Bio-organoclays Based on Phospholipids as Immobilization Hosts for

Jan 25, 2010 - A new type of hybrid biomaterials based on the clay minerals montmorillonite and sepiolite as well as phosphatidylcholine, acting as en...
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Bio-organoclays Based on Phospholipids as Immobilization Hosts for Biological Species Bernd Wicklein, Margarita Darder, Pilar Aranda, and Eduardo Ruiz-Hitzky* Instituto de Ciencia de Materiales de Madrid, CSIC, Cantoblanco, 28049 Madrid, Spain Received September 30, 2009. Revised Manuscript Received December 3, 2009 A new type of hybrid biomaterials based on the clay minerals montmorillonite and sepiolite as well as phosphatidylcholine, acting as environment-friendly biomodifier, was prepared. The biohybrids were characterized by sampling of adsorption isotherms in different organic solvents. The results suggest bilayer formation both on the external sepiolite surface as well as in the intracrystalline space of the montmorillonite. The obtained supported lipid membranes were further investigated by X-ray diffraction, multinuclear solid state NMR, Fourier transformed IR spectroscopy and thermal analysis. From these results an adsorption model based on electrostatic interaction between the polar phospholipid headgroups and the silicate surface could be postulated. The versatility of bio-organoclays as immobilization host for biological species was demonstrated in a mycotoxin retention study.

Introduction The most commonly applied and well-studied organic modifiers for clay minerals are long-chain quaternary alkylammonium cations.1 Ion exchange with interlayer cations in smectites may result in various arrangements of densely packed alkylammonium chains such as monolayers, bilayers, or paraffin-type structures whereas the surrounding silicate framework offers robust accommodation.2 These highly stable organophilic clays find a broad range of industrial applications, including, for instance, polymer-clay nanocomposites.3 However, a severe drawback in the expansion of possible applications in biological fields is the toxicity and lack of biocompatibility of the quaternary alkylammonium salts, which are cationic surfactant agents. Attempts to enhance the biocompatibility of clay minerals have recently been undertaken, and different types of clays have been functionalized by assembly with different biopolymers.4-6 In this work, we introduce a new type of environmentally friendly bio-organoclay using phospholipids as a long-chain surfactant of biological origin. The lipid phosphatidylcholine (PC) is the main constituent of cell membranes and provides its structural framework.7 Two fatty acid chains are connected via a glycerol backbone to a zwitterionic headgroup constituting the amphiphilic nature of phosphatidylcholine (Figure 1). Therefore, PC has the ability to form self-assembled structures such as micelles, liposomes, organogels, or supported artificial *Corresponding author. Tel: þ34 91 334 9000. Fax: þ34 91 372 0623. E-mail: [email protected]. (1) Lagaly, G. Solid State Ionics 1986, 22, 43. (2) Ruiz-Hitzky, E.; Aranda, P.; Serratosa, J. M. In Handbook of Layered Materials; Auerbach, S. M., Carrado, K. A., Dutta, P., Eds.; Marcel Dekker: New York, 2004; Chapter 3, pp 91-154. (3) Ruiz-Hitzky, E.; Van Meerbeeck, A. Clay Mineral and OrganoclayPolymer Nanocomposites. In Handbook of Clay Science; Bergaya, F., Theng, B. K. G., Lagaly, G., Eds.; Elsevier Science: Amsterdam, 2006; Chapter 10.3, pp 583-621. (4) Darder, M.; Aranda, P.; Ruiz-Hitzky, E. Adv. Mater. 2007, 19, 1309. (5) Ruiz-Hitzky, E.; Darder, M.; Aranda, P. J. Mater. Chem. 2005, 15, 3650. (6) Ruiz-Hitzky, E., Ariga, K., Lvov Y., Eds. Bio-Inorganic Hybrid Nanomaterials: Strategies, Syntheses, Characterization and Applications; Wiley-VCH: Weinheim, Germany, 2007. (7) van Meer, G.; Voelker, D. R.; Feigenson, G. W. Nat. Rev. Mol. Cell Biol. 2008, 9, 112. (8) Shchipunov, Yu. A. Colloids Surf., A 2001, 183-185, 541. (9) Menger, F. M.; Chlebowski, M. E.; Galloway, A. L.; Lu, H.; Seredyuk, V. A.; Sorrells, J. L.; Zhang, H. Langmuir 2005, 21, 10336.

