipitation process.29 The encapsulation efficiencies of the process were 86 ± 0.5 and 83 ± 0.8%, and the loading contents were 22.3 ± 0.2 and 17 ± 0.4% for the EC and the acrylate-EC carriers, respectively. Particle sizes estimated from SEM images were 223 ± 50 and 232 ± 62 nm for the clarithromycin-loaded EC and the clarithromycin-loaded acrylate-EC spheres, respectively (Figure 5). These clarithromycin-loaded particles were used in the in vivo treatment of mice infected with H. pylori. Six treated groups of the H. pylori infected mice, (i) control (distilled water), (ii) free clarithromycin, (iii) unloaded EC particles, (iv) clarithromycin-loaded EC particles, (v) unloaded acrylate-EC particles + vit C, and (vi) clarithromycin-loaded acrylate-EC particles + vit C, were immunohistochemically evaluated for the presence of H. pylori after the treatment (Table 1). High densities of short curved and coccoid forms of H. pylori were observed in the fundus and pylorus of all mice in the positive control group and the adhesion and the invasion of bacteria at the stomach epithelial cells could be observed clearly (Figure 5A). Unloaded EC particles gave a comparable result to the positive control group, indicating that the EC particles
Table 1. Immunohistochemical Staining Results for the Detection of H. pyloria group H. pylori positive control (distilled water) free clarithromycin unloaded EC particles clarithromycin-loaded EC particles unloaded acrylate-EC particles + vit C clarithromycin-loaded acrylate-EC particles + vit C
grading of positive staining +++ ++ − +++ +++ ++ ++ − +++ +
a The positive immunoreactivity: −, negative; +, low; ++, moderate; ++ +, numerous.
possessed no activity toward H. pylori. The number of positive immunostaining bacteria were reduced from numerous in the control group to moderate-high in the group treated with the blank acrylate-EC particles + vit C (Figure 5B). We speculated that this small effect might be from the high acidity of vit C. Nevertheless, the effect was not pronounced so we did not pursue further investigation. The free clarithromycin treatment appeared to cause quite small reduction in H. pylori infection level. This low efficacy of the free clarithromycin was likely a result of poor drug retention on the stomach surface. The numbers of positive immunostaining bacteria were reduced from numerous in the control group to moderate in the group treated with clarithromycin-loaded EC particles (Figure 5C). This agrees well with our previous report on a good stomach adhesion of EC particles and an improved H. pylori treatment using the EC particles to deliver antibacterial agents.29,31 However, significantly greater improvement was observed in the clarithromycin-loaded acrylate-EC particles + vit C treatment group, that is, all mice in this group showed low numbers of positive immunostaining bacteria. In addition, both the epithelial surface and the adjacent mucus layer appeared well-defined and healthy with no damage and no bacterial H
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invasion (Figure 5C). It was likely that Michael thiol−ene reaction between acrylate moieties of the acrylate-EC carriers and the thiol groups (generated by vit C) on both the mucus layer and the surface of the gastric epithelial cells took place, resulting in the stomach surface covalently linked with the drug particles. The covalently linked particles with clarithromycin loaded inside helped maintaining high clarithromycin concentration around the infected area through a sustained release of clarithromycin into the surrounding tissue. This resulted in the effective treatment of the H. pylori infection (see model in Figure 5D). Small effect of vit C might also contribute to this improved treatment, but the covalent attachment of the clickable carriers to the stomach lining was likely the main reason. The healthy and H. pylori free epithelial surface observed in all the immunostained stomach tissues of the clarithromycinloaded acrylate-EC + vit C treated group implied a high drug concentration at the mucosal layer and the epithelial surface during the treatment. Agitation and contraction of the stomach probably helped facilitating distribution of both the administered carriers and the vit C around the stomach lining. In addition, the 232 ± 62 nm size of the particles, together with the decorated PEO chains, conformed well to the features required for good mucus distribution of the nanoparticles.35 The in vivo results discussed above clearly showed the superiority of the clickable carrier (acrylate-EC particles) over the carrier that relied on noncovalent adsorption (EC particles) under the real condition in which the mucus was continuously shed. Since the turnover period of the mucus in gastrointestinal tracts is approximately 4−6 h in rat and human,36 the covalently bound drug particles in the mucus layer should have approximately 4−6 h of a residence time at the stomach lining. In addition, we also speculate some direct attachments of the drug carriers to the surface of stomach epithelial cells, which theoretically should retain the drug carriers for a longer period.
Article
ASSOCIATED CONTENT
S Supporting Information *
1
H NMR spectra of PEG-acrylate, COOH-PEO-acrylate, EC, acrylate-EC, and ECTAMRA; FTIR spectra of PEG-acrylate, COOH-PEO-acrylate, EC, and acrylate-EC; XPS (C 1s) spectra of the EC and acrylate-EC particles; 1H NMR spectra of (1) the mixture of acrylate-EC and porcine mucin at various post mixing times, (2) the mixture of acrylate-EC, porcine mucin, and TCEP, (3) the mixture of acrylate-EC, mucin, and vitamin C at various post-mixing times, (4) the mixture of PEG-acrylate and TCEP at various post mixing times, and (5) the mixture of PEG-acrylate and vitamin C. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the Royal Golden Jubilee Ph.D. Program (PHD50K0361) from the Thailand Research Fund; the Ratchadapiseksompot Endowment Fund from Chulalongkorn University (Postdoctoral Fellowship and RES560530097-Adv Mat); the Nanotec-CU Center of Excellence on Food and Agriculture; the Center of Excellence on Petrochemical and Materials Technology, Chulalongkorn University.
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REFERENCES
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CONCLUSIONS The drug carrier with tethering acrylate moieties on its surface was successfully synthesized by attaching PEO-acrylates onto EC polymer chains and allowing the obtained acrylate-EC polymer to self-assemble into spherical particles. Covalently attaching the acrylate-EC carriers onto mucosal layers, HEp-2 cells and stomach surfaces was accomplished using an edible vit C or a relatively nontoxic TCEP to reduce disulfide bridges on those three biomaterials into thiols, so that the Michael thiol− ene reaction could proceed. Comparing to EC carriers with no tethering acrylate moieties, the use of acrylate-EC carriers in conjunction with a reducing agent could significantly increase the carrier retentions on those three biological surfaces. Similarly, when capsaicin was delivered onto ex vivo porcine stomachs using the acrylate-EC carriers in a presence of a reducing agent, the higher capsaicin concentration in stomach tissues was attained. Application of this acrylate-EC carrier/vit C combination to combat H. pylori in the infected C57BL/6 mice gave an impressive result. Although demonstrated here on EC carriers, the platform of decorating drug carrier surfaces with acrylate groups can be applied to carriers made from other materials. Furthermore, cystine disulfide bridges are common on various biological surfaces, hence, paving the way for applications of this novel strategy of drug delivery on the vast biological sites. I
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