- Email: [email protected]
Preparation and characterization of soluble branched ionic β-cyclodextrins and their inclusion complexes with triclosan
Carbohydrate Polymers 142 (2016) 149–157
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Preparation and characterization of soluble branched ionic -cyclodextrins and their inclusion complexes with triclosan Flor Gómez-Galván a , Leyre Pérez-Álvarez a,b,∗ , Janire Matas a , Arturo Álvarez-Bautista c,d , Joana Poejo c,d , Catarina M. Duarte c,d , Leire Ruiz-Rubio a , Jose Luis Vila-Vilela a , Luis M. León a,b a Departamento de Química Física (Laboratorio de Química Macromolecular), Universidad del País Vasco (UPV/EHU), B◦ Sarriena s/n, 48940 Leioa, Vizcaya, Spain b BCMaterials, Building 500-1st Floor, Bizkaia Science and Technology Park, 48160 Derio, Spain c Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal d iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
a r t i c l e
i n f o
Article history: Received 24 September 2015 Received in revised form 19 January 2016 Accepted 21 January 2016 Available online 23 January 2016 Keywords: Branched ionic cyclodextrins Epichlorohydrin Cytotoxicity Water solubility
a b s t r a c t This study aims to synthesize, characterize and investigate the water solubility and cytotoxicity of branched anionic/cationic -cyclodextrins (bCDs) obtained by reaction with epichlorohydrin and chloroacetic acid or choline chloride, respectively, by a single step polycondensation reaction. Obtained ionic bCDs were investigated as an attempt to comparatively study anionic and cationic bCDs. Water solubility of both ionic derivatives was similar (400 mg/mL) at neutral and basic pHs and remarkably higher than that of their neutral homologues. Additionally, a pH-dependent solubility of anionic bCDs was observed. Cytotoxicity of ionic bCDs was evaluated on Human colon carcinoma Caco-2 cells and high cell viability (>99%) was observed in the range of 0–100 mg/mL for anionic and cationic samples, in the same range of that of neutral and parent -CDs. Additionally, complexes formation capacity with triclosan, a poor water soluble antimicrobial agent, was confirmed by several techniques observing a complexation limit around 4 mg/mL for both systems and higher stability constant for anionic bCDs than cationic derivatives. © 2016 Published by Elsevier Ltd.
1. Introduction Cyclodextrins (CDs) are cyclic oligosaccharides produced from starch and which contain six (␣-CDs), seven (-CDs) or eight (␥-CDs) glucopyranose units linked by ␣-1,4 glucosidic bonds (Breslow, 2010, chap. 2). The -form is the most accessible and widely used cyclodextrin. As a result of the restricted rotation of linked glucopyranose units CDs are toroidal or truncated cone shaped molecules with an internal cavity whose size is around 6.0–7.0 A˚ for -CDs (Loftsson & Brewster, 1996). CDs have hydrophilic hydroxyl groups on their outer surface, which is hydrophilic, but a hydrophobic cavity in the center, which provides an apolar matrix. As a result, CDs have a special capability to entrap
∗ Corresponding author at: Departamento de Química Física, Facultad de Ciencia y Tecnología (Laboratorio de Química Macromolecular), Universidad del País Vasco (UPV/EHU), B◦ Sarriena s/n, 48940 Leioa, Bizkaia, Spain. E-mail address: [email protected] (L. Pérez-Álvarez). http://dx.doi.org/10.1016/j.carbpol.2016.01.046 0144-8617/© 2016 Published by Elsevier Ltd.
hydrophobic guests (Szejtli, 1988, chap. 2; Singh, Bharti, Madan, & Hiremath, 2010). This property has received increasing attention in the last years and has been extensively exploited to form host–guest inclusion complexes with a variety of drugs (Uekama, Hirayama, & Irie, 1998), food additives (Kayaci & Uyar, 2012), and antibacterial agents (Hill, Gomes, & Taylor, 2013), in different fields such as analytical chemistry (Szente & Szeman, 2013), catalysis (Easton, 2007), pharmacy (Davis & Brewster, 2004), food industries (Astray, Gonzalez-Barreiro, Mejuto, Rial-Otero, & Simal-Gándara, 2009) and the treatment of wastewater (Crinia & Morcellet, 2002). Over the last decades, CDs have been used as drug and antimicrobials carriers showing that they are able to enhance water solubility, chemical stability, bioavailability and decrease unfavorable sideeffects of the active agents (Blomberg, Kumpulainen, David, & Amiel, 2004). However, parent -CDs have poor water solubility as a consequence of the intra molecular hydrogen bonding of the secondary hydroxyl groups which limits their further applications. To overcome this drawback, various CDs derivatives have been developed
150
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
by substitution of their hydroxyl groups, such as, methylated CDs, hydroxyalkylated CDs, sulfobutyl ether CDs, which are examples of commercially available CDs derivatives (Szente & Szejtli, 1999). On the other hand, substituted -CDs, such as CD-based polymers, which are the most studied substituted -CDs and refer to those structures containing two or more covalently linked CDs units, are of interest because they exhibit enhanced aqueous solubility (Qian, Guan, & Xiao, 2008). Harada, Furue, and Nozakura (1976) reported the synthesis of polymeric CDs by radical polymerization of acrylate derivatives of CDs or monomers that form complexes with CDs. Later, Renard, Deratani, Volet, and Sebille (1997) studied a simpler synthetic route by polycondensation of CDs, with bi-functional agents such as epichlorohydrin (EP) and reported a systematic study of the reaction conditions on the polycondensation reaction under basic conditions (Renard, Barnathan, Deratani, & Sebille, 1997). The primary product of this reaction is not a true polymer but a heterogeneous mixture of various CD glyceryl ethers with crosslinked structures including CD moieties, linking bridges and side-chain substituents. Particularly, these polycyclodextrins prepared by polymer crosslinking with EP have shown be more efficient than acrylate derivatives in binding larger guest molecules with the help of the hydrophobic EP fractions and adjacent CD units (Harada, Furue, & Mozakura, 1981; Szeman, Fenyvesi, & Szejtli, 1987). Another approach to increase CDs water solubility is introducing ionic fragments on CDs that has resulted in an increase of the water solubility and in a lower cytotoxicity. Although water solubility improvement of ionic CDs have been reported, improving water solubility does not necessarily lead to a reduced cytotoxicity, namely, methylated -CDs show enhanced water solubility but high toxicity on Caco-2 cells and human erythrocytes (Li, Xiao, Li, & Zhong, 2004). Certainly, although CDs have been extensively studied there is limited information about their cytotoxic effects (Kiss et al., 2010). Up today, the in vivo cytotoxicity of CDs has been correlated with their binding and extraction capacity of membrane cholesterol which alters the permeability properties of the membrane and leads to hemolysis (Li et al., 2004). Additionally, charged CDs have different complexation and solubilization capabilities in comparison with other CD derivatives and the electrostatic interaction outside the cavity seems to enhance the solubilization of the hydrophobic active agent. In order to combine the good values of charged CDs and substituted CD, different investigations have focused on cationic cyclodextrin polymers which have been synthesized (Blomberg et al., 2004; Huang, Xin, Guo, & Li, 2010; Li & Loh, 2008; Xin et al., 2010; Zhang et al., 2010) by polycondensation reaction with EP to constitute typically polymer/oligomers and choline chloride (CC) or more recently with glycidyltrimetrylammonium chloride, to provide cationic groups (Junthip, Tabary, Leclercq, & Martel, 2015). These cationic polycyclodextrins have been reported to exhibit better hemocompatibility to human erythrocytes, no cytotoxicity, good drug inclusion and dissolution abilities than that of CDs. Meanwhile, in recent research branched CpCDs have also shown to be safe enough as non-viral gene vectors (Huang et al., 2010; Li, Guo, Xin, Zhao, & Xiao, 2010). These CpCDs have been employed to form inclusion complexes with antibiotics and their structure and antimicrobial activities have been studied, suggesting better association and antimicrobial activity with small antibiotic molecules (Qian et al., 2008). Regarding anionic substituted CDs, Martel, Ruffin, Weltrowski, Lekchiri, and Morcellet (2005) obtained anionic polycyclodextrins by a polyesterification reaction of CDs and some of their derivatives with di and poly(carboxylic acid)s obtaining gels and soluble forms. However, this research concluded the unsuccessful use of dicarboxylic acids and that obtaining soluble form required soft conditions that led to a low yield and high cytotoxicity, while
citric acid was an appropriate crosslinking that lets to obtain soluble derivatives. This anionic CDs derivatives, in which negative charge corresponds to the crosslinking agent, have been employed recently in the development of multilayer systems by complexes formation with chitosan onto polyester fibers (Martin, Tabary, Chai, et al., 2013; Martin, Tabary, Leclercq, et al., 2013). Besides, Yang, Hoonor, Jin, and Kim (2013) attempted to isolate and characterize cationic and anionic -cyclodextrin polymers through one step polycondensation process with EP using choline chloride (CC) for cationic polymer and chloroacetic acid (CAA) for obtaining anionic polymer. The detailed structural analysis of these ionic -CD compounds was done confirming the formation of branched -CD monomers and oligomers with a few units of -CD. Reaction parameters were studied in order to eliminate the undesirable crosslinking, and increase reaction yield and molecular weight. Despite the authors claim the water soluble properties of the prepared ionic substituted CDs no studies were made regarding water solubility. Triclosan (TR), [5-chloro-2-(2,4-dichlorophenoxy)phenol], is an antimicrobial agent widely used in consumer and medical products such as mouthwashes, toothpastes, surgical scrubs and suture materials. TR inhibits the growth of a wide range of microorganisms blocking the synthesis of lipids and by the inhibition of enoylacyl carrier protein reductase, which is an enzyme responsible for fatty acid biosynthesis (Heath, Roland, & Rock, 2000). Additionally, TR has revealed remarkable anti-inflammatory properties (Skaare, Kjaerheim, Barkvoll, & Rolla, 1997). However, TR is hydrophobic and its solubility in water is extremely low (10–20 mg/L). On the other hand, recent studies suggest the potential health risk of TR overuse because it may be an endocrine disruptor (Veldhoen et al., 2006) and could increase the proliferation of antibiotic-resistant bacteria. Additionally, due to its worldwide widespread use over the last four decades, TR recovery from aquifers is becoming a concerning issue. To overcome this, in the last decades different investigations have explored the possibility of incorporating this compound into modified cyclodextrins (Fidaleo, Zuorro, & Lavecchia, 2013; Jug, Maestrelli, & Mura, 2012). These investigations suggest that an inclusion complex of 1:1 stoichiometry is formed and indicate a considerably high stability and water solubility (Kayaci, Umu, Tekinay, & Uyar, 2013; Qian et al., 2008). Qian et al. (2008) prepared and studied inclusion complexes of TR and cationic polymeric cyclodextrins and observed excellent antimicrobial properties after the complexation. Jug, Kosalec, Maestrellic, and Mura (2011) studied comparatively the interaction of TR with parent -CD and with its water-soluble neutral epichlorohydrin polymer by several analytical techniques observing a higher affinity of polymers than -CD for the interaction with TR, resulting in products with superior dissolution properties. Conversely, the same authors studied the TR solubilizing efficiency of anionic carboxymethylated -cyclodextrin-epichlorohydrin polymer and observed less stable complexation compared to that of the nonionic polymeric CDs (Jug, Kosalec, Maestrellic, & Mura, 2012). This research presents a further characterization of bCDs synthesized by polycondensation reaction with EP and CC or CA and the study of water solubility and cytotoxic properties of the obtained cationic and anionic bCDs. Ionic bCDs revealed that exhibit a remarkably higher solubility compared with their neutral homologues. Besides, inclusion complex formation between the obtained ionic bCDs and the bactericidal TR was studied and the water solubility capability of TR/bCDs complexes was analyzed. This paper aims to be a comparative study between anionic and cationic bCDs that led to a wider view on anionic bCDs and their ability to form inclusion complexes with a no ionic model host molecule, as TR. These CD-derivatives can be potentially useful on base on their soluble and ionic nature in the development of new materials for the recovery of TR in water treatment
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