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membranes.8,9 PC liposomes have attained interest as biomimetic supports for pharmaceutical drug carriers10 or immobilizing biological species such as membrane proteins via electrostatic interactions.11 However, its zwitterionic structure makes PC prone to electrostatic interaction with clay minerals,12-14 which provide a robust support and an easy-to-handle carrier system. Clay minerals are fine-grained silicates with either lamellar or fibrous morphology. Their unique characteristics such as colloidal behavior, ion-exchange capacity, and adsorption properties, together with a general nonhazardous nature and even biocompatibility, make them suitable as host structures for biological species. The clays used in this work are the phyllosilicate montmorillonite, belonging to the smectite family, and sepiolite, a microfibrous mineral (Figure 2). In the case of montmorillonite, a 2:1 layer structure is formed by the repetition of octahedral alumina sheets sandwiched by two tetrahedral silica sheets. Magnesium and other cations can be present in the octahedral layers, replacing aluminum cations originating in negative charged layers that are compensated for by exchangeable cations in the intracrystalline space between two consecutive layers.2 The typical cation exchange capacity (CEC) of montmorillonite is in the 70-100 meq/100 g range. A common feature of smectites is their swelling capability by the stepwise hydration of the interlayer cations. Furthermore, these cations can be easily exchanged with other cations, including organic cations and positively charged biomolecules.15 The resulting materials are referred to as intercalation compounds, being stable and functionalized organoclays.2 Sepiolite is a hydrated magnesium silicate of 2-10 μm particle size with Si12O30Mg8(OH,F)4(H2O)4 3 8H2O as the unit cell formula.16,17 It is composed of ribbons of a 2:1 phyllosilicate (10) N€assander, U. K.; Storm, G. P.; Peeters, A. M.; Crommelin, D. J. A. In Biodegradable Polymers as Drug Delivery Systems; Chasin, M., Langer, R., Eds.; Marcel Dekker: New York, 1990; pp 261-338. (11) Sastry, M. Trends Biotechnol. 2002, 20, 185. (12) Sahai, N. J. Colloid Interface Sci. 2002, 252, 309. (13) Wiegart, L.; Struth, B.; Tolan, M.; Terech, P. Langmuir 2005, 21, 7349. (14) Rapuano, R.; Carmona-Ribeiro, A. M. J. Colloid Interface Sci. 2000, 226, 299. (15) Szabo, T.; Mitea, R.; Leeman, H.; Premachandra, G. S.; Johnston, C. T.; Szekeres, M.; Dekany, I.; Schoonheydt, R. A. Clays Clay Miner. 2008, 56, 494. (16) Brauner, K.; Preisinger, A. Miner. Petr. Mitt. 1956, 6, 120. (17) Santaren, J.; Sanz, J.; Ruiz-Hitzky, E. Clay Miner. 1990, 38, 63.

Published on Web 01/25/2010

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Figure 1. Representation of the molecular structure of phosphatidylcholine.

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additives in animal feeds because of their nontoxicity and biocompatibility. AfB1 sequestration of the newly developed bio-organoclays was compared to commercial alkylammonium organoclay cloisite B30 and to cetyltrimethylammonium (CTA)exchanged sepiolite (SEP-12 wt % CTA). (The synthesis procedure can be found elsewhere.26) The present work reports on the preparation and characterization of new biohybrid nanomaterials based on the assembling of PC and clay minerals with assays showing their ability to act as convenient supports for biological species.

Experimental Section Starting Materials and Reagents. Montmorillonite (MMT)

Figure 2. Schematic representation of the structures of montmorillonite (A) and sepiolite (B).

structure with a discontinuous sheet of octahedral magnesium oxide hydroxide being sandwiched by continuous sheets of silica tetrahedra. These building blocks form channels and tunnels of 1.1  0.4 nm that are accessible to water and other small molecules.18 Sepiolite exhibits free Si-OH silanol groups along the rims of the channels and at the end of the tunnels, respectively. These silanol groups represent good anchor points for the functionalization of sepiolite because they are susceptible to interaction with functional groups via hydrogen bonding interactions2 or grafting methods.19 The sepiolite structure possesses a relatively small charge deficit resulting in a low cation exchanger material with a CEC close to 15 meq/100 g.14 Combining the good PC adsorption behavior on clays and its biointerface properties, PC-modified clay systems are promising materials for agricultural, clinical, and biotechnological applications. Examples are the sequestration of fungi-produced mycotoxins as shown in a preliminary study.20 Recently, the preparation of clay liposomes from aqueous media has been reported. Herbicide-containing PC vesicles adsorbed externally on colloidal montmorillonite platelets render a supported planar bilayer (SPB).21 On homoionic smectites, however, ion exchange is considered to be an important adsorption mechanism2 providing the possibility to form intercalated lipid-clay composites. To demonstrate the versatility of phospholipid-modified clays as potential hosts for immobilizing biological species, a mycotoxin retention study was carried out. Mycotoxins are toxic compounds produced as secondary metabolites by many species of fungi after infesting grain and other food crops. This represents a true threat to the health of both animals and humans. Produced by Aspergillus flavus, aflatoxin B1 is a toxin that is considered to be among the most carcinogenic substances known.22,23 Although mycotoxins threaten food supplies, these compounds are strongly retained by soil materials24,25 which are usually employed as (18) Ruiz-Hitzky, E. J. Mater. Chem. 2001, 11, 86. (19) Ruiz-Hitzky, E. Organic-Inorganic Materials: From Intercalation Chemistry to Devices. In Functional Hybrid Materials; Gomez-Romero, P., Sanchez, C., Eds.; Wiley-VCH: Weinheim, Germany, 2004; Chapter 2, pp 15-49. (20) Wicklein, B.; Darder, M.; Aranda, P.; Ruiz-Hitzky, E. Macla 2008, 9, 257. (21) Sanchez-Verdejo, T.; Undabeytia, T.; Nir, S.; Maqueda, C.; Morillo, E. Environ. Sci. Technol. 2008, 42, 5779. (22) Heathcote, J. G.; Hibbert, J. R. Aflatoxins: Chemical and Biological Aspects; Elsevier Scientific: Amsterdam, 1978. (23) Betina V. Mycotoxins: Chemical, Biological and Environmental Aspects; Elsevier Scientific: Amsterdam, 1989; pp 114-144. (24) Desheng, Q.; Fan, L.; Yanhu, Y.; Niya, Z. Poult. Sci. 2005, 84, 959. (25) Jaynes, W. F.; Zartman, R. E.; Hudnall, W. H. Appl. Clay Sci. 2006, 36, 197.