processes as well as for local and restricted antibacterial action of TR. 2. Materials and methods 2.1. Materials -Cyclodextrin (-CD), epichlorohydrin (EP), choline chloride (CC) chloroacetic acid (CAA) and triclosan (TR) were purchased from Sigma Aldrich. Sodium hydroxide (NaOH, ≥98%, anhydrous, pellets) and hydrochloric acid (37%) were obtained from Panreac QP. All cell culture media and supplements namely, RPMI1640 medium, Fetal Bovine Serum (FBS), Penicillin–Streptomycin (PS) and trypsin/ethylenediamine tetraacetic acid (EDTA) were obtained from Invitrogen (Invitrogen Corporation, Paisley, UK). For cytotoxicity assays phosphate buffered saline (PBS) powder and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, USA). Dimethyl sulfoxide (DMSO) was obtained from Carlo Erba Reagents (Italy). All reagents were of AR grade and used as received without further purification. Distilled water was used throughout. 2.2. Synthesis of branched ionic ˇ-CD Branched ionic -CD were synthesized by one-step condensation reaction (Li et al., 2004; Qian et al., 2008; Renard, Barnathan, et al., 1997; Xin et al., 2010; Yang et al., 2013). In this work, the molar ratio of the reactants was -CD/EP/CC (CAA) = 1/10/2 and NaOH 22%w/w. 5 g (4.4 mmol) of -CD was dissolved in 8 mL of NaOH (22% w/w) solution and the mixture was magnetically stirred at 25 ◦ C for 24 h. Then, 1.228 g of CC in the synthesis of cationic bCD, or 0.832 g of CAA for the anionic bCDs, was fed into the solution. For the neutral branched cyclodextrin this step is suppressed. The mixture was heated at 30 ◦ C and EP (3.445 mL) was added rapidly. The temperature and stirring (600 rpm) were kept constant during the reaction. The reaction was stopped after 3 h 30 min by addition of acetone. After decantation, acetone was removed and the solution was neutralized with hydrochloric acid (6 N). The solution obtained was kept at 50 ◦ C overnight. After cooling, the solution was dialyzed for 3 days with a dialysis membrane of molecular weight cut-off of 3500 Da to remove unreacted EP and CC/CAA. After dialysis, the aqueous solution was evaporated and the solid triturated with acetone. The white product was isolated by filtration and dried under vacuum. 2.3. Preparation of TR/ionic bˇCD complexes The formation of inclusion complexes of TR was performed by a coprecipitation method. The molar ratio of TR to CD used was 1:1. Initially, TR was stirred in 2 mL of water and because TR is not water soluble, a suspension was obtained in each vial. Then, bCD was dissolved in water at 60 ◦ C, respectively. The resulting bCD solution was added into the aqueous TR suspension dropwise and heated at 60 ◦ C for 1 h and then stirred overnight at room temperature. TR/bCD solution was turbid and precipitation was observed subsequent to stirring overnight, indicating that TR form an inclusion complex with bCD. The resulting complexes were filtered and washed with water several times to remove uncomplexed CD. Finally, the solid TR/bCD was vacuum dried (Kayaci et al., 2013). 2.4. Characterization of ionic bˇCDs and their complexes with TR Gel permeation chromatography. Gel permeation chromatography (pump: Waters 600E System Controller; detector: Waters 410 Differential Refractometer) was carried out with Ultrahydrogel 250 and 500 columns at 40 ◦ C and the flow rate 0.7 mL/min. Water
151
was used as an eluent. Samples were used with concentration of 0.2–0.4% (w/v) and filtrated with a 0.45 m Nylon Cameo filtersyringe prior to the use. NMR spectroscopy. 1 H NMR and bidimensional 1 H–13 C NMR spectra of bCDs and TR/bCDs complexes dissolved in deuterium oxide were recorded using a spectrometer Bruker Avance operated at 500 MHz. Chemical shifts (ı) are reported relative to water (ı = 4.8). FTIR spectroscopy. Fourier transform infrared spectroscopy was performed on Nexus 470 FT-IR (Thermo Nicolet Company), running from 4000 to 400 cm−1 in KBr pellets of samples, at a resolution of 4 cm−1 and 32 scans per spectrum. UV/Vis spectroscopy. The solubility of bCDs was measured at 20 ◦ C as follows. Saturated solutions of samples were prepared and mechanically stirred at room temperature during 48 h. The obtained solutions were filtered through a 0.22 m Nylon Cameo filter-syringe. The filtrate was dried until constant weight and the solubility was estimated in terms of the weight of samples in the saturated solution and solution volume. Besides, solubility at different pH values was investigated by visible spectrophotometry (Shidmazu-Multispec1501). bCDs were dispersed in distilled water (5 mg/mL) acidified with HCl (1 M) and the pH of the dispersions was adjusted adding 2 M NaOH solution. The transmittance of the dispersions/solutions was measured at 515 nm as a measure of the turbidity of the obtained solutions or dispersions. Two repeats were conducted. Additionally, the solubility of TR in water and in bCD solutions was measured at 20 ◦ C as follows: an excess of TR was added to 5 mL of bCD solutions with different concentration (2–9 g/mL). The mixtures were shaken in a water bath shaker at 300 rpm during 72 h. After the equilibration, an aliquot was withdrawn and filtered by a 450 m filter (Millipore). The concentration of the bactericidal in the supernatants was determined by measuring their ultraviolet absorbance at a wavelength of 280 nm and compared with the calibration curve determined in a mixture methanol: water (95:5). Three repeats were conducted in this case. Zeta potential measurements. The zeta potential of bCDs solutions was measured with a zeta potential analyzer (Zeta-Sizer IV, Malvern Instruments). After dispersing bCDs in acid water solution (5 mg/mL), the pH was adjusted by adding of 2 M NaOH solution and the electrophoretic mobility of each sample was measured. Conductimetric/Potentiometric titrations. The amount of protonable (carboxylic) groups of anionic bCDs was measured by potentiometric and conductimetric titration. In this method, a known amount of HCl solution (0.02 mol/L) is added, in excess, into a solution containing a known quantity of anionic bCDs, allowing enough time to charge all protonable groups. The resulting solution was then titrated using a solution of NaOH (10 mmol/L). X-ray diffractometry (XRD). X-ray diffraction patterns were obtained by a Transmission STOE Stadip X-ray diffractometer with a goniometer speed of 0.1◦ /90 s. The range of diffraction angle 2 was 0–100◦ . 2.5. Cytotoxicity assay Cell toxicity assays were performed using Human colon carcinoma Caco-2 cells which are a good model of the intestinal barrier (Sambuy et al., 2005). Caco-2 cell line was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and cells were routinely grown in a standard medium: RPMI-1640 supplemented with 10% (v/v) of inactivated FBS and 5000 Units of PS. Stock cells were maintained as monolayers in 80 cm2 culture flasks and subcultured every week at a split ratio of 1–4 by treatment with 0.1% trypsin/0.02% EDTA and incubated at 37 ◦ C in a 5% CO2 humidified atmosphere.
152
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
Cytotoxic experiments were carried out as previously described with some modifications (Kiss et al., 2010). Briefly, Caco-2 (between passage 30 and 40) were seeded in 96-well plates at a density of 2 × 104 cells/well and the medium was changed every 48 h. After reaching confluence (6–10 days), cells were exposed to increasing concentrations of cationic bCD or anionic bCD (0–100 mg/mL) dissolved in PBS and incubated at 37 ◦ C and 5% CO2 . After 30 min of incubation, cells were washed two times with PBS and 100 L of MTT solution was added to each well at a final concentration of 0.5 mg/mL. MTT, a yellowish solution, is converted to dark blue, water-insoluble MTT formazan crystals by mitochondrial dehydrogenases of living cells and therefore the amount of formazan product is proportional to the number of viable cells. After 3 h of incubation, crystals were solubilized by adding 150 L of DMSO and the quantity of formazan produced was quantified spectrophotometrically at 570 nm in a microplate reader (EPOCH, Bio-Tek, USA). Results were expressed in terms of percentage of cellular viability relative to the untreated control (cells with PBS). Experiments were performed in triplicate in three independent assays.
3. Results
Fig. 1. FT-IR spectra in the region of 4000–500 cm−1 of -CDs, neutral bCDs, anionic bCDS and cationic bCDs.
3.1. Synthesis and characterization of ionic bˇCD Branched ionic CDs were synthesized following the procedure described in the experimental part by the polycondensation reaction with EP in the presence of CC or CAA. As has been commented above, the polycondensation with EP through the epoxy ring opening by the hydroxyl groups of the CD is the most used and simple synthetic way for the substitution of CDs. It is well known that this reaction route leads to branched and heterogeneous structures of low molecular weight. This was the case of prepared samples that had a weight average molecular weight of 1800, 1700, and 1600 g/mol for neutral, cationic and anionic bCDs determined by size exclusion chromatography, demonstrating that monomeric and branched CD derivatives were synthesized and no polymerization took place. This reaction by EP polycondensation leads to a mixture of heterogeneous structures with different length of 2-methoxypropyl ether repeating fragments as tails and different substitution degree, as is schematized in the general formula showed in Fig. 2A (Renard, Deratani, et al., 1997). However, it is known that in highly basic conditions, when NaOH/CD > 10, as described in this paper, substitutions take places preferably in the more accessible hydroxyls (primary, O-6), and only in case NaOH/CD < 10 the three positions (0–2, 0–3 and 0–6) would be substituted (Renard, Deratani, et al., 1997). When branched ionic CDs are synthesized by addition of CC or CAA substitution reactions take place together with the EP polycondensations, limiting EP tails growth and generating charged groups as pending group in cyclodextrin derivatives (Yang et al., 2013). This is in accordance with the slightly higher weight average molecular weight determined for neutral bCDs than ionic bCDs. The obtained bCDs were characterized by FTIR spectroscopy. The FTIR spectra of bCDs are different from that of CD (Fig. 1). FTIR spectra of branched -CDs showed the typical attenuation of the three characteristic bands of the monomer in the region of 1000–1200 cm−1 corresponding to the coupled C C and stretching vibration of C O bonds (1035 and 1081 cm−1 ) and the antisymmetric stretching vibration of C O C glycosidic bond (1154 cm−1 ), that indicates the advance of the modification reaction (Li et al., 2004; Qian et al., 2008). Besides FTIR spectra of branched CDs show that the stretching vibration of OH at 3422 cm−1 is reduced and the asymmetric stretching vibration of CH2 at 2876 cm−1 increases in comparison with the spectrum of monomeric CDs. These changes have been previously reported as obvious evidences for reaction
between CD and epichlorohydrin (Li et al., 2010; Qian et al., 2008). Also, the successful introduction of choline chloride on branched CD was evidenced by the presence of a new peak at 1480 cm−1 corresponding to the methyl groups of the quaternary ammonium (Junthip et al., 2015). At 1700 cm−1 may be visualized a combination of the elongation of COOH and COOR groups of anionic bCD (Martin, Tabary, Leclercq, et al., 2013). NMR spectroscopy allowed verifying the incorporation of cationic and anionic moieties into the -CDs. Fig. 2A shows the 1 H NMR spectra of neutral and branched ionic CDs, where the resonance signal of the pyranose ring protons (H-2, 3, 4, 5 and 6) at 3.4–4.1 ppm and that of the anomeric proton H-1, which appears at 5.0 ppm, could be observed. The quantification of the cationic substitution could be done by the integration of the peak that appears at 3.12 ppm, which corresponds to the methylic protons of quaternary ammonium moiety (Li et al., 2004; Qian et al., 2008; Yang et al., 2013) relative to the signal of H-1 appearing at 5.0 ppm. The number of cationic groups per anhydroglucose unit calculated was 0.64 ± 0.05. Based on the comparison between the relation among the areas of the peaks at 5.1 and 3.4–4.1 of the 1 H NMR spectra of -CD, and the relation between those integral values for the samples, EP/-CD relation of each sample was estimated, resulting in 0.41 ± 0.14, 0.31 ± 0.04 and 0.08 ± 0.05, for neutral bCDs, anionic bCD and cationic bCD. These data suggest that structures with less branched tails are obtained for cationic samples. This might be related with the electrostatic attraction between cationic moieties and negative charges of growing tails during polycondensation reactions. In the case of the anionic bCDs 1 H–13 C bidimensional NMR spectrum (Fig. 2B) allowed to corroborate the incorporation of COOH groups into bCDs. A weak resonance signal correlating with the 1 H signal at 4.1 ppm is observed around 170 ppm in the 13 C NMR spectrum. This signal could be assigned to the carbon atoms of carbonyl groups adjacent to CH2 protons, which could correspond to the carboxyl groups introduced with the anionic modification. The quantification of acidic groups introduced in the anionic bCD was accomplished by conductimetric and potentiometric titration and 0.62 ± 0.01 carboxyl groups per cyclodextrin ring were calculated. This value is close to that of cationic bCDs, indicating that electrostatic attraction or repulsion forces do not have a significant effect in the overall content of introduced ionic moiety in
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
153
Fig. 2. (A) 1 H NMR spectra of -CDs, cationic bCD, neutral bCD and anionic bCD in D2 O and (B) 1 H NMR and 13 C NMR bidimensional spectra of anionic bCD in D2 O.
the bCD. This could be understood by the fact that ionic groups are incorporated as tails to the side chains of EP. 3.2. Water solubility properties of ionic bˇCD As has been commented above, the low water solubility of -CD, which limits their applicability, is attributed to intermolecular hydrogen bonds that exist between the secondary hydroxyl groups, which are unfavorable for interaction between CDs and surrounding water molecules. Thus, the introduction of an ionic group originates electrostatic repulsions disrupting these intermolecular hydrogen bonds and increasing aqueous solubility. Water solubility properties of cationic derivatives of polymeric -CDs had been studied before (Li et al., 2004; Qian et al., 2008; Yang et al., 2013), however, to the best of our knowledge, the solubility of anionic bCD obtained by polycondensation with EP has not been extensively investigated.
Therefore, the water solubility of obtained bCDs was studied monitoring the transmittance of aqueous solutions at 550 nm. As can be observed in Fig. 3, ionic derivatives show a clear increase in solubility over the neutral samples due to the removal of hydrogen bonding network by the modification of primary hydroxyl groups which is enhanced in the case of ionic derivatives by the electrostatic repulsions. Likewise, it was observed that the solubility of anionic bCDs varies as a function of the pH of the solution, while the solubility of cationic bCDs remains constant. This is explained by the fact that anionic groups resulted of the ionization of the carboxyl groups that is pH dependent; at neutral and basic pHs the ionization degree is higher and consequently the solubility increases. However, in cationic derivatives since ammonium groups are not ionizable, pH dependence was not observed. The diffraction patterns of the ionic bCDs and the zeta potential of their solutions were studied in order to verify this behavior.
154
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
This decrease is in accordance with water solubility results and could be attributed to the opposing effect of high concentrations of counterions. Zeta potential analysis showed that at neutral pHs there was not a significant difference between the net charge of prepared cationic and anionic bCDs that is reflected in a similar solubility. Li et al. (2004) when studied the aqueous solubility of cationic bCDs concluded that it was also insensitive to the modification degree and the charge density due to the fact that ionic group does not combine directly to the -CD but to the EP chain. Certainly, when the X-ray diffraction pattern of the obtained bCDs (Fig. 4B) was compared with that of the pure -CD, it can be observed that the chemical modification resulted in the loss of crystallinity in the samples which were essentially amorphous, included neutral derivatives which showed lower solubility. This result reinforced the role of ionic moieties in order to obtain soluble bCDs samples on the basis of the repulsive interactions of charged groups. 3.3. Cytotoxicity on Caco-2 cells Fig. 3. Solubility of prepared (䊐) neutral, () anionic and (♦) cationic bCDs and transmittance of their solutions (5 mg/mL) at pH = 3, 7 and 10.
The study of the zeta potential of solutions of the synthesized bCDs (Fig. 4A) corroborated the ionic character of the branched CDs, obtaining positive zeta potential values for cationic bCDs and negative values for anionic derivatives. Besides, it could be observed a small negative charge for neutral bCDs, as it was reported in other investigations (Li et al., 2004; Müller & Brauns, 1985). This suggests an inherent negative charge present in all the bCDs derivatives and that despite the overall positive charge of cationic bCDs, negative charges could also exist in cationic bCDs. On the other hand, cationic charge is independent of the pH of the medium, but anionic charge varies according to the ionization equilibrium of carboxyl groups, so when the external pH is increased from 3 to 7 the negative charge increases, that agrees with the results observed in the solubility study. However, for high pH values, pH = 10, a decrease of negative zeta potential was observed.
It is reported that the ionic groups in -CD derivatives can significantly decrease the cytotoxic effect (Kiss et al., 2010). However, cytotoxicity of modified ionic derivatives has not been sufficiently evaluated. One study about the hemolytic effect of cationic polymeric cyclodextrins (Li et al., 2004), revealed lower hemolysis for substituted cyclodextrins than parent monomer and a decreasing hemolysis as cationic charge number increases. Studies about ionic, methylated and both ionic and methylated -CDs showed less cytotoxicity for ionic samples than the corresponding electroneutral methylated ones, but no comparison with parent or neutral but no methylated -CDs was made (Kiss et al., 2010). In this paper, the cytotoxicity of ionic and neutral bCDs were evaluated on Human colon Caco-2 cells after incubation of increasing concentrations of bCDs (0–100 mg/mL) during 30 min and it was compared with that of parent -CDs. The results of toxicity experiments (Fig. 5), in the concentrations tested, 58.8 mM and 62.5 mM for cationic bCD and anionic bCD, respectively, revealed that ionic bCDs behave as neutral and parent -CD and
Fig. 4. (A) Zeta potential of (䊐) neutral, (♦) cationic and () anionic bCDs at different pH values and (B) XRD of -CD, TR, physical mixture of TR and bCDs, neutral, cationic and anionic bCDs.
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
155
3.4. TR/bˇCDs complex formation
Fig. 5. Cytotoxicity of (䊐) cationic bCD, () anionic bCD, () neutral bCD and () -CD on human colon carcinoma Caco-2 cells during 30 min of incubation. Cell viability was expressed as the percentage of untreated control. Data are expressed as mean ± SD (n = 3).
did not show an inherent cytotoxicity derived from their ionic nature and possible interactions with cellular membranes. Indeed, it has been reported that cytotoxicity of -CDs derivatives seem to be directly related to the cholesterol solubilizing properties, and therefore it varies with the presence of ionic groups (Kiss et al., 2010). In this way, it is believed that ionic charges tend to enhance the steric hindrance which could decrease the inclusion capability to cholesterol and consequently cytotoxicity. On the other hand, a higher cell viability of substituted and branched CDs was claimed based on the fact that increasing the carbohydrate size seems to reduce cytotoxicity (Srinivasachari & Reineke, 2009). However, in the present work major differences were not observed among the cytotoxic properties of parent, neutral or ionic derivatives. These results are consistent with those separately reported for commercial ionic (Kiss et al., 2010) and parent CDs (Castagne et al., 2009).
Fig. 6.
1
The widely used antibacterial agent TR was complexed with the synthesized ionic bCDs by a coprecipitation procedure, as has been described above. The 1 H NMR spectra of TR, TR/bCDs complexes and chemical shifts are shown in the figure below (Fig. 6). Slight shift of chemical shifts of the protons (Ha-f) after inclusion of TR in TR/bCD complexes are observed. The H-3 and H-5 atoms of bCDs in TR/bCD complexes showed a significant upfield shift in comparison with uncomplexed bCDs. This results might indicate that these changes are due to the correct complex formation with the branched ionic sample. Regarding DSC analysis (Fig. 7A), it can be observed that the thermogram of pure TR presents an endothermic peak with onset temperature of 62.9 ◦ C, which corresponds to the melting process. The TR fusion peak disappeared completely for anionic bCD derivatives and in case of cationic bCD samples its intensity was significantly reduced and shifted to lower temperature (56.3 ◦ C), reflecting the interaction between bCD and TR. FTIR and X-ray diffraction analysis of the plain components and the complexes were also performed in order to further investigate the existence of TR/branched sample complexation, confirming the formation of TR/bCD complexes. The obtained FTIR spectra are showed in Fig. 7B. In the case of complexes samples, characteristic peaks of TR and bCD were observed, confirming the presence of both in these samples. In addition, the characteristic peaks of TR at 1505, 1472, and 1417 cm−1 shifted to 1508, 1474, and 1419 cm−1 , respectively, for the TR/bCD samples (Fidaleo et al., 2013). X-ray diffraction analysis also confirmed above results (Fig. 4B). In the Xray diffractograms of TR/bCD physical mixtures, all the peaks of TR peaks remain visible with the amorphous pattern of bCs, while they disappeared almost completely in the TR/bCD complexes samples.
3.5. Solubility of TR/bˇCDs complexes In order to assess the ability of prepared ionic bCDs solubilizing TR, phase solubility studies were carried out according to the method described by Higuchi and Connors (Higuchi & Connors,
H NMR spectra of (A) TR/bCDs complexes, (B) TR, (C) expanded region for TR/bCDs complexes and 1 H chemical shifts of TR and TR/bCDs complex.
156
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
Fig. 7. (A) DSC thermograms of: TR, cationic bCDs, anionic bCDs and the complexes TR/cationic bCDs and TR/anionic bCDs, (B) FTIR spectra of TR, cationic bCDs, anionic bCDs and their complexes TR/cationic bCDs and TR/anionic bCDs.
1965). Fig. 8 shows a graphical representation of the results of phase-solubility studies. When the aqueous solubility of TR was studied in the presence of increasing bCDs concentrations the same trend was observed in the presence of both ionic derivatives. It can be observed an increase in the solubility of TR as concentration of bCD increases, confirming the complex formation and revealing that TR/bCDs complexation takes place and leads to an increase in the apparent water solubility of TR of up to 20-fold. The resulting phase-solubility diagram could be classified as BS type according to Higuchi and Connors, which is typical of monomeric CD derivatives. The aqueous solubility of TR increased linearly with the bCD concentration for low bCD concentration values until solubility limit is reached (Hernández-Sánchez, López-Miranda, Lucas-Abellán, & ˜ Núnez-Delicado, 2012) at 2.2 mg/mL TR with 4.0 mg/mL anionic bCDs and 1.4 mg/mL TR with 3.8 mg/mL cationic bCDs concentrations. Further addition of CDs resulted in the formation of less soluble inclusion complexes as has been previously described by Higuchi (Grant & Higuchi, 1990). This low complexation limit could be ascribed to the steric hindrance that arises from the heterogeneous structure of EP-substituted CDs with many side chains (Frömming & Szejtli, 1994, chap. 2).
The slope of solubility diagram was less than one; it was therefore assumed that the solubility increase could be attributed to the formation of the 1:1 complex (Loftsson, Jarho, Másson & Järvinen, 2005). In these conditions the apparent stability constant (Ks ) of the complex can be calculated from the slope and the intrinsic solubility (s0 ) of the drug in the aqueous media using Eq. (1) according to Higuchi and Connors (Higuchi & Connors, 1965): Ks =
slope s0 (1 − slope)
(1)
Apparent stability constant value was 1465 M−1 and 4729 M−1 for cationic bCD and anionic bCDs respectively, which indicate that the complexes are adequately stable (Higuchi & Connors, 1965). Besides, the Ks of cationic bCDs is lower than that of anionic bCDs. This fact could be ascribed to the decrease in drug accessibility through the -CD internal cavity as consequence of the steric hindrance of the introduction of quaternary ammonium, as has been postulate by other authors when complexation of hydrophobic drugs with cationic polymeric -CDs and -CDs were studied (Li et al., 2004).
4. Conclusions
Fig. 8. Phase-solubility diagrams for TR included in (䊐) cationic bCDs and (䊉) anionic bCDs.