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from Crook County, WY, with a cationic exchange capacity of 76 meq/100 g (according to the specifications of its producer) was obtained from the Source Clay Minerals Repository of the Clay Minerals Society, Columbia, MO. Homoionic Naþ-montmorillonite with a 95% purity sold commercially as Pangel S9 with a CEC value close to 15 meq/100 g was furnished by TOLSA and used as supplied. Phospholipids (PL) from egg yolk with 60% (TLC) and 99% (TLC) L-R-phosphatidylcholine (PC) content were supplied by Aldrich. Among the 40% are lipids belonging to the ethanolamine and lyso family as well as cholesterol. The fatty acid composition of this egg yolk phosphatidylcholine lot consists of approximately 27% 16:0, 13% 18:0, 31% 18:1, 17% 18:2, and 5% 20:4 (other fatty acids being minor contributors), which would give an average molecular weight of approximately 773 g/mol. Accordingly, the average number of acyl-chain carbon atoms is n = 17.5. Aflatoxin B1 (AfB1) from A. flavus was supplied by Sigma. Methanol and ethanol (both absolute grade) were purchased from Scharlau. Deionized water (resistivity >18.2 MΩ cm) was obtained from a Maxima ultrapure water system from Elga. Synthesis Procedures. For adsorption isotherms, phospholipid solutions of 10 mL of methanol and ethanol with a lipid concentration ranging from 0.1 to 12.5 mM, respectively, were prepared with consideration of the PL purity. In each PL solution, 20 mg of Naþ-montmorillonite and sepiolite was immersed. Accordingly, the initial clay to lipid ratio was between 27:1 and 0.2:1. The suspensions were stirred for 24 h at ambient temperature (approximately 25 °C). The resultant biocomposites were collected by means of a centrifuge (8000 rpm, 15 min ), vacuum dried at room temperature, and ground to powder for further analysis. The lipid content of the biohybrids is denoted as mmol of PC per 100 g of clay if not stated otherwise. Because PC is considered to be the main adsorbate from PL solutions, the obtained materials are henceforth designated as clay PC. Mycotoxin Adsorption. Methanolic stock solutions of 500 μg of AfB1/mL were prepared, and aliquots were diluted in bidistilled water. Adsorption isotherms of AfB1 on modified clays were obtained from 5 mL of 1 mg/mL sorbent dispersions and the addition of 4-100 μL of AfB1 stock solution aliquots. Employed sorbent materials were MMT-24 wt % PC and SEP modified with 11 and 23 wt % PC and as reference materials Cloisite 30B (CLO 30B, Southern Clay Products, Inc.) and SEP-12 wt % CTA. All mixtures were agitated on a magnetic stirrer for 20 h at ambient temperature and then centrifuged for 10 min at 8000 rpm. The adsorbed amount of toxin was estimated from the difference in the initial toxin concentration and the concentration in the supernatant. The toxin concentration was determined by means of a UV-vis spectrometer using the adsorbance value of AfB1 at (26) Aranda, P.; Kun, R.; Martı´ n-Luengo, M. A.; Letaı¨ ef, S.; Dekany, I.; RuizHitzky, E. Chem. Mater. 2008, 20, 84.

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362 nm (ε = 0.0589 mL/μg), which was derived from calibrations curves. (Caution! Mycotoxins are extremely toxic compounds.) Characterization. Adsorption isotherms were calculated from data obtained by chemical analysis (PerkinElmer 2400 series II CHNS/O elemental analyzer) of the organic content in the solids. The thermal stability of the biocomposites was investigated by thermogravimetric analysis (TG) and differential thermal analysis (DTA) on a SSC/5200 Seiko analyzer. 29Si CP MAS NMR and 31P proton-coupled MAS NMR spectra of PC-SEP were obtained on a Bruker Avance 400 spectrometer, using a standard cross-polarization pulse sequence. In the case of the MMT-based biohybrids, the paramagnetism of Fe impeded the recording of useful NMR spectra. Powder samples were spun at 10 kHz whereas PL was measured in ethanol solution. Spectrometer frequencies were set to 79.49 and 161.97 MHz for 29Si and 31 P, respectively. A contact time of 2 ms and a period between successive accumulations of 5-10 s were used. The number of scans was 400-800. Chemical shift values were referenced to tetramethylsilane (TMS) and 85% phosphoric acid (H3PO4). The sodium content was semiquantitatively determined by energydispersive X-ray spectroscopy with a TypeSDD Apollo 10 EDAX detector mounted on a FEI NOVA NANOSEM 230 field-emission scanning electron microscope. The data acquisition conditions were kept constant at a working distance 6.3 mm, a spot size of 2.5, a landing energy of 6.0 keV, and an accumulation time of 100 s. Structural information was also provided from X-ray powder diffraction (Bruker D8, with Cu anode and Ni filter), and Fourier transform infrared spectroscopy (FTIR) was recorded on a NICOLET 20SXC spectrometer. Contact angle measurements of the sepiolite hybrids were performed on a Kr€ uss drop shape analysis system. For angles of 30°, the contact angle was measured according to the Laplace-Young method.

Results and Discussion Adsorption Processes. As with conventional alkylammonium organoclays, the new type of bio-organoclay was prepared via a cation exchange mechanism where adsorption processes are dominating. The adsorption isotherms at 25 °C of the studied PC-clay systems are presented in Figure 3 together with the fitting curves if procurable. Phospholipid was adsorbed from organic solution on Naþmontmorillonite and sepiolite, respectively. The initial slope of the PC-MMT isotherm, obtained in MeOH, (Figure 3A), can be well fitted (R = 0.9969) to the L-type (Langmuir) adsorption isotherms27 Γ ¼

bxm Cs 1 þ bCs

ð1Þ

where Γ is the adsorbed amount of lipid, b is the affinity constant between lipid and clay interaction sites, xm is the maximum adsorbed amount, and Cs is the equilibrium lipid concentration. The fitting reveals an affinity constant of 0.58 mmol-1 L. For ionic species, b can be related to the Gibbs free adsorption energy ΔGads through the relation K = (bF/4)2 where F is the ratio of the solvent (methanol) density to its molecular weight, F ≈ 247 mol L-1 28 ΔGads ¼ -RT ln K

ð2Þ

where R is the gas constant and T is the adsorption temperature. The Gibbs free energy of adsorption was calculated to be (27) Giles, C. H.; MacEwan, T. H.; Nakhwa, S. N.; Smith, D. J. Chem. Soc. 1960, 111, 3973. (28) Miller, R.; Fainerman, V. B.; M€ohwald, H. J. Colloid Interface Sci. 2002, 247, 193.

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Figure 3. Adsorption isotherms from methanol and ethanol of a phospholipid on montmorillonite (A) and sepiolite (B), respectively. Solid lines represent fitting curves. Adsorption took place at 25 °C for 24 h under the agitation of a magnetic stirrer.

-52.1 kJ mol-1. This value is comparable to results obtained by microcalometry for the intercalation of macrocyclic compounds in MMT, which is known to form very stable composites.29 Hence, the calculated value in this work is indicative of the high affinity of phospholipids for the mineral surface and strong adsorption. The saturation amount of the intercalated phospholipid is 82 mmol/100 g, which is in accordance with the CEC (76 meq/100 g) of Wyoming montmorillonite. This finding supports sodium cation exchange as the basic adsorption mechanism that was also indicated by semiquantitative EDX analysis of the sodium content of the clay upon lipid adsorption (Figure 4). Zwitterionic lipid molecules exchanged with sodium cations can be tentatively related to uptake by the anionic phosphate group of protons from highly polarized water molecules in the intracrystalline space.30 It has been discussed by Petelska et al.31 that the cationic form of PC dominates when hydrogen ions are present in excess.31 Thus, it can be proposed that cationic PC molecules in the specific environment of Na-MMT may well participate in a cation exchange mechanism. Similar zwitterion sorption mechanisms accompanied by proton uptake have been recently reported for the intercalation of other organic species.32 The spatial occupation per PC molecule can be estimated from the assumption that 41 mmol of PC/100 g of MMT is adsorbed on each layer side. Thus, a packing density of 1.4 nm2/molecule is calculated, which is about twice the molecular cross-sectional area (29) Aranda, P.; Casal, B.; Fripiat, J. J.; Ruiz-Hitzky, E. Langmuir 1994, 10, 1207. (30) Fripiat, J.; Chaussidon, J.; Jelli, A. Chimie-Physique de Phenomenes de Surface; Masson et Cie: Paris, 1971. (31) Petelska, A. D; Figaszewski, Z. A. Biophys. J. 2000, 78, 812. (32) Figueroa, R. A.; Leonard, A.; Mackay, A. A. Environ. Sci. Technol. 2004, 38, 476.

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Figure 4. From EDX measurements, obtained sodium to silicon ratios in atom % as a function of PC content in mmol/100 g of the composite. The measurement conditions were held constant in order to compare the results within a series of samples (working distance, 6.3 mm; spot size, 2.5; landing energy, 6.0 keV; accumulation time, 100 s).

in PC monolayers at the air-water interface, which is estimated to be 0.7 nm2.14 The second slope, observed in Figure 3A, can be attributed to the adsorption of phospholipids on the external clay surface. This can be reasoned from the fact that the basal spacing remains constant (Figure 5B), thus no more lipid molecules are introduced into the intracrystalline space. Figure 5A shows the d001 spacing of PC-MMT obtained from methanol solution. The layer expansion of the first slope is about 1.45 nm, which can be attributed to a monolayer arrangement of lipid molecules. The observed maximum expansion is 5.1 nm. Taking into account the silicate layer thickness of 0.96 nm,29 an increase in the interlayer distance of 4.2 nm can be estimated. This value is slightly lower than the 4.6 nm thickness of the lipid bilayer calculated with 1.64 þ 0.17n,33 where n is the average number of carbon atoms in the acyl chain of the used 60% TLC egg yolk PC (n = 17.5). Longchain alkylammonium ions with higher intercalation loading typically tent to arrange into tilted bimolecular films.1 On the basis of this assumption, a tilt angle of about 66° with respect to the interlayer surface can be calculated. A smaller quantity of PL from ethanol solution adsorbs onto montmorillonite, forming a plateau at about 40 mmol/100 g (Figure 3B). Again, two slopes can be distinguished, indicating strong intracrystalline adsorption until the first plateau is reached and external adsorption as the basal spacing remains constant for the second slope (Figure 5D). Fitting to a Langmuir isotherm (R = 0.9696) gives a Gibbs adsorption energy of -57.4 kJ/mol. XRD patterns are presented in Figure 5C where the maximum d001 value is 4.5 nm, giving an interlayer expansion of 3.6 nm. This value indicates a tilt angle of ca. 50° for the lipid bilayer. Phospholipids in the monolayer arrangement show a comparable basal expansion to that in the methanol system. In both MMT-PL systems, the adsorption isotherms coincide with the interlayer expansion curves. A possible explanation for the significantly lower total adsorption quantity in ethanol might be related to the competition between the solvent and the adsorbing lipid layer. The formation of the first layer is mainly governed by the electrostatic interaction between the silicate surface and the polar PC headgroup. Further adsorption involves intermolecular attraction between the lipid molecules, mainly van der Waals forces, because these are generally weaker as H-bonding and the polarity of the surrounding solvent gain in importance. Ethanol (ε = 25.3) is more lipophilic (33) Karlovska, J.; Uhrikova, D.; Kucerka, N.; Teixeira, J.; Devinsky, F.; Lacko, I.; Balgavy, P. Biophys. Chem. 2006, 119, 69.