Substitution reaction of -CDs with EP and CC or CAA led to branched ionic -CDs with similar ionic moiety content but less EP content in case of cationic derivatives, that might be ascribed to the electrostatic interaction between the positive charge of CAA and negative charge growing side chains. These ionic bCDs showed similar water solubility at neutral and basic pHs, that was 10 fold higher water solubility than that of neutral bCDs due to the incorporation of ionic moieties. Water solubility of anionic bCDs was pH-dependent accordingly with the ionization of their carboxyl groups and contrary to cationic bCDs. Both prepared ionic bCDs showed high cell viability with human colon carcinoma Caco-2 cells and formed inclusion complexes with the antibacterial TR. TR/bCDs complexation led to an increase in the apparent water solubility of TR of up to 20-fold but with a complexation limit around 4.0 mg/mL. TR solubility at complexation limit and apparent stability constant value were lower for cationic bCDs than anionic samples, reflecting the higher steric hindrance of the ammonium moiety in comparison with carboxylic groups of cationic bCDs and anionic bCDs, respectively.
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
Acknowledgments Authors are grateful to the Government of Mexico (CONACYT) and to theGovernment of the Basque Country for financial support (ACTIMAT project, ETORTEK program) (Grupos de Investigación, IT350-10) and technical and human support provided by SGIKER (UPV/EHU, MICINN, GV/EJ, ERDF, and ESF). References Astray, G., Gonzalez-Barreiro, C., Mejuto, J. C., Rial-Otero, R., & Simal-Gándara, J. (2009). A review on the use of cyclodextrins in foods. Food Hydrocolloids, 23, 1631–1640. Blomberg, E., Kumpulainen, A., David, C., & Amiel, C. (2004). Polymer bilayer formation due to specific interactions between -cyclodextrin and adamantane: A surface force study. Langmuir, 20, 10449–10454. Breslow, R. (2010). Cyclodextrins. In Molecular encapsulation: Organic reactions in constrained systems. UK: John Wiley & Sons. Castagne, D., Fillet, M., Delattre, L., Evrard, B., Nusgens, B., & Piel, G. (2009). Study of the cholesterol extraction capacity of -cyclodextrin and its derivatives, relationships with their effects on endothelial cell viability and on membrane models. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 63, 225–2231. Easton, C. J. (2005). Cyclodextrin-based catalysts and molecular reactors. Pure Applied Chemistry, 77, 1865–1871. Crinia, G., & Morcellet, M. (2002). Synthesis and applications of adsorbents containing cyclodextrins. Journal of Separation Science, 25, 789–813. Davis, M. E., & Brewster, M. E. (2004). Cyclodextrin-based pharmaceutics: Past, present and future. Nature Reviews Drug Discovery, 3, 1023–1035. Fidaleo, M., Zuorro, A., & Lavecchia, R. (2013). Enhanced antibacterial and anti-quorum sensing activities of triclosan by complexation with modified -cyclodextrins. World Journal of Microbiology and Biotechnology, 29, 1731–1736. Frömming, K.-H., & Szejtli, J. (1994). Cyclodextrins in pharmacy. The Netherlands: Springer Science+ Business Media B.V. Grant, D., & Higuchi, T. (1990). Solubility behaviour of organics compounds. Techniques of chemistry. pp. 21. New York: Wiley Interscience Publication. Harada, A., Furue, M., & Nozakura, S.-I. (1976). Cyclodextrin-containing polymers. 1. Preparation of polymers. Macromolecules, 9, 701–704. Harada, A., Furue, M., & Nozakura, S.-I. (1981). Inclusion of aromatic compounds by a -cyclodextrin-epichlorohydrin polymer. Polymer Journal, 13, 777–781. Heath, R. J., Li, J., Roland, G. E., & Rock, C. O. (2000). Inhibition of the staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene. Journal of Biological Chemistry, 275, 4654–4659. Higuchi, T., & Connors, K. A. (1965). Phase solubility techniques. Advanced Analytical Chemical Instrumentation, 4, 117–212. ˜ Hernández-Sánchez, P., López-Miranda, S., Lucas-Abellán, C., & Núnez-Delicado, E. (2012). Complexation of eugenol (EG), as main component of clove oil and as pure compound, with ˇ- and HP-ˇ-CDs. Food and Nutrition Sciences, 3, 716–723. Hill, L. E., Gomes, C., & Taylor, T. M. (2013). Characterization of beta-cyclodextrin inclusion complexes containing essential oils (trans-cinnamaldehyde, eugenol, cinnamon bark, and clove bud extracts) for antimicrobial delivery applications. LWT - Food Science and Technology, 51, 86–93. Huang, L., Xin, J., Guo, Y., & Li, J. (2010). A novel insulin oral delivery system assisted by cationic -cyclodextrin polymers. Journal of Applied Polymer Science, 115, 1371–1379. Jug, M., Kosalec, I., Maestrellic, F., & Mura, P. (2011). Analysis of triclosan inclusion complexes with -cyclodextrin and its water-soluble polymeric derivative. Journal of Pharmaceutical and Biomedical Analysis, 54, 1030–1039. Jug, M., Kosalec, I., Maestrellic, F., & Mura, P. (2012). Development of low methoxy amidated pectin-based mucoadhesive patches for buccal delivery of triclosan: Effect of cyclodextrin complexation. Carbohydrate Polymers, 90, 1794–1803. Jug, M., Maestrelli, F., & Mura, P. (2012). Native and polymeric -cyclodextrins in performance improvement of chitosan films aimed for buccal delivery of poorly soluble drugs. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 74, 87–97. Junthip, J., Tabary, N., Leclercq, L., & Martel, B. (2015). Cationic -cyclodextrin polymer applied to a dual cyclodextrinpolyelectrolyte multilayer system. Carbohydrate Polymers, 126, 156–167. Kayaci, F., Umu, O. C. O., Tekinay, T., & Uyar, T. (2013). Antibacterial electrospun poly(lactic acid) (PLA) nanofibrous webs incorporating triclosan/cyclodextrin inclusion complexes. Journal of Agricultural and Food Chemistry, 61, 3901–3908. Kayaci, F., & Uyar, T. (2012). Encapsulation of vanillin/cyclodextrin inclusion complex in electrospun polyvinyl alcohol (PVA) nanowebs: Prolonged shelf life and high temperature stability of vanillin. Food Chemistry, 133, 641–649.
157
Kiss, T., Fenyvesi, F., Bácskay, I., Váradi, J., Fenyvesi, E., Iványi, R., et al. (2010). Evaluation of the cytotoxicity of beta-cyclodextrin derivatives: Evidence for the role of cholesterol extraction. European Journal of Pharmaceutical Science, 40, 376–380. Li, J., Guo, Z., Xin, J., Zhao, G., & Xiao, H. (2010). Arm star polymers with different cationic groups based on cyclodextrin core for DNA delivery. Carbohydrate Polymers, 79, 277–283. Li, J., & Loh, X. (2008). Cyclodextrin-based supramolecular architectures: Syntheses, structures, and applications for drug and gene delivery. Advanced Drug Delivery Reviews, 60, 1000–1017. Li, J., Xiao, H., Li, J., & Zhong, Y. (2004). Drug carrier systems based on water-soluble cationic -cyclodextrin polymers. International Journal of Pharmaceutics, 278, 329–342. Loftsson, T., & Brewster, M. E. (1996). Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. Journal of Pharmaceutical Sciences, 85, 1017–1025. Loftsson, T., Jarho, P., Másson, M., & Järvinen, T. (2005). Cyclodextrins in drug delivery. Expert Opinion on Drug Delivery, 2, 335–351. Martel, B., Ruffin, D., Weltrowski, M., Lekchiri, Y., & Morcellet, M. (2005). Water-soluble polymers and gels from the polycondensation between cyclodextrins and poly(carboxylic acid)s: A study of the preparation parameters. Journal of Applied Polymer Science, 97, 433–442. Martin, A., Tabary, N., Chai, F., Leclercq, L., Junthip, J., Aubert-Viard, F., et al. (2013). Build-up of an antimicrobial multilayer coating on a textile support based on a methylene blue–poly(cyclodextrin) complex. Biomedical Materials, 8, 1–14, 065006. Martin, A., Tabary, N., Leclercq, L., Junthipa, J., Degoutin, S., Aubert-Viard, F., et al. (2013). Multilayered textile coating based on a -cyclodextrin polyelectrolyte for the controlled release of drugs. Carbohydrate Polymers, 93, 718–730. Müller, B. W., & Brauns, U. (1985). Solubilization of drugs by modified -cyclodextrins. International Journal of Pharmaceutics, 26, 77–88. Renard, E., Barnathan, G., Deratani, A., & Sebille, B. (1997). Polycondensation of cyclodextrins with epichlorohydrin. Influence of reaction conditions on the polymer structure. Macromolecular Symposia, 122, 229–234. Renard, E., Deratani, A., Volet, G., & Sebille, B. (1997). Preparation and characterization of water soluble high molecular weight -cyclodextrin-epichlorohydrin polymers. European Polymer Journal, 33, 49–57. Qian, L., Guan, Y., & Xiao, H. (2008). Preparation and characterization of inclusion complexes of a cationic-cyclodextrin polymer with butylparaben or triclosan. International Journal of Pharmaceutics, 357, 244–251. Sambuy, Y., Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A., & Zucco, F. (2005). The Caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biology and Toxicology, 21, 1–2. Singh, R., Bharti, N., Madan, J., & Hiremath, S. N. (2010). Characterization of cyclodextrin inclusion complexes – A review. Journal of Pharmaceutical Science and Technology, 2, 171–183. Skaare, A. B., Kjaerheim, V., Barkvoll, P., & Rolla, G. (1997). Does the nature of the solvent affect the anti-inflammatory capacity of triclosan? An experimental study. Journal of Clinical Periodontology, 24, 124–128. Szejtli. (1988). Cyclodextrins technology. Budapest: Springer-Science+Business Media B.V. Szeman, J., Fenyvesi, E., & Szejtli, J. (1987). Water soluble cyclodextrin polymers: Their interaction with drugs. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 5, 427–431. Szente, L., & Szejtli, J. (1999). Highly soluble cyclodextrin derivatives: Chemistry, properties, and trends in development. Advanced Drug Delivery Reviews, 36, 17–28. Szente, L., & Szeman, J. (2013). Cyclodextrins in analytical chemistry: Host–guest type molecular recognition. Analytical Chemistry, 85, 8024–8030. Srinivasachari, S., & Reineke, T. M. (2009). Versatile supramolecular pDNA vehicles via “click polymerization” of -cyclodextrin with oligoethyleneamines. Biomaterials, 30, 928–938. Uekama, K., Hirayama, F., & Irie, T. (1998). Cyclodextrin drug carrier systems. Chemical Reviews, 98, 2045–2076. Veldhoen, N., Skirrow, R. C., Osachoff, H., Wigmore, H., Clapson, D. J., Gunderson, M. P., et al. (2006). The bactericidal agent triclosan modulates thyroid hormone associated gene expression and disrupts postembryonic anuran development. Aquatic Toxicology, 80(3), 217–227. Xin, J., Guo, Z., Chen, X., Jiang, W., Li, J., & Li, M. (2010). Study of branched cationic -cyclodextrin polymer/indomethacin complex and its release profile from alginate hydrogel. International Journal of Pharmaceutics, 386, 221–228. Yang, S. Y., Hoonor, R., Jin, H.-S., & Kim, J. (2013). Synthesis and characterization of cationic and anionic cyclodextrin oligomers and their use in layer-by-layer film formation. Bulletin of Korean Chemical Society, 34, 2016–2022. Zhang, N., Li, J., Jiang, W., Ren, C., Li, J., Xin, X., et al. (2010). Effective protection and controlled release of insulin by cationic -cyclodextrin polymers from alginate/chitosan nanoparticles. International Journal of Pharmaceutics, 393, 212–218.