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than methanol (ε = 33),34 hence pulling the adsorption equilibrium between the PL layer and dissolved lipid molecules to the solvent side. On the contrary, lipid-lipid attraction in slightly less lipophilic methanol seems to exceed the solvation force of methanol, resulting in a higher adsorption amount. The influence of intracrystalline water on the adsorption behavior of PL on Naþ-MMT was also investigated. Prior to adsorption, Naþ-MMT powder samples were exposed for 10 days to different relative humidity of 0, 33, 55, and 98%. The results suggest a trend in which adsorbed PL quantities increase with increasing relative humidity (Figure 6). This finding indicates an important influence of intracrystalline water on the adsorption mechanism. It is known that water molecules on mineral surfaces may act as water bridges for PL molecules, forming an interlayer between the lipid layer and the silicate surface.13,14 The water molecules enhance the electrostatic interaction of the headgroup moieties with the mineral surface. In this way, the affinity of the phospholipids toward Naþ-montmorillonite is increased. PL adsorption quantities on sepiolite were generally lower compared to that of MMT samples. In contrast to the Naþ-montmorillonite system, adsorption amounts from ethanol were higher compared to those from methanol (Figure 3B). However, the characteristic isotherm shape, indicative of bilayer formation, was also observed. The adsorption curve from methanol was found to fit well to a Freundlich isotherm (R = 0.9994), with the empirical constants 1/n = 0.48 and K = 9.6 mmol/100 g. Freundlich isotherms generally imply surface energy heterogeneity of the solid adsorbent,35 lateral molecule interaction, and thus a change in motion in the state of being adsorbed.36,37 Both assumptions match the PC-SEP system. Apart from the ion-exchange contribution, coordinated water molecules and silanol groups of sepiolite can also act as adsorption sites.18 Therefore, sepiolite possesses distinct adsorption sites and surface energies. Additionally, the lipid molecules interact strongly via van der Waals attraction of their fatty acid chains, enabling membrane assembly. These considerations are essentially neglected in Langmuir-type isotherms. A tentative hypothesis for the observed adsorption behavior of PC on sepiolite to explain the solvent influence might be based on competitive adsorption. Methanol as the more polar molecule of the two solvents interacts more strongly with coordinated water at the external surface of the silicate (i.e., water molecules bonded to Mg at the external channels of the silicate). Because these molecules are strongly polarized, the zwitterions easily associate with these sites. Accordingly, the FTIR spectra (Figure 7) show a strongly perturbed δHOH(coord) band at 1625 cm-1 with increasing PC content. However, as shown by Serna et at.,38 methanol can facilely replace these water molecules, which in turn is disadvantageous to the adsorption of PC in the presence of methanol compared to ethanol. Hence, the more coordinated water molecules that are substituted, the less PC that adsorbs on sepiolite. The possibility of lipid penetration into the tunnel system of sepiolite can be ruled out because of the exceeding molecular size of PC and results from FTIR measurements. The diameter of a lipid molecule is about 0.9 nm whereas the smallest dimension of a sepiolite tunnel is 0.4 nm. Furthermore, FTIR spectroscopy of PC-clay composites shows an almost unperturbed absorption band of zeolitic water molecules at 1660 cm-1 (Figure 7). This can (34) Lide, D. R. CRC Handbook of Chemistry and Physics, 86th ed.; CRC Press: Boca Raton, FL, 2009; pp 15-13. (35) Laidler, K. J. In Catalysis; Emmett, P. H. Eds; Reinhold: New York, 1954; Vol. 1, pp 75, 99, 101, 103-107. (36) Brunauer, S. The Adsorption of Gases and Vapors; University Press: Princeton, NJ, 1945; Vol. 1, pp 54, 55, 57, 82, 83, 223, 261, 262. (37) Oscik, J. Adsorption; Ellis Horwood: Chichester, 1982. pp. 32, 102. (38) Serna, J. C. Ph.D. Thesis, University of Madrid, 1973.

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Figure 5. XRD patterns of MMT-lipid composites with increasing amounts of adsorbed phospholipid, given in mmol/100 g, as obtained from methanol (A) with (a) 2, (b) 6, (c) 12, (d) 18, (e) 59, (f) 84, (g) 105, and (h) 125 and ethanol solutions (C) with (a) 5, (b) 14, (c) 20, (d) 32, (e) 41, (f) 55, (g) 61, and (h) 82. The basal d001 spacing was plotted with the corresponding adsorption isotherms from methanol (B) and ethanol (D).

Figure 6. Adsorption in methanol of phospholipid on Naþmontmorillonite upon exposure to different relative humidity prior to adsorption.

be interpreted as a strong indication of the absence of interaction with tunnel-penetrating lipid molecules. Characterization of the Bio-organoclays. The molecular interaction between phospholipids and the clay mineral surface was further investigated by multinuclear MAS NMR and FTIR. In the case of sepiolite, a special focus was assigned to the functional silanol groups, which represent one of the principal adsorption sites on this mineral. Figure 8A shows the 29Si CP/MAS NMR spectra of the starting sepiolite. The three characteristic signals at -92.3, -94.7, and -98.3 ppm are assigned to the near-edge, center, and edge crystallographic positions of Si atoms in sepiolite. The low-intensity signal at -85.2 ppm has been attributed to the Q2(Si-OH) group, situated (39) Barron, P. F.; Frost, R. L. Am. Mineral. 1985, 70, 758.

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Figure 7. FTIR spectra showing the band of the lipid carboxyl group and the two vibrations of zeolitic and coordinated water molecules of neat sepiolite (a) and sepiolite-PC composites with increasing amounts of adsorbed phospholipids in mmol/100 g; (b) 7, (c) 16, (d) 34, and (e) 40. The spectra were recorded from cast films.

at the edges and ends of the sepiolite channels.39-41 Upon PL adsorption, this signal diminished, as can be seen in Figure 8A. This is indicative of the interaction of the silanol group with other polar species. These findings were also supported by FTIR. Stretching vibrations of structural hydroxyl groups are depicted in Figure 8B. (40) Sanz, J. Distribution of Ions in Phyllosilicates by NMR Spectroscopy. In Absorption Spectroscopy in Mineralogy; Moltana, A., Burragato, F., Eds.; Elsevier Science Publishers: Amsterdam, 1990; pp 103-144. (41) Weir, M. R.; Kuang, W.; Facey, G. A.; Detellier, C. Clays Clay Miner. 2002, 50, 240.

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Figure 8.

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29

Si CP/MAS NMR (A) and FTIR (B) of neat sepiolite and sepiolite-lipid hybrids. The wavenumber range of the OH stretching vibrations of Si-OH and Mg-OH is depicted in B with (a) neat sepiolite, (b) 7 mmol/100 g lipid, and (c) 16 mmol/100 g lipid in the composite. All samples were prepared from ethanol, and the FTIR spectra of pure cast composite films were recorded.

The 3718 cm-1 band is assigned to the OH stretching vibration νSi-OH of the silanol group in natural sepiolite.42-44 This band is shifted toward lower frequencies with increasing PC content and eventually disappeared because of the overlap of adjacent bands.18,26 A possible explanation for this observation is the strong interaction of the freely accessible silanol groups with the polar headgroup of adsorbing phospholipids. The band at 3688 cm-1 corresponds to the stretching vibration of magnesium hydroxyl groups,18 which remained unaltered during the course of PL adsorption because they are located inside the sepiolite structure and hence are inaccessible to lipid molecules. The molecular assembly and morphology of the phospholipids on the sepiolite surface were investigated by contact angle measurements, 31P MAS NMR, and FTIR. Contact angle measurements were conducted to follow the change in surface hydrophilicity upon PL adsorption (Figure 9). Until the first plateau of the adsorption isotherm, the contact angle increased slightly, whereas during the subsequent slope the contact angle decreased. The rise in the contact angle can be related to the increased hydrophobicity of the sepiolite fibers. This is the case for lipid monolayer coverage with the hydrocarbon chains oriented bottom-up. With beginning bilayer formation, more and more polar headgroups are exposed at the air interface of the upper membrane leaflet, hence the hydrophilicity increases. Further evidence of controlled lipidic mono- and bilayer formation on sepiolite was provided by solid-state 31P MAS NMR. This technique became a powerful, well-established tool for studying lipid-lipid and lipid-substrate interactions; furthermore, the line shape is characteristic of the different lipid phases.45,46 The protonnondecoupled 31P MAS NMR spectra of two sepiolite-lipid composites (prepared from ethanol with 9 and 28 mmol of PC/ 100 g) and the reference spectra of PL dissolved in ethanol are displayed in Figure 10A. A sharp, isotropic signal at -1.01 ppm is obtained from dissolved PC. This signal is shifted and broadened with increasing amounts of adsorbed PC in the composite because of (i) proton-phosphorus dipolar interaction and (ii) chemical shift anisotropy of the phosphorus nuclei,46 whereas these contributions are averaged out in the case of dissolved PL by diffusion of the lipid molecules. The spectrum of SEP-28PC displays a maximum at (42) (43) (44) (45) (46)

Ahlrichs, J. L.; Serna, C.; Serratosa, J. M. Clays Clay Miner. 1975, 23, 119. Nagata, H.; Shimoda, S.; Toshio, S. Clays Clay Miner. 1974, 22, 285. Frost, R. L.; Ding, Z. Thermochim. Acta 2003, 397, 119. Auger, M. Biophys. Chem. 1997, 68, 233. Seelig, J. Biochim. Biophys. Acta 1978, 515, 105.

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Figure 9. Contact angles of water on cast SEP-PC films are plotted together with the corresponding adsorption isotherm of PL on sepiolite obtained in ethanol.

Figure 10.

31

P MAS NMR (A) of neat sepiolite and sepiolitelipid composites containing 9 and 28 mmol of PC/100 g of clay, respectively. FTIR spectra (B) showing the νCdO band of phosphatidylcholine in (a) neat sepiolite and (b) 7, (c) 17, (d) 25, (e) 34, and (f) 40 mmol/100 g of lipid and (g) PC of 60% TLC purity. The spectra were recorded of pure cast composite films. The reference 31P NMR spectrum of phospholipid (60% TLC) was obtained from a 10 mM ethanol solution.