Contents lists available at ScienceDirect
Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol
Preparation and characterization of soluble branched ionic -cyclodextrins and their inclusion complexes with triclosan Flor Gómez-Galván a , Leyre Pérez-Álvarez a,b,∗ , Janire Matas a , Arturo Álvarez-Bautista c,d , Joana Poejo c,d , Catarina M. Duarte c,d , Leire Ruiz-Rubio a , Jose Luis Vila-Vilela a , Luis M. León a,b a Departamento de Química Física (Laboratorio de Química Macromolecular), Universidad del País Vasco (UPV/EHU), B◦ Sarriena s/n, 48940 Leioa, Vizcaya, Spain b BCMaterials, Building 500-1st Floor, Bizkaia Science and Technology Park, 48160 Derio, Spain c Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal d iBET, Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 Oeiras, Portugal
a r t i c l e
i n f o
Article history: Received 24 September 2015 Received in revised form 19 January 2016 Accepted 21 January 2016 Available online 23 January 2016 Keywords: Branched ionic cyclodextrins Epichlorohydrin Cytotoxicity Water solubility
a b s t r a c t This study aims to synthesize, characterize and investigate the water solubility and cytotoxicity of branched anionic/cationic -cyclodextrins (bCDs) obtained by reaction with epichlorohydrin and chloroacetic acid or choline chloride, respectively, by a single step polycondensation reaction. Obtained ionic bCDs were investigated as an attempt to comparatively study anionic and cationic bCDs. Water solubility of both ionic derivatives was similar (400 mg/mL) at neutral and basic pHs and remarkably higher than that of their neutral homologues. Additionally, a pH-dependent solubility of anionic bCDs was observed. Cytotoxicity of ionic bCDs was evaluated on Human colon carcinoma Caco-2 cells and high cell viability (>99%) was observed in the range of 0–100 mg/mL for anionic and cationic samples, in the same range of that of neutral and parent -CDs. Additionally, complexes formation capacity with triclosan, a poor water soluble antimicrobial agent, was confirmed by several techniques observing a complexation limit around 4 mg/mL for both systems and higher stability constant for anionic bCDs than cationic derivatives. © 2016 Published by Elsevier Ltd.
1. Introduction Cyclodextrins (CDs) are cyclic oligosaccharides produced from starch and which contain six (␣-CDs), seven (-CDs) or eight (␥-CDs) glucopyranose units linked by ␣-1,4 glucosidic bonds (Breslow, 2010, chap. 2). The -form is the most accessible and widely used cyclodextrin. As a result of the restricted rotation of linked glucopyranose units CDs are toroidal or truncated cone shaped molecules with an internal cavity whose size is around 6.0–7.0 A˚ for -CDs (Loftsson & Brewster, 1996). CDs have hydrophilic hydroxyl groups on their outer surface, which is hydrophilic, but a hydrophobic cavity in the center, which provides an apolar matrix. As a result, CDs have a special capability to entrap
∗ Corresponding author at: Departamento de Química Física, Facultad de Ciencia y Tecnología (Laboratorio de Química Macromolecular), Universidad del País Vasco (UPV/EHU), B◦ Sarriena s/n, 48940 Leioa, Bizkaia, Spain. E-mail address: [email protected] (L. Pérez-Álvarez). http://dx.doi.org/10.1016/j.carbpol.2016.01.046 0144-8617/© 2016 Published by Elsevier Ltd.
hydrophobic guests (Szejtli, 1988, chap. 2; Singh, Bharti, Madan, & Hiremath, 2010). This property has received increasing attention in the last years and has been extensively exploited to form host–guest inclusion complexes with a variety of drugs (Uekama, Hirayama, & Irie, 1998), food additives (Kayaci & Uyar, 2012), and antibacterial agents (Hill, Gomes, & Taylor, 2013), in different fields such as analytical chemistry (Szente & Szeman, 2013), catalysis (Easton, 2007), pharmacy (Davis & Brewster, 2004), food industries (Astray, Gonzalez-Barreiro, Mejuto, Rial-Otero, & Simal-Gándara, 2009) and the treatment of wastewater (Crinia & Morcellet, 2002). Over the last decades, CDs have been used as drug and antimicrobials carriers showing that they are able to enhance water solubility, chemical stability, bioavailability and decrease unfavorable sideeffects of the active agents (Blomberg, Kumpulainen, David, & Amiel, 2004). However, parent -CDs have poor water solubility as a consequence of the intra molecular hydrogen bonding of the secondary hydroxyl groups which limits their further applications. To overcome this drawback, various CDs derivatives have been developed
150
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
by substitution of their hydroxyl groups, such as, methylated CDs, hydroxyalkylated CDs, sulfobutyl ether CDs, which are examples of commercially available CDs derivatives (Szente & Szejtli, 1999). On the other hand, substituted -CDs, such as CD-based polymers, which are the most studied substituted -CDs and refer to those structures containing two or more covalently linked CDs units, are of interest because they exhibit enhanced aqueous solubility (Qian, Guan, & Xiao, 2008). Harada, Furue, and Nozakura (1976) reported the synthesis of polymeric CDs by radical polymerization of acrylate derivatives of CDs or monomers that form complexes with CDs. Later, Renard, Deratani, Volet, and Sebille (1997) studied a simpler synthetic route by polycondensation of CDs, with bi-functional agents such as epichlorohydrin (EP) and reported a systematic study of the reaction conditions on the polycondensation reaction under basic conditions (Renard, Barnathan, Deratani, & Sebille, 1997). The primary product of this reaction is not a true polymer but a heterogeneous mixture of various CD glyceryl ethers with crosslinked structures including CD moieties, linking bridges and side-chain substituents. Particularly, these polycyclodextrins prepared by polymer crosslinking with EP have shown be more efficient than acrylate derivatives in binding larger guest molecules with the help of the hydrophobic EP fractions and adjacent CD units (Harada, Furue, & Mozakura, 1981; Szeman, Fenyvesi, & Szejtli, 1987). Another approach to increase CDs water solubility is introducing ionic fragments on CDs that has resulted in an increase of the water solubility and in a lower cytotoxicity. Although water solubility improvement of ionic CDs have been reported, improving water solubility does not necessarily lead to a reduced cytotoxicity, namely, methylated -CDs show enhanced water solubility but high toxicity on Caco-2 cells and human erythrocytes (Li, Xiao, Li, & Zhong, 2004). Certainly, although CDs have been extensively studied there is limited information about their cytotoxic effects (Kiss et al., 2010). Up today, the in vivo cytotoxicity of CDs has been correlated with their binding and extraction capacity of membrane cholesterol which alters the permeability properties of the membrane and leads to hemolysis (Li et al., 2004). Additionally, charged CDs have different complexation and solubilization capabilities in comparison with other CD derivatives and the electrostatic interaction outside the cavity seems to enhance the solubilization of the hydrophobic active agent. In order to combine the good values of charged CDs and substituted CD, different investigations have focused on cationic cyclodextrin polymers which have been synthesized (Blomberg et al., 2004; Huang, Xin, Guo, & Li, 2010; Li & Loh, 2008; Xin et al., 2010; Zhang et al., 2010) by polycondensation reaction with EP to constitute typically polymer/oligomers and choline chloride (CC) or more recently with glycidyltrimetrylammonium chloride, to provide cationic groups (Junthip, Tabary, Leclercq, & Martel, 2015). These cationic polycyclodextrins have been reported to exhibit better hemocompatibility to human erythrocytes, no cytotoxicity, good drug inclusion and dissolution abilities than that of CDs. Meanwhile, in recent research branched CpCDs have also shown to be safe enough as non-viral gene vectors (Huang et al., 2010; Li, Guo, Xin, Zhao, & Xiao, 2010). These CpCDs have been employed to form inclusion complexes with antibiotics and their structure and antimicrobial activities have been studied, suggesting better association and antimicrobial activity with small antibiotic molecules (Qian et al., 2008). Regarding anionic substituted CDs, Martel, Ruffin, Weltrowski, Lekchiri, and Morcellet (2005) obtained anionic polycyclodextrins by a polyesterification reaction of CDs and some of their derivatives with di and poly(carboxylic acid)s obtaining gels and soluble forms. However, this research concluded the unsuccessful use of dicarboxylic acids and that obtaining soluble form required soft conditions that led to a low yield and high cytotoxicity, while
citric acid was an appropriate crosslinking that lets to obtain soluble derivatives. This anionic CDs derivatives, in which negative charge corresponds to the crosslinking agent, have been employed recently in the development of multilayer systems by complexes formation with chitosan onto polyester fibers (Martin, Tabary, Chai, et al., 2013; Martin, Tabary, Leclercq, et al., 2013). Besides, Yang, Hoonor, Jin, and Kim (2013) attempted to isolate and characterize cationic and anionic -cyclodextrin polymers through one step polycondensation process with EP using choline chloride (CC) for cationic polymer and chloroacetic acid (CAA) for obtaining anionic polymer. The detailed structural analysis of these ionic -CD compounds was done confirming the formation of branched -CD monomers and oligomers with a few units of -CD. Reaction parameters were studied in order to eliminate the undesirable crosslinking, and increase reaction yield and molecular weight. Despite the authors claim the water soluble properties of the prepared ionic substituted CDs no studies were made regarding water solubility. Triclosan (TR), [5-chloro-2-(2,4-dichlorophenoxy)phenol], is an antimicrobial agent widely used in consumer and medical products such as mouthwashes, toothpastes, surgical scrubs and suture materials. TR inhibits the growth of a wide range of microorganisms blocking the synthesis of lipids and by the inhibition of enoylacyl carrier protein reductase, which is an enzyme responsible for fatty acid biosynthesis (Heath, Roland, & Rock, 2000). Additionally, TR has revealed remarkable anti-inflammatory properties (Skaare, Kjaerheim, Barkvoll, & Rolla, 1997). However, TR is hydrophobic and its solubility in water is extremely low (10–20 mg/L). On the other hand, recent studies suggest the potential health risk of TR overuse because it may be an endocrine disruptor (Veldhoen et al., 2006) and could increase the proliferation of antibiotic-resistant bacteria. Additionally, due to its worldwide widespread use over the last four decades, TR recovery from aquifers is becoming a concerning issue. To overcome this, in the last decades different investigations have explored the possibility of incorporating this compound into modified cyclodextrins (Fidaleo, Zuorro, & Lavecchia, 2013; Jug, Maestrelli, & Mura, 2012). These investigations suggest that an inclusion complex of 1:1 stoichiometry is formed and indicate a considerably high stability and water solubility (Kayaci, Umu, Tekinay, & Uyar, 2013; Qian et al., 2008). Qian et al. (2008) prepared and studied inclusion complexes of TR and cationic polymeric cyclodextrins and observed excellent antimicrobial properties after the complexation. Jug, Kosalec, Maestrellic, and Mura (2011) studied comparatively the interaction of TR with parent -CD and with its water-soluble neutral epichlorohydrin polymer by several analytical techniques observing a higher affinity of polymers than -CD for the interaction with TR, resulting in products with superior dissolution properties. Conversely, the same authors studied the TR solubilizing efficiency of anionic carboxymethylated -cyclodextrin-epichlorohydrin polymer and observed less stable complexation compared to that of the nonionic polymeric CDs (Jug, Kosalec, Maestrellic, & Mura, 2012). This research presents a further characterization of bCDs synthesized by polycondensation reaction with EP and CC or CA and the study of water solubility and cytotoxic properties of the obtained cationic and anionic bCDs. Ionic bCDs revealed that exhibit a remarkably higher solubility compared with their neutral homologues. Besides, inclusion complex formation between the obtained ionic bCDs and the bactericidal TR was studied and the water solubility capability of TR/bCDs complexes was analyzed. This paper aims to be a comparative study between anionic and cationic bCDs that led to a wider view on anionic bCDs and their ability to form inclusion complexes with a no ionic model host molecule, as TR. These CD-derivatives can be potentially useful on base on their soluble and ionic nature in the development of new materials for the recovery of TR in water treatment
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