-0.86 ppm and a shoulder at -4.18 ppm. The shape of this spectrum is indicative of lamellar lipid phases.46 This presumption can also be supported by the adsorption isotherm of PC-SEP/EtOH (Figure 3B), suggesting beginning bilayer formation for the SEP28PC sample. However, the absence of the characteristic lamellar line shape of the SEP-9PC spectrum indicates incomplete bilayer morphology. The additional signal at -2.89 ppm may be related to molecules that are in electrostatic interaction with the silicate surface, as also suggested from silica-associated lipid films.47,48 The nature of these interactions is supposedly hydrogen bonding between the phosphate group and the silanol groups, as reasoned from the diminishment of the Q2(Si-OH) signal in Figure 8A. This interaction was also reported by Jiazuan et al.49 as deduced from 1 H and 2D NOESY NMR data for dipalmitylphosphatidylcholine adsorbed on silica. The fact that the SEP-28PC spectrum does not show this signal is assumed to be associated with the strong overlap with the lamellar line shape of the lipid bilayer. (47) Murray, D. K.; Harrison, J. C.; Wallace, W. E. J. Colloid Interface Sci. 2005, 288, 166. (48) Snyder, J. A.; Madura, J. D. J. Phys. Chem. B 2008, 112, 7095. (49) Chunbo, Y.; Daqing, Z.; Aizhuo, L.; Jiazuan, N. J. Colloid Interface Sci. 1995, 172, 536.

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Figure 11. Thermal gravimetry curves of (A) the sepiolite hybrid (16 mmol/100 g of PC adsorbed from ethanol) and (B) the montmorillonite hybrid (22 mmol/100 g of PC adsorbed from methanol) in the temperature range of 20-900 °C recorded under airflow conditions at a heating rate of 10 K/min. XRD patterns of MMT-22PC as prepared and annealed at 400 °C (C) and the FTIR spectra (obtained from KBr pellets) of the same samples (D).

Another indication of hydrogen bonding can be concluded from stretching vibrations of the carbonyl group (Figure 10B). The unperturbed νCdO band of PC appears at 1741 cm-1, whereas this band is shifted to 1725 cm-1 for the SEP-7PC sample. In subsequent spectra of samples with increased PC amounts, this band is being shifted back again toward the initial vibrational frequency of pure PC. At low PC content, all lipid molecules are in close contact with the sepiolite surface and interact with the silanol groups and coordinated water molecules, causing a significant shift of the νCdO band. As the lipid content increases, the additionally adsorbed molecules are not able to be in direct interaction with the adsorption centers because they are already occupied. The upper molecules then adsorb via intermolecular interaction, namely, van der Waals forces, and thus the carbonyl group is less perturbed. According to the synthesis of the clay-PC materials from PL, an approach to ascertain the composition of the resultant compounds can be proposed by means of thermal analysis. Figure 11A displays the thermal gravimetric (TG) and differential thermogravimetry (DTG) curves (range of 20-900 °C under airflow conditions) of SEP-16PC (equivalent to 10 wt % PC as estimated from chemical analysis) and the PC references of 99 and 60% purity, respectively. The TA curve of the sepiolite-lipid hybrid displays several weight losses. From room temperature up to 178 °C, two weight losses are detected with maxima at 62 and 147 °C, respectively. The first process can be attributed to the elimination of physically adsorbed water whereas the latter one can be associated with zeolitic water.50

This fact is supported by the presence of endothermic peaks in the corresponding DTA curve (data not shown). The weight loss at 328 °C can be related to the combustion of L-R-phosphatidylcholine (exothermic peak in DTA curve), as suggested from the comparison with the reference DTG curve of the 99% PC sample. The second peak at 380 °C of the 60% PC sample in the DTG curve is likely to be associated with the presence of other species in PL, such as cholesterol and proteins, because this phospholipid sample is of lower purity. The absence of this peak in the sepiolitephospholipid DTG curve strongly indicates preferential adsorption of L-R-phosphatidylcholine on the mineral, thus offering fairly pure sepiolite-PC composites. The main PC elimination occurs from 293 to 543 °C with a weight loss of 9.7 wt %. Considering the loss of the coordinated water that happens in the same temperature range,51 a residual lipid fraction on sepiolite can be assumed. The last weight loss at 820 °C is characteristic of the dehydroxylation of internal Mg-OH.42-44 The thermal stability of the MMT-lipid hybrids was also investigated (Figure 11B). The TG curve of composite sample MMT-22PC (equivalent to 15 wt % PC as estimated from chemical analysis) shows three weight losses with maxima at 63, 292-316, and 629 °C. The first weight loss from room temperature until 100 °C (endothermic effect) is attributed to the removal of residual interlayer water molecules. This process occurs at slightly higher temperatures than in neat montmorillonite, possibly being related to the hindered escape of the intracrystalline water as a result of the presence of intercalated lipid molecules.

(50) Jones, B. F.; Galan, E. In Reviews in Mineralogy; Bailey, S. W., Ed.; Mineralogical Society of America: Washington, DC, 1988; Vol. 19, pp 631-674.

(51) Kuang, W.; Facey, G. A.; Detellier, C.; Casal, B.; Serratosa, J. M.; RuizHitzky, E. Chem. Mater. 2003, 15, 4956.

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It is assumed that PC interacts strongly with these water molecules that in turn are bridging to residual sodium ions in the interlayer space. The elimination of PC seems to occur in three stages: first, combustion (exothermic peak in the DTA curve, not shown) centered at around 300 °C and accompanied by a weight loss of 5.0 wt %; second, a constant weight loss of 4.4 wt % until 580 °C; third, the last elimination step assumingly superimposed on the dehydroxylation of montmorillonite, which appears at 629 °C. This is about 40 °C lower than in pristine montmorillonite (TG curve in Figure 11B), which is known to occur when the interlayer cation nature is changed or organic species are intercalated into smectites.52 The superposition of these two events is reasoned from the higher weight loss of the MMT-lipid composite as compared to neat montmorillonite (5.5 vs 3.7 wt %). As a summation of all of the PC assigned weight losses, a residual lipid fraction of ca. 4 wt % is estimated. This value exceeds the theoretical ash fraction of 2.7 wt % as estimated from the TG of nonsupported phospholipids. This fact indicates an additional entrapment effect by the collapse of thermally treated lamellar montmorillonite. Similar observations were also found elsewhere for organomontmorillonite composites.29 Supporting evidence was also obtained from further investigation by XRD and FTIR spectroscopy. The MMT-22PC sample was annealed at 400 °C for 10 min prior to characterization. Its XRD pattern shows a decrease in the d001 basal spacing from 1.55 to 1.23 nm (Figure 11C), resulting in a residual 0.3 nm layer expansion. The FTIR spectrum (Figure 11D) reveals the diminished intensity of the stretching and scissoring bands of the carbonyl group νCdO as well as the acyl chain νC-H and δC-H. Both facts suggest the incomplete combustion of lipid molecules. Applications of the Bio-organoclays: Mycotoxin Retention. The prepared PC-clay systems can act as a sequestration agent for biological species such as mycotoxins and enzymes. To demonstrate these properties, the adsorption of aflatoxin B1 (AfB1) has been tested and compared to that of organoclays incorporating alkylammonium surfactants. AfB1 adsorption isotherms of the modified sepiolite and montmorillonite as well as of the corresponding pristine clays are given in Figure 12A,B, respectively. Most curves could be fit to the Langmuir-type isotherm. According to the fitting, the two SEP-PC composites adsorb AfB1 to a similar extent (about 1440 mg/100 g), indicating a toxin saturation level. The MMT 24 wt % PC system adsorbed AfB1 following the L3-type Giles classification27 with the first threshold of 1915 mg/100 g and subsequent strong adsorption (Figure 12B). Reference materials CLO 30B and SEP-CTA displayed similar Langmuirian adsorption characteristics, however, with a smaller retention capacity. The question of whether AfB1 penetrates the intracrystalline space of NaMMT is a controversially discussed topic in the literature.24,53,54 Interestingly, however, lipid-modified montmorillonites increase the retention of AfB1. This greater AfB1 adsorption is assumed to be related to AfB1 penetration into the hydrophobic region of the lipid layer (hydrophobic interactions) rather than hydrogen bonding and van der Waals and other interactions, as can be postulated for pristine smectite. The calculation of the Gibbs adsorption energy (Table 1) was based on an approach for toxic dyes.55 Lower ΔGads values were estimated for increased lipid content in the PC composites, hence (52) Schultz, L. G. Clays Clay Miner. 1969, 17, 115. (53) Phillips, T. D.; Lemke, S. L.; Grant, P. G. Adv. Exp. Med. Biol. 2002, 504, 157. (54) Kannewischer, I.; Arvide, M. G. T.; White, G. N.; Dixon, J. B. Clay Sci. Jpn. 2006, 12(Supplement 2), 199. (55) Mittal, A. J. Hazard. Mater. 2006, B133, 196.

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Figure 12. (A) Aflatoxin B1 adsorption isotherms and corresponding Langmuir fits if procurable. The isotherms were obtained at ambient temperature after 20 h of magnetic stirring. The modified clays were MMT-24PC, SEP-23PC, SEP-11PC, SEP-12CTA, and CLO 30B (modifier content in wt %). (B) The entire isotherm of MMT-24PC is presented with the inset showing the low-concentration tail. Table 1. Results of Langmuir Isotherm Fitting of AfB1 Adsorption on Modified Claysa clay hybrids

xmax (mg/100 g)

ΔGads (kJ/mol)

R2

MMT-24PC 1915 -38.0 0.881 SEP-23PC 1442 -39.7 0.997 SEP-11PC 1473 -33.2 0.988 SEP-12CTA 1376 -36.1 0.976 CLO 30B 966 -43.2 0.939 a For the calculation of the Gibbs adsorption energy, an approach for toxic dyes was used. The PC content is given in wt %.

suggesting an enhanced toxin affinity of the clay hybrids with increased lipid content. According to the calculated xmax values, the toxin retention of the montmorillonite-PC hybrid was up to 100% higher than for alkyammonium organoclays CLO 30B and SEP-12CTA.

Conclusions In this work, we report a new type of clay-phospholipid material displaying lipid molecules associated with the external surface of sepiolite and the internal surface of montmorillonite. The controlled adsorption of phospholipid molecules on Naþhomoionic montmorillonite and sepiolite was carried out in methanolic and ethanolic solutions. PC intercalation in montmorillonite causes an interlayer swelling of about 4.2 nm, which is compatible with an intracrystalline PC arrangement in form of a lipid bilayer being protected by the rigid silicate framework. The adsorption mechanism in the case of Naþmontmorillonite was found to be a cation exchange process where intracrystalline water enhances the lipid’s affinity for the clay. Langmuir 2010, 26(7), 5217–5225

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The sepiolite-lipid hybrids also include other interaction mechanisms such as hydrogen bonding of the lipid headgroup moieties with the Si-OH groups and the coordinated water molecules located at the external channels of this silicate. The obtained bio-organoclays have been tested for potential applications in a mycotoxin retention study. They demonstrated a superior aflatoxin B1 sequestration capacity as compared to that of the commercial alkylammonium organoclay and at the same time were less harmful to the environment.

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Acknowledgment. This work was supported by the CICYT (Spain, MAT2006-03356 and MAT2009-09960), the Comunidad Autonoma de Madrid (Spain, S-0505/MAT/0027), and the CSIC (Spain, PIF08-018). M.D. and B.W. acknowledge the Spanish MICINN for the award of a Ramon y Cajal contract and the Comunidad de Madrid for a Personal Investigador de Apoyo contract, respectively. We also thank J. Sanz for fruitful discussions of the NMR data, A. Valera for EDX measurements and E. Peiteado from the ICV-CSIC for the contact angle measurements.

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