processes as well as for local and restricted antibacterial action of TR. 2. Materials and methods 2.1. Materials -Cyclodextrin (-CD), epichlorohydrin (EP), choline chloride (CC) chloroacetic acid (CAA) and triclosan (TR) were purchased from Sigma Aldrich. Sodium hydroxide (NaOH, ≥98%, anhydrous, pellets) and hydrochloric acid (37%) were obtained from Panreac QP. All cell culture media and supplements namely, RPMI1640 medium, Fetal Bovine Serum (FBS), Penicillin–Streptomycin (PS) and trypsin/ethylenediamine tetraacetic acid (EDTA) were obtained from Invitrogen (Invitrogen Corporation, Paisley, UK). For cytotoxicity assays phosphate buffered saline (PBS) powder and thiazolyl blue tetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, USA). Dimethyl sulfoxide (DMSO) was obtained from Carlo Erba Reagents (Italy). All reagents were of AR grade and used as received without further purification. Distilled water was used throughout. 2.2. Synthesis of branched ionic ˇ-CD Branched ionic -CD were synthesized by one-step condensation reaction (Li et al., 2004; Qian et al., 2008; Renard, Barnathan, et al., 1997; Xin et al., 2010; Yang et al., 2013). In this work, the molar ratio of the reactants was -CD/EP/CC (CAA) = 1/10/2 and NaOH 22%w/w. 5 g (4.4 mmol) of -CD was dissolved in 8 mL of NaOH (22% w/w) solution and the mixture was magnetically stirred at 25 ◦ C for 24 h. Then, 1.228 g of CC in the synthesis of cationic bCD, or 0.832 g of CAA for the anionic bCDs, was fed into the solution. For the neutral branched cyclodextrin this step is suppressed. The mixture was heated at 30 ◦ C and EP (3.445 mL) was added rapidly. The temperature and stirring (600 rpm) were kept constant during the reaction. The reaction was stopped after 3 h 30 min by addition of acetone. After decantation, acetone was removed and the solution was neutralized with hydrochloric acid (6 N). The solution obtained was kept at 50 ◦ C overnight. After cooling, the solution was dialyzed for 3 days with a dialysis membrane of molecular weight cut-off of 3500 Da to remove unreacted EP and CC/CAA. After dialysis, the aqueous solution was evaporated and the solid triturated with acetone. The white product was isolated by filtration and dried under vacuum. 2.3. Preparation of TR/ionic bˇCD complexes The formation of inclusion complexes of TR was performed by a coprecipitation method. The molar ratio of TR to CD used was 1:1. Initially, TR was stirred in 2 mL of water and because TR is not water soluble, a suspension was obtained in each vial. Then, bCD was dissolved in water at 60 ◦ C, respectively. The resulting bCD solution was added into the aqueous TR suspension dropwise and heated at 60 ◦ C for 1 h and then stirred overnight at room temperature. TR/bCD solution was turbid and precipitation was observed subsequent to stirring overnight, indicating that TR form an inclusion complex with bCD. The resulting complexes were filtered and washed with water several times to remove uncomplexed CD. Finally, the solid TR/bCD was vacuum dried (Kayaci et al., 2013). 2.4. Characterization of ionic bˇCDs and their complexes with TR Gel permeation chromatography. Gel permeation chromatography (pump: Waters 600E System Controller; detector: Waters 410 Differential Refractometer) was carried out with Ultrahydrogel 250 and 500 columns at 40 ◦ C and the flow rate 0.7 mL/min. Water
151
was used as an eluent. Samples were used with concentration of 0.2–0.4% (w/v) and filtrated with a 0.45 m Nylon Cameo filtersyringe prior to the use. NMR spectroscopy. 1 H NMR and bidimensional 1 H–13 C NMR spectra of bCDs and TR/bCDs complexes dissolved in deuterium oxide were recorded using a spectrometer Bruker Avance operated at 500 MHz. Chemical shifts (ı) are reported relative to water (ı = 4.8). FTIR spectroscopy. Fourier transform infrared spectroscopy was performed on Nexus 470 FT-IR (Thermo Nicolet Company), running from 4000 to 400 cm−1 in KBr pellets of samples, at a resolution of 4 cm−1 and 32 scans per spectrum. UV/Vis spectroscopy. The solubility of bCDs was measured at 20 ◦ C as follows. Saturated solutions of samples were prepared and mechanically stirred at room temperature during 48 h. The obtained solutions were filtered through a 0.22 m Nylon Cameo filter-syringe. The filtrate was dried until constant weight and the solubility was estimated in terms of the weight of samples in the saturated solution and solution volume. Besides, solubility at different pH values was investigated by visible spectrophotometry (Shidmazu-Multispec1501). bCDs were dispersed in distilled water (5 mg/mL) acidified with HCl (1 M) and the pH of the dispersions was adjusted adding 2 M NaOH solution. The transmittance of the dispersions/solutions was measured at 515 nm as a measure of the turbidity of the obtained solutions or dispersions. Two repeats were conducted. Additionally, the solubility of TR in water and in bCD solutions was measured at 20 ◦ C as follows: an excess of TR was added to 5 mL of bCD solutions with different concentration (2–9 g/mL). The mixtures were shaken in a water bath shaker at 300 rpm during 72 h. After the equilibration, an aliquot was withdrawn and filtered by a 450 m filter (Millipore). The concentration of the bactericidal in the supernatants was determined by measuring their ultraviolet absorbance at a wavelength of 280 nm and compared with the calibration curve determined in a mixture methanol: water (95:5). Three repeats were conducted in this case. Zeta potential measurements. The zeta potential of bCDs solutions was measured with a zeta potential analyzer (Zeta-Sizer IV, Malvern Instruments). After dispersing bCDs in acid water solution (5 mg/mL), the pH was adjusted by adding of 2 M NaOH solution and the electrophoretic mobility of each sample was measured. Conductimetric/Potentiometric titrations. The amount of protonable (carboxylic) groups of anionic bCDs was measured by potentiometric and conductimetric titration. In this method, a known amount of HCl solution (0.02 mol/L) is added, in excess, into a solution containing a known quantity of anionic bCDs, allowing enough time to charge all protonable groups. The resulting solution was then titrated using a solution of NaOH (10 mmol/L). X-ray diffractometry (XRD). X-ray diffraction patterns were obtained by a Transmission STOE Stadip X-ray diffractometer with a goniometer speed of 0.1◦ /90 s. The range of diffraction angle 2 was 0–100◦ . 2.5. Cytotoxicity assay Cell toxicity assays were performed using Human colon carcinoma Caco-2 cells which are a good model of the intestinal barrier (Sambuy et al., 2005). Caco-2 cell line was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) and cells were routinely grown in a standard medium: RPMI-1640 supplemented with 10% (v/v) of inactivated FBS and 5000 Units of PS. Stock cells were maintained as monolayers in 80 cm2 culture flasks and subcultured every week at a split ratio of 1–4 by treatment with 0.1% trypsin/0.02% EDTA and incubated at 37 ◦ C in a 5% CO2 humidified atmosphere.
152
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
Cytotoxic experiments were carried out as previously described with some modifications (Kiss et al., 2010). Briefly, Caco-2 (between passage 30 and 40) were seeded in 96-well plates at a density of 2 × 104 cells/well and the medium was changed every 48 h. After reaching confluence (6–10 days), cells were exposed to increasing concentrations of cationic bCD or anionic bCD (0–100 mg/mL) dissolved in PBS and incubated at 37 ◦ C and 5% CO2 . After 30 min of incubation, cells were washed two times with PBS and 100 L of MTT solution was added to each well at a final concentration of 0.5 mg/mL. MTT, a yellowish solution, is converted to dark blue, water-insoluble MTT formazan crystals by mitochondrial dehydrogenases of living cells and therefore the amount of formazan product is proportional to the number of viable cells. After 3 h of incubation, crystals were solubilized by adding 150 L of DMSO and the quantity of formazan produced was quantified spectrophotometrically at 570 nm in a microplate reader (EPOCH, Bio-Tek, USA). Results were expressed in terms of percentage of cellular viability relative to the untreated control (cells with PBS). Experiments were performed in triplicate in three independent assays.
3. Results
Fig. 1. FT-IR spectra in the region of 4000–500 cm−1 of -CDs, neutral bCDs, anionic bCDS and cationic bCDs.
3.1. Synthesis and characterization of ionic bˇCD Branched ionic CDs were synthesized following the procedure described in the experimental part by the polycondensation reaction with EP in the presence of CC or CAA. As has been commented above, the polycondensation with EP through the epoxy ring opening by the hydroxyl groups of the CD is the most used and simple synthetic way for the substitution of CDs. It is well known that this reaction route leads to branched and heterogeneous structures of low molecular weight. This was the case of prepared samples that had a weight average molecular weight of 1800, 1700, and 1600 g/mol for neutral, cationic and anionic bCDs determined by size exclusion chromatography, demonstrating that monomeric and branched CD derivatives were synthesized and no polymerization took place. This reaction by EP polycondensation leads to a mixture of heterogeneous structures with different length of 2-methoxypropyl ether repeating fragments as tails and different substitution degree, as is schematized in the general formula showed in Fig. 2A (Renard, Deratani, et al., 1997). However, it is known that in highly basic conditions, when NaOH/CD > 10, as described in this paper, substitutions take places preferably in the more accessible hydroxyls (primary, O-6), and only in case NaOH/CD < 10 the three positions (0–2, 0–3 and 0–6) would be substituted (Renard, Deratani, et al., 1997). When branched ionic CDs are synthesized by addition of CC or CAA substitution reactions take place together with the EP polycondensations, limiting EP tails growth and generating charged groups as pending group in cyclodextrin derivatives (Yang et al., 2013). This is in accordance with the slightly higher weight average molecular weight determined for neutral bCDs than ionic bCDs. The obtained bCDs were characterized by FTIR spectroscopy. The FTIR spectra of bCDs are different from that of CD (Fig. 1). FTIR spectra of branched -CDs showed the typical attenuation of the three characteristic bands of the monomer in the region of 1000–1200 cm−1 corresponding to the coupled C C and stretching vibration of C O bonds (1035 and 1081 cm−1 ) and the antisymmetric stretching vibration of C O C glycosidic bond (1154 cm−1 ), that indicates the advance of the modification reaction (Li et al., 2004; Qian et al., 2008). Besides FTIR spectra of branched CDs show that the stretching vibration of OH at 3422 cm−1 is reduced and the asymmetric stretching vibration of CH2 at 2876 cm−1 increases in comparison with the spectrum of monomeric CDs. These changes have been previously reported as obvious evidences for reaction
between CD and epichlorohydrin (Li et al., 2010; Qian et al., 2008). Also, the successful introduction of choline chloride on branched CD was evidenced by the presence of a new peak at 1480 cm−1 corresponding to the methyl groups of the quaternary ammonium (Junthip et al., 2015). At 1700 cm−1 may be visualized a combination of the elongation of COOH and COOR groups of anionic bCD (Martin, Tabary, Leclercq, et al., 2013). NMR spectroscopy allowed verifying the incorporation of cationic and anionic moieties into the -CDs. Fig. 2A shows the 1 H NMR spectra of neutral and branched ionic CDs, where the resonance signal of the pyranose ring protons (H-2, 3, 4, 5 and 6) at 3.4–4.1 ppm and that of the anomeric proton H-1, which appears at 5.0 ppm, could be observed. The quantification of the cationic substitution could be done by the integration of the peak that appears at 3.12 ppm, which corresponds to the methylic protons of quaternary ammonium moiety (Li et al., 2004; Qian et al., 2008; Yang et al., 2013) relative to the signal of H-1 appearing at 5.0 ppm. The number of cationic groups per anhydroglucose unit calculated was 0.64 ± 0.05. Based on the comparison between the relation among the areas of the peaks at 5.1 and 3.4–4.1 of the 1 H NMR spectra of -CD, and the relation between those integral values for the samples, EP/-CD relation of each sample was estimated, resulting in 0.41 ± 0.14, 0.31 ± 0.04 and 0.08 ± 0.05, for neutral bCDs, anionic bCD and cationic bCD. These data suggest that structures with less branched tails are obtained for cationic samples. This might be related with the electrostatic attraction between cationic moieties and negative charges of growing tails during polycondensation reactions. In the case of the anionic bCDs 1 H–13 C bidimensional NMR spectrum (Fig. 2B) allowed to corroborate the incorporation of COOH groups into bCDs. A weak resonance signal correlating with the 1 H signal at 4.1 ppm is observed around 170 ppm in the 13 C NMR spectrum. This signal could be assigned to the carbon atoms of carbonyl groups adjacent to CH2 protons, which could correspond to the carboxyl groups introduced with the anionic modification. The quantification of acidic groups introduced in the anionic bCD was accomplished by conductimetric and potentiometric titration and 0.62 ± 0.01 carboxyl groups per cyclodextrin ring were calculated. This value is close to that of cationic bCDs, indicating that electrostatic attraction or repulsion forces do not have a significant effect in the overall content of introduced ionic moiety in
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
153
Fig. 2. (A) 1 H NMR spectra of -CDs, cationic bCD, neutral bCD and anionic bCD in D2 O and (B) 1 H NMR and 13 C NMR bidimensional spectra of anionic bCD in D2 O.
the bCD. This could be understood by the fact that ionic groups are incorporated as tails to the side chains of EP. 3.2. Water solubility properties of ionic bˇCD As has been commented above, the low water solubility of -CD, which limits their applicability, is attributed to intermolecular hydrogen bonds that exist between the secondary hydroxyl groups, which are unfavorable for interaction between CDs and surrounding water molecules. Thus, the introduction of an ionic group originates electrostatic repulsions disrupting these intermolecular hydrogen bonds and increasing aqueous solubility. Water solubility properties of cationic derivatives of polymeric -CDs had been studied before (Li et al., 2004; Qian et al., 2008; Yang et al., 2013), however, to the best of our knowledge, the solubility of anionic bCD obtained by polycondensation with EP has not been extensively investigated.
Therefore, the water solubility of obtained bCDs was studied monitoring the transmittance of aqueous solutions at 550 nm. As can be observed in Fig. 3, ionic derivatives show a clear increase in solubility over the neutral samples due to the removal of hydrogen bonding network by the modification of primary hydroxyl groups which is enhanced in the case of ionic derivatives by the electrostatic repulsions. Likewise, it was observed that the solubility of anionic bCDs varies as a function of the pH of the solution, while the solubility of cationic bCDs remains constant. This is explained by the fact that anionic groups resulted of the ionization of the carboxyl groups that is pH dependent; at neutral and basic pHs the ionization degree is higher and consequently the solubility increases. However, in cationic derivatives since ammonium groups are not ionizable, pH dependence was not observed. The diffraction patterns of the ionic bCDs and the zeta potential of their solutions were studied in order to verify this behavior.
154
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
This decrease is in accordance with water solubility results and could be attributed to the opposing effect of high concentrations of counterions. Zeta potential analysis showed that at neutral pHs there was not a significant difference between the net charge of prepared cationic and anionic bCDs that is reflected in a similar solubility. Li et al. (2004) when studied the aqueous solubility of cationic bCDs concluded that it was also insensitive to the modification degree and the charge density due to the fact that ionic group does not combine directly to the -CD but to the EP chain. Certainly, when the X-ray diffraction pattern of the obtained bCDs (Fig. 4B) was compared with that of the pure -CD, it can be observed that the chemical modification resulted in the loss of crystallinity in the samples which were essentially amorphous, included neutral derivatives which showed lower solubility. This result reinforced the role of ionic moieties in order to obtain soluble bCDs samples on the basis of the repulsive interactions of charged groups. 3.3. Cytotoxicity on Caco-2 cells Fig. 3. Solubility of prepared (䊐) neutral, () anionic and (♦) cationic bCDs and transmittance of their solutions (5 mg/mL) at pH = 3, 7 and 10.
The study of the zeta potential of solutions of the synthesized bCDs (Fig. 4A) corroborated the ionic character of the branched CDs, obtaining positive zeta potential values for cationic bCDs and negative values for anionic derivatives. Besides, it could be observed a small negative charge for neutral bCDs, as it was reported in other investigations (Li et al., 2004; Müller & Brauns, 1985). This suggests an inherent negative charge present in all the bCDs derivatives and that despite the overall positive charge of cationic bCDs, negative charges could also exist in cationic bCDs. On the other hand, cationic charge is independent of the pH of the medium, but anionic charge varies according to the ionization equilibrium of carboxyl groups, so when the external pH is increased from 3 to 7 the negative charge increases, that agrees with the results observed in the solubility study. However, for high pH values, pH = 10, a decrease of negative zeta potential was observed.
It is reported that the ionic groups in -CD derivatives can significantly decrease the cytotoxic effect (Kiss et al., 2010). However, cytotoxicity of modified ionic derivatives has not been sufficiently evaluated. One study about the hemolytic effect of cationic polymeric cyclodextrins (Li et al., 2004), revealed lower hemolysis for substituted cyclodextrins than parent monomer and a decreasing hemolysis as cationic charge number increases. Studies about ionic, methylated and both ionic and methylated -CDs showed less cytotoxicity for ionic samples than the corresponding electroneutral methylated ones, but no comparison with parent or neutral but no methylated -CDs was made (Kiss et al., 2010). In this paper, the cytotoxicity of ionic and neutral bCDs were evaluated on Human colon Caco-2 cells after incubation of increasing concentrations of bCDs (0–100 mg/mL) during 30 min and it was compared with that of parent -CDs. The results of toxicity experiments (Fig. 5), in the concentrations tested, 58.8 mM and 62.5 mM for cationic bCD and anionic bCD, respectively, revealed that ionic bCDs behave as neutral and parent -CD and
Fig. 4. (A) Zeta potential of (䊐) neutral, (♦) cationic and () anionic bCDs at different pH values and (B) XRD of -CD, TR, physical mixture of TR and bCDs, neutral, cationic and anionic bCDs.
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
155
3.4. TR/bˇCDs complex formation
Fig. 5. Cytotoxicity of (䊐) cationic bCD, () anionic bCD, () neutral bCD and () -CD on human colon carcinoma Caco-2 cells during 30 min of incubation. Cell viability was expressed as the percentage of untreated control. Data are expressed as mean ± SD (n = 3).
did not show an inherent cytotoxicity derived from their ionic nature and possible interactions with cellular membranes. Indeed, it has been reported that cytotoxicity of -CDs derivatives seem to be directly related to the cholesterol solubilizing properties, and therefore it varies with the presence of ionic groups (Kiss et al., 2010). In this way, it is believed that ionic charges tend to enhance the steric hindrance which could decrease the inclusion capability to cholesterol and consequently cytotoxicity. On the other hand, a higher cell viability of substituted and branched CDs was claimed based on the fact that increasing the carbohydrate size seems to reduce cytotoxicity (Srinivasachari & Reineke, 2009). However, in the present work major differences were not observed among the cytotoxic properties of parent, neutral or ionic derivatives. These results are consistent with those separately reported for commercial ionic (Kiss et al., 2010) and parent CDs (Castagne et al., 2009).
Fig. 6.
1
The widely used antibacterial agent TR was complexed with the synthesized ionic bCDs by a coprecipitation procedure, as has been described above. The 1 H NMR spectra of TR, TR/bCDs complexes and chemical shifts are shown in the figure below (Fig. 6). Slight shift of chemical shifts of the protons (Ha-f) after inclusion of TR in TR/bCD complexes are observed. The H-3 and H-5 atoms of bCDs in TR/bCD complexes showed a significant upfield shift in comparison with uncomplexed bCDs. This results might indicate that these changes are due to the correct complex formation with the branched ionic sample. Regarding DSC analysis (Fig. 7A), it can be observed that the thermogram of pure TR presents an endothermic peak with onset temperature of 62.9 ◦ C, which corresponds to the melting process. The TR fusion peak disappeared completely for anionic bCD derivatives and in case of cationic bCD samples its intensity was significantly reduced and shifted to lower temperature (56.3 ◦ C), reflecting the interaction between bCD and TR. FTIR and X-ray diffraction analysis of the plain components and the complexes were also performed in order to further investigate the existence of TR/branched sample complexation, confirming the formation of TR/bCD complexes. The obtained FTIR spectra are showed in Fig. 7B. In the case of complexes samples, characteristic peaks of TR and bCD were observed, confirming the presence of both in these samples. In addition, the characteristic peaks of TR at 1505, 1472, and 1417 cm−1 shifted to 1508, 1474, and 1419 cm−1 , respectively, for the TR/bCD samples (Fidaleo et al., 2013). X-ray diffraction analysis also confirmed above results (Fig. 4B). In the Xray diffractograms of TR/bCD physical mixtures, all the peaks of TR peaks remain visible with the amorphous pattern of bCs, while they disappeared almost completely in the TR/bCD complexes samples.
3.5. Solubility of TR/bˇCDs complexes In order to assess the ability of prepared ionic bCDs solubilizing TR, phase solubility studies were carried out according to the method described by Higuchi and Connors (Higuchi & Connors,
H NMR spectra of (A) TR/bCDs complexes, (B) TR, (C) expanded region for TR/bCDs complexes and 1 H chemical shifts of TR and TR/bCDs complex.
156
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
Fig. 7. (A) DSC thermograms of: TR, cationic bCDs, anionic bCDs and the complexes TR/cationic bCDs and TR/anionic bCDs, (B) FTIR spectra of TR, cationic bCDs, anionic bCDs and their complexes TR/cationic bCDs and TR/anionic bCDs.
1965). Fig. 8 shows a graphical representation of the results of phase-solubility studies. When the aqueous solubility of TR was studied in the presence of increasing bCDs concentrations the same trend was observed in the presence of both ionic derivatives. It can be observed an increase in the solubility of TR as concentration of bCD increases, confirming the complex formation and revealing that TR/bCDs complexation takes place and leads to an increase in the apparent water solubility of TR of up to 20-fold. The resulting phase-solubility diagram could be classified as BS type according to Higuchi and Connors, which is typical of monomeric CD derivatives. The aqueous solubility of TR increased linearly with the bCD concentration for low bCD concentration values until solubility limit is reached (Hernández-Sánchez, López-Miranda, Lucas-Abellán, & ˜ Núnez-Delicado, 2012) at 2.2 mg/mL TR with 4.0 mg/mL anionic bCDs and 1.4 mg/mL TR with 3.8 mg/mL cationic bCDs concentrations. Further addition of CDs resulted in the formation of less soluble inclusion complexes as has been previously described by Higuchi (Grant & Higuchi, 1990). This low complexation limit could be ascribed to the steric hindrance that arises from the heterogeneous structure of EP-substituted CDs with many side chains (Frömming & Szejtli, 1994, chap. 2).
The slope of solubility diagram was less than one; it was therefore assumed that the solubility increase could be attributed to the formation of the 1:1 complex (Loftsson, Jarho, Másson & Järvinen, 2005). In these conditions the apparent stability constant (Ks ) of the complex can be calculated from the slope and the intrinsic solubility (s0 ) of the drug in the aqueous media using Eq. (1) according to Higuchi and Connors (Higuchi & Connors, 1965): Ks =
slope s0 (1 − slope)
(1)
Apparent stability constant value was 1465 M−1 and 4729 M−1 for cationic bCD and anionic bCDs respectively, which indicate that the complexes are adequately stable (Higuchi & Connors, 1965). Besides, the Ks of cationic bCDs is lower than that of anionic bCDs. This fact could be ascribed to the decrease in drug accessibility through the -CD internal cavity as consequence of the steric hindrance of the introduction of quaternary ammonium, as has been postulate by other authors when complexation of hydrophobic drugs with cationic polymeric -CDs and -CDs were studied (Li et al., 2004).
4. Conclusions
Fig. 8. Phase-solubility diagrams for TR included in (䊐) cationic bCDs and (䊉) anionic bCDs.
Substitution reaction of -CDs with EP and CC or CAA led to branched ionic -CDs with similar ionic moiety content but less EP content in case of cationic derivatives, that might be ascribed to the electrostatic interaction between the positive charge of CAA and negative charge growing side chains. These ionic bCDs showed similar water solubility at neutral and basic pHs, that was 10 fold higher water solubility than that of neutral bCDs due to the incorporation of ionic moieties. Water solubility of anionic bCDs was pH-dependent accordingly with the ionization of their carboxyl groups and contrary to cationic bCDs. Both prepared ionic bCDs showed high cell viability with human colon carcinoma Caco-2 cells and formed inclusion complexes with the antibacterial TR. TR/bCDs complexation led to an increase in the apparent water solubility of TR of up to 20-fold but with a complexation limit around 4.0 mg/mL. TR solubility at complexation limit and apparent stability constant value were lower for cationic bCDs than anionic samples, reflecting the higher steric hindrance of the ammonium moiety in comparison with carboxylic groups of cationic bCDs and anionic bCDs, respectively.
F. Gómez-Galván et al. / Carbohydrate Polymers 142 (2016) 149–157
Acknowledgments Authors are grateful to the Government of Mexico (CONACYT) and to theGovernment of the Basque Country for financial support (ACTIMAT project, ETORTEK program) (Grupos de Investigación, IT350-10) and technical and human support provided by SGIKER (UPV/EHU, MICINN, GV/EJ, ERDF, and ESF). References Astray, G., Gonzalez-Barreiro, C., Mejuto, J. C., Rial-Otero, R., & Simal-Gándara, J. (2009). A review on the use of cyclodextrins in foods. Food Hydrocolloids, 23, 1631–1640. Blomberg, E., Kumpulainen, A., David, C., & Amiel, C. (2004). Polymer bilayer formation due to specific interactions between -cyclodextrin and adamantane: A surface force study. Langmuir, 20, 10449–10454. Breslow, R. (2010). Cyclodextrins. In Molecular encapsulation: Organic reactions in constrained systems. UK: John Wiley & Sons. Castagne, D., Fillet, M., Delattre, L., Evrard, B., Nusgens, B., & Piel, G. (2009). Study of the cholesterol extraction capacity of -cyclodextrin and its derivatives, relationships with their effects on endothelial cell viability and on membrane models. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 63, 225–2231. Easton, C. J. (2005). Cyclodextrin-based catalysts and molecular reactors. Pure Applied Chemistry, 77, 1865–1871. Crinia, G., & Morcellet, M. (2002). Synthesis and applications of adsorbents containing cyclodextrins. Journal of Separation Science, 25, 789–813. Davis, M. E., & Brewster, M. E. (2004). Cyclodextrin-based pharmaceutics: Past, present and future. Nature Reviews Drug Discovery, 3, 1023–1035. Fidaleo, M., Zuorro, A., & Lavecchia, R. (2013). Enhanced antibacterial and anti-quorum sensing activities of triclosan by complexation with modified -cyclodextrins. World Journal of Microbiology and Biotechnology, 29, 1731–1736. Frömming, K.-H., & Szejtli, J. (1994). Cyclodextrins in pharmacy. The Netherlands: Springer Science+ Business Media B.V. Grant, D., & Higuchi, T. (1990). Solubility behaviour of organics compounds. Techniques of chemistry. pp. 21. New York: Wiley Interscience Publication. Harada, A., Furue, M., & Nozakura, S.-I. (1976). Cyclodextrin-containing polymers. 1. Preparation of polymers. Macromolecules, 9, 701–704. Harada, A., Furue, M., & Nozakura, S.-I. (1981). Inclusion of aromatic compounds by a -cyclodextrin-epichlorohydrin polymer. Polymer Journal, 13, 777–781. Heath, R. J., Li, J., Roland, G. E., & Rock, C. O. (2000). Inhibition of the staphylococcus aureus NADPH-dependent enoyl-acyl carrier protein reductase by triclosan and hexachlorophene. Journal of Biological Chemistry, 275, 4654–4659. Higuchi, T., & Connors, K. A. (1965). Phase solubility techniques. Advanced Analytical Chemical Instrumentation, 4, 117–212. ˜ Hernández-Sánchez, P., López-Miranda, S., Lucas-Abellán, C., & Núnez-Delicado, E. (2012). Complexation of eugenol (EG), as main component of clove oil and as pure compound, with ˇ- and HP-ˇ-CDs. Food and Nutrition Sciences, 3, 716–723. Hill, L. E., Gomes, C., & Taylor, T. M. (2013). Characterization of beta-cyclodextrin inclusion complexes containing essential oils (trans-cinnamaldehyde, eugenol, cinnamon bark, and clove bud extracts) for antimicrobial delivery applications. LWT - Food Science and Technology, 51, 86–93. Huang, L., Xin, J., Guo, Y., & Li, J. (2010). A novel insulin oral delivery system assisted by cationic -cyclodextrin polymers. Journal of Applied Polymer Science, 115, 1371–1379. Jug, M., Kosalec, I., Maestrellic, F., & Mura, P. (2011). Analysis of triclosan inclusion complexes with -cyclodextrin and its water-soluble polymeric derivative. Journal of Pharmaceutical and Biomedical Analysis, 54, 1030–1039. Jug, M., Kosalec, I., Maestrellic, F., & Mura, P. (2012). Development of low methoxy amidated pectin-based mucoadhesive patches for buccal delivery of triclosan: Effect of cyclodextrin complexation. Carbohydrate Polymers, 90, 1794–1803. Jug, M., Maestrelli, F., & Mura, P. (2012). Native and polymeric -cyclodextrins in performance improvement of chitosan films aimed for buccal delivery of poorly soluble drugs. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 74, 87–97. Junthip, J., Tabary, N., Leclercq, L., & Martel, B. (2015). Cationic -cyclodextrin polymer applied to a dual cyclodextrinpolyelectrolyte multilayer system. Carbohydrate Polymers, 126, 156–167. Kayaci, F., Umu, O. C. O., Tekinay, T., & Uyar, T. (2013). Antibacterial electrospun poly(lactic acid) (PLA) nanofibrous webs incorporating triclosan/cyclodextrin inclusion complexes. Journal of Agricultural and Food Chemistry, 61, 3901–3908. Kayaci, F., & Uyar, T. (2012). Encapsulation of vanillin/cyclodextrin inclusion complex in electrospun polyvinyl alcohol (PVA) nanowebs: Prolonged shelf life and high temperature stability of vanillin. Food Chemistry, 133, 641–649.
157
Kiss, T., Fenyvesi, F., Bácskay, I., Váradi, J., Fenyvesi, E., Iványi, R., et al. (2010). Evaluation of the cytotoxicity of beta-cyclodextrin derivatives: Evidence for the role of cholesterol extraction. European Journal of Pharmaceutical Science, 40, 376–380. Li, J., Guo, Z., Xin, J., Zhao, G., & Xiao, H. (2010). Arm star polymers with different cationic groups based on cyclodextrin core for DNA delivery. Carbohydrate Polymers, 79, 277–283. Li, J., & Loh, X. (2008). Cyclodextrin-based supramolecular architectures: Syntheses, structures, and applications for drug and gene delivery. Advanced Drug Delivery Reviews, 60, 1000–1017. Li, J., Xiao, H., Li, J., & Zhong, Y. (2004). Drug carrier systems based on water-soluble cationic -cyclodextrin polymers. International Journal of Pharmaceutics, 278, 329–342. Loftsson, T., & Brewster, M. E. (1996). Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. Journal of Pharmaceutical Sciences, 85, 1017–1025. Loftsson, T., Jarho, P., Másson, M., & Järvinen, T. (2005). Cyclodextrins in drug delivery. Expert Opinion on Drug Delivery, 2, 335–351. Martel, B., Ruffin, D., Weltrowski, M., Lekchiri, Y., & Morcellet, M. (2005). Water-soluble polymers and gels from the polycondensation between cyclodextrins and poly(carboxylic acid)s: A study of the preparation parameters. Journal of Applied Polymer Science, 97, 433–442. Martin, A., Tabary, N., Chai, F., Leclercq, L., Junthip, J., Aubert-Viard, F., et al. (2013). Build-up of an antimicrobial multilayer coating on a textile support based on a methylene blue–poly(cyclodextrin) complex. Biomedical Materials, 8, 1–14, 065006. Martin, A., Tabary, N., Leclercq, L., Junthipa, J., Degoutin, S., Aubert-Viard, F., et al. (2013). Multilayered textile coating based on a -cyclodextrin polyelectrolyte for the controlled release of drugs. Carbohydrate Polymers, 93, 718–730. Müller, B. W., & Brauns, U. (1985). Solubilization of drugs by modified -cyclodextrins. International Journal of Pharmaceutics, 26, 77–88. Renard, E., Barnathan, G., Deratani, A., & Sebille, B. (1997). Polycondensation of cyclodextrins with epichlorohydrin. Influence of reaction conditions on the polymer structure. Macromolecular Symposia, 122, 229–234. Renard, E., Deratani, A., Volet, G., & Sebille, B. (1997). Preparation and characterization of water soluble high molecular weight -cyclodextrin-epichlorohydrin polymers. European Polymer Journal, 33, 49–57. Qian, L., Guan, Y., & Xiao, H. (2008). Preparation and characterization of inclusion complexes of a cationic-cyclodextrin polymer with butylparaben or triclosan. International Journal of Pharmaceutics, 357, 244–251. Sambuy, Y., Angelis, I., Ranaldi, G., Scarino, M. L., Stammati, A., & Zucco, F. (2005). The Caco-2 cell line as a model of the intestinal barrier: Influence of cell and culture-related factors on Caco-2 cell functional characteristics. Cell Biology and Toxicology, 21, 1–2. Singh, R., Bharti, N., Madan, J., & Hiremath, S. N. (2010). Characterization of cyclodextrin inclusion complexes – A review. Journal of Pharmaceutical Science and Technology, 2, 171–183. Skaare, A. B., Kjaerheim, V., Barkvoll, P., & Rolla, G. (1997). Does the nature of the solvent affect the anti-inflammatory capacity of triclosan? An experimental study. Journal of Clinical Periodontology, 24, 124–128. Szejtli. (1988). Cyclodextrins technology. Budapest: Springer-Science+Business Media B.V. Szeman, J., Fenyvesi, E., & Szejtli, J. (1987). Water soluble cyclodextrin polymers: Their interaction with drugs. Journal of Inclusion Phenomena and Macrocyclic Chemistry, 5, 427–431. Szente, L., & Szejtli, J. (1999). Highly soluble cyclodextrin derivatives: Chemistry, properties, and trends in development. Advanced Drug Delivery Reviews, 36, 17–28. Szente, L., & Szeman, J. (2013). Cyclodextrins in analytical chemistry: Host–guest type molecular recognition. Analytical Chemistry, 85, 8024–8030. Srinivasachari, S., & Reineke, T. M. (2009). Versatile supramolecular pDNA vehicles via “click polymerization” of -cyclodextrin with oligoethyleneamines. Biomaterials, 30, 928–938. Uekama, K., Hirayama, F., & Irie, T. (1998). Cyclodextrin drug carrier systems. Chemical Reviews, 98, 2045–2076. Veldhoen, N., Skirrow, R. C., Osachoff, H., Wigmore, H., Clapson, D. J., Gunderson, M. P., et al. (2006). The bactericidal agent triclosan modulates thyroid hormone associated gene expression and disrupts postembryonic anuran development. Aquatic Toxicology, 80(3), 217–227. Xin, J., Guo, Z., Chen, X., Jiang, W., Li, J., & Li, M. (2010). Study of branched cationic -cyclodextrin polymer/indomethacin complex and its release profile from alginate hydrogel. International Journal of Pharmaceutics, 386, 221–228. Yang, S. Y., Hoonor, R., Jin, H.-S., & Kim, J. (2013). Synthesis and characterization of cationic and anionic cyclodextrin oligomers and their use in layer-by-layer film formation. Bulletin of Korean Chemical Society, 34, 2016–2022. Zhang, N., Li, J., Jiang, W., Ren, C., Li, J., Xin, X., et al. (2010). Effective protection and controlled release of insulin by cationic -cyclodextrin polymers from alginate/chitosan nanoparticles. International Journal of Pharmaceutics, 393, 212–218